BACKGROUND:Mpox continues to spread across east and central Africa, with Uganda among the most affected countries. The diagnostic reference standard, centralised real-time quantitative PCR (qPCR), requires specialised infrastructure and sample-transport logistics, producing extended turnaround times that delay public health responses, particularly in remote or underserved settings. Point-of-care molecular diagnostics could address this, but prospective clinical evaluation data from affected regions remain limited. METHODS:We conducted a prospective diagnostic accuracy study across six health facilities in Kampala and Wakiso, Uganda, within routine outpatient and inpatient pathways. Individuals of any age were consecutively enrolled if they presented at a participating site during the enrolment period with signs or symptoms meeting the WHO suspected-case definition for mpox and had at least one active cutaneous lesion amenable to swabbing. Cutaneous lesion swabs were tested at the point-of-care with Dragonfly, a sample-to-result molecular platform using a dual-target design to detect orthopoxvirus (OPXV) and monkeypox virus (MPXV), followed by confirmatory qPCR. The primary outcome was the diagnostic accuracy (sensitivity and specificity) of Dragonfly for MPXV and OPXV against qPCR. Usability and acceptability were assessed in a focus group discussion with front-line users. Clinical and epidemiological associations were examined among concordant participants using a prespecified, literature-informed binary feature set with Fisher's exact tests and Benjamini-Hochberg correction. FINDINGS:Between Sept 17 and Nov 28, 2025, of 300 enrolled participants, 196 (65%) were positive for MPXV by qPCR (median cycle threshold 21·1 [IQR 19·1-23·9]). Among confirmed MPXV cases, 108 (55%) were male and 88 (45%) were female by self-report. For the primary outcome, Dragonfly showed 98·7% (95% CI 96·6-99·5) overall agreement with qPCR, with results available in under 40 min. For MPXV, sensitivity was 98·5% (95·6-99·5) and specificity was 96·2% (90·5-98·5); for OPXV, sensitivity was 100% (98·1-100) and specificity 96·2% (90·6-98·5). Front-line users reported high acceptability, attributing this to avoidance of centralised laboratory logistics, while noting training and supply-chain requirements for routine use. INTERPRETATION:Dragonfly showed high sensitivity and specificity for mpox detection across a broad range of viral loads in the field, supporting its potential as a point-of-care diagnostic in high-burden, resource-limited settings (eg, low-income and middle-income countries, remote environments, or small clinics). Further research should assess integration with existing diagnostics, cost-effectiveness, and performance across clades and key populations. FUNDING:UK Biotechnology and Biological Sciences Research Council, UK Medical Research Council, and Wellcome Trust funded Centres for Antimicrobial Optimisation Network programme.

ABSTRACT In 2022, the World Health Organization declared the worldwide outbreak of mpox to be a public health emergency of international concern. The causative monkeypox virus (MPXV) belonged to clade IIb and is transmitted through sexual contact with a low case fatality rate (0.1%), which, together with under-detection, all contributed to a rapid global spread particularly within the MSM (men who have sex with men) community. As MPXV clade II remains circulating worldwide, a new outbreak of the more fatal clade I disease has been declared in Central and East Africa, and remains uncontrolled in part due to the lack of point-of-care (POC) diagnostics for rapid decisions on treatment and self-isolation. To address the lack of POC solutions for mpox, we have designed and evaluated an orthopoxvirus-specific lateral flow device (LFD) that could be used for the diagnosis of mpox. Using an LFD comprising four monoclonal antibodies against the A27 protein, we demonstrate sensitivity to 3 × 105 pfu/mL. This sensitivity is expected to be sufficient for the detection of MPXV from lesion sites and may also be sufficient for other sample types such as saliva and urine. We found that the presence of guanidinium thiocyanate, a common ingredient in inactivating viral transport media, masked the LFD antigen, resulting in false negatives. POC diagnosis of mpox may be possible using an LFD to reduce delays arising from sample shipment to centralized laboratory testing facilities. In order to achieve this, our work demonstrates that an LFD-optimized buffer is required, as the sample collection buffer may have a detrimental impact on sensitivity for clinical material.IMPORTANCEMpox cases have dramatically increased both in traditionally monkeypox virus endemic countries and also worldwide. This increase comes at a time when immunity derived from smallpox vaccination is no longer available. Diagnosis of mpox is complicated due to both disease presentation and the availability of local diagnostic laboratories. The availability of a point-of-care diagnostic tool such as an lateral flow device (LFD) would play an important role to both diagnose and prevent onward transmission. This manuscript provides developers and assessors with key data for defining true sensitivity and specificity of a successful LFD in addition to buffer conditions for sample collection.

We propose a new nomenclature for mpox virus lineages with sustained human-to-human transmission to improve tracking, communication and public health response.

