Abstract As part of preparedness activities supporting pathogens classified under the UK High Consequence Infectious Diseases (HCID) framework, we previously evaluated both a whole-genome tiling amplicon sequencing scheme and a pan-viral hybridisation capture approach (TWIST-CVRP) for sequencing Andes virus (ANDV). In light of the recent outbreak, we make available viral sequencing datasets generated using a historical ANDV isolate (Chile, 1997). In addition, we provide an evaluation of tiling amplicon scheme performance and present recommended primer updates informed by in silico comparison with the recently released outbreak genome. These datasets are intended to support benchmarking, validation, and optimisation of bioinformatic pipelines across the community.

Introduction. Lateral flow tests have played a key role in the response to the COVID-19 pandemic and are likely to be a major component of diagnostic strategies to combat future outbreaks of infectious disease. Gap Statement. One challenge posed by widescale use of lateral flow tests in the community is the loss of sequence information to track virus evolution and epidemiology. Aim. Beyond their primary diagnostic function, it has been demonstrated that recovery of viral RNA from positive lateral flow devices (LFDs) for genome sequencing purposes is possible. We, therefore, aimed to assess the robustness and broader applicability of this process. Methodology. We evaluated SARS-CoV-2 RNA recovery and subsequent sequencing from eluates from the four major LFDs in use in the UK, testing both cultured virus and residual clinical nasal swab samples. Results. Our results demonstrated that sequencing from LFD eluates is possible, at clinically relevant titres, within a reasonable processing time frame post-use, and gave sequences concordant with routine methods. However, results varied across the four devices used. Conclusion. Our results highlight that if sequencing from LFD eluates is to be put into routine use, there is the requirement for refinement of existing or second-generation designs, optimized for sequencing as an intended output from positive tests.

Ixodes ricinus is a key vector of several bacterial pathogens, including Borrelia and Rickettsia species. To assess bacterial prevalence and diversity, we characterised the microbiome of ticks collected from a single UK woodland, comparing different woodland types, life stages (adult male, female, and nymph), and sampling years (spring 2019-2021). Unlike previous studies that target short regions of the 16S rRNA gene, we used Nanopore sequencing to generate full-length 16S reads, allowing for higher taxonomic resolution. Our results showed significant microbiome differences across life stages, but not by woodland type or year. Rickettsia was the most abundant taxon, with prevalence varying by life stage and year. Borrelia was present in all life stages but absent in 2021 samples. Anaplasma abundance varied significantly by year and life stage, peaking in nymphs from Pine woodland. Bartonella was found across all woodland types, with the highest abundance in nymphs from Oak-dominated areas.

Background Genomic epidemiology has helped reconstruct the global and regional movement of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there is still a lack of understanding of SARS-CoV-2 spread in some of the world's least developed countries (LDCs). Methods To begin to address this disparity, we studied the transmission dynamics of the virus in Bangladesh during the country's first COVID-19 wave by analysing case reports and whole-genome sequences from all eight divisions of the country. Results We detected > 50 virus introductions to the country during the period, including during a period of national lockdown. Additionally, through discrete phylogeographic analyses, we identified that geographical distance and population -density and/or -size influenced virus spatial dispersal in Bangladesh. Conclusions Overall, this study expands our knowledge of SARS-CoV-2 genomic epidemiology in Bangladesh, shedding light on crucial transmission characteristics within the country, while also acknowledging resemblances and differences to patterns observed in other nations.

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 the UK there are several tick vectors of Borrelia burgdorferi s.l. complex, Rickettsia spp and Anaplasma spp. Currently, PCR and ELISA are the most used methods to detect these pathogens, but they only confirm absence or presence of a target, with cost and DNA extract needed for testing both limiting factors. A molecular based sequencing panel has been developed to detect and characterise the bacterial microbiome in ticks, their last blood meal and the species of tick, validated using Oxford Nanopore long read sequencing and bioinformatic pipeline to analyse results. This multiplexed panel was tested on tick samples, both fed and questing, as well as comparing microbiome characterisation with nine targets of the 16S rRNA gene to full length 16S sequencing. Results from this show the potential for the panel to be used for routine surveillance of pathogens, to detect invasive tick species and to give an insight into the host preference of ticks. An example dataset of wildlife and game fed ticks show that Ixodes ricinus is the most abundant tick species feeding on animal and bird hosts, other species detected were I. hexagonus and I. frontalis. Source of last host was confirmed molecularly in 88% of ticks, as well as identifying other possible hosts from previous developmental stages. Microbiome was characterised and results reveal the presence of pathogenic genera, with some blood fed ticks showing evidence of co-infections, giving insight into the role that wildlife and game animals have as reservoir hosts in the UK.

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.

