Prolonged Viable Viral Shedding in Immunocompromised Patients with Covid-19

Background: Immunocompromised patients with COVID-19 tend to shed viable virus for a prolonged period. Therefore, for moderately or severely immunocompromised patients with COVID-19, CDC recommends an isolation period of at least 20 days and ending isolation in conjunction with serial testing and consultation with an infectious disease specialist. However, data on viral kinetics and risk factors for prolonged viral shedding in these patients are limited. Method(s): From February 1, 2022 to April 1, 2022, we collected weekly saliva samples from immunocompromised patients with COVID-19 admitted to a tertiary hospital in Seoul, South Korea. Genomic and subgenomic RNAs were measured, and virus culture was performed. Result(s): A total of 41 patients were enrolled;29 (70%) were receiving chemotherapy against hematologic malignancies and the remaining 12 (30%) had undergone solid organ transplantation. Of the 41 patients, 14 (34%) had received 3 doses or more of COVID-19 vaccines. Real-time RT-PCR revealed that 7 (17%) were infected with Omicron BA.1, and 33 (80%) with Omicron BA.2. The median duration of viable virus shedding was 4 weeks (IQR 3-6). Patients undergoing B-cell depleting therapy shed viable virus for longer than the comparator (p=0.01). Multivariable analysis showed that 3-dose or more vaccination (HR 0.33, 95% CI 0.12 - 0.93, p = 0.04) and B-cell depleting therapy (HR 12.50, 95% CI 2.44 - 100.00, p = 0.003) independently affected viable virus shedding of SARS-CoV-2. Conclusion(s): Immunocompromised patients with COVID-19 shed viable virus for median 4 weeks. B-cell depleting therapy increases the risk of prolonged viable viral shedding, while completion of a primary vaccine series reduces this risk. Overall distribution of samples according to genomic viral copy number and culture positivity. Red dot indicates positive culture results, whereas blue dot indicated negative culture results. (Figure Presented).

HIGH FAT AND SUGAR DIET INDUCES GUT LEUKOCYTE DISRUPTIONS EXACERBATED BY SARS-CoV-2

Background: The disparity in COVID-19 disease burden between European, Asian, and African countries is notable, with considerably higher morbidity and mortality in many European countries as well as the U.S. Dietary differences between regions could play a role in differential COVID-19 pathogenesis, as Western diets high in fat and sugar have been implicated in enhancing gut damage and pathogenesis during viral infections. Here we investigate the effect of diet on gut immunity and SARS-CoV-2 infection. Method(s): Six pigtail macaques were fed a commercial monkey chow diet, then transitioned to a high fat and sugar chow diet (HFD) for approximately two months prior to infection with Delta strain SARS-CoV-2. Animals were sampled prior to HFD initiation, during HFD administration but prior to infection, and for approximately one month post-infection. HFD was maintained following infection and animals were euthanized at the study conclusion. Result(s): Viral RNA was detected for up to 28 days post-infection in nose swabs, with peak viral load at day 2 at a mean of 8.2x109 copies/mL of swab fluid. Subgenomic RNA (sgRNA, indicating viral replication) decayed more rapidly, with all animals having undetectable sgRNA by day 21, and a lower peak of 2.6x109 copies/mL swab fluid on day 2. Viral RNA load was approximately 3.5 logs greater and sgRNA load approximately 3 logs higher at day 2 than in rhesus macaques infected with WA2020 SARS-CoV-2 and fed standard monkey chow. Mucosal rectal biopsies indicated significantly lower B cell frequencies from baseline to approximately two months following HFD administration (p=0.04, Dunn's), and frequencies had not recovered approximately one month postinfection. GI tract-resident IgG+ B cells were nearly absent at necropsy, with mean frequency 0.03% of total B cells. B cell loss was coupled with modest T cell expansion during HFD administration, though frequencies declined following infection. Furthermore, NK cell frequencies tended to decline from baseline throughout HFD administration, and were further depleted at necropsy one month post-infection. Conclusion(s): SARS-CoV-2 infection can induce lymphopenia, and our sampling of gut mucosal tissue indicates B cell depletion and NK cell loss with a HFD that is further exacerbated by SARS-CoV-2 infection. Excess dietary fat and sugar may disrupt gut barrier integrity and immunity, in turn predisposing the tissue to pathology of viral infection.

