Genetic associations from the Long COVID-19 Host Genetics Initiative contribute to understanding of the genetics of prolonged symptoms in COVID-19

Background/Objectives: One of the most remarkable features of SARS-CoV-2 infection is that a large proportion of individuals are asymptomatic while others experience progressive, even lifethreatening acute respiratory distress syndrome, and some suffer from prolonged symptoms (long COVID). The contribution of host genetics to susceptibility and severity of infectious disease is well-documented, and include rare monogenic inborn errors of immunity as well as common genetic variation. Studies on genetic risk factors for long COVID have not yet been published. Method(s): We compared long COVID (1534) to COVID-19 patients (96,692) and population controls (800,353) using both questionnaire and EHR- based studies. First meta-analysis of 11 GWAS studies from 8 countries did not show genome-wide significant associations. Result(s): Testing 24 variants earlier associated to COVID-19 susceptibility or severity by COVID-19 Host Genetics Initiative showed genetic variation in rs505922, an intronic variant in ABO blood group gene, to be associated with long COVID compared to population controls (OR = 1.16, 95% CI: 1.07-1.27, p = 0.033). (Within-COVID analysis gave similar OR, but was not significant after conservative Bonferroni correction (OR = 1.17, 95% CI: 1.06-1.30, p = 092)). Conclusion(s): The first data freeze of the Long COVID Host Genetics Initiative suggests that the O blood group is associated with a 14% reduced risk for long COVID. The following data freezes with growing sample sizes will possibly elucidate long COVID pathophysiology and pave the way for possible treatments for long lasting COVID symptoms.

Association of Angiotensin Converting Enzyme 2 (ACE2) Gene Polymorphisms with Disease Severity in Indian Covid-19 Patients: A Next-gen Sequencing Approach

The clinical picture of COVID-19 ranges from asymptomatic to mild, moderate or severe disease sometimes leading to death. Differences in the interaction between SARS-CoV-2 Spike (S) protein and angiotensin converting enzyme 2 (ACE2) protein may lead to differences in disease severity. We studied whether ACE2 polymorphisms are associated with disease severity and outcome. We recruited 114 patients between July 2020 - March 2022 confirmed positive by RTPCR for COVID-19 with different degrees of severity (21 mild, 29 moderate, 34 severe, 30 death) and 30 controls (10 non-vaccinated+ 20 vaccinated) who were RT-PCR negative inspite of high-risk contact. Next-gen sequencing was done on MiSeq (Illumina) using amplicon-based targeted sequencing approach using a custom-designed panel to sequence all the exons of ACE2 gene. SPSS ver.26 was used for analysis. The following ACE2 variants were identified on the Local Run Manager (LRM) software from Illumina: (i) rs2285666 (c.439+4G>A) splice region variant, in controls (60%) and Patients (45.8%), (ii) rs4646140 (c.802+24G>A) intronic variant in 4/114 patients and 1/30 controls, (iii) rs41303171 ( c.2158A>G) missense variant in 2/114 patients, (iv) rs536749578 (c.2114+9T>C) intron variant, (v) rs763994205 (c.868A>C) missense variant and (vi) rs7595907 (c.656G>A) missense variant in 1/114 patient each only. rs2285666 was observed in equal frequency ( 60%) in vaccinated and non-vaccinated controls. rs2285666 was observed amongst different severity groups: Mild (80.95%), Moderate (37.93%), Severe (44.11%), and Death (56.67%) revealing association with disease severity, probably having a protective effect. However, these results need to be confirmed on larger sample sizes.

