Development of Multiplex Molecular Assays for Simultaneous Detection of Dengue Serotypes and Chikungunya Virus for Arbovirus Surveillance.

The major arboviruses mainly belong to the Bunyaviridae, Togaviridae, and Flaviviridae families, among which the chikungunya virus and dengue virus have emerged as global public health problems. The main objective of this study was to develop specific, sensitive, and cost-effective molecular multiplex RT-PCR and RT-qPCR assays for the rapid and simultaneous detection of CHIKV and the four serotypes of DENV for arbovirus surveillance. Specific primers for all viruses were designed, and one-step multiplex RT-PCR (mRT-PCR) and RT-qPCR (mRT-qPCR) were developed using reference strains of the CHIKV and DENV serotypes. The specificity of the test for all the viruses was confirmed through sequencing. The standard curves showed a high correlation coefficient, R2 = 0.99, for DENV-2 and DENV-3; R2 = 0.98, for DENV-4; and CHIKV; R2 = 0.93, for DENV-1. The limits of detection were calculated to be 4.1 × 10-1 copies/reaction for DENV-1, DENV-3, and CHIKV and 4.1 × 101 for DENV-2 and DENV-4. The specificity and sensitivity of the newly developed mRT-PCR and mRT-qPCR were validated using positive serum samples collected from India and Burkina Faso. The sensitivity of mRT-PCR and mRT-qPCR are 91%, and 100%, respectively. The specificity of both assays was 100%. mRT-PCR and mRT-qPCR assays are low-cost, and a combination of both will be a useful tool for arbovirus surveillance.

Phytochemical rich Himalayan Rhododendron arboreum petals inhibit SARS-CoV-2 infection in vitro.

Phytochemicals with potential to competitively bind to the host receptors or inhibit SARS-CoV-2 replication, may prove to be useful as adjunct therapeutics for COVID-19. We profiled and investigated the phytochemicals of Rhododendron arboreum petals sourced from Himalayan flora, undertook in vitro studies and found it as a promising candidate against SARS-CoV-2. The phytochemicals were reported in various scientific investigations to act against a range of virus in vitro and in vivo, which prompted us to test against SARS-CoV-2. In vitro assays of R. arboreum petals hot aqueous extract confirmed dose dependent reduction in SARS-CoV-2 viral load in infected Vero E6 cells (80% inhibition at 1 mg/ml; IC50 = 173 µg/ml) and phytochemicals profiled were subjected to molecular docking studies against SARS CoV-2 target proteins. The molecules 5-O-Feruloyl-quinic acid, 3-Caffeoyl-quinic acid, 5-O-Coumaroyl-D-quinic acid, Epicatechin and Catechin showed promising binding affinity with SARS-CoV-2 Main protease (MPro; PDB ID: 6LU7; responsible for viral replication) and Human Angiotensin Converting Enzyme-2 (ACE2; PDB ID: 1R4L; mediate viral entry in the host). Molecular dynamics (MD) simulation of 5-O-Feruloyl-quinic acid, an abundant molecule in the extract complexed with the target proteins showed stable interactions. Taken together, the phytochemical profiling, in silico analysis and in vitro anti-viral assay revealed that the petals extract act upon MPro and may be inhibiting SARS-CoV-2 replication. This is the first report highlighting R. arboreum petals as a reservoir of antiviral phytochemicals with potential anti-SARS-CoV-2 activity using an in vitro system.

SARS-CoV-2 variants: Impact on biological and clinical outcome.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that was first identified in December 2019, in Wuhan, China was found to be the etiological agent for a novel respiratory infection that led to a Coronavirus Induced Disease named COVID-19. The disease spread to pandemic magnitudes within a few weeks and since then we have been dealing with several waves across the world, due to the emergence of variants and novel mutations in this RNA virus. A direct outcome of these variants apart from the spike of cases is the diverse disease presentation and difficulty in employing effective diagnostic tools apart from confusing disease outcomes. Transmissibility rates of the variants, host response, and virus evolution are some of the features found to impact COVID-19 disease management. In this review, we will discuss the emerging variants of SARS-CoV-2, notable mutations in the viral genome, the possible impact of these mutations on detection, disease presentation, and management as well as the recent findings in the mechanisms that underlie virus-host interaction. Our aim is to invigorate a scientific debate on how pathogenic potential of the new pandemic viral strains contributes toward development in the field of virology in general and COVID-19 disease in particular.