Reliable, easy-to-handle phenotypic screening platforms are needed for the identification of anti-SARS-CoV-2 compounds. Here, we present caspase 3/7 activity as a readout for monitoring the replication of SARS-CoV-2 isolates from different variants, including a remdesivir-resistant strain, and of other coronaviruses in numerous cell culture models, independently of cytopathogenic effect formation. Compared to other models, the Caco-2 subline Caco-2-F03 displayed superior performance. It possesses a stable SARS-CoV-2 susceptibility phenotype and does not produce false-positive hits due to drug-induced phospholipidosis. A proof-of-concept screen of 1,796 kinase inhibitors identified known and novel antiviral drug candidates including inhibitors of phosphoglycerate dehydrogenase (PHGDH), CDC like kinase 1 (CLK-1), and colony stimulating factor 1 receptor (CSF1R). The activity of the PHGDH inhibitor NCT-503 was further increased in combination with the hexokinase II (HK2) inhibitor 2-deoxy-D-glucose, which is in clinical development for COVID-19. In conclusion, caspase 3/7 activity detection in SARS-CoV-2-infected Caco-2-F03 cells provides a simple phenotypic high-throughput screening platform for SARS-CoV-2 drug candidates that reduces false-positive hits.

New vaccines, therapeutics and immunity elicited by natural infection create evolutionary pressure on SARS-CoV-2 to evolve and adapt to evade vaccine-induced and infection-elicited immunity. Vaccine and therapeutics developers thus find themselves in an “arms race” with the virus. The ongoing assessment of emerging SARS-CoV-2 variants remains essential as the global community transitions from an emergency response to a long-term management plan. Here, we describe how an authentic virus neutralisation assay using low passage clinical virus isolates has been employed to monitor resistance of emerging virus variants to neutralising antibodies from humans and experimentally infected hamsters. Sera and plasma from people who received three doses of a vaccine as well as those who received a bivalent booster were assessed against SARS-CoV-2 variants, up to and including JN.1. Contemporary or recent virus variants showed substantial resistance to neutralisation by antibodies from those who had received three doses of an ancestral vaccine but were still effectively neutralised by antibodies from individuals who had received a bivalent booster (ancestral/BA.1). In our recent studies, however, the JN.1 VOI was found to be significantly more resistant to neutralisation by antibodies from those who had received the ancestral/BA.1 bivalent boost. Convalescent sera from hamsters that had been experimentally infected with one of seven virus variants (ancestral, BA.1, BA.4, BA.5.2.1, XBB.1.5, XBB.1.16, XBB.2.3) were also tested here. The recent contemporary variant, BA.2.86, was effectively neutralised by sera from hamsters infected with XBB.1.5 and XBB.1.16 but it was not neutralised by sera from those infected with BA.5.2.1. These data support the recommendations given by the WHO that a new vaccine was required and should consist of an XBB sub-lineage antigen.### Competing Interest StatementThe authors have declared no competing interest.

The mutation profile of the SARS-CoV-2 Omicron (lineage BA.1) variant posed a concern for naturally acquired and vaccine-induced immunity. We investigated the ability of prior infection with an early SARS-CoV-2 ancestral isolate (Australia/VIC01/2020, VIC01) to protect against disease caused by BA.1. We established that BA.1 infection in naïve Syrian hamsters resulted in a less severe disease than a comparable dose of the ancestral virus, with fewer clinical signs including less weight loss. We present data to show that these clinical observations were almost absent in convalescent hamsters challenged with the same dose of BA.1 50 days after an initial infection with ancestral virus. These data provide evidence that convalescent immunity against ancestral SARS-CoV-2 is protective against BA.1 in the Syrian hamster model of infection. Comparison with published pre-clinical and clinical data supports consistency of the model and its predictive value for the outcome in humans. Further, the ability to detect protection against the less severe disease caused by BA.1 demonstrates continued value of the Syrian hamster model for evaluation of BA.1-specific countermeasures.

In the summer of 2020, it became clear that the genetic composition of SARS-CoV-2 was changing rapidly. This was highlighted by the rapid emergence of the D614G mutation at that time. In the autumn of 2020, the project entitled "Agility" was initiated with funding from the Coalition for Epidemic Preparedness Innovations (CEPI) to assess new variants of SARS-CoV-2. The project was designed to reach out and intercept swabs containing live variant viruses in order to generate highly characterised master and working stocks, and to assess the biological consequences of the rapid genetic changes using both in vitro and in vivo approaches. Since November 2020, a total of 21 variants have been acquired and tested against either a panel of convalescent sera from early in the pandemic, and/or a panel of plasma from triple-vaccinated participants. A pattern of continuous evolution of SARS-CoV-2 has been revealed. Sequential characterisation of the most globally significant variants available to us, generated in real-time, indicated that the most recent Omicron variants appear to have evolved in a manner that avoids immunological recognition by convalescent plasma from the era of the ancestral virus when analysed in an authentic virus neutralisation assay.

