Analysis of SARS-CoV-2 omicron mutations that emerged during long-term replication in a lung cancer xenograft mouse model.

SARS-CoV-2 Omicron has the largest number of mutations among all the known SARS-CoV-2 variants. The presence of these mutations might explain why Omicron is more infectious and vaccines have lower efficacy to Omicron than other variants, despite lower virulence of Omicron. We recently established a long-term in vivo replication model by infecting Calu-3 xenograft tumors in immunodeficient mice with parental SARS-CoV-2 and found that various mutations occurred majorly in the spike protein during extended replication. To investigate whether there are differences in the spectrum and frequency of mutations between parental SARS-CoV-2 and Omicron, we here applied this model to Omicron. At 30 days after infection, we found that the virus was present at high titers in the tumor tissues and had developed several rare sporadic mutations, mainly in ORF1ab with additional minor spike protein mutations. Many of the mutant isolates had higher replicative activity in Calu-3 cells compared with the original SARS-CoV-2 Omicron virus, suggesting that the novel mutations contributed to increased viral replication. Serial propagation of SARS-CoV-2 Omicron in cultured Calu-3 cells resulted in several rare sporadic mutations in various viral proteins with no mutations in the spike protein. Therefore, the genome of SARS-CoV-2 Omicron seems largely stable compared with that of the parental SARS-CoV-2 during extended replication in Calu-3 cells and xenograft model. The sporadic mutations and modified growth properties observed in Omicron might explain the emergence of Omicron sublineages. However, we cannot exclude the possibility of some differences in natural infection.

A mouse xenograft long-term replication yields a SARS-CoV-2 Delta mutant with increased lethality.

We recently established a long-term SARS-CoV-2 infection model using lung-cancer xenograft mice and identified mutations that arose in the SARS-CoV-2 genome during long-term propagation. Here, we applied our model to the SARS-CoV-2 Delta variant, which has increased transmissibility and immune escape compared with ancestral SARS-CoV-2. We observed limited mutations in SARS-CoV-2 Delta during long-term propagation, including two predominant mutations: R682W in the spike protein and L330W in the nucleocapsid protein. We analyzed two representative isolates, Delta-10 and Delta-12, with both predominant mutations and some additional mutations. Delta-10 and Delta-12 showed lower replication capacity compared with SARS-CoV-2 Delta in cultured cells; however, Delta-12 was more lethal in K18-hACE2 mice compared with SARS-CoV-2 Delta and Delta-10. Mice infected with Delta-12 had higher viral titers, more severe histopathology in the lungs, higher chemokine expression, increased astrocyte and microglia activation, and extensive neutrophil infiltration in the brain. Brain tissue hemorrhage and mild vacuolation were also observed, suggesting that the high lethality of Delta-12 was associated with lung and brain pathology. Our long-term infection model can provide mutant viruses derived from SARS-CoV-2 Delta and knowledge about the possible contributions of emergent mutations to the properties of new variants.

Omicron BA.2 breakthrough infection elicits CD8+ T cell responses recognizing the spike of later Omicron subvariants.

Here, we examine peripheral blood memory T cell responses against the SARS-CoV-2 BA.4/BA.5 variant spike among vaccinated individuals with or without Omicron breakthrough infections. We provide evidence supporting a lack of original antigenic sin in CD8+ T cell responses targeting the spike. We show that BNT162b2-induced memory T cells respond to the BA.4/BA.5 spike. Among individuals with BA.1/BA.2 breakthrough infections, IFN-γ-producing CD8+ T cell responses against the BA.4/BA.5 spike increased. In a subgroup with BA.2 breakthrough infections, IFN-γ-producing CD8+ T cell responses against the BA.2-mutated spike region increased and correlated directly with responses against the BA.4/BA.5 spike, indicating that BA.2 spike-specific CD8+ T cells elicited by BA.2 breakthrough infection cross-react with the BA.4/BA.5 spike. We identified CD8+ T cell epitope peptides that are present in the spike of BA.2 and BA.4/BA.5 but not the original spike. These peptides are fully conserved in the spike of now-dominant XBB lineages. Our study shows that breakthrough infection by early Omicron subvariants elicits CD8+ T cell responses that recognize epitopes within the spike of newly emerging subvariants.

