A Temperature-Sensitive Recombinant of Avian Coronavirus Infectious Bronchitis Virus Provides Complete Protection against Homologous Challenge

Avian coronavirus infectious bronchitis virus (IBV) is the etiological agent of infectious bronchitis, an acute highly contagious economically relevant respiratory disease of poultry. Vaccination is used to control IBV infections, with live-attenuated vaccines generated via serial passage of a virulent field isolate through embryonated hens' eggs. A fine balance must be achieved between attenuation and the retention of immunogenicity. The exact molecular mechanism of attenuation is unknown, and vaccines produced in this manner present a risk of reversion to virulence as few consensus level changes are acquired. Our previous research resulted in the generation of a recombinant IBV (rIBV) known as M41-R, based on a pathogenic strain M41-CK. M41-R was attenuated in vivo by two amino acid changes, Nsp10-Pro85Leu and Nsp14-Val393Leu; however, the mechanism of attenuation was not determined. Pro85 and Val393 were found to be conserved among not only IBV strains but members of the wider coronavirus family. This study demonstrates that the same changes are associated with a temperature-sensitive (ts) replication phenotype at 41°C in vitro, suggesting that the two phenotypes may be linked. Vaccination of specific-pathogen-free chickens with M41-R induced 100% protection against clinical disease, tracheal ciliary damage, and challenge virus replication following homologous challenge with virulent M41-CK. Temperature sensitivity has been used to rationally attenuate other viral pathogens, including influenza, and the identification of amino acid changes that impart both a ts and an attenuated phenotype may therefore offer an avenue for future coronavirus vaccine development. IMPORTANCE Infectious bronchitis virus is a pathogen of economic and welfare concern for the global poultry industry. Live-attenuated vaccines against are generated by serial passage of a virulent isolate in embryonated eggs until attenuation is achieved. The exact mechanisms of attenuation are unknown, and vaccines produced have a risk of reversion to virulence. Reverse genetics provides a method to generate vaccines that are rationally attenuated and are more stable with respect to back selection due to their clonal origin. Genetic populations resulting from molecular clones are more homogeneous and lack the presence of parental pathogenic viruses, which generation by multiple passage does not. In this study, we identified two amino acids that impart a temperature-sensitive replication phenotype. Immunogenicity is retained and vaccination results in 100% protection against homologous challenge. Temperature sensitivity, used for the development of vaccines against other viruses, presents a method for the development of coronavirus vaccines

The Evolution, Spread and Global Threat of H6Nx Avian Influenza Viruses

Avian influenza viruses of the subtype H6Nx are being detected globally with increasing frequency. Some H6Nx lineages are becoming enzootic in Asian poultry and sporadic incursions into European poultry are occurring more frequently. H6Nx viruses that contain mammalian adaptation motifs pose a zoonotic threat and have caused human cases. Although currently understudied globally, H6Nx avian influenza viruses pose a substantial threat to both poultry and human health. In this review we examine the current state of knowledge of H6Nx viruses including their global distribution, tropism, transmission routes and human health risk

Identification of Amino Acids within Nonstructural Proteins 10 and 14 of the Avian Coronavirus Infectious Bronchitis Virus That Result in Attenuation In Vivo and In Ovo

