Infection kinetics, syncytia formation, and inflammatory biomarkers as predictive indicators for the pathogenicity of SARS-CoV-2 Variants of Concern in Calu-3 cells.

The ongoing COVID-19 pandemic has led to the emergence of new SARS-CoV-2 variants as a result of continued host-virus interaction and viral genome mutations. These variants have been associated with varying levels of transmissibility and disease severity. We investigated the phenotypic profiles of six SARS-CoV-2 variants (WT, D614G, Alpha, Beta, Delta, and Omicron) in Calu-3 cells, a human lung epithelial cell line. In our model demonstrated that all variants, except for Omicron, had higher efficiency in virus entry compared to the wild-type. The Delta variant had the greatest phenotypic advantage in terms of early infection kinetics and marked syncytia formation, which could facilitate cell-to-cell spreading, while the Omicron variant displayed slower replication and fewer syncytia formation. We also identified the Delta variant as the strongest inducer of inflammatory biomarkers, including pro-inflammatory cytokines/chemokines (IP-10/CXCL10, TNF-α, and IL-6), anti-inflammatory cytokine (IL-1RA), and growth factors (FGF-2 and VEGF-A), while these inflammatory mediators were not significantly elevated with Omicron infection. These findings are consistent with the observations that there was a generally more pronounced inflammatory response and angiogenesis activity within the lungs of COVID-19 patients as well as more severe symptoms and higher mortality rate during the Delta wave, as compared to less severe symptoms and lower mortality observed during the current Omicron wave in Thailand. Our findings suggest that early infectivity kinetics, enhanced syncytia formation, and specific inflammatory mediator production may serve as predictive indicators for the virulence potential of future SARS-CoV-2 variants.

Keratin protein nanofibers from merino wool yarn: a top-down approach for the disintegration of hierarchical wool architecture to extract α-keratin protein nanofibers.

We report the extraction of keratin nanofibers from the medulla of a parent yarn after denaturing the cuticle and cortex microstructures of a merino wool yarn. Controlled alkaline hydrolysis, followed by high-speed blending in acetic acid, allowed for the extraction of keratin protein nanofibers with an average diameter of 25 nm and a length of less than 3 µm. SEM and AFM analyses showed the removal of cuticle cells from the yarn. FT-IR and DSC analyses confirmed the hydrolysis and denaturation of the sheet protein matrix of cuticle cells. XPS analysis provided strong evidence for the gradual removal of the epicuticle, cuticle cells, and cortex of the hierarchical wool structure with an increase in alkaline hydrolysis conditions. It was confirmed that the merino wool yarn subjected to hydrolysis under alkaline conditions exposed its internal fibrillar surface. In an acetic acid medium, these fibrillar surfaces obtained a surface charge, which further supported the defibrillation of the structure into its individual nanofibrils during high-speed blending. The extracted nanostructures constitute mainly α-helical proteins. The morphology of the nanofibers is composed of a uniform circular cross-section based on the images obtained using AFM, TEM, and SEM. The extracted nanofibers were successfully fabricated into transparent sheets that can be used in several applications.

Systematic Exploration of SARS-CoV-2 Adaptation to Vero E6, Vero E6/TMPRSS2, and Calu-3 Cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread globally, and scientists around the world are currently studying the virus intensively in order to fight against the on-going pandemic of the virus. To do so, SARS-CoV-2 is typically grown in the lab to generate viral stocks for various kinds of experimental investigations. However, accumulating evidence suggests that such viruses often undergo cell culture adaptation. Here, we systematically explored cell culture adaptation of two SARS-CoV-2 variants, namely the B.1.36.16 variant and the AY.30 variant, a sub lineage of the B.1.617.2 (Delta) variant, propagated in three different cell lines, including Vero E6, Vero E6/TMPRSS2, and Calu-3 cells. Our analyses detected numerous potential cell culture adaptation changes scattering across the entire virus genome, many of which could be found in naturally circulating isolates. Notable ones included mutations around the spike glycoprotein's multibasic cleavage site, and the Omicron-defining H655Y mutation on the spike glycoprotein, as well as mutations in the nucleocapsid protein's linker region, all of which were found to be Vero E6-specific. Our analyses also identified deletion mutations on the non-structural protein 1 and membrane glycoprotein as potential Calu-3-specific adaptation changes. S848C mutation on the non-structural protein 3, located to the protein's papain-like protease domain, was also identified as a potential adaptation change, found in viruses propagated in all three cell lines. Our results highlight SARS-CoV-2 high adaptability, emphasize the need to deep-sequence cultured viral samples when used in intricate and sensitive biological experiments, and illustrate the power of experimental evolutionary study in shedding lights on the virus evolutionary landscape.

Detectable Duration of Viable SARS-CoV-2, Total and Subgenomic SARS-CoV-2 RNA in Noncritically Ill COVID-19 Patients: a Prospective Cohort Study.

Determination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity is important in guiding the infection control and differentiating between reinfection and persistent viral RNA. Although viral culture is the gold standard to determine viral infectivity, the method is not practical. We studied the kinetics of SARS-CoV-2 total RNAs and subgenomic RNAs (sgRNAs) and their potential role as surrogate markers of viral infectivity. The kinetics of SARS-CoV-2 sgRNAs compared to those of the culture and total RNA shedding in a prospective cohort of patients diagnosed with coronavirus disease 2019 (COVID-19) were investigated. A total of 260 nasopharyngeal swabs from 36 patients were collected every other day after entering the study until the day of viral total RNA clearance, as measured by reverse transcription PCR (RT-PCR). Time to cessation of viral shedding was in order from shortest to longest: by viral culture, sgRNA RT-PCR, and total RNA RT-PCR. The median time (interquartile range) to negativity of viral culture, subgenomic N transcript, and N gene were 7 (5 to 9), 11 (9 to 16), and 18 (13 to 21) days, respectively (P < 0.001). Further analysis identified the receipt of steroid as the factors associated with longer duration of viral infectivity (hazard ratio, 3.28; 95% confidence interval, 1.02 to 10.61; P = 0.047). We propose the potential role of the detection of SARS-CoV-2 subgenomic RNA as the surrogate marker of viral infectivity. Patients with negative subgenomic N RNA RT-PCR could be considered for ending isolation. IMPORTANCE Our study, combined with existing evidence, suggests the feasibility of the use of subgenomic RNA RT-PCR as a surrogate marker for SARS-CoV-2 infectivity. The kinetics of SARS-CoV-2 subgenomic RNA should be further investigated in immunocompromised patients.

Target Enrichment Metagenomics Reveals Human Pegivirus-1 in Pediatric Hematopoietic Stem Cell Transplantation Recipients.

Human pegivirus-1 (HPgV-1) is a lymphotropic human virus, typically considered nonpathogenic, but its infection can sometimes cause persistent viremia both in immunocompetent and immunosuppressed individuals. In a viral discovery research program in hematopoietic stem cell transplant (HSCT) pediatric patients, HPgV-1 was detected in 3 out of 14 patients (21.4%) using a target enrichment next-generation sequencing method, and the presence of the viruses was confirmed by agent-specific qRT-PCR assays. For the first time in this patient cohort, complete genomes of HPgV-1 were acquired and characterized. Phylogenetic analyses indicated that two patients had HPgV-1 genotype 2 and one had HPgV-1 genotype 3. Intra-host genomic variations were described and discussed. Our results highlight the necessity to screen HSCT patients and blood and stem cell donors to reduce the potential risk of HPgV-1 transmission.