High-resolution phylogeographical surveillance of Hantaan orthohantavirus using rapid amplicon-based Flongle sequencing, Republic of Korea.

Orthohantaviruses, etiological agents of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome, pose a critical public health threat worldwide. Hantaan orthohantavirus (HTNV) outbreaks are particularly endemic in Gyeonggi Province in northern area of the Republic of Korea (ROK). Small mammals were collected from three regions in the Gyeonggi Province during 2017 and 2018. Serological and molecular prevalence of HTNV was 25/201 (12.4%) and 10/25 (40%), respectively. A novel nanopore-based diagnostic assay using a cost-efficient Flongle chip was developed to rapidly and sensitively detect HTNV infection in rodent specimens within 3 h. A rapid phylogeographical surveillance of HTNV at high-resolution phylogeny was established using the amplicon-based Flongle sequencing. In total, seven whole-genome sequences of HTNV were newly obtained from wild rodents collected in Paju-si (Gaekhyeon-ri) and Yeoncheon-gun (Hyeonga-ri and Wangnim-ri), Gyeonggi Province. Phylogenetic analyses revealed well-supported evolutionary divergence and genetic diversity, enhancing the resolution of the phylogeographic map of orthohantaviruses in the ROK. Incongruences in phylogenetic patterns were identified among HTNV tripartite genomes, suggesting differential evolution for each segment. These findings provide crucial insights into on-site diagnostics, genome-based surveillance, and the evolutionary dynamics of orthohantaviruses to mitigate hantaviral outbreaks in HFRS-endemic areas in the ROK.

Exploring the Genetic Diversity and Molecular Evolution of Seoul and Hantaan Orthohantaviruses.

Seoul (SEOV) and Hantaan (HTNV) orthohantaviruses are significant zoonotic pathogens responsible for hemorrhagic fever with renal syndrome. Here, we investigated the molecular evolution of SEOV and HTNV through phylogenetic and bioinformatic analyses using complete genome sequences of their large (L), medium (M), and small (S) gene segments. Despite similar epizootic cycles and clinical symptoms, SEOV and HTNV exhibited distinct genetic and evolutionary dynamics. The phylogenetic trees of each segment consistently showed major genetic clades associated with the geographical distribution of both viruses. Remarkably, SEOV M and S segments exhibit higher evolutionary rates, rapidly increasing genetic diversity, and a more recent origin in contrast to HTNV. Reassortment events were infrequent, but both viruses appear to utilize the M gene segment in genetic exchanges. SEOV favors the L or M segment reassortment, while HTNV prefers the M or S segment exchange. Purifying selection dominates in all three gene segments of both viruses, yet SEOV experiences an elevated positive selection in its glycoprotein Gc ectodomain. Key amino acid differences, including a positive 'lysine fence' (through residues K77, K82, K231, K307, and K310) located at the tip of the Gn, alongside the physical stability around an RGD-like motif through M108-F334 interaction, may contribute to the unique antigenic properties of SEOV. With the increasing global dispersion and potential implications of SEOV for the global public health landscape, this study highlights the unique evolutionary dynamics and antigenic properties of SEOV and HTNV in informing vaccine design and public health preparedness.

Analysis of spike protein variants evolved in a novel in vivo long-term replication model for SARS-CoV-2.

