Porcine Epidemic Diarrhea Virus nsp7 Inhibits Interferon-Induced JAK-STAT Signaling through Sequestering the Interaction between KPNA1 and STAT1.

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus that causes high mortality in piglets. Interferon (IFN) responses are the primary defense mechanism against viral infection; however, viruses always evolve elaborate strategies to antagonize the antiviral action of IFN. Previous study showed that PEDV nonstructural protein 7 (nsp7), a component of the viral replicase polyprotein, can antagonize ploy(I:C)-induced type I IFN production. Here, we found that PEDV nsp7 also antagonized IFN-α-induced JAK-STAT signaling and the production of IFN-stimulated genes. PEDV nsp7 did not affect the protein and phosphorylation levels of JAK1, Tyk2, STAT1, and STAT2 or the formation of the interferon-stimulated gene factor 3 (ISGF3) complex. However, PEDV nsp7 prevented the nuclear translocation of STAT1 and STAT2. Mechanistically, PEDV nsp7 interacted with the DNA binding domain of STAT1/STAT2, which sequestered the interaction between karyopherin α1 (KPNA1) and STAT1, thereby blocking the nuclear transport of ISGF3. Collectively, these data reveal a new mechanism developed by PEDV to inhibit type I IFN signaling pathway. IMPORTANCE In recent years, an emerging porcine epidemic diarrhea virus (PEDV) variant has gained attention because of serious outbreaks of piglet diarrhea in China and the United States. Coronavirus nonstructural protein 7 (nsp7) has been proposed to act with nsp8 as part of an RNA primase to generate RNA primers for viral RNA synthesis. However, accumulating evidence indicates that coronavirus nsp7 can also antagonize type I IFN production. Our present study extends previous findings and demonstrates that PEDV nsp7 also antagonizes IFN-α-induced IFN signaling by competing with KPNA1 for binding to STAT1, thereby enriching the immune regulation function of coronavirus nsp7.

Antiviral Activities of Carbazole Derivatives against Porcine Epidemic Diarrhea Virus In Vitro.

Porcine epidemic diarrhea virus (PEDV), an enteric coronavirus, causes neonatal pig acute gastrointestinal infection with a characterization of severe diarrhea, vomiting, high morbidity, and high mortality, resulting in tremendous damages to the swine industry. Neither specific antiviral drugs nor effective vaccines are available, posing a high priority to screen antiviral drugs. The aim of this study is to investigate anti-PEDV effects of carbazole alkaloid derivatives. Eighteen carbazole derivatives (No.1 to No.18) were synthesized, and No.5, No.7, and No.18 were identified to markedly reduce the replication of enhanced green fluorescent protein (EGFP) inserted-PEDV, and the mRNA level of PEDV N. Flow cytometry assay, coupled with CCK8 assay, confirmed No.7 and No.18 carbazole derivatives displayed high inhibition effects with low cell toxicity. Furthermore, time course analysis indicated No.7 and No.18 carbazole derivatives exerted inhibition at the early stage of the viral life cycle. Collectively, the analysis underlines the benefit of carbazole derivatives as potential inhibitors of PEDV, and provides candidates for the development of novel therapeutic agents.

Features of capsule endoscopy in COVID-19 patients with a six-month follow-up: A prospective observational study.

Recently, the coronavirus disease 2019 (COVID-19) has caused a global pandemic. Several studies indicate that the digestive system can also be affected by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, patients with digestive symptoms should have a capsule endoscopy (CE). COVID-19 patients with gastrointestinal (GI) symptoms who underwent CE were recruited from March 2020 to April 2020. We collected patients' data and performed a prospective follow-up study for 6 months. All 11 COVID-19 cases with GI symptoms who underwent CE presented gastritis. Eight cases (72.7%) had intestinal mucosa inflammation. Among them, two cases showed intestinal ulcers or erosions. Moreover, two cases displayed colonic mucositis. One case was lost during follow-up. At 3-6 months after hospital discharge, five patients underwent CE again, presenting gastrointestinal lesions. Five of the 10 cases had GI symptoms, such as abdominal pain, diarrhea, constipation, and others. Among these five cases, the GI symptoms of three patients disappeared at the last follow-up and two patients still presented diarrhea symptoms. Overall, we observed damaged digestive tract mucosa that could be caused by SARS-CoV-2. Moreover, after discharge, some patients still presented intestinal lesions and GI symptoms.

