Evolutionary adaptation of bovine coronavirus (BCoV): Screening of natural recombinations across the complete genomes.

Bovine coronavirus (BCoV) is a member of pathogenic Betacoronaviruses that has been circulating for several decades in multiple host species. Given the similarity between BCoV and human coronaviruses, the current study aimed to review the complete genomes of 107 BCoV strains available on the GenBank database, collected between 1983 and 2017 from different countries. The maximum-likelihood based phylogenetic analysis revealed three main BCoV genogroups: GI, GII, and GIII. GI is further divided into nine subgenogroups: GI-a to GI-i. The GI-a to GI-d are restricted to Japan, and GI-e to GI-i to the USA. The evolutionary relationships were also inferred using phylogenetic network analysis, revealing two major distinct networks dominated by viruses identified in the USA and Japan, respectively. The USA strains-dominated Network Cluster includes two sub-branches: France/Germany and Japan/China in addition to the United States, while Japan strains-dominated Network Cluster is limited to Japan. Twelve recombination events were determined, including 11 intragenogroup (GI) and one intergenogroup (GII vs. GI-g). The breakpoints of the recombination events were mainly located in ORF1ab and the spike glycoprotein ORF. Interestingly, 10 of 12 recombination events occurred between Japan strains, one between the USA strains, and one from intercontinental recombination (Japan vs. USA). These findings suggest that geographical characteristics, and population density with closer contact, might significantly impact the BCoV infection and co-infection and boost the emergence of more complex virus lineages.

Is adherence to Mediterranean and vegetarian dietary patterns associated with traditional and novel markers of inflammation?

Numerous inflammatory biomarkers have been identified in processes leading to cardiac events, including C-reactive protein (hs-CRP), but more novel markers of inflammation including platelet activating factor (PAF) and lipoprotein phospholipase A2 (Lp-PLA2) may be more specific indicators of chronic cardiovascular inflammatory processes.(1) Healthy diet influences cardiovascular disease (CVD) risk and modulating inflammation is a proposed mechanism for risk reduction. Plant-based dietary patterns have been associated with reduced CVD risk.(1) This study aimed to investigate whether adherence to Mediterranean or vegetarian dietary pattern is associated with traditional and novel markers of inflammation in healthy adults. In a cross-sectional study, 100 healthy adults (49 +/- 13 years, 69% female) were recruited and categorised as either high (n = 68) or low risk for CVD (n = 32). Fasted plasma was analysed for hs-CRP, PAF and Lp-PLA2. Dietary intake was assessed using a validated food frequency questionnaire. Validated tools(2,3) were used to calculate Mediterranean and vegetarian dietary pattern adherence scores. Pearson's and Spearman's rank correlation were used to assess associations between each marker of inflammation and dietary pattern scores. T-tests assessed differences in markers between high and low adherers (based on median cut-off) to each dietary pattern. In the total sample, hs-CRP had a medium, negative correlation with vegetarian dietary pattern score (r = -0.445, p < .001), which remained significant for those at high (r = -0.424, p < .001) but not low (r = -0.233, p = .199) risk of CVD. Those with high vegetarian dietary pattern adherence had significantly lower mean hs-CRP (1.62 +/- 3.75 mg/L) compared to those with low adherence (3.23 +/- 4.03 mg/L, p < 0.001) There was a small, negative correlation between Mediterranean dietary pattern score and hs-CRP (r = -0.276, p = .006). Again the correlation was only significant for those at high (r = -.296, p = .015) but not low (r = -.088, p = .633) CVD risk. Those with high Mediterranean dietary pattern adherence had significantly lower mean hs-CRP (1.35 +/- 1.36) than those with low adherence (3.41 +/- 5.13, p = .006). Results for PAF showed a small, positive but nonsignificant correlation with both dietary pattern scores Lp-PLA2 had small, negative, nonsignificant correlations with both dietary pattern scores. There was no difference in PAF or Lp-PLA2 levels between those with high versus low adherence for either dietary pattern. We can conclude that hs-CRP, a traditional marker of inflammation, is correlated with Mediterranean and Vegetarian dietary pattern adherence. Whilst there was no correlation with novel markers of inflammation, this could be due to the timing of COVID-19 vaccinations which coincided with outcome measures Larger studies are needed to determine whether a true relationship exists between diet scores and markers of inflammation.

