Multiple introductions of monkeypox virus to Ireland during the international mpox outbreak, May 2022 to October 2023.

BackgroundMpox, caused by monkeypox virus (MPXV), was considered a rare zoonotic disease before May 2022, when a global epidemic of cases in non-endemic countries led to the declaration of a Public Health Emergency of International Concern. Cases of mpox in Ireland, a country without previous mpox reports, could reflect extended local transmission or multiple epidemiological introductions.AimTo elucidate the origins and molecular characteristics of MPXV circulating in Ireland between May 2022 and October 2023.MethodsWhole genome sequencing of MPXV from 75% of all Irish mpox cases (182/242) was performed and compared to sequences retrieved from public databases (n = 3,362). Bayesian approaches were used to infer divergence time between sequences from different subclades and evaluate putative importation events from other countries.ResultsOf 242 detected mpox cases, 99% were males (median age: 35 years; range: 15-60). All 182 analysed genomes were assigned to Clade IIb and, presence of 12 distinguishable subclades suggests multiple introductions into Ireland. Estimation of time to divergence of subclades further supports the hypothesis for multiple importation events from numerous countries, indicative of extended and sustained international spread of mpox. Further analysis of sequences revealed that 92% of nucleotide mutations were from cytosine to thymine (or from guanine to adenine), leading to a high number of non-synonymous mutations across subclades; mutations associated with tecovirimat resistance were not observed.ConclusionWe provide insights into the international transmission dynamics supporting multiple introductions of MPXV into Ireland. Such information supported the implementation of evidence-informed public health control measures.

Antisense oligonucleotide targeting hepatic Serum Amyloid A limits the progression of angiotensin II-induced abdominal aortic aneurysm formation.

BACKGROUND AND AIMS: Obesity increases the risk for abdominal aortic aneurysms (AAA) in humans and enhances angiotensin II (AngII)-induced AAA formation in C57BL/6 mice. We reported that deficiency of Serum Amyloid A (SAA) significantly reduces AngII-induced inflammation and AAA in both hyperlipidemic apoE-deficient and obese C57BL/6 mice. The aim of this study is to investigate whether SAA plays a role in the progression of early AAA in obese C57BL/6 mice. METHODS: Male C57BL/6J mice were fed a high-fat diet (60% kcal as fat) throughout the study. After 4 months of diet, the mice were infused with AngII until the end of the study. Mice with at least a 25% increase in the luminal diameter of the abdominal aorta after 4 weeks of AngII infusion were stratified into 2 groups. The first group received a control antisense oligonucleotide (Ctr ASO), and the second group received ASO that suppresses SAA (SAA-ASO) until the end of the study. RESULTS: Plasma SAA levels were significantly reduced by the SAA ASO treatment. While mice that received the control ASO had continued aortic dilation throughout the AngII infusion periods, the mice that received SAA-ASO had a significant reduction in the progression of aortic dilation, which was associated with significant reductions in matrix metalloprotease activities, decreased macrophage infiltration and decreased elastin breaks in the abdominal aortas. CONCLUSIONS: We demonstrate for the first time that suppression of SAA protects obese C57BL/6 mice from the progression of AngII-induced AAA. Suppression of SAA may be a therapeutic approach to limit AAA progression.

A phased strengthening of laboratory capacity in the Eastern Mediterranean Region during the COVID-19 pandemic.

The Eastern Mediterranean Region (EMR) faces ongoing challenges in its public health system due to limited resources, logistical issues, and political disruptions. The COVID-19 pandemic accelerated the need for stronger laboratory capacities to handle the increased demand for testing. In a phased response, EMR countries utilized the National Influenza Centers to rapidly establish and scale molecular testing for SARS-CoV-2, the causative agent of COVID-19. The expansion of capacity included strong collaborations between public health bodies and private and academic sectors to decentralize and expand testing to the subnational level. To ensure that the quality of testing was not impacted by rapid expansion, national and subnational laboratories were enrolled in external quality assurance programs for the duration of the response. Implementation of genomic surveillance was prioritized for variant tracking, leading to the establishment of regional sequencing reference laboratories and the distribution of MinION sequencing platforms to complex emergency countries who previously had limited experience with pathogen sequencing. Challenges included a lack of technical expertise, including in implementing novel diagnostic assays and sequencing, a lack of bioinformatics expertise in the region, and significant logistical and procurement challenges. The collaborative approach, coordinated through the WHO Eastern Mediterranean Regional Office, enabled all 22 countries to achieve SARS-CoV-2 diagnostic capabilities, highlighting the pivotal role of laboratories in global health security.

