Antiviral Efficacies of FDA-Approved Drugs against SARS-CoV-2 Infection in Ferrets.

Due to the urgent need of a therapeutic treatment for coronavirus (CoV) disease 2019 (COVID-19) patients, a number of FDA-approved/repurposed drugs have been suggested as antiviral candidates at clinics, without sufficient information. Furthermore, there have been extensive debates over antiviral candidates for their effectiveness and safety against severe acute respiratory syndrome CoV 2 (SARS-CoV-2), suggesting that rapid preclinical animal studies are required to identify potential antiviral candidates for human trials. To this end, the antiviral efficacies of lopinavir-ritonavir, hydroxychloroquine sulfate, and emtricitabine-tenofovir for SARS-CoV-2 infection were assessed in the ferret infection model. While the lopinavir-ritonavir-, hydroxychloroquine sulfate-, or emtricitabine-tenofovir-treated group exhibited lower overall clinical scores than the phosphate-buffered saline (PBS)-treated control group, the virus titers in nasal washes, stool specimens, and respiratory tissues were similar between all three antiviral-candidate-treated groups and the PBS-treated control group. Only the emtricitabine-tenofovir-treated group showed lower virus titers in nasal washes at 8 days postinfection (dpi) than the PBS-treated control group. To further explore the effect of immune suppression on viral infection and clinical outcome, ferrets were treated with azathioprine, an immunosuppressive drug. Compared to the PBS-treated control group, azathioprine-immunosuppressed ferrets exhibited a longer period of clinical illness, higher virus titers in nasal turbinate, delayed virus clearance, and significantly lower serum neutralization (SN) antibody titers. Taken together, all antiviral drugs tested marginally reduced the overall clinical scores of infected ferrets but did not significantly affect in vivo virus titers. Despite the potential discrepancy of drug efficacies between animals and humans, these preclinical ferret data should be highly informative to future therapeutic treatment of COVID-19 patients.IMPORTANCE The SARS-CoV-2 pandemic continues to spread worldwide, with rapidly increasing numbers of mortalities, placing increasing strain on health care systems. Despite serious public health concerns, no effective vaccines or therapeutics have been approved by regulatory agencies. In this study, we tested the FDA-approved drugs lopinavir-ritonavir, hydroxychloroquine sulfate, and emtricitabine-tenofovir against SARS-CoV-2 infection in a highly susceptible ferret infection model. While most of the drug treatments marginally reduced clinical symptoms, they did not reduce virus titers, with the exception of emtricitabine-tenofovir treatment, which led to diminished virus titers in nasal washes at 8 dpi. Further, the azathioprine-treated immunosuppressed ferrets showed delayed virus clearance and low SN titers, resulting in a prolonged infection. As several FDA-approved or repurposed drugs are being tested as antiviral candidates at clinics without sufficient information, rapid preclinical animal studies should proceed to identify therapeutic drug candidates with strong antiviral potential and high safety prior to a human efficacy trial.

Construction of SARS-CoV-2 virus-like particles in plant.

The pandemic of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused a public health emergency, and research on the development of various types of vaccines is rapidly progressing at an unprecedented development speed internationally. Some vaccines have already been approved for emergency use and are being supplied to people around the world, but there are still many ongoing efforts to create new vaccines. Virus-like particles (VLPs) enable the construction of promising platforms in the field of vaccine development. Here, we demonstrate that non-infectious SARS-CoV-2 VLPs can be successfully assembled by co-expressing three important viral proteins membrane (M), envelop (E) and nucleocapsid (N) in plants. Plant-derived VLPs were purified by sedimentation through a sucrose cushion. The shape and size of plant-derived VLPs are similar to native SARS-CoV-2 VLPs without spike. Although the assembled VLPs do not have S protein spikes, they could be developed as formulations that can improve the immunogenicity of vaccines including S antigens, and further could be used as platforms that can carry S antigens of concern for various mutations.

Age-dependent pathogenic characteristics of SARS-CoV-2 infection in ferrets.

