The Architecture of SARS-CoV-2 Transcriptome.

SARS-CoV-2 is a betacoronavirus responsible for the COVID-19 pandemic. Although the SARS-CoV-2 genome was reported recently, its transcriptomic architecture is unknown. Utilizing two complementary sequencing techniques, we present a high-resolution map of the SARS-CoV-2 transcriptome and epitranscriptome. DNA nanoball sequencing shows that the transcriptome is highly complex owing to numerous discontinuous transcription events. In addition to the canonical genomic and 9 subgenomic RNAs, SARS-CoV-2 produces transcripts encoding unknown ORFs with fusion, deletion, and/or frameshift. Using nanopore direct RNA sequencing, we further find at least 41 RNA modification sites on viral transcripts, with the most frequent motif, AAGAA. Modified RNAs have shorter poly(A) tails than unmodified RNAs, suggesting a link between the modification and the 3' tail. Functional investigation of the unknown transcripts and RNA modifications discovered in this study will open new directions to our understanding of the life cycle and pathogenicity of SARS-CoV-2.

The Association Between Antibody Responses and Prolonged Viable Severe Acute Respiratory Syndrome Coronavirus 2 Shedding in Immunocompromised Patients: A Prospective Cohort Study.

Immunocompromised patients with coronavirus disease 2019 were prospectively enrolled from March to November 2022 to understand the association between antibody responses and severe acute respiratory syndrome coronavirus 2 shedding. A total of 62 patients were analyzed, and the results indicated a faster decline in genomic and subgenomic viral RNA in patients with higher neutralizing and S1-specific immunoglobulin G (IgG) antibodies (both P < .001). Notably, high neutralizing antibody levels were associated with a significantly faster decrease in viable virus cultures (P = .04). Our observations suggest the role of neutralizing antibodies in prolonged virus shedding in immunocompromised patients, highlighting the potential benefits of enhancing their humoral immune response through vaccination or monoclonal antibody treatments.

Differential beta-coronavirus infection dynamics in human bronchial epithelial organoids.

The lower respiratory system serves as the target and barrier for beta-coronavirus (beta-CoV) infections. In this study, we explored beta-CoV infection dynamics in human bronchial epithelial (HBE) organoids, focusing on HCoV-OC43, SARS-CoV, MERS-CoV, and SARS-CoV-2. Utilizing advanced organoid culture techniques, we observed robust replication for all beta-CoVs, particularly noting that SARS-CoV-2 reached peak viral RNA levels at 72 h postinfection. Through comprehensive transcriptomic analysis, we identified significant shifts in cell population dynamics, marked by an increase in goblet cells and a concurrent decrease in ciliated cells. Furthermore, our cell tropism analysis unveiled distinct preferences in viral targeting: HCoV-OC43 predominantly infected club cells, while SARS-CoV had a dual tropism for goblet and ciliated cells. In contrast, SARS-CoV-2 primarily infected ciliated cells, and MERS-CoV showed a marked affinity for goblet cells. Host factor analysis revealed the upregulation of genes encoding viral receptors and proteases. Notably, HCoV-OC43 induced the unfolded protein response pathway, which may facilitate viral replication. Our study also reveals a complex interplay between inflammatory pathways and the suppression of interferon responses during beta-CoV infections. These findings provide insights into host-virus interactions and antiviral defense mechanisms, contributing to our understanding of beta-CoV infections in the respiratory tract.

Experimental Infection and Transmission of SARS-CoV-2 Delta and Omicron Variants among Beagle Dogs.

We assessed susceptibility of dogs to SARS-COV-2 Delta and Omicron variants by experimentally inoculating beagle dogs. Moreover, we investigated transmissibility of the variants from infected to naive dogs. The dogs were susceptible to infection without clinical signs and transmitted both strains to other dogs through direct contact.

Virological characteristics and the rapid antigen test as deisolation criteria in immunocompromised patients with COVID-19: A prospective cohort study.