Molecular genetic markers associated with course of severe COVID-19 pneumonia. Different pathways study

Introduction: It is known that the development of COVID-19 in the human body consists of complex system of biological mechanisms underlying the complex interplay between infectious agents and the human host. This raised the question about hosts' genetic variants as predictors of clinical phenotype. The aim of our study was to analyze the effect of the NOS3 gene (VNTR intron 4 a/b), NR3C1 gene (C647G, rs41423247) and the SFTPB gene (C1580T, rs11130866) variants on the course of severe COVID-19 pneumonia in patients. Material(s) and Method(s): The study group included 20 patients (13 men and 7 women) with diagnosis "viral COVID19 pneumonia" treated at the intensive care unit. Investigation of the NOS3, NR3C1 and SFTPB genes variants was carried out by a molecular method using PCR-RFLP and allele-specific PCR, respectively. Result(s): The correlation analysis showed a significant association of the NOS3 gene variants and level of SpO2 (rS=-0.488, p=0.029;SpO2=93.1+/-2.4% for b/b and SpO2=82.0+/-1.1% for a/a genotypes). Also a significant positive correlation was between NR3C1 gene variants and duration of nasal intermittent positive pressure ventilation (nIPP) therapy (rS=0.454, p=0.044;for 647CC - 1.5+/-1.0 days and for 674GG - 3.9+/-2.5 days), presence of fever (need for antipyretics) (rS=0.525, p=0.017;647C vs 647G alleles - chi2=5.8, p=0.016). No significant correlations were found for the variants of SFTPB gene. The obtained results support a hypothesis about the combined influence of different pathways genes variants (NOS3 and NR3C1) on severity of COVID-19. However, in order to draw definite conclusions, further multifaceted research in this area are need.

How to study a highly toxic protein to bacteria: A case of voltage sensor domain of mouse sperm-specific sodium/proton exchanger

Heterologous expression systems have been used as a powerful experimental strategy to study the function of many proteins, particularly ion transporters. For this experiment, it is fundamental to prepare an expression vector encoding a protein of interest. However, we encountered problems in vector preparation of the voltage sensor domain (VSD) of murine sperm-specific Na+/H+ exchanger (sNHE) due to its severe toxicity to bacteria. We overcame the problems by insertion of an amber stop codon or a synthetic intron into the coding sequence of the VSD in the expression vectors. Both methods allowed us to express the protein of interest in HEK293 cells (combined with a stop codon suppression system for amber codon). The VSD of mouse sNHE generates voltage-dependent outward ionic currents, which is a probable cause of toxicity to bacteria. We propose these two strategies as practical solutions to study the function of any protein toxic to bacteria.

Discovery of a tRNA-like base sequence in the coronavirus genome and possible mechanism of action.

BACKGROUND: The question of whether the coronavirus genome contain as-yetununderstood genetic component. PURPOSE (OBJECTIVE): Elucidate the novel functions of the discovered tRNA-like base sequence and lead to the development of novel therapeutic agents. METHODS: A novel tRNA-like base sequence was found in the sequences complementary to the genomes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-CoV. By comparing mutations in the tRNA-like base sequences of these two viruses, it was found that base pairing in the cloverleaf model of SARS-CoV-2 was more robust than that of SARS-CoV. RESULTS: The results of homology search between a short sequence of the coronavirus tRNA-like base sequence and human genes suggest that the molecule produced by this novel tRNA-like sequence may be involved in the splicing of human messenger RNA. CONCLUSIONS: Experimental molecular evidence of the tRNA-like base sequence discovered in this study is urgently needed.

Mechanisms of primary and secondary resistance to RET inhibitors in patients with RET-positive advanced NSCLC