Cumulative effects of ambient particulate matter pollution on deaths: A multicity analysis of mortality displacement.

BACKGROUND: Systematic evaluations of the cumulative effects and mortality displacement of ambient particulate matter (PM) pollution on deaths are lacking. We aimed to discern the cumulative effect profile of PM exposure, and investigate the presence of mortality displacement in a large-scale population. METHODS: We conducted a time-series analysis with different exposure-lag models on 13 cities in Jiangsu, China, to estimate the effects of PM pollution on non-accidental, cardiovascular, and respiratory mortality (2015-2019). Over-dispersed Poisson generalized additive models were integrated with distributed lag models to estimate cumulative exposure effects, and assess mortality displacement. RESULTS: Pooled cumulative effect estimates with lags of 0-7 and 0-14 days were substantially larger than those with single-day and 2-day moving average lags. For each 10 µg/m3 increment in PM2.5 concentration with a cumulative lag of 0-7 days, we estimated an increase of 0.50 % (95 % CI: 0.29, 0.72), 0.63 % (95 % CI: 0.38, 0.88), and 0.50 % (95 % CI: 0.01, 1.01) in pooled estimates of non-accidental, cardiovascular, and respiratory mortality, respectively. Both PM10 and PM2.5 were associated with significant increases in non-accidental and cardiovascular mortality with a cumulative lag of 0-14 days. We observed mortality displacement within 30 days for non-accidental, cardiovascular, and respiratory deaths. CONCLUSIONS: Our findings suggest that risk assessment based on single-day or 2-day moving average lag structures may underestimate the adverse effects of PM pollution. The cumulative effects of PM exposure on non-accidental and cardiovascular mortality can last up to 14 days. Evidence of mortality displacement for non-accidental, cardiovascular, and respiratory deaths was found.

Emergence of a young case infected with avian influenza A (H5N6) in Anhui Province, East China during the COVID-19 pandemic.

In the context of the coronavirus disease 2019 pandemic, we investigated the epidemiological and clinical characteristics of a young patient infected by avian influenza A (H5N6) virus in Anhui Province, East China, and analyzed genomic features of the pathogen in 2020. Through the cross-sectional investigation of external environment monitoring (December 29-31, 2020), 1909 samples were collected from Fuyang City. It was found that the positive rate of H5N6 was higher than other areas obviously in Tianma poultry market, where the case appeared. In addition, dual coinfections were detected with a 0.057% polymerase chain reaction positive rate the surveillance years. The virus was the clade 2.3.4.4, which was most likely formed by genetic reassortment between H5N6 and H9N2 viruses. This study found that the evolution rates of the hemagglutinin and neuraminidase genes of the virus were higher than those of common seasonal influenza viruses. The virus was still highly pathogenic to poultry and had a preference for avian receptor binding.

Breath-, air- and surface-borne SARS-CoV-2 in hospitals.