Introduction: The spectrum of SARS-CoV-2 mutations have increased over time, resulting in the emergence of several variants of concern. Persistent infection is assumed to be involved in the evolution of the variants. Calu-3 human lung cancer cells persistently grow without apoptosis and release low virus titers after infection. Methods: We established a novel in vivo long-term replication model using xenografts of Calu-3 human lung cancer cells in immunodeficient mice. Virus replication in the tumor was monitored for 30 days and occurrence of mutations in the viral genome was determined by whole-genome deep sequencing. Viral isolates with mutations were selected after plaque forming assays and their properties were determined in cells and in K18-hACE2 mice. Results: After infection with parental SARS-CoV-2, viruses were found in the tumor tissues for up to 30 days and acquired various mutations, predominantly in the spike (S) protein, some of which increased while others fluctuated for 30 days. Three viral isolates with different combination of mutations produced higher virus titers than the parental virus in Calu-3 cells without cytopathic effects. In K18-hACE2 mice, the variants were less lethal than the parental virus. Infection with each variant induced production of cross-reactive antibodies to the receptor binding domain of parental SARS-CoV-2 S protein and provided protective immunity against subsequent challenge with parental virus. Discussion: These results suggest that most of the SARS-CoV-2 variants acquired mutations promoting host adaptation in the Calu-3 xenograft mice. This model can be used in the future to further study SARS-CoV-2 variants upon long-term replication in vivo.

Molecular evolution and targeted recombination of SARS-CoV-2 in South Korea.

SARS-CoV-2 variants have continuously emerged globally, including in South Korea. To characterize the molecular evolution of SARS-CoV-2 in South Korea, we performed phylogenetic and genomic recombination analyses using more than 12,000 complete genome sequences collected until October 2022. The variants in South Korea originated from globally identified variants of concern and harbored genetic clade-common and clade-specific amino acid mutations mainly around the N-terminal domain (NTD) or receptor binding domain (RBD) in the spike protein. Several point mutation residues in key antigenic sites were under positive selection persistently with changing genetic clades of SARS-CoV-2. Furthermore, we detected 17 potential genomic recombinants and 76.4% (13/17) retained the mosaic NTD or RBD genome. Our results suggest that point mutations and genomic recombination in the spike contributed to the molecular evolution of SARS-CoV-2 in South Korea, which will form an integral part of global prevention and control measures against SARS-CoV-2.

Genome-informed investigation of the molecular evolution and genetic reassortment of severe fever with thrombocytopenia syndrome virus.

BACKGROUND: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a viral pathogen causing significant clinical signs from mild fever with thrombocytopenia to severe hemorrhages. World Health Organization has paid special attention to the dramatic increase in human SFTS cases in China, Japan, and South Korea since the 2010s. The present study investigated the molecular evolution and genetic reassortment of SFTSVs using complete genomic sequences. METHODS/PRINCIPAL FINDING: We collected the complete genome sequences of SFTSVs globally isolated until 2019 (L segment, n = 307; M segment, n = 326; and S segment, n = 564) and evaluated the evolutionary profiles of SFTSVs based on phylogenetic and molecular selection pressure analyses. By employing a time-scaled Bayesian inference method, we found the geographical heterogeneity of dominant SFTSV genotypes in China, Japan, and South Korea around several centuries before and locally spread by tick-born spillover with infrequent long-distance transmission. Purifying selection predominated the molecular evolution of SFTSVs with limited gene reassortment and fixed substitution, but almost all three gene segments appeared to harbor at least one amino acid residue under positive selection. Specifically, the nonstructural protein and glycoprotein (Gn/Gc) genes were preferential selective targets, and the Gn region retained the highest number of positively selected residues. CONCLUSION/SIGNIFICANCE: Here, the large-scale genomic analyses of SFTSVs improved prior knowledge of how this virus emerged and evolved in China, Japan, and South Korea. Our results highlight the importance of SFTSV surveillance in both human and non-human reservoirs at the molecular level to fight against fatal human infection with the virus.