While the seroprevalence of SARS-CoV-2 in healthy people does not differ significantly among age groups, those aged 65 years or older exhibit strikingly higher COVID-19 mortality compared to younger individuals. To further understand differing COVID-19 manifestations in patients of different ages, three age groups of ferrets are infected with SARS-CoV-2. Although SARS-CoV-2 is isolated from all ferrets regardless of age, aged ferrets (≥3 years old) show higher viral loads, longer nasal virus shedding, and more severe lung inflammatory cell infiltration, and clinical symptoms compared to juvenile (≤6 months) and young adult (1-2 years) groups. Furthermore, direct contact ferrets co-housed with the virus-infected aged group shed more virus than direct-contact ferrets co-housed with virus-infected juvenile or young adult ferrets. Transcriptome analysis of aged ferret lungs reveals strong enrichment of gene sets related to type I interferon, activated T cells, and M1 macrophage responses, mimicking the gene expression profile of severe COVID-19 patients. Thus, SARS-CoV-2-infected aged ferrets highly recapitulate COVID-19 patients with severe symptoms and are useful for understanding age-associated infection, transmission, and pathogenesis of SARS-CoV-2.

Neutralizing Antibody Production in Asymptomatic and Mild COVID-19 Patients, in Comparison with Pneumonic COVID-19 Patients.

OBJECTIVES: To investigate antibody production in asymptomatic and mild COVID-19 patients. METHODS: Sera from asymptomatic to severe COVID-19 patients were collected. Microneutralization (MN), fluorescence immunoassay (FIA), and enzyme-linked immunosorbent assay (ELISA) were performed. RESULTS: A total of 70 laboratory-confirmed COVID-19 patients were evaluated, including 15 asymptomatic/anosmia, 49 mild symptomatic, and 6 pneumonia patients. The production of the neutralizing antibody was observed in 100% of pneumonia, 93.9% of mild symptomatic, and 80.0% of asymptomatic/anosmia groups. All the patients in the pneumonia group showed high MN titer (≥1:80), while 36.7% of mild symptomatic and 20.0% of asymptomatic/anosmia groups showed high titer (p < 0.001). Anti-SARS-CoV-2 antibodies could be more sensitively detected by FIA IgG (98.8%) and ELISA (97.6%) in overall. For the FIA IgG test, all patients in the pneumonia group exhibited a high COI value (≥15.0), while 89.8% of mild symptomatic and 73.3% of asymptomatic/anosmia groups showed a high value (p = 0.049). For the ELISA test, all patients in the pneumonia group showed a high optical density (OD) ratio (≥3.0), while 65.3% of mild symptomatic and 53.3% of asymptomatic/anosmia groups showed a high ratio (p = 0.006). CONCLUSIONS: Most asymptomatic and mild COVID-19 patients produced the neutralizing antibody, although the titers were lower than pneumonia patients. ELISA and FIA sensitively detected anti-SARS-CoV-2 antibodies.

Single-cell transcriptome of bronchoalveolar lavage fluid reveals sequential change of macrophages during SARS-CoV-2 infection in ferrets.

Few studies have used a longitudinal approach to describe the immune response to SARS-CoV-2 infection. Here, we perform single-cell RNA sequencing of bronchoalveolar lavage fluid cells longitudinally obtained from SARS-CoV-2-infected ferrets. Landscape analysis of the lung immune microenvironment shows distinct changes in cell proportions and characteristics compared to uninfected control, at 2 and 5 days post-infection (dpi). Macrophages are classified into 10 distinct subpopulations with transcriptome changes among monocyte-derived infiltrating macrophages and differentiated M1/M2 macrophages, notably at 2 dpi. Moreover, trajectory analysis reveals gene expression changes from monocyte-derived infiltrating macrophages toward M1 or M2 macrophages and identifies a macrophage subpopulation that has rapidly undergone SARS-CoV-2-mediated activation of inflammatory responses. Finally, we find that M1 or M2 macrophages show distinct patterns of gene modules downregulated by immune-modulatory drugs. Overall, these results elucidate fundamental aspects of the immune response dynamics provoked by SARS-CoV-2 infection.

Differences in seroprevalence between epicenter and non-epicenter areas of the COVID-19 outbreak in South Korea.