The Gammacoronavirus infectious bronchitis virus (IBV) is a highly contagious global pathogen prevalent in all types of poultry flocks. IBV is responsible for economic losses and welfare issues in domestic poultry, resulting in a significant risk to food security. IBV vaccines are currently generated by serial passage of virulent IBV field isolates through embryonated hens' eggs. The different patterns of genomic variation accumulated during this process means that the exact mechanism of attenuation is unknown and presents a risk of reversion to virulence. Additionally, the passaging process adapts the virus to replicate in chicken embryos, increasing embryo lethality. Vaccines produced in this manner are therefore unsuitable for in ovo application. We have developed a reverse genetics system, based on the pathogenic IBV strain M41, to identify genes which can be targeted for rational attenuation. During the development of this reverse genetics system, we identified four amino acids, located in nonstructural proteins (nsps) 10, 14, 15, and 16, which resulted in attenuation both in vivo and in ovo. Further investigation highlighted a role of amino acid changes, Pro85Leu in nsp 10 and Val393Leu in nsp 14, in the attenuated in vivo phenotype observed. This study provides evidence that mutations in nsps offer a promising mechanism for the development of rationally attenuated live vaccines against IBV, which have the potential for in ovo application. IMPORTANCE The Gammacoronavirus infectious bronchitis virus (IBV) is the etiological agent of infectious bronchitis, an acute, highly contagious, economically important disease of poultry. Vaccination is achieved using a mixture of live attenuated vaccines for young chicks and inactivated vaccines as boosters for laying hens. Live attenuated vaccines are generated through serial passage in embryonated hens' eggs, an empirical process which achieves attenuation but retains immunogenicity. However, these vaccines have a risk of reversion to virulence, and they are lethal to the embryo. In this study, we identified amino acids in the replicase gene which attenuated IBV strain M41, both in vivo and in ovo. Stability assays indicate that the attenuating amino acids are stable and unlikely to revert. The data in this study provide evidence that specific modifications in the replicase gene offer a promising direction for IBV live attenuated vaccine development, with the potential for in ovo application

Prevalence and architecture of de novo mutations in developmental disorders.

The genomes of individuals with severe, undiagnosed developmental disorders are enriched in damaging de novo mutations (DNMs) in developmentally important genes. Here we have sequenced the exomes of 4,293 families containing individuals with developmental disorders, and meta-analysed these data with data from another 3,287 individuals with similar disorders. We show that the most important factors influencing the diagnostic yield of DNMs are the sex of the affected individual, the relatedness of their parents, whether close relatives are affected and the parental ages. We identified 94 genes enriched in damaging DNMs, including 14 that previously lacked compelling evidence of involvement in developmental disorders. We have also characterized the phenotypic diversity among these disorders. We estimate that 42% of our cohort carry pathogenic DNMs in coding sequences; approximately half of these DNMs disrupt gene function and the remainder result in altered protein function. We estimate that developmental disorders caused by DNMs have an average prevalence of 1 in 213 to 1 in 448 births, depending on parental age. Given current global demographics, this equates to almost 400,000 children born per year

Psychosocial impact of undergoing prostate cancer screening for men with BRCA1 or BRCA2 mutations.

OBJECTIVES: To report the baseline results of a longitudinal psychosocial study that forms part of the IMPACT study, a multi-national investigation of targeted prostate cancer (PCa) screening among men with a known pathogenic germline mutation in the BRCA1 or BRCA2 genes. PARTICPANTS AND METHODS: Men enrolled in the IMPACT study were invited to complete a questionnaire at collaborating sites prior to each annual screening visit. The questionnaire included sociodemographic characteristics and the following measures: the Hospital Anxiety and Depression Scale (HADS), Impact of Event Scale (IES), 36-item short-form health survey (SF-36), Memorial Anxiety Scale for Prostate Cancer, Cancer Worry Scale-Revised, risk perception and knowledge. The results of the baseline questionnaire are presented. RESULTS: A total of 432 men completed questionnaires: 98 and 160 had mutations in BRCA1 and BRCA2 genes, respectively, and 174 were controls (familial mutation negative). Participants' perception of PCa risk was influenced by genetic status. Knowledge levels were high and unrelated to genetic status. Mean scores for the HADS and SF-36 were within reported general population norms and mean IES scores were within normal range. IES mean intrusion and avoidance scores were significantly higher in BRCA1/BRCA2 carriers than in controls and were higher in men with increased PCa risk perception. At the multivariate level, risk perception contributed more significantly to variance in IES scores than genetic status. CONCLUSION: This is the first study to report the psychosocial profile of men with BRCA1/BRCA2 mutations undergoing PCa screening. No clinically concerning levels of general or cancer-specific distress or poor quality of life were detected in the cohort as a whole. A small subset of participants reported higher levels of distress, suggesting the need for healthcare professionals offering PCa screening to identify these risk factors and offer additional information and support to men seeking PCa screening

