Virus-derived Synthetic Riboswitches for Real-time Coronaviruses Sensing in Live Cells (preprint)

Real-time sensing of viral infection in live cells are crucial for virology research and antiviral development. However, existing methods face challenges of low signal sensitivity and the necessity for viral manipulation and cell fixation. Here, we develop a viral riboswitch (vRibo) approach that employs the viral replicase to induce transgene expression upon viral infection. The vRibo is designed to detect viral real-time transcription and replication in live cells, which in response triggers the translation of reporter and therapeutic genes. By integrating a viral packaging sequence, vRibo can be transmitted to neighboring cells through progeny virions, effectively acting as a "Trojan Horse". The negative-strand vRibo elements demonstrated effective detection of several coronaviruses, including 229E and OC43, due to the conservation of cis-acting RNA structures across coronaviruses. Notably, vRibo functions as a dual-purpose system, acting both as an infection detector and inducible antiviral system. vRibo has the potential for basic virology research applications and can be adopted in improving the inducible expression of mRNA medicines for future coronaviruses.

Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles (preprint)

The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelle where the replication of viral genomic RNA (vgRNA) occurs. To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain vgRNA clusters along with viral double-stranded RNA (dsRNA) clusters and the replication enzyme, encapsulated by membranes derived from the host endoplasmic reticulum (ER). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of ER labels and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are enclosed by DMVs at early infection stages which then merge into vesicle packets as infection progresses. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.

Stable expression of large transgenes via the knock-in of an integrase-deficient lentivirus.

The targeted insertion and stable expression of a large genetic payload in primary human cells demands methods that are robust, efficient and easy to implement. Large payload insertion via retroviruses is typically semi-random and hindered by transgene silencing. Leveraging homology-directed repair to place payloads under the control of endogenous essential genes can overcome silencing but often results in low knock-in efficiencies and cytotoxicity. Here we report a method for the knock-in and stable expression of a large payload and for the simultaneous knock-in of two genes at two endogenous loci. The method, which we named CLIP (for 'CRISPR for long-fragment integration via pseudovirus'), leverages an integrase-deficient lentivirus encoding a payload flanked by homology arms and 'cut sites' to insert the payload upstream and in-frame of an endogenous essential gene, followed by the delivery of a CRISPR-associated ribonucleoprotein complex via electroporation. We show that CLIP enables the efficient insertion and stable expression of large payloads and of two difficult-to-express viral antigens in primary T cells at low cytotoxicity. CLIP offers a scalable and efficient method for manufacturing engineered primary cells.

SARS-CoV-2 escapes direct NK cell killing through Nsp1-mediated downregulation of ligands for NKG2D.

Natural killer (NK) cells are cytotoxic effector cells that target and lyse virally infected cells; many viruses therefore encode mechanisms to escape such NK cell killing. Here, we interrogate the ability of SARS-CoV-2 to modulate NK cell recognition and lysis of infected cells. We find that NK cells exhibit poor cytotoxic responses against SARS-CoV-2-infected targets, preferentially killing uninfected bystander cells. We demonstrate that this escape is driven by downregulation of ligands for the activating receptor NKG2D (NKG2D-L). Indeed, early in viral infection, prior to NKG2D-L downregulation, NK cells are able to target and kill infected cells; however, this ability is lost as viral proteins are expressed. Finally, we find that SARS-CoV-2 non-structural protein 1 (Nsp1) mediates downregulation of NKG2D-L and that Nsp1 alone is sufficient to confer resistance to NK cell killing. Collectively, our work demonstrates that SARS-CoV-2 evades direct NK cell cytotoxicity and describes a mechanism by which this occurs.

