Hyaluronan in mesenchymal stromal cell lineage differentiation from human pluripotent stem cells: application in serum free culture.

BACKGROUND: Hyaluronan (HA) is an extracellular glycosaminoglycan polysaccharide with widespread roles throughout development and in healthy and neoplastic tissues. In pluripotent stem cell culture it can support both stem cell renewal and differentiation. However, responses to HA in culture are influenced by interaction with a range of cognate factors and receptors including components of blood serum supplements, which alter results. These may contribute to variation in cell batch production yield and phenotype as well as heighten the risks of adventitious pathogen transmission in the course of cell processing for therapeutic applications. MAIN: Here we characterise differentiation of a human embryo/pluripotent stem cell derived Mesenchymal Stromal Cell (hESC/PSC-MSC)-like cell population by culture on a planar surface coated with HA in serum-free media qualified for cell production for therapy. Resulting cells met minimum criteria of the International Society for Cellular Therapy for identification as MSC by expression of. CD90, CD73, CD105, and lack of expression for CD34, CD45, CD14 and HLA-II. They were positive for other MSC associated markers (i.e.CD166, CD56, CD44, HLA 1-A) whilst negative for others (e.g. CD271, CD71, CD146). In vitro co-culture assessment of MSC associated functionality confirmed support of growth of hematopoietic progenitors and inhibition of mitogen activated proliferation of lymphocytes from umbilical cord and adult peripheral blood mononuclear cells, respectively. Co-culture with immortalized THP-1 monocyte derived macrophages (Mɸ) concurrently stimulated with lipopolysaccharide as a pro-inflammatory stimulus, resulted in a dose dependent increase in pro-inflammatory IL6 but negligible effect on TNFα. To further investigate these functionalities, a bulk cell RNA sequence comparison with adult human bone marrow derived MSC and hESC substantiated a distinctive genetic signature more proximate to the former. CONCLUSION: Cultivation of human pluripotent stem cells on a planar substrate of HA in serum-free culture media systems is sufficient to yield a distinctive developmental mesenchymal stromal cell lineage with potential to modify the function of haematopoietic lineages in therapeutic applications.

Asymptomatic immunodeficiency-associated vaccine-derived poliovirus infections in two UK children.

Increasing detections of vaccine-derived poliovirus (VDPV) globally, including in countries previously declared polio free, is a public health emergency of international concern. Individuals with primary immunodeficiency (PID) can excrete polioviruses for prolonged periods, which could act as a source of cryptic transmission of viruses with potential to cause neurological disease. Here, we report on the detection of immunodeficiency-associated VDPVs (iVDPV) from two asymptomatic male PID children in the UK in 2019. The first child cleared poliovirus with increased doses of intravenous immunoglobulin, the second child following haematopoetic stem cell transplantation. We perform genetic and phenotypic characterisation of the infecting strains, demonstrating intra-host evolution and a neurovirulent phenotype in transgenic mice. Our findings highlight a pressing need to strengthen polio surveillance. Systematic collection of stool from asymptomatic PID patients who are at high risk for poliovirus excretion could improve the ability to detect and contain iVDPVs.

Intrinsic host susceptibility among multiple species to intranasal SARS-CoV-2 identifies diverse virological, biodistribution and pathological outcomes.

SARS-CoV-2 exhibits a diverse host species range with variable outcomes, enabling differential host susceptibility studies to assess suitability for pre-clinical countermeasure and pathogenesis studies. Baseline virological, molecular and pathological outcomes were determined among multiple species-one Old World non-human primate (NHP) species (cynomolgus macaques), two New World NHP species (red-bellied tamarins; common marmosets) and Syrian hamsters-following single-dose, atraumatic intranasal administration of SARS-CoV-2/Victoria-01. After serial sacrifice 2, 10 and 28-days post-infection (dpi), hamsters and cynomolgus macaques displayed differential virus biodistribution across respiratory, gastrointestinal and cardiovascular systems. Uniquely, New World tamarins, unlike marmosets, exhibited high levels of acute upper airway infection, infectious virus recovery associated with mild lung pathology representing a host previously unrecognized as susceptible to SARS-CoV-2. Across all species, lung pathology was identified post-clearance of virus shedding (antigen/RNA), with an association of virus particles within replication organelles in lung sections analysed by electron microscopy. Disrupted cell ultrastructure and lung architecture, including abnormal morphology of mitochondria 10-28 dpi, represented on-going pathophysiological consequences of SARS-CoV-2 in predominantly asymptomatic hosts. Infection kinetics and host pathology comparators using standardized methodologies enables model selection to bridge differential outcomes within upper and lower respiratory tracts and elucidate longer-term consequences of asymptomatic SARS-CoV-2 infection.