Viral enteritis in cattle: To well known viruses and beyond

Livestock products supply about 13 percent of energy and 28 percent of protein in diets consumed worldwide. Diarrhea is a leading cause of sickness and death of beef and dairy calves in their first month of life and also affecting adult cattle, resulting in large economic losses and a negative impact on animal welfare. Despite the usual multifactorial origin, viruses are generally involved, being among the most important causes of diarrhea. There are several viruses that have been confirmed as etiological agents (i.e., rotavirus and coronavirus), and some viruses that are not yet confirmed as etiological agents. This review summarizes the viruses that have been detected in the enteric tract of cattle and tries to deepen and gather knowledge about them.Copyright © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Features of liver function in pediatric patients infected with Delta variant versus Omicron variant of severe acute respiratory syndrome Coronavirus 2

Objective: To summarize and analyze the features of liver function in pediatric patients infected with Delta variant versus Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: In this study, an analysis was performed for the liver function test results of the locally transmitted or imported pediatric patients with SARS-CoV-2 infection during isolation who were admitted to Guangzhou Eighth People's Hospital, Guangzhou Medical University, since May 21, 2021, and the clinical data and the constituent ratio of liver injury were compared between the pediatric patients infected with Delta variant and those infected with Omicron variant. The independent samples t-test or the Mann-Whitney U test was used for comparison of continuous data between two groups, and the chi-square test or the Fisher's exact test was used for comparison of categorical data between two groups. Results: A total of 85 pediatric patients infected with SARS-CoV-2 were enrolled, among whom there were 32 (37.6%) pediatric patients infected with Delta variant and 53 (62.4%) pediatric patients infected with Omicron variant, and there were no significant differences between the two groups in age, sex, body height, body weight, and comorbidities (all P > 0.05). There were no significant differences between the two groups in elating aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase, total bilirubin, albumin, and cholinesterase (all P > 0.05), and the pediatric patients infected with Omicron variant had a significantly higher level of total bile acid (TBA) than those infected with Delta variant (Z=-2.336, P=0.020). However, the median values of TBA were within the normal range and the ratios of abnormal TBA were no significant difference between the two groups (P > 0.05). Among the 85 pediatric patients, 10 (11.8%) had a mild increase in liver function parameters, among whom 7 had an increase in TBA, 1 had an increase in ALT, 1 had increases in ALT and AST, and 1 had an increase in ALP. The analysis of liver injury in the pediatric patients infected with Delta variant or Omicron variant showed that there was no significant difference in the constituent ratio of liver injury caused by the two variants (6.3% vs 15.1%, X2=0.691, P=0.406). Conclusion: Mild liver injury is observed in pediatric patients infected with Delta and Omicron variants of SARS-CoV-2, but further studies are needed to evaluate the long-term influence of such infection on liver function.

Identification of Natural Lead Compounds against Hemagglutinin-Esterase Surface Glycoprotein in Human Coronaviruses Investigated via MD Simulation, Principal Component Analysis, Cross-Correlation, H-Bond Plot and MMGBSA.

The pandemic outbreak of human coronavirus is a global health concern that affects people of all ages and genders, but there is currently still no effective, approved and potential drug against human coronavirus, as many other coronavirus vaccines have serious side effects while the development of small antiviral inhibitors has gained tremendous attention. For this research, HE was used as a therapeutic target, as the spike protein displays a high binding affinity for both host ACE2 and viral HE glycoprotein. Molecular docking, pharmacophore modelling and virtual screening of 38,000 natural compounds were employed to find out the best natural inhibitor against human coronaviruses with more efficiency and fewer side effects and further evaluated via MD simulation, PCA, DCCR and MMGBSA. The lead compound 'Calceolarioside B' was identified on the basis of pharmacophoric features which depict favorable binding (ΔGbind -37.6799 kcal/mol) with the HE(5N11) receptor that describes positive correlation movements in active site residues with better stability, a robust H-bond network, compactness and reliable ADMET properties. The Fraxinus sieboldiana Blume plant containing the Calceolarioside B compound could be used as a potential inhibitor that shows a higher efficacy and potency with fewer side effects. This research work will aid investigators in the testing and identification of chemicals that are effective and useful against human coronavirus.