To compare the standardized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroprevalence of high epicenter region with non-epicenter region, serological studies were performed with a total of 3,268 sera from Daegu City and 3,981 sera from Chungbuk Province. Indirect immunofluorescence assay (IFA) for SARS-CoV-2 IgG results showed a high seroprevalence rate in the Daegu City (epicenter) compared with a non-epicenter area (Chungbuk Province) (1.27% vs. 0.91%, P = 0.0358). It is noteworthy that the highest seroprevalence in Daegu City was found in elderly patients (70's) whereas young adult patients (20's) in Chungbuk Province showed the highest seroprevalence. Neutralizing antibody (NAb) titers were found in three samples from Daegu City (3/3, 268, 0.09%) while none of the samples from Chungbuk Province were NAb positive. These results demonstrated that even following the large outbreak, the seropositive rate of SARS-CoV-2 in the general population remained low in South Korea.

A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein.

Vaccines and therapeutics are urgently needed for the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we screen human monoclonal antibodies (mAb) targeting the receptor binding domain (RBD) of the viral spike protein via antibody library constructed from peripheral blood mononuclear cells of a convalescent patient. The CT-P59 mAb potently neutralizes SARS-CoV-2 isolates including the D614G variant without antibody-dependent enhancement effect. Complex crystal structure of CT-P59 Fab/RBD shows that CT-P59 blocks interaction regions of RBD for angiotensin converting enzyme 2 (ACE2) receptor with an orientation that is notably different from previously reported RBD-targeting mAbs. Furthermore, therapeutic effects of CT-P59 are evaluated in three animal models (ferret, hamster, and rhesus monkey), demonstrating a substantial reduction in viral titer along with alleviation of clinical symptoms. Therefore, CT-P59 may be a promising therapeutic candidate for COVID-19.

Critical role of neutralizing antibody for SARS-CoV-2 reinfection and transmission.

Cases of laboratory-confirmed SARS-CoV-2 reinfection have been reported in a number of countries. Further, the level of natural immunity induced by SARS-CoV-2 infection is not fully clear, nor is it clear if a primary infection is protective against reinfection. To investigate the potential association between serum antibody titres and reinfection of SARS-CoV-2, ferrets with different levels of NAb titres after primary SARS-CoV-2 infection were subjected to reinfection with a heterologous SARS-CoV-2 strain. All heterologous SARS-CoV-2 reinfected ferrets showed active virus replication in the upper respiratory and gastro-intestinal tracts. However, the high NAb titre group showed attenuated viral replication and rapid viral clearance. In addition, direct-contact transmission was observed only from reinfected ferrets with low NAb titres (<20), and not from other groups. Further, lung histopathology demonstrated the presence of limited inflammatory regions in the high NAb titre groups compared with control and low NAb groups. This study demonstrates a close correlation between a low NAb titre and SARS-CoV-2 reinfection in a recovered ferret reinfection model.

Serologic Evaluation of Healthcare Workers Caring for COVID-19 Patients in the Republic of Korea.

The safety of healthcare workers (HCWs) against severe acute respiratory syndrome virus 2 (SARS-CoV-2) transmission is an important aspect of managing the coronavirus disease 2019 (COVID-19) pandemic. In the South Korea, highly stringent infection prevention and control (IPC) guidelines are implemented, and reports of healthcare-associated SARS-CoV-2 transmission among HCWs are limited. However, subclinical infections may have been missed by the current symptom-based screening strategy. To evaluate the risk of undetected SARS-CoV-2 transmissions from COVID-19 patients to HCWs, we conducted a multicenter seroprevalence study after the first surge of the COVID-19 outbreak. A total of 432 HCWs were evaluated, comprising 309 HCWs designated to laboratory-confirmed COVID-19 patient care and 123 non-designated HCWs. Designated HCWs wore personal protective equipment including an N95 respirator, eye protection, hooded overalls, shoe covers, and inner and outer gloves. Use of a powered air-purifying respirator was recommended for aerosol-generating procedures or long-duration care activities. A high-sensitivity (99.1%) fluorescence immunoassay immunoglobulin G (IgG) kit was used as the initial screening test, and two enzyme-linked immunosorbent assay kits for total and IgG antibodies were used to confirm the test results. A microneutralization test was additionally performed to evaluate the neutralizing activity of positive specimens. Among the evaluated HCWs, none of the non-designated HCWs had a positive result, while one of the HCWs designated for COVID-19 patient care (1/309, 0.3%) was seropositive for SARS-CoV-2 with confirmed neutralizing activity (1:40). This finding suggests that subclinical seroconversion may occur among HCWs caring for COVID-19 patients, although the risk is low under strict IPC guidance.

