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OBJECTIVE: The social distancing measures governments implemented in response to the COVID-19 pandemic have had substantial impacts. For some communities, these impacts will be disproportionate, with those communities experiencing inequalities, marginalisation or discrimination facing specific challenges. Lesbian, gay, bisexual, queer and allied (LGBQ+) communities experience a range of well-being inequalities that may have been impacted by the pandemic. The study aimed to assess the comparative impact of the UK's response to COVID-19 on LGBQ+ communities. DESIGN: A mixed-method explanatory sequential study of the general population using a cross-sectional online survey and semistructured interviews. SETTING: Community, North West of England. PARTICIPANTS: Adults aged 18 years and over; 1540 participated in the survey (192, 12%, LGBQ+) with 49 undergoing semistructured interviews (15 LGBQ+) during spring and summer of 2020. RESULTS: Survey findings indicated that LGBQ+ people experienced similar positive and negative impacts to the rest of the population, but some negative impacts were more marked among the LGBQ+ community. LGBQ+ participants were more likely to disagree that 'the government considered the impact on people like you' when preparing guidance. They were significantly more likely to report being unable to access sufficient food and required medication, eating less healthily, exercising less regularly, experiencing poorer quality sleep and taking more pain medicine than usual. Interview data supported these differences; isolation, being unable to access social networks and concerns about health were commonly discussed by the LGBQ+ participants. Positive impacts, including better work-life balance, were similar across both groups. CONCLUSIONS: The findings indicate LGBQ+ communities' wellbeing inequalities have been compounded by the social distancing restrictions, for example, by impacts on social networks increasing loneliness. Preparedness planning for future pandemics should include equality impact assessments for potential interventions.

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Microglia, the primary immune cells of the central nervous system (CNS), are derived from the yolk sac and populate the brain during development. Once microglia migrate to the CNS, they are self-renewing and require CSF1R signaling for their maintenance. Pexidartinib (PLX3397, PLX), a small molecule inhibitor of the CSF1R, has been shown to effectively deplete microglia since microglial maintenance is CSF1R-dependent. There have, however, been several conflicting reports that have shown the potential off-target effects of PLX on peripheral immune cells particularly those of lymphoid origin. Given this controversy in the use of the PLX family of drugs, it has become important to ascertain to what extent PLX affects the peripheral immune profile in lymphoid (spleen, and bone marrow) and non-lymphoid (kidney, lungs, and heart) organs. PLX3397 chow treatment at 660 mg/kg for 7 days significantly reduced CD45+ macrophages, CX3CR1-GFP cells, CD11b+CD45intermediate cells, and P2RY12 expression in the brain. However, there were minimal effects on peripheral immune cells from both lymphoid and non-lymphoid organs except in the heart where there was a significant decrease in CD3+ cells, inflammatory and patrolling monocytes, and CD11b+Ly6G+ neutrophils. We then stimulated the immune system with 1 mg/kg of LPS which resulted in a significant reduction in the number of innate immune cells. In this context, PLX did not alter the cytokine profile in the serum and the brain of naïve mice but did so in the LPS-stimulated group resulting in a significant reduction in TNFα, IL-1α, IFN-γ and IL-1ß. Furthermore, PLX did not alter locomotor activity in the open field test suggesting that microglia do not contribute to LPS-induced sickness behavior. Our results provide an assessment of immune cell populations with PLX3397 treatment on brain, lymphoid and non-lymphoid organs without and during LPS treatment that can serve as a resource for understanding consequences of such approaches.

