Evaluating Z-FA-FMK, a host cathepsin L protease inhibitor, as a potent and broad-spectrum antiviral therapy against SARS-CoV-2 and related coronaviruses.

Even though the World Health Organization announced the end of the COVID-19 pandemic as a global public health emergency on May 5, 2023, SARS-CoV-2 continues to pose a significant health threat worldwide, resulting in substantial numbers of infections and fatalities. This study investigated the antiviral potential of Z-FA-FMK (FMK), a novel host cathepsin L protease inhibitor, against SARS-CoV-2 infection using both in vitro and in vivo models. In vitro assessments of FMK against a diverse set of SARS-CoV-2 strains, including the Wuhan-like strain and nine variants, demonstrated potent inhibition with EC50 values ranging from 0.55 to 2.41 µM, showcasing similar or superior efficacy compared to FDA-approved antivirals nirmatrelvir (NTV) and molnupiravir (MPV). In vivo experiments using orally administered FMK (25 mg/kg) in SARS-CoV-2-infected K18 hACE2 transgenic mice revealed improved survival rates of 60% and accelerated recovery compared to NTV and MPV treatments. Additionally, FMK displayed a longer half-life (17.26 ± 8.89 h) than NTV and MPV in the mouse model. Due to its host-targeting mechanism, FMK offers potential advantages such as reduced drug resistance and broad-spectrum antiviral activity against multiple coronaviruses. These findings indicate that FMK may serve as a promising candidate for further clinical evaluation in the fight against SARS-CoV-2.

Reciprocal enhancement of SARS-CoV-2 and influenza virus replication in human pluripotent stem cell-derived lung organoids1.

Patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (FLUAV) coinfections were associated with severe respiratory failure and more deaths. Here, we developed a model for studying SARS-CoV-2 and FLUAV coinfection using human pluripotent stem cell-induced alveolar type II organoids (hiAT2). hiAT2 organoids were susceptible to infection by both viruses and had features of severe lung damage. A single virus markedly enhanced the susceptibility to other virus infections. SARS-CoV-2 delta variants upregulated α-2-3-linked sialic acid, while FLUAV upregulated angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). Moreover, coinfection by SARS-CoV-2 and FLUAV caused hyperactivation of proinflammatory and immune-related signaling pathways and cellular damage compared to a respective single virus in hiAT2 organoids. This study provides insight into molecular mechanisms underlying enhanced infectivity and severity in patients with co-infection of SARS-CoV-2 and FLUAV, which may aid in the development of therapeutics for such co-infection cases.

Combination therapy with nirmatrelvir and molnupiravir improves the survival of SARS-CoV-2 infected mice.

As the SARS-CoV-2 pandemic remains uncontrolled owing to the continuous emergence of variants of concern, there is an immediate need to implement the most effective antiviral treatment strategies, especially for risk groups. Here, we evaluated the therapeutic potency of nirmatrelvir, remdesivir and molnupiravir, and their combinations in SARS-CoV-2 infected K18-hACE2 transgenic mice. Systemic treatment of mice with each drug (20 mg/kg) resulted in slightly enhanced antiviral efficacy and yielded an increased life expectancy of only about 20-40% survival. However, combination therapy with nirmatrelvir (20 mg/kg) and molnupiravir (20 mg/kg) in lethally infected mice showed profound inhibition of SARS-CoV-2 replication in both the lung and brain and synergistically improved survival rates up to 80% compared to those with nirmatrelvir (36%, P < 0.001) and molnupiravir (43%, P < 0.001) administered alone. This combination therapy effectively reduced clinical severity score, virus-induced tissue damage, and viral distribution compared to those in animals treated with these monotherapies. Furthermore, all these assessments associated with this combination were also significantly higher than that of mice receiving remdesivir monotherapy (P < 0.001) and the nirmatrelvir (20 mg/kg) and remdesivir (20 mg/kg) combination (P < 0.001), underscored the clinical significance of this combination. By contrast, the nirmatrelvir and remdesivir combination showed less antiviral efficacy, with lower survival compared to nirmatrelvir monotherapy due to the insufficient plasma exposure of the remdesivir, demonstrating the inefficient therapeutic effect of this combination in the mouse model. The combination therapy with nirmatrelvir and molnupiravir contributes to alleviated morbidity and mortality, which can serve as a basis for the design of clinical studies of this combination in the treatment of COVID-19 patients.