Sustained detection of type 2 poliovirus in London sewage between February and July, 2022, by enhanced environmental surveillance.

BACKGROUND: The international spread of poliovirus exposes all countries to the risk of outbreaks and is designated a Public Health Emergency of International Concern by WHO. This risk can be exacerbated in countries using inactivated polio vaccine, which offers excellent protection against paralysis but is less effective than oral vaccine against poliovirus shedding, potentially allowing circulation without detection of paralytic cases for long periods of time. Our study investigated the molecular properties of type 2 poliovirus isolates found in sewage with an aim to detect virus transmission in the community. METHODS: We performed environmental surveillance in London, UK, testing sewage samples using WHO recommended methods that include concentration, virus isolation in cell culture, and molecular characterisation. We additionally implemented direct molecular detection and determined whole-genome sequences of every isolate using novel nanopore protocols. FINDINGS: 118 genetically linked poliovirus isolates related to the serotype 2 Sabin vaccine strain were detected in 21 of 52 sequential sewage samples collected in London between Feb 8 and July 4, 2022. Expansion of environmental surveillance sites in London helped localise transmission to several boroughs in north and east London. All isolates have lost two key attenuating mutations, are recombinants with a species C enterovirus, and an increasing proportion (20 of 118) meet the criterion for a vaccine-derived poliovirus, having six to ten nucleotide changes in the gene coding for VP1 capsid protein. INTERPRETATION: Environmental surveillance allowed early detection of poliovirus importation and circulation in London, permitting a rapid public health response, including enhanced surveillance and an inactivated polio vaccine campaign among children aged 1-9 years. Whole-genome sequences generated through nanopore sequencing established linkage of isolates and confirmed transmission of a unique recombinant poliovirus lineage that has now been detected in Israel and the USA. FUNDING: Medicines and Healthcare products Regulatory Agency, UK Health Security Agency, Bill & Melinda Gates Foundation, and National Institute for Health Research Medical Research Council.

Developing whole cell standards for the microbiome field.

BACKGROUND: Effective standardisation of the microbiome field is essential to facilitate global translational research and increase the reproducibility of microbiome studies. In this study, we describe the development and validation of a whole cell reference reagent specific to the gut microbiome by the UK National Institute for Biological Standards and Control. We also provide and test a two-step reporting framework to allow microbiome researchers to quickly and accurately validate choices of DNA extraction, sequencing, and bioinformatic pipelines. RESULTS: Using 20 strains that are commonly found in the gut, we developed a whole cell reference reagent (WC-Gut RR) for the evaluation of the DNA extraction protocols commonly used in microbiome pipelines. DNA was first analysed using the physicochemical measures of yield, integrity, and purity, which demonstrated kits widely differed in the quality of the DNA they produced. Importantly, the combination of the WC-Gut RR and the three physicochemical measures allowed us to differentiate clearly between kit performance. We next assessed the ability of WC-Gut RR to evaluate kit performance in the reconstitution of accurate taxonomic profiles. We applied a four-measure framework consisting of Sensitivity, false-positive relative abundance (FPRA), Diversity, and Similarity as previously described for DNA reagents. Using the WC-Gut RR and these four measures, we could reliably identify the DNA extraction kits' biases when using with both 16S rRNA sequencing and shotgun sequencing. Moreover, when combining this with complementary DNA standards, we could estimate the relative bias contributions of DNA extraction kits vs bioinformatic analysis. Finally, we assessed WC-Gut RR alongside other commercially available reagents. The analysis here clearly demonstrates that reagents of lower complexity, not composed of anaerobic and hard-to-lyse strains from the gut, can artificially inflate the performance of microbiome DNA extraction kits and bioinformatic pipelines. CONCLUSIONS: We produced a complex whole cell reagent that is specific for the gut microbiome and can be used to evaluate and benchmark DNA extractions in microbiome studies. Used alongside a DNA standard, the NIBSC DNA-Gut-Mix RR helps estimating where biases occur in microbiome pipelines. In the future, we aim to establish minimum thresholds for data quality through an interlaboratory collaborative study. Video Abstract.