The COVID-19 pandemic has brought an unprecedented crisis to the global health sector. When discharging COVID-19 patients in accordance with throat or nasal swab protocols using RT-PCR, the potential risk of reintroducing the infection source to humans and the environment must be resolved. Here, 14 patients including 10 COVID-19 subjects were recruited; exhaled breath condensate (EBC), air samples and surface swabs were collected and analyzed for SARS-CoV-2 using reverse transcription-polymerase chain reaction (RT-PCR) in four hospitals with applied natural ventilation and disinfection practices in Wuhan. Here we discovered that 22.2% of COVID-19 patients (n = 9), who were ready for hospital discharge based on current guidelines, had SARS-CoV-2 in their exhaled breath (~105 RNA copies/m3). Although fewer surface swabs (3.1%, n = 318) tested positive, medical equipment such as face shield frequently contacted/used by healthcare workers and the work shift floor were contaminated by SARS-CoV-2 (3-8 viruses/cm2). Three of the air samples (n = 44) including those collected using a robot-assisted sampler were detected positive by a digital PCR with a concentration level of 9-219 viruses/m3. RT-PCR diagnosis using throat swab specimens had a failure rate of more than 22% in safely discharging COVID-19 patients who were otherwise still exhaling the SARS-CoV-2 by a rate of estimated ~1400 RNA copies per minute into the air. Direct surface contact might not represent a major transmission route, and lower positive rate of air sample (6.8%) was likely due to natural ventilation (1.6-3.3 m/s) and regular disinfection practices. While there is a critical need for strengthening hospital discharge standards in preventing re-emergence of COVID-19 spread, use of breath sample as a supplement specimen could further guard the hospital discharge to ensure the safety of the public and minimize the pandemic re-emergence risk.

Clinical efficacy of low-dose emetine for patients with COVID-19: a real-world study.

OBJECTIVE: Emetine, an isoquinoline alkaloid that is enriched at high concentrations in the lung, has shown potent in vitro activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The aim of this study was to better understand the effectiveness of low-dose emetine for patients with coronavirus disease 2019 (COVID-19). METHODS: In this real-world study, 63 patients with mild or common COVID-19 were recruited from Wuhan Fangcang Shelter Hospital and five COVID-19-designated hospitals in Anhui Province, China from February to March 2020. Thirty-nine patients from Wuhan Fangcang Shelter Hospital were assigned to a pragmatic randomized controlled clinical trial, and 24 patients from the 5 COVID-19-designated hospitals in Anhui Province underwent a real-world study. The medication course of emetine was less than 10 days. The main symptoms and adverse reactions of all patients were observed and recorded. The primary outcome measure was the time required for a negative SARS-CoV-2 RNA result or the negative result rate on day 10. Secondary outcomes included axillary temperature, transcutaneous oxygen saturation, and respiratory frequency recovery. The study was approved by the Ethics Committee of The First Affiliated Hospital of Anhui Medical University on February 20, 2019 (approval No. PJ2020-03-19) and was registered with the Chinese Clinical Trial Registry on February 20, 2019 (registration number: ChiCTR2000030022). RESULTS: The oxygen saturation values were higher in the treatment group than in the control group on the first day after enrollment for patients treated at Fangcang Shelter Hospital. The axillary body temperature, respiratory rate, and oxygen saturation among patients in Fangcang Shelter Hospital were related to the time effect but not to the intervention measures. The respiratory rate and oxygen saturation of patients in the Anhui designated hospitals were related to the intervention measures but not to the time effect. The axillary body temperature of patients in Anhui designated hospitals was related to the time effect but not to the intervention measures. CONCLUSION: Our preliminary study shows that low-dose emetine combined with basic conventional antiviral drugs improves clinical symptoms in patients with mild and common COVID-19 without apparent adverse effects, suggesting that moderately increased doses of emetine may have good potential for treatment and prevention of COVID-19.

Health-related quality of life of COVID-19 patients after discharge: A multicenter follow-up study.