Deficiency of Acute-Phase Serum Amyloid A Exacerbates Sepsis-Induced Mortality and Lung Injury in Mice.

Serum amyloid A (SAA) is a family of proteins, the plasma levels of which may increase >1000-fold in acute inflammatory states. We investigated the role of SAA in sepsis using mice deficient in all three acute-phase SAA isoforms (SAA-TKO). SAA deficiency significantly increased mortality rates in the three experimental sepsis mouse models: cecal ligation and puncture (CLP), cecal slurry (CS) injection, and lipopolysaccharide (LPS) treatments. SAA-TKO mice had exacerbated lung pathology compared to wild-type (WT) mice after CLP. A bulk RNA sequencing performed on lung tissues excised 24 h after CLP indicated significant enrichment in the expression of genes associated with chemokine production, chemokine and cytokine-mediated signaling, neutrophil chemotaxis, and neutrophil migration in SAA-TKO compared to WT mice. Consistently, myeloperoxidase activity and neutrophil counts were significantly increased in the lungs of septic SAA-TKO mice compared to WT mice. The in vitro treatment of HL-60, neutrophil-like cells, with SAA or SAA bound to a high-density lipoprotein (SAA-HDL), significantly decreased cellular transmigration through laminin-coated membranes compared to untreated cells. Thus, SAA potentially prevents neutrophil transmigration into injured lungs, thus reducing exacerbated tissue injury and mortality. In conclusion, we demonstrate for the first time that endogenous SAA plays a protective role in sepsis, including ameliorating lung injury.

Implementation and expansion of laboratory capacity for molecular diagnostics in response to COVID-19 and preparedness for other emerging infectious diseases in the Islamic Emirate of Afghanistan.

Background: Afghanistan experienced various outbreaks before and during the Covid-19 pandemic, including dengue, Crimean Congo hemorrhagic fever (CCHF), measles, and acute watery diarrhea (AWD). Diagnostic and surveillance support was limited, with only the Central Public Health Laboratory equipped to handle outbreak responses. This article highlights initiatives taken to improve diagnostic capabilities for COVID-19 and other outbreaks of public health concern encountered during the pandemic. Background: The World Health Organization (WHO) Afghanistan Country Office collaborated with the WHO Eastern Mediterranean Regional Office (EMRO), Central Public Health Laboratory (CPHL), and National Influenza Center (NIC) to enhance COVID-19 diagnostic capacity at national and subnational facilities. To alleviate pressure on CPHL, a state-of-the-art laboratory was established at the National Infectious Disease Hospital (NIDH) in Kabul in 2021-2022, while WHO EMRO facilitated the regionalization of testing to subnational facilities for dengue, CCHF, and AWD in 2022-2023. Results: COVID-19 testing capacity expanded nationwide to 34 Biosafety Level II labs, improving diagnosis time. Daily testing rose from 1000 in 2020 to 9200 in 2023, with 848,799 cumulative tests. NIDH identified 229 CCHF cases and 45 cases nationally. Dengue and CCHF testing, decentralized to Nangarhar and Kandahar labs, identified 338 dengue and 18 CCHF cases. AWD testing shifted to NIDH and five subnational facilities (Kandahar, Paktia, Balkh, Herat, and Nangarhar labs), while measles testing also decentralized to nine subnational facilities. Conclusion: Afghanistan implemented a remarkable, multisectoral response to priority pathogens. The nation now possesses diagnostic expertise at national and subnational levels, supported by genomic surveillance. Future efforts should concentrate on expanding and sustaining this capacity to enhance public health responses.

