Study on the differences in tissue tropism and innate immune response with different Avian coronavirus strains

Infectious bronchitis(IB) has been one of the most important diseases that endanger the world's poultry industry. Frequent mutations of IBV lead to the emergence of many different pathogenic strains. The appearance of different pathogenic IBV strains may be related to the activation of the immune system after infection. The study on IBV tissue tropism and the immune mechanism after infection is of great significance to the prevention and control of epidemic diseases. The purpose of this study is to investigate the relationship between the tissue tropism and innate immune responses of different strains of chicken infectious bronchitis virus(IB)in avian coronavirus. Different IBV strains were inoculated into primary cells, chicken embryos, and SPF chickens to identify the tissue tropism to each organ. TLR3, TLR7, MAVS, MDA5, IFN-beta, and IFN-P and other cytokines were measured by fluorescence quantitative methods to find out different expressions of mRNA in different tissues and organs of SPF chicken including trachea,kidney,stomach,lung, spleen and bursa. The results showed that besides H52,a known vaccine strain, the AH strain is highly pathogenic to the urinary system and the TM strain is pathogenic to the digestive system. The infection experiment showed that in trachea, the mRNA expression of MDA5 in AH infection group was significantly down-regulated, the mRNA expression of IFN-beta and IFN-P in TM group was significantly up-regulated, and the expression of MAVS and IFN-beta in H52 group was up-regulated. In lung tissues, TLR3, TLR7, IFN-beta, and IFN-P were significantly upregulated in AH group. MDA5 and IFN-P expressions were significantly increased in the TM group, and TLR3, TLR7, MDA5, IFN-beta and IFN-P were up-regulated in the H52 group. In the kidney, the expressions of IFNbeta and TLR7 were down-regulated in AH and TM groups, and the expressions of TLR7, MAVS, IFN-beta, and IFN-P were up-regulated in the H52 group. In the glandular stomach,TLR7, MAVS, MDA5, IFN-beta, and IFN-P expression were up-regulated in AH group,TM strain IFN-P expression was up-regulated,TLR7 expression in H52 group was significantly down-regulated, MAVS, IFN-beta, and IFN-P expression were upregulated. In the spleen, MAVS expression was up-regulated in AH group, and TLR3,TLR7 and MDA5 expression in TM group significantly increased. The expressions of TLR7 and IFN-P in H52 group also significantly increased. In the bursal, there was no significant difference in the expression of each factor in the AH group, the expressions of TLR7, MDA5 and IFN-P in the TM group were up-regulated, and in H52 group, TLR3, TLR7, MAVS, MDA5, IFN-beta and IFN-P expressions were all significantly up-regulated. The expression levels of cytokines in infection with different IBV strains are different in different tissues and organs. The innate immune responses of different tissues are diverse and are related to the expression level of immune molecules. This experiment lays the theoretical foundation for the research and application of different IBV strain infection mechanisms, vaccine development, and pathogen-cell interactions. Reset.

Combinations of Host- and Virus-Targeting Antiviral Drugs Confer Synergistic Suppression of SARS-CoV-2.