Background: RET fusions are found in 1-2% of patients (pts) with advanced non-small cell lung cancer (aNSCLC). Targeted therapy with RET inhibitors (RETi) significantly improved prognosis. Molecular mechanisms of resistance are still incompletely characterized. Methods: This multicentric retrospective study included 24 centres. Eligible pts had a RET+ aNSCLC, were treated with a RETi and had at least one molecular profile by next-generation sequencing (NGS), performed before and/or after RETi, on tissue and/or plasma samples. Primary resistance under RETi was defined as disease progression (PD) within 6 months of therapy. Results: 95 patients were included with 112 biopsies: 93 at baseline, 19 at PD. 17 patients had paired NGS (baseline and PD). Median age was 65 years (range 56-72);62% were female, 54% were never smokers, 17% had brain metastasis (BM) at diagnosis. 55 patients received pralsetinib, 36 selpercatinib, 4 other RETi. Overall, median PFS under RETi was 17.1 months (95%CI 12.6-28). Primary resistance to RETi occurred in 22 (23%) patients. Primary resistant versus durable responders to RETi had non-adenocarcinoma histology in 9% vs 46% (p=0.61), smoking history in 57% vs 40% (p=0.21), BM in 5% vs 21% (p=0.1), TP53 mutations in 37% vs 22% (p=0.23). KRAS G12V mutation and SMARCA4 alterations were found only in poor responders (4.5% vs 0%, p=0.2;and 25% vs 0%, p=0.04, respectively). Among biopsies at PD (N=19, 13 liquid and 6 tissue biopsies), 7/13 (54%) liquid biopsies failed due to insufficient ctDNA. In 12 evaluable pts, 3 (25%) acquired secondary RET mutations (2 G810S and 1 S904F), 3 (25%) had novel RET rearrangements (2 in intron 11, 1 RET-DOCK1, 1 RET-CSGALNACT2) and 3 (25%) pts had off-target alterations (2 MET and 1 MYC amplification). Three pts (25%) developed novel TP53 mutations, while 3 (25%) had no novel identifiable alterations at PD. Conclusions: SMARCA4 and KRAS co-mutations may have a role in primary resistance to RETi. Secondary RET mutations, novel RET rearrangements and MET/MYC amplifications were identified after treatment with RETi. More than half of pts had insufficient ctDNA at PD, making tissue biopsy essential to identify resistance mechanisms. Legal entity responsible for the study: Institut Gustave Roussy. Funding: Has not received any funding. Disclosure: V. Fallet: Financial Interests, Personal, Advisory Board: AstraZeneca, BMS, Takeda, Roche, Pfizer, Sanofi, Sandoz, Jansen;Financial Interests, Personal, Invited Speaker: AstraZeneca, BMS, Takeda, Pfizer, MSD;Financial Interests, Personal, Expert Testimony: GSK, Boehringer. C. Audigier-Valette: Financial Interests, Personal, Advisory Role: AbbVie, AstraZeneca, Boehringer Ingelheim, Bristol Myers Squibb, Ipsen, Eli Lilly, Novartis, Pfizer, and Roche. A. Russo: Financial Interests, Personal, Advisory Board: Pfizer, AstraZeneca, MSD, Novartis;Financial Interests, Personal, Writing Engagements: AstraZeneca, Novartis. A. Calles Blanco: Financial Interests, Personal, Advisory Board: AstraZeneca, Boehringer Ingelheim, Pfizer, Roche, Lilly, Merck Sharp & Dohme, Novartis, Bristol-Myers Squibb, Takeda, Sanofi;Financial Interests, Personal, Other, Speaker honoraria: Bayer;Financial Interests, Institutional, Research Grant, Drug-only for Investigator-initiated trial: Merck Sharp & Dohme. P. Iranzo Gomez: Financial Interests, Personal, Advisory Role: Bristol-Myers Squibb Recipient, F. Hoffmann, La Roche AG, Merck Sharp & Dohme, Boehringer Ingelheim, MSD Oncology, Rovi, Yowa Kirin, Grunenthal Pharma S.A., Pfizer. M. Tagliamento: Financial Interests, Personal, Other, medical writer: Novartis, Amgen;Financial Interests, Personal, Invited Speaker, travel/accommodation: Roche, Bristol-Myers Squibb, AstraZeneca, Takeda. L. Mezquita: Financial Interests, Personal, Advisory Board: Takeda, AstraZeneca, Roche;Financial Interests, Personal, Invited Speaker: Roche, BMS, AstraZeneca, Takeda;Financial Interests, Personal, Research Grant, SEOM Beca Retorno 2019: BI;Financial Interests, Personal, Research Grant, ESMO TR Research Fellowship 2019: BMS;Financial Interests, Institutional, Research Grant, COVID research Grant: Amgen;Financial Interests, Institutional, Invited Speaker: Inivata, Stilla. C. Lindsay: Financial Interests, Institutional, Principal Investigator: Roche, Amgen, BI;Financial Interests, Personal, Advisory Role: CBPartners, Amgen. S. Ponce: Financial Interests, Institutional, Principal Investigator: Merck Sharp and Dohme, F. Hoffmann-La Roche, Foundation Medicine, PharmaMar. Personal fees: Merck Sharp and Dohme, Bristol-Myers Squibb, F. Hoffmann-La Roche, Foundation Medicine, AstraZeneca, Boehringer Ingelheim, Eli Lilly, Pfizer, Amgen, Celgene.;Financial Interests, Personal, Advisory Board: Merck Sharp and Dohme, Bristol-Myers Squibb, F. Hoffmann-La Roche, Foundation Medicine, AstraZeneca, Boehringer Ingelheim, Eli Lilly, Pfizer, Amgen, Celgene.;Non-Financial Interests, Personal, Other: Merck Sharp and Dohme, Bristol-Myers Squibb, F. Hoffmann-La Roche. M. Aldea: Financial Interests, Personal, Invited Speaker, travel/accommodation: Sandoz. All other authors have declared no conflicts of interest.