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BACKGROUND: The estimated global prevalence and burden of non-alcoholic fatty liver disease (NAFLD) and its advanced stage, non-alcoholic steatohepatitis (NASH), is increasing. Yet, NAFLD remains largely underdiagnosed. In addition to hepatic morbidity and mortality, NAFLD is associated with increased cardiovascular complications, warranting a multidisciplinary approach. Despite its rapidly increasing prevalence, knowledge of NAFLD among healthcare workers is limited, especially with specialists outside the field of hepatology and gastroenterology. OBJECTIVES: To investigate knowledge, practice and opinions/attitudes of healthcare workers towards diagnosis and management of NAFLD/NASH. METHODS: The survey was designed in collaboration with a multidisciplinary scientific committee established especially for this study. The survey was disseminated to healthcare workers from seven different disciplines through four collaborating societies, social media and at a cardiology-themed conference from February to June 2022. Median and interquartile range were mentioned for numeric responses and proportions for categorical responses or responses on a Likert scale. Likert scale responses were treated as ordinal data and analysed with the appropriate tests. RESULTS: The full dataset included 613 respondents from 88 different countries (including 488 physicians). 64% of the surveyed physicians underestimated the prevalence of NAFLD. General practitioners and cardiologists underestimated the prevalence most often (74% and 77%, respectively). Compared to the other disciplines, cardiologists were least familiar with the symptoms and diagnostic criteria and felt least confident in diagnosing and managing NAFLD. Overall, 65% of physicians reported regularly using evidence-based guidelines for managing NAFLD, yet 72% reported challenges in providing lifestyle recommendations. A lack of awareness was the most common reported reason for the lack of screening for NAFLD (68% respectively). CONCLUSIONS: Despite the growing burden of NAFLD, there is a significant gap in awareness, knowledge, and management among physicians treating patients with cardiometabolic comorbidities, particularly cardiologists. Hepatologists and gastroenterologists could play a role in educating their fellow physicians.

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BACKGROUND AND OBJECTIVE: Macular thickness fluctuations (MTF) over time may be more predictive of visual outcomes than absolute macular thickness in patients with diabetic macular edema (DME) treated with anti-vascular endothelial growth factor (anti-VEGF). It is unclear whether this association exists in DME patients treated with intravitreal steroids or whether steroids confer reduced MTF versus anti-VEGF treatments. PATIENTS AND METHODS: MTF was compared before and after initiation of steroids in DME patients treated with intravitreal steroids. A mixed-effects linear regression model was used to determine the association between MTF and best-corrected visual acuity (BCVA). RESULTS: Mean 12-month MTF significantly decreased after steroid initiation (61.1 µm versus 53.5 µm, P = 0.04, n = 105 eyes). Mean BCVA after 12 months was not significantly different from baseline. No significant association between post-steroid MTF and 12-month BCVA was found. CONCLUSION: Steroid treatment decreases MTF while BCVA remains stable in DME patients previously treated with anti-VEGF. [Ophthalmic Surg Lasers Imaging Retina 2023;54:454-460.].

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Background and Aims: The impacts of the COVID-19 pandemic have not been equal, with a disproportionate impact among ethnic minority communities. Structural inequalities in social determinants of health such as housing and employment have contributed to COVID-19's impact on deprived communities, including many ethnic minority communities. To compare (1) how the UK government's "social distancing" restrictions and guidance were perceived and implemented by ethnic minority populations compared to white populations, (2) the impact of restrictions and guidance upon these groups. Methods: An explanatory sequential mixed methods study incorporated a quantitative survey and qualitative semi-structured interviews to explore individual perceptions and experiences of COVID-19 and the national restrictions. Survey participants (n = 1587) were recruited from North West England; 60 (4%) participants were from ethnic minority communities. Forty-nine interviews were conducted; 19 (39%) participants were from ethnic minority communities. Interviews were transcribed verbatim and analysed using a thematic approach. Data collection was between April and August 2020. Results: Significant differences in demographics and household overcrowding were observed between white vs ethnic minority survey respondents, who were also significantly less confident in their knowledge of COVID-19, less likely to be high-risk drinkers, and marginally more likely to have experienced job loss and/or reduced household income. There were no group differences in wellbeing, perceptions, or nonfinancial impacts. Two inter-related themes included: (1) government guidance, incorporating people's knowledge and understanding of the guidance and their confusion/frustration over messaging; (2) the impacts of restrictions on keyworkers, home-schooling, working from home and changes in lifestyle/wellbeing. Conclusions: Further research is needed on the long-term impacts of COVID-19 on ethnic minority communities. If policy responses to COVID-19 are to benefit ethnic minority communities, there is a need for future studies to consider fundamental societal issues, such as the role of housing and economic disadvantage.