There are limited data supporting current Centers for Disease Control and Prevention guidelines for the isolation period in moderate to severely immunocompromised patients with coronavirus disease 2019 (COVID-19). Adult COVID-19 patients who underwent solid organ transplantation (SOT) or received active chemotherapy against hematologic malignancy were enrolled and weekly respiratory samples were collected. Samples with positive genomic real-time polymerase chain reaction results underwent virus culture and rapid antigen testing (RAT). A total of 65 patients (40 with hematologic malignancy and 25 SOT) were enrolled. The median duration of viable virus shedding was 4 weeks (interquartile range: 3-7). Multivariable analysis revealed that B-cell depletion (hazard ratio [HR]: 4.76) was associated with prolonged viral shedding, and COVID-19 vaccination (≥3 doses) was negatively associated with prolonged viral shedding (HR: 0.22). The sensitivity, specificity, positive predictive value, and negative predictive value of RAT for viable virus shedding were 79%, 76%, 74%, and 81%, respectively. The negative predictive value of RAT was only 48% (95% confidence interval [CI]: 33-65) in the samples from those with symptom onset ≤20 days, but it was as high as 92% (95% CI: 85-96) in the samples from those with symptom onset >20 days. About half of immunocompromised COVID-19 patients shed viable virus for ≥4 weeks from the diagnosis, and virus shedding was prolonged especially in unvaccinated patients with B-cell-depleting therapy treatment. RAT beyond 20 days in immunocompromised patients had a relatively high negative predictive value for viable virus shedding.

Short-Term Effectiveness of Oral Nirmatrelvir/Ritonavir Against the SARS-CoV-2 Omicron Variant and Culture-Positive Viral Shedding.

BACKGROUND: Information on the effectiveness of nirmatrelvir/ritonavir against the omicron is limited. The clinical response and viral kinetics to therapy in the real world need to be evaluated. METHODS: Mild to moderate coronavirus disease 2019 (COVID-19) patients with risk factors for severe illness were prospectively enrolled as a treatment group with nirmatrelvir/ritonavir therapy versus a control group with supportive care. Serial viral load and culture from the upper respiratory tract were evaluated for seven days, and clinical responses and adverse reactions were evaluated for 28 days. RESULTS: A total of 51 patients were analyzed including 40 in the treatment group and 11 in the control group. Faster symptom resolution during hospitalization (P = 0.048) was observed in the treatment group. Only minor adverse reactions were reported in 27.5% of patients. The viral load on Day 7 was lower in the treatment group (P = 0.002). The viral culture showed a positivity of 67.6% (25/37) vs. 100% (6/6) on Day 1, 0% (0/37) vs. 16.7 (1/6) on Day 5, and 0% (0/16) vs. 50.0% (2/4) on Day 7 in the treatment and control groups, respectively. CONCLUSIONS: Nirmatrelvir/ritonavir against the omicron was safe and resulted in negative viral culture conversion after Day 5 of treatment with better symptomatic resolution.

Duration of Humoral Immunity and Cross-Neutralizing Activity Against the Alpha, Beta, and Delta Variants After Wild-Type Severe Acute Respiratory Syndrome Coronavirus 2 Infection: A Prospective Cohort Study.

A prospective cohort study was conducted for adults with a diagnosis of with coronavirus disease 2019 (COVID-19). Convalescent blood samples were obtained 4, 6, and 11 months after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The seropositivity of anti-spike antibody was maintained in all patients (100%) until 11 months after COVID-19 diagnosis. Neutralizing antibody levels against wild-type SARS-CoV-2 gradually decreased but remained positive in >50% of patients 11 months after diagnosis: in 98.5% (67 of 68) at 4 months, 86.8% (46 of 53) at 6 months, and 58.8% (40 of 68) at 11 months. However, cross-neutralizing activity against the Beta and Delta variants was attenuated 2.53-fold and 2.93-fold, respectively, compared with the wild-type strain.

Longitudinal Analysis of Memory T-Cell Responses in Survivors of Middle East Respiratory Syndrome.

BACKGROUND: Middle East respiratory syndrome (MERS) is a highly lethal respiratory disease caused by a zoonotic betacoronavirus. The development of effective vaccines and control measures requires a thorough understanding of the immune response to this viral infection. METHODS: We investigated cellular immune responses up to 5 years after infection in a cohort of 59 MERS survivors by performing enzyme-linked immunospot assay and intracellular cytokine staining after stimulation of peripheral blood mononuclear cells with synthetic viral peptides. RESULTS: Memory T-cell responses were detected in 82%, 75%, 69%, 64%, and 64% of MERS survivors from 1-5 years post-infection, respectively. Although the frequency of virus-specific interferon gamma (IFN-γ)-secreting T cells tended to be higher in moderately/severely ill patients than in mildly ill patients during the early period of follow-up, there was no significant difference among the different clinical severity groups across all time points. While both CD4+ and CD8+ T cells were involved in memory T-cell responses, CD4+ T cells persisted slightly longer than CD8+ T cells. Both memory CD4+ and CD8+ T cells recognized the E/M/N proteins better than the S protein and maintained their polyfunctionality throughout the period examined. Memory T-cell responses correlated positively with antibody responses during the initial 3-4 years but not with maximum viral loads at any time point. CONCLUSIONS: These findings advance our understanding of the dynamics of virus-specific memory T-cell immunity after MERS-coronavirus infection, which is relevant to the development of effective T cell-based vaccines.

