Comparison of the pathogenesis of SARS-CoV-2 infection in K18-hACE2 mouse and Syrian golden hamster models.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, causes life-threatening disease. This novel coronavirus enters host cells via the respiratory tract, promoting the formation of severe pulmonary lesions and systemic disease. Few animal models can simulate the clinical signs and pathology of COVID-19 patients. Diverse preclinical studies using K18-hACE2 mice and Syrian golden hamsters, which are highly permissive to SARS-CoV-2 in the respiratory tract, are emerging; however, the systemic pathogenesis and cellular tropism of these models remain obscure. We intranasally infected K18-hACE2 mice and Syrian golden hamsters with SARS-CoV-2, and compared the clinical features, pathogenesis, cellular tropism and infiltrated immune-cell subsets. In K18-hACE2 mice, SARS-CoV-2 persistently replicated in alveolar cells and caused pulmonary and extrapulmonary disease, resulting in fatal outcomes. Conversely, in Syrian golden hamsters, transient SARS-CoV-2 infection in bronchial cells caused reversible pulmonary disease, without mortality. Our findings provide comprehensive insights into the pathogenic spectrum of COVID-19 using preclinical models.

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.

Evaluation of a biotin-based surrogate virus neutralization test for detecting postvaccination antibodies against SARS-CoV-2 variants in sera.

A severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) surrogate virus neutralization test (sVNT) was used to determine the degree of inhibition of binding between human angiotensin converting enzyme 2 (hACE2) and the receptor binding domain (RBD) of spike protein by neutralizing antibodies in a biosafety level 2 facility. Here, to improve the sensitivity and specificity of the commercial sVNT, we developed a new biotin based sVNT using biotinylated RBD and HRP conjugated streptavidin instead of HRP conjugated RBD for direct detection in an ELISA assay that strongly correlated to the FDA approved cPass sVNT commercial kit (R2 = 0.8521) and pseudo virus neutralization test (R2 = 0.9006) (pVNT). The biotin based sVNT was evaluated in 535 postvaccination serum samples corresponding to second and third boosts of AZD1222 and BNT162b2 vaccines of the wild type strain. We confirmed that the neutralizing antibodies against SARS-CoV-2 variants in second vaccination sera decreased after a median of 141.5 days. Furthermore, vaccination sera from BNT162b2-BNT162b2 vaccines maintained neutralizing antibodies for longer than those of AZD1222 only vaccination. In addition, both vaccines maintained high neutralizing antibodies in third vaccination sera against Omicron BA.2 after a median of 27 days, but neutralizing antibodies significantly decreased after a median of 141.5 days. Along with the cPass sVNT commercial kit, biotin based sVNTs may also be suitable for specifically detecting neutralizing antibodies against multiple SARS-CoV-2 variants; however, to initially monitor the neutralizing antibodies in vaccinated sera using high throughput screening, conventional PRNT could be replaced by sVNT to circumvent the inconvenience of a long test time.

Temporal Transcriptome Analysis of SARS-CoV-2-Infected Lung and Spleen in Human ACE2-Transgenic Mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible and potentially fatal virus. So far, most comprehensive analyses encompassing clinical and transcriptional manifestation have concentrated on the lungs. Here, we confirmed evident signs of viral infection in the lungs and spleen of SARS-CoV-2-infected K18-hACE2 mice, which replicate the phenotype and infection symptoms in hospitalized humans. Seven days post viral detection in organs, infected mice showed decreased vital signs, leading to death. Bronchopneumonia due to infiltration of leukocytes in the lungs and reduction in the spleen lymphocyte region were observed. Transcriptome profiling implicated the meticulous regulation of distress and recovery from cytokine-mediated immunity by distinct immune cell types in a time-dependent manner. In lungs, the chemokine-driven response to viral invasion was highly elevated at 2 days post infection (dpi). In late infection, diseased lungs, post the innate immune process, showed recovery signs. The spleen established an even more immediate line of defense than the lungs, and the cytokine expression profile dropped at 7 dpi. At 5 dpi, spleen samples diverged into two distinct groups with different transcriptome profile and pathophysiology. Inhibition of consecutive host cell viral entry and massive immunoglobulin production and proteolysis inhibition seemed that one group endeavored to survive, while the other group struggled with developmental regeneration against consistent viral intrusion through the replication cycle. Our results may contribute to improved understanding of the longitudinal response to viral infection and development of potential therapeutics for hospitalized patients affected by SARS-CoV-2.