A natural variation in the RNA polymerase of severe fever with thrombocytopenia syndrome virus enhances viral replication and in vivo virulence.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease with high mortality in Eastern Asia. The disease is caused by the SFTS virus (SFTSV), also known as Dabie bandavirus, which has a segmented RNA genome consisting of L, M, and S segments. Previous studies have suggested differential viral virulence depending on the genotypes of SFTSV; however, the critical viral factor involved in the differential viral virulence is unknown. Here, we found a significant difference in viral replication in vitro and virulence in vivo between two Korean isolates belonging to the F and B genotypes, respectively. By generating viral reassortants using the two viral strains, we demonstrated that the L segment, which encodes viral RNA-dependent RNA polymerase (RdRp), is responsible for the enhanced viral replication and virulence. Comparison of amino acid sequences and viral replication rates revealed a point variation, E251K, on the surface of RdRp to be the most significant determinant for the enhanced viral replication rate and in vivo virulence. The effect of the variation was further confirmed using recombinant SFTSV generated by reverse genetic engineering. Therefore, our results indicate that natural variations affecting the viral replicase activity could significantly contribute to the viral virulence of SFTSV.

Stabilization of RNA G-quadruplexes in the SARS-CoV-2 genome inhibits viral infection via translational suppression.

The G-quadruplex (G4) formed in single-stranded DNAs or RNAs plays a key role in diverse biological processes and is considered as a potential antiviral target. In the genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 25 putative G4-forming sequences are predicted; however, the effects of G4-binding ligands on SARS-CoV-2 replication have not been studied in the context of viral infection. In this study, we investigated whether G4-ligands suppressed SARS-CoV-2 replication and whether their antiviral activity involved stabilization of viral RNA G4s and suppression of viral gene expression. We found that pyridostatin (PDS) suppressed viral gene expression and genome replication as effectively as the RNA polymerase inhibitor remdesivir. Biophysical analyses revealed that the 25 predicted G4s in the SARS-CoV-2 genome formed a parallel G4 structure. In particular, G4-644 and G4-3467 located in the 5' region of ORF1a, formed a G4 structure that could be effectively stabilized by PDS. We also showed that PDS significantly suppressed translation of the reporter genes containing these G4s. Taken together, our results demonstrate that stabilization of RNA G4s by PDS in the SARS-CoV-2 genome inhibits viral infection via translational suppression, highlighting the therapeutic potential of G4-ligands in SARS-CoV-2 infection.

Comparison of culture-competent virus shedding duration of SARS-CoV-2 Omicron variant in regard to vaccination status: A prospective cohort study.

Previous studies have shown that fully vaccinated patients with SARS-CoV-2 Delta variants has shorter viable viral shedding period compared to unvaccinated or partially vaccinated patients. However, data about effects of vaccination against the viable viral shedding period in patients with SARS-CoV-2 Omicron variants were limited. We compared the viable viral shedding period of SARS-CoV-2 omicron variant regard to vaccination status. Saliva samples were obtained daily from patients with SARS-CoV-2 Omicron variant, and genomic assessments and virus culture was performed to those samples. We found no difference in viable viral shedding period between fully vaccinated and not or partially vaccinated, nor between 1st boostered vs non-boostered patients with SARS-CoV-2 Omicron variant.

Performance evaluation of newly developed surrogate virus neutralization tests for detecting neutralizing antibodies against SARS-CoV-2.