Genetic Characterization of Novel Oral Polio Vaccine Type 2 Viruses During Initial Use Phase Under Emergency Use Listing - Worldwide, March-October 2021.

The emergence and international spread of neurovirulent circulating vaccine-derived polioviruses (cVDPVs) across multiple countries in Africa and Asia in recent years pose a major challenge to the goal of eradicating all forms of polioviruses. Approximately 90% of all cVDPV outbreaks are caused by the type 2 strain of the Sabin vaccine, an oral live, attenuated vaccine; cVDPV outbreaks typically occur in areas of persistently low immunization coverage (1). A novel type 2 oral poliovirus vaccine (nOPV2), produced by genetic modification of the type 2 Sabin vaccine virus genome (2), was developed and evaluated through phase I and phase II clinical trials during 2017-2019. nOPV2 was demonstrated to be safe and well-tolerated, have noninferior immunogenicity, and have superior genetic stability compared with Sabin monovalent type 2 (as measured by preservation of the primary attenuation site [domain V in the 5' noncoding region] and significantly lower neurovirulence of fecally shed vaccine virus in transgenic mice) (3-5). These findings indicate that nOPV2 could be an important tool in reducing the risk for generating vaccine-derived polioviruses (VDPVs) and the risk for vaccine-associated paralytic poliomyelitis cases. Based on the favorable preclinical and clinical data, and the public health emergency of international concern generated by ongoing endemic wild poliovirus transmission and cVDPV type 2 outbreaks, the World Health Organization authorized nOPV2 for use under the Emergency Use Listing (EUL) pathway in November 2020, allowing for its first use for outbreak response in March 2021 (6). As required by the EUL process, among other EUL obligations, an extensive plan was developed and deployed for obtaining and monitoring nOPV2 isolates detected during acute flaccid paralysis (AFP) surveillance, environmental surveillance, adverse events after immunization surveillance, and targeted surveillance for adverse events of special interest (i.e., prespecified events that have the potential to be causally associated with the vaccine product), during outbreak response, as well as through planned field studies. Under this monitoring framework, data generated from whole-genome sequencing of nOPV2 isolates, alongside other virologic data for isolates from AFP and environmental surveillance systems, are reviewed by the genetic characterization subgroup of an nOPV working group of the Global Polio Eradication Initiative. Global nOPV2 genomic surveillance during March-October 2021 confirmed genetic stability of the primary attenuating site. Sequence data generated through this unprecedented global effort confirm the genetic stability of nOPV2 relative to Sabin 2 and suggest that nOPV2 will be an important tool in the eradication of poliomyelitis. nOPV2 surveillance should continue for the duration of the EUL.

Detection of Enterovirus D68 in Wastewater Samples from the UK between July and November 2021.

Infection with enterovirus D68 (EV-D68) has been linked with severe neurological disease such as acute flaccid myelitis (AFM) in recent years. However, active surveillance for EV-D68 is lacking, which makes full assessment of this association difficult. Although a high number of EV-D68 infections were expected in 2020 based on the EV-D68's known biannual circulation patterns, no apparent increase in EV-D68 detections or AFM cases was observed during 2020. We describe an upsurge of EV-D68 detections in wastewater samples from the United Kingdom between July and November 2021 mirroring the recently reported rise in EV-D68 detections in clinical samples from various European countries. We provide the first publicly available 2021 EV-D68 sequences showing co-circulation of EV-D68 strains from genetic clade D and sub-clade B3 as in previous years. Our results show the value of environmental surveillance (ES) for the early detection of circulating and clinically relevant human viruses. The use of a next-generation sequencing (NGS) approach helped us to estimate the prevalence of EV-D68 viruses among EV strains from other EV serotypes and to detect EV-D68 minor variants. The utility of ES at reducing gaps in virus surveillance for EV-D68 and the possible impact of nonpharmaceutical interventions introduced to control the COVID-19 pandemic on EV-D68 transmission dynamics are discussed.

A customised target capture sequencing tool for molecular identification of Aloe vera and relatives.

