Sequential Infection with Influenza A Virus Followed by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Leads to More Severe Disease and Encephalitis in a Mouse Model of COVID-19.

COVID-19 is a spectrum of clinical symptoms in humans caused by infection with SARS-CoV-2. The coalescence of SARS-CoV-2 with seasonal respiratory viruses, particularly influenza viruses, is a global health concern. To understand this, transgenic mice expressing the human ACE2 receptor (K18-hACE2) were infected with influenza A virus (IAV) followed by SARS-CoV-2 and the host response and effect on virus biology was compared to K18-hACE2 mice infected with IAV or SARS-CoV-2 alone. The sequentially infected mice showed reduced SARS-CoV-2 RNA synthesis, yet exhibited more rapid weight loss, more severe lung damage and a prolongation of the innate response compared to the singly infected or control mice. Sequential infection also exacerbated the extrapulmonary encephalitic manifestations associated with SARS-CoV-2 infection. Conversely, prior infection with a commercially available, multivalent live-attenuated influenza vaccine (Fluenz Tetra) elicited the same reduction in SARS-CoV-2 RNA synthesis, albeit without the associated increase in disease severity. This suggests that the innate immune response stimulated by IAV inhibits SARS-CoV-2. Interestingly, infection with an attenuated, apathogenic influenza vaccine does not result in an aberrant immune response and enhanced disease severity. Taken together, the data suggest coinfection ('twinfection') is deleterious and mitigation steps should be instituted as part of the comprehensive public health and management strategy of COVID-19.

Characterizing the Impact of Cyanobacterial Blooms on the Photoreactivity of Surface Waters from New York Lakes: A Combined Statewide Survey and Laboratory Investigation.

Cyanobacterial blooms introduce autochthonous dissolved organic matter (DOM) into aquatic environments, but their impact on surface water photoreactivity has not been investigated through collaborative field sampling with comparative laboratory assessments. In this work, we quantified the apparent quantum yields (Φapp,RI) of reactive intermediates (RIs), including excited triplet states of dissolved organic matter (3DOM*), singlet oxygen (1O2), and hydroxyl radicals (•OH), for whole water samples collected by citizen volunteers from more than 100 New York lakes. Multiple comparisons tests and orthogonal partial least-squares analysis identified the level of cyanobacterial chlorophyll a as a key factor in explaining the enhanced photoreactivity of whole water samples sourced from bloom-impacted lakes. Laboratory recultivation of bloom samples in bloom-free lake water demonstrated that apparent increases in Φapp,RI during cyanobacterial growth were likely driven by the production of photoreactive moieties through the heterotrophic transformation of freshly produced labile bloom exudates. Cyanobacterial proliferation also altered the energy distribution of 3DOM* and contributed to the accelerated transformation of protriptyline, a model organic micropollutant susceptible to photosensitized reactions, under simulated sunlight conditions. Overall, our study provides insights into the relationship between the photoreactivity of surface waters and the limnological characteristics and trophic state of lakes and highlights the relevance of cyanobacterial abundance in predicting the photoreactivity of bloom-impacted surface waters.

SARS-CoV-2 NSP12 associates with TRiC and the P323L substitution acts as a host adaption.

IMPORTANCE: SARS-CoV-2 has caused a worldwide health and economic crisis. During the course of the pandemic, genetic changes occurred in the virus, which have resulted in new properties of the virus-particularly around gains in transmission and the ability to partially evade either natural or vaccine-acquired immunity. Some of these viruses have been labeled Variants of Concern (VoCs). At the root of all VoCs are two mutations, one in the viral spike protein that has been very well characterized and the other in the virus polymerase (NSP12). This is the viral protein responsible for replicating the genome. We show that NSP12 associates with host cell proteins that act as a scaffold to facilitate the function of this protein. Furthermore, we found that different variants of NSP12 interact with host cell proteins in subtle and different ways, which affect function.