LATE ONSET PRIMARY HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS in AN INDIAN ADOLESCENT BOY during A PEAK of POST COVID-19 MULTISYSTEM INFLAMMATORY SYNDROME

Background: Familial Hemophagocytic lymphohistiocytosis (fHLH) categorized as FHL2 (PRF1), FHL3 (UNC13D), FHL4 (STX11), and FHL5 (STXBP2) encoding for Perforin, Munc13-4, Syntaxin11, and Syntaxin binding protein 2, respectively. There is limited information available about the clinical and mutational spectrum of FHL patients in Indian population. Objectives: To delineate clinical and laboratory features of late onset familial Hemophagocytic Lymphohistiocytosis. Methods: A 12-years-old well nourished sick looking boy, born to a non-consanguineous parents with normal birth, development and immunization history with uneventful past presented to us with 6 days history of high fever, cough, breathing difficulty and severe headache. He had occasional vomiting, abdominal pain, polyarthragia & chest pain from last 10 days. Mother also had given history of throat pain, backache & some non-specifc papular rashes over face before the onset of fever. His vitals were normal. Examination revealed faint diffuse fxed erythematous rash all over the body, pallor, icterus and hepatos-plenomegaly. Musculoskeletal examination was unremarkable. Lab evaluation revealed HB 8.9gm%, TLC 4700/cumm with neutrophils 40% and lymphocytes 56% with 8-9% activated lympocytes. Further evaluation showed low ESR 6mm/hr, fbrinogen 97mg% and albumin 2.2 gm% with elevated CRP 40mg/L, ferritin 2000ng/ml, LDH 658IU/L, SGPT 110IU/L, SGOT 221 IU/L, total bilirubin 6mg%, D-dimer 4355 ng:EFU/ml and Triglycerides 441mg%. His blood, urine, CSF and bone marrow cultures were sterile for endemic bacterial and viral infections in our area. His EBV PCR, CoVID RT PCR and CoVID antibody (Total & IgG) test were negative. His immunoglobulin leves were normal. HRCT Chest showed bilateral mild-moderate plural effusions, mild interstitial thickening in both the lower lobes, few fbrotic opacities & old areas of consolidation bilaterally. 2D echo showed mild pericardial effusion. Bone marrow examination showed Hypercellular marrow with iron depletion and occasional hemophagocytosis with CD8 T lymphocytes proliferation (55.2%) and double positive CD4 & CD8 (1.2%). He was initially commenced on supportive therapy, oxygen & intravenous antibiotics. In view of most probable non-infectious, non-malignant hemophagocytic lymphohistiocytosis, he was fnally given intravenous immunoglobulin (2gm/kg) and intravenous pulse methylprednisolone (30mg/kg). He responded well to above regimen within 3 days. He was discharged with tapering steroids over few weeks. Clinical exome by NGS revealed Homozygous Mutation in STXBP2 gene Intron 14, c.1280-1G>C (3' Splice Site) His parents has been counselled for hematopoietic stem cell transplantation and their decision is still pending. Results: We compared our patietnt with a reference to the largest Indian series of pediatric HLH1. Conclusion: Primary HLH type 5 can present frst time during childhood and adolescence. Any child presenting with unexplained HLH features should undergo genetic analysis irrespective of person's past and family history.

