Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity.

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant

Hospital admission and emergency care attendance risk for SARS-CoV-2 delta (B.1.617.2) compared with alpha (B.1.1.7) variants of concern: a cohort study

Background: The SARS-CoV-2 delta (B.1.617.2) variant was first detected in England in March, 2021. It has since rapidly become the predominant lineage, owing to high transmissibility. It is suspected that the delta variant is associated with more severe disease than the previously dominant alpha (B.1.1.7) variant. We aimed to characterise the severity of the delta variant compared with the alpha variant by determining the relative risk of hospital attendance outcomes. Methods: This cohort study was done among all patients with COVID-19 in England between March 29 and May 23, 2021, who were identified as being infected with either the alpha or delta SARS-CoV-2 variant through whole-genome sequencing. Individual-level data on these patients were linked to routine health-care datasets on vaccination, emergency care attendance, hospital admission, and mortality (data from Public Health England's Second Generation Surveillance System and COVID-19-associated deaths dataset; the National Immunisation Management System; and NHS Digital Secondary Uses Services and Emergency Care Data Set). The risk for hospital admission and emergency care attendance were compared between patients with sequencing-confirmed delta and alpha variants for the whole cohort and by vaccination status subgroups. Stratified Cox regression was used to adjust for age, sex, ethnicity, deprivation, recent international travel, area of residence, calendar week, and vaccination status. Findings: Individual-level data on 43 338 COVID-19-positive patients (8682 with the delta variant, 34 656 with the alpha variant; median age 31 years [IQR 17–43]) were included in our analysis. 196 (2·3%) patients with the delta variant versus 764 (2·2%) patients with the alpha variant were admitted to hospital within 14 days after the specimen was taken (adjusted hazard ratio [HR] 2·26 [95% CI 1·32–3·89]). 498 (5·7%) patients with the delta variant versus 1448 (4·2%) patients with the alpha variant were admitted to hospital or attended emergency care within 14 days (adjusted HR 1·45 [1·08–1·95]). Most patients were unvaccinated (32 078 [74·0%] across both groups). The HRs for vaccinated patients with the delta variant versus the alpha variant (adjusted HR for hospital admission 1·94 [95% CI 0·47–8·05] and for hospital admission or emergency care attendance 1·58 [0·69–3·61]) were similar to the HRs for unvaccinated patients (2·32 [1·29–4·16] and 1·43 [1·04–1·97]; p=0·82 for both) but the precision for the vaccinated subgroup was low. Interpretation: This large national study found a higher hospital admission or emergency care attendance risk for patients with COVID-19 infected with the delta variant compared with the alpha variant. Results suggest that outbreaks of the delta variant in unvaccinated populations might lead to a greater burden on health-care services than the alpha variant. Funding: Medical Research Council; UK Research and Innovation; Department of Health and Social Care; and National Institute for Health Research

Changes in symptomatology, reinfection, and transmissibility associated with the SARS-CoV-2 variant B.1.1.7: an ecological study