The SARS-CoV-2 protein ORF3c is a mitochondrial modulator of innate immunity.

The SARS-CoV-2 genome encodes a multitude of accessory proteins. Using comparative genomic approaches, an additional accessory protein, ORF3c, has been predicted to be encoded within the ORF3a sgmRNA. Expression of ORF3c during infection has been confirmed independently by ribosome profiling. Despite ORF3c also being present in the 2002-2003 SARS-CoV, its function has remained unexplored. Here we show that ORF3c localizes to mitochondria, where it inhibits innate immunity by restricting IFN-ß production, but not NF-κB activation or JAK-STAT signaling downstream of type I IFN stimulation. We find that ORF3c is inhibitory after stimulation with cytoplasmic RNA helicases RIG-I or MDA5 or adaptor protein MAVS, but not after TRIF, TBK1 or phospho-IRF3 stimulation. ORF3c co-immunoprecipitates with the antiviral proteins MAVS and PGAM5 and induces MAVS cleavage by caspase-3. Together, these data provide insight into an uncharacterized mechanism of innate immune evasion by this important human pathogen.

Adenovirus Transcriptome in Human Cells Infected with ChAdOx1-Vectored Candidate HIV-1 Vaccine Is Dominated by High Levels of Correctly Spliced HIVconsv1&62 Transgene RNA.

We develop candidate HIV-1 vaccines, of which two components, ChAdOx1.tHIVconsv1 (C1) and ChAdOx1.HIVconsv62 (C62), are delivered by the simian adenovirus-derived vaccine vector ChAdOx1. Aberrant adenovirus RNA splicing involving transgene(s) coding for the SARS-CoV-2 spike was suggested as an aetiology of rare adverse events temporarily associated with the initial deployment of adenovirus-vectored vaccines during the COVID-19 pandemic. Here, to eliminate this theoretically plausible splicing phenomenon from the list of possible pathomechanisms for our HIV-1 vaccine candidates, we directly sequenced mRNAs in C1- and C62-infected nonpermissive MRC-5 and A549 and permissive HEK293 human cell lines. Our two main observations in nonpermissive human cells, which are most similar to those which become infected after the intramuscular administration of vaccines into human volunteers, were that (i) the dominant adenovirus vector-derived mRNAs were the expected transcripts coding for the HIVconsvX immunogens and (ii) atypical splicing events within the synthetic open reading frame of the two transgenes are rare. We conclude that inadvertent RNA splicing is not a safety concern for the two tested candidate HIV-1 vaccines.

The P323L substitution in the SARS-CoV-2 polymerase (NSP12) confers a selective advantage during infection.

BACKGROUND: The mutational landscape of SARS-CoV-2 varies at the dominant viral genome sequence and minor genomic variant population. During the COVID-19 pandemic, an early substitution in the genome was the D614G change in the spike protein, associated with an increase in transmissibility. Genomes with D614G are accompanied by a P323L substitution in the viral polymerase (NSP12). However, P323L is not thought to be under strong selective pressure. RESULTS: Investigation of P323L/D614G substitutions in the population shows rapid emergence during the containment phase and early surge phase during the first wave. These substitutions emerge from minor genomic variants which become dominant viral genome sequence. This is investigated in vivo and in vitro using SARS-CoV-2 with P323 and D614 in the dominant genome sequence and L323 and G614 in the minor variant population. During infection, there is rapid selection of L323 into the dominant viral genome sequence but not G614. Reverse genetics is used to create two viruses (either P323 or L323) with the same genetic background. L323 shows greater abundance of viral RNA and proteins and a smaller plaque morphology than P323. CONCLUSIONS: These data suggest that P323L is an important contribution in the emergence of variants with transmission advantages. Sequence analysis of viral populations suggests it may be possible to predict the emergence of a new variant based on tracking the frequency of minor variant genomes. The ability to predict an emerging variant of SARS-CoV-2 in the global landscape may aid in the evaluation of medical countermeasures and non-pharmaceutical interventions.