Three directly acting antivirals (DAAs) demonstrated substantial reduction in COVID-19 hospitalizations and deaths in clinical trials. However, these agents did not completely prevent severe illness and are associated with cases of rebound illness and viral shedding. Combination regimens can enhance antiviral potency, reduce the emergence of drug-resistant variants, and lower the dose of each component in the combination. Concurrently targeting virus entry and virus replication offers opportunities to discover synergistic drug combinations. While combination antiviral drug treatments are standard for chronic RNA virus infections, no antiviral combination therapy has been approved for SARS-CoV-2. Here, we demonstrate that combining host-targeting antivirals (HTAs) that target TMPRSS2 and hence SARS-CoV-2 entry, with the DAA molnupiravir, which targets SARS-CoV-2 replication, synergistically suppresses SARS-CoV-2 infection in Calu-3 lung epithelial cells. Strong synergy was observed when molnupiravir, an oral drug, was combined with three TMPRSS2 (HTA) oral or inhaled inhibitors: camostat, avoralstat, or nafamostat. The combination of camostat plus molnupiravir was also effective against the beta and delta variants of concern. The pyrimidine biosynthesis inhibitor brequinar combined with molnupiravir also conferred robust synergistic inhibition. These HTA+DAA combinations had similar potency to the synergistic all-DAA combination of molnupiravir plus nirmatrelvir, the protease inhibitor found in paxlovid. Pharmacodynamic modeling allowed estimates of antiviral potency at all possible concentrations of each agent within plausible therapeutic ranges, suggesting possible in vivo efficacy. The triple combination of camostat, brequinar, and molnupiravir further increased antiviral potency. These findings support the development of HTA+DAA combinations for pandemic response and preparedness. IMPORTANCE Imagine a future viral pandemic where if you test positive for the new virus, you can quickly take some medicines at home for a few days so that you do not get too sick. To date, only single drugs have been approved for outpatient use against SARS-CoV-2, and we are learning that these have some limitations and may succumb to drug resistance. Here, we show that combinations of two oral drugs are better than the single ones in blocking SARS-CoV-2, and we use mathematical modeling to show that these drug combinations are likely to work in people. We also show that a combination of three oral drugs works even better at eradicating the virus. Our findings therefore bode well for the development of oral drug cocktails for at home use at the first sign of an infection by a coronavirus or other emerging viral pathogens.

Drug Combinations as a First Line of Defense against Coronaviruses and Other Emerging Viruses.

The world was unprepared for coronavirus disease 2019 (COVID-19) and remains ill-equipped for future pandemics. While unprecedented strides have been made developing vaccines and treatments for COVID-19, there remains a need for highly effective and widely available regimens for ambulatory use for novel coronaviruses and other viral pathogens. We posit that a priority is to develop pan-family drug cocktails to enhance potency, limit toxicity, and avoid drug resistance. We urge cocktail development for all viruses with pandemic potential both in the short term (<1 to 2 years) and longer term with pairs of drugs in advanced clinical testing or repurposed agents approved for other indications. While significant efforts were launched against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in vitro and in the clinic, many studies employed solo drugs and had disappointing results. Here, we review drug combination studies against SARS-CoV-2 and other viruses and introduce a model-driven approach to assess drug pairs with the highest likelihood of clinical efficacy. Where component agents lack sufficient potency, we advocate for synergistic combinations to achieve therapeutic levels. We also discuss issues that stymied therapeutic progress against COVID-19, including testing of agents with low likelihood of efficacy late in clinical disease and lack of focus on developing virologic surrogate endpoints. There is a need to expedite efficient clinical trials testing drug combinations that could be taken at home by recently infected individuals and exposed contacts as early as possible during the next pandemic, whether caused by a coronavirus or another viral pathogen. The approach herein represents a proactive plan for global viral pandemic preparedness.

Development and validation of prognosis model of mortality risk in patients with COVID-19.

This study aimed to identify clinical features for prognosing mortality risk using machine-learning methods in patients with coronavirus disease 2019 (COVID-19). A retrospective study of the inpatients with COVID-19 admitted from 15 January to 15 March 2020 in Wuhan is reported. The data of symptoms, comorbidity, demographic, vital sign, CT scans results and laboratory test results on admission were collected. Machine-learning methods (Random Forest and XGboost) were used to rank clinical features for mortality risk. Multivariate logistic regression models were applied to identify clinical features with statistical significance. The predictors of mortality were lactate dehydrogenase (LDH), C-reactive protein (CRP) and age based on 500 bootstrapped samples. A multivariate logistic regression model was formed to predict mortality 292 in-sample patients with area under the receiver operating characteristics (AUROC) of 0.9521, which was better than CURB-65 (AUROC of 0.8501) and the machine-learning-based model (AUROC of 0.4530). An out-sample data set of 13 patients was further tested to show our model (AUROC of 0.6061) was also better than CURB-65 (AUROC of 0.4608) and the machine-learning-based model (AUROC of 0.2292). LDH, CRP and age can be used to identify severe patients with COVID-19 on hospital admission.