Monitoring the quality of SARS-CoV-2 virus detection in molecular diagnostic laboratories in the Eastern Mediterranean Region during the COVID-19 pandemic.

INTRODUCTION: The COVID-19 pandemic placed unprecedented stress on laboratories in the Eastern Mediterranean Region. Building on existing capacity for influenza diagnostics, countries introduced COVID-19 diagnostic support to ~100% regional coverage. A key challenge during the expansion was maintaining quality testing in laboratories, ensuring that correct results were shared with medical facilities. METHODS: WHO organized two rounds of independently monitored severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) external quality assurance programs (EQAP). The Public Health Laboratory (PHL) division of WHO supplied external quality assurance (EQA) panels, from the Royal College of Pathologists of Australasia Quality Assurance Programme (RCPAQAP) Australia to laboratories not enrolled in recurring Global Influenza Surveillance and Response System (GISRS) quality assurance programs, in which national influenza centers routinely participate. RESULTS: Fifteen and 14 countries participated in PHL/EQAP for SARS-CoV-2 between 2020 and 2022. Concordance was consistent between rounds, reaching 96.4% and 89.9%. A separate assessment of GISRS/EQAP to national-level laboratories identified high levels of response and concordance for SARS-CoV-2 (100% response, 93% concordance), which was reduced for influenza (50% response rate, 80% concordance), reflecting the challenge of prioritizing pathogens during outbreaks. CONCLUSION: The proliferation of laboratories in response to COVID-19 was a success story from the pandemic. However, monitoring the quality of laboratories was challenging via existing EQAP. The addition of PHL/EQAP provided a mechanism to monitor performance of laboratories that were not designated as national influenza centers. While a high proportion of laboratories attained good results, continual emphasis on quality and enrollment in EQAP is key to ensuring sustainability of laboratory testing in future.

Key aspects defining the development and implementation of a regional genomic surveillance strategy for the Eastern Mediterranean Region.

The COVID-19 pandemic highlighted the critical role of pathogen sequencing in making informed public health decisions. Initially, the Eastern Mediterranean Region faced limitations in sequencing capacity. However, with robust WHO and stakeholder support, the situation significantly improved. By 2022, COVID-19 sequencing was underway in 22 out of 23 regional countries, with varying throughput and capacity. Notably, three genomic hubs were established in Oman, UAE, and Morocco, playing a key role in providing expanded genomics training and support across the region. While primarily for COVID-19 surveillance, this sequencing capacity offers an opportunity to integrate genomic surveillance into existing networks. This integration can enable early detection and response to high-threat pathogens with pandemic potential. To advance this, WHO/EMRO collaborated with stakeholders to formulate the Eastern Mediterranean Regional Genomic Surveillance Strategy for Emerging Pathogens of Pandemic Concern. Consultative meetings with regional and international genomic surveillance experts identified strategy focal points, key partners, priority pathogens, and implementation steps. As the strategy awaits member states' ratification in Q4 2023, this manuscript outlines pivotal facets defined by member states and the strategic document's key deliverables and opportunities. These efforts aim to yield a substantial positive impact within the region.

A model for public-private partnership during the COVID-19 pandemic: Lessons from Biolab and public laboratories working in the Hashemite Kingdom of Jordan.