Poxviruses are dsDNA viruses infecting a wide range of cell types, where they need to contend with multiple host antiviral pathways, including DNA and RNA sensing. Accordingly, poxviruses encode a variety of immune antagonists, most of which are expressed early during infection from within virus cores before uncoating and genome release take place. Amongst these antagonists, the poxvirus immune nuclease (poxin) counteracts the cyclic 2'3'-GMP-AMP (2'3'-cGAMP) synthase (cGAS)/stimulator of interferon genes DNA sensing pathway by degrading the immunomodulatory cyclic dinucleotide 2'3'-cGAMP, the product of activated cGAS. Here, we use poxviruses engineered to lack poxin to investigate how virus infection triggers the activation of STING and its downstream transcription factor interferon-responsive factor 3 (IRF3). Our results demonstrate that poxin-deficient vaccinia virus (VACV) and ectromelia virus (ECTV) induce IRF3 activation in primary fibroblasts and differentiated macrophages, although to a lower extent in VACV compared to ECTV. In fibroblasts, IRF3 activation was detectable at 10 h post-infection (hpi) and was abolished by the DNA replication inhibitor cytosine arabinoside (AraC), indicating that the sensing was mediated by replicated genomes. In macrophages, IRF3 activation was detectable at 4 hpi, and this was not affected by AraC, suggesting that the sensing in this cell type was induced by genomes released from incoming virions. In agreement with this, macrophages expressing short hairpin RNA (shRNA) against the virus uncoating factor D5 showed reduced IRF3 activation upon infection. Collectively, our data show that the viral genome is sensed by cGAS prior to and during genome replication, but immune activation downstream of it is effectively suppressed by poxin. Our data also support the model where virus uncoating acts as an immune evasion strategy to simultaneously cloak the viral genome and allow the expression of early immune antagonists.

Background In the face of the COVID-19 pandemic, the Defence Science and Technology Laboratory (Dstl) and Defence Pathology combined to form the Defence Clinical Lab (DCL), an accredited (ISO/IEC 17025:2017) high-throughput SARS-CoV-2 PCR screening capability for military personnel. Laboratory structure and resource The DCL was modular in organisation, with laboratory modules and supporting functions combining to provide the accredited SARS-CoV-2 (envelope (E)-gene) PCR assay. The DCL was resourced by Dstl scientists and military clinicians and biomedical scientists. Laboratory results Over 12 months of operation, the DCL was open on 289 days and tested over 72 000 samples. Six hundred military SARS-CoV-2-positive results were reported with a median E-gene quantitation cycle (Cq) value of 30.44. The lowest Cq value for a positive result observed was 11.20. Only 64 samples (0.09%) were voided due to assay inhibition after processing started. Conclusions Through a sustained effort and despite various operational issues, the collaboration between Dstl scientific expertise and Defence Pathology clinical expertise provided the UK military with an accredited high-throughput SARS-CoV-2 PCR test capability at the height of the COVID-19 pandemic. The DCL helped facilitate military training and operational deployments contributing to the maintenance of UK military capability. In offering a bespoke capability, including features such as testing samples in unit batches and oversight by military consultant microbiologists, the DCL provided additional benefits to the UK Ministry of Defence that were potentially not available from other SARS-CoV-2 PCR laboratories. The links between Dstl and Defence Pathology have also been strengthened, benefitting future research activities and operational responses.

Eastern equine encephalitis virus (EEEV) usually cycles between Culiseta melanura mosquitoes and birds; however, it can also infect humans. EEEV has a positive-sense RNA genome that, in infected cells, serves as an mRNA for the P1234 polyprotein. P1234 undergoes a series of precise cleavage events producing four nonstructural proteins (nsP1-4) representing subunits of the RNA replicase. Here, we report the construction and properties of a trans-replicase for EEEV. The template RNA of EEEV was shown to be replicated by replicases of diverse alphaviruses. The EEEV replicase, on the other hand, demonstrated limited ability in replicating template RNAs originating from alphaviruses of the Semliki Forest virus complex. The replicase of EEEV was also successfully reconstructed from P123 and nsP4 components. The ability of EEEV P123 to form functional RNA replicases with heterologous nsP4s was more efficient using EEEV template RNA than heterologous alphavirus template RNA. This finding indicates that unlike with previously studied Semliki Forest complex alphaviruses, P123 and/or its processing products have a leading role in EEEV template RNA recognition. Infection of HEK293T cells harboring the EEEV template RNA with EEEV or Western equine encephalitis virus prominently activated expression of a reporter encoded in the template RNA; the effect was much smaller for infection with other alphaviruses and not detectable upon flavivirus infection. At the same time, EEEV infection resulted only in a limited activation of the template RNA of chikungunya virus. Thus, cells harboring reporter-carrying template RNAs can be used as sensitive and selective biosensors for different alphaviruses. IMPORTANCE Infection of EEEV in humans can cause serious neurologic disease with an approximately 30% fatality rate. Although human infections are rare, a record-breaking number was documented in 2019. The replication of EEEV has a unique requirement for host factors but is poorly studied, partly because the virus requires biosafety level 3 facilities which can limit the scope of experiments; at the same time, these studies are crucial for developing antiviral approaches. The EEEV trans-replicase developed here contributes significantly to research on EEEV, providing a safe and versatile tool for studying the virus RNA replication. Using this system, the compatibility of EEEV replicase components with counterparts from other alphaviruses was analyzed. The obtained data can be used to develop unique biosensors that provide alternative methods for detection, identification, quantitation, and neutralization of viable alphaviruses that are compatible with high throughput, semiautomated approaches.