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BACKGROUND: Bacterial infections cause substantial pain and disability among people who inject drugs. We described time trends in hospital admissions for injecting-related infections in England. METHODS: We analyzed hospital admissions in England between January 2002 and December 2021. We included patients with infections commonly caused by drug injection, including cutaneous abscesses, cellulitis, endocarditis, or osteomyelitis, and a diagnosis of opioid use disorder. We used Poisson regression to estimate seasonal variation and changes associated with coronavirus disease 2019 (COVID-19) response. RESULTS: There were 92 303 hospital admissions for injection-associated infections between 2002 and 2021. Eighty-seven percent were skin, soft-tissue, or vascular infections; 72% of patients were male; and the median age increased from 31 years in 2002 to 42 years in 2021. The rate of admissions reduced from 13.97 per day (95% confidence interval [CI], 13.59-14.36) in 2003 to 8.94 (95% CI, 8.64-9.25) in 2011, then increased to 18.91 (95% CI, 18.46-19.36) in 2019. At the introduction of COVID-19 response in March 2020, the rate of injection-associated infections reduced by 35.3% (95% CI, 32.1-38.4). Injection-associated infections were also seasonal; the rate was 1.21 (95% CI, 1.18-1.24) times higher in July than in February. CONCLUSIONS: This incidence of opioid injection-associated infections varies within years and reduced following COVID-19 response measures. This suggests that social and structural factors such as housing and the degree of social mixing may contribute to the risk of infection, supporting investment in improved social conditions for this population as a means to reduce the burden of injecting-related infections.

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BACKGROUND: Peroxisome proliferator-activated receptor (PPAR) agonists may have favorable outcomes on non-alcoholic fatty liver disease. This study serves as proof of concept to evaluate whether dual PPAR-α/γ agonists improve non-invasive tests of liver steatosis and fibrosis. METHODS: This is a post-hoc analysis of a randomized, double-blind, placebo-controlled, multi-center trial comprising 7226 patients with type 2 diabetes mellitus and recent coronary artery disease randomized to receive aleglitazar, a PPAR-α/γ agonists, or placebo for two years. Main outcomes were change in non-invasive tests for liver steatosis and fibrosis: Liver Fat Score (LFS), Liver Accumulation Product (LAP), Fibrosis-4 (FIB-4), and NAFLD Fibrosis Score (NFS). RESULTS: LFS, LAP and FIB-4 decreased upon treatment, whereas scores in the placebo group remained the same or increased (P<0.001). NFS responded differently but remained consistently lower than placebo. In the treatment group more participants shifted to a lower FIB-4 and NFS category, or improved in respect to the LAP cut-off values compared to the placebo group (P<0.001 for FIB-4 and LAP, P<0.004 for NFS). LFS had a low discriminative power in this study. CONCLUSION: This post-hoc analysis showed improvement of non-invasive tests of liver steatosis and fibrosis after starting dual PPAR-α/γ agonist treatment, adding to the evidence that this pathway has potential in non-alcoholic fatty liver disease treatment.