INTRODUCTION: The global COVID-19 pandemic overwhelmed national public health and laboratory capacity in Jordan and globally. In response, Biolab, a private laboratory group with 27 branches across Jordan, assisted with testing. Biolab was equipped to quickly increase molecular testing capacity without compromising quality or turnaround time, allowing them to contribute to national COVID-19 surveillance efforts. METHODS: Biolab expanded testing in Jordan by operationalizing automated testing platforms at various locations, including 16 branches, 2 drive-through and 2 walk-through centres, and entry points for airports and marine passenger arrivals. Genomic and molecular testing were implemented to track variants. Information technology platforms were introduced for sample management, registration, and commercial sample payments. Data were directly provided to the Ministry of Health through these platforms to support public health decision-making and responses. Biolab prioritized staff well-being by providing mental, financial, and physical health support during the pandemic. RESULTS: Biolab processed more than two million samples, with a turnaround time of ~1.5 h. Results were transmitted directly to key stakeholders in near real time. Biolab conducted variant evaluations on >1.4 million samples using molecular variant testing and >1000 samples using whole genome sequencing. Biolab prioritized staff well-being, improving staff satisfaction from 74% to 91%, a remarkable achievement when many laboratory systems experienced staff burnout and dissatisfaction. CONCLUSION: The collaboration between public and private laboratories during COVID-19 established a model for future joint efforts to prevent outbreaks from becoming pandemics. Biolab's focus on efficiency, quality, and staff well-being enabled consistent, high-quality performance. The introduction of innovative information technology platforms ensured swift information dissemination. Biolab plans to continue investing in these platforms and expand pathogen testing, creating a top-tier testing infrastructure in Jordan with a demonstrated ability to cooperate with the government for public benefit.

Antisense Oligonucleotide Targeting Hepatic Serum Amyloid A Limits the Progression of Angiotensin II-Induced Abdominal Aortic Aneurysm Formation.

OBJECTIVE: Obesity increases the risk for abdominal aortic aneurysms (AAA) in humans and enhances angiotensin II (AngII)-induced AAA formation in C57BL/6 mice. Obesity is also associated with increases in serum amyloid A (SAA). We previously reported that deficiency of SAA significantly reduces AngII-induced inflammation and AAA in both hyperlipidemic apoE-deficient and obese C57BL/6 mice. In this study, we investigated whether SAA plays a role in the progression of early AAA in obese C57BL/6 mice. APPROACH AND RESULTS: Male C57BL/6J mice were fed a high-fat diet (60% kcal as fat) throughout the study. After 4 months of diet, the mice were infused with angiotensin II (AngII) until the end of the study. Mice with at least a 25% increase in the luminal diameter of the abdominal aorta after 4 weeks of AngII infusion were stratified into 2 groups. The first group received a control antisense oligonucleotide (Ctr ASO), and the second group received ASO that suppresses SAA (SAA-ASO) until the end of the study. Plasma SAA levels were significantly reduced by the SAA ASO treatment. While mice that received the control ASO had continued aortic dilation throughout the AngII infusion periods, the mice that received SAA-ASO had a significant reduction in the progression of aortic dilation, which was associated with significant reductions in matrix metalloprotease activities, decreased macrophage infiltration and decreased elastin breaks in the abdominal aortas. CONCLUSION: We demonstrate for the first time that suppression of SAA protects obese C57BL/6 mice from the progression of AngII-induced AAA. Suppression of SAA may be a therapeutic approach to limit AAA progression.

Patterns of within-host genetic diversity in SARS-CoV-2.

Monitoring the spread of SARS-CoV-2 and reconstructing transmission chains has become a major public health focus for many governments around the world. The modest mutation rate and rapid transmission of SARS-CoV-2 prevents the reconstruction of transmission chains from consensus genome sequences, but within-host genetic diversity could theoretically help identify close contacts. Here we describe the patterns of within-host diversity in 1181 SARS-CoV-2 samples sequenced to high depth in duplicate. 95.1% of samples show within-host mutations at detectable allele frequencies. Analyses of the mutational spectra revealed strong strand asymmetries suggestive of damage or RNA editing of the plus strand, rather than replication errors, dominating the accumulation of mutations during the SARS-CoV-2 pandemic. Within- and between-host diversity show strong purifying selection, particularly against nonsense mutations. Recurrent within-host mutations, many of which coincide with known phylogenetic homoplasies, display a spectrum and patterns of purifying selection more suggestive of mutational hotspots than recombination or convergent evolution. While allele frequencies suggest that most samples result from infection by a single lineage, we identify multiple putative examples of co-infection. Integrating these results into an epidemiological inference framework, we find that while sharing of within-host variants between samples could help the reconstruction of transmission chains, mutational hotspots and rare cases of superinfection can confound these analyses.