Immunogenicity and Safety of Anti-SARS-CoV-2 mRNA Vaccines in a Cohort of Patients with Hereditary Angioedema.

Many factors may trigger hereditary angioedema (HAE) attacks. This study aims to gain insights into the benefits and potential risks of COVID-19 vaccination in HAE patients, focusing particularly on the possibility of triggering attacks. We enrolled 31 patients with HAE undergoing two doses of the SARS-CoV-2 mRNA Comirnaty-BioNTech/Pfizer vaccine. To evaluate the possible influence of the vaccine on disease control and attack frequency, we administered the angioedema control test (AECT) 4-week version before (T0), 21 days after the first dose (T1), and between 21 and 28 days after the second dose (T2). Despite 5 patients (16.1%) experiencing attacks within 72 h of the first dose administration, no significant variation in attack frequency was observed before and after vaccination [F(2,60) = 0.123; p = 0.799]. In addition, patients reported higher AECT scores at T1 and T2 compared to T0 [F(2,44) = 6.541; p < 0.05; post hoc p < 0.05)], indicating that the disease was rather more controlled after vaccinations than in the previous period. All patients showed a positive serological response to the vaccine without significant differences from healthy controls (U = 162; p = 0.062). These observations suggest that the vaccine administration is safe and effective in HAE patients.

A CASE OF SARS-COV-2 ASSOCIATED WITH QUADRUPLE BACTERIAL COINFECTION

SESSION TITLE: COVID-19 Co-Infections SESSION TYPE: Rapid Fire Case Reports PRESENTED ON: 10/19/2022 12:45 pm - 1:45 pm INTRODUCTION: SARS-CoV-2 has been associated with co-infecting pathogens, such as bacteria, viruses, and fungi. Little has been reported about community acquired atypical bacterial co-infections with SARS-CoV-2. We present a case of a patient with recent COVID-19 pneumonia and diagnosis of Legionella and Mycoplasma pneumonia, in addition of E. coli and C. perfringens bacteremia, that emphasizes SARS-CoV-2 impact in human immunity and the need to consider community acquired infections. CASE PRESENTATION: A 64-year-old male with history of hypertension, alcohol use disorder, iron deficiency anemia, and recent COVID-19 pneumonia presented to the ED with shortness of breath, dark urine, and increased confusion. The patient was admitted to the hospital a week prior with COVID-19 pneumonia and acute kidney injury. He received dexamethasone, remdesivir, and IV fluids. After 8 days, he was discharged home. Upon evaluation, he was afebrile and normotensive, but tachycardic, 129/min, on 4 L of nasal cannula sating 100%. On exam, the patient was oriented only to person and had decreased breath sounds bilaterally. Labs revealed an elevated WBC, 15.3 K/mcL, with left shift, low Hgb, 7.8 g/dL, with low MCV, 61 fL, increased BUN/Cr, 56 mg/dL and 2.8 mg/dL, and an abnormal hepatic panel, AST 121 U/L, ALT 45 U/L, alkaline phosphatase 153 U/L. Ammonia, GGT, CPK and lactic acid were within normal range;but the D-dimer and procalcitonin were elevated, 4618 ng/mL and 25.12 ng/mL, respectively. A urinalysis showed gross pyuria, positive leukocyte esterase and mild proteinuria. CT head showed no acute abnormalities, but the chest X-Ray revealed a hazy opacity in the left mid and lower lung, followed by a CT chest that demonstrated peripheral and lower lobe ground glass opacities and a CT abdomen that showed right sided perinephric and periureteral stranding. Given increased risk for thromboembolism, a VQ scan was done being negative for pulmonary embolism. The patient was admitted with acute metabolic encephalopathy, acute kidney injury, transaminitis, pyelonephritis and concern for hospital acquired pneumonia. Vancomycin, cefepime and metronidazole were ordered. HIV screen was negative. COVID-19 PCR, Legionella urine antigen and Mycoplasma IgG and IgM serologies were positive. Blood cultures grew E. coli and C. perfringens. Infectious Disease and Gastroenterology were consulted. The patient was started on azithromycin and a colonoscopy was done showing only diverticulosis. After an extended hospital course, the patient was cleared for discharge, without oxygen needs, to a nursing home with appropriate follow up. DISCUSSION: Co-infection with bacteria causing atypical pneumonia and bacteremia should be considered in patients with recent or current SARS-CoV-2. CONCLUSIONS: Prompt identification of co-existing pathogens can promote a safe and evidence-based approach to the treatment of patients with SARS-CoV-2. Reference #1: Alhuofie S. (2021). An Elderly COVID-19 Patient with Community-Acquired Legionella and Mycoplasma Coinfections: A Rare Case Report. Healthcare (Basel, Switzerland), 9(11), 1598. https://doi.org/10.3390/healthcare9111598 Reference #2: Hoque, M. N., Akter, S., Mishu, I. D., Islam, M. R., Rahman, M. S., Akhter, M., Islam, I., Hasan, M. M., Rahaman, M. M., Sultana, M., Islam, T., & Hossain, M. A. (2021). Microbial co-infections in COVID-19: Associated microbiota and underlying mechanisms of pathogenesis. Microbial pathogenesis, 156, 104941. https://doi.org/10.1016/j.micpath.2021.104941 Reference #3: Zhu, X., Ge, Y., Wu, T., Zhao, K., Chen, Y., Wu, B., Zhu, F., Zhu, B., & Cui, L. (2020). Co-infection with respiratory pathogens among COVID-2019 cases. Virus research, 285, 198005. https://doi.org/10.1016/j.virusres.2020.198005 DISCLOSURES: No relevant relationships by Albert Chang No relevant relationships by Eric Chang No relevant relationships by KOMAL KAUR No relevant relationships by Katiria Pintor Jime ez