Plant molecular identification studies have, until recently, been limited to the use of highly conserved markers from plastid and other organellar genomes, compromising resolution in highly diverse plant clades. Due to their higher evolutionary rates and reduced paralogy, low-copy nuclear genes overcome this limitation but are difficult to sequence with conventional methods and require high-quality input DNA. Aloe vera and its relatives in the Alooideae clade (Asphodelaceae, subfamily Asphodeloideae) are of economic interest for food and health products and have horticultural value. However, pressing conservation issues are increasing the need for a molecular identification tool to regulate the trade. With > 600 species and an origin of ± 15 million years ago, this predominantly African succulent plant clade is a diverse and taxonomically complex group for which low-copy nuclear genes would be desirable for accurate species discrimination. Unfortunately, with an average genome size of 16.76 pg, obtaining high coverage sequencing data for these genes would be prohibitively costly and computationally demanding. We used newly generated transcriptome data to design a customised RNA-bait panel targeting 189 low-copy nuclear genes in Alooideae. We demonstrate its efficacy in obtaining high-coverage sequence data for the target loci on Illumina sequencing platforms, including degraded DNA samples from museum specimens, with considerably improved phylogenetic resolution. This customised target capture sequencing protocol has the potential to confidently indicate phylogenetic relationships of Aloe vera and related species, as well as aid molecular identification applications.

Changing composition of SARS-CoV-2 lineages and rise of Delta variant in England.

BACKGROUND: Since its emergence in Autumn 2020, the SARS-CoV-2 Variant of Concern (VOC) B.1.1.7 (WHO label Alpha) rapidly became the dominant lineage across much of Europe. Simultaneously, several other VOCs were identified globally. Unlike B.1.1.7, some of these VOCs possess mutations thought to confer partial immune escape. Understanding when and how these additional VOCs pose a threat in settings where B.1.1.7 is currently dominant is vital. METHODS: We examine trends in the prevalence of non-B.1.1.7 lineages in London and other English regions using passive-case detection PCR data, cross-sectional community infection surveys, genomic surveillance, and wastewater monitoring. The study period spans from 31st January 2021 to 15th May 2021. FINDINGS: Across data sources, the percentage of non-B.1.1.7 variants has been increasing since late March 2021. This increase was initially driven by a variety of lineages with immune escape. From mid-April, B.1.617.2 (WHO label Delta) spread rapidly, becoming the dominant variant in England by late May. INTERPRETATION: The outcome of competition between variants depends on a wide range of factors such as intrinsic transmissibility, evasion of prior immunity, demographic specificities and interactions with non-pharmaceutical interventions. The presence and rise of non-B.1.1.7 variants in March likely was driven by importations and some community transmission. There was competition between non-B.1.17 variants which resulted in B.1.617.2 becoming dominant in April and May with considerable community transmission. Our results underscore that early detection of new variants requires a diverse array of data sources in community surveillance. Continued real-time information on the highly dynamic composition and trajectory of different SARS-CoV-2 lineages is essential to future control efforts. FUNDING: National Institute for Health Research, Medicines and Healthcare products Regulatory Agency, DeepMind, EPSRC, EA Funds programme, Open Philanthropy, Academy of Medical Sciences Bill,Melinda Gates Foundation, Imperial College Healthcare NHS Trust, The Novo Nordisk Foundation, MRC Centre for Global Infectious Disease Analysis, Community Jameel, Cancer Research UK, Imperial College COVID-19 Research Fund, Medical Research Council, Wellcome Sanger Institute.

Rapid Increase of SARS-CoV-2 Variant B.1.1.7 Detected in Sewage Samples from England between October 2020 and January 2021.