Factors Associated With Viral Load Kinetics of Middle East Respiratory Syndrome Coronavirus During the 2015 Outbreak in South Korea.

We conducted a retrospective study of Middle East respiratory syndrome coronavirus (MERS-CoV) viral load kinetics using data from patients hospitalized with MERS-CoV infection between 19 May and 20 August 2015. Viral load trajectories were considered over the hospitalization period using 1714 viral load results measured in serial respiratory specimens of 185 patients. The viral load levels were significantly higher among nonsurvivors than among survivors (P = .003). Healthcare workers (P = .001) and nonspreaders (P < .001) had significantly lower viral loads. Viral RNA was present on the day of symptom onset and peaked 4-10 days after symptom onset.

Longitudinal Assessment of Antisevere Acute Respiratory Syndrome Coronavirus 2 Immune Responses for Six Months Based on the Clinical Severity of Coronavirus Disease 2019.

BACKGROUND: There is insufficient data on the longevity of immunity acquired after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: We aimed to evaluate the duration of SARS-CoV-2-specific humoral and cellular immunity according to the clinical severity of coronavirus disease 2019 (COVID-19). The study population comprised asymptomatic (n = 14), symptomatic/nonpneumonic (n = 42), and pneumonic (n = 41) patients. RESULTS: The anti-SARS-CoV-2 immunoglobulin class G and neutralizing antibody (NAb) titers lasted until 6 months after diagnosis, with positivity rates of 66.7% and 86.9%, respectively. Older age, prolonged viral shedding, and accompanying pneumonia were more frequently found in patients with sustained humoral immunity. Severe acute respiratory syndrome coronavirus 2-specific T-cell response was strongly observed in pneumonic patients and prominent in individuals with sustained humoral immunity. CONCLUSIONS: In conclusion, most (>85%) patients carry NAb until 6 months after diagnosis of SARS-CoV-2 infection, providing insights for establishing vaccination strategies against COVID-19.

Sustained Responses of Neutralizing Antibodies Against Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in Recovered Patients and Their Therapeutic Applicability.

BACKGROUND: Zoonotic coronaviruses have emerged as a global threat by causing fatal respiratory infections. Given the lack of specific antiviral therapies, application of human convalescent plasma retaining neutralizing activity could be a viable therapeutic option that can bridges this gap. METHODS: We traced antibody responses and memory B cells in peripheral blood collected from 70 recovered Middle East respiratory syndrome coronavirus (MERS-CoV) patients for 3 years after the 2015 outbreak in South Korea. We also used a mouse infection model to examine whether the neutralizing activity of collected sera could provide therapeutic benefit in vivo upon lethal MERS-CoV challenge. RESULTS: Anti-spike-specific IgG responses, including neutralizing activity and antibody-secreting memory B cells, persisted for up to 3 years, especially in MERS patients who suffered from severe pneumonia. Mean antibody titers gradually decreased annually by less than 2-fold. Levels of antibody responses were significantly correlated with fever duration, viral shedding periods, and maximum viral loads observed during infection periods. In a transgenic mice model challenged with lethal doses of MERS-CoV, a significant reduction in viral loads and enhanced survival was observed when therapeutically treated with human plasma retaining a high neutralizing titer (> 1/5000). However, this failed to reduce pulmonary pathogenesis, as revealed by pathological changes in lungs and initial weight loss. CONCLUSIONS: High titers of neutralizing activity are required for suppressive effect on the viral replication but may not be sufficient to reduce inflammatory lesions upon fatal infection. Therefore, immune sera with high neutralizing activity must be carefully selected for plasma therapy of zoonotic coronavirus infection.

Risk factors for transmission of Middle East respiratory syndrome coronavirus infection during the 2015 outbreak in South Korea.