Modeling tourists’ preference between hotels and peer-to-peer (P2P) sharing accommodation: a pre- and post-COVID-19 comparison

Purpose: With the rapid development of sharing economy, travelers are facing choices between conventional hotels and the peer-to-peer sharing accommodation in urban tourism. The purpose of this study is to examine how travelers form their preferences in such choice situations and whether/how their preference formation mode would change with the COVID-19 pandemic. Design/methodology/approach: A relative preference model was constructed and estimated for both domestic and outbound tourists, based on two waves of survey data collected before and after the COVID-19. The results of this study were compared to derive the evolution of preference formation patterns. Findings: A set of 15 key value attributes and personal traits was identified, together with their differential effects with the pandemic. Their divergent effects between domestic and outbound trips were also delineated. Based on these findings, the competitive edges and advantageous market profiles were depicted for both hotel and sharing accommodation sectors. Originality/value: This study contributes to the knowledge of tourists’ preference between accommodation types and adds empirical evidences to the impact of the pandemic on tourist behavior patterns. Both hotel and sharing accommodation practitioners can benefit from the findings to enhance their competitiveness. © 2022, Emerald Publishing Limited.

[Epidemiological characteristics of a local cluster epidemic caused by 2019-nCoV Delta variant in Ningbo, 2021].

Objective: To understand the epidemiological characteristics of a local clustered epidemic caused by 2019-nCoV Delta variant in Ningbo and provide reference for the improvement of COVID-19 epidemic prevention and control. Methods: Case finding was conducted based on case definitions, and field epidemiological investigation of COVID-19 cases was carried out. In which Nasal and oropharyngeal swabs of the cases were collected for pathogen testing, and the results were analyzed with descriptive epidemiological methods. Results: A total of 74 COVID-19 cases were reported in this epidemic, and the cases were mainly mild ones, accounting for 87.84% (65/74), and there were no severe or critical cases. The epidemic curve showed a human-to-human transmission mode, indicating that a transmission for at least six generations had occurred. The age of the COVID-19 patients ranged from 2 years to 80 years, and 27.03% (20/74) of the cases were older than 60 years. The cases were mainly workers (55.41%, 41/74) and housework/the unemployed (27.03%, 20/74). The COVID-19 epidemic was limited, and no further spread to other areas occurred. The transmission chain among the cases was clear, and the gene sequencing results confirmed that the current epidemic was caused by 2019-nCoV Delta variant, which was highly homologous to the strains from other province. Conclusion: The local COVID-19 epidemic in Ningbo was caused by imported cases of COVID-19 from other province, and local community spread occurred through daily contacts between cases and contacts.

PATIENT PREFERENCES FOR RECEIVING CLINICAL VIDEO TELEHEALTH VISITS AT THE VETERANS HEALTH ADMINISTRATION

STATEMENT OF PROBLEM/QUESTION: There is a need to understand patients' experiences of the unprecedented expansion in telehealth services during Covid-19 at the Veterans Health Administration (VHA) to maintain its sustainability. DESCRIPTION OF PROGRAM/INTERVENTION: To evaluate Veterans' experiences with clinical video telehealth visits (CVT) and its impact on preferences for future CVT utilization, we conducted a quality improvement self-administered mailed survey Veterans from New York harbor (NYH) and San Diego (SD) VHA sites, as well as interviews (N=20) with clinical providers and VHA leadership from both sites. MEASURES OF SUCCESS: Provider and system-related barriers and facilitators were evaluated using interviews. Patient satisfaction and preferences for receiving CVT in comparison to in-person visits were identified. We evaluated these measures, as well as contrasted the differences from the information gathered from the survey and interviews. FINDINGS TO DATE: Veterans who received at least one CVT were identified through EHR. Using standardized tools, we assessed barriers to and facilitators of use, satisfaction, and preferences for CVT utilization in comparison to in person visits in different scenarios among N=308 from NYH (53%) and SD (47%). Our sample was mostly males (83%), with half being non-Hispanic Whites (50.6%), with mean age of 62.5 years (SD = + 13.6, range= 26-88). Satisfaction CVT was high overall (8.4 on a scale from 0-10, SD= + 2.0). The proportion of Veterans reporting positive experiences with CVT was high (Range: 94-98%) overall (e.g., ability to ask all needed questions, provider spending enough time, ability to communicate all health concerns). However, less Veterans (35.4 %) reported CVT preference (i.e., preferred CVT or found no difference in contrast to in person visits) compared to in person visits (64.6%). Overall, use of video calls (e.g., FaceTime) (79.9%), and having a device to access internet (97.1%) were high with no difference between groups. Veterans endorsing CVT were more likely to report intention to have future CVT for managing chronic illness (62% vs 38 %, p <.001), meeting with a specialist such as a dermatologist (41% vs 25.3%, p=0.0067), having followup care from an in person visit (90% vs 66%;p <.001), less discouraged by provider's inability to perform physical examination (15.5% vs 63.1%;p=0.0006), and reported that CVT saved time (91.8% vs 66.2%;p=0.0019). Providers viewed CVT as a complement to in-person visits;easier to schedule with less missed appointments;and easier to assess patient environment and speak with family members. Barriers included too many steps for scheduling (e.g., clerk needs to schedule and then send out links);and technological trouble shooting is not immediately accessible, often leading to switch to phone. KEY LESSONS FOR DISSEMINATION: There is very high satisfaction with CVT. However, most veterans seem to prefer in person visits, which varied by reason for visit. Our mixed methods approach delineated a potential discrepancy between process of CVT and provider perceptions of CVT barriers.

