Neuroimmune mechanisms in autism etiology - untangling a complex problem using human cellular models.

Autism spectrum disorder (ASD) affects 1 in 36 people and is more often diagnosed in males than in females. Core features of ASD are impaired social interactions, repetitive behaviors and deficits in verbal communication. ASD is a highly heterogeneous and heritable disorder, yet its underlying genetic causes account only for up to 80% of the cases. Hence, a subset of ASD cases could be influenced by environmental risk factors. Maternal immune activation (MIA) is a response to inflammation during pregnancy, which can lead to increased inflammatory signals to the fetus. Inflammatory signals can cross the placenta and blood brain barriers affecting fetal brain development. Epidemiological and animal studies suggest that MIA could contribute to ASD etiology. However, human mechanistic studies have been hindered by a lack of experimental systems that could replicate the impact of MIA during fetal development. Therefore, mechanisms altered by inflammation during human pre-natal brain development, and that could underlie ASD pathogenesis have been largely understudied. The advent of human cellular models with induced pluripotent stem cell (iPSC) and organoid technology is closing this gap in knowledge by providing both access to molecular manipulations and culturing capability of tissue that would be otherwise inaccessible. We present an overview of multiple levels of evidence from clinical, epidemiological, and cellular studies that provide a potential link between higher ASD risk and inflammation. More importantly, we discuss how stem cell-derived models may constitute an ideal experimental system to mechanistically interrogate the effect of inflammation during the early stages of brain development.

A Novel Approach to Facial Reanimation and Restoration Following Radical Parotidectomies.

Background: Parotidectomies are indicated for a variety of reasons. Regardless of the indication for surgery, facial reanimation may be required because of facial nerve sacrifice or iatrogenic damage. In these cases, facial restoration performed concurrently with ablative surgery is considered the gold standard, and delayed reanimation is usually not attempted. Methods: A retrospective review of all patients who underwent parotidectomies from 2009 to 2022 in a single institution was performed. Indications, surgical techniques, and outcomes of an algorithmic template were applied to these cases using the Sunnybrook, Terzis scores, and Smile Index. A comparison was made between immediate vs. late repairs. Results: Of a total of 90 patients who underwent parotidectomy, 17 (15.3%) had a radical parotidectomy, and 73 (84.7%) had a total or superficial parotidectomy. Among those who underwent complete removal of the gland and nerve sacrifice, eight patients (47.1%) had facial restoration. There were four patients each in the immediate (n = 4) and late repair (n = 4) groups. Surgical techniques ranged from cable grafts to vascularized cross facial nerve grafts (sural communicating nerve flap as per the Koshima procedure) and vascularized nerve flaps (chimeric vastus lateralis and anterolateral thigh flaps, and superficial circumflex perforator flap with lateral femoral cutaneous nerve). Conclusions: The algorithm between one technique and another should take into consideration age, comorbidities, soft tissue defects, presence of facial nerve branches for reinnervation, and donor site morbidity. While immediate facial nerve repair is ideal, there is still benefit in performing a delayed repair in this algorithm.

Admissions to paediatric medical wards with a primary mental health diagnosis: a systematic review of the literature.

OBJECTIVE: To systematically review the literature describing children and young people (CYP) admissions to paediatric general wards because of primary mental health (MH) reasons, particularly in MH crisis. DESIGN: PubMed, Embase, PsycINFO, Web of Science and Google Scholar were searched, with no restriction on country or language. We addressed five search questions to inform: trends and/or the number of admissions, the risk factors for adverse care, the experiences of CYP, families/carers and healthcare professionals (HCPs) and the evidence of interventions aimed at improving the care during admissions.Two reviewers independently assessed the relevance of abstracts identified, extracted data and undertook quality assessment. This review was registered with PROSPERO (CRD42022350655). RESULTS: Thirty-two studies met the inclusion criteria. Eighteen addressed trends and/or numbers/proportions of admissions, 12 provided data about the views/experiences of HCPs, two provided data about CYP's experiences and four explored improving care. We were unable to identify studies examining risk factors for harm during admissions, but studies did report the length of stay in general paediatric/adult settings while waiting for specialised care, which could be considered a risk factor while caring for this group. CONCLUSIONS: MH admissions to children's wards are a long-standing issue and are increasing. CYP will continue to need to be admitted in crisis, with paediatric wards a common location while waiting for assessment. For services to be delivered effectively and for CYP and their families/carers to feel supported and HCPs to feel confident, we need to facilitate more integrated physical and MH pathways of care. PROSPERO REGISTRATION NUMBER: CRD42022350655.