Efficacy of Qingfei Paidu Decoction on Patients with COVID-19 Pneumonia in Wuhan, China: A Propensity Score Matching Study.

BACKGROUND: In view of the global efforts to develop effective treatments for the current worldwide coronavirus 2019 (COVID-19) pandemic, Qingfei Paidu decoction (QPD), a novel traditional Chinese medicine (TCM) prescription, was formulated as an optimized combination of constituents of classic prescriptions used to treat numerous febrile and respiratory-related diseases. This prescription has been used to treat patients with COVID-19 pneumonia in Wuhan, China. Hypothesis/Purpose. We hypothesized that QPD would have beneficial effects on patients with COVID-19. We aimed to prove this hypothesis by evaluating the efficacy of QPD in patients with COVID-19 pneumonia. METHODS: In this single-center, retrospective, observational study, we identified eligible participants who received a laboratory diagnosis of COVID-19 between January 15 and March 15, 2020, in the west campus of Union Hospital in Wuhan, China. QPD was supplied as an oral liquid packaged in 200-mL containers, and patients were orally administered one package twice daily 40 minutes after a meal. The primary outcome was death, which was compared between patients who did and did not receive QPD (QPD and NoQPD groups, respectively). Propensity score matching (PSM) was used to identify cohorts. RESULTS: In total, 239 and 522 participants were enrolled in the QPD and NoQPD groups, respectively. After PSM at a 1 : 1 ratio, 446 patients meeting the criteria were included in the analysis with 223 in each arm. In the QPD and NoQPD groups, 7 (3.2%) and 29 (13.0%) patients died, and those in the QPD group had a significantly lower risk of death (hazard ratio (HR) 0.29, 95% CI: 0.13-0.67) than those in the NoQPD group (p = 0.004). Furthermore, the survival time was significantly longer in the QPD group than in the NoQPD group (p < 0.001). CONCLUSION: The use of QPD may reduce the risk of death in patients with COVID-19 pneumonia.

Expression of Human ACE2 N-terminal Domain, Part of the Receptor for SARS-CoV-2, in Fusion With Maltose-Binding Protein, E. coli Ribonuclease I and Human RNase A.

The SARS-CoV-2 viral genome contains a positive-strand single-stranded RNA of ∼30 kb. Human ACE2 protein is the receptor for SARS-CoV-2 virus attachment and infection. We propose to use ribonucleases (RNases) as antiviral agents to destroy the viral genome in vitro. In the virions, the RNA is protected by viral capsid proteins, membrane proteins, and nucleocapsid proteins. To utilize RNases as antiviral strategy, we set out to construct RNase fusion with human ACE2 receptor N-terminal domain (ACE2NTD). We expressed six proteins in E. coli cells: (1) MBP-ACE2NTD, (2) ACE2NTD-GFP, (3) RNase I (6×His), (4) RNase III (6×His), (5) RNase I-ACE2NTD (6×His), and (6) human RNase A-ACE2NTD (6×His). We evaluated fusion expression in different E. coli strains, partially purified MBP-ACE2NTD protein from the soluble fraction of bacterial cell lysate, and refolded MBP-ACE2NTD protein from inclusion body. The engineered RNase I-ACE2NTD (6×His) and hRNase A-ACE2NTD (6×His) fusions are active in cleaving SARS-CoV-2 RNA fragment in vitro. The recombinant RNase I (6×His) and RNase III (6×His) are active in cleaving RNA and dsRNA in test tube. This study provides a proof-of-concept for construction of fusion protein between human receptor and nuclease that may be used to degrade viral nucleic acids.

Cross-Sectional Seroepidemiologic Study of Coronavirus Disease 2019 (COVID-19) among Close Contacts, Children, and Migrant Workers in Shanghai.