SARS-CoV-2 variants with multiple amino acid mutations in the spike protein are emerging in different parts of the world, raising concerns regarding their possible impact on human immune response and vaccine efficacy against the virus. Recently, a variant named lineage B.1.1.7 was detected and shown to be rapidly spreading across the UK since November 2020. As surveillance for these SARS-CoV-2 variants of concern (VOCs) becomes critical, we have investigated the use of environmental surveillance (ES) for the rapid detection and quantification of B.1.1.7 viruses in sewage as a way of monitoring its expansion that is independent on the investigation of identified clinical cases. Next-generation sequencing analysis of amplicons synthesized from sewage concentrates revealed the presence of B.1.1.7 mutations in viral sequences, first identified in a sample collected in London on 10 November 2020 and shown to rapidly increase in frequency to >95% in January 2021, in agreement with clinical data over the same period. We show that ES can provide an early warning of VOCs becoming prevalent in the population and that, as well as B.1.1.7, our method can detect VOCs B.1.351 and P.1, first identified in South Africa and Brazil, respectively, and other viruses carrying critical spike mutation E484K, known to have an effect on virus antigenicity. Although we did not detect such mutation in viral RNAs from sewage, we did detect mutations at amino acids 478, 490, and 494, located close to amino acid 484 in the spike protein structure and known to also have an effect on antigenicity. IMPORTANCE The recent appearance and growth of new SARS-CoV-2 variants represent a major challenge for the control of the COVID-19 pandemic. These variants of concern contain mutations affecting antigenicity, which raises concerns on their possible impact on human immune response to the virus and vaccine efficacy against them. Here, we show how environmental surveillance for SARS-CoV-2 can be used to help us understand virus transmission patterns and provide an early warning of variants becoming prevalent in the population. We describe the detection and quantification of variant B.1.1.7, first identified in southeast England in sewage samples from London (UK) before widespread transmission of this variant was obvious from clinical cases. Variant B.1.1.7 was first detected in a sample from early November 2020, with the frequency of B.1.1.7 mutations detected in sewage rapidly increasing to >95% in January 2021, in agreement with increasing SARS-CoV-2 infections associated with B.1.1.7 viruses.

Tracking SARS-CoV-2 in Sewage: Evidence of Changes in Virus Variant Predominance during COVID-19 Pandemic.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), responsible for the ongoing coronavirus disease (COVID-19) pandemic, is frequently shed in faeces during infection, and viral RNA has recently been detected in sewage in some countries. We have investigated the presence of SARS-CoV-2 RNA in wastewater samples from South-East England between 14th January and 12th May 2020. A novel nested RT-PCR approach targeting five different regions of the viral genome improved the sensitivity of RT-qPCR assays and generated nucleotide sequences at sites with known sequence polymorphisms among SARS-CoV-2 isolates. We were able to detect co-circulating virus variants, some specifically prevalent in England, and to identify changes in viral RNA sequences with time consistent with the recently reported increasing global dominance of Spike protein G614 pandemic variant. Low levels of viral RNA were detected in a sample from 11th February, 3 days before the first case was reported in the sewage plant catchment area. SARS-CoV-2 RNA concentration increased in March and April, and a sharp reduction was observed in May, showing the effects of lockdown measures. We conclude that viral RNA sequences found in sewage closely resemble those from clinical samples and that environmental surveillance can be used to monitor SARS-CoV-2 transmission, tracing virus variants and detecting virus importations.

Developing standards for the microbiome field.

BACKGROUND: Effective standardisation of methodologies to analyse the microbiome is essential to the entire microbiome community. Despite the microbiome field being established for over a decade, there are no accredited or certified reference materials available to the wider community. In this study, we describe the development of the first reference reagents produced by the National Institute for Biological Standards and Control (NIBSC) for microbiome analysis by next-generation sequencing. These can act as global working standards and will be evaluated as candidate World Health Organization International Reference Reagents. RESULTS: We developed the NIBSC DNA reference reagents Gut-Mix-RR and Gut-HiLo-RR and a four-measure framework for evaluation of bioinformatics tool and pipeline bias. Using these reagents and reporting system, we performed an independent evaluation of a variety of bioinformatics tools by analysing shotgun sequencing and 16S rRNA sequencing data generated from the Gut-Mix-RR and Gut-HiLo-RR. We demonstrate that key measures of microbiome health, such as diversity estimates, are largely inflated by the majority of bioinformatics tools. Across all tested tools, biases were present, with a clear trade-off occurring between sensitivity and the relative abundance of false positives in the final dataset. Using commercially available mock communities, we investigated how the composition of reference reagents may impact benchmarking studies. Reporting measures consistently changed when the same bioinformatics tools were used on different community compositions. This was influenced by both community complexity and taxonomy of species present. Both NIBSC reference reagents, which consisted of gut commensal species, proved to be the most challenging for the majority of bioinformatics tools tested. Going forward, we recommend the field uses site-specific reagents of a high complexity to ensure pipeline benchmarking is fit for purpose. CONCLUSIONS: If a consensus of acceptable levels of error can be agreed on, widespread adoption of these reference reagents will standardise downstream gut microbiome analyses. We propose to do this through a large open-invite collaborative study for multiple laboratories in 2020. Video Abstract.