Comparative single-cell genomics of Atribacterota JS1 in the Japan Trench hadal sedimentary biosphere

Deep-sea and subseafloor sedimentary environments host heterotrophic microbial communities that contribute to Earth’s carbon cycling. However, the potential metabolic functions of individual microorganisms and their biogeographical distributions in hadal ocean sediments remain largely unexplored. In this study, we conducted single-cell genome sequencing on sediment samples collected from six sites (7,445–8,023 m water depth) along an approximately 500 km transect of the Japan Trench during the International Ocean Discovery Program Expedition 386. A total of 1,886 single-cell amplified genomes (SAGs) were obtained, offering comprehensive genetic insights into sedimentary microbial communities in surface sediments (<1 m depth) above the sulfate-methane transition zone along the Japan Trench. Our genome data set included 269 SAGs from Atribacterota JS1, the predominant bacterial clade in these hadal environments. Phylogenetic analysis classified SAGs into nine distinct phylotypes, whereas metagenome-assembled genomes were categorized into only two phylotypes, advancing JS1 diversity coverage through a single cell-based approach. Comparative genomic analysis of JS1 lineages from different habitats revealed frequent detection of genes related to organic carbon utilization, such as extracellular enzymes like clostripain and α-amylase, and ABC transporters of oligopeptide from Japan Trench members. Furthermore, specific JS1 phylotypes exhibited a strong correlation with in situ methane concentrations and contained genes involved in glycine betaine metabolism. These findings suggest that the phylogenomically diverse and novel Atribacterota JS1 is widely distributed in Japan Trench sediment, playing crucial roles in carbon cycling within the hadal sedimentary biosphere

Heterozygous Variants in KMT2E Cause a Spectrum of Neurodevelopmental Disorders and Epilepsy.

We delineate a KMT2E-related neurodevelopmental disorder on the basis of 38 individuals in 36 families. This study includes 31 distinct heterozygous variants in KMT2E (28 ascertained from Matchmaker Exchange and three previously reported), and four individuals with chromosome 7q22.2-22.23 microdeletions encompassing KMT2E (one previously reported). Almost all variants occurred de novo, and most were truncating. Most affected individuals with protein-truncating variants presented with mild intellectual disability. One-quarter of individuals met criteria for autism. Additional common features include macrocephaly, hypotonia, functional gastrointestinal abnormalities, and a subtle facial gestalt. Epilepsy was present in about one-fifth of individuals with truncating variants and was responsive to treatment with anti-epileptic medications in almost all. More than 70% of the individuals were male, and expressivity was variable by sex; epilepsy was more common in females and autism more common in males. The four individuals with microdeletions encompassing KMT2E generally presented similarly to those with truncating variants, but the degree of developmental delay was greater. The group of four individuals with missense variants in KMT2E presented with the most severe developmental delays. Epilepsy was present in all individuals with missense variants, often manifesting as treatment-resistant infantile epileptic encephalopathy. Microcephaly was also common in this group. Haploinsufficiency versus gain-of-function or dominant-negative effects specific to these missense variants in KMT2E might explain this divergence in phenotype, but requires independent validation. Disruptive variants in KMT2E are an under-recognized cause of neurodevelopmental abnormalities

Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)

Multidisciplinary characterisation and modelling of a small upland catchment in Scotland

A combination of conventional surveying and non-invasive techniques have been applied to characterising the geomorphology, soils and shallow substrates of a typical small catchment in the Southern Uplands in Scotland, in three dimensions. Integration of geospatial, geophysical and geotechnical data, in the resulting digital 3D model, enable the nature and extent of individual components of the landscape to be measured and their relationships at depth to interpreted and visualised. This type of baseline data is fundamental to understanding past, and monitoring and measuring the impacts of future environmental changes in environmentally sensitive areas