(1) Background: Along with an increasing risk caused by migrant workers returning to the urban areas for the resumption of work and production and growing epidemiological evidence of possible transmission during the incubation period, a study of Coronavirus Disease 2019 (COVID-19) is warranted among key populations to determine the serum antibody against the SARS-CoV-2 and the carrying status of SARS-CoV-2 to identify potential asymptomatic infection and to explore the risk factors. (2) Method: This is a cross-sectional seroepidemiologic study. Three categories of targeted populations (close contacts, migrant workers who return to urban areas for work, and school children) will be included in this study as they are important for case identification in communities. A multi-stage sampling method will be employed to acquire an adequate sample size. Assessments that include questionnaires and blood, nasopharyngeal specimens, and feces collection will be performed via home-visit survey. (3) Ethics and Dissemination: The study was approved by the Institute Review Board of School of Public Health, Fudan University (IRB#2020-04-0818). Before data collection, written informed consent will be obtained from all participants. The manuscripts from this work will be submitted for publication in quality peer-reviewed journals and presented at national or international conferences.

Clinical Manifestation and Neonatal Outcomes of Pregnant Patients With Coronavirus Disease 2019 Pneumonia in Wuhan, China.

BACKGROUND: Clinical manifestation and neonatal outcomes of pregnant women with coronavirus disease 2019 (COVID-19) were unclear in Wuhan, China. METHODS: We retrospectively analyzed clinical characteristics of pregnant and nonpregnant women with COVID-19 aged from 20 to 40, admitted between January 15 and March 15, 2020 at Union Hospital, Wuhan, and symptoms of pregnant women with COVID-19 and compared the clinical characteristics and symptoms to historic data previously reported for H1N1. RESULTS: Among 64 patients, 34 (53.13%) were pregnant, with higher proportion of exposure history (29.41% vs 6.67%) and more pulmonary infiltration on computed tomography test (50% vs 10%) compared to nonpregnant women. Of pregnant patients, 27 (79.41%) completed pregnancy, 5 (14.71%) had natural delivery, 18 (52.94%) had cesarean section, and 4 (11.76%) had abortion; 5 (14.71%) patients were asymptomatic. All 23 newborns had negative reverse-transcription polymerase chain results, and an average 1-minute Apgar score was 8-9 points. Pregnant and nonpregnant patients show differences in symptoms such as fever, expectoration, and fatigue and on laboratory tests such as neurophils, fibrinogen, D-dimer, and erythrocyte sedimentation rate. Pregnant patients with COVID-19 tend to have more milder symptoms than those with H1N1. CONCLUSIONS: Clinical characteristics of pregnant patients with COVID-19 are less serious than nonpregnant. No evidence indicated that pregnant women may have fetal infection through vertical transmission of COVID-19. Pregnant patients with H1N1 had more serious condition than those with COVID-19.

In-hospital cardiac arrest outcomes among patients with COVID-19 pneumonia in Wuhan, China.

OBJECTIVE: To describe the characteristics and outcomes of patients with severe COVID-19 and in-hospital cardiac arrest (IHCA) in Wuhan, China. METHODS: The outcomes of patients with severe COVID-19 pneumonia after IHCA over a 40-day period were retrospectively evaluated. Between January 15 and February 25, 2020, data for all cardiopulmonary resuscitation (CPR) attempts for IHCA that occurred in a tertiary teaching hospital in Wuhan, China were collected according to the Utstein style. The primary outcome was restoration of spontaneous circulation (ROSC), and the secondary outcomes were 30-day survival, and neurological outcome. RESULTS: Data from 136 patients showed 119 (87.5%) patients had a respiratory cause for their cardiac arrest, and 113 (83.1%) were resuscitated in a general ward. The initial rhythm was asystole in 89.7%, pulseless electrical activity (PEA) in 4.4%, and shockable in 5.9%. Most patients with IHCA were monitored (93.4%) and in most resuscitation (89%) was initiated <1 min. The average length of hospital stay was 7 days and the time from illness onset to hospital admission was 10 days. The most frequent comorbidity was hypertension (30.2%), and the most frequent symptom was shortness of breath (75%). Of the patients receiving CPR, ROSC was achieved in 18 (13.2%) patients, 4 (2.9%) patients survived for at least 30 days, and one patient achieved a favourable neurological outcome at 30 days. Cardiac arrest location and initial rhythm were associated with better outcomes. CONCLUSION: Survival of patients with severe COVID-19 pneumonia who had an in-hospital cardiac arrest was poor in Wuhan.