Laboratory information management system for COVID-19 non-clinical efficacy trial data.

BACKGROUND: As the number of large-scale studies involving multiple organizations producing data has steadily increased, an integrated system for a common interoperable format is needed. In response to the coronavirus disease 2019 (COVID-19) pandemic, a number of global efforts are underway to develop vaccines and therapeutics. We are therefore observing an explosion in the proliferation of COVID-19 data, and interoperability is highly requested in multiple institutions participating simultaneously in COVID-19 pandemic research. RESULTS: In this study, a laboratory information management system (LIMS) approach has been adopted to systemically manage various COVID-19 non-clinical trial data, including mortality, clinical signs, body weight, body temperature, organ weights, viral titer (viral replication and viral RNA), and multiorgan histopathology, from multiple institutions based on a web interface. The main aim of the implemented system is to integrate, standardize, and organize data collected from laboratories in multiple institutes for COVID-19 non-clinical efficacy testings. Six animal biosafety level 3 institutions proved the feasibility of our system. Substantial benefits were shown by maximizing collaborative high-quality non-clinical research. CONCLUSIONS: This LIMS platform can be used for future outbreaks, leading to accelerated medical product development through the systematic management of extensive data from non-clinical animal studies.

Genomic Comparisons of Alphacoronaviruses and Betacoronaviruses from Korean Bats.

Coronaviruses are well known as a diverse family of viruses that affect a wide range of hosts. Since the outbreak of severe acute respiratory syndrome, a variety of bat-associated coronaviruses have been identified in many countries. However, they do not represent all the specific geographic locations of their hosts. In this study, full-length genomes representing newly identified bat coronaviruses in South Korea were obtained using an RNA sequencing approach. The analysis, based on genome structure, conserved replicase domains, spike gene, and nucleocapsid genes revealed that bat Alphacoronaviruses are from three different viral species. Among them, the newly identified B20-97 strain may represent a new putative species, closely related to PEDV. In addition, the newly-identified MERS-related coronavirus exhibited shared genomic nucleotide identities of less than 76.4% with other Merbecoviruses. Recombination analysis and multiple alignments of spike and RBD amino acid sequences suggested that this strain underwent recombination events and could possibly use hDPP4 molecules as its receptor. The bat SARS-related CoV B20-50 is unlikely to be able to use hACE2 as its receptor and lack of an open reading frame in ORF8 gene region. Our results illustrate the diversity of coronaviruses in Korean bats and their evolutionary relationships. The evolution of the bat coronaviruses related ORF8 accessory gene is also discussed.

Severe acute respiratory syndrome coronavirus 2 and influenza A virus co-infection alters viral tropism and haematological composition in Syrian hamsters.

The ongoing coronavirus disease 2019 pandemic and its overlap with the influenza season lead to concerns over severe disease caused by the influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) co-infections. Using a Syrian hamster co-infection model with SARS-CoV-2 and the pandemic influenza virus A/California/04/2009 (H1N1), we found (a) more severe disease in co-infected animals, compared to those infected with influenza virus alone but not SARS-CoV-2 infection alone; (b) altered haematological changes in only co-infected animals and (c) altered influenza virus tropism in the respiratory tracts of co-infected animals. Overall, our study revealed that co-infection with SARS-CoV-2 and influenza virus is associated with altered disease severity and tissue tropism, as well as haematological changes, compared to infection with either virus alone.

Relationship between SARS-CoV-2 antibody titer and the severity of COVID-19.