The COVID-19 pandemic has had major health impacts across the globe. The scientific community has focused much attention on finding ways to monitor how the virus responsible for the pandemic, SARS-CoV-2, spreads. One option is to perform genetic tests, known as sequencing, on SARS-CoV-2 samples to determine the genetic code of the virus and to find any differences or mutations in the genes between the viral samples. Viruses mutate within their hosts and can develop into variants that are able to more easily transmit between hosts. Genetic sequencing can reveal how genetically similar two SARS-CoV-2 samples are. But tracking how SARS-CoV-2 moves from one person to the next through sequencing can be tricky. Even a sample of SARS-CoV-2 viruses from the same individual can display differences in their genetic material or within-host variants. Could genetic testing of within-host variants shed light on factors driving SARS-CoV-2 to evolve in humans? To get to the bottom of this, Tonkin-Hill, Martincorena et al. probed the genetics of SARS-CoV-2 within-host variants using 1,181 samples. The analyses revealed that 95.1% of samples contained within-host variants. A number of variants occurred frequently in many samples, which were consistent with mutational hotspots in the SARS-CoV-2 genome. In addition, within-host variants displayed mutation patterns that were similar to patterns found between infected individuals. The shared within-host variants between samples can help to reconstruct transmission chains. However, the observed mutational hotspots and the detection of multiple strains within an individual can make this challenging. These findings could be used to help predict how SARS-CoV-2 evolves in response to interventions such as vaccines. They also suggest that caution is needed when using information on within-host variants to determine transmission between individuals.

Superspreaders drive the largest outbreaks of hospital onset COVID-19 infections.

SARS-CoV-2 is notable both for its rapid spread, and for the heterogeneity of its patterns of transmission, with multiple published incidences of superspreading behaviour. Here, we applied a novel network reconstruction algorithm to infer patterns of viral transmission occurring between patients and health care workers (HCWs) in the largest clusters of COVID-19 infection identified during the first wave of the epidemic at Cambridge University Hospitals NHS Foundation Trust, UK. Based upon dates of individuals reporting symptoms, recorded individual locations, and viral genome sequence data, we show an uneven pattern of transmission between individuals, with patients being much more likely to be infected by other patients than by HCWs. Further, the data were consistent with a pattern of superspreading, whereby 21% of individuals caused 80% of transmission events. Our study provides a detailed retrospective analysis of nosocomial SARS-CoV-2 transmission, and sheds light on the need for intensive and pervasive infection control procedures.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, presents a global public health challenge. Hospitals have been at the forefront of this battle, treating large numbers of sick patients over several waves of infection. Finding ways to manage the spread of the virus in hospitals is key to protecting vulnerable patients and workers, while keeping hospitals running, but to generate effective infection control, researchers must understand how SARS-CoV-2 spreads. A range of factors make studying the transmission of SARS-CoV-2 in hospitals tricky. For instance, some people do not present any symptoms, and, amongst those who do, it can be difficult to determine whether they caught the virus in the hospital or somewhere else. However, comparing the genetic information of the SARS-CoV-2 virus from different people in a hospital could allow scientists to understand how it spreads. Samples of the genetic material of SARS-CoV-2 can be obtained by swabbing infected individuals. If the genetic sequences of two samples are very different, it is unlikely that the individuals who provided the samples transmitted the virus to one another. Illingworth, Hamilton et al. used this information, along with other data about how SARS-CoV-2 is transmitted, to develop an algorithm that can determine how the virus spreads from person to person in different hospital wards. To build their algorithm, Illingworth, Hamilton et al. collected SARS-CoV-2 genetic data from patients and staff in a hospital, and combined it with information about how SARS-CoV-2 spreads and how these people moved in the hospital . The algorithm showed that, for the most part, patients were infected by other patients (20 out of 22 cases), while staff were infected equally by patients and staff. By further probing these data, Illingworth, Hamilton et al. revealed that 80% of hospital-acquired infections were caused by a group of just 21% of individuals in the study, identifying a 'superspreader' pattern. These findings may help to inform SARS-CoV-2 infection control measures to reduce spread within hospitals, and could potentially be used to improve infection control in other contexts.