Differential transcriptomic landscapes of SARS-CoV-2-infected and bystander lung cells reveal proviral genes selectively upregulated by viral replication

Bulk transcriptomic analyses of virus-cell interactions are commonly performed on mixed populations of infected and uninfected bystander cells and may thus lead to inaccurate interpretations. Moreover, they generally focus on the expression of the coding genome and not on the total transcriptome, which is largely composed of long non-coding RNAs (lncRNAs). We performed polyA+ and whole transcriptome analysis of lung epithelial A549 cells infected with SARS-CoV-2, which were sorted based on the expression of the viral protein Spike (S). To increase the sequencing depth and ameliorate the robustness of the analysis, the samples were depleted of viral transcripts, which constituted up to 85% of total reads in the S-positive cells. Results highlighted a high number of down-regulated genes upon infection, an indication of massive host transcription shutdown. We also noticed an increased level of intronic reads in infected cells as compared to control cells, suggesting a defect in mRNA splicing. Among the upregulated coding genes in S-positive cells, we recovered candidates previously identified by analysis performed on non-sorted cells, such as CXCL8 and CCL20, but also novel candidates, including IL- 32, ITGAM and the down-regulated FEN1. Comparison of mRNA abundances of few coding, non-coding and unannotated genes between non-sorted cells and sorted ones confirmed the accuracy of the approach. We also observed that S-negative bystander cells and mock-infected control cells exhibited very similar transcriptomic profiles. This was not due to a lack of communication between infected and bystander cells: transcripts related to inflammatory cytokines underwent normal splicing and maturation in infected cells, with concomitant high levels of protein secretion. The observed lack of major transcriptomic changes in the bystander population can be linked to the insufficient interferon response in infected cells. Finally, we explored the functional implications of genes selectively upregulated in the infected subpopulation and observed previously uncharacterized proviral activities of lncRNA ADIRFAS1 and endogenous Interleukin 32 (IL-32), making them attractive targets for putative therapeutic strategies. Thus, analyzing the whole transcriptome of pure populations of infected lung cells allowed the accurate identification of cellular functions that are directly affected by infection and recovery of coding and non-coding genes relevant for SARS-CoV-2 replication.

Identification of Complement-Related Missense Variants in Pediatric Patients with Acute and Post COVID-19 Syndromes