Background The SARS-CoV-2 variant B.1.1.7 was first identified in December, 2020, in England. We aimed to investigate whether increases in the proportion of infections with this variant are associated with differences in symptoms or disease course, reinfection rates, or transmissibility. Methods We did an ecological study to examine the association between the regional proportion of infections with the SARS-CoV-2 B.1.1.7 variant and reported symptoms, disease course, rates of reinfection, and transmissibility. Data on types and duration of symptoms were obtained from longitudinal reports from users of the COVID Symptom Study app who reported a positive test for COVID-19 between Sept 28 and Dec 27, 2020 (during which the prevalence of B.1.1.7 increased most notably in parts of the UK). From this dataset, we also estimated the frequency of possible reinfection, defined as the presence of two reported positive tests separated by more than 90 days with a period of reporting no symptoms for more than 7 days before the second positive test. The proportion of SARS-CoV-2 infections with the B.1.1.7 variant across the UK was estimated with use of genomic data from the COVID-19 Genomics UK Consortium and data from Public Health England on spike-gene target failure (a non-specific indicator of the B.1.1.7 variant) in community cases in England. We used linear regression to examine the association between reported symptoms and proportion of B.1.1.7. We assessed the Spearman correlation between the proportion of B.1.1.7 cases and number of reinfections over time, and between the number of positive tests and reinfections. We estimated incidence for B.1.1.7 and previous variants, and compared the effective reproduction number, Rt, for the two incidence estimates. Findings From Sept 28 to Dec 27, 2020, positive COVID-19 tests were reported by 36 920 COVID Symptom Study app users whose region was known and who reported as healthy on app sign-up. We found no changes in reported symptoms or disease duration associated with B.1.1.7. For the same period, possible reinfections were identified in 249 (0·7% [95% CI 0·6–0·8]) of 36 509 app users who reported a positive swab test before Oct 1, 2020, but there was no evidence that the frequency of reinfections was higher for the B.1.1.7 variant than for pre-existing variants. Reinfection occurrences were more positively correlated with the overall regional rise in cases (Spearman correlation 0·56–0·69 for South East, London, and East of England) than with the regional increase in the proportion of infections with the B.1.1.7 variant (Spearman correlation 0·38–0·56 in the same regions), suggesting B.1.1.7 does not substantially alter the risk of reinfection. We found a multiplicative increase in the Rt of B.1.1.7 by a factor of 1·35 (95% CI 1·02–1·69) relative to pre-existing variants. However, Rt fell below 1 during regional and national lockdowns, even in regions with high proportions of infections with the B.1.1.7 variant. Interpretation The lack of change in symptoms identified in this study indicates that existing testing and surveillance infrastructure do not need to change specifically for the B.1.1.7 variant. In addition, given that there was no apparent increase in the reinfection rate, vaccines are likely to remain effective against the B.1.1.7 variant. Funding Zoe Global, Department of Health (UK), Wellcome Trust, Engineering and Physical Sciences Research Council (UK), National Institute for Health Research (UK), Medical Research Council (UK), Alzheimer's Society

Genomic assessment of quarantine measures to prevent SARS-CoV-2 importation and transmission

Mitigation of SARS-CoV-2 transmission from international travel is a priority. We evaluated the effectiveness of travellers being required to quarantine for 14-days on return to England in Summer 2020. We identified 4,207 travel-related SARS-CoV-2 cases and their contacts, and identified 827 associated SARS-CoV-2 genomes. Overall, quarantine was associated with a lower rate of contacts, and the impact of quarantine was greatest in the 16–20 age-group. 186 SARS-CoV-2 genomes were sufficiently unique to identify travel-related clusters. Fewer genomically-linked cases were observed for index cases who returned from countries with quarantine requirement compared to countries with no quarantine requirement. This difference was explained by fewer importation events per identified genome for these cases, as opposed to fewer onward contacts per case. Overall, our study demonstrates that a 14-day quarantine period reduces, but does not completely eliminate, the onward transmission of imported cases, mainly by dissuading travel to countries with a quarantine requirement

Genomic epidemiology of SARS-CoV-2 in a UK university identifies dynamics of transmission

AbstractUnderstanding SARS-CoV-2 transmission in higher education settings is important to limit spread between students, and into at-risk populations. In this study, we sequenced 482 SARS-CoV-2 isolates from the University of Cambridge from 5 October to 6 December 2020. We perform a detailed phylogenetic comparison with 972 isolates from the surrounding community, complemented with epidemiological and contact tracing data, to determine transmission dynamics. We observe limited viral introductions into the university; the majority of student cases were linked to a single genetic cluster, likely following social gatherings at a venue outside the university. We identify considerable onward transmission associated with student accommodation and courses; this was effectively contained using local infection control measures and following a national lockdown. Transmission clusters were largely segregated within the university or the community. Our study highlights key determinants of SARS-CoV-2 transmission and effective interventions in a higher education setting that will inform public health policy during pandemics.</jats:p