AIMS AND OBJECTIVES: To determine the health-related quality of life (HRQoL) of COVID-19 patients after discharge and its predicting factors. BACKGROUND: COVID-19 has caused a worldwide pandemic and led a huge impact on the health of human and daily life. It has been demonstrated that physical and psychological conditions of hospitalised COVID-19 patients are impaired, but the studies focus on physical and psychological conditions of COVID-19 patients after discharge from hospital are rare. DESIGN: A multicentre follow-up study. METHODS: This was a multicentre follow-up study of COVID-19 patients who had discharged from six designated hospitals. Physical symptoms and HRQoL were surveyed at first follow-up (the third month after discharge). The latest multiple laboratory findings were collected through medical examination records. This study was performed and reported in accordance with STROBE checklist. RESULTS: Three hundred eleven patients (57.6%) were reported with one or more physical symptoms. The scores of HRQoL of COVID-19 patients at third month after discharge, except for the dimension of general health, were significantly lower than Chinese population norm (p < .001). Results of logistic regression showed that female (odds ratio (OR): 1.79, 95% confidence interval (CI): 1.04-3.06), older age (≥60 years) (OR: 2.44, 95% CI: 1.33-4.47) and the physical symptom after discharge (OR: 40.15, 95% CI: 9.68-166.49) were risk factors for poor physical component summary; the physical symptom after discharge (OR: 6.68, 95% CI: 4.21-10.59) was a risk factor for poor mental component summary. CONCLUSIONS: Health-related quality of life of discharged COVID-19 patients did not come back to normal at third month after discharge and affected by age, sex and the physical symptom after discharge. RELEVANCE TO CLINICAL PRACTICE: Healthcare workers should pay more attention to the physical and psychological rehabilitation of discharged COVID-19 patients. Long-term follow-up on COVID-19 patients after discharge is needed to determine the long-term impact of COVID-19.

Application of cell surface-fluorescence immunosorbent assay in rapid screening of a hybridoma secreting monoclonal antibodyagainst PEDV N protein

To verify the effect of cell surface-fluorescence immunosorbent assay (CSFIA) on screening the highly active hybridoma cells, 6-8 week-old female BALB/c mice were immunized with recombinant porcine epidemic diarrhea virus (PEDV) N protein, and then spleen cells isolated from mice were fused with SP2/0 cells to generate hybridomas. The hybridomas secreting MAbs against PEDV N protein were screened by indirect ELISA and cell surface-fluorescence immunosorbent assay (CSFIA), respectively. The titer of MAbs secreted by each hybridoma was determined. Using these methods, two strains of hybridoma celllines secreting MAbs against PEDV N protein, designated as 3D3 and 1B1, respectively, were obtained and characterized. The results showed that the titer of 1B1 produced by CSFIA was 10 times higher than that of 3D3, and the preparation time was shortened by about 30 days on account of the direct selection of hybridoma through the fluorescence intensity without the need of multiple subcloning procedures. This study indicates that CSFIA is a powerful method to screen the hybridomas secreting higher titer MAb in a rapid one-step process, which would be valuable alternative for a rapid production of MAbs.

Rapid manipulation of the porcine epidemic diarrhea virus genome by CRISPR/Cas9 technology.

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus causing lethal watery diarrhea in suckling piglets. Reverse genetics is a valuable tool to study the functions of viral genes and to generate vaccine candidates. In this study, a full-length infectious cDNA clone of the highly virulent PEDV strain AJ1102 was assembled in a bacterial artificial chromosome (BAC). The rescued virus (rAJ1102) exhibited similar proliferation characteristics in vitro to the wildtype AJ1102. Using CRISPR/Cas9 technology, a recombinant virus rAJ1102-ΔORF3-EGFP in which the ORF3 gene was replaced with an EGFP gene, was successfully generated, and its proliferation characteristics were compared with the parental rAJ1102. Importantly, it just took one week to construct the recombinant PEDV rAJ1102-ΔORF3-EGFP using this method, providing a more efficient platform for PEDV genome manipulation, which could also be applied to other RNA viruses.

Environmental virus detection associated with asymptomatic SARS-CoV-2-infected individuals with positive anal swabs.

In the fight against the outbreak of COVID-19 in China, we treated some asymptomatic infected individuals. This study aimed to detect pathogens in biological and environmental samples of these asymptomatic infected individuals and analyse their association. Using a cross-sectional study design, we collected biological and environmental samples from 19 patients treated in the isolation ward of Nanjing No.2 Hospital. Biological samples included saliva, pharyngeal swabs, blood, anal swabs, and exhaled breath condensate. Swab samples from the ward environment included inside masks, outside masks, palm swabs, bedside handrails, bedside tables, cell phone screens, toilet cell phone shelves, toilet pads and toilet lids. We also obtained some samples from public areas. We used RT-PCR to detect pathogens and colloidal gold to detect antibodies. As results, 19 asymptomatic infected individuals participated in the survey, with 8 positives for pathogens and 11 positives only for antibodies. Three positive samples were detected from among 96 environmental samples, respectively, from a cell phone surface, a cell phone shelf and a bedside handrail. No positive samples were detected in the exhaled breath condensate in this work. All patients identified pathogens in the environment had positive anal swabs. There was a statistical association between positive anal swabs and positive environmental samples. The association of positive samples from the surrounding of asymptomatically infected patients with positive anal swabs suggested that patients might secrete the virus for a more extended period.