The origin and hazardous potential of human mpox is obscured by a lack of genomic data between the 2018, when exportations from Nigeria were recorded, and 2022 when the global outbreak started. Here, 18 genomes from patients across southern Nigeria in 2019/20 reveal multiple lineages of Monkeypox virus have achieved sustained human-to-human transmission, co-existing in humans for several years and accumulating mutations consistent with APOBEC3 activity suggesting the virus in humans is now segregated from its natural reservoir. Remarkably, three genomes have disruptions in the A46R gene, which contributes to innate immune modulation. The data demonstrates that the A.2 lineage, multiply exported to North America since 2021 independently of the global outbreak, has persisted in Nigeria for more than two years prior to its latest exportation. One-Sentence Summary Mpox is now a human diseae evolving in humans with multiple variants taking separate paths towards adaptation, some analogous to those of Variola

In May, 2015, WHO recommended best practices for naming new infectious diseases to avoid offense or economic effect for any ethnic, regional, or other groups.1WHOWorld Health Organization best practices for the naming of new human infectious diseases.https://www.who.int/publications/i/item/WHO-HSE-FOS-15.1Date: May 15, 2015Date accessed: January 31, 2023Google Scholar Although mpox (formerly known as monkeypox) is not new, WHO has endorsed mpox as the new name for this re-emerging disease and backed the scientific community to agree on neutral nomenclature for variants of viruses. The first report of mpox that led to the discovery of the global outbreak was made to WHO on May 13, 2022. The outbreak spread to 110 countries2WHO2022 monkeypox outbreak: global trends.https://worldhealthorg.shinyapps.io/mpx_global/Date: 2022Date accessed: January 31, 2023Google Scholar and was declared a public health emergency of international concern. The Director-General of WHO called on member states to ensure respect for human rights and to address stigma and discrimination.3WHOWHO Director-General declares the ongoing monkeypox outbreak a public health emergency of international concern.https://www.who.int/europe/news/item/23-07-2022-who-director-general-declares-the-ongoing-monkeypox-outbreak-a-public-health-event-of-international-concernDate: July 23, 2022Date accessed: January 31, 2023Google Scholar As of Jan 31, 2023, there were 85 549 confirmed cases of mpox reported by 110 countries, including 89 deaths.2WHO2022 monkeypox outbreak: global trends.https://worldhealthorg.shinyapps.io/mpx_global/Date: 2022Date accessed: January 31, 2023Google Scholar Mpox is caused by the species monkeypox virus (MPXV), genus Orthopoxvirus, discovered in 1958 in a primate research facility in Denmark, with the first human case reported in 1970.4von Magnus P Andersen EK Birkum Petersen K Birch-Andersen A A pox-like disease in cynomolgus monkeys.Acta Pathol Microbiol Scand. 1959; 46: 156-176Crossref Scopus (354) Google Scholar Two virus clades were identified: the Congo Basin (or central African) clade and the west African clade.5Likos AM Sammons SA Olson VA et al.A tale of two clades: monkeypox viruses.J Gen Virol. 2005; 86: 2661-2672Crossref PubMed Scopus (402) Google Scholar Although stigma became a concern during outbreaks in Africa,6Oyebanji O Ofonagoro U Akande O et al.Lay media reporting of monkeypox in Nigeria.BMJ Glob Health. 2019; 4e002019Crossref PubMed Scopus (7) Google Scholar the 2022 global outbreak reignited discussion with proposals to rename virus clades.7Happi C Adetifa I Mbala P et al.Urgent need for a non-discriminatory and non-stigmatizing nomenclature for monkeypox virus.PLoS Biol. 2022; 20e3001769Crossref PubMed Scopus (81) Google Scholar Although the nomenclature of virus variants is the remit of scientists, reaching consensus quickly was important. On Aug 8, 2022, WHO convened an ad-hoc expert meeting to discuss characteristics of MPXV clades and propose names for them. Participants included orthopoxvirologists, evolutionary biologists, and other scientists from (1) WHO collaborating centres on orthopoxviruses at the US Centers for Disease Control and Prevention and the Russian State Research Centre of Virology and Biotechnology; (2) the WHO Technical Advisory Group on SARS-CoV-2 Virus Evolution; (3) the WHO Advisory Committee on Variola Virus Research; (4) the Poxviridae study group of the International Committee on the Taxonomy of Viruses; (5) research and public health institutes in Africa and around the world; and (6) public virus-sequence databases. The meeting reviewed the phylogeny and characteristics of MPXVs and proposed a neutral naming convention.7Happi C Adetifa I Mbala P et al.Urgent need for a non-discriminatory and non-stigmatizing nomenclature for monkeypox virus.PLoS Biol. 2022; 20e3001769Crossref PubMed Scopus (81) Google Scholar MPXV phylogeny shows two distinct clusters corresponding to the previously recognised clades. Consensus was reached for nomenclature of a Roman numeral for each clade with lowercase Latin characters for subclades; the Congo Basin clade became Clade I and the west African clade became Clade II, encompassing two phylogenetically distinct subclades, IIa and IIb.8WHOMonkeypox: experts give virus variants new names.https://www.who.int/news/item/12-08-2022-monkeypox-experts-give-virus-variants-new-namesDate: Aug 12, 2022Date accessed: October 9, 2022Google Scholar There are appreciable genetic differences between Clades I and II, showing nearly twice the divergence as that between subclades IIa and IIb. Nonetheless, both subclades include genomes from the 1960s and 1970s and appear to have evolved separately from a most recent common ancestor dating back hundreds of years. Neither subclade is descended from the other.5Likos AM Sammons SA Olson VA et al.A tale of two clades: monkeypox viruses.J Gen Virol. 2005; 86: 2661-2672Crossref PubMed Scopus (402) Google Scholar Although the current global outbreak is related primarily to Clade IIb, new cases related to Clade IIa continue to be reported, requiring the tracking of numerous clades and lineages. To distinguish emerging lineages, nomenclature that encodes genealogical relationships between variants was proposed. Lineage labels would follow the convention used for SARS-CoV-2, with an uppercase Latin character followed by a period, and a number representing the nth descendant of the Latin character (eg, Clade IIb.A.1).9Rambaut A Holmes EC O'Toole Á et al.A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology.Nat Microbiol. 2020; 5: 1403-1407Crossref PubMed Scopus (1403) Google Scholar The assignment of virus lineage will help to identify epidemiological links within and across geographic regions and support the understanding of evolutionary dynamics. Parallel discussions considered changing the disease name in the WHO International Classification of Diseases (ICD). WHO issued a public call for new name suggestions for monkeypox in August, 2022. Over 200 proposals received on the ICD platform were reviewed with criteria such as rationale, appropriateness, current usage, scientific accuracy, pronounceability, translatability, potential for confusion, and the guidance for the naming of new diseases. Consultations involved the ICD Medical and Scientific Advisory Committee, the Classification and Statistics Advisory Committee with representation from WHO member states, and the WHO Family of International Classifications. The review recommended the use of mpox as a synonym or inclusion name for monkeypox. The new name was proposed by a men's health community organisation, endorsed by WHO after all suggestions were considered,10WHOWHO recommends new name for monkeypox disease.https://www.who.int/news/item/28-11-2022-who-recommends-new-name-for-monkeypox-diseaseDate: Nov 28, 2022Date accessed: December 1, 2022Google Scholar and is being phased in as a synonym to become the preferred term of the ICD after December, 2023. Discussions on terminology in other languages will continue throughout 2023, providing member states with a choice of preferred term in their language for national usage and statistics. Mpox is now included in ICD-10 and ICD-11, effective as of January, 2023.10WHOWHO recommends new name for monkeypox disease.https://www.who.int/news/item/28-11-2022-who-recommends-new-name-for-monkeypox-diseaseDate: Nov 28, 2022Date accessed: December 1, 2022Google Scholar WHO encourages all member states and stakeholders to follow these recommendations on mpox and its virus clades. EJL was supported by the US National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number U24AI162625. All other authors declare no competing interests. The working group thanks African scientists who advocated for new clade names for MPXV, and front-line health workers and advocates at Rézo, a Montreal community based organisation for gay, bisexual, queer, cis, and trans men who have sex with men, who led a grassroots movement to rename mpox. DU chaired the ad-hoc experts meeting on MPXV clades and variants, which involved authors AA, JB, KK, EJL, MRM, TdO, JO, AR, and RFL, and experts Ifedayo Adetifa, Oluwatoni Akinola, Olajumoke Babatunde, Sylvie Briand, Clarissa Damaso, Inger Damon, Drew Endy, Delia Enria, Mariano Esteban, Bradley Hersh, Christina Hutson, Yu Li, Abdi Mahamud, Sandy Mak, Peter Mala, Colin McInnes, Jean-Vivien Mombouli, Richard Neher, Nnaemeka Ndodo, Mark Perkins, Mike Ryan, Jilian Sacks, Soumya Swaminathan, Henda Triki. We thank Meng Zhang, Co-Chair of the ICD Classification and Statistics Advisory Committee, all participants in these discussions, and Maria Van Kerkhove for ongoing support. The findings and conclusions in this Comment are those of the authors and do not necessarily represent the views of their respective institutions.