A luciferase-based approach for measuring HBGA blockade antibody titers against human norovirus.

BACKGROUND: Noroviruses are the most common cause of viral gastroenteritis worldwide, yet there is a deficit in the understanding of protective immunity. Surrogate neutralization assays have been widely used that measure the ability of antibodies to block virus-like particle (VLP) binding to histo-blood group antigens (HBGAs). However, screening large sample sets against multiple antigens using the traditional HBGA blocking assay requires significant investment in terms of time, equipment, and technical expertise, largely associated with the generation of purified VLPs. METHODS: To address these issues, a luciferase immunoprecipitation system (LIPS) assay was modified to measure the norovirus-specific HBGA blockade activity of antibodies. The assay (designated LIPS-Blockade) was validated using a panel of well-characterized homotypic and heterotypic hyperimmune sera as well as strain-specific HBGA blocking monoclonal antibodies. RESULTS: The LIPS-Blockade assay was comparable in specificity to a standard HBGA blocking protocol performed with VLPs. Using time-ordered patient sera, the luciferase-based approach was also able to detect changes in HBGA blocking titers following viral challenge and natural infection with norovirus. CONCLUSION: In this study we developed a rapid, robust, and scalable surrogate neutralization assay for noroviruses that circumvented the need for purified VLPs. This LIPS-Blockade assay should streamline the process of large-scale immunological studies, ultimately aiding in the characterization of protective immunity to human noroviruses.

Genomic epidemiology of COVID-19 in care homes in the east of England.

COVID-19 poses a major challenge to care homes, as SARS-CoV-2 is readily transmitted and causes disproportionately severe disease in older people. Here, 1167 residents from 337 care homes were identified from a dataset of 6600 COVID-19 cases from the East of England. Older age and being a care home resident were associated with increased mortality. SARS-CoV-2 genomes were available for 700 residents from 292 care homes. By integrating genomic and temporal data, 409 viral clusters within the 292 homes were identified, indicating two different patterns - outbreaks among care home residents and independent introductions with limited onward transmission. Approximately 70% of residents in the genomic analysis were admitted to hospital during the study, providing extensive opportunities for transmission between care homes and hospitals. Limiting viral transmission within care homes should be a key target for infection control to reduce COVID-19 mortality in this population.

A novel antiviral formulation inhibits a range of enveloped viruses.

Some free fatty acids derived from milk and vegetable oils are known to have potent antiviral and antibacterial properties. However, therapeutic applications of short- to medium-chain fatty acids are limited by physical characteristics such as immiscibility in aqueous solutions. We evaluated a novel proprietary formulation based on an emulsion of short-chain caprylic acid, ViroSAL, for its ability to inhibit a range of viral infections in vitro and in vivo. In vitro, ViroSAL inhibited the enveloped viruses Epstein-Barr, measles, herpes simplex, Zika and orf parapoxvirus, together with Ebola, Lassa, vesicular stomatitis and severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) pseudoviruses, in a concentration- and time-dependent manner. Evaluation of the components of ViroSAL revealed that caprylic acid was the main antiviral component; however, the ViroSAL formulation significantly inhibited viral entry compared with caprylic acid alone. In vivo, ViroSAL significantly inhibited Zika and Semliki Forest virus replication in mice following the inoculation of these viruses into mosquito bite sites. In agreement with studies investigating other free fatty acids, ViroSAL had no effect on norovirus, a non-enveloped virus, indicating that its mechanism of action may be surfactant disruption of the viral envelope. We have identified a novel antiviral formulation that is of great interest for the prevention and/or treatment of a broad range of enveloped viruses, particularly those of the skin and mucosal surfaces.

Rapid implementation of SARS-CoV-2 sequencing to investigate cases of health-care associated COVID-19: a prospective genomic surveillance study.