Background: Transmission heterogeneity was observed during the 2015 South Korean outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Only 22 of 186 cases transmitted the infection, and 5 super-spreading events caused 150 transmissions. We investigated the risk factors for MERS-CoV transmission. Methods: Epidemiological reports were used to classify patients as non-spreaders, spreaders (1-4 transmission), or those associated with super-spreading event (≥4 transmissions). Logistic regression analyses were used to evaluate the factors that influenced MERS-CoV transmission. Results: Compared to non-spreaders, spreaders exhibited a longer interval from symptom onset to isolation (7 days vs. 3 days) and more frequent pre-isolation pneumonia diagnoses (68.2% vs. 17.1%). Spreaders also exhibited higher values for pre-isolation contacts (149 vs. 17.5), pre-isolation hospitalization (68.2% vs. 16.5%), and emergency room visits (50% vs. 7.3%). Spreaders exhibited lower cycle thresholds for the upE and ORF1a genes (22.7 vs. 27.2 and 23.7 vs. 27.9, respectively). Transmission was independently associated with the cycle threshold (odds ratio [OR]: 0.84, 95% confidence interval [CI]: 0.75-0.96) and pre-isolation hospitalization or emergency room visits (OR: 6.82, 95% CI: 2.06-22.84). The spreaders with ≥4 transmissions exhibited higher values for pre-isolation contacts (777 vs. 78), pre-isolation emergency room visits (100% vs. 35.3%), and doctor-shopping (100% vs. 47.1%), compared to other spreaders. Conclusions: These findings indicate that transmission is determined by host infectivity and the number of contacts, whereas super-spreading events were determined by the number of contacts and hospital visits. These relationships highlight the importance of rapidly enforcing infection control measures to prevent outbreaks.

Viral RNA in Blood as Indicator of Severe Outcome in Middle East Respiratory Syndrome Coronavirus Infection.

We evaluated the diagnostic and clinical usefulness of blood specimens to detect Middle East respiratory syndrome coronavirus infection in 21 patients from the 2015 outbreak in South Korea. Viral RNA was detected in blood from 33% of patients at initial diagnosis, and the detection preceded a worse clinical course.

Variations in Spike Glycoprotein Gene of MERS-CoV, South Korea, 2015.

An outbreak of nosocomial infections with Middle East respiratory syndrome coronavirus occurred in South Korea in May 2015. Spike glycoprotein genes of virus strains from South Korea were closely related to those of strains from Riyadh, Saudi Arabia. However, virus strains from South Korea showed strain-specific variations.

Genetic characteristics of mumps viruses isolated in Korea from 2007 to 2012.

Mumps is a vaccine-preventable viral disease. Despite vaccine coverage of >95%, the incidence of mumps has increased in Korea since 2007. This study aimed to genetically characterize mumps virus (MuV) strains that circulated in Korea between 2007 and 2012 to determine the factors underlying mumps outbreaks. MuV was isolated from 175 clinical specimens between 2007 and 2012 in Korea. Upon analysis of the SH gene in Korean mumps virus isolates, three different genotypes were identified: I, H, and F. The MuV genotypes I and H co-circulated in Korea, and eight isolates of Korean genotype F were found within the same time period in 2008. An analysis of HN amino-acid sequence data showed that Korean isolates had no changes in their glycosylation sites. At putative neutralizing epitope sites, the Jeryl-Lynn strain showed 4-5 different amino acid sequences from those observed in Korean isolates. Korean isolates of genotypes I and H shared distinctive point mutations on putative neutralizing epitope positions in each genotype. This report describes the genetic characteristics of MuV strains circulating in Korea and provides information on endemic mumps infections. This information may be important to help prevent mumps and control outbreaks of mumps in Korea. J. Med. Virol. 88:1479-1486, 2016. © 2016 Wiley Periodicals, Inc.

Middle East Respiratory Syndrome in 3 Persons, South Korea, 2015.

In May 2015, Middle East respiratory syndrome coronavirus infection was laboratory confirmed in South Korea. Patients were a man who had visited the Middle East, his wife, and a man who shared a hospital room with the index patient. Rapid laboratory confirmation will facilitate subsequent prevention and control for imported cases.

The host protease KLK5 primes and activates spike proteins to promote human betacoronavirus replication and lung inflammation.

Coronaviruses rely on host proteases to activate the viral spike protein, which facilitates fusion with the host cell membrane and the release of viral genomic RNAs into the host cell cytoplasm. The distribution of specific host proteases in the host determines the host, tissue, and cellular tropism of these viruses. Here, we identified the kallikrein (KLK) family member KLK5 as a major host protease secreted by human airway cells and exploited by multiple human betacoronaviruses. KLK5 cleaved both the priming (S1/S2) and activation (S2') sites of spike proteins from various human betacoronaviruses in vitro. In contrast, KLK12 and KLK13 displayed preferences for either the S2' or S1/S2 site, respectively. Whereas KLK12 and KLK13 worked in concert to activate SARS-CoV-2 and MERS-CoV spike proteins, KLK5 by itself efficiently activated spike proteins from several human betacoronaviruses, including SARS-CoV-2. Infection of differentiated human bronchial epithelial cells (HBECs) with human betacoronaviruses induced an increase in KLK5 that promoted virus replication. Furthermore, ursolic acid and other related plant-derived triterpenoids that inhibit KLK5 effectively suppressed the replication of SARS-CoV, MERS-CoV, and SARS-CoV-2 in HBECs and mitigated lung inflammation in mice infected with MERS-CoV or SARS-CoV-2. We propose that KLK5 is a pancoronavirus host factor and a promising therapeutic target for current and future coronavirus-induced diseases.