Historically, mpox has been characterized as an endemic zoonotic disease that transmits through contact with the reservoir rodent host in West and Central Africa. However, in May 2022, human cases of mpox were detected spreading internationally beyond countries with known endemic reservoirs. When the first cases from 2022 were sequenced, they shared 42 nucleotide differences from the closest mpox virus (MPXV) previously sampled. Nearly all these mutations are characteristic of the action of APOBEC3 deaminases, host enzymes with antiviral function. Assuming APOBEC3 editing is characteristic of human MPXV infection, we developed a dual-process phylogenetic molecular clock that-inferring a rate of ~6 APOBEC3 mutations per year-estimates that MPXV has been circulating in humans since 2016. These observations of sustained MPXV transmission present a fundamental shift to the perceived paradigm of MPXV epidemiology as a zoonosis and highlight the need for revising public health messaging around MPXV as well as outbreak management and control.

The 2022 global mpox outbreak raises questions about how this zoonotic disease established effective human-to-human transmission and its potential for further adaptation. The 2022 outbreak virus is related to an ongoing outbreak in Nigeria originally reported in 2017, but the evolutionary path linking the two remains unclear due to a lack of genomic data between 2018, when virus exportations from Nigeria were first recorded, and 2022, when the global mpox outbreak began. Here, 18 viral genomes obtained from patients across southern Nigeria in 2019-2020 reveal multiple lineages of monkeypox virus (MPXV) co-circulated in humans for several years before 2022, with progressive accumulation of mutations consistent with APOBEC3 activity over time. We identify Nigerian A.2 lineage isolates, confirming the lineage that has been multiply exported to North America independently of the 2022 outbreak originated in Nigeria, and that it has persisted by human-to-human transmission in Nigeria for more than 2 years before its latest exportation. Finally, we identify a lineage-defining APOBEC3-style mutation in all A.2 isolates that disrupts gene A46R, encoding a viral innate immune modulator. Collectively, our data demonstrate MPXV capacity for sustained diversification within humans, including mutations that may be consistent with established mechanisms of poxvirus adaptation.