Short communication: Virological and B cell profiles of chikungunya and Dengue virus co-infections in Delhi during 2017-2019.

With explosive epidemics of chikungunya in India since 2004, chikungunya virus (CHIKV) now co-circulates in geographical areas where Dengue virus (DENV) is already endemic and thus provides opportunity for the same mosquito to be infected with both viruses. Although there are excellent studies that have addressed the clinical of mono and co-infection, we have little to no knowledge on the current viral sequences that pre-dominate co-infections, and the B cell response elicited. In this study, we analyzed febrile patients that were confirmed to have DENV-CHIKV co-infections and asked the following questions: 1) what is the frequency of co-infections found in a single cycle of transmission; 2) what are the viral sequences associated with them; 3) what does the antibody secreting cell / plasmablast response look like in patients that are co-infected with both viruses. We report those co-infections occur at a frequency of 6.7% in the transmission cycle, and while DENV-3 is now frequently detected, we do not see a serotype bias in the patients that are co-infected with ESCA strain of CHIKV. Moreover, the effector B cell response (plasmablasts) observed are specific to both infecting viruses indicating no overt bias. Further studies to associate whether any of these properties have a bearing on clinical disease manifestation will be both timely and important.

Zinc2+ ion inhibits SARS-CoV-2 main protease and viral replication in vitro.

Zinc deficiency is linked to poor prognosis in COVID-19 patients while clinical trials with zinc demonstrate better clinical outcomes. The molecular targets and mechanistic details of the anti-coronaviral activity of zinc remain obscure. We show that zinc not only inhibits the SARS-CoV-2 main protease (Mpro) with nanomolar affinity, but also viral replication. We present the first crystal structure of the Mpro-Zn2+ complex at 1.9 Å and provide the structural basis of viral replication inhibition. We show that Zn2+ coordinates with the catalytic dyad at the enzyme active site along with two previously unknown water molecules in a tetrahedral geometry to form a stable inhibited Mpro-Zn2+ complex. Further, the natural ionophore quercetin increases the anti-viral potency of Zn2+. As the catalytic dyad is highly conserved across SARS-CoV, MERS-CoV and all variants of SARS-CoV-2, Zn2+ mediated inhibition of Mpro may have wider implications.

The Immunomodulatory CEA Cell Adhesion Molecule 6 (CEACAM6/CD66c) Is a Protein Receptor for the Influenza a Virus.

To establish a productive infection in host cells, viruses often use one or multiple host membrane glycoproteins as their receptors. For Influenza A virus (IAV) such a glycoprotein receptor has not been described, to date. Here we show that IAV is using the host membrane glycoprotein CD66c as a receptor for entry into human epithelial lung cells. Neuraminidase (NA), a viral spike protein, binds to CD66c on the cell surface during IAV entry into the host cells. Lung cells overexpressing CD66c showed an increase in virus binding and subsequent entry into the cell. Upon comparison, CD66c demonstrated higher binding capacity than other membrane glycoproteins (EGFR and DC-SIGN) reported earlier to facilitate IAV entry into host cells. siRNA mediated knockdown of CD66c from lung cells inhibited virus binding on cell surface and entry into cells. Blocking CD66c by antibody on the cell surface resulted in decreased virus entry. We found that CD66c is a specific glycoprotein receptor for influenza A virus that did not affect entry of non-IAV RNA virus (Hepatitis C virus). Finally, IAV pre-incubated with recombinant CD66c protein when administered intranasally in mice showed decreased cytopathic effects in mice lungs. This publication is the first to report CD66c (Carcinoembryonic cell adhesion molecule 6 or CEACAM6) as a glycoprotein receptor for Influenza A virus.

