Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2,3,4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

A novel serological assay for detection of rabies virus neutralising antibodies

We have developed retroviral vectors that can be used in neutralisation assays to determine antibody titres for biohazard level 3/4 pathogens. This assay has two advantages over existing methods: (1) Retroviral pseudotypes can be handled in biohazard level 2 laboratories; (2) The use of either green fluorescent protein, luciferase or β-galactosidase as a reporter allows the assay to be used at low cost in laboratories throughout the world. G-protein sequences from CVS-11, EBLV-1, EBLV-2 and a human street genotype-1 virus (RV61) cDNA were cloned and co-expressed along with HIV/MLV gag-pol and a GFP or luciferase in human 293T cells. The resulting pseudotyped viruses were able to infect a number of target cells and, with the exception of RV61, produced viral titres similar to the well characterised Vesicular Stomatitis Virus pseudotype. Using blinded sera, neutralisation titres were comparable to those using OIE’s gold standard fluorescent antibody virus neutralisation test and assays also revealed cross-neutralisation of EBLV-1, EBLV-2 and CVS-11. This development of an inexpensive and robust high-throughput microassay for the measurement of rabies virus neutralising antibodies in animal and human serum samples will be widely applicable for use in wildlife vaccine campaigns and in human post-vaccination antibody monitoring

Investigating the antibody neutralisation potential of a rabies virus lentiviral pseudotype: an international inter-laboratory trial

Rabies infection causes an acute viral encephalitis that is almost invariably fatal. Transmission most commonly occurs following the bite of an infected dog, and annually >55,000 people die from this disease, the majority being located in the developing world. Dog vaccination and responsible dog ownership are key to the control and elimination of rabies and the ability to detect virus neutralising antibodies is key to the evaluation of vaccination status following pre-immunisation. Current diagnostic tests are expensive, and as such are not readily performed in resource limited settings. To overcome this, lentiviral pseudotypes have been applied as an alternative platform for post vaccinal assessment and serosurveillance. The assay requires a small amount of serum in comparison to the standard fluorescent virus neutralisation assay (FAVN). Furthermore, the ability to tailor the assay according to facilities available through flexibility of reporter gene expression enables transfer of this technology across areas where the virus remains endemic, i.e. Africa and Asia. These reporter gene detection systems eliminate the need for expensive equipment and facilities allowing the assay to be performed in laboratories throughout the world. An inter laboratory ring trial has shown that the pseudotype assay for rabies virus is both reliable and reproducible

Development of a novel serological assay for the detection of rabies virus neutralising antibodies using lentiviral pseudotypes

Rabies virus (RABV) causes an acute encephalitis that is almost invariably fatal. Whilst effective vaccines and post exposure prophylaxis are available it is estimated that over 55,000 human fatalities occur worldwide each year. This figure is, however, believed to be a gross underestimation of the actual number of human deaths, as a disproportionate number of fatalities occur across the developing world where reporting systems are mainly absent. The detection of virus neutralising antibodies (VNA) is key to the evaluation of vaccination status following pre-immunisation. Current tests for RABV VNAs require expensive reagents and equipment, along with biosafety containment level 3 facilities, something not easily achieved in rabies endemic areas. The use of lentiviral pseudotypes as a vector for gene therapy is well documented. These have proven to be highly sensitive yet flexible platforms which can be adapted to allow the evaluation of vaccines and antiviral drugs against highly pathogenic viruses without the need for high level containment facilities or great expense. We have adapted this technology to allow the determination of serum neutralising antibody titres against highly pathogenic rabies and related lyssaviruses. The pseudotype assay only requires a small amount of serum in comparison to the standard fluorescent virus neutralisation assay (FAVN), and the use of different reporter genes, such as green fluorescent protein, luciferase, or β-galactosidase, makes it possible for the assay to be undertaken at low cost in laboratories throughout the world. G-protein sequences from viral isolates representing each lyssavirus genotype and the newly classified Eurasian strains were cloned and co-expressed with lentiviral gag-pol and different reporter genes. The pseudotypes infected a number of target cell lines and produced titres almost equivalent to VSV-G protein pseudotypes. To date, over 500 sera have been evaluated concurrently using genotype 1, CVS-11 live virus (FAVN) and pseudotype neutralisation assays. Comparison of antibody titres reveals a 96.4% sensitivity and 100% specificity (r=0.83, p<0.001). Neutralisation assays using pseudotypes with glycoproteins from other lyssavirus genotypes suggest that they provide a greater sensitivity compared to the current virus neutralization tests and will therefore allow a more accurate determination of serological response to these highly pathogenic infections. Importantly for the use of this assay in countries where the cold chain cannot be maintained, CVS-11 pseudotypes were highly stable during freeze-thaw cycles and storage at room temperature. These results suggest that the proposed pseudotype assay is a practical, effective and robust solution for rapid lyssavirus serosurveillance wherever it is needed