Detection and Isolation of SARS-CoV-2 in Serum, Urine, and Stool Specimens of COVID-19 Patients from the Republic of Korea.

OBJECTIVES: Coronavirus Disease-19 (COVID-19) is a respiratory infection characterized by the main symptoms of pneumonia and fever. It is caused by the novel coronavirus severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2), which is known to spread via respiratory droplets. We aimed to determine the rate and likelihood of SARS-CoV-2 transmission from COVID-19 patients through non-respiratory routes. METHODS: Serum, urine, and stool samples were collected from 74 hospitalized patients diagnosed with COVID-19 based on the detection of SARS-CoV-2 in respiratory samples. The SARS-CoV-2 RNA genome was extracted from each specimen and real-time reverse transcription polymerase chain reaction performed. CaCo-2 cells were inoculated with the specimens containing the SARS-COV-2 genome, and subcultured for virus isolation. After culturing, viral replication in the cell supernatant was assessed. RESULTS: Of the samples collected from 74 COVID-19 patients, SARS-CoV-2 was detected in 15 serum, urine, or stool samples. The virus detection rate in the serum, urine, and stool samples were 2.8% (9/323), 0.8% (2/247), and 10.1% (13/129), and the mean viral load was 1,210 ± 1,861, 79 ± 30, and 3,176 ± 7,208 copy/µL, respectively. However, the SARS-CoV-2 was not isolated by the culture method from the samples that tested positive for the SARS-CoV-2 gene. CONCLUSION: While the virus remained detectable in the respiratory samples of COVID-19 patients for several days after hospitalization, its detection in the serum, urine, and stool samples was intermittent. Since the virus could not be isolated from the SARS-COV-2-positive samples, the risk of viral transmission via stool and urine is expected to be low.

Development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) thermal inactivation method with preservation of diagnostic sensitivity.

Various treatments and agents had been reported to inactivate RNA viruses. Of these, thermal inactivation is generally considered an effective and cheap method of sample preparation for downstream assays. The purpose of this study is to establish a safe inactivation method for SARS-CoV-2 without compromising the amount of amplifiable viral genome necessary for clinical diagnoses. In this study, we demonstrate the infectivity and genomic stability of SARSCoV- 2 by thermal inactivation at both 56°C and 65°C. The results substantiate that viable SARS-CoV-2 is readily inactivated when incubated at 56°C for 30 min or at 65°C for 10 min. qRT-PCR of specimens heat-inactivated at 56°C for 30 min or 65°C for 15 min revealed similar genomic RNA stability compared with non-heat inactivated specimens. Further, we demonstrate that 30 min of thermal inactivation at 56°C could inactivate viable viruses from clinical COVID-19 specimens without attenuating the qRT-PCR diagnostic sensitivity. Heat treatment of clinical specimens from COVID-19 patients at 56°C for 30 min or 65°C for 15 min could be a useful method for the inactivation of a highly contagious agent, SARS-CoV-2. Use of this method would reduce the potential for secondary infections in BSL2 conditions during diagnostic procedures. Importantly, infectious virus can be inactivated in clinical specimens without compromising the sensitivity of the diagnostic RT-PCR assay.

Viable SARS-CoV-2 in various specimens from COVID-19 patients.