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BACKGROUND AND AIMS: Mortality and drug treatment data suggest that the median age of people who inject drugs is increasing. We aimed to describe changes in the characteristics of people injecting drugs in the United Kingdom (UK). DESIGN: Repeat cross-sectional surveys and modelling. SETTING: Low-threshold services in the United Kingdom such as needle and syringe programmes. PARTICIPANTS: A total of 79 900 people who recently injected psychoactive drugs in the United Kingdom, recruited as part of the Unlinked Anonymous Monitoring Survey (England, Wales, Northern Ireland, 1990-2019) and Needle Exchange Surveillance Initiative (Scotland, 2008-2019). MEASUREMENTS: Age of people currently injecting, age at first injection, duration of injecting (each 1990-2019) and estimates of new people who started injecting (1980-2019). FINDINGS: In England, Wales and Northern Ireland between 1990 and 2019, the median age of people injecting increased from 27 (interquartile range [IQR], 24-31) to 40 (IQR, 34-46); median age at first injection increased from 22 (IQR, 19-25) to 33 (IQR, 28-39); and median years of injecting increased from 7 (IQR, 3-11) to 18 (IQR, 9-23). Values in Scotland and England were similar after 2008. The estimated number that started injecting annually in England increased from 5470 (95% prediction interval [PrI] 3120-6940) in 1980 to a peak of 10 270 (95% PrI, 8980-12 780) in 1998, and then decreased to 2420 (95% PrI, 1320-5580) in 2019. The number in Scotland followed a similar pattern, increasing from 1220 (95% PrI, 740-2430) in 1980 to a peak of 3080 (95% PrI, 2160-3350) in 1998, then decreased to a 270 (95% PrI, 130-600) in 2018. The timing of the peak differed between regions, with earlier peaks in London and the North West of England. CONCLUSIONS: In the United Kingdom, large cohorts started injecting psychoactive drugs in the 1980s and 1990s and many still inject today. Relatively few people started in more recent years. This has led to changes in the population injecting drugs, including an older average age and longer injecting histories.

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SIGNIFICANCE: Teleophthalmology became widely used during the coronavirus 2019 pandemic; however, the quality of this care remains to be understood. PURPOSE: This study aimed to compare patient satisfaction levels from virtual and in-person visits based on post-visit surveys, as well as investigate demographic characteristics that may predict patient satisfaction with virtual visits. METHODS: Virtual (n = 2943) and in-person (n = 56,175) visits from March 19, 2020, to July 31, 2020, were identified using the electronic health record system. For in-person visits, a random subset of 3000 visits was acquired using a random number generator. Of these, 2266 virtual and 2590 in-person visits met the inclusion criteria. Patients who completed the Telemedicine for Medical Practice Survey and Medical Practice Survey were analyzed in this report. Nonparametric Mann-Whitney test was used to compare scores between groups. RESULTS: Two hundred eleven virtual patients (9.31%; 82 phone, 115 video, 14 hybrid) and 307 in-person patients (11.85%) completed the Telemedicine for Medical Practice Survey and Medical Practice Survey, respectively. Satisfaction scores were similar and high in both groups-virtual visit satisfaction scores averaged 4.82, whereas in-person visit satisfaction averaged 4.85 (P = .80, θ = 0.501 [0.493 to 0.509]). Only one question yielded significantly different satisfaction scores, and no demographic variables were significant predictors of satisfaction scores. CONCLUSIONS: Patient satisfaction is comparable between virtual and in-person visits, validating the continued usage of telemedicine for eye care visits.

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Individual reactions to traumatic stress vary dramatically, yet the biological basis of this variation remains poorly understood. Recent studies demonstrate the surprising plasticity of oligodendrocytes and myelin with stress and experience, providing a potential mechanism by which trauma induces aberrant structural and functional changes in the adult brain. In this study, we utilized a translational approach to test the hypothesis that gray matter oligodendrocytes contribute to traumatic-stress-induced behavioral variation in both rats and humans. We exposed adult, male rats to a single, severe stressor and used a multimodal approach to characterize avoidance, startle, and fear-learning behavior, as well as oligodendrocyte and myelin basic protein (MBP) content in multiple brain areas. We found that oligodendrocyte cell density and MBP were correlated with behavioral outcomes in a region-specific manner. Specifically, stress-induced avoidance positively correlated with hippocampal dentate gyrus oligodendrocytes and MBP. Viral overexpression of the oligodendrogenic factor Olig1 in the dentate gyrus was sufficient to induce an anxiety-like behavioral phenotype. In contrast, contextual fear learning positively correlated with MBP in the amygdala and spatial-processing regions of the hippocampus. In a group of trauma-exposed US veterans, T1-/T2-weighted magnetic resonance imaging estimates of hippocampal and amygdala myelin associated with symptom profiles in a region-specific manner that mirrored the findings in rats. These results demonstrate a species-independent relationship between region-specific, gray matter oligodendrocytes and differential behavioral phenotypes following traumatic stress exposure. This study suggests a novel mechanism for brain plasticity that underlies individual variance in sensitivity to traumatic stress.