In 2017, human monkeypox was detected in Nigeria for the first time since 1971, with sporadic outbreaks outside Africa, including in the UK.1Adler H Gould S Hine P et al.Clinical features and management of human monkeypox: a retrospective observational study in the UK.Lancet Infect Dis. 2022; (published online May 24.)https://doi.org/10.1016/S1473-3099(22)00228-6Summary Full Text Full Text PDF PubMed Scopus (645) Google Scholar In May, 2022, the number of cases outside Africa increased substantially with clear extended human-to-human transmission apparently involving intimate contact and with the possibility of transmission by other routes, such as fomites or droplets. This finding indicates that monkeypox virus is using a new route of transmission to overcome barriers that have prevented its emergence as a non-zoonotic human pathogen. Although the current outbreak appears to be mild in adults, young children and immunocompromised adults are at pronounced risk. UK Health Security Agency guidance2UK Health Security AgencyMonkeypox cases confirmed in England—latest updates.https://www.gov.uk/government/news/monkeypox-cases-confirmed-in-england-latest-updatesDate: 2022Date accessed: June 27, 2022Google Scholar recommends that patients isolate until the rash has cleared, and for contacts of a confirmed case to isolate for 21 days after exposure. Rapid point-of-care diagnostics will be an important tool for controlling the spread of infection. Here, we report the characteristics of a prototype lateral flow assay that uses a cocktail of four monoclonal antibodies specific for Old World orthopoxviruses.3Ulaeto DO, Pulford D, Smith J, et al. Differential recognition of orthopoxviruses by antibodies to vaccinia IMV proteins. XIII International Poxvirus and Iridovirus Symposium; Sept 2–6, 2000 (abstr P29).Google Scholar The monoclonal antibodies do not recognise New World orthopoxviruses or members of the Parapoxvirus, Leporipoxvirus, or Suipoxvirus genera.3Ulaeto DO, Pulford D, Smith J, et al. Differential recognition of orthopoxviruses by antibodies to vaccinia IMV proteins. XIII International Poxvirus and Iridovirus Symposium; Sept 2–6, 2000 (abstr P29).Google Scholar Few lateral flow assays were available for preliminary testing using sucrose gradient-purified Modified Vaccinia Ankara in assay sample buffer. Initial titration established a sensitivity limit of between 104·5 and 105 plaque-forming units (appendix p 1). Testing conducted in clinical sample buffer, taken from a standard COVID-19 rapid test kit (BBI Solutions, Crumlin, UK), showed the same sensitivity limit. Testing in human saliva resulted in approximately 1 log reduction in sensitivity. However, if saliva was diluted 1:4 (v/v) in clinical sample buffer the reduction in sensitivity was around 0·5 log. Considering the high concentration of monkeypox virus and the antigen expected on skin and in oral lesions, the sensitivity limit of this assay should be applicable to virus detection in a clinical setting. Our data show the potential of this lateral flow assay for rapid sensitive detection of Orthopoxvirus, which needs to be urgently confirmed with clinical samples from patients with monkeypox. Vaccinia and cowpox viruses are associated with the Orthopoxvirus genus known to infect humans. However, these infections are very rare and have a different clinical presentation to monkeypox. Consequently, by corroborating with clinical presentation, the lateral flow assay could provide a powerful point-of-care diagnostic for monkeypox, which will enhance disease control efforts. MWC received funding from the US FDA to provide materials for this research. PH received the tecoviramat drug from Siga Technologies for an expanded access protocol for monkeypox treatment in Central African Republic. All other authors declare no competing interests. Download .pdf (.17 MB) Help with pdf files Supplementary appendix