Coronavirus: History, Genome Structure and Pathogenesis

Background: The positive sense and inordinate large RNA genome enclosed by helical nu-cleocapsids along with an outermost layer belongs to the family Coronaviridae. The phylogenetic tree of this family has been classified into Class1 as alpha, Class 2 as beta, Class 3 as gamma, and Class 4 as delta CoV. The mammalian respiratory and gastrointestinal tracts are the main target organs of this en-veloped virus with misperceived mechanisms. The relevance of this virus family has considerably in-creased by the recent emergence of the Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS), which are caused by viruses that belong to the beta-CoV group. Aim(s): Aforesaid illustrations of the emergence of coronavirus diseases over the past two decades, SARS (2002;2003) and MERS (2012 to present)-the ongoing COVID-19 outbreak has pressurized the WHO to take innovative measures for public health, research and medical communities. The aim of the present review is to have proficiency in the coronavirus replication and transcription process which is still in its infancy. Conclusion(s): As an outcome of epidemics, it is being recognized as one of the most advancing viruses by the virtue of high genomic nucleotide substitution rates and recombination. The hallmark of coronavirus replication is discontinuous transcription resulting in the production of multiple subgenomic mRNAs having sequences complementary to both ends of the genome. Therefore, the complete genome sequence of coronavirus will be used as a frame of reference for comprehending this classical phenome-non of the RNA replication process. Finally, research on the pathogenesis of coronaviruses and the host immunopathological response will aid in designing vaccines and minimizing the mortality rate.Copyright © 2021 Bentham Science Publishers.

Detection of subgenomic mRNA from endemic human coronavirus OC43 and NL63 compared to viral genomic loads, single virus detection and clinical manifestations in children with respiratory tract infections.

BACKGROUND: The importance of endemic human coronavirus (HCoV) in children has been insufficiently elucidated upon. Our aims were to develop subgenomic (sg) mRNA tests for HCoV species OC43 and NL63, and to evaluate the relationships to HCoV genomic loads, single HCoV detections and clinical manifestations. METHODS: We have used an 11-yearlong cohort study of children admitted with respiratory tract infection (RTI) and hospital controls. Nasopharyngeal aspirates were analyzed for HCoV subtypes OC43 and NL63 with in-house diagnostic PCR. Positive samples were tested with newly developed real-time PCRs targeting sg mRNA coding for the nucleocapsid protein. RESULTS: OC43 sg mRNA was detected in 86% (105/122) of available OC43-positive samples in the RTI group, and in 63% (12/19) of control samples. NL63 sg mRNA was detected in 72% (71/98) and 71% (12/17) of available NL63-positive patient and control samples, respectively. In RTI samples, sg mRNA detection was strongly associated with a Ct value <32 in both diagnostic PCR tests (OC43: OR = 54, 95% CI [6.8-428]; NL63: OR = 42, 95% CI [9.0-198]) and single NL63 detections (OR = 6.9, 95% CI [1.5-32]). Comparing RTI and controls, only OC43 was associated with RTI when adjusted for age (aOR = 3.2, 95% CI [1.1-9.4]). CONCLUSION: We found strong associations between OC43 and NL63 sg mRNA and high viral genomic loads. sg mRNA for OC43 was associated with RTI. The association between sg mRNA and clinical manifestations needs further evaluation.

Analysis of SARS-CoV-2 known and novel subgenomic mRNAs in cell culture, animal model, and clinical samples using LeTRS, a bioinformatic tool to identify unique sequence identifiers.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a complex strategy for the transcription of viral subgenomic mRNAs (sgmRNAs), which are targets for nucleic acid diagnostics. Each of these sgmRNAs has a unique 5' sequence, the leader-transcriptional regulatory sequence gene junction (leader-TRS junction), that can be identified using sequencing. High-resolution sequencing has been used to investigate the biology of SARS-CoV-2 and the host response in cell culture and animal models and from clinical samples. LeTRS, a bioinformatics tool, was developed to identify leader-TRS junctions and can be used as a proxy to quantify sgmRNAs for understanding virus biology. LeTRS is readily adaptable for other coronaviruses such as Middle East respiratory syndrome coronavirus or a future newly discovered coronavirus. LeTRS was tested on published data sets and novel clinical samples from patients and longitudinal samples from animal models with coronavirus disease 2019. LeTRS identified known leader-TRS junctions and identified putative novel sgmRNAs that were common across different mammalian species. This may be indicative of an evolutionary mechanism where plasticity in transcription generates novel open reading frames, which can then subject to selection pressure. The data indicated multiphasic abundance of sgmRNAs in two different animal models. This recapitulates the relative sgmRNA abundance observed in cells at early points in infection but not at late points. This pattern is reflected in some human nasopharyngeal samples and therefore has implications for transmission models and nucleic acid-based diagnostics. LeTRS provides a quantitative measure of sgmRNA abundance from sequencing data. This can be used to assess the biology of SARS-CoV-2 (or other coronaviruses) in clinical and nonclinical samples, especially to evaluate different variants and medical countermeasures that may influence viral RNA synthesis.