OBJECTIVES: The aim was to determine whether various clinical specimens obtained from COVID-19 patients contain the infectious virus. METHODS: To demonstrate whether various clinical specimens contain the viable virus, we collected naso/oropharyngeal swabs and saliva, urine and stool samples from five COVID-19 patients and performed a quantitative polymerase chain reaction (qPCR) to assess viral load. Specimens positive with qPCR were subjected to virus isolation in Vero cells. We also used urine and stool samples to intranasally inoculate ferrets and evaluated the virus titres in nasal washes on 2, 4, 6 and 8 days post infection. RESULTS: SARS-CoV-2 RNA was detected in all naso/oropharyngeal swabs and saliva, urine and stool samples collected between days 8 and 30 of the clinical course. Notably, viral loads in urine, saliva and stool samples were almost equal to or higher than those in naso/oropharyngeal swabs (urine 1.08 ± 0.16-2.09 ± 0.85 log10 copies/mL, saliva 1.07 ± 0.34-1.65 ± 0.46 log10 copies/mL, stool 1.17 ± 0.32 log10 copies/mL, naso/oropharyngeal swabs 1.18 ± 0.12-1.34 ± 0.30 log10 copies/mL). Further, viable SARS-CoV-2 was isolated from naso/oropharyngeal swabs and saliva of COVID-19 patients, as well as nasal washes of ferrets inoculated with patient urine or stool. DISCUSSION: Viable SARS-CoV-2 was demonstrated in saliva, urine and stool samples from COVID-19 patients up to days 11-15 of the clinical course. This result suggests that viable SARS-CoV-2 can be secreted in various clinical samples and respiratory specimens.

Neutralizing Antibody Production in Asymptomatic and Mild COVID-19 Patients, in Comparison with Pneumonic COVID-19 Patients

Objectives: To investigate antibody production in asymptomatic and mild COVID-19 patients. Methods: Sera from asymptomatic to severe COVID-19 patients were collected. Microneutralization (MN), fluorescence immunoassay (FIA), and enzyme-linked immunosorbent assay (ELISA) were performed. Results: A total of 70 laboratory-confirmed COVID-19 patients were evaluated, including 15 asymptomatic/anosmia, 49 mild symptomatic, and 6 pneumonia patients. The production of the neutralizing antibody was observed in 100% of pneumonia, 93.9% of mild symptomatic, and 80.0% of asymptomatic/anosmia groups. All the patients in the pneumonia group showed high MN titer (≥1:80), while 36.7% of mild symptomatic and 20.0% of asymptomatic/anosmia groups showed high titer (p <0.001). Anti-SARS-CoV-2 antibodies could be more sensitively detected by FIA IgG (98.8%) and ELISA (97.6%) in overall. For the FIA IgG test, all patients in the pneumonia group exhibited a high COI value (≥15.0), while 89.8% of mild symptomatic and 73.3% of asymptomatic/anosmia groups showed a high value (p = 0.049). For the ELISA test, all patients in the pneumonia group showed a high optical density (OD) ratio (≥3.0), while 65.3% of mild symptomatic and 53.3% of asymptomatic/anosmia groups showed a high ratio (p = 0.006). Conclusions: Most asymptomatic and mild COVID-19 patients produced the neutralizing antibody, although the titers were lower than pneumonia patients. ELISA and FIA sensitively detected anti-SARS-CoV-2 antibodies.

Infection and Rapid Transmission of SARS-CoV-2 in Ferrets.

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China and rapidly spread worldwide. To prevent SARS-CoV-2 dissemination, understanding the in vivo characteristics of SARS-CoV-2 is a high priority. We report a ferret model of SARS-CoV-2 infection and transmission that recapitulates aspects of human disease. SARS-CoV-2-infected ferrets exhibit elevated body temperatures and virus replication. Although fatalities were not observed, SARS-CoV-2-infected ferrets shed virus in nasal washes, saliva, urine, and feces up to 8 days post-infection. At 2 days post-contact, SARS-CoV-2 was detected in all naive direct contact ferrets. Furthermore, a few naive indirect contact ferrets were positive for viral RNA, suggesting airborne transmission. Viral antigens were detected in nasal turbinate, trachea, lungs, and intestine with acute bronchiolitis present in infected lungs. Thus, ferrets represent an infection and transmission animal model of COVID-19 that may facilitate development of SARS-CoV-2 therapeutics and vaccines.