The international outbreak of monkeypox that was recognised in May, 2022 represents a new transmission route for monkeypox virus. Since it was first recognised as a zoonotic pathogen in the 1970s, this virus has frequently jumped from its rodent reservoir hosts into people, predominantly in the Democratic Republic of the Congo. Such zoonotic events are estimated at several thousand per year, but are characterised by low human-to-human transmission.1Bunge EM Hoet B Chen L et al.The changing epidemiology of human monkeypox—a potential threat? A systematic review.PLoS Negl Trop Dis. 2022; 16e0010141Crossref PubMed Scopus (695) Google Scholar, 2Rimoin AW Mulembakani PM Johnston SC et al.Major increase in human monkeypox incidence 30 years after smallpox vaccination campaigns cease in the Democratic Republic of Congo.Proc Natl Acad Sci USA. 2010; 107: 16262-16267Crossref PubMed Scopus (439) Google Scholar In the Democratic Republic of the Congo, zoonosis is predominantly seen in school-age boys in rural areas, who engage in hunting small game.3Nolen LD Osadebe L Katomba J et al.Introduction of monkeypox into a community and household: risk factors and zoonotic reservoirs in the Democratic Republic of the Congo.Am J Trop Med Hyg. 2015; 93: 410-415Crossref PubMed Scopus (76) Google Scholar By contrast, the outbreak that began in Nigeria in 2017 predominantly involved men aged 25–40 years in urban or periurban areas, with no obvious connection to suspected animal reservoirs.4Ogoina D Iroezindu M James HI et al.Clinical course and outcome of human monkeypox in Nigeria.Clin Infect Dis. 2020; 71: e210-e214Crossref PubMed Scopus (221) Google Scholar The presentation of monkeypox in the current outbreak is also new. Traditionally, human monkeypox presents as a generalised monomorphic pustular rash, and genital lesions are rare.5Ježek Z Fenner F Human monkeypox. Karger Publishers, Basel1988Google Scholar, 6Huhn GD Bauer AM Yorita K et al.Clinical characteristics of human monkeypox, and risk factors for severe disease.Clin Infect Dis. 2005; 41: 1742-1751Crossref PubMed Scopus (325) Google Scholar, 7Ježek Z Szczeniowski M Paluku K Mutombo M Human monkeypox: clinical features of 282 patients.J Infect Dis. 1987; 156: 293-298Crossref PubMed Scopus (337) Google Scholar In the current international outbreak, including in Nigeria, an ulcerating genital rash develops in the majority of cases. For clinical presentations outside Africa, the genital rash precedes the generalised pustular rash, which is often minor.4Ogoina D Iroezindu M James HI et al.Clinical course and outcome of human monkeypox in Nigeria.Clin Infect Dis. 2020; 71: e210-e214Crossref PubMed Scopus (221) Google Scholar, 8Antinori A Mazzotta V Vita S et al.Epidemiological, clinical and virological characteristics of four cases of monkeypox support transmission through sexual contact, Italy, May 2022.Euro Surveill. 2022; 272200421Crossref PubMed Scopus (303) Google Scholar, 9Vaughan A Aarons E Astbury J et al.Two cases of monkeypox imported to the United Kingdom, September 2018.Euro Surveill. 2018; 231800509Crossref PubMed Scopus (226) Google Scholar, 10Erez N Achdout H Milrot E et al.Diagnosis of imported monkeypox, Israel, 2018.Emerg Infect Dis. 2019; 25: 980-983Crossref PubMed Scopus (217) Google Scholar, 11Hammerschlag Y MacLeod G Papadakis G et al.Monkeypox infection presenting as genital rash, Australia, May 2022.Euro Surveill. 2022; 272200411Crossref PubMed Scopus (98) Google Scholar, 12Duque MP Ribeiro S Martins JV et al.Ongoing monkeypox virus outbreak, Portugal, 29 April to 23 May 2022.Euro Surveill. 2022; 272200424PubMed Google Scholar This presentation suggests that the genital area is a site of primary infection, giving rise to a localised rash, which is then sometimes followed by a secondary disseminated infection. Interestingly, reports from Nigeria up to 2020 and the USA in 2003 describe most patients as having monomorphic lesions, whereas in the current outbreak clinicians in the UK anecdotally report predominantly pleiomorphic lesions at different stages of eruption at the same time. In the current outbreak, monkeypox virus seems to be transmitting via a primary localised rash (appendix). Transmission via primary rash (primary transmission) removes the requirement for the virus to establish a general disseminated infection, and this could facilitate the evolution of variants. Such a transmission route could also facilitate the co-transmission of multiple variants, as primary lesions avoid the bottleneck of secondary dissemination. If monkeypox virus is adapting to human transmission by this novel route, the adaptations should most readily be seen in genomes that are sequenced from primary rash lesions. Although transmission outside central Africa might be predominantly via primary rash, secondary disseminated rashes are still observed. If multiple genomes are transmitted via the primary rash, then the current situation could involve the co-transmission of two syndromes—a localised or primary monkeypox and a generalised or secondary monkeypox—that have different in-host selection pressures and pose different transmission hazards. Although both syndromes are initially caused by the same virus, primary monkeypox is expected to favour variants that are adapted for primary transmission, whereas generalised monkeypox would favour variants that are capable of disseminated infection. Importantly, primary transmission chains are likely to be accelerated relative to transmissions via secondary rash. Also, if the current international spread reflects, or facilitates, adaptation for primary transmission, the incidence of disseminated infections might reduce over time. A reduction in disseminated infections would reduce the risk of transmission by fomites or droplets, but might also lead to a higher proportion of unrecognised infections, increasing the difficulty of breaking the transmission chain. Additionally, if primary transmission facilitates variants that are better adapted to this transmission route, we might expect further evolution as adaptation to humans is refined by natural selection. Monitoring by health authorities for the emergence of variants that are adapted for primary transmission is therefore important. In generalised rashes caused by Orthopoxvirus species, each lesion is thought to be clonal.13Li G Chen N Feng Z et al.Genomic sequence and analysis of a vaccinia virus isolate from a patient with a smallpox vaccine-related complication.Virol J. 2006; 3: 88Crossref PubMed Scopus (22) Google Scholar, 14Qin L Upton C Hazes B Evans DH Genomic analysis of the vaccinia virus strain variants found in Dryvax vaccine.J Virol. 2011; 85: 13049-13060Crossref PubMed Scopus (57) Google Scholar As such, a genome sequence taken from a single lesion might not be representative of the population within the patient, although this might not be the case for lesions that represent a primary focus of infection. If viruses closer to the zoonotic parent are more fit for disseminated infection, then we will expect to see more of these parental sequences recovered from secondary rash lesions, and a higher proportion of adaptive mutations in genomes recovered from primary rash lesions. To gain a full understanding of the evolution and adaptation of monkeypox virus in this international outbreak, sequencing genomes from multiple lesions from both the primary and secondary rash of individual patients is of crucial importance. In practical terms, and particularly in the current outbreaks in Europe, this approach could mean sampling genital and perianal lesions in addition to lesions elsewhere on the body. Studies such as the ISARIC–WHO Clinical Characterisation Protocol for Severe Emerging Infections,15International Severe Acute Respiratory and Emerging Infection ConsortiumMonkeypox response.https://isaric.org/research/monkeypox-response/Date accessed: June 14, 2022Google Scholar which was recently adapted for monkeypox, provide opportunities to obtain longitudinal samples from multiple sites and compartments for virus characterisation. Such an approach enables the comparison of longitudinal virology results with respective exposure histories and clinical descriptions of the rash illness, to explore hypotheses about primary and secondary transmission. We declare no competing interests. Download .pdf (.28 MB) Help with pdf files Supplementary appendix