Mental Health Admissions to Paediatric Wards Study (MAPS): protocol of a prospective study of mental health admissions to paediatric wards in England using surveillance and qualitative methods.

INTRODUCTION: Children and young people (CYP) presenting with a mental health (MH) crisis are frequently admitted to general acute paediatric wards as a place of safety. Prior to the pandemic, a survey in England showed that CYP occupied 6% of general paediatric inpatient beds due to an MH crisis, and there have been longstanding concerns about the quality of care to support these patients in this setting. MAPS aims to generate a Theory of Change (ToC) model to improve the quality of care for CYP admitted to acute paediatric services after presenting with an MH crisis. Here, we describe work packages (WPs) 2 and 3 of the study, which have been granted ethics approval. METHODS AND ANALYSIS: We will undertake a national (England), sequential, mixed-methods study to inform a ToC framework alongside a stakeholder group consisting of patients, families/carers and healthcare professionals (HCPs). Our study consists of four WPs undertaken over 30 months. WP2 is limited to working with stakeholders to develop a data collection instrument and then use this in a prospective study of MH admissions over 6 months in 15 purposively recruited acute paediatric wards across England. WP3 consists of gathering the views of CYP, their families/carers and HCPs during admissions using semistructured interviews. ETHICS AND DISSEMINATION: WP2 and WP3 received ethical approval (ref: 23/LO/0349). We will publish the overall synthesis of data and the final ToC to improve care of CYP with MH crisis admitted to general acute paediatric settings. As co-producers of the ToC, we will work with our stakeholder group to ensure wide dissemination of findings. Potential impacts will be upon service development, new models of care, training and workforce planning. PROSPERO REGISTRATION NUMBER: CRD42022350655.

Mental Health Admissions to Paediatric Wards Study (MAPS): a protocol for the analysis of Hospital Episode Statistics (HES) data.

INTRODUCTION: Children and young people (CYP) presenting with a mental health (MH) crisis are frequently admitted to general acute paediatric wards as a place of safety. Prior to the pandemic, a survey in England showed that CYP occupied 6% of general paediatric inpatient beds due to an MH crisis, and there have been longstanding concerns about the quality of care to support these patients in this setting. Mental Health Admissions to Paediatric Wards Study aims to generate a theory of change (ToC) model to improve the quality of care for CYP admitted to acute paediatric services after presenting in a MH crisis. METHODS AND ANALYSIS: We will undertake a national (England), sequential, mixed methods study to inform a ToC framework alongside a stakeholder group consisting of patients, families/carers and healthcare professionals (HCPs). Our study consists of four work packages (WP) undertaken over 30 months. WP1 is limited to using national routine administrative data to identify and characterise trends in MH admissions in acute paediatric wards in England between 2015- 2022. ETHICS AND DISSEMINATION: WP1 received ethical approval (Ref 23/NW/0192). We will publish the overall synthesis of data and the final ToC to improve care of CYP with MH crisis admitted to general acute paediatric settings. As coproducers of the ToC, we will work with our stakeholder group to ensure wide dissemination of findings. Potential impacts will be on service development, new models of care, training and workforce planning.