A robust lentiviral pseudotype neutralisation assay for rabies and lyssavirus serology

The inflexibility of existing serological techniques for rabies can make it difficult to monitor the impact of current control strategies. We have developed a neutralisation assay that uses lentiviral pseudotypes (comprising the outer envelope of rabies virus, human immunodeficiency virus core and a packageable reporter gene) as antigen vectors. The assay can be used for vaccine evaluation, serosurveillance and antiviral screening in low-containment laboratories within developed and resource-limited countries. Post-vaccination serum antibody titres determined using the fluorescent antibody virus neutralisation assay and pseudotype assay show a specificity of 100%, sensitivity of 96.4% and strong correlation (r=0.85; p<0.001). To increase the assay’s utility we incorporated the envelope glycoprotein of lyssavirus isolates from genotypes 1-7 and a range of different reporter genes. Genotype 1 (CVS-11) pseudotypes were highly stable during freeze-thaw cycles and storage at room temperature suggesting the pseudotype assay is a suitable option for undertaking lyssavirus serosurveillance in areas most affected by these infections

Molecular Epidemiology of Bat Lyssaviruses in Europe

Bat rabies cases in Europe are principally attributed to two lyssaviruses, namely European bat lyssavirus type 1 (EBLV-1) and European bat lyssavirus type 2 (EBLV-2). Between 1977 and 2011, 961 cases of bat rabies were reported to Rabies Bulletin Europe, with the vast majority (>97%) being attributed to EBLV-1. There have been 25 suspected cases of EBLV-2, of which 22 have been confirmed. In addition, two single isolations of unique lyssaviruses from European insectivorous bats were reported in south-west Russia in 2002 (West Caucasian bat virus) and in Germany in 2010 (Bokeloh bat lyssavirus). In this review, we present phylogenetic analyses of the EBLV-1 and EBLV-2 using partial nucleoprotein (N) gene sequences. In particular, we have analysed all EBLV-2 cases for which viral sequences (N gene, 400 nucleotides) are available (n = 21). Oropharyngeal swabs collected from two healthy Myotis daubentonii during active surveillance programmes in Scotland and Switzerland also yielded viral RNA (EBLV-2). Despite the relatively low number of EBLV-2 cases, a surprisingly large amount of anomalous data has been published in the scientific literature and Genbank, which we have collated and clarified. For both viruses, geographical relationships are clearly defined on the phylogenetic analysis. Whilst there is no clear chronological clustering for either virus, there is some evidence for host specific relationships, particularly for EBLV-1 where more host variation has been observed. Further genomic regions must be studied, in particular for EBLV-1 isolates from Spain and the EBLV-2 isolates to provide support for the existence of sublineages

Quantifying antigenic relationships among the lyssaviruses

All lyssaviruses cause fatal encephalitis in mammals. There is sufficient antigenic variation within the genus to cause variable vaccine efficacy, but this variation is difficult to characterize quantitatively: sequence analysis cannot yet provide detailed antigenic information, and antigenic neutralization data have been refractory to high-resolution robust interpretation. Here, we address these issues by using state-of-the-art antigenic analyses to generate a high-resolution antigenic map of a global panel of 25 lyssaviruses. We compared the calculated antigenic distances with viral glycoprotein ectodomain sequence data. Although 67% of antigenic variation was predictable from the glycoprotein amino acid sequence, there are in some cases substantial differences between genetic and antigenic distances, thus highlighting the risk of inferring antigenic relationships solely from sequence data at this time. These differences included epidemiologically important antigenic differences between vaccine strains and wild-type rabies viruses. Further, we quantitatively assessed the antigenic relationships measured by using rabbit, mouse, and human sera, validating the use of nonhuman experimental animals as a model for determining antigenic variation in humans. The use of passive immune globulin is a crucial component of rabies postexposure prophylaxis, and here we also show that it is p