A collection of over 3000 independent, full-length SARS-CoV-2 sequences deriving from posited or confirmed chronic infections is assembled and 14 distinct mutation patterns (MPs) that repeatedly appear in these sequences are described, including four CD8 T cell-escape MPs and two MPs that represent adaptation to tissue compartments outside the upper-respiratory tract.
Abstract
The SARS-CoV-2 pandemic has been marked by two outstanding features that have had major impacts on global public health: the repeated emergence and growth of highly divergent saltation variants with no known close relatives and the development of adverse health effects extending beyond the period of acute infection, called long Covid, in a proportion of the population. Chronic infections in immunocompromised hosts are the most likely explanation for the emergence of saltation variants, and some evidence indicates that viral persistence contributes to long Covid. Knowledge of intrahost evolution during prolonged SARS-CoV-2 infection is therefore vital for understanding the global evolution of SARS-CoV-2 and for deciphering the nature of long Covid and promising avenues for treatment. We assembled a collection of over 3000 independent, full-length SARS-CoV-2 sequences deriving from posited or confirmed chronic infections. We describe 14 distinct mutation patterns (MPs) that repeatedly appear in these sequences—each involving mutations in multiple genomic regions—including four CD8 T cell-escape MPs and two MPs that represent adaptation to tissue compartments outside the upper-respiratory tract. The existence of these MPs promises new insights into the life cycle and evolution of SARS-CoV-2 and the nature of persistent SARS-CoV-2 infection.
BACKGROUND
Since the beginning of the COVID-19 pandemic, a large number of SARS-CoV-2 variants have emerged from the reference strain (Wuhan-Hu-1, NC_045512.2). Using these variants to understand the genetic diversity and temporal dynamics of SARS-CoV-2 in Niger is essential for developing and implementing effective public health interventions.
METHOD
SARS-CoV-2 detection was performed on nasopharyngeal samples collected from travelers and symptomatic patients at the National Reference Laboratory in Niger from March 2020 to March 2023 using RT-qPCR techniques. A subset of samples with a cycle threshold <28 were selected for whole-genome sequencing and genomic analysis, which included phylogenetic reconstruction to study the evolutionary relationships of SARS-CoV-2 variants detected in Niger.
RESULTS
Among 286,872 samples from suspected cases tested, we confirmed 8266 positives for SARS-CoV-2, resulting in a positivity rate of 2.88%. Of the positive cases, 268 (3.24%) resulted in death and 5577 (67.48%) were travelers. All the WHO-designated Variants of Concern (VOCs), including Alpha B.1.1.7 (n = 2), Beta B.1.351 (n = 1), Delta B.1.617 (n = 20) and Omicron B.1.1.529 (n = 95) circulated in Niger during the study period. Among the Variants of Interest (VOIs) and Variants Under Monitoring (VUMs), the A27 lineage (n = 49) was the predominant virus detected and co-circulated with Eta lineage B.1.525 (n = 21). Phylogeographic analysis identified at least 60 independent introductions of SARS-CoV-2 into Niger, the majority of which were closely related to published sequences from other African countries (n = 25) and European countries (n = 24). Phylogenetic analysis of the four predominant variants revealed a temporal progression of SARS-CoV-2 lineages in Niger, with well-supported clades. A high number of mutations were observed, mainly located in the spike protein, ORF1a, and ORF1b genes.
CONCLUSION
This study helps to understand the SARS-CoV-2 evolution and dynamics in Niger Republic, where diagnostic capacities and containment measures were challenging.