Rapid and demonstrable inactivation of SARS-CoV-2 is crucial to ensure operator safety during high-throughput testing of clinical samples. The inactivation efficacy of SARS-CoV-2 was evaluated using commercially available lysis buffers from three viral RNA extraction kits used on two high-throughput (96-well) RNA extraction platforms (Qiagen QIAcube HT and the Thermo Fisher KingFisher Flex) in combination with thermal treatment. Buffer volumes and sample ratios were chosen for their optimised suitability for RNA extraction rather than inactivation efficacy and tested against a representative sample type: SARS-CoV-2 spiked into viral transport medium (VTM). A lysis buffer mix from the MagMAX Pathogen RNA/DNA kit (Thermo Fisher), used on the KingFisher Flex, which included guanidinium isothiocyanate (GITC), a detergent, and isopropanol, demonstrated a minimum inactivation efficacy of 1 × 105 tissue culture infectious dose (TCID)50/ml. Alternative lysis buffer mixes from the MagMAX Viral/Pathogen Nucleic Acid kit (Thermo Fisher) also used on the KingFisher Flex and from the QIAamp 96 Virus QIAcube HT Kit (Qiagen) used on the QIAcube HT (both of which contained GITC and a detergent) reduced titres by 1 × 104 TCID50/ml but did not completely inactivate the virus. Heat treatment alone (15 min, 68°C) did not completely inactivate the virus, demonstrating a reduction of 1 × 103 TCID50/ml. When inactivation methods included both heat treatment and addition of lysis buffer, all methods were shown to completely inactivate SARS-CoV-2 inactivation against the viral titres tested. Results are discussed in the context of the operation of a high-throughput diagnostic laboratory.

The three encephalitic alphaviruses, namely, the Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, and WEEV), are classified by the Centers for Disease Control and Prevention (CDC) as biothreat agents. Currently, no licensed medical countermeasures (MCMs) against these viruses are available for humans. Neutralizing antibodies (NAbs) are fast-acting and highly effective MCMs for use in both pre- and post-exposure settings against biothreat agents. While significant work has been done to identify anti-VEEV NAbs, less has been done to identify NAbs against EEEV and WEEV. In order to develop anti-EEEV or -WEEV NAbs, mice were immunized using complementary strategies with a variety of different EEEV or WEEV immunogens to maximize the generation of NAbs to each of these viruses. Of the hybridomas generated, three anti-EEEV and seven anti-WEEV monoclonal antibodies were identified with in vitro neutralization activity. The most potent neutralizers (two anti-EEEV NAbs and three anti-WEEV NAbs) were further evaluated for neutralization activity against additional strains of EEEV, a single strain of Madariaga virus (formerly South American EEEV), or WEEV. Of these, G1-2-H4 and G1-4-C3 neutralized all three EEEV strains and the Madariaga virus strain, whereas G8-2-H9 and 12 WA neutralized six out of eight WEEV strains. To determine the protective efficacy of these NAbs, the five most potent neutralizers were evaluated in respective mouse aerosol challenge models. All five NAbs demonstrated various levels of protection when administered at doses of 2.5 mg/kg or 10 mg/kg 24 h before the respective virus exposure via the aerosol route. Of these, anti-EEEV NAb G1-4-C3 and anti-WEEV NAb 8C2 provided 100% protection at both doses and all surviving mice were free of clinical signs throughout the study. Additionally, no virus was detected in the brain 14 days post virus exposure. Taken together, efficacious NAbs were developed that demonstrate the potential for the development of cross-strain antibody-based MCMs against EEEV and WEEV infections.

Vaccinia virus produces two types of virions known as single-membraned intracellular mature virus (MV) and double-membraned extracellular enveloped virus (EV). EV production peaks earlier when initial MVs are further wrapped and secreted to spread infection within the host. However, late during infection, MVs accumulate intracellularly and become important for host-to-host transmission. The process that regulates this switch remains elusive and is thought to be influenced by host factors. Here, we examined the hypothesis that EV and MV production are regulated by the virus through expression of F13 and the MV-specific protein A26. By switching the promoters and altering the expression kinetics of F13 and A26, we demonstrate that A26 expression downregulates EV production and plaque size, thus limiting viral spread. This process correlates with A26 association with the MV surface protein A27 and exclusion of F13, thus reducing EV titers. Thus, MV maturation is controlled by the abundance of the viral A26 protein, independently of other factors, and is rate limiting for EV production. The A26 gene is conserved within vertebrate poxviruses but is strikingly lost in poxviruses known to be transmitted exclusively by biting arthropods. A26-mediated virus maturation thus has the appearance to be an ancient evolutionary adaptation to enhance transmission of poxviruses that has subsequently been lost from vector-adapted species, for which it may serve as a genetic signature. The existence of virus-regulated mechanisms to produce virions adapted to fulfill different functions represents a novel level of complexity in mammalian viruses with major impacts on evolution, adaptation, and transmission. IMPORTANCE Chordopoxviruses are mammalian viruses that uniquely produce a first type of virion adapted to spread within the host and a second type that enhances transmission between hosts, which can take place by multiple ways, including direct contact, respiratory droplets, oral/fecal routes, or via vectors. Both virion types are important to balance intrahost dissemination and interhost transmission, so virus maturation pathways must be tightly controlled. Here, we provide evidence that the abundance and kinetics of expression of the viral protein A26 regulates this process by preventing formation of the first form and shifting maturation toward the second form. A26 is expressed late after the initial wave of progeny virions is produced, so sufficient viral dissemination is ensured, and A26 provides virions with enhanced environmental stability. Conservation of A26 in all vertebrate poxviruses, but not in those transmitted exclusively via biting arthropods, reveals the importance of A26-controlled virus maturation for transmission routes involving environmental exposure.