Recent Emergence of Bovine Coronavirus Variants with Mutations in the Hemagglutinin-Esterase Receptor Binding Domain in U.S. Cattle.

Bovine coronavirus (BCoV) has spilled over to many species, including humans, where the host range variant coronavirus OC43 is endemic. The balance of the opposing activities of the surface spike (S) and hemagglutinin-esterase (HE) glycoproteins controls BCoV avidity, which is critical for interspecies transmission and host adaptation. Here, 78 genomes were sequenced directly from clinical samples collected between 2013 and 2022 from cattle in 12 states, primarily in the Midwestern U.S. Relatively little genetic diversity was observed, with genomes having >98% nucleotide identity. Eleven isolates collected between 2020 and 2022 from four states (Nebraska, Colorado, California, and Wisconsin) contained a 12 nucleotide insertion in the receptor-binding domain (RBD) of the HE gene similar to one recently reported in China, and a single genome from Nebraska collected in 2020 contained a novel 12 nucleotide deletion in the HE gene RBD. Isogenic HE proteins containing either the insertion or deletion in the HE RBD maintained esterase activity and could bind bovine submaxillary mucin, a substrate enriched in the receptor 9-O-acetylated-sialic acid, despite modeling that predicted structural changes in the HE R3 loop critical for receptor binding. The emergence of BCoV with structural variants in the RBD raises the possibility of further interspecies transmission.

SARS-CoV-2 Infection and C1-Esterase Inhibitor: Camouflage Pattern and New Perspective.

In Covid-19, the pathological effect of SARS-CoV-2 infection is arbitrated through direct viral toxicity, unusual immune response, endothelial dysfunction, deregulated renin-angiotensin system [RAS], and thrombo-inflammation, leading to acute lung injury (ALI), with a succession of acute respiratory distress syndrome (ARDS) in critical conditions. C1 esterase inhibitor (C1INH) is a protease inhibitor that inhibits the spontaneous activation of complement and contact systems and kinin pathway, clotting, and fibrinolytic systems. Therefore, targeting the complement system through activation of C1INH might be a novel therapeutic modality in the treatment of Covid-19. Therefore, this study aims to illustrate the potential nexus between C1INH and the pathophysiology of SARS-CoV-2 infection. C1INH is highly dysregulated in Covid-19 due to inflammatory and coagulation disorders. C1INH is up-regulated in Covid-19 and sepsis as an acute phase response, but this increase is insufficient to block the activated complement system. In addition, the C1INH serum level predicts the development of ARDS in Covid-19 patients, as its up-regulation is associated with the development of cytokine storm. In Covid-19, C1INH might be inhibited or dysregulated by SARS-CoV-2, leading to propagation of complement system activation with subsequent uncontrolled immunological stimulation due to activation of bradykinin and FXII with sequential activation of coagulation cascades and polymerization of fibrin. Thus, suppression of C1INH by SARS-CoV-2 infection leads to thrombosis and excessive inflammation due to uncontrolled activation of complements and contact systems.