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BACKGROUND AND OBJECTIVES: This study aimed to evaluate the necessity of the postoperative day-1 (POD1) review after pars plana vitrectomy. PATIENTS AND METHODS: The analysis included available literature that documented medical and surgical interventions performed on POD1 review after pars plana vitrectomy. A meta-analysis of proportions was conducted using a binomial-normal model to analyze three data sets consisting of all interventions, medical interventions, and surgical interventions. Heterogeneity and publication bias analyses were performed. RESULTS: POD1 reviews of 2,262 patients across 14 studies were examined to yield a total POD1 intervention rate estimate of 4.7% (95% confidence interval [CI], 3.0-13.9). When stratified by medical or surgical intervention, the intervention rate estimates were 4.1% (95% CI, 1.4-11.6) and 0.7% (95% CI, 0.3-1.3), respectively. The most common complication requiring postoperative intervention was elevated intraocular pressure. CONCLUSION: Given the wide confidence intervals of the estimated intervention rates, variability in postoperative practices, and range of interventions performed, the POD1 review cannot be discarded in its entirety. [Ophthalmic Surg Lasers Imaging Retina. 2021;52:513-518.].

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Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate cytopathic events induced by SARS-CoV-2 with virus replication processes in frozen-hydrated cells, we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. Here we report critical SARS-CoV-2 structural events - e.g. viral RNA transport portals, virus assembly intermediates, virus egress pathway, and native virus spike structures, in the context of whole-cell volumes revealing drastic cytppathic changes. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.

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INTRODUCTION: Chemsex is a specific form of sexualised drug use (SDU) that is an emerging public health issue among men who have sex with men (MSM). Although the recent focus on chemsex is a reflection of the associated harms it is important to understand SDU more broadly and its associations with risk behaviours. Additionally, some of the reasons suggested for MSM engagement in SDU are also likely to apply to women who have sex with women (WSW) and trans people. The aim of this review was to investigate SDU, including chemsex, among lesbian, gay, bisexual and trans (LGBT) people internationally in relation to sexual health outcomes (HIV status, STI diagnosis, condom use). METHODS: Papers that were published between January 2010 and June 2020 reporting SDU in MSM, WSW, or trans people were identified through Medline, PsycINFO, CINAHL Plus and Web of Science. Results were synthesised using a narrative approach. RESULTS: The search identified 2,710 publications, of which 75 were included in the final synthesis. The majority of studies measured SDU among MSM (n = 71), and four studies measured SDU among trans people. Research into SDU had been conducted in 55 countries and 32 countries had recorded the use of a chemsex drug among MSM, although the drugs used to define chemsex varied. Among studies that researched MSM, SDU was most commonly investigated in relation to condomless anal intercourse (n = 42), followed by HIV prevalence (n = 35), and then STI diagnoses (n = 27). Drug use was generally associated with sexual health outcomes, but particularly in chemsex studies. CONCLUSIONS: SDU research is lacking among WSW and trans people, despite trans women having a high HIV prevalence. Among MSM, most drugs were associated with sexual health outcomes, and therefore it is important to include both chemsex drugs and other drugs in SDU research.