BACKGROUND: The burden and influence of health-care associated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is unknown. We aimed to examine the use of rapid SARS-CoV-2 sequencing combined with detailed epidemiological analysis to investigate health-care associated SARS-CoV-2 infections and inform infection control measures. METHODS: In this prospective surveillance study, we set up rapid SARS-CoV-2 nanopore sequencing from PCR-positive diagnostic samples collected from our hospital (Cambridge, UK) and a random selection from hospitals in the East of England, enabling sample-to-sequence in less than 24 h. We established a weekly review and reporting system with integration of genomic and epidemiological data to investigate suspected health-care associated COVID-19 cases. FINDINGS: Between March 13 and April 24, 2020, we collected clinical data and samples from 5613 patients with COVID-19 from across the East of England. We sequenced 1000 samples producing 747 high-quality genomes. We combined epidemiological and genomic analysis of the 299 patients from our hospital and identified 35 clusters of identical viruses involving 159 patients. 92 (58%) of 159 patients had strong epidemiological links and 32 (20%) patients had plausible epidemiological links. These results were fed back to clinical, infection control, and hospital management teams, leading to infection-control interventions and informing patient safety reporting. INTERPRETATION: We established real-time genomic surveillance of SARS-CoV-2 in a UK hospital and showed the benefit of combined genomic and epidemiological analysis for the investigation of health-care associated COVID-19. This approach enabled us to detect cryptic transmission events and identify opportunities to target infection-control interventions to further reduce health-care associated infections. Our findings have important implications for national public health policy as they enable rapid tracking and investigation of infections in hospital and community settings. FUNDING: COVID-19 Genomics UK funded by the Department of Health and Social Care, UK Research and Innovation, and the Wellcome Sanger Institute.

Rapid in-country sequencing of whole virus genomes to inform rabies elimination programmes.

Genomic surveillance is an important aspect of contemporary disease management but has yet to be used routinely to monitor endemic disease transmission and control in low- and middle-income countries. Rabies is an almost invariably fatal viral disease that causes a large public health and economic burden in Asia and Africa, despite being entirely vaccine preventable. With policy efforts now directed towards achieving a global goal of zero dog-mediated human rabies deaths by 2030, establishing effective surveillance tools is critical. Genomic data can provide important and unique insights into rabies spread and persistence that can direct control efforts. However, capacity for genomic research in low- and middle-income countries is held back by limited laboratory infrastructure, cost, supply chains and other logistical challenges. Here we present and validate an end-to-end workflow to facilitate affordable whole genome sequencing for rabies surveillance utilising nanopore technology. We used this workflow in Kenya, Tanzania and the Philippines to generate rabies virus genomes in two to three days, reducing costs to approximately £60 per genome. This is over half the cost of metagenomic sequencing previously conducted for Tanzanian samples, which involved exporting samples to the UK and a three- to six-month lag time. Ongoing optimization of workflows are likely to reduce these costs further. We also present tools to support routine whole genome sequencing and interpretation for genomic surveillance. Moreover, combined with training workshops to empower scientists in-country, we show that local sequencing capacity can be readily established and sustainable, negating the common misperception that cutting-edge genomic research can only be conducted in high resource laboratories. More generally, we argue that the capacity to harness genomic data is a game-changer for endemic disease surveillance and should precipitate a new wave of researchers from low- and middle-income countries.

Pharmacokinetics of TKM-130803 in Sierra Leonean patients with Ebola virus disease: plasma concentrations exceed target levels, with drug accumulation in the most severe patients.