BACKGROUND: It remains unclear whether high titers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies aggravate clinical manifestations in patients or whether severe clinical manifestations result in high antibody titers. Thus, we investigated the cause-effect relationship between SARS-CoV-2 antibody titers and disease severity. METHODS: We prospectively enrolled patients admitted with the diagnosis of coronavirus disease-19 (COVID-19) from February 2020 to August 2020. We measured SARS-CoV-2 antibody titers, namely anti-receptor-binding domain (RBD) antibody and neutralizing antibody (NAb), from blood samples and calculated the chest radiograph (CXR) scores of the patients to evaluate the severity of COVID-19. RESULTS: Overall, 40 patients with COVID-19 were enrolled. Pneumonia was observed in more than half of the patients (25/40, 60%). SARS-CoV-2 antibody titers were higher in patients who were aged >60 years (anti-RBD antibodies, P = 0.003 and NAb, P = 0.009), presented with pneumonia (P = 0.006 and 0.007, respectively), and required oxygen therapy (P = 0.003 and 0.004, respectively) than in those who were not. CXR scores peaked (at 15-21 days after the onset of symptoms) statistically significantly earlier than SARS-CoV-2 antibody titers (at 22-30 days for NAb and at 31-70 days for anti-RBD antibody). There was a close correlation between the maximum CXR score and the maximum SAR-CoV-2 antibody titer. CONCLUSIONS: Based on the comparison of the peak time of SARS-CoV-2 antibody titers with the CXR score after symptom onset, we suggest that severe clinical manifestations result in high titers of SARS-CoV-2 antibodies.

Diagnostic performance and clinical feasibility of a novel one-step RT-qPCR assay for simultaneous detection of multiple severe acute respiratory syndrome coronaviruses.

Coronavirus disease 2019 (COVID-19) is an acute respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Other coronaviruses (CoVs) can also infect humans, although the majority cause only mild respiratory symptoms. Because early diagnosis of SARS-CoV-2 is critical for preventing further transmission events and improving clinical outcomes, it is important to be able to distinguish SARS-CoV-2 from other SARS-related CoVs in respiratory samples. Therefore, we developed and evaluated a novel reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay targeting the genes encoding the spike (S) and membrane (M) proteins to enable the rapid identification of SARS-CoV-2, including several new circulating variants and other emerging SARS-like CoVs. By analysis of in vitro-transcribed mRNA, we established multiplex RT-qPCR assays capable of detecting 5 × 10° copies/reaction. Using RNA extracted from cell culture supernatants, our multiple simultaneous SARS-CoV-2 assays had a limit of detection of 1 × 10° TCID50/mL and showed no cross-reaction with human CoVs or other respiratory viruses. We also validated our method using human clinical samples from patients with COVID-19 and healthy individuals, including nasal swab and sputum samples. This novel one-step multiplex RT-qPCR assay can be used to improve the laboratory diagnosis of human-pathogenic CoVs, including SARS-CoV-2, and may be useful for the identification of other SARS-like CoVs of zoonotic origin.

Long-term surveillance of bat coronaviruses in Korea: Diversity and distribution pattern.

Bats harbour diverse coronaviruses (CoVs), some of which are associated with zoonotic infections, as well as inter-species transmission. In this study, a total of 512 bat faecal samples from the bat habitats at different geographical locations in South Korea were investigated between 2016 and 2019. Seventy-eight samples were positive for coronaviruses (15.2%), comprising 68 alphacoronaviruses (13.3%) and 10 betacoronaviruses (2.0%). The positive rates tended to increase during the awakening (April) period. Notably, betacoronaviruses were only found in the site where Rhinolophus ferrumequinum was the major species of bats, and were related to SARS- and MERS-related CoVs identified in China and South Korea, respectively. No betacoronaviruses were closely related to SARS-CoV-2 in this study. Alphacoronaviruses were detected in the sites where Hypsugo alaschanicus, Miniopterus fuliginosus, Miniopterus schreibersii, Rhinolophus ferrumequinum, Myotis bombinus, Myotis macrodactylus and Myotis petax were found to be the major bat species. Furthermore, alphacoronaviruses had higher genetic diversity than betacoronaviruses and had a wider distribution in Korea. Considering that different bat species are co-roosting in crowded conditions in the same habitat, the diverse coronaviruses in Korean bats are likely to undergo cross-species transmission events due to the richness in host species. Therefore, continuous monitoring should be performed, especially at the awakening time of the hibernating bats in the habitats where diverse bat species co-roost, to better understand the evolution of coronaviruses in bats.