SARS-CoV-2 spike trimer vaccine expressed in Nicotiana benthamiana adjuvanted with Alum elicits protective immune responses in mice.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has spurred rapid development of vaccines as part of the public health response. However, the general strategy used to construct recombinant trimeric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) proteins in mammalian cells is not completely adaptive to molecular farming. Therefore, we generated several constructs of recombinant S proteins for high expression in Nicotiana benthamiana. Intramuscular injection of N. benthamiana-expressed Sct vaccine (NSct Vac) into Balb/c mice elicited both humoral and cellular immune responses, and booster doses increased neutralizing antibody titres. In human angiotensin-converting enzyme knock-in mice, two doses of NSct Vac induced anti-S and neutralizing antibodies, which cross-neutralized Alpha, Beta, Delta and Omicron variants. Survival rates after lethal challenge with SARS-CoV-2 were up to 80%, without significant body weight loss, and viral titres in lung tissue fell rapidly, with no infectious virus detectable at 7-day post-infection. Thus, plant-derived NSct Vac could be a candidate COVID-19 vaccine.

An infectious cDNA clone of a growth attenuated Korean isolate of MERS coronavirus KNIH002 in clade B.

The MERS-CoV isolated during the 2015 nosocomial outbreak in Korea showed distinctive differences in mortality and transmission patterns compared to the prototype MERS-CoV EMC strain belonging to clade A. We established a BAC-based reverse genetics system for a Korean isolate of MERS-CoV KNIH002 in the clade B phylogenetically far from the EMC strain, and generated a recombinant MERS-CoV expressing red fluorescent protein. The virus rescued from the infectious clone and KNIH002 strain displayed growth attenuation compared to the EMC strain. Consecutive passages of the rescued virus rapidly generated various ORF5 variants, highlighting its genetic instability and calling for caution in the use of repeatedly passaged virus in pathogenesis studies and for evaluation of control measures against MERS-CoV. The infectious clone for the KNIH002 in contemporary epidemic clade B would be useful for better understanding of a functional link between molecular evolution and pathophysiology of MERS-CoV by comparative studies with EMC strain.

Antiviral activity of digoxin and ouabain against SARS-CoV-2 infection and its implication for COVID-19.

The current coronavirus (COVID-19) pandemic is exacerbated by the absence of effective therapeutic agents. Notably, patients with COVID-19 and comorbidities such as hypertension and cardiac diseases have a higher mortality rate. An efficient strategy in response to this issue is repurposing drugs with antiviral activity for therapeutic effect. Digoxin (DIG) and ouabain (OUA) are FDA drugs for heart diseases that have antiviral activity against several coronaviruses. Thus, we aimed to assess antiviral activity of DIG and OUA against SARS-CoV-2 infection. The half-maximal inhibitory concentrations (IC50) of DIG and OUA were determined at a nanomolar concentration. Progeny virus titers of single-dose treatment of DIG, OUA and remdesivir were approximately 103-, 104- and 103-fold lower (> 99% inhibition), respectively, than that of non-treated control or chloroquine at 48 h post-infection (hpi). Furthermore, therapeutic treatment with DIG and OUA inhibited over 99% of SARS-CoV-2 replication, leading to viral inhibition at the post entry stage of the viral life cycle. Collectively, these results suggest that DIG and OUA may be an alternative treatment for COVID-19, with potential additional therapeutic effects for patients with cardiovascular disease.

Characterization of Severe Acute Respiratory Syndrome Coronavirus 2 Stability in Multiple Water Matrices.

The advent of the global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) necessitates a thorough study of the stability and transmissibility in the environment. We characterized the stability of SARS-CoV-2 in three water matrices: fresh, tap, and seawater. The minimum infective dose of SARS-CoV-2 in Vero cells was confirmed to be 10³ PFU/mL. The stability of SARS-CoV-2 varied according to the water matrix: infective SARS-CoV-2 was undetectable after treatment with fresh water and seawater, but remained detectable for 2 days in tap water, when starting with an initial concentration of 104 PFU/mL. When the starting concentration was increased to 105 PFU/mL, a similar trend was observed. In addition, viral RNA persisted longer than infectious virus in all water matrices. This study was conducted in stagnant water containing a significantly high titer of virus, thus, human-to-human transmission of SARS-CoV-2 through the actual aquatic environment is expected to be rare.