Adamou Lagaré, Issaka Aboubacar Adakal, A. Yacouba et al.· Infection, Genetics and Evol...· 0 citations
In addition to causing cold and flu-like symptoms, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) can also cause chronic longer-term diseases. Antiviral drugs, especially used combinatorially, have the potential to reduce the severity of individual infections and prevent the development of chronic disease. One of the safest and most versatile reverse genetics systems for SARS-CoV-2 studies is a bacterial artificial chromosome (BAC)-based system harboring the WA1 strain full-length genome and attenuating deletions in the accessory open reading frame 3a and 7b proteins (ORF3a and ORF7b, respectively). Here, a scarless genome engineering technique called En Passant mutagenesis was used to change one amino acid in the viral main protease (Mpro P132) into the residue present in contemporary Omicron strains (H132), in order to more accurately study protease inhibitors and resistance mechanisms. This recombinant, attenuated viral system yields antiviral EC50 values for the active component of approved drugs including nirmatrelvir (Paxlovid) and ensitrelvir (Xocova) and, importantly, also enables a parallel assessment of drug efflux. For instance, the antiviral potency of nirmatrelvir improves 50-fold by inhibiting the P-Glycoprotein (P-Gp) transporter with ritonavir or tariquidar, whereas the potency of ensitrelvir is unaffected. This system also enables the safe isolation and characterization of viral variants with reduced sensitivity to drugs, as evidenced by Mpro M49L compromising the efficacy of ensitrelvir. Together, these systems combine to provide safe, reliable, and quantitative approaches for Mpro variant analysis and drug testing without the biosafety concerns of conducting these experiments using wildtype isolates. IMPORTANCE Safe genetic systems for studying coronavirus biology and developing next generation antivirals are important. One of the most versatile systems leverages a bacterial artificial chromosome to efficiently propagate and engineer a full-length SARS-CoV-2 genome. This system is also safe because it has crippling deletion mutations that limit virus replication to a small number of cell lines. Here, we use a genome engineering technology to change a single amino acid in the viruses’ main protease enzyme to match that of circulating Omicron isolates. The resulting attenuated virus was also used to demonstrate antiviral efficacy of approved drugs and uncover mutants with reduced drug sensitivity. The emergent mutants match those in a subset of circulating strains further demonstrating broad relevance.
SARS-CoV-2 persistence has been proposed as a potential contributor to the pathogenesis of long COVID, with reservoir tissues potentially serving as sites for viral persistence, intra-host evolution, and intermittent viral shedding. Here, we used experimentally infected Syrian hamsters to investigate long-term SARS-CoV-2 persistence across tissues, viral infectivity, and associated immunological and metabolic alterations. Syrian hamsters (Mesocricetus auratus) were intranasally infected with a SARS-CoV-2 parental strain or Gamma and Delta variants and monitored for up to one year, with samples collected at 3, 15, 30, 90, 150, and 365 days post-infection (dpi). During the acute phase, infected animals exhibited significant weight loss, viral shedding, and marked pulmonary inflammation, accompanied by increased expression of pro-inflammatory cytokines at 3 dpi. Infection was confirmed by seroconversion, with sustained IgG responses and low-titer neutralizing antibodies against Omicron. Viral nucleoprotein was detected in multiple tissues up to 365 dpi, while RT-qPCR identified persistent low-level viral RNA in the lungs, brain, spleen, and thymus throughout the observation period, without evidence of productive viral replication. Immune gene expression displayed organ-specific temporal patterns: acute pulmonary inflammation transitioned into broad late-stage suppression, except for sustained TGF-β expression; the brain exhibited a late chemokine signature at 365 dpi; and the thymus showed a delayed immune activation peak at 150 dpi, particularly in Delta-infected animals. Metabolomic profiling revealed a shared acute-phase metabolic signature across variants that largely resolved by 365 dpi, whereas Delta-infected animals retained distinct residual metabolic alterations. Collectively, these findings establish a model of long-term SARS-CoV-2 tissue persistence characterized by organ-specific immune and metabolic signatures, providing a platform to investigate mechanisms underlying post-acute sequelae and evaluate potential therapeutic strategies.
Thais Melquiades de Lima, Carlos Eduardo Capelini Eli Lopes, Maria Vitoria Oliveira de Souza et al.· bioRxiv· 0 citations
Background Improved understanding of how variants cause breakthrough infection, despite pre-existing immunity, is needed to advance development of next-generation SARS-CoV-2 vaccines, including those that may provide cross-variant protection or block transmission. SARS-CoV-2 controlled human infection models (CHIMs) may therefore identify correlates of protection and accelerate the development of new interventions. Methods Healthy vaccinated adults aged 18-30 years were inoculated intranasally in a stepwise dose-escalation CHIM with doses from 1x102 TCID50 to 1x106 TCID50 of SARS-CoV-2 Delta variant. Within the 1x106 TCID50 group, participants were selected for serum neutralising antibody titres (NT50) less than or equal to 1:80. Post-inoculation, participants were quarantined for up to 14 days. Outpatient follow-up continued for 12 months. The primary aim was to elicit safe, well-tolerated Delta SARS-CoV-2 breakthrough infection at a rate of over 50%. Findings Forty-six participants were inoculated; 22 during dose-escalation with no resultant infections, and 24 at the highest dose, of whom 18 were screened for low serum neutralising antibodies. Sustained infection with mild-to-moderate symptoms occurred in 33% (6/18) of the sero-selected group, with highly variable viral loads, viral emissions and symptoms. Serum neutralising antibody, anti-N IgG and to a lesser extent, mucosal anti-S IgA and N-specific T cells most strongly predicted protection from virologically-defined infection. Higher neutralisation, serum and nasal anti-N IgG level, and N-specific T cell responses correlated with lower viral load, while baseline nasal anti-S IgA was associated with lower symptom scores. Transient infection was additionally observed in 6 participants and was associated with higher baseline N-specific T cell responses than those that developed sustained infection. Interpretation Susceptibility to SARS-CoV-2 breakthrough infection in those with hybrid immunity is strongly associated with low levels of pre-existing antibody, but the diversity of immune markers associated with protection implies that additional benefits may be conferred by multi-pronged immunity.