Voluntary risk mitigation behaviour can reduce impact of SARS-CoV-2: a real-time modelling study of the January 2022 Omicron wave in England.

BACKGROUND: Predicting the likely size of future SARS-CoV-2 waves is necessary for public health planning. In England, voluntary "plan B" mitigation measures were introduced in December 2021 including increased home working and face coverings in shops but stopped short of restrictions on social contacts. The impact of voluntary risk mitigation behaviours on future SARS-CoV-2 burden is unknown. METHODS: We developed a rapid online survey of risk mitigation behaviours ahead of the winter 2021 festive period and deployed in two longitudinal cohort studies in the UK (Avon Longitudinal Study of Parents and Children (ALSPAC) and TwinsUK/COVID Symptom Study (CSS) Biobank) in December 2021. Using an individual-based, probabilistic model of COVID-19 transmission between social contacts with SARS-CoV-2 Omicron variant parameters and realistic vaccine coverage in England, we predicted the potential impact of the SARS-CoV-2 Omicron wave in England in terms of the effective reproduction number and cumulative infections, hospital admissions and deaths. Using survey results, we estimated in real-time the impact of voluntary risk mitigation behaviours on the Omicron wave in England, if implemented for the entire epidemic wave. RESULTS: Over 95% of survey respondents (NALSPAC = 2686 and NTwins = 6155) reported some risk mitigation behaviours, with vaccination and using home testing kits reported most frequently. Less than half of those respondents reported that their behaviour was due to "plan B". We estimate that without risk mitigation behaviours, the Omicron variant is consistent with an effective reproduction number between 2.5 and 3.5. Due to the reduced vaccine effectiveness against infection with the Omicron variant, our modelled estimates suggest that between 55% and 60% of the English population could be infected during the current wave, translating into between 12,000 and 46,000 cumulative deaths, depending on assumptions about severity and vaccine effectiveness. The actual number of deaths was 15,208 (26 November 2021-1 March 2022). We estimate that voluntary risk reduction measures could reduce the effective reproduction number to between 1.8 and 2.2 and reduce the cumulative number of deaths by up to 24%. CONCLUSIONS: Predicting future infection burden is affected by uncertainty in disease severity and vaccine effectiveness estimates. In addition to biological uncertainty, we show that voluntary measures substantially reduce the projected impact of the SARS-CoV-2 Omicron variant but that voluntary measures alone would be unlikely to completely control transmission.

Direct RNA sequencing of respiratory syncytial virus infected human cells generates a detailed overview of RSV polycistronic mRNA and transcript abundance.

To characterize species of viral mRNA transcripts generated during respiratory syncytial virus (RSV) infection, human fibroblast-like MRC-5 lung cells were infected with subgroup A RSV for 6, 16 and 24 hours. In addition, we characterised the viral transcriptome in infected Calu-3 lung epithelial cells at 48 hours post infection. Total RNA was harvested and polyadenylated mRNA was enriched and sequenced by direct RNA sequencing using an Oxford nanopore device. This platform yielded over 450,000 direct mRNA transcript reads which were mapped to the viral genome and analysed to determine the relative mRNA levels of viral genes using our in-house ORF-centric pipeline. We examined the frequency of polycistronic readthrough mRNAs were generated and assessed the length of the polyadenylated tails for each group of transcripts. We show a general but non-linear decline in gene transcript abundance across the viral genome, as predicted by the model of RSV gene transcription. However, the decline in transcript abundance is not uniform. The polyadenylate tails generated by the viral polymerase are similar in length to those generated by the host polyadenylation machinery and broadly declined in length for most transcripts as the infection progressed. Finally, we observed that the steady state abundance of transcripts with very short polyadenylate tails less than 20 nucleotides is less for N, SH and G transcripts in both cell lines compared to NS1, NS2, P, M, F and M2 which may reflect differences in mRNA stability and/or translation rates within and between the cell lines.