Toilets dominate environmental detection of severe acute respiratory syndrome coronavirus 2 in a hospital.

Respiratory and fecal aerosols play confirmed and suspected roles, respectively, in transmitting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). An extensive environmental sampling campaign of both toilet and non-toilet environments was performed in a dedicated hospital building for patients with coronavirus disease 2019 (COVID-19), and the associated environmental factors were analyzed. In total, 107 surface samples, 46 air samples, two exhaled condensate samples, and two expired air samples were collected within and beyond four three-bed isolation rooms. The data of the COVID-19 patients were collected. The building environmental design and the cleaning routines were reviewed. Field measurements of airflow and CO2 concentrations were conducted. The 107 surface samples comprised 37 from toilets, 34 from other surfaces in isolation rooms, and 36 from other surfaces outside the isolation rooms in the hospital. Four of these samples were positive, namely two ward door handles, one bathroom toilet seat cover, and one bathroom door handle. Three were weakly positive, namely one bathroom toilet seat, one bathroom washbasin tap lever, and one bathroom ceiling exhaust louver. Of the 46 air samples, one collected from a corridor was weakly positive. The two exhaled condensate samples and the two expired air samples were negative. The fecal-derived aerosols in patients' toilets contained most of the detected SARS-CoV-2 in the hospital, highlighting the importance of surface and hand hygiene for intervention.

Digestive Symptoms in COVID-19 Patients With Mild Disease Severity: Clinical Presentation, Stool Viral RNA Testing, and Outcomes.

OBJECTIVES: Coronavirus disease 2019 (COVID-19) most commonly presents with respiratory symptoms, including cough, shortness of breath, and sore throat. However, digestive symptoms also occur in patients with COVID-19 and are often described in outpatients with less severe disease. In this study, we sought to describe the clinical characteristics of COVID-19 patients with digestive symptoms and mild disease severity. METHODS: We identified COVID-19 patients with mild disease and one or more digestive symptoms (diarrhea, nausea, and vomiting), with or without respiratory symptoms, and compared them with a group presenting solely with respiratory symptoms. We followed up patients clinically until they tested negative for COVID-19 on at least 2 sequential respiratory tract specimens collected ≥24 hours apart. We then compared the clinical features between those with digestive symptoms and those with respiratory symptoms. RESULTS: There were 206 patients with low severity COVID-19, including 48 presenting with a digestive symptom alone, 69 with both digestive and respiratory symptoms, and 89 with respiratory symptoms alone. Between the 2 groups with digestive symptoms, 67 presented with diarrhea, of whom 19.4% experienced diarrhea as the first symptom in their illness course. The diarrhea lasted from 1 to 14 days, with an average duration of 5.4 ± 3.1 days and a frequency of 4.3 ± 2.2 bowel movements per day. Concurrent fever was found in 62.4% of patients with a digestive symptom. Patients with digestive symptoms presented for care later than those with respiratory symptoms (16.0 ± 7.7 vs 11.6 ± 5.1 days, P < 0.001). Nevertheless, patients with digestive symptoms had a longer duration between symptom onset and viral clearance (P < 0.001) and were more likely to be fecal virus positive (73.3% vs 14.3%, P = 0.033) than those with respiratory symptoms. DISCUSSION: We describe a unique subgroup of COVID-19 patients with mild disease severity marked by the presence of digestive symptoms. These patients are more likely to test positive for viral RNA in stool, to have a longer delay before viral clearance, and to experience delayed diagnosis compared with patients with only respiratory symptoms.