The density of Borrelia burgdorferi-infected Ixodes ricinus nymphs (DIN) was investigated during 2013-2017 across a Lyme disease-endemic landscape in southern England. The density of nymphs (DON), nymph infection prevalence (NIP), and DIN varied across five different natural habitats, with the highest DIN in woodland edge and high biodiversity woodlands. DIN was significantly lower in scrub grassland compared to the woodland edge, with low DON and no evidence of infection in ticks in non-scrub grassland. Over the 5 years, DON, NIP and DIN were comparable within habitats, except in 2014, with NIP varying three-fold and DIN significantly lower compared to 2015-2017. Borrelia garinii was most common, with bird-associated Borrelia (B. garinii/valaisiana) accounting for ~70% of all typed sequences. Borrelia burgdorferi sensu stricto was more common than B. afzelii. Borrelia afzelii was more common in scrub grassland than woodland and absent in some years. The possible impact of scrub on grazed grassland, management of ecotonal woodland margins with public access, and the possible role of birds/gamebirds impacting NIP are discussed. Mean NIP was 7.6%, highlighting the potential risk posed by B. burgdorferi in this endemic area. There is a need for continued research to understand its complex ecology and identify strategies for minimizing risk to public health, through habitat/game management and public awareness.

Seven years after the declaration of the first epidemic of Ebola virus disease in Guinea, the country faced a new outbreak-between 14 February and 19 June 2021-near the epicentre of the previous epidemic1,2. Here we use next-generation sequencing to generate complete or near-complete genomes of Zaire ebolavirus from samples obtained from 12 different patients. These genomes form a well-supported phylogenetic cluster with genomes from the previous outbreak, which indicates that the new outbreak was not the result of a new spillover event from an animal reservoir. The 2021 lineage shows considerably lower divergence than would be expected during sustained human-to-human transmission, which suggests a persistent infection with reduced replication or a period of latency. The resurgence of Zaire ebolavirus from humans five years after the end of the previous outbreak of Ebola virus disease reinforces the need for long-term medical and social care for patients who survive the disease, to reduce the risk of re-emergence and to prevent further stigmatization.

ABSTRACTRecently, a new variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) designated VOC 202012/01 (or B.1.1.7 lineage) has become highly prevalent in several countries, after first being described in the United Kingdom (UK). Its rate of transmission has been estimated to be increased compared to other lineages. In the present study, we show the emergence, dominance and the rapid spread of the B.1.1.7 lineage in Lebanon.

BackgroundThe public health impact of Chikungunya virus (CHIKV) is often underestimated. Usually considered a mild condition of short duration, recent outbreaks have reported greater incidence of severe illness, fatality, and longer-term disability. In 2018/19, Eastern Sudan experienced the largest epidemic of CHIKV in Africa to date, affecting an estimated 487,600 people. Known locally as Kankasha, this study examines clinical characteristics, risk factors, and phylogenetics of the epidemic in Kassala City.Methodology/principal findingsA prospective cohort of 102 adults and 40 children presenting with chikungunya-like illness were enrolled at Kassala Teaching Hospital in October 2018. Clinical information, socio-demographic data, and sera samples were analysed to confirm diagnosis, characterise illness, and identify viral strain. CHIKV infection was confirmed by real-time reverse transcription-PCR in 84.5% (120/142) of participants. Nine (7.5%) CHIKV-positive participants had concurrent Dengue virus (DENV) infection; 34/118 participants (28.8%) had a positive Rapid Diagnostic Test for Plasmodium falciparum; six (5.0%) had haemorrhagic symptoms including two children with life-threatening bleeding. One CHIKV-positive participant died with acute renal injury. Age was not associated with severity of illness although CHIKV-infected participants were younger (p = 0.003). Two to four months post-illness, 63% of adults available for follow-up (30) were still experiencing arthralgia in one or more joints, and 11% remained moderately disabled on Rapid3 assessment. Phylogenetic analysis showed all CHIKV sequences from this study belonged to a single clade within the Indian Ocean Lineage (IOL) of the East/Central/South African (ECSA) genotype. History of contact with an infected person was the only factor associated with infection (p = 0.01), and likely related to being in the same vector environment.Conclusions/significanceVulnerability to CHIKV remains in Kassala and elsewhere in Sudan due to widespread Aedes aegypti presence and mosquito-fostering household water storage methods. This study highlights the importance of increasing awareness of the severity and impact of CHIKV outbreaks, and the need for urgent actions to reduce transmission risk in households.