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BACKGROUND: Chemsex and sexualised drug use (SDU) among men who have sex with men (MSM) has become a public health issue because of its associated sexual risks. Some MSM engaging in SDU require further help and support, but it is not clear if this need is being met. This research seeks to understand MSM and service provider (SP) perspectives of the current standard of service provision for MSM engaging in SDU. METHOD: Semi-structured interviews were conducted with 13 MSM and 16 SPs in England (January-December 2019). MSM who reported taking one of ten substances for sex in the past 12 months were recruited from a mailing list or a community organisation providing chemsex support. Potential SP participants were recruited via an email sent from their local healthcare NHS Trust. Data for MSM and SPs were analysed separately using thematic analysis. RESULTS: MSM participants had a median age of 34 years (range 23-66).The majority of SPs recruited were genitourinary medicine (GUM) consultants. We found that the main reason for engagement in SDU was because of the enhanced sexual experience. Most MSM recruited were satisfied with their sexual health service provision, but barriers to care were highlighted by both MSM and SPs. Four themes relating to barriers to care were identified: accessibility of sexual health services, funding of services, SP's attitudes towards MSM who engage in SDU, and services outside sexual healthcare. CONCLUSION: SPs appeared to have adopted a harm reduction approach to MSM engaging in SDU, but for MSM wanting further help and assistance in relation to their SDU additional barriers to care existed. Training healthcare providers outside of sexual health services regarding chemsex and SDU may reduce some of these barriers, but funding for sexual health services needs to be improved to maintain and further develop services.

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A letter to the editor to discuss several uses of brain magnetic resonance imaging (MRI) in the investigation of neurological manifestations of covid-19. Described several situations in which the MRI is needed. Brain MRI is an important diagnostic method in the covid-19 scenario, to investigate possible neurological complications of the disease.

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BACKGROUND: The use of image and performance enhancing drugs (IPEDs) among men who have sex with men (MSM) and women who have sex with women (WSW) is rarely studied, with most of this research focused on anabolic androgenic steroid use among MSM. To address this gap, the extent of recent IPED use and the associated factors are explored in a community-recruited sample of MSM and WSW METHODS: Data from the UK LGBT Sex and Lifestyles Survey was used, which recruited through social-media advertising and community organisations (April-June 2018). Participants were asked if they had taken any IPEDs (e.g. anabolic steroids, growth hormone, hCG, Melanotan, non-prescribed diet pills) in the past 12 months. Factors associated with recent (in past 12 months) IPED use among MSM and WSW were investigated using stepwise binary logistic regression. RESULTS: 1,658 MSM and 1,507 WSW were included in the analysis. Among MSM, 3.4% (n = 57) had recently taken IPEDs, 60% of those taking IPEDs had used psychoactive drugs. In the multivariable analysis, IPED use among MSM was associated with psychoactive drug use, Viagra use, higher body dissatisfaction, and lower sexual satisfaction. Among WSW, 4.1% (n = 62) had recently taken IPEDs, and 50% of those taking IPEDs had used psychoactive drugs. In the multivariable analysis, IPED use among WSW was associated with being aged 45 years and over, recent STI diagnosis, recent sexual contact without consent, and higher body dissatisfaction. CONCLUSION: IPED use was reported by around 1-in-25 MSM and WSW, and was associated with a number of health and psychological problems. Research to better understand the drivers and impacts of IPED use among MSM and WSW, and how this compares to use among heterosexual people is needed.