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant

Hypertension-induced inflammation: from cellular mechanisms to disease

The work presented in this thesis focused on high intraluminal pressure and its role in vascular inflammation. This was done in the context of hypertension and its contribution to the progression of atherosclerosis, the major underlying pathology in coronary artery disease. While a large body of work have explored the role of immune cells in the pathogenesis of hypertension, very few studies have investigated the opposite; whether hypertension plays a role in the aetiology of inflammation. Studies which have previously attempted to investigate the effect of high blood pressure and inflammation have been thwarted by the difficulty of trying to separate the consequences of the various neurohumoral factors associated with hypertension compared to those induced by the increase in pressure itself. This is particularly true of in vivo protocols. To address this, we developed a customised ex vivo vessel chamber that enabled the recording of leukocyte adhesion to the endothelium in real time, in intact vessels under various pressures. Using this technique, vessels exposed to high intraluminal pressure demonstrated greater leukocyte adhesion, adhesion molecule gene expression and endothelial microparticle production. Interestingly, we found this to be independent of the renin-angiotensin system. Several mechanisms involved in this process are presented in Chapters 4 & 5. In Chapter 4, the production of reactive oxygen species (ROS), endothelial nitric oxide synthase (eNOS), arginase, and the transcription factor, nuclear factor κB (NFκB), all known to be implicated in both hypertension and vascular inflammation, were explored as possible mechanisms. High intraluminal pressure was shown to increase ROS production, arginase II expression and activity as well as NFκB expression, while eNOS was unaltered by acute increases in pressure. Furthermore, functional studies indicated a possible role for NADPH oxidase and the mitochondria as the sources of the ROS mediating response. To tease out the mechanical forces responsible for the pressure-induced response, in Chapter 5, we observed the effect of different shear rates on leukocyte adhesion in pressurised vessels. High intraluminal pressure (circumferential stretch) together with low shear stress produced the greatest response, while high stress reduced the effect of high pressure. Caveolae, membrane invaginations that have been shown to mediate many intracellular signalling pathways, were explored as possible mechanosensors involved in pressure-induced inflammatory response. Cav1-/- (Caveolin-1) mice and Cav1 KD cells had reduced leukocyte adhesion and adhesion molecule gene expression in response to TNFα stimulation. These results suggest caveolae play an important role in the inflammatory response. Furthermore, when the effect of pressure on caveolae structure was further explored, we showed, for the first time, that high intraluminal pressure reduced caveolae number. We speculate this may be due to the dissociation of the caveolae proteins Cav1 and cavin-1. In Chapter 6, we examined the effect of hypertension on atherosclerosis in an in vivo setting. Hypertensive atherosclerotic prone BPHxApoe-/- placed on a high fat diet for 12 weeks demonstrated no change in plaque size. However, detrimental changes in plaque morphology with increased lipid and macrophage content and reduced collagen were observed suggestive of reduced plaque stability. We also demonstrated that blockade of P-selectin, which mediates recruitment of leukocytes to the endothelium, partially improved this stability. In conclusion, the studies described in this thesis provide evidence that high intraluminal pressure induces vascular inflammation. We demonstrated that the mechanical forces exerted by high pressure promote caveolae flattening, NADPH oxidase dependent ROS production, arginase II activation and NFκB translocation, which all contribute to endothelial activation, adhesion molecule expression and enhanced leukocyte adhesion, all hallmarks of inflammation. We also showed in vivo that hypertension may result in atherosclerotic plaque instability, which may be partially improved by blocking leukocyte recruitment. Therefore, this thesis has established fundamental concepts on how high intraluminal pressure can alter cellular biological responses that may lead to new paradigms in the management of the increased risk of cardiovascular complications seen in hypertensive individuals