We evaluated newly developed surrogate virus neutralization tests (sVNT) for detecting neutralizing antibodies (NAbs) against the receptor binding domain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). VERI-Q SARS-CoV-2 Neutralizing Antibody Detection ELISA Kit (MiCo BioMed, Gyeonggi-do, Republic of Korea, hereafter, "eCoV-CN") is an enzyme-linked immunosorbent assay-based sVNT, and VERI-Q SARS-CoV-2 Neutralizing Antibody Rapid Test Kit (MiCo BioMed, hereafter, "rCoV-RN") is a point-of-care lateral-flow immunochromatography test with auto-scanner. A total of 411 serum samples were evaluated. Both evaluations used a 50% plaque reduction neutralization test (PRNT50) as the gold standard. Compared with PRNT50, the eCoV-CN showed 98.7% positive percent agreement (PPA), 96.8% negative percent agreement (NPA), 97.4% total percent agreement (TPA), with kappa values of 0.942. The rCoV-RN showed 98.7% PPA, 97.4% NPA, 97.8% TPA, and kappa values of 0.951, comparing to PRNT50. Neither assay indicated cross-reactivity for other pathogens, and the signal indexes were statistically significantly correlated to the PRNT50 titer. The two evaluated sVNTs show comparable performances to the PRNT50 with the advantages of technical simplicity, speed, and do not require cell culture facilities.

In vitro and in vivo suppression of SARS-CoV-2 replication by a modified, short, cell-penetrating peptide targeting the C-terminal domain of the viral spike protein.

Peptides are promising therapeutic agents for COVID-19 because of their specificity, easy synthesis, and ability to be fine-tuned. We previously demonstrated that a cell-permeable peptide corresponding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike C-terminal domain (CD) inhibits the interaction between viral spike and nucleocapsid proteins that results in SARS-CoV-2 replication in vitro. Here, we used docking studies to design R-t-Spike CD(D), a more potent short cell-penetrating peptide composed of all D-form amino acids and evaluated its inhibitory effect against the replication of SARS-CoV-2 S clade and other variants. R-t-Spike CD(D) was internalized into Vero cells and Calu-3 cells and suppressed the replication of SARS-CoV-2 S clade, delta variant, and omicron variant with higher potency than the original peptide. In hemizygous K18-hACE2 mice, intratracheal administration of R-t-Spike CD(D) effectively delivered the peptide to the trachea and lungs, whereas intranasal administration delivered the peptide mostly to the upper respiratory system and stomach, and a small amount to the lungs. Administration by either route reduced viral loads in mouse lungs and turbinates. Furthermore, intranasally administered R-t-Spike CD(D) mitigated pathological change in the lungs and increased the survival of mice after infection with the SARS-CoV-2 S clade or delta variant. Our data suggest that R-t-Spike CD(D) has potential as a therapeutic agent against SARS-CoV-2 infection.

Humoral and cellular immunogenicity of homologous and heterologous booster vaccination in Ad26.COV2.S-primed individuals: Comparison by breakthrough infection.

Background: Whether or not a single-dose Ad26.COV2.S prime and boost vaccination induces sufficient immunity is unclear. Concerns about the increased risk of breakthrough infections in the Ad26.COV2.S-primed population have also been raised. Methods: A prospective cohort study was conducted. Participants included healthy adults who were Ad26.COV2.S primed and scheduled to receive a booster vaccination with BNT162b2, mRNA-1273, or Ad26.COV2.S. The IgG anti-receptor binding domain (RBD) antibody titers, neutralizing antibody (NAb) titers (against wild type [WT] and Omicron [BA.1 and BA.5]), and Spike-specific interferon-γ responses of the participants were estimated at baseline, 3-4 weeks, 3 months, and 6 months after booster vaccination. Results: A total of 89 participants were recruited (26 boosted with BNT162b2, 57 with mRNA-1273, and 7 with Ad26.COV2.S). The IgG anti-RBD antibody titers of all participants were significantly higher at 6 months post-vaccination than at baseline. The NAb titers against WT at 3 months post-vaccination were 359, 258, and 166 in the participants from the BNT162b2-, mRNA-1273-, and Ad26.COV2.S-boosted groups, respectively. Compared with those against WT, the NAb titers against BA.1/BA.5 were lower by 23.9/10.9-, 16.6/7.4-, and 13.8/7.2-fold in the participants from the BNT162b2-, mRNA-1273-, and Ad26.COV2.S-boosted groups, respectively, at 3 months post-vaccination. Notably, the NAb titers against BA.1 were not boosted after Ad26.COV2.S vaccination. Breakthrough infections occurred in 53.8%, 62.5%, and 42.9% of the participants from the BNT162b2-, mRNA-1273-, and Ad26.COV2.S-boosted groups, respectively. No significant difference in humoral and cellular immunity was found between individuals with and without SARS-CoV-2 breakthrough infections. Conclusion: Booster vaccination elicited acceptable humoral and cellular immune responses in Ad26.COV2.S-primed individuals. However, the neutralizing activities against Omicron subvariants were negligible, and breakthrough infection rates were remarkably high at 3 months post-booster vaccination, irrespective of the vaccine type. A booster dose of a vaccine containing the Omicron variant antigen would be required.