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INTRODUCTION AND AIMS: Historically, people who inject image and performance enhancing drugs (IPED) were not perceived as being at high risk of HIV or hepatitis C virus (HCV) infection. However, recent studies indicate HCV and HIV prevalences are elevated, with many HCV infections undiagnosed. DESIGN AND METHODS: Men who inject IPEDs recruited from community settings and specialist services, including needle-syringe programs, across UK during 2016 self-completed a questionnaire. Multivariate analyses examined factors associated with HCV/HIV testing. RESULTS: The participants' (n=562; 24% service recruited) median age was 31 years, 4% identified as gay or bisexual, 18% had ever been imprisoned and 6% had ever injected a psychoactive drug. Those community recruited more often reported sharing drugs vials (16% vs. 8%, P=0.021) and, among those with 2+ sexual partners, poor condom use (50% vs. 36%, P=0.063), than those service recruited. Overall, one-third had ever been tested for HCV (31%) and/or HIV (34%). Testing uptake was associated with other risk factors for HCV/HIV, being recruited through services and having received metabolic tests. Participants' motivations for using IPEDs were associated with recruitment setting and HIV/HCV testing uptake. DISCUSSION AND CONCLUSIONS: The majority were untested for HCV/HIV. HCV/HIV testing and risks were associated with recruitment through services. Previous needle and syringe program-based studies have potentially overestimated testing uptake and underestimated risk. Targeted interventions are needed, particularly for those not accessing services. The association between HCV/HIV testing uptake and receipt of metabolic tests suggests that developing a combined offer of these tests as part of health monitoring could improve uptake.

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Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. However, investigation of the SARS-CoV-2 infection in the native cellular context is scarce, and there is a lack of comprehensive knowledge on SARS-CoV-2 replicative cycle. Understanding the genome replication, assembly and egress of SARS-CoV-2, a multistage process that involves different cellular compartments and the activity of many viral and cellular proteins, is critically important as it bears the means of medical intervention to stop infection. Here, we investigated SARS-CoV-2 replication in Vero cells under the near-native frozen-hydrated condition using a unique correlative multi-modal, multi-scale cryo-imaging approach combining soft X-ray cryo-tomography and serial cryoFIB/SEM volume imaging of the entire SARS-CoV-2 infected cell with cryo-electron tomography (cryoET) of cellular lamellae and cell periphery, as well as structure determination of viral components by subtomogram averaging. Our results reveal at the whole cell level profound cytopathic effects of SARS-CoV-2 infection, exemplified by a large amount of heterogeneous vesicles in the cytoplasm for RNA synthesis and virus assembly, formation of membrane tunnels through which viruses exit, and drastic cytoplasm invasion into nucleus. Furthermore, cryoET of cell lamellae reveals how viral RNAs are transported from double-membrane vesicles where they are synthesized to viral assembly sites; how viral spikes and RNPs assist in virus assembly and budding; and how fully assembled virus particles exit the cell, thus stablishing a model of SARS-CoV-2 genome replication, virus assembly and egress pathways.

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Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. However, investigation of the SARS-CoV-2 infection in the native cellular context is scarce, and there is a lack of comprehensive knowledge on SARS-CoV-2 replicative cycle. Understanding the genome replication, assembly and egress of SARS-CoV-2, a multistage process that involves different cellular compartments and the activity of many viral and cellular proteins, is critically important as it bears the means of medical intervention to stop infection. Here, we investigated SARS-CoV-2 replication in Vero cells under the near-native frozen-hydrated condition using a unique correlative multi-modal, multi-scale cryo-imaging approach combining soft X-ray cryo-tomography and serial cryoFIB/SEM volume imaging of the entire SARS-CoV-2 infected cell with cryo-electron tomography (cryoET) of cellular lamellae and cell periphery, as well as structure determination of viral components by subtomogram averaging. Our results reveal at the whole cell level profound cytopathic effects of SARS-CoV-2 infection, exemplified by a large amount of heterogeneous vesicles in the cytoplasm for RNA synthesis and virus assembly, formation of membrane tunnels through which viruses exit, and drastic cytoplasm invasion into nucleus. Furthermore, cryoET of cell lamellae reveals how viral RNAs are transported from double-membrane vesicles where they are synthesized to viral assembly sites; how viral spikes and RNPs assist in virus assembly and budding; and how fully assembled virus particles exit the cell, thus stablishing a model of SARS-CoV-2 genome replication, virus assembly and egress pathways.