BACKGROUND: TKM-130803 is a specific anti-EBOV therapeutic comprised of two small interfering RNAs (siRNA) siLpol-2 and siVP35-2. The pharmacokinetics (PK) of these siRNAs was defined in Ebola virus disease (EVD) patients, with reference to efficacy (ET) and toxicology thresholds (TT). The relationship between PK and patient survival was explored. METHODS: Pharmacokinetic (PK) and pharmacodynamic (PD) data were available for seven participants with EVD in Sierra Leone who received 0·3 mg/kg of TKM-130803 by intravenous infusion over 2 h daily for up to 7 days. Plasma concentration of siRNA was compared to survival at 14 days. PK data were fitted to two-compartment models then Monte Carlo simulated PK profiles were compared to ET (Cmax 0·04-0·57 ng/mL and mean concentration 1·43 ng/mL), and TT (3000 ng/mL). FINDINGS: Viral loads (VL) were not significantly different at treatment onset or during treatment (p = 0·1) in subjects who survived or died. siRNA was in quantitative excess of virus genomes throughout treatment, but the 95% percentile exceeded TT. The maximum AUC for which the 95% percentile remained under TT was a continuous infusion of 0·15 mg/kg/day. Plasma concentration of both siRNAs were higher in subjects who died compared to subjects who survived (p<0·025 both siRNAs). INTERPRETATION: TKM-130803 was circulating in molar excess of circulating virus; a level considered needed for efficacy. Given extremely high viral loads it seems likely that the patients died because they were physiologically beyond the point of no return. Subjects who died exhibited some indication of impaired drug clearance, justifying caution in dosing strategies for such patients. This analysis has given a useful insight into the pharmacokinetics of the siRNA in the disease state and illustrates the value of designing PKPD studies into future clinical trials in epidemic situations. FUNDING: This work was supported by the Wellcome Trust of Great Britain (grant number 106491/Z/14/Z and 097997/Z/11/A) and by the EU FP7 project PREPARE (602525). The PHE laboratory was funded by the UK Department for International Development. The funders had no role in trial design, data collection or analysis. The views expressed are those of the authors and not necessarily those of Public Health England, the Department of Health, or the EU. TRIAL REGISTRATION: Pan African Clinical Trials Registry PACTR201501000997429.

COMRADES determines in vivo RNA structures and interactions.

The structural flexibility of RNA underlies fundamental biological processes, but there are no methods for exploring the multiple conformations adopted by RNAs in vivo. We developed cross-linking of matched RNAs and deep sequencing (COMRADES) for in-depth RNA conformation capture, and a pipeline for the retrieval of RNA structural ensembles. Using COMRADES, we determined the architecture of the Zika virus RNA genome inside cells, and identified multiple site-specific interactions with human noncoding RNAs.

Neurodevelopmental protein Musashi-1 interacts with the Zika genome and promotes viral replication.

A recent outbreak of Zika virus in Brazil has led to a simultaneous increase in reports of neonatal microcephaly. Zika targets cerebral neural precursors, a cell population essential for cortical development, but the cause of this neurotropism remains obscure. Here we report that the neural RNA-binding protein Musashi-1 (MSI1) interacts with the Zika genome and enables viral replication. Zika infection disrupts the binding of MSI1 to its endogenous targets, thereby deregulating expression of factors implicated in neural stem cell function. We further show that MSI1 is highly expressed in neural progenitors of the human embryonic brain and is mutated in individuals with autosomal recessive primary microcephaly. Selective MSI1 expression in neural precursors could therefore explain the exceptional vulnerability of these cells to Zika infection.

Virus genomes reveal factors that spread and sustained the Ebola epidemic.

The 2013-2016 West African epidemic caused by the Ebola virus was of unprecedented magnitude, duration and impact. Here we reconstruct the dispersal, proliferation and decline of Ebola virus throughout the region by analysing 1,610 Ebola virus genomes, which represent over 5% of the known cases. We test the association of geography, climate and demography with viral movement among administrative regions, inferring a classic 'gravity' model, with intense dispersal between larger and closer populations. Despite attenuation of international dispersal after border closures, cross-border transmission had already sown the seeds for an international epidemic, rendering these measures ineffective at curbing the epidemic. We address why the epidemic did not spread into neighbouring countries, showing that these countries were susceptible to substantial outbreaks but at lower risk of introductions. Finally, we reveal that this large epidemic was a heterogeneous and spatially dissociated collection of transmission clusters of varying size, duration and connectivity. These insights will help to inform interventions in future epidemics.