C1 esterase inhibitor-mediated immunosuppression in COVID-19: friend or foe?

From asymptomatic to severe, SARS-CoV-2, causative agent of COVID-19, elicits varying disease severities. Moreover, understanding innate and adaptive immune responses to SARS-CoV-2 is imperative since variants such as Omicron negatively impact adaptive antibody neutralization. Severe COVID-19 is, in part, associated with aberrant activation of complement and Factor XII (FXIIa), initiator of contact system activation. Paradoxically, a protein that inhibits the three known pathways of complement activation and FXIIa, C1 esterase inhibitor (C1-INH), is increased in COVID-19 patient plasma and is associated with disease severity. Here we review the role of C1-INH in the regulation of innate and adaptive immune responses. Additionally, we contextualize regulation of C1-INH and SERPING1, the gene encoding C1-INH, by other pathogens and SARS viruses and propose that viral proteins bind to C1-INH to inhibit its function in severe COVID-19. Finally, we review the current clinical trials and published results of exogenous C1-INH treatment in COVID-19 patients.

In silico Drug Repurposing for the Identification of Antimalarial Drugs as Candidate Inhibitors of SARS-CoV-2

Background: Coronavirus disease (COVID-19) is a severe acute respiratory condition that has affected millions of people worldwide, indicating a global health emergency. Despite the deteriorating trends of COVID-19, no drugs are validated to have substantial efficacy in the potential treatment of COVID-19 patients in large-scale trials. Methods: This study aimed at identifying potential antimalarial candidate molecules for the treatment of COVID and evaluating the possible mechanism of action by in silico screening method. In silico screening studies on various antimalarial compounds, like amodiaquine, chloroquine, hydroxychloroquine, mefloquine, primaquine, and atovaquone, were conducted using PyRx and AutoDoc 1.5.6 tools against ACE 2 receptor, 3CL protease, hemagglutinin esterase, spike protein of SARS HR1 motif, and papain-like protease virus proteins. Results: Based on PyRx results, mefloquine and atovaquone were found to have higher docking affinity scores against virus proteins compared to other antimalarial compounds. Screening report of atovaquone exhibited affirmative inhibition constant for spike protein of SARS HR1 motif, 3CL protease, and papain-like protease. Conclusion: In silico analysis reported atovaquone as a promising candidate for COVID 19 therapy.

A Deadly Embrace: Hemagglutination Mediated by SARS-CoV-2 Spike Protein at Its 22 N-Glycosylation Sites, Red Blood Cell Surface Sialoglycoproteins, and Antibody.

Rouleaux (stacked clumps) of red blood cells (RBCs) observed in the blood of COVID-19 patients in three studies call attention to the properties of several enveloped virus strains dating back to seminal findings of the 1940s. For COVID-19, key such properties are: (1) SARS-CoV-2 binds to RBCs in vitro and also in the blood of COVID-19 patients; (2) although ACE2 is its target for viral fusion and replication, SARS-CoV-2 initially attaches to sialic acid (SA) terminal moieties on host cell membranes via glycans on its spike protein; (3) certain enveloped viruses express hemagglutinin esterase (HE), an enzyme that releases these glycan-mediated bindings to host cells, which is expressed among betacoronaviruses in the common cold strains but not the virulent strains, SARS-CoV, SARS-CoV-2 and MERS. The arrangement and chemical composition of the glycans at the 22 N-glycosylation sites of SARS-CoV-2 spike protein and those at the sialoglycoprotein coating of RBCs allow exploration of specifics as to how virally induced RBC clumping may form. The in vitro and clinical testing of these possibilities can be sharpened by the incorporation of an existing anti-COVID-19 therapeutic that has been found in silico to competitively bind to multiple glycans on SARS-CoV-2 spike protein.