Protocol for the EACH trial: a multicentre phase II study evaluating the safety and antitumour activity of the combination of avelumab, an anti-PD-L1 agent, and cetuximab, as any line treatment for patients with recurrent/metastatic head and neck squamous cell cancer (HNSCC) in the UK.

INTRODUCTION: Head and neck cancer is the eighth most common cancer in the UK. Current standard of care treatment for patients with recurrent/metastatic squamous cell head and neck carcinoma (HNSCC) is platinum-based chemotherapy combined with the anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody, cetuximab. However, most patients will have poor median overall survival (OS) of 6-9 months despite treatment. HNSCC tumours exhibit an immune landscape poised to respond to immunotherapeutic approaches, with most tumours expressing the immunosuppressive receptor programmed death-ligand 1 (PD-L1). We undertook the current study to determine the safety and efficacy of avelumab, a monoclonal antibody targeting the interaction between PD-L1 and its receptor on cytotoxic T-cells, in combination with cetuximab. METHODS AND ANALYSIS: This is a multi-centre, single-arm dose de-escalation phase II safety and efficacy study of avelumab combined with cetuximab; the study was to progress to a randomised phase II trial, however, the study will now complete after the safety run-in component. Up to 16 participants with histologically/cytologically recurrent/metastatic squamous cell carcinoma (including HNSCC) who have not received cetuximab previously will be recruited. All patients will receive 10 mg/kg avelumab and cetuximab (500, 400 or 300 mg/m2 depending on the cohort open at time of registration) on days 1 and 15 of 4-week cycles for up to 1 year, (avelumab not given cycle 1 day 1). A modified continual reassessment method will be used to determine dose de-escalation. The primary objective is to establish the safety of the combination and to determine the optimum dose of cetuximab. Secondary objectives include assessing evidence of antitumour activity by evaluating response rates and disease control rates at 6 and 12 months as well as progression-free and OS. ETHICS AND DISSEMINATION: Approval granted by City and East REC (18/LO/0021). Findings will be published in peer-reviewed journals and disseminated at conferences. TRIAL REGISTRATION NUMBER: NCT03494322.

Pediatric Hospitalizations and ICU Admissions Due to COVID-19 and Pediatric Inflammatory Multisystem Syndrome Temporally Associated With SARS-CoV-2 in England.

Importance: Investigating how the risk of serious illness after SARS-CoV-2 infection in children and adolescents has changed as new variants have emerged is essential to inform public health interventions and clinical guidance. Objective: To examine risk factors associated with hospitalization for COVID-19 or pediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS) among children and adolescents during the first 2 years of the COVID-19 pandemic and change in risk factors over time. Design, Setting, and Participants: This population-level analysis of hospitalizations after SARS-CoV-2 infection in England among children and adolescents aged 0 to 17 years was conducted from February 1, 2020, to January 31, 2022. National data on hospital activity were linked with data on SARS-CoV-2 testing, SARS-CoV-2 vaccination, pediatric intensive care unit (PICU) admissions, and mortality. Children and adolescents hospitalized with COVID-19 or PIMS-TS during this time were included. Maternal, elective, and injury-related hospitalizations were excluded. Exposures: Previous medical comorbidities, sociodemographic factors, and timing of hospitalization when different SARS-CoV-2 variants (ie, wild type, Alpha, Delta, and Omicron) were dominant in England. Main Outcomes: PICU admission and death within 28 days of hospitalization with COVID-19 or PIMS-TS. Results: A total of 10 540 hospitalizations due to COVID-19 and 997 due to PIMS-TS were identified within 1 125 010 emergency hospitalizations for other causes. The number of hospitalizations due to COVID-19 and PIMS-TS per new SARS-CoV-2 infections in England declined during the second year of the COVID-19 pandemic. Among 10 540 hospitalized children and adolescents, 448 (4.3%) required PICU admission due to COVID-19, declining from 162 of 1635 (9.9%) with wild type, 98 of 1616 (6.1%) with Alpha, and 129 of 3789 (3.4%) with Delta to 59 of 3500 (1.7%) with Omicron. Forty-eight children and adolescents died within 28 days of hospitalization due to COVID-19, and no children died of PIMS-TS (PIMS-S data were limited to November 2020 onward). Risk of severe COVID-19 in children and adolescents was associated with medical comorbidities and neurodisability regardless of SARS-CoV-2 variant. Results were similar when children and adolescents with prior SARS-CoV-2 exposure or vaccination were excluded. Conclusions: In this study of data across the first 2 years of the COVID-19 pandemic, risk of severe disease from SARS-CoV-2 infection in children and adolescents in England remained low. Children and adolescents with multiple medical problems, particularly neurodisability, were at increased risk and should be central to public health measures as further variants emerge.