[Analysis on infection risk in secondary close contacts of COVID-19 patients].

Objective: To study the infection rate of secondary close contacts of COVID-19 patients, and assess the infection risk in the contacts. Methods: COVID-19 patients' close contacts (with a clear exposure time to index case) with negative nucleic acid test results and secondary close contacts were surveyed in continuous isolation and medical observation in this prospective study. The dynamic nucleic acid test results of the close contacts and secondary contacts of COVID-19 patients were collected to assess their risk of infection. Results: A total of 4 533 close contacts were surveyed, in whom 14 were confirmed as COVID-19 patients with overall secondary attack rate of 0.31%, and 4 201 secondary contacts were tracked, in whom no subsequent infections occurred. Conclusion: Close contacts of COVID-19 patients entered in centralized isolation for medical observation with negative nucleic acid tese results,the secondary close contacts of COVID-19 patients have no risk of infection.

[Transmission chains of local epidemic of COVID-19 caused by 2019-nCoV Delta variant in Zhenhai district, Ningbo].

Objective: To investigate the local epidemic of COVID-19 caused by 2019-nCoV Delta variant in Zhenhai district of Ningbo, identify the transmission chain and provide reference for the prevention and control of COVID-19 epidemic. Methods: The incidence data of COVID-19 in Zhenhai from 6 to 18 December, 2021 were collected in field investigation. Field epidemiological investigation was conducted to understand the epidemiological characteristics of COVID-19 cases and analyze the transmission chains. Results: The first case might be infected with 2019-nCoV through direct or indirect exposure when passing through a medium-risk area, then a family cluster was caused, and the epidemic spread through close contacts of family members with others such as work, daily life, and moxibustion. The epidemic lasted for 14 days, and 74 confirmed COVID-19 cases were reported. The median incubation period was 4.0(3.0,5.8)d. All the cases were in a chain of transmission for more than 6 generations, and the intergenerational interval was 3.5(2.0,5.3)d. The gene sequencing result indicated that the pathogen was Delta AY.4 variant of 2019-nCoV. Both the epidemiological investigation and the gene sequencing results supported that the local COVID-19 epidemic in Zhenhai was associated with the COVID-19 epidemic in Shanghai. Conclusions: The transmission chain of this epidemic was clear. Delta AY.4 variant has obvious characteristic to cause case clusters in families, places with poor ventilation, and residential communities. It is suggested to strengthen the health management in key areas and key populations, and increase the frequency of nucleic acid testing.

Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro.

A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies.

[Analysis on infectivity of COVID-19 patients before and after last negative nucleic acid test].