A. Singanayagam, H. Wagstaffe, L. J. Slater et al.· medRxiv· 0 citations
COVID-19, caused by the SARS-CoV-2 virus, has a wide spectrum of clinical presentations even among individuals with similar demographics. Disease severity has been linked with viral-mediated expression of interferons and interferon-stimulated genes. Among the interferon-stimulated genes are members of adenosine deaminases acting on the RNA (ADAR) family that contribute to transcriptome diversity and modulate immune response. Previous studies identified altered ADAR expression and editing patterns during SARS-CoV-2 infection, although it remains unclear whether ADAR expression and activity differ between patients with varying severities of COVID-19. We used whole-blood transcriptomes from individuals with critical or non-critical COVID-19 and matched for age, sex, and presence of comorbidities. Results show differential expression of numerous genes, including those involved in neutrophil degranulation, and upregulation of ADAR1 and its isoform ADARp110 in patients with critical COVID-19. We identified severity-specific editing events, including nonsynonymous edits, within distinct biological pathways. Differentially edited sites—that could serve as molecular markers for COVID-19 severity—were found within genes enriched in signal transduction, RNA and protein metabolism, and inflammatory pathways. Our results demonstrate differences in expression and editing patterns of ADARs between critical and non-critical patients, supporting a potential role of ADAR editing in COVID-19 pathogenesis.
Aiswarya Mukundan Nair, H. Piontkivska· International Journal of Mol...· 0 citations
Introduction: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) shows continuous genetic variation, resulting in the emergence of multiple viral lineages worldwide. These mutations can influence viral transmissibility, pathogenic potential, and immune escape, resulting in changed disease pattern. Genomic surveillance allows systematic tracking of viral evolution and circulating variants; thus, monitoring is essential for informed public health planning and clinical preparedness.
Aim: To detect the emergence of variants of Omicron lineage in the hilly state of Himachal Pradesh, India.
Materials and Methods: This cross-sectional study was conducted at the Shri Lal Bahadur Shastri Medical College, Mandi, Himachal Pradesh, India from January 2024 to June 2024. All the SARS-CoV-2 positive samples with Ct value ≤30 received across the state from the four Virus Research and Diagnostic Laboratories (VRDLs) of the state were included. Ribonucleic acid (RNA) extraction was performed using the Q-Line Molecular Viral RNA Extraction Kit, and Whole Genome Sequencing (WGS) was carried out on the Oxford Nanopore MinION platform. Consensus sequences were generated using Medaka and Nanopolish for variant identification and phylogenetic analysis. All demographic and clinical data, like age, sex, clinical presentation, and vaccination status, were recorded and tabulated in Microsoft Excel 2021. The study focused on genomic sequences generated, and the data were presented as numbers and percentages.
Results: A total of 30 SARS-CoV-2 positive samples were received. Among SARS-CoV-2 positive cases, adult females constituted the highest proportion, 17 (56.7%), with most patients aged 19-60 years, 17 (56.7%), and the majority of samples originating from Shimla, 11 (36.7%). The study identified JN.1 sublineages in 19 (63.3%) as the predominant cause of infection. Most cases, 20 (66.7%) reported to the outpatient department. Mild symptoms such as fever and cough were seen in 7 (35%) cases. Even though 23 (76.7%) of cases were vaccinated with the COVID-19 vaccine, 10 (33.3%) required admissions. However, no mortality was reported.
Conclusion: Tracking SARS-CoV-2 variants revealed the predominance of the JN.1 sublineage of Omicron with generally mild symptoms. Continuous genetic evolution of SARS CoV-2 highlights the need for robust, ongoing genomic surveillance integrated with epidemiological data to enable early detection of emerging variants and strengthen control strategies.
Priyanka Rao, S. Ganju, Lata R. Chandel· Journal of Clinical and Diag...· 0 citations