A highly discriminatory RNA strand-specific assay to facilitate analysis of the role of cis-acting elements in foot-and-mouth disease virus replication.

Foot-and-mouth-disease virus (FMDV), the aetiological agent responsible for foot-and-mouth disease (FMD), is a member of the genus Aphthovirus within the family Picornavirus. In common with all picornaviruses, replication of the single-stranded positive-sense RNA genome involves synthesis of a negative-sense complementary strand that serves as a template for the synthesis of multiple positive-sense progeny strands. We have previously employed FMDV replicons to examine viral RNA and protein elements essential to replication, but the factors affecting differential strand production remain unknown. Replicon-based systems require transfection of high levels of RNA, which can overload sensitive techniques such as quantitative PCR, preventing discrimination of specific strands. Here, we describe a method in which replicating RNA is labelled in vivo with 5-ethynyl uridine. The modified base is then linked to a biotin tag using click chemistry, facilitating purification of newly synthesised viral genomes or anti-genomes from input RNA. This selected RNA can then be amplified by strand-specific quantitative PCR, thus enabling investigation of the consequences of defined mutations on the relative synthesis of negative-sense intermediate and positive-strand progeny RNAs. We apply this new approach to investigate the consequence of mutation of viral cis-acting replication elements and provide direct evidence for their roles in negative-strand synthesis.

Thrombin cleavage of the hepatitis E virus polyprotein at multiple conserved locations is required for genome replication.

The genomes of positive-sense RNA viruses encode polyproteins that are essential for mediating viral replication. These viral polyproteins must undergo proteolysis (also termed polyprotein processing) to generate functional protein units. This proteolysis can be performed by virally-encoded proteases as well as host cellular proteases, and is generally believed to be a key step in regulating viral replication. Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis. The positive-sense RNA genome is translated to generate a polyprotein, termed pORF1, which is necessary and sufficient for viral genome replication. However, the mechanism of polyprotein processing in HEV remains to be determined. In this study, we aimed to understand processing of this polyprotein and its role in viral replication using a combination of in vitro translation experiments and HEV sub-genomic replicons. Our data suggest no evidence for a virally-encoded protease or auto-proteolytic activity, as in vitro translation predominantly generates unprocessed viral polyprotein precursors. However, seven cleavage sites within the polyprotein (suggested by bioinformatic analysis) are susceptible to the host cellular protease, thrombin. Using two sub-genomic replicon systems, we demonstrate that mutagenesis of these sites prevents replication, as does pharmacological inhibition of serine proteases including thrombin. Overall, our data supports a model where HEV uses host proteases to support replication and could have evolved to be independent of a virally-encoded protease for polyprotein processing.

Germline de novo mutations in families with Mendelian cancer syndromes caused by defects in DNA repair.