Objective: To investigate the infection rate in close contacts of COVID-19 patients before and after the last negative nucleic acid test, evaluate the effect of dynamic nucleic acid test in determining the infectivity of COVID-19 patients. Methods: Dynamic nucleic acid test results of COVID-19 cases were collected in a retrospective cohort study. COVID-19 cases with negative nucleic acid test results before their first positive nucleic acid tests were selected as study subjects. Close contacts of the index cases and the secondary close contacts were kept isolation for medical observation to assess their risk of infection. Results: This study included 89 confirmed cases from two local COVID-19 epidemics in Ningbo. A total of 5 609 close contacts were surveyed, the overall infection rate was 0.20%. No close contacts of the COVID-19 cases before the last negative nucleic acid test were infected, and the infection rate in the close contacts of the COVID-19 cases after the last negative nucleic acid test was 1.33%, all of these close contacts lived together with the index cases. No secondary close contacts were infected. Conclusion: COVID-19 patient becomes infectious after the last nucleic acid is negative, and has no infectivity before the last nucleic acid negative.

SARS-CoV-2 escapes direct NK cell killing through Nsp1-mediated downregulation of ligands for NKG2D (preprint)

Natural killer (NK) cells are cytotoxic effector cells that respond rapidly to viral infection by targeting and lysing infected cells, and many viruses encode mechanisms to escape such NK cell killing. Here, we sought to investigate the ability of SARS-CoV-2 to modulate NK cell recognition and lysis of infected cells. We found that NK cells exhibit poor cytotoxic responses against SARS-CoV-2-infected targets, preferentially killing uninfected bystander cells. We demonstrate that this escape is driven by strong downregulation of ligands for the activating receptor NKG2D on SARS-CoV-2-infected cells. Indeed, in the initial stages of viral infection, prior to NKG2D-ligand downregulation, NK cells are able to successfully target and kill infected cells; however, this ability is lost as viral proteins are expressed within infected cells. Finally, we found that SARS-CoV-2 non-structural protein 1 (Nsp1) mediates the downregulation of NKG2D ligands and that transfection with Nsp1 alone is sufficient to confer resistance to NK cell killing. Collectively, our work reveals that SARS-CoV-2 evades NK cell cytotoxic responses and describes a mechanism by which this occurs.

Multi-color super-resolution imaging to study human coronavirus RNA during cellular infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third human coronavirus within 20 years that gave rise to a life-threatening disease and the first to reach pandemic spread. To make therapeutic headway against current and future coronaviruses, the biology of coronavirus RNA during infection must be precisely understood. Here, we present a robust and generalizable framework combining high-throughput confocal and super-resolution microscopy imaging to study coronavirus infection at the nanoscale. Using the model human coronavirus HCoV-229E, we specifically labeled coronavirus genomic RNA (gRNA) and double-stranded RNA (dsRNA) via multi-color RNA immunoFISH and visualized their localization patterns within the cell. The 10-nm resolution achieved by our approach uncovers a striking spatial organization of gRNA and dsRNA into three distinct structures and enables quantitative characterization of the status of the infection after antiviral drug treatment. Our approach provides a comprehensive imaging framework that will enable future investigations of coronavirus fundamental biology and therapeutic effects.

A retrospective study of the clinical characteristics of COVID-19 patients with gastrointestinal symptoms