Absconding During Inpatient Care from a Tertiary Psychiatric Hospital: A Comparative Study.

BACKGROUND: Absconding from psychiatric hospitals is of great concern for patients and caregivers. Absconding affects not only the treatment and safety of these patients but also patient's caregivers and the community. Further investigation is needed to examine the pattern of this event and the characteristics of patients who abscond. Hence, our study was aimed to examine the sociodemographic and clinical profiles of inpatients who absconded from a psychiatric hospital in five years and to compare them with matched controls. METHODS: A retrospective chart review of inpatients who absconded and matched control inpatients during the specified period of five years from January 2014 to December 2018 was done at a psychiatric hospital. Each control was matched with a corresponding absconding case on the following order: (a) admission ward, (b) admission period, (c) diagnosis, and (d) age. Results: Among 20,052 adult admissions during the specified period, 38 patients absconded, with a rate of 1.8 per 1,000 admissions. Most of them were male, from a younger age group, diagnosed with schizophrenia or mood disorder, and having comorbid substance use disorder, irritable affect, impaired judgment, and absent insight. Most of the events occurred within the first two weeks of admission. About 11% of them had a history of prior absconding from the hospital. CONCLUSION: Knowledge about the associated sociodemographic and clinical profile would help clinicians and mental health care professionals to prevent absconding. Further risk assessment using a patient's profile would help to reduce absconding events from psychiatric hospitals in the future.

Host Lipid Rafts Play a Major Role in Binding and Endocytosis of Influenza A Virus.

Influenza still remains one of the most challenging diseases, posing a significant threat to public health. Host lipid rafts play a critical role in influenza A virus (IAV) assembly and budding, however, their role in polyvalent IAV host binding and endocytosis had remained elusive until now. In the present study, we observed co-localization of IAV with a lipid raft marker ganglioside, GM1, on the host surface. Further, we isolated the lipid raft micro-domains from IAV infected cells and detected IAV protein in the raft fraction. Finally, raft disruption using Methyl-ß-Cyclodextrin revealed significant reduction in IAV host binding, suggesting utilization of host rafts for polyvalent binding on the host cell surface. In addition to this, cyclodextrin mediated inhibition of raft-dependent endocytosis showed significantly reduced IAV internalization. Interestingly, exposure of cells to cyclodextrin two hours post-IAV binding showed no such reduction in IAV entry, indicating use of raft-dependent endocytosis for host entry. In summary, this study demonstrates that host lipid rafts are selected by IAV as a host attachment factors for multivalent binding, and IAV utilizes these micro-domains to exploit raft-dependent endocytosis for host internalization, a virus entry route previously unknown for IAV.

Effect of Recumbent Body Positions on Dynamic Lung Function Parameters in Healthy Young Subjects.

INTRODUCTION: The change in body position can alter pulmonary functions parameters, therefore it is important to understand the physiological basis of these alteration. Ideally, spirometry is done in sitting position until the subject is unable to do so. Hospitalized patients often assume recumbent body positions irrespective of underlying pathology. Hence, need arises to find out best recumbent body positions for the benefit of these patients to make breathing comfortable for them. AIM: The aim of this study was to find out whether the change from the supine position to crook lying and Fowler's position (45° dorsal elevation) causes change in spirometric parameters. MATERIALS AND METHODS: The present work was carried out at Department of Physiology, King George's Medical University, Lucknow. A total 131 apparently healthy individuals were enrolled in this cross-sectional study. Lung function was assessed using a PC-based spirometer according to American Thoracic Society guideline in the supine, crook lying and Fowler's position (45° dorsal elevation). RESULTS: The study consisted of 131 subjects (male 66%, female 34%), with mean age of 20.15±2.71 years and BMI 21.20±3.28 Kg/m2. Repeated measures ANOVA with post hoc Bonferroni test was used to compare the mean values between each body position. Compared with the other two positions, Fowler's position showed significantly (p<0.05) higher values for FVC, FEV1, PEF, FEF25-75%. CONCLUSION: Recumbent body position influences spirometric parameters in young healthy subjects. We demonstrated that spirometric values are higher in the Fowler's position than in the supine or crook lying position. The results of this study will help in the selection of the best alternative position for the spirometry in bed ridden patients.