Characterization of a human monoclonal antibody generated from a B-cell specific for a prefusion-stabilized spike protein of Middle East respiratory syndrome coronavirus.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory infection and continues to infect humans, thereby contributing to a high mortality rate (34.3% in 2019). In the absence of an available licensed vaccine and antiviral agent, therapeutic human antibodies have been suggested as candidates for treatment. In this study, human monoclonal antibodies were isolated by sorting B cells from patient's PBMC cells with prefusion stabilized spike (S) probes and a direct immunoglobulin cloning strategy. We identified six receptor-binding domain (RBD)-specific and five S1 (non-RBD)-specific antibodies, among which, only the RBD-specific antibodies showed high neutralizing potency (IC50 0.006-1.787 µg/ml) as well as high affinity to RBD. Notably, passive immunization using a highly potent antibody (KNIH90-F1) at a relatively low dose (2 mg/kg) completely protected transgenic mice expressing human DPP4 against MERS-CoV lethal challenge. These results suggested that human monoclonal antibodies isolated by using the rationally designed prefusion MERS-CoV S probe could be considered potential candidates for the development of therapeutic and/or prophylactic antiviral agents for MERS-CoV human infection.

The effect of mutations derived from mouse-adapted H3N2 seasonal influenza A virus to pathogenicity and host adaptation.

Elucidating the genetic basis of influenza A viruses (IAVs) is important to understand which mutations will determine the virulence and the host range of mammals. Here, seasonal H3N2 influenza was adapted in mice by serial passage and four mutants, each carrying amino acid substitutions related to mouse adaptation in either the PB2, HA, NP, or NA protein, were generated. To confirm the contribution of each gene to enhanced pathogenicity and mouse adaptation, mice were inoculated with the respective variants, and virulence, replication, histopathology, and infectivity were examined. The virus harboring HA mutations displayed increased infection efficiency and replication competence, resulting in higher mortality in mice relative to those infected with wild-type virus. By contrast, the NP D34N mutation caused rapid and widespread infection in multiple organs without presenting virulent symptoms. Additionally, the PB2 F323L mutation presented delayed but elevated replication competence in the respiratory tract, whereas the S331R mutation in NA showed no considerable effects on mouse adaptation. These results suggested that mouse-adapted changes in HA are major factors in increased pathogenicity and that mutations in NP and PB2 also contribute to cross-species adaptability. Our findings offer a better understanding of the molecular basis for IAV pathogenicity and adaptation in a new host.

Drug Discovery Platform Targeting M. tuberculosis with Human Embryonic Stem Cell-Derived Macrophages.

A major limitation in anti-tuberculosis drug screening is the lack of reliable and scalable models for homogeneous human primary macrophage cells of non-cancer origin. Here we report a modified protocol for generating homogeneous populations of macrophage-like cells from human embryonic stem cells. The induced macrophages, referred to as iMACs, presented similar transcriptomic profiles and characteristic immunological features of classical macrophages and were permissive to viral and bacterial infection, in particular Mycobacterium tuberculosis (Mtb). More importantly, iMAC production was amenable to scale up. To evaluate iMAC efficiency in high-throughput anti-tuberculosis drug screening, we performed a phenotypic screening against intracellular Mtb, involving a library of 3,716 compounds that included FDA-approved drugs and other bioactive compounds. Our primary screen identified 120 hits, which were validated in a secondary screen by dose-intracellular and -extracellular Mtb assays. Our confirmatory studies identified a novel anti-Mtb compound, 10-DEBC, also showing activity against drug-resistant strains.

Erratum: Seasonal Cycle and Relationship of Seasonal Rhino- and Influenza Virus Epidemics With Episodes of Asthma Exacerbation in Different Age Groups.

This corrects the article on p. 517 in vol. 9, PMID: 28913991.

Nation-wide surveillance of human acute respiratory virus infections between 2013 and 2015 in Korea.