DNA repair defects underlie many cancer syndromes. We tested whether de novo germline mutations (DNMs) are increased in families with germline defects in polymerase proofreading or base excision repair. A parent with a single germline POLE or POLD1 mutation, or biallelic MUTYH mutations, had 3-4 fold increased DNMs over sex-matched controls. POLE had the largest effect. The DNMs carried mutational signatures of the appropriate DNA repair deficiency. No DNM increase occurred in offspring of MUTYH heterozygous parents. Parental DNA repair defects caused about 20-150 DNMs per child, additional to the ~60 found in controls, but almost all extra DNMs occurred in non-coding regions. No increase in post-zygotic mutations was detected, excepting a child with bi-allelic MUTYH mutations who was excluded from the main analysis; she had received chemotherapy and may have undergone oligoclonal haematopoiesis. Inherited DNA repair defects associated with base pair-level mutations increase DNMs, but phenotypic consequences appear unlikely.

The Warburg micro syndrome protein RAB3GAP1 modulates neuronal morphogenesis and interacts with axon elongation end ER-Golgi trafficking factors.

RAB3GAP1 is GTPase activating protein localized to the ER and Golgi compartments. In humans, mutations in RAB3GAP1 are the most common cause of Warburg Micro syndrome, a neurodevelopmental disorder associated with intellectual disability, microcephaly, and agenesis of the corpus callosum. We found that downregulation of RAB3GAP1 leads to a reduction in neurite outgrowth and complexity in human stem cell derived neurons. To further define the cellular function of RAB3GAP1, we sought to identify novel interacting proteins. We used a combination of mass spectrometry, co-immunoprecipitation and colocalization analysis and identified two novel interactors of RAB3GAP1: the axon elongation factor Dedicator of cytokinesis 7 (DOCK7) and the TATA modulatory factor 1 (TMF1) a modulator of Endoplasmic Reticulum (ER) to Golgi trafficking. To define the relationship between RAB3GAP1 and its two novel interactors, we analyzed their localization to different subcellular compartments in neuronal and non-neuronal cells with loss of RAB3GAP1. We find that RAB3GAP1 is important for the sub-cellular localization of TMF1 and DOCK7 across different compartments of the Golgi and endoplasmic reticulum. In addition, we find that loss of function mutations in RAB3GAP1 lead to dysregulation of pathways that are activated in response to the cellular stress like ATF6, MAPK, and PI3-AKT signaling. In summary, our findings suggest a novel role for RAB3GAP1 in neurite outgrowth that could encompass the regulation of proteins that control axon elongation, ER-Golgi trafficking, as well as pathways implicated in response to cellular stress.

Studies towards the Synthesis of (+)-Dictyoxetane.

The dolabellane-type diterpene dictyoxetane represents a significant challenge to synthetic organic chemistry. Methodology directed towards the total synthesis of naturally occurring (+)-dictyoxetane is reported. Catalytic asymmetric synthesis of the trans-hydrindane ring system is achieved through chemoselective deoxygenation of the Hajos-Parrish ketone. An alternative to the Garst-Spencer furan annulation is developed for the synthesis of a 2,5-dimethyl, tetrasubstituted furan, employing a tandem 5-exo-dig alcohol to alkyne cyclisation/aromatisation reaction as a key step. The (4+3) cycloaddition reaction of an oxyallyl cation with a tetrasubstituted furan is established on a cyclohexanone-derived model system, and a range of related (4+3) cycloadditions investigated on a homochiral, trans-hydrindane-fused furan, where regio- and diastereoselectivity is required for the natural product synthesis. In an alternative (4+2) Diels-Alder approach, a C2 -symmetric vinyl sulfoxide-based chiral ketene equivalent is used to prepare oxanorbornenes with the same oxygen bridge stereochemistry found in the 2,7-dioxatricyclo[4.2.1.03,8 ]nonane ring system of the natural product.

Key Features of a Multi-Disciplinary Hospital-Based Rehabilitation Program for Children and Adolescents with Moderate to Severe Myalgic Encephalomyelitis/Chronic Fatigue Syndrome ME/CFS.