SummaryLamPORE is a novel diagnostic platform for the detection of SARS-CoV-2 RNA that combines loop-mediated isothermal amplification with nanopore sequencing, which could potentially be used to analyse thousands of samples per day on a single instrument. We evaluated the performance of LamPORE against RT-PCR using RNA extracted from spiked respiratory samples and from stored nose and throat swabs collected at two UK hospitals. The limit of detection of LamPORE was 7-10 genome copies/µl of extracted RNA. This is above the limit achievable by RT-PCR but was not associated with a significant reduction of sensitivity in clinical samples. Positive clinical specimens came mostly from patients with acute symptomatic infection, and among these LamPORE had a diagnostic sensitivity of 99.1% (226/228 [95% CI 96.9–99.9%]). Among negative clinical specimens, including 153 with other respiratory pathogens detected, LamPORE had a diagnostic specificity of 99.6% (278/279 [98.0–100.0%]). Overall, 1.4% (7/514 [0.5–2.9]) of samples produced an indeterminate result on first testing, and repeat LamPORE testing on the same RNA extract had a reproducibility of 96.8% (478/494 [94.8–98.1]). This indicates that LamPORE has a similar performance to RT-PCR for the diagnosis of SARS-CoV-2 infection in symptomatic patients, and offers a promising approach to high-throughput testing.

There is a vital need for authentic COVID-19 animal models to enable the pre-clinical evaluation of candidate vaccines and therapeutics. Here we report a dose titration study of SARS-CoV-2 in the ferret model. After a high (5 × 10 6 pfu) and medium (5 × 10 4 pfu) dose of virus is delivered, intranasally, viral RNA shedding in the upper respiratory tract (URT) is observed in 6/6 animals, however, only 1/6 ferrets show similar signs after low dose (5 × 10 2 pfu) challenge. Following sequential culls pathological signs of mild multifocal bronchopneumonia in approximately 5–15% of the lung is seen on day 3, in high and medium dosed groups. Ferrets re-challenged, after virus shedding ceased, are fully protected from acute lung pathology. The endpoints of URT viral RNA replication & distinct lung pathology are observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease.

The source of the COVID-19 pandemic is unknown, but the natural host of the progenitor sarbecovirus is thought to be Asian horseshoe (rhinolophid) bats. We identified and sequenced a novel sarbecovirus (RhGB01) from a British horseshoe bat, at the western extreme of the rhinolophid range. Our results extend both the geographic and species ranges of sarbecoviruses and suggest their presence throughout the horseshoe bat distribution. Within the spike protein receptor binding domain, but excluding the receptor binding motif, RhGB01 has a 77% (SARS-CoV-2) and 81% (SARS-CoV) amino acid homology. While apparently lacking hACE2 binding ability, and hence unlikely to be zoonotic without mutation, RhGB01 presents opportunity for SARS-CoV-2 and other sarbecovirus homologous recombination. Our findings highlight that the natural distribution of sarbecoviruses and opportunities for recombination through intermediate host co-infection are underestimated. Preventing transmission of SARS-CoV-2 to bats is critical with the current global mass vaccination campaign against this virus.

BackgroundInfluenza virus presents a considerable challenge to public health by causing seasonal epidemics and occasional pandemics. Nanopore metagenomic sequencing has the potential to be deployed for near-patient testing, providing rapid infection diagnosis, rationalising antimicrobial therapy, and supporting infection-control interventions.AimTo evaluate the applicability of this sequencing approach as a routine laboratory test for influenza in clinical settings.MethodsWe conducted Oxford Nanopore Technologies (Oxford, United Kingdom (UK)) metagenomic sequencing for 180 respiratory samples from a UK hospital during the 2018/19 influenza season, and compared results to routine molecular diagnostic standards (Xpert Xpress Flu/RSV assay; BioFire FilmArray Respiratory Panel 2 assay). We investigated drug resistance, genetic diversity, and nosocomial transmission using influenza sequence data.ResultsCompared to standard testing, Nanopore metagenomic sequencing was 83% (75/90) sensitive and 93% (84/90) specific for detecting influenza A viruses. Of 59 samples with haemagglutinin subtype determined, 40 were H1 and 19 H3. We identified an influenza A(H3N2) genome encoding the oseltamivir resistance S331R mutation in neuraminidase, potentially associated with an emerging distinct intra-subtype reassortant. Whole genome phylogeny refuted suspicions of a transmission cluster in a ward, but identified two other clusters that likely reflected nosocomial transmission, associated with a predominant community-circulating strain. We also detected other potentially pathogenic viruses and bacteria from the metagenome.ConclusionNanopore metagenomic sequencing can detect the emergence of novel variants and drug resistance, providing timely insights into antimicrobial stewardship and vaccine design. Full genome generation can help investigate and manage nosocomial outbreaks.