Hypertension-induced inflammation: from cellular mechanisms to disease

The work presented in this thesis focused on high intraluminal pressure and its role in vascular inflammation. This was done in the context of hypertension and its contribution to the progression of atherosclerosis, the major underlying pathology in coronary artery disease. While a large body of work have explored the role of immune cells in the pathogenesis of hypertension, very few studies have investigated the opposite; whether hypertension plays a role in the aetiology of inflammation. Studies which have previously attempted to investigate the effect of high blood pressure and inflammation have been thwarted by the difficulty of trying to separate the consequences of the various neurohumoral factors associated with hypertension compared to those induced by the increase in pressure itself. This is particularly true of in vivo protocols. To address this, we developed a customised ex vivo vessel chamber that enabled the recording of leukocyte adhesion to the endothelium in real time, in intact vessels under various pressures. Using this technique, vessels exposed to high intraluminal pressure demonstrated greater leukocyte adhesion, adhesion molecule gene expression and endothelial microparticle production. Interestingly, we found this to be independent of the renin-angiotensin system. Several mechanisms involved in this process are presented in Chapters 4 & 5. In Chapter 4, the production of reactive oxygen species (ROS), endothelial nitric oxide synthase (eNOS), arginase, and the transcription factor, nuclear factor κB (NFκB), all known to be implicated in both hypertension and vascular inflammation, were explored as possible mechanisms. High intraluminal pressure was shown to increase ROS production, arginase II expression and activity as well as NFκB expression, while eNOS was unaltered by acute increases in pressure. Furthermore, functional studies indicated a possible role for NADPH oxidase and the mitochondria as the sources of the ROS mediating response. To tease out the mechanical forces responsible for the pressure-induced response, in Chapter 5, we observed the effect of different shear rates on leukocyte adhesion in pressurised vessels. High intraluminal pressure (circumferential stretch) together with low shear stress produced the greatest response, while high stress reduced the effect of high pressure. Caveolae, membrane invaginations that have been shown to mediate many intracellular signalling pathways, were explored as possible mechanosensors involved in pressure-induced inflammatory response. Cav1-/- (Caveolin-1) mice and Cav1 KD cells had reduced leukocyte adhesion and adhesion molecule gene expression in response to TNFα stimulation. These results suggest caveolae play an important role in the inflammatory response. Furthermore, when the effect of pressure on caveolae structure was further explored, we showed, for the first time, that high intraluminal pressure reduced caveolae number. We speculate this may be due to the dissociation of the caveolae proteins Cav1 and cavin-1. In Chapter 6, we examined the effect of hypertension on atherosclerosis in an in vivo setting. Hypertensive atherosclerotic prone BPHxApoe-/- placed on a high fat diet for 12 weeks demonstrated no change in plaque size. However, detrimental changes in plaque morphology with increased lipid and macrophage content and reduced collagen were observed suggestive of reduced plaque stability. We also demonstrated that blockade of P-selectin, which mediates recruitment of leukocytes to the endothelium, partially improved this stability. In conclusion, the studies described in this thesis provide evidence that high intraluminal pressure induces vascular inflammation. We demonstrated that the mechanical forces exerted by high pressure promote caveolae flattening, NADPH oxidase dependent ROS production, arginase II activation and NFκB translocation, which all contribute to endothelial activation, adhesion molecule expression and enhanced leukocyte adhesion, all hallmarks of inflammation. We also showed in vivo that hypertension may result in atherosclerotic plaque instability, which may be partially improved by blocking leukocyte recruitment. Therefore, this thesis has established fundamental concepts on how high intraluminal pressure can alter cellular biological responses that may lead to new paradigms in the management of the increased risk of cardiovascular complications seen in hypertensive individuals

Caveolae:A role in endothelial inflammation and mechanotransduction?

Vascular inflammation and disease progression, such as atherosclerosis, are in part a consequence of haemodynamic forces generated by changes in blood flow. The haemodynamic forces, such as shear stress or stretch, interact with vascular endothelial cells, which transduce the mechanical stimuli into biochemical signals via mechanosensors, which can induce an upregulation in pathways involved in inflammatory signaling. However, it is unclear how these mechanosensors respond to shear stress and most significantly what cellular mechanisms are involved in sensing the haemodynamic stimuli. This review explores the transition from shear forces, stretch and pressure to endothelial inflammation and the process of mechanotransduction, specifically highlighting evidence to suggest that caveolae play as a role as mechanosensors