PURPOSE OF THE STUDY: There is limited published data on treatment or outcomes of children and young people (CYP) with moderate or severe Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). Here, we describe outcomes of moderate and severe ME/CFS in CYP treated in a tertiary adolescent service. This information is useful when planning services for CYP and families affected by moderate/severe ME/CFS and to guide future management trials and commissioning decisions. STUDY DESIGN: A retrospective review was conducted of medical records of the 27 CYP who received ward-based treatment in 2015. Notes were retrospectively reviewed to assess progress in four markers of wellbeing over the period of treatment: (i) mobility, (ii) education, (iii) sleep and (iv) involvement in social/recreational activities. RESULTS: A total of 23/27 (85%) showed improvement in one or more domains over their period of ward-based therapy. 19/27 (70%) of patients showed improvement in physical ability. In 15/23 patients (65%), there was an improvement in ability to access education, in 12/24 (50%) sleep improved, and 16/27 (59%) demonstrated an improvement in socialising/ability perform recreational activities. CONCLUSION/IMPLICATIONS: A multidisciplinary hospital-based rehabilitation programme for moderate and severe ME/CFS was associated with improvement in at least one area of wellbeing in 85% of the CYP we reviewed. These data may be used as a baseline to evaluate the impact of other models of delivering care for this patient group. It may be useful when considering other groups such as those affected by Post-COVID Syndrome.

Dynamic Changes in the NK-, Neutrophil-, and B-cell Immunophenotypes Relevant in High Metastatic Risk Post Neoadjuvant Chemotherapy-Resistant Early Breast Cancers.

PURPOSE: To identify potential immune targets in post-neoadjuvant chemotherapy (NAC)-resistant triple-negative breast cancer (TNBC) and ER+HER2- breast cancer disease. EXPERIMENTAL DESIGN: Following pathology review, 153 patients were identified as having residual cancer burden (RCB) II/III disease (TNBC n = 80; ER+HER2-n = 73). Baseline pre-NAC samples were available for evaluation for 32 of 80 TNBC and 36 of 73 ER+HER2- cases. Bright-field hematoxylin and eosin assessment allowed for tumor-infiltrating lymphocyte (TIL) evaluation in all cases. Multiplexed immunofluorescence was used to identify the abundance and distribution of immune cell subsets. Levels of checkpoints including PD-1/PD-L1 expression were also quantified. Findings were then validated using expression profiling of cancer and immune-related genes. Cytometry by time-of-flight characterized the dynamic changes in circulating immune cells with NAC. RESULTS: RCB II/III TNBC and ER+HER2- breast cancer were immunologically "cold" at baseline and end of NAC. Although the distribution of immune cell subsets across subtypes was similar, the mRNA expression profiles were both subtype- and chemotherapy-specific. TNBC RCB II/III disease was enriched with genes related to neutrophil degranulation, and displayed strong interplay across immune and cancer pathways. We observed similarities in the dynamic changes in B-cell biology following NAC irrespective of subtype. However, NAC induced changes in the local and circulating tumor immune microenvironment (TIME) that varied by subtype and response. Specifically, in TNBC residual disease, we observed downregulation of stimulatory (CD40/OX40L) and inhibitory (PD-L1/PD-1) receptor expression and an increase in NK cell populations (especially non-cytolytic, exhausted CD56dimCD16-) within both the local TIME and peripheral white cell populations. CONCLUSIONS: This study identifies several potential immunologic pathways in residual disease, which may be targeted to benefit high-risk patients.

Positive strand RNA viruses differ in the constraints they place on the folding of their negative strand.