Demonstration of virulence factors of Trichosporon species isolated from clinical samples during the COVID 19 pandemic - A study from a tertiary care medical college hospital from South India

Background:Genus Tricosporon consists of basidiomycetous yeast that form a part of the human microbiota.They cause an array of human diseases, both in imunocompetant and immunocompromised patients. They are the second most commonly reported cause of disseminated infections in humans. Their ability to form Biofilms make them a potential agent of Catheter associated Fungemia. They exhibit virulence factors like Biofilm formation, hemolysis and produce enzymes like proteases and phospholipases that increases the pathogenicity by breaking down the proteins and dis- rupting the host cell membranes. The proteolytic activity plays an important role in its pathogenicity by facilitating inva- sion through degradation of keratin and collagen. Methods:This observational period study was performed at our hospital from 20/06/2020 to 20/10/2020.The isolates were subjected to tests to demonstrate virulence factors. Determination of phospholipase activity was performed using using egg yolk agar, hemolytic activity by SDA with 7% sheep blood, esterase activity was determined by using Tween -80 opacity test medium and the production of Biofilm formation was done by micro titre plate method. All results were recorded. The isolates were confirmed by MALDI TOF mass spectrometry. Results:1.Colony morphology 2.Urease production using christensons urea agar (2%) 3.Phenotypic and Biochemical test result were per- formed and tabulated. Proteomic confirmation results are awaited. [Formula presented] Conclusions:Knowing the virulence factor and the commonly occurring Trichosporon species aids in understanding the pathogenesis and to plan strategic treatment modalities

Known Cellular and Receptor Interactions of Animal and Human Coronaviruses: A Review.

This article aims to review all currently known interactions between animal and human coronaviruses and their cellular receptors. Over the past 20 years, three novel coronaviruses have emerged that have caused severe disease in humans, including SARS-CoV-2 (severe acute respiratory syndrome virus 2); therefore, a deeper understanding of coronavirus host-cell interactions is essential. Receptor-binding is the first stage in coronavirus entry prior to replication and can be altered by minor changes within the spike protein-the coronavirus surface glycoprotein responsible for the recognition of cell-surface receptors. The recognition of receptors by coronaviruses is also a major determinant in infection, tropism, and pathogenesis and acts as a key target for host-immune surveillance and other potential intervention strategies. We aim to highlight the need for a continued in-depth understanding of this subject area following on from the SARS-CoV-2 pandemic, with the possibility for more zoonotic transmission events. We also acknowledge the need for more targeted research towards glycan-coronavirus interactions as zoonotic spillover events from animals to humans, following an alteration in glycan-binding capability, have been well-documented for other viruses such as Influenza A.

Target genes used for biosensor development in COVID-19 diagnosis.

In a published review entitled "COVID-19 diagnosis -A review of current methods", the authors considered hemagglutinin esterase as one of the structural proteins of SARS-CoV-2 and also they did not represent ORF3b, ORF9b, and ORF9c in SARS-CoV-2 genome structure. However, according to the scientific evidence, among coronaviruses only some betacoronaviruses (Embecovirus subgenera) contain HE, and the genome of most of the coronaviruses such as SARS-CoV-2, SARS-CoV, and MERS-CoV lack the HE gene. In addition, the genome of SARS-CoV-2 contains several accessory proteins ORFs including ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF9c, and ORF10.

Reverse Genetics with a Full-Length Infectious cDNA Clone of Bovine Torovirus.

Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified in the new family Tobaniviridae. While reverse genetics systems have been established for various CoVs, none exist for ToVs. Here, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and hemagglutinin (HA)-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated a recombinant virus (rEGFP) wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. rEGFP expressed EGFP in infected cells but showed significantly lower levels of viral growth than wtBToV. Moreover, rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in nonstructural proteins (NSPs) that emerged during passage exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a phenotype similar to that of these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. IMPORTANCE ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae. Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, pathogenesis, and BToV vaccine development.