Background: Complement dysregulation has been documented in the molecular pathophysiology of COVID-19 and recently implicated in the relevant pediatric patient inflammatory responses. Aims: Based on our previous data in adults, we hypothesized that signatures of complement genetic variants would also be detectable in pediatric patients exhibiting COVID-19 signs and symptoms. Methods: We prospectively studied consecutive pediatric patients from our COVID-19 Units (November 2020-March 2021). COVID-19 was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). Patient data were recorded by treating physicians that followed patients up to discharge. DNA was obtained from peripheral blood samples. Probes were designed using the Design studio (Illumina). Amplicons cover exons of complement-associated genes (C3, C5, CFB, CFD, CFH, CFHR1, CFI, CD46, CD55, MBL2, MASP1, MASP2, COLEC11, FCN1, FCN3 as well as ADAMTS13 and ΤHBD) spanning 15 bases into introns. We used 10ng of initial DNA material. Libraries were quantified using Qubit and sequenced on a MiniSeq System in a 2x150 bp run. Analysis was performed using the TruSeq Amplicon application (BaseSpace). Alignment was based on the banded Smith-Waterman algorithm in the targeted regions (specified in a manifest file). We performed variant calling with the Illumina-developed Somatic Variant Caller in germline mode and variant allele frequency higher than 20%. Both Ensembl and Refseq were used for annotation of the output files. A preliminary analysis (A) for the identification of variants of clinical significance was based on annotated ClinVar data, while a further and more selective analysis (B) was performed to identify missense complement coding variants that may biochemically contribute to the deregulation of innate responses during infection. This analysis was mainly based on the dbSNP and UniProt databases and available literature. Results: We studied 80 children and adolescents, 8 of whom developed inflammatory syndromes (MIS-C group). Among them, 41 were hospitalized and eventually all survived. 1. In our preliminary analysis, patients exhibited heterogeneous variant profiles including pathogenic, benign, likely benign, and variants of unknown significance (median number of variants: 97, range: 61-103). We found a variant of ADAMTS13 (rs2301612, missense) in 39 patients. We also detected two missense risk factor variants, previously detected in complement-related diseases: rs2230199 in C3 (33 patients);and rs800292 in CFH (36 patients). Among them, 40 patients had a combination of these characterized variants. This combination was significantly associated with the presence of dyspnea (p=0.031) and cough (p=0.042). Furthermore, 27 patients had a pathogenic variant in MBL2 (rs1800450), and 7 a pathogenic deletion in FCN3 that have been previously associated with inflammatory syndromes. 2. The results of our further analysis are summarized in Figure. We identified common variants, some well represented by relatively high frequencies (>70%) (rs11098044 in CFI, rs1061170 in CFH and rs12711521 in MASP2) and others less abundant, but varying considerably between the hospitalized group, the non-admitted group and the MIS-C individuals (rs2230199 in C3, rs1065489 in CFH, rs12614 and rs641153 in CFB, rs1800450 in MBL2, rs2273346 and rs72550870 in MASP2, rs72549154 in MASP3 and rs7567833 in COLEC11, all highlighted in Figure in red).). Structurally, the majority of these common variants of interest encode charge reversal mutations. These may influence protein-protein interactions in complex formations that are important for complement activation and/or regulation. Conclusion: In pediatric COVID-19 we have detected a novel set of complement missense coding variants some of which have been implicated earlier in inflammatory syndromes and endothelial stress responses. Certain combinations of mutations of alternative and/or lectin pathway components may increase the threshold dynamics of complement consumption and therefore alter COVID-19 phenotypes. [Formula prese ted] Disclosures: Gavriilaki: Alexion, Omeros, Sanofi Corporation: Consultancy;Gilead Corporation: Honoraria;Pfizer Corporation: Research Funding. Anagnostopoulos: Abbvie: Other: clinical trials;Sanofi: Other: clinical trials;Ocopeptides: Other: clinical trials;GSK: Other: clinical trials;Incyte: Other: clinical trials;Takeda: Other: clinical trials;Amgen: Other: clinical trials;Janssen: Other: clinical trials;novartis: Other: clinical trials;Celgene: Other: clinical trials;Roche: Other: clinical trials;Astellas: Other: clinical trials.

Durable Response to Crizotinib in a Patient with Pulmonary Adenocarcinoma Harboring MET Intron 14 Mutation: A Case Report.