Host protein kinases required for SARS-CoV-2 nucleocapsid phosphorylation and viral replication.

Multiple coronaviruses have emerged independently in the past 20 years that cause lethal human diseases. Although vaccine development targeting these viruses has been accelerated substantially, there remain patients requiring treatment who cannot be vaccinated or who experience breakthrough infections. Understanding the common host factors necessary for the life cycles of coronaviruses may reveal conserved therapeutic targets. Here, we used the known substrate specificities of mammalian protein kinases to deconvolute the sequence of phosphorylation events mediated by three host protein kinase families (SRPK, GSK-3, and CK1) that coordinately phosphorylate a cluster of serine and threonine residues in the viral N protein, which is required for viral replication. We also showed that loss or inhibition of SRPK1/2, which we propose initiates the N protein phosphorylation cascade, compromised the viral replication cycle. Because these phosphorylation sites are highly conserved across coronaviruses, inhibitors of these protein kinases not only may have therapeutic potential against COVID-19 but also may be broadly useful against coronavirus-mediated diseases.

Analysis of SARS-CoV-2 known and novel subgenomic mRNAs in cell culture, animal model, and clinical samples using LeTRS, a bioinformatic tool to identify unique sequence identifiers.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a complex strategy for the transcription of viral subgenomic mRNAs (sgmRNAs), which are targets for nucleic acid diagnostics. Each of these sgmRNAs has a unique 5' sequence, the leader-transcriptional regulatory sequence gene junction (leader-TRS junction), that can be identified using sequencing. High-resolution sequencing has been used to investigate the biology of SARS-CoV-2 and the host response in cell culture and animal models and from clinical samples. LeTRS, a bioinformatics tool, was developed to identify leader-TRS junctions and can be used as a proxy to quantify sgmRNAs for understanding virus biology. LeTRS is readily adaptable for other coronaviruses such as Middle East respiratory syndrome coronavirus or a future newly discovered coronavirus. LeTRS was tested on published data sets and novel clinical samples from patients and longitudinal samples from animal models with coronavirus disease 2019. LeTRS identified known leader-TRS junctions and identified putative novel sgmRNAs that were common across different mammalian species. This may be indicative of an evolutionary mechanism where plasticity in transcription generates novel open reading frames, which can then subject to selection pressure. The data indicated multiphasic abundance of sgmRNAs in two different animal models. This recapitulates the relative sgmRNA abundance observed in cells at early points in infection but not at late points. This pattern is reflected in some human nasopharyngeal samples and therefore has implications for transmission models and nucleic acid-based diagnostics. LeTRS provides a quantitative measure of sgmRNA abundance from sequencing data. This can be used to assess the biology of SARS-CoV-2 (or other coronaviruses) in clinical and nonclinical samples, especially to evaluate different variants and medical countermeasures that may influence viral RNA synthesis.

Nanopore ReCappable sequencing maps SARS-CoV-2 5' capping sites and provides new insights into the structure of sgRNAs.

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested subgenomic RNAsused to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5' cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.

Structural insights in cell-type specific evolution of intra-host diversity by SARS-CoV-2.

As the global burden of SARS-CoV-2 infections escalates, so does the evolution of viral variants with increased transmissibility and pathology. In addition to this entrenched diversity, RNA viruses can also display genetic diversity within single infected hosts with co-existing viral variants evolving differently in distinct cell types. The BriSΔ variant, originally identified as a viral subpopulation from SARS-CoV-2 isolate hCoV-19/England/02/2020, comprises in the spike an eight amino-acid deletion encompassing a furin recognition motif and S1/S2 cleavage site. We elucidate the structure, function and molecular dynamics of this spike providing mechanistic insight into how the deletion correlates to viral cell tropism, ACE2 receptor binding and infectivity of this SARS-CoV-2 variant. Our results reveal long-range allosteric communication between functional domains that differ in the wild-type and the deletion variant and support a view of SARS-CoV-2 probing multiple evolutionary trajectories in distinct cell types within the same infected host.