Establishment of multicenter COVID-19 therapeutics preclinical test system in Republic of Korea.

Throughout the recent COVID-19 pandemic, South Korea led national efforts to develop vaccines and therapeutics for SARS-CoV-2. The project proceeded as follows: 1) evaluation system setup (including Animal Biosafety Level 3 (ABSL3) facility alliance, standardized nonclinical evaluation protocol, and laboratory information management system), 2) application (including committee review and selection), and 3) evaluation (including expert judgment and reporting). After receiving 101 applications, the selection committee reviewed pharmacokinetics, toxicity, and efficacy data and selected 32 final candidates. In the nonclinical efficacy test, we used golden Syrian hamsters and human angiotensin-converting enzyme 2 transgenic mice under a cytokeratin 18 promoter to evaluate mortality, clinical signs, body weight, viral titer, neutralizing antibody presence, and histopathology. These data indicated eight new drugs and one repositioned drug having significant efficacy for COVID-19. Three vaccine and four antiviral drugs exerted significant protective activities against SARS-CoV-2 pathogenesis. Additionally, two anti-inflammatory drugs showed therapeutic effects on lung lesions and weight loss through their mechanism of action but did not affect viral replication. Along with systematic verification of COVID-19 animal models through large-scale studies, our findings suggest that ABSL3 multicenter alliance and nonclinical evaluation protocol standardization can promote reliable efficacy testing against COVID-19, thus expediting medical product development.

Comparison of secondary attack rate and viable virus shedding between patients with SARS-CoV-2 Delta and Omicron variants: A prospective cohort study.

There are limited data comparing the transmission rates and kinetics of viable virus shedding of the Omicron variant to those of the Delta variant. We compared these rates in hospitalized patients infected with Delta and Omicron variants. We prospectively enrolled adult patients with COVID-19 admitted to a tertiary care hospital in South Korea between September 2021 and May 2022. Secondary attack rates were calculated by epidemiologic investigation, and daily saliva samples were collected to evaluate viral shedding kinetics. Genomic and subgenomic SARS-CoV-2 RNA was measured by PCR, and virus culture was performed from daily saliva samples. A total of 88 patients with COVID-19 who agreed to daily sampling and were interviewed, were included. Of the 88 patients, 48 (59%) were infected with Delta, and 34 (41%) with Omicron; a further 5 patients gave undetectable or inconclusive RNA PCR results and 1 was suspected of being coinfected with both variants. Omicron group had a higher secondary attack rate (31% [38/124] vs. 7% [34/456], p < 0.001). Survival analysis revealed that shorter viable virus shedding period was observed in Omicron variant compared with Delta variant (median 4, IQR [1-7], vs. 8.5 days, IQR [5-12 days], p < 0.001). Multivariable analysis revealed that moderate-to-critical disease severity (HR: 1.96), and immunocompromised status (HR: 2.17) were independent predictors of prolonged viral shedding, whereas completion of initial vaccine series or first booster-vaccinated status (HR: 0.49), and Omicron infection (HR: 0.44) were independently associated with shorter viable virus shedding. Patients with Omicron infections had higher transmission rates but shorter periods of transmissible virus shedding than those with Delta infections.