The rs2070895 (-250G/A) Single Nucleotide Polymorphism in Hepatic Lipase (HL) Gene and the Risk of Coronary Artery Disease in North Indian Population: A Case-Control Study.

INTRODUCTION: Several Single Nucleotide Polymorphisms (SNPs) in lipid transport genes have been shown to be associated with Coronary Artery Disease (CAD). The Hepatic Lipase (HL)glycoprotein is a key component that catalyzes the hydrolysis of triglycerides and phospholipids in all major classes of lipoproteins. AIM: We studied whether the HL gene-250G/A polymorphism affect blood lipid level and the CAD in a North Indian population. MATERIALS AND METHODS: A total number of 477 subjects were enrolled in the study after approval of the Institutional Ethics Committee. Out of 477 subjects, 233 were with coronary artery disease as study group and 244 subjects without coronary artery disease as control group. All subjects recruited with matched ethnicity in age group of 40-70 years. Blood samples were collected in EDTA vials and genomic DNA was extracted from blood using the phenol-chloroform method. Lipid profile was estimated by using a commercially available kit. Polymorphisms in the HL (-250 G/A) gene were analysed by using restriction fragment length polymorphism-polymerase chain reaction (PCR-RFLP) method. The effect of this polymorphism on plasma lipids, lipoproteins and coronary artery disease was determined. RESULTS: In Human Hepatic Lipase (LIPC)-250G/A genotype, the frequencies of GG, GA and AA genotype in CAD group was 80.69%, 15.45% and 3.86%, respectively; in the control group, the corresponding frequencies were 90.16%, 9.02% and 0.82%, respectively. A significant difference was found in the genotype (LIPC-250G/A) distribution between the two groups. Further logistic regression analysis indicated that the GA and AA genotypes in SNP-250G/A were significantly associated with CAD in all genetic models (In codominant model- GA vs. GG, OR=1.91, 95% CI=1. 09-3.37, p=0. 03 and AA vs. GG, OR= 5.26, 95% CI= 1.10-24.60, p=0.04; in dominant model- GA+AA vs. GG, OR=2.19, p=0.004 and in recessive model- AA vs. GG+GA, OR=5.26, p=0.04 whereas, A allele at nucleotide -250G/A in the LIPC gene had an association with increased risk of CAD (OR=2.33, p=<0.008). CONCLUSION: Our findings indicated that the higher frequency of a dominant model (GA+AA) as well as mutant allele A of LIPC-250 G/A polymorphism is significantly associated with risk of CAD and the lipid profile can be used as a predictor of CAD.

Cardiac Output Assessment by Transthoracic Electrical Bio-impedance in Patients of Acute Myocardial Infarction: Comparative Analysis with Echocardiography.

Objectives: Transthoracic electrical bio-impedance (TEB) has been proposed as a non-invasive and continuous method of cardiac output (CO) measurement, but it still has not found wide usages in clinics. The present study measured CO, using a new instrument NICOMON, and compared it with Echocardiography (ECHO) in acute myocardial infarction (AMI) patients. Methods: In the present study 100 patients of AMI were assessed by both ECHO and NICOMON for cardiac output and ECHO is considered as a reference method for comparison. TEB CO was measured by passing an alternating current and measuring the bio-impedance across the thorax. End diastolic volume (EDV), End systolic volume (ESV) & Left ventricular outflow tract (LVOT) diameter, measured by ECHO were used to calculate CO. Various statistical methods like "t"-test & correlation coefficient (r) were used where found suitable. Results: Results: Mean TEB-CO (4.03±1.11 l/min) was significantly higher (p<0.001) than mean ECHO-CO (3.80±1.28 l/min) with a mean difference of 0.25±1.02 l/min. Conclusions: NICOMON measures CO non-invasively but, it needs more elaborative studies on a larger sample to establish it as an alternative method of ECHO for cardiac output measurement on regular basis.