Development of an Isavirus minigenome system to study the function of the pocket RNA-binding domain of the viral nucleoprotein (NP) in salmon cells.

The Isavirus is an orthomyxovirus with a genome composed of eight segments of negative single-strand RNA (-ssRNA). It has been proposed that the eight genomic segments of the Isavirus are organized as a ribonucleoprotein (RNP) complex called a minigenome, which contains all the viral RNA segments, a viral heterotrimeric polymerase and multiple copies of the viral nucleoprotein (NP). Here, we develop an Isavirus minigenome system and show the importance of the formation of active RNPs and the role of viral NP R189, R194, R302 and K325 residues in the NP RNA-binding domain in the context of RNPs. The results indicate it is possible to generate a minigenome in salmon cells, a composite ISAV RNPs with EGFP-based chimeric vRNA with heterotrimeric polymerase (PB1, PB2, PA) and NP protein using CMV-based auxiliary plasmids. It was also shown that NP R189, R194, R302 and K325 residues are important to generate viral mRNA from the constituted RNPs and a detectable reporter protein. This work is the first salmon cell-based minigenome assay for the Isavirus, which was evaluated by a bioinformatic and functional study of the NP protein in viral RNPs, which showed that correct NP-vRNA interaction is key to the functioning of RNPs.

Differential Methylation in APOE (Chr19; Exon Four; from 44,909,188 to 44,909,373/hg38) and Increased Apolipoprotein E Plasma Levels in Subjects with Mild Cognitive Impairment.

BACKGROUND: Biomarkers are essential for identification of individuals at high risk of mild cognitive impairment (MCI) for potential prevention of dementia. We investigated DNA methylation in the APOE gene and apolipoprotein E (ApoE) plasma levels as MCI biomarkers in Colombian subjects with MCI and controls. METHODS: In total, 100 participants were included (71% women; average age, 70 years; range, 43⁻91 years). MCI was diagnosed by neuropsychological testing, medical and social history, activities of daily living, cognitive symptoms and neuroimaging. Using multivariate logistic regression models adjusted by age and gender, we examined the risk association of MCI with plasma ApoE and APOE methylation. RESULTS: MCI was diagnosed in 41 subjects (average age, 66.5 ± 9.6 years) and compared with 59 controls. Elevated plasma ApoE and APOE methylation of CpGs 165, 190, and 198 were risk factors for MCI (p < 0.05). Higher CpG-227 methylation correlated with lower risk for MCI (p = 0.002). Only CpG-227 was significantly correlated with plasma ApoE levels (correlation coefficient = -0.665; p = 0.008). CONCLUSION: Differential APOE methylation and increased plasma ApoE levels were correlated with MCI. These epigenetic patterns require confirmation in larger samples but could potentially be used as biomarkers to identify early stages of MCI.

Development of a reverse genetic system for infectious salmon anemia virus: rescue of recombinant fluorescent virus by using salmon internal transcribed spacer region 1 as a novel promoter.

Infectious salmon anemia (ISA) is a serious disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), belonging to the genus Isavirus, family Orthomyxoviridae. There is an urgent need to understand the virulence factors and pathogenic mechanisms of ISAV and to develop new vaccine approaches. Using a recombinant molecular biology approach, we report the development of a plasmid-based reverse genetic system for ISAV, which includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). Salmon cells cotransfected with pSS-URG-based vectors expressing the eight viral RNA segments and four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex allowed the generation of infectious recombinant ISAV (rISAV). We generated three recombinant viruses, wild-type rISAV(901_09) and rISAVr(S6-NotI-HPR) containing a NotI restriction site and rISAV(S6/EGFP-HPR) harboring the open reading frame of enhanced green fluorescent protein (EGFP), both within the highly polymorphic region (HPR) of segment 6. All rescued viruses showed replication activity and cytopathic effect in Atlantic salmon kidney-infected cells. The fluorescent recombinant viruses also showed a characteristic cytopathic effect in salmon cells, and the viruses replicated to a titer of 6.5105 PFU/ml,similar to that of the wild-type virus. This novel reverse genetics system offers a powerful tool to study the molecular biology of ISAV and to develop a new generation of ISAV vaccines to prevent and mitigate ISAV infection, which has had a profound effect on the salmon industry.

The Comorbidities, Radiographic Findings, Age, and Lymphopenia (CORAL) Tool: A Diagnostic Ally for Emergency Physicians Created for the COVID-19 Crisis and Beyond.

BACKGROUND: This study aimed to develop a novel clinical approach to predict intensive care unit (ICU) admission and mortality among coronavirus disease 2019 (COVID-19) patients in the emergency department (ED). METHODS: A retrospective cohort study was conducted including adults ≥ 18 years diagnosed with COVID-19 in the emergency department and admitted to the ICU between March and July 2020 in an academic hospital. The outcome variables were mortality and ICU admission. Additional variables that were collected included sex, age, comorbidities, symptom phenotype, and laboratory (lymphopenia) and imaging findings. A logistic regression model was used to construct and validate the risk models. RESULTS: A total of 808 patients were included in the study; 61.9% were men. The mean age was 57.8 ± 15.9 years, and high blood pressure (HBP) was the most prevalent comorbidity (31.8%). Seventy-six (9.4%) patients were admitted to the ICU. Age ≥ 60 years, chronic obstructive pulmonary disease (COPD), lymphopenia, and imaging findings correlated with mortality. Age ≥ 60 years, lymphopenia (<1,000 cells per microliter), and hypothyroidism correlated with ICU admission. These variables were incorporated into a scoring system (Comorbidities, Radiographic findings, Age, and Lymphopenia (CORAL) tool) to predict mortality and ICU admission. CONCLUSIONS: Our Comorbidities, Radiographic findings, Age, and Lymphopenia (CORAL) tool is a practical tool for different clinical settings independent of access to advanced medical resources or technologies. CORAL is suitable for emergency physicians in low- and middle-income countries.