BACKGROUND: For patients with non-epidermal non-small-cell lung cancer (NSCLC), molecular alterations should always be investigated, especially in non-smokers, who have a very high frequency of targetable alterations (EGFR 52%; ALK 8% in particular). MET exon 14 alterations are identified in 3-4% of NSCLCs and MET gene amplification and high protein expression are associated with a poor prognosis. The French recommendations only authorize the use of capmatinib and crizotinib if the mutation concerns exon 14. However, several different types of mutation in exon 14 of MET and its flanking introns can induce a jump in exon 14, activate the MET gene and thus be sensitive to anti-MET tyrosine kinase inhibitors. CASE SUMMARY: This case concerns a 76-year-old Caucasian male with a medical history including idiopathic thrombocytopenic purpura, chronic myelomonocytic leukemia (CMML), atrial fibrillation, arterial hypertension, obesity (BMI 36kg/m2), and a 5-10 pack-per-year smoking history. A left upper lobe pulmonary nodule of 12.4 mm was discovered in March 2019. The patient received adjuvant chemotherapy with carboplatin AUC 5 and vinorelbine 25.00 mg/m2. At the end of the adjuvant treatment, the patient was in complete remission for 5 months. In February 2020, the CT scan revealed a mediastinal lymph node progression. A complementary molecular analysis was realized on the initial surgical specimen. A c.3082+3A>T mutation in the MET gene was identified. This mutation confers susceptibility to anti-MET tyrosine kinase inhibitors. Treatment with crizotinib was initiated with an initial dose of 250 mg/day for 15 days and then increased to 250 mg twice a day. After 7 months of treatment with crizotinib, the disease was still stable according to RECIST 1.1. CONCLUSION: We report here the original case of a patient presenting a lung adenocarcinoma with an intron 14 mutation and having a durable TKI response.

Different Neutralization Sensitivity of SARS-CoV-2 Cell-to-Cell and Cell-Free Modes of Infection to Convalescent Sera.

The COVID-19 pandemic caused by SARS-CoV-2 has posed a global threat to human lives and economics. One of the best ways to determine protection against the infection is to quantify the neutralizing activity of serum antibodies. Multiple assays have been developed to validate SARS-CoV-2 neutralization; most of them utilized lentiviral or vesicular stomatitis virus-based particles pseudotyped with the spike (S) protein, making them safe and acceptable to work with in many labs. However, these systems are only capable of measuring infection with purified particles. This study has developed a pseudoviral assay with replication-dependent reporter vectors that can accurately quantify the level of infection directly from the virus producing cell to the permissive target cell. Comparative analysis of cell-free and cell-to-cell infection revealed that the neutralizing activity of convalescent sera was more than tenfold lower in cell cocultures than in the cell-free mode of infection. As the pseudoviral system could not properly model the mechanisms of SARS-CoV-2 transmission, similar experiments were performed with replication-competent coronavirus, which detected nearly complete SARS-CoV-2 cell-to-cell infection resistance to neutralization by convalescent sera. These findings suggest that the cell-to-cell mode of SARS-CoV-2 transmission, for which the mechanisms are largely unknown, could be of great importance for treatment and prevention of COVID-19.

Complexities in viral replication strategies as a potential explanation for prevalence of asymptomatic carriers in Covid-19 infections: analytical observation on SARS-Cov2 genome characteristics.

Analytical observations (in silico) indicate molecular features of SARS-Cov2 genome that potentially explains the high prevalence of asymptomatic cases in Covid-19 pandemic. We observed that the virus maintains a low preference for 'GGG' codon for glycine (3%) in its genome. We also observed multiple putative introns of 26-44 nucleotide (nt) length in the genomic region between the coding regions of Nsp10 and RPol in the viral ORF1ab, like several other beta-coronaviruses of similar infectivity levels. It appears that the virus employs a dual strategy to ensure unhindered replication within the host. One of the strategies employ a (- )1 frameshift translation event through programmed ribosomal slippage at the ribosomal slippage site in the ORF1ab. The alternate strategy relies on intron excision to generate a read through frame. The presence of 'GGG' in this conserved ribosomal slippage site ensures adequate tRNA in cytoplasm to match the codon, implying no additional frameshift translation due to ribosomal stalling. With fewer replication events, viral load remains low and resulting in asymptomatic cases. We suggest that this strategy is the primary reason for the prevalence of asymptomatic cases in the disease, enabling the virus to spread rapidly.