The prevalence of eight respiratory viruses detected in patients with acute respiratory infections (ARIs) in Korea was investigated through analysis of data recorded by the Korea Influenza and Respiratory Viruses Surveillance System (KINRESS) from 2013 to 2015. Nasal aspirate and throat swabs specimens were collected from 36 915 patients with ARIs, and viral nucleic acids were detected by real-time (reverse-transcription) polymerase chain reaction for eight respiratory viruses, including human respiratory syncytial viruses (HRSVs), influenza viruses (IFVs), human parainfluenza viruses (HPIVs), human coronaviruses (HCoVs), human rhinovirus (HRV), human adenovirus (HAdV), human bocavirus (HBoV), and human metapneumovirus (HMPV). The overall positive rate of patient specimens was 49.4% (18 236/36 915), 5% of which carried two or more viruses simultaneously. HRV (15.6%) was the most predominantly detected virus, followed by IFVs (14.6%), HAdV (7.5%), HPIVs (5.8%), HCoVs (4.2%), HRSVs (3.6%), HBoV (1.9%), and HMPV (1.6%). Most of the ARIs were significantly correlated with clinical symptoms of fever, cough, and runny nose. Although HRV and HAdV were frequently detected throughout the year in patients, other respiratory viruses showed apparent seasonality. HRSVs and IFVs were the major causative agents of acute respiratory diseases in infants and young children. Overall, this study demonstrates a meaningful relationship between viral infection and typical manifestations of known clinical features as well as seasonality, age distribution, and co-infection among respiratory viruses. Therefore, these data could provide useful information for public health management and to enhance patient care for primary clinicians.

Seasonal Cycle and Relationship of Seasonal Rhino- and Influenza Virus Epidemics With Episodes of Asthma Exacerbation in Different Age Groups.

PURPOSE: Seasonal variations in asthma exacerbation (AE) are associated with respiratory virus outbreaks and the return of children to school after vacation. This study aims to elucidate the period, phase, and amplitude of seasonal cycles of AE in 5 different age groups with regard to rhino- and influenza virus epidemics in Korea. METHODS: The number of daily emergency department (ED) visits for AE in all age groups of Korea and the nationwide weekly incidence of rhino- and influenza virus, were obtained for 2008-2012. Fourier regression was used to model rhythmicity, and the Cosinor method was used to determine the amplitude and phase of the cycles in each age group. The cross-correlation function (CCF) between AE and the rhino- and influenza virus epidemics was also calculated. RESULTS: There were 157,559 events of AE (0.62 events/1,000 individuals/year) during the study period. There were spring and fall peaks of AE in children and adults, but only 1 winter peak in the elderly. The amplitude of the AE peak in infants was higher in spring than in fall (9.16 vs 3.04, P<0.010), and the fall peak was approximately 1 month later in infants than in school children (October 11 vs November 13, P<0.010). The association between AE and rhinovirus was greatest in school children (rho=0.331), and the association between AE and influenza virus was greatest in those aged ≥60 years (rho=0.682). CONCLUSIONS: The rhythmicity, amplitude, and phase of the annual cycle of AE differed among different age groups. The patterns of AE were related to the annual rhino- and influenza virus epidemics.

Oseltamivir-resistant influenza viruses isolated in South Korea from 2005 to 2010.

South Korean isolates of oseltamivir-resistant influenza viruses from 2005-2010 were investigated with a total 491 influenza viruses identified from 1702 specimens. Neuraminidase genes from 342 influenza viruses (71 A/H1N1, 74 pandemic A/H1N1 2009, 117 A/H3N2, and 80 B) were analyzed by RT-PCR with molecular markers for oseltamivir resistance. The H274Y mutation in the NA protein was identified in 100 % (n=40) of A/H1N1 viruses circulating in 2008-2009. Influenza A/H1N1 viruses harboring the H274Y substitution exhibited, on average, a 626-fold reduction in oseltamivir susceptibility and clustered with the A/Norway/1736/2007 strain. Close and timely monitoring for resistance to clinically available influenza antivirals should be consistently performed.

Development and evaluation of multiplex real-time RT-PCR assays for seasonal, pandemic A/H1pdm09 and avian A/H5 influenza viruses detection.

Since the pandemic influenza A (H1N1) 2009 ((H1N1)pdm09) virus spread all over the world, the (H1N1)pdm09 virus has been circulating with seasonal influenza viruses. We developed rapid and sensitive one-step multiplex real-time RT-PCR assays (rRT-PCR) for simultaneous detection of influenza viruses currently circulating in humans, and the avian A/H5 virus. The detection limit of each assay was 4.8 to 1 copies per reaction and no cross-reactivity with other major respiratory pathogens was found. Analytical positive predictive value (PPV), negative predictive value (NPV) sensitivity and specificity were 100%, 94.1%, 93.7% and 100%, respectively. Clinical evaluation revealed that 1,976 (16.5%) of 11,963 throat swabs from patients with respiratory symptoms were confirmed as 1,651 (83.6%) A/H1pdm09, 308 (15.6%) A/H3 and 17 (0.8%) B virus during the 2010-2011 influenza season. Collectively, the multiplex rRT-PCR assays described here provide a practical tool for reliable implementation of influenza surveillance and diagnosis.