Coupling Suspect and Nontarget Screening with Mass Balance Modeling to Characterize Organic Micropollutants in the Onondaga Lake-Three Rivers System.

Characterizing the occurrence, sources, and fate of organic micropollutants (OMPs) in lake-river systems serves as an important foundation for constraining the potential impacts of OMPs on the ecosystem functions of these critical landscape features. In this work, we combined suspect and nontarget screening with mass balance modeling to investigate OMP contamination in the Onondaga Lake-Three Rivers system of New York. Suspect and nontarget screening enabled by liquid chromatography-high-resolution mass spectrometry led to the confirmation and quantification of 105 OMPs in water samples collected throughout the lake-river system, which were grouped by their concentration patterns into wastewater-derived and mixed-source clusters via hierarchical cluster analysis. Four of these OMPs (i.e., galaxolidone, diphenylphosphinic acid, N-butylbenzenesulfonamide, and triisopropanolamine) were prioritized and identified by nontarget screening based on their characteristic vertical distribution patterns during thermal stratification in Onondaga Lake. Mass balance modeling performed using the concentration and discharge data highlighted the export of OMPs from Onondaga Lake to the Three Rivers as a major contributor to the OMP budget in this lake-river system. Overall, this work demonstrated the utility of an integrated screening and modeling framework that can be adapted for OMP characterization, fate assessment, and load apportionment in similar surface water systems.

TMPRSS2 promotes SARS-CoV-2 evasion from NCOA7-mediated restriction.

Interferons play a critical role in regulating host immune responses to SARS-CoV-2, but the interferon (IFN)-stimulated gene (ISG) effectors that inhibit SARS-CoV-2 are not well characterized. The IFN-inducible short isoform of human nuclear receptor coactivator 7 (NCOA7) inhibits endocytic virus entry, interacts with the vacuolar ATPase, and promotes endo-lysosomal vesicle acidification and lysosomal protease activity. Here, we used ectopic expression and gene knockout to demonstrate that NCOA7 inhibits infection by SARS-CoV-2 as well as by lentivirus particles pseudotyped with SARS-CoV-2 Spike in lung epithelial cells. Infection with the highly pathogenic, SARS-CoV-1 and MERS-CoV, or seasonal, HCoV-229E and HCoV-NL63, coronavirus Spike-pseudotyped viruses was also inhibited by NCOA7. Importantly, either overexpression of TMPRSS2, which promotes plasma membrane fusion versus endosomal fusion of SARS-CoV-2, or removal of Spike's polybasic furin cleavage site rendered SARS-CoV-2 less sensitive to NCOA7 restriction. Collectively, our data indicate that furin cleavage sensitizes SARS-CoV-2 Spike to the antiviral consequences of endosomal acidification by NCOA7, and suggest that the acquisition of furin cleavage may have favoured the co-option of cell surface TMPRSS proteases as a strategy to evade the suppressive effects of IFN-induced endo-lysosomal dysregulation on virus infection.