Generation of a lethal mouse model expressing human ACE2 and TMPRSS2 for SARS-CoV-2 infection and pathogenesis.

Mouse models expressing human ACE2 for coronavirus disease 2019 have been frequently used to understand its pathogenesis and develop therapeutic strategies against SARS-CoV-2. Given that human TMPRSS2 supports viral entry, replication, and pathogenesis, we established a double-transgenic mouse model expressing both human ACE2 and TMPRSS2 for SARS-CoV-2 infection. Co-overexpression of both genes increased viral infectivity in vitro and in vivo. Double-transgenic mice showed significant body weight loss, clinical disease symptoms, acute lung injury, lung inflammation, and lethality in response to viral infection, indicating that they were highly susceptible to SARS-CoV-2. Pretreatment with the TMPRSS2 inhibitor, nafamostat, effectively reduced virus-induced weight loss, viral replication, and mortality in the double-transgenic mice. Moreover, the susceptibility and differential pathogenesis of SARS-CoV-2 variants were demonstrated in this animal model. Together, our results demonstrate that double-transgenic mice could provide a highly susceptible mouse model for viral infection to understand SARS-CoV-2 pathogenesis and evaluate antiviral therapeutics against coronavirus disease 2019.

Rise in broadly cross-reactive adaptive immunity against human ß-coronaviruses in MERS-recovered patients during the COVID-19 pandemic.

To develop a universal coronavirus (CoV) vaccine, long-term immunity against multiple CoVs, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, Middle East respiratory syndrome (MERS)-CoV, and future CoV strains, is crucial. Following the 2015 Korean MERS outbreak, we conducted a long-term follow-up study and found that although neutralizing antibodies and memory T cells against MERS-CoV declined over 5 years, some recovered patients exhibited increased antibody levels during the COVID-19 pandemic. This likely resulted from cross-reactive immunity induced by SARS-CoV-2 vaccines or infections. A significant correlation in antibody responses across various CoVs indicates shared immunogenic epitopes. Two epitopes-the spike protein's stem helix and intracellular domain-were highly immunogenic after MERS-CoV infection and after SARS-CoV-2 vaccination or infection. In addition, memory T cell responses, especially polyfunctional CD4+ T cells, were enhanced during the pandemic, correlating significantly with MERS-CoV spike-specific antibodies and neutralizing activity. Therefore, incorporating these cross-reactive and immunogenic epitopes into pan-CoV vaccine formulations may facilitate effective vaccine development.

Evaluation of antiviral drugs against newly emerged SARS-CoV-2 Omicron subvariants.

Ongoing emergence of SARS-CoV-2 Omicron subvariants and their rapid worldwide spread pose a threat to public health. From November 2022 to February 2023, newly emerged Omicron subvariants, including BQ.1.1, BF.7, BA.5.2, XBB.1, XBB.1.5, and BN.1.9, became prevalent global strains (>5% global prevalence). These Omicron subvariants are resistant to several therapeutic antibodies. Thus, the antiviral activity of current drugs such as remdesivir, molnupiravir, and nirmatrelvir, which target highly conserved regions of SARS-CoV-2, against newly emerged Omicron subvariants need to be evaluated. We assessed the antiviral efficacy of the drugs using the half-maximal inhibitory concentration (IC50) against human isolates of 23 Omicron subvariants and four former SARS-CoV-2 variants of concern (VOCs) and compared it with the antiviral efficacy of these drugs against the SARS-CoV-2 reference strain (hCoV/Korea/KCDC03/2020). Maximal IC50-fold changes of remdesivir, molnupiravir, and nirmatrelvir were 1.9 (BA.2.75.2), 1.2 (B.1.627.2), and 1.4 (BA.2.3), respectively, compared to median IC50 values of the reference strain. Moreover, median IC50-fold changes of remdesivir, molnupiravir, and nirmatrelvir against the Omicron variants were 0.96, 0.4, and 0.62, respectively, similar to the 1.02, 0.88, and 0.67, respectively, median IC50-fold changes for previous VOCs. Although K90R and P132H in Nsp 5, and P323L, A529V, G671S, V405F, and ins823D in Nsp 12 mutations were identified, these amino acid substitutions did not affect drug antiviral activity. These results indicate that current antivirals retain antiviral efficacy against newly emerged Omicron subvariants. It is important to continue active surveillance and testing of new variants for drug resistance to enable early identification of drug-resistant strains.