Objective: To investigate the gastrointestinal (GI) manifestations of COVID-19 and retrospectively analyze the clinical characteristics of COVID-19 patients with GI symptoms. Methods: Data of 137 COVID-19 inpatients treated in Renmin Hospital of Wuhan University from February 1 to February 29, 2020 were collected. Patients were divided into GI group and Non-GI group according to the presence of digestive system abnormalities and gastrointestinal symptoms before and during admission. General data, clinical data, and relevant laboratory examination results of 137 patients were collected. SPSS 23.0 software was used for statistical analysis to compare the differences of various indicators between the two groups. Results: There was no statistically significant difference between the clinical data and the clinical manifestations of fever and dry cough between the GI group and the Non-GI group (P>0.05). The proportion of patients with fatigue in the GI group was higher than that in the Non-GI group (P<0.001). The proportion of critically ill patients was greater than that of the Non-GI group (P=0.011), and the proportion of GI combined with hypertension, diabetes, cardiovascular disease and chronic liver disease was higher than that of the Non-GI group (all P<0.05). GI group mortality rate was much higher than that of Non-GI group (P<0.001). Patients with GI group had higher white blood cell count and neutrophil count than Non-GI (P<0.001). The proportion of neutrophils, lymphocytes as well as lymph between the two groups had no statistical difference (P>0.05). The proportion of monocytes in the GI group was lower than that in the Non-GI group (P=0.033). There was no statistical difference in platelet count and C-reactive protein level between the two groups (P>0.05). LDH, TBIL, and Urea levels of GI group were higher than those of Non-GI group (P<0.05). There was no statistical difference in other cardiac, liver and kidney function tests, PT and APTT values between the two groups (P>0.05), but D-dimer in GI group was higher than in Non-GI group (P<0.001). Conclusion: Gastrointestinal symptoms are common in COVID-19 patients, and patients with other underlying diseases are at greater risk for developing gastrointestinal symptoms. COVID-19 patients with gastrointestinal symptoms progress more rapidly, have a higher mortality rate, and exhibit certain concomitant symptoms and laboratory tests that are specific to COVID-19. Therefore, more attention should be paid to the digestive system abnormalities and gastrointestinal symptoms in COVID-19 patients during clinical work. © 2021, Editorial Board of Medical Journal of Wuhan University. All right reserved.

Clinical characteristics of patients with COVID-19 combined with cardiovascular disease

Objective: To retrospectively analyze the clinical characteristics of coronavirus disease 2019 (COVID-19) and the impact of cardiovascular disease (CVD) on the clinical manifestations of COVID-19. Methods: A total of 128 patients diagnosed with COVID-19 in Renmin Hospital of Wuhan University from February 1 to February 29, 2020 were divided into CVD group (n=62) and non-CVD group (n=66). The general data, admission symptoms and laboratory examination results including blood routine, immunity, heart, liver and kidney function were obtained and statistically analyzed by SPSS 22.0 statistical software. The differences of various indexes between CVD group and non-CVD group were compared. Results: There was no significant difference in gender between CVD group and non-CVD group(P>0.05).The average age of CVD group was higher than that of non-CVD group (P<0.001). The proportion of fever and cough, severe and critical patients was higher than that respectively of non-CVD group (all P<0.05). There was no significant difference in the incidence of fatigue, dyspnea, and asymptomatic between the two groups (all P>0.05). The average levels of eukocyte count, neutrophil ratio, neutrophil count, monocyte count, and C-reactive protein in CVD group were higher than those in non-CVD group (all P<0.05), while the average lymphocyte proportion in CVD group was lower than that in non-CVD group (P<0.05). There was no significant difference in lymphocyte count and platelet count between the two groups (both P>0.05). The average LDH, myohemoglobin, CK-MB, NT-proBNP, TBIL, and Urea in CVD group were higher than those in non-CVD group (all P<0.05), but there was no significant difference in ALT, AST, and Cr between the two groups (all P>0.05). Conclusion: Compared with non-CVD patients with COVID-19, CVD patients with COVID-19 are older, have more obvious symptoms, with a higher risksin heart, liver, and kidney injury, but the mechanism is not clear yet. © 2021, Editorial Board of Medical Journal of Wuhan University. All right reserved.

Multi-color super-resolution imaging to study human coronavirus RNA during cellular infection (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third human coronavirus within 20 years that gave rise to a life-threatening disease and the first to reach pandemic spread. To make therapeutic headway against current and future coronaviruses, the biology of coronavirus RNA during infection must be precisely understood. Here, we present a robust and generalizable framework combining high-throughput confocal and super-resolution microscopy imaging to study coronavirus infection at the nanoscale. Employing the model human coronavirus HCoV-229E, we specifically labeled coronavirus genomic RNA (gRNA) and double-stranded RNA (dsRNA) via multicolor RNA-immunoFISH and visualized their localization patterns within the cell. The exquisite resolution of our approach uncovers a striking spatial organization of gRNA and dsRNA into three distinct structures and enables quantitative characterization of the status of the infection after antiviral drug treatment. Our approach provides a comprehensive framework that supports investigations of coronavirus fundamental biology and therapeutic effects.