A small-scale study with Mosi-guard Natural spray, an insect repellent containing Citriodiol, was performed to determine if it has virucidal activity against SARS-CoV-2. A liquid test examined the activity of the insect repellent and the individual components for virucidal activity. A surface contact test looked at the activity of the insect repellent when impregnated on a latex surface as a synthetic skin for potential topical prophylactic application. Both Mosi-guard Natural spray and Citriodiol, as well as other components of the repellent, had virucidal activity in the liquid contact test. On a latex surface used to simulate treated skin, the titre of SARS-CoV-2 was less over time on the Mosi-guard Natural-treated surface but virus was still recovered.

Smallpox eradication, coordinated by the WHO and certified 40 years ago, led to the cessation of routine smallpox vaccination in most countries. It is estimated that over 70% of the world's population is no longer protected against smallpox, and through cross-immunity, to closely related orthopox viruses such as monkeypox. Monkeypox is now a re-emerging disease. Monkeypox is endemic in as yet unconfirmed animal reservoirs in sub-Saharan Africa, while its human epidemiology appears to be changing. Monkeypox in small animals imported from Ghana as exotic pets was at the origin of an outbreak of human monkeypox in the USA in 2003. Travellers infected in Nigeria were at the origin of monkeypox cases in the UK in 2018 and 2019, Israel in 2018 and Singapore in2019. Together with sporadic reports of human infections with other orthopox viruses, these facts invite speculation that emergent or re-emergent human monkeypox might fill the epidemiological niche vacated by smallpox. An ad-hoc and unofficial group of interested experts met to consider these issues at Chatham House, London in June 2019, in order to review available data and identify monkeypox-related research gaps. Gaps identified by the experts included:The experts further agreed on the need for a better understanding of the genomic evolution and changing epidemiology of orthopox viruses, the usefulness of in-field genomic diagnostics, and the best disease control strategies, including the possibility of vaccination with new generation non-replicating smallpox vaccines and treatment with recently developed antivirals.

Western equine encephalitis virus (WEEV) naturally cycles between mosquitos and birds or rodents, with a case fatality rate of up to 15% in humans during epizootic outbreaks. There are no medical countermeasures to treat WEEV infection, and accidental aerosol exposure increases the case fatality rate up to 40%. Understanding the pathogenesis of infection is required to develop and assess medical countermeasures. This study describes the clinical and pathological findings of mice infected with WEEV by the aerosol route, and use as a model for WEEV infection in humans. Balb/c mice were infected by the aerosol route with a dose range of high-virulence WEEV strain Fleming to establish the median lethal dose (MLD). The disease course was acute, culminating in severe clinical signs, neuroinvasion, and dose-dependent mortality. Further groups of mice were exposed by the aerosol route, periodically sacrificed, and tissues excised for histopathological examination and virology. Viral titres peaked four days post-challenge in the brain and lungs, corresponding with severe bilateral lesions in rostroventral regions of the encephalon, especially in the olfactory bulb and piriform cortex. Recapitulation of the most serious clinical presentations of human WEEV disease in mice may prove a useful tool in the evaluation of medical countermeasures.

In recent years concern has mounted regarding the possibility of a re-emergence of smallpox through biowarfare or bioterrorism. There is also concern over the incidence of human monkeypox in endemic areas and the potential for monkeypox to be accidentally transported to non-endemic areas. In the event of re-emergence of smallpox or emergence of monkeypox, the accepted route of administration for live replicating smallpox vaccine is dermal scarification, which generates a virus-shedding lesion that persists for several days at the vaccination site. The lesion is a potential source of contact transmission of vaccine to individuals who may be contra-indicated for receipt of the live vaccine. In this study, we compare dermal scarification with intramuscular vaccination for replicating smallpox vaccine in a mouse lethal challenge model. Comparisons are made over multiple vaccine and challenge doses and data recorded for lethality, disease severity, and antibody responses. Qualitative and quantitative differences between the two routes are observed, and for the intramuscular route the febrile response is not suppressed after subsequent virulent vaccinia virus challenge. However both routes generate an immune response and protect from severe disease and death. Although dermal scarification is the preferred route of vaccination for the general population, intramuscular vaccination may be an option for people who are not contraindicated for the live vaccine, but who are close contacts of people who are contraindicated for the live vaccine, in an emergency situation.

Smallpox vaccination carries a high risk of adverse events in recipients with a variety of contra-indications for live vaccines. Although alternative non-replicating vaccines have been described in the form of replication-deficient vaccine viruses, DNA vaccines, and subunit vaccines, these are less efficacious than replicating vaccines in animal models. DNA and subunit vaccines in particular have not been shown to give equivalent protection to the traditional replicating smallpox vaccine. We show here that combinations of the orthopoxvirus A27, A33, B5 and L1 proteins give differing levels of protection when administered in different combinations with different adjuvants. In particular, the combination of B5 and A27 proteins adjuvanted with CpG oligodeoxynucleotides (ODN) gives a level of protection in mice that is equivalent to the Lister traditional vaccine in a lethal vaccinia virus challenge model.