Functional Activity of the Complement System in Hospitalized COVID-19 Patients: A Prospective Cohort Study.

AIMS: Although the exact factors promoting disease progression in COVID-19 are not fully elucidated, unregulated activation of the complement system (CS) seems to play a crucial role in the pathogenesis of acute lung injury (ALI) induced by SARS-CoV-2. In particular, the lectin pathway (LP) has been implicated in previous autopsy studies. The primary purpose of our study is to investigate the role of the CS in hospitalized COVID-19 patients with varying degrees of disease severity. METHODS: In a single-center prospective observational study, 154 hospitalized patients with PCR-confirmed SARS-CoV-2 infection were included. Serum samples on admission to the COVID-19 ward were collected for analysis of CS pathway activities and concentrations of LP proteins [mannose-binding lectin (MBL) and ficolin-3 (FCN-3)] & C1 esterase inhibitor (C1IHN). The primary outcome was mechanical ventilation or in-hospital death. RESULTS: The patients were predominately male and had multiple comorbidities. ICU admission was required in 16% of the patients and death (3%) or mechanical ventilation occurred in 23 patients (15%). There was no significant difference in LP activity, MBL and FCN-3 concentrations according to different peak disease severities. The median alternative pathway (AP) activity was significantly lower (65%, IQR 50-94) in patients with death/invasive ventilation compared to patients without (87%, IQR 68-102, p=0.026). An optimal threshold of <65.5% for AP activity was derived from a ROC curve resulting in increased odds for death or mechanical ventilation (OR 4,93; 95% CI 1.70-14.33, p=0.003) even after adjustment for confounding factors. Classical pathway (CP) activity was slightly lower in patients with more severe disease (median 101% for death/mechanical ventilation vs 109%, p=0.014). C1INH concentration correlated positively with length of stay, inflammatory markers and disease severity on admission but not during follow-up. CONCLUSION: Our results point to an overactivated AP in critically ill COVID-19 patients in vivo leading to complement consumption and consequently to a significantly reduced AP activity in vitro. The LP does not seem to play a role in the progression to severe COVID-19. Apart from its acute phase reaction the significance of C1INH in COVID-19 requires further studies.

Determination of camostat and its metabolites in human plasma - Preservation of samples and quantification by a validated UHPLC-MS/MS method.

OBJECTIVES: Camostat mesilate is a drug that is being repurposed for new applications such as that against COVID-19 and prostate cancer. This induces a need for the development of an analytical method for the quantification of camostat and its metabolites in plasma samples. Camostat is, however, very unstable in whole blood and plasma due to its two ester bonds. The molecule is readily hydrolysed by esterases to 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA) and further to 4-guanidinobenzoic acid (GBA). For reliable quantification of camostat, a technique is required that can instantly inhibit esterases when blood samples are collected. DESIGN AND METHODS: An ultra-high-performance liquid chromatography-tandem mass spectrometry method (UHPLC-ESI-MS/MS) using stable isotopically labelled analogues as internal standards was developed and validated. Different esterase inhibitors were tested for their ability to stop the hydrolysis of camostat ester bonds. RESULTS: Both diisopropylfluorophosphate (DFP) and paraoxon were discovered as efficient inhibitors of camostat metabolism at 10 mM concentrations. No significant changes in camostat and GBPA concentrations were observed in fluoride-citrate-DFP/paraoxon-preserved plasma after 24 h of storage at room temperature or 4 months of storage at -20 °C and -80 °C. The lower limits of quantification were 0.1 ng/mL for camostat and GBPA and 0.2 ng/mL for GBA. The mean true extraction recoveries were greater than 90%. The relative intra-laboratory reproducibility standard deviations were at a maximum of 8% at concentrations of 1-800 ng/mL. The trueness expressed as the relative bias of the test results was within ±3% at concentrations of 1-800 ng/mL. CONCLUSIONS: A methodology was developed that preserves camostat and GBPA in plasma samples and provides accurate and sensitive quantification of camostat, GBPA and GBA by UHPLC-MS/MS.