Amplicon and Metagenomic Analysis of Middle East Respiratory Syndrome (MERS) Coronavirus and the Microbiome in Patients with Severe MERS.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic infection that emerged in the Middle East in 2012. Symptoms range from mild to severe and include both respiratory and gastrointestinal illnesses. The virus is mainly present in camel populations with occasional zoonotic spill over into humans. The severity of infection in humans is influenced by numerous factors, and similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), underlying health complications can play a major role. Currently, MERS-CoV and SARS-CoV-2 are coincident in the Middle East and thus a rapid way of sequencing MERS-CoV to derive genotype information for molecular epidemiology is needed. Additionally, complicating factors in MERS-CoV infections are coinfections that require clinical management. The ability to rapidly characterize these infections would be advantageous. To rapidly sequence MERS-CoV, an amplicon-based approach was developed and coupled to Oxford Nanopore long read length sequencing. This and a metagenomic approach were evaluated with clinical samples from patients with MERS. The data illustrated that whole-genome or near-whole-genome information on MERS-CoV could be rapidly obtained. This approach provided data on both consensus genomes and the presence of minor variants, including deletion mutants. The metagenomic analysis provided information of the background microbiome. The advantage of this approach is that insertions and deletions can be identified, which are the major drivers of genotype change in coronaviruses. IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in late 2012 in Saudi Arabia. The virus is a serious threat to people not only in the Middle East but also in the world and has been detected in over 27 countries. MERS-CoV is spreading in the Middle East and neighboring countries, and approximately 35% of reported patients with this virus have died. This is the most severe coronavirus infection so far described. Saudi Arabia is a destination for many millions of people in the world who visit for religious purposes (Umrah and Hajj), and so it is a very vulnerable area, which imposes unique challenges for effective control of this epidemic. The significance of our study is that clinical samples from patients with MERS were used for rapid in-depth sequencing and metagenomic analysis using long read length sequencing.

The furin cleavage site in the SARS-CoV-2 spike protein is required for transmission in ferrets.

SARS-CoV-2 entry requires sequential cleavage of the spike glycoprotein at the S1/S2 and the S2' cleavage sites to mediate membrane fusion. SARS-CoV-2 has a polybasic insertion (PRRAR) at the S1/S2 cleavage site that can be cleaved by furin. Using lentiviral pseudotypes and a cell-culture-adapted SARS-CoV-2 virus with an S1/S2 deletion, we show that the polybasic insertion endows SARS-CoV-2 with a selective advantage in lung cells and primary human airway epithelial cells, but impairs replication in Vero E6, a cell line used for passaging SARS-CoV-2. Using engineered spike variants and live virus competition assays and by measuring growth kinetics, we find that the selective advantage in lung and primary human airway epithelial cells depends on the expression of the cell surface protease TMPRSS2, which enables endosome-independent virus entry by a route that avoids antiviral IFITM proteins. SARS-CoV-2 virus lacking the S1/S2 furin cleavage site was shed to lower titres from infected ferrets and was not transmitted to cohoused sentinel animals, unlike wild-type virus. Analysis of 100,000 SARS-CoV-2 sequences derived from patients and 24 human postmortem tissues showed low frequencies of naturally occurring mutants that harbour deletions at the polybasic site. Taken together, our findings reveal that the furin cleavage site is an important determinant of SARS-CoV-2 transmission.

SARS-CoV-2 vaccine ChAdOx1 nCoV-19 infection of human cell lines reveals low levels of viral backbone gene transcription alongside very high levels of SARS-CoV-2 S glycoprotein gene transcription.

BACKGROUND: ChAdOx1 nCoV-19 is a recombinant adenovirus vaccine against SARS-CoV-2 that has passed phase III clinical trials and is now in use across the globe. Although replication-defective in normal cells, 28 kbp of adenovirus genes is delivered to the cell nucleus alongside the SARS-CoV-2 S glycoprotein gene. METHODS: We used direct RNA sequencing to analyse transcript expression from the ChAdOx1 nCoV-19 genome in human MRC-5 and A549 cell lines that are non-permissive for vector replication alongside the replication permissive cell line, HEK293. In addition, we used quantitative proteomics to study over time the proteome and phosphoproteome of A549 and MRC5 cells infected with the ChAdOx1 nCoV-19 vaccine. RESULTS: The expected SARS-CoV-2 S coding transcript dominated in all cell lines. We also detected rare S transcripts with aberrant splice patterns or polyadenylation site usage. Adenovirus vector transcripts were almost absent in MRC-5 cells, but in A549 cells, there was a broader repertoire of adenoviral gene expression at very low levels. Proteomically, in addition to S glycoprotein, we detected multiple adenovirus proteins in A549 cells compared to just one in MRC5 cells. CONCLUSIONS: Overall, the ChAdOx1 nCoV-19 vaccine's transcriptomic and proteomic repertoire in cell culture is as expected. The combined transcriptomic and proteomics approaches provide a detailed insight into the behaviour of this important class of vaccine using state-of-the-art techniques and illustrate the potential of this technique to inform future viral vaccine vector design.