Genome replication of positive strand RNA viruses requires the production of a complementary negative strand RNA that serves as a template for synthesis of more positive strand progeny. Structural RNA elements are important for genome replication, but while they are readily observed in the positive strand, evidence of their existence in the negative strand is more limited. We hypothesized that this was due to viruses differing in their capacity to allow this latter RNA to adopt structural folds. To investigate this, ribozymes were introduced into the negative strand of different viral constructs; the expectation being that if RNA folding occurred, negative strand cleavage and suppression of replication would be seen. Indeed, this was what happened with hepatitis C virus (HCV) and feline calicivirus (FCV) constructs. However, little or no impact was observed for chikungunya virus (CHIKV), human rhinovirus (HRV), hepatitis E virus (HEV), and yellow fever virus (YFV) constructs. Reduced cleavage in the negative strand proved to be due to duplex formation with the positive strand. Interestingly, ribozyme-containing RNAs also remained intact when produced in vitro by the HCV polymerase, again due to duplex formation. Overall, our results show that there are important differences in the conformational constraints imposed on the folding of the negative strand between different positive strand RNA viruses.

Light-induced asymmetries in embryonic retinal gene expression are mediated by the vascular system and extracellular matrix.

Left-right asymmetries in the nervous system (lateralisation) influence a broad range of behaviours, from social responses to navigation and language. The role and pathways of endogenous and environmental mechanisms in the ontogeny of lateralisation remains to be established. The domestic chick is a model of both endogenous and experience-induced lateralisation driven by light exposure. Following the endogenous rightward rotation of the embryo, the asymmetrical position in the egg results in a greater exposure of the right eye to environmental light. To identify the genetic pathways activated by asymmetric light stimulation, and their time course, we exposed embryos to different light regimes: darkness, 6 h of light and 24 h of light. We used RNA-seq to compare gene expression in the right and left retinas and telencephalon. We detected differential gene expression in right vs left retina after 6 h of light exposure. This difference was absent in the darkness condition and had already disappeared by 24 h of light exposure, suggesting that light-induced activation is a self-terminating phenomenon. This transient effect of light exposure was associated with a downregulation of the sensitive-period mediator gene DIO2 (iodothyronine deiodinase 2) in the right retina. No differences between genes expressed in the right vs. left telencephalon were detected. Gene networks associated with lateralisation were connected to vascularisation, cell motility, and the extracellular matrix. Interestingly, we know that the extracellular matrix-including the differentially expressed PDGFRB gene-is involved in morphogenesis, sensitive periods, and in the endogenous chiral mechanism of primary cilia, that drives lateralisation. Our data show a similarity between endogenous and experience-driven lateralisation, identifying functional gene networks that affect lateralisation in a specific time window.

The RNA pseudoknots in foot-and-mouth disease virus are dispensable for genome replication, but essential for the production of infectious virus.

Non-coding regions of viral RNA (vRNA) genomes are critically important in the regulation of gene expression. In particular, pseudoknot (PK) structures, which are present in a wide range of RNA molecules, have a variety of roles. The 5' untranslated region (5' UTR) of foot-and-mouth disease virus (FMDV) vRNA is considerably longer than in other viruses from the picornavirus family and consists of a number of distinctive structural motifs that includes multiple (2, 3 or 4 depending on the virus strain) putative PKs linked in tandem. The role(s) of the PKs in the FMDV infection are not fully understood. Here, using bioinformatics, sub-genomic replicons and recombinant viruses we have investigated the structural conservation and importance of the PKs in the FMDV lifecycle. Our results show that despite the conservation of two or more PKs across all FMDVs, a replicon lacking PKs was replication competent, albeit at reduced levels. Furthermore, in competition experiments, GFP FMDV replicons with less than two (0 or 1) PK structures were outcompeted by a mCherry FMDV wt replicon that had 4 PKs, whereas GFP replicons with 2 or 4 PKs were not. This apparent replicative advantage offered by the additional PKs correlates with the maintenance of at least two PKs in the genomes of FMDV field isolates. Despite a replicon lacking any PKs retaining the ability to replicate, viruses completely lacking PK were not viable and at least one PK was essential for recovery of infections virus, suggesting a role for the PKs in virion assembly. Thus, our study points to roles for the PKs in both vRNA replication and virion assembly, thereby improving understanding the molecular biology of FMDV replication and the wider roles of PK in RNA functions.