Impact of influenza virus escape-mutations on influenza detection by the rapid influenza diagnostic test.

During the influenza pandemic of 2009-2010, rapid influenza diagnostic tests (RIDTs) were used to detect influenza viral infections because they are quick and simple to use. However, retrospective studies showed that RIDTs performed poorly when used to diagnose pandemic viral infections. Determining how amino acid sequence changes in pandemic or epidemic influenza viral antigens impact clinical value of RIDTs has not been possible, because the viral epitopes recognized by RIDTs have been not mapped. In this study, the effect of escape-variations or mutations in influenza viral antigens upon the sensitivity and specificity of an RIDT was investigated by characterizing the immunological properties of the antibodies used in the RIDT. Escape-mutants were generated by cultivating A/Korea/01/2009 in the presence of an excess of the same antibodies used in the RIDT. Escape-mutants not recognized by the RIDT were selected. Epitopes recognized by the RIDT were mapped by comparing the sequence and immunological analysis of the escape-variants and wild-type isolates. The RIDT antibodies recognized epitopes on the Sa antigenic site and in the F subdomain in hemagglutinin. Variants bearing mutations in these epitopes were not detected by the RIDT. The frequency of escape-variants emerging since the 2009-2010 pandemic was calculated as 1.27% using in silico surveillance of influenza sequence databases. These results suggest that mapping the relevant epitopes of RIDTs and making such information available to clinics would be helpful for determining whether RIDTs match newly emergent strains and subtypes prior to retrospective re-evaluation of the RIDTs using clinical specimens.

High prevalence of amantadine-resistant influenza A virus isolated in Gyeonggi Province, South Korea, during 2005-2010.

Amantadine resistance among influenza A viruses was investigated in South Korea in 2005-2010. Of 308 influenza A viruses examined, 229 had the S31N substitution in the M2 protein. The frequency of amantadine resistance was 30 %, 100 %, and 76 % in influenza A/H1N1, pandemic A/H1N1 2009(A/H1N1pdm), and A/H3N2 subtypes, respectively. The amantadine-resistant influenza A/H1N1pdm and A/H3N2 viruses were circulating continuously from 2008 to 2009 and from 2005 to 2006, respectively. Amantadine resistance among influenza A viruses increased dramatically during the 5-year study period, and this has diminished the usefulness of this class of drugs.

Avian influenza a (H5N1) virus antibodies in poultry cullers, South Korea, 2003-2004.

Transmission of influenza (H5N1) virus from birds to humans is a serious public health threat. In South Korea, serologic investigation among 2,512 poultry workers exposed during December 2003-March 2004 to poultry with confirmed or suspected influenza (H5N1) virus infection found antibodies in 9. Frequency of bird-to-human transmission was low.

Oseltamivir-resistant pandemic (H1N1) 2009 virus, South Korea.

To identify oseltamivir resistance, we analyzed neuraminidase H275Y mutations in samples from 10 patients infected with pandemic (H1N1) 2009 virus in South Korea who had influenza that was refractory to antiviral treatment with this drug. A neuraminidase I117M mutation that might influence oseltamivir susceptibility was detected in sequential specimens from 1 patient.

Replication and pathogenesis of the pandemic (H1N1) 2009 influenza virus in mammalian models.

This study aimed to characterize the replication and pathogenic properties of a Korean pandemic (H1N1) 2009 influenza virus isolate in ferrets and mice. Ferrets infected with A/Korea/01/2009 (H1N1) virus showed mild clinical signs. The virus replicated well in lungs and slightly in brains with no replication in any other organs. Severe bronchopneumonia and thickening of alveolar walls were detected in the lungs. Viral antigens were detected in the bronchiolar epithelial cells, in peribronchial glands with severe peribronchitis and in cells present in the alveoli. A/Korea/01/2009 (H1N1) virus-infected mice showed weight loss and pathological lung lesions including perivascular cuffing, interstitial pneumonia and alveolitis. The virus replicated highly in the lungs and slightly in the nasal tissues. Viral antigens were detected in bronchiolar epithelial cells, pneumocytes and interstitial macrophages. However, seasonal H1N1 influenza virus did not replicate in the lungs of ferrets, and viral antigens were not detected. Thus, this Korean pandemic (H1N1) 2009 isolate infected the lungs of ferrets and mice successfully and caused more pathological lesions than did the seasonal influenza virus.