Analysis of an Ebola virus disease survivor whose host and viral markers were predictive of death indicates the effectiveness of medical countermeasures and supportive care.

BACKGROUND: Ebola virus disease (EVD) is an often-fatal infection where the effectiveness of medical countermeasures is uncertain. During the West African outbreak (2013-2016), several patients were treated with different types of anti-viral therapies including monoclonal antibody-based cocktails that had the potential to neutralise Ebola virus (EBOV). However, at the time, the efficacy of these therapies was uncertain. Given the scale of the outbreak, several clinical phenotypes came to the forefront including the ability of the same virus to cause recrudescence in the same patient-perhaps through persisting in immune privileged sites. Several key questions remained including establishing if monoclonal antibody therapy was effective in humans with severe EVD, whether virus escape mutants were selected during treatment, and what is the potential mechanism(s) of persistence. This was made possible through longitudinal samples taken from a UK patient with EVD. METHODS: Several different sample types, plasma and cerebrospinal fluid, were collected and sequenced using Illumina-based RNAseq. Sequence reads were mapped both to EBOV and the human genome and differential gene expression analysis used to identify changes in the abundance of gene transcripts as infection progressed. Digital Cell Quantitation analysis was used to predict the immune phenotype in samples derived from blood. RESULTS: The findings were compared to equivalent data from West African patients. The study found that both virus and host markers were predictive of a fatal outcome. This suggested that the extensive supportive care, and most likely the application of the medical countermeasure ZMab (a monoclonal antibody cocktail), contributed to survival of the UK patient. The switch from progression to a 'fatal' outcome to a 'survival' outcome could be seen in both the viral and host markers. The UK patient also suffered a recrudescence infection 10 months after the initial infection. Analysis of the sequencing data indicated that the virus entered a period of reduced or minimal replication, rather than other potential mechanisms of persistence-such as defective interfering genomes. CONCLUSIONS: The data showed that comprehensive supportive care and the application of medical countermeasures are worth pursuing despite an initial unfavourable prognosis.

Organic Micropollutants in New York Lakes: A Statewide Citizen Science Occurrence Study.

The widespread occurrence of organic micropollutants (OMPs) is a challenge for aquatic ecosystem management, and closing the gaps in risk assessment of OMPs requires a data-driven approach. One promising tool for increasing the spatiotemporal coverage of OMP data sets is through the active involvement of citizen volunteers to expand the scale of OMP monitoring. Working collaboratively with volunteers from the Citizens Statewide Lake Assessment Program (CSLAP), we conducted the first statewide study on OMP occurrence in surface waters of New York lakes. Samples collected by CSLAP volunteers were analyzed for OMPs by a suspect screening method based on mixed-mode solid-phase extraction and liquid chromatography-high resolution mass spectrometry. Sixty-five OMPs were confirmed and quantified in samples from 111 lakes across New York. Hierarchical clustering of OMP occurrence data revealed the relevance of 11 most frequently detected OMPs for classifying the contamination status of lakes. Partial least squares regression and multiple linear regression analyses prioritized three water quality parameters linked to agricultural and developed land uses (i.e., total dissolved nitrogen, specific conductance, and a wastewater-derived fluorescent organic matter component) as the best combination of predictors that partly explained the interlake variability in OMP occurrence. Lastly, the exposure-activity ratio approach identified the potential for biological effects associated with detected OMPs that warrant further biomonitoring studies. Overall, this work demonstrated the feasibility of incorporating citizen science approaches into the regional impact assessment of OMPs.