Inhibition of a broad range of SARS-CoV-2 variants by antiviral phytochemicals in hACE2 mice.

Although several vaccines and antiviral drugs against SARS-CoV-2 are currently available, control and prevention of COVID-19 through these interventions is limited due to inaccessibility and economic issues in some regions and countries. Moreover, incomplete viral clearance by ineffective therapeutics may lead to rapid genetic evolution, resulting in the emergence of new SARS-CoV-2 variants that may escape the host immune system as well as currently available COVID-19 vaccines. Here, we report that phytochemicals extracted from Chlorella spp. and Psidium guajava possess broad-spectrum antiviral activity against a range of SARS-CoV-2 variants. Through chromatography-based screening, we identified four bioactive compounds and subsequently demonstrated their potential antiviral activities in vivo. Interestingly, in hACE2 mice, treatment with these compounds significantly attenuates SARS-CoV-2-induced proinflammatory responses, demonstrating their potential anti-inflammatory activity. Collectively, our study suggests that phytochemicals from edible plants may be readily available therapeutics and prophylactics against multiple SARS-CoV-2 strains and variants.

Transmissibility and pathogenicity of SARS-CoV-2 variants in animal models.

As of February 2022, SARS-CoV-2 is still one of the most serious public health threats due to its high mortality rate and rapid spread of novel variants. Since the first outbreak in 2019, general understanding of SARS-CoV-2 has been improved through basic and clinical studies; however, knowledge gaps still exist in our understanding of the emerging novel SARSCoV-2 variants, which impacts the corresponding development of vaccines and therapeutics. Especially, accumulation of mutations in SARS-CoV-2 and rapid spread in populations with previous immunity has resulted in selection of variants that evade the host immune response. This phenomenon threatens to render current SARS-CoV-2 vaccines ineffective for controlling the pandemic. Proper animal models are essential for detailed investigations into the viral etiology, transmission and pathogenesis mechanisms, as well as evaluation of the efficacy of vaccine candidates against recent SARS-CoV-2 variants. Further, the choice of animal model for each research topic is important for researchers to gain better knowledge of recent SARS-CoV-2 variants. Here, we review the advantages and limitations of each animal model, including mice, hamsters, ferrets, and non-human primates, to elucidate variant SARS-CoV-2 etiology and transmission and to evaluate therapeutic and vaccine efficacy.

Coinfection with SARS-CoV-2 and Influenza A Virus Increases Disease Severity and Impairs Neutralizing Antibody and CD4+ T Cell Responses.

Given the current coronavirus disease 2019 (COVID-19) pandemic, coinfection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (IAV) is a major concern for public health. However, the immunopathogenic events occurring with coinfections of SARS-CoV-2 and IAV remain unclear. Here, we report the pathogenic and immunological consequences of SARS-CoV-2 and IAV H1N1 coinfection in the K18-hACE2 transgenic mouse model. Compared with a single infection with SARS-CoV-2 or IAV, coinfections not only prolonged the primary virus infection period but also increased immune cell infiltration and inflammatory cytokine levels in bronchoalveolar lavage fluid leading to severe pneumonia and lung damage. Moreover, coinfections caused severe lymphopenia in peripheral blood, resulting in reduced total IgG, neutralizing antibody titers, and CD4+ T cell responses against each virus. This study sheds light on the immunopathogenesis of SARS-CoV-2 and IAV coinfection, which may guide the development of effective therapeutic strategies for the treatment of patients coinfected with these viruses. IMPORTANCE The cocirculation of influenza virus merging with the COVID-19 pandemic raises a potentially severe threat to public health. Recently, increasing numbers of SARS-CoV-2 and influenza virus coinfection have been reported from many countries. It is a worrisome issue that SARS-CoV-2 coinfection with other pathogens may worsen the clinical outcome and severity of COVID-19 and increase fatality. Here, we evaluated SARS-CoV-2 and IAV coinfection using the K18-hACE2 mouse model. Coinfected mice exhibited increased mortality with prolonged IAV shedding. Furthermore, coinfected mice showed a higher level of cytokines and chemokines than a single infection condition. Interestingly, our data show that coinfected mice showed significantly fewer virus-specific and neutralizing antibodies than the mice with a single infection. Overall, this study suggests that coinfection aggravates viral pathology by impaired neutralizing antibody response.

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.

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.

Experimental Animal Models of Coronavirus Infections: Strengths and Limitations.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since the emergence of SARS-CoV-2 in the human population in late 2019, it has spread on an unprecedented scale worldwide leading to the first coronavirus pandemic. SARS-CoV-2 infection results in a wide range of clinical manifestations from asymptomatic to fatal cases. Although intensive research has been undertaken to increase understanding of the complex biology of SARS-CoV-2 infection, the detailed mechanisms underpinning the severe pathogenesis and interactions between the virus and the host immune response are not well understood. Thus, the development of appropriate animal models that recapitulate human clinical manifestations and immune responses against SARS-CoV-2 is crucial. Although many animal models are currently available for the study of SARS-CoV-2 infection, each has distinct advantages and disadvantages, and some models show variable results between and within species. Thus, we aim to discuss the different animal models, including mice, hamsters, ferrets, and non-human primates, employed for SARS-CoV-2 infection studies and outline their individual strengths and limitations for use in studies aimed at increasing understanding of coronavirus pathogenesis. Moreover, a significant advantage of these animal models is that they can be tailored, providing unique options specific to the scientific goals of each researcher.

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.

Development of Spike Receptor-Binding Domain Nanoparticles as a Vaccine Candidate against SARS-CoV-2 Infection in Ferrets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a causative agent of the CoV disease 2019 (COVID-19) pandemic, enters host cells via the interaction of its receptor-binding domain (RBD) of the spike protein with host angiotensin-converting enzyme 2 (ACE2). Therefore, the RBD is a promising vaccine target to induce protective immunity against SARS-CoV-2 infection. In this study, we report the development of an RBD protein-based vaccine candidate against SARS-CoV-2 using self-assembling Helicobacter pylori-bullfrog ferritin nanoparticles as an antigen delivery system. RBD-ferritin protein purified from mammalian cells efficiently assembled into 24-mer nanoparticles. Sixteen- to 20-month-old ferrets were vaccinated with RBD-ferritin nanoparticles (RBD nanoparticles) by intramuscular or intranasal inoculation. All vaccinated ferrets with RBD nanoparticles produced potent neutralizing antibodies against SARS-CoV-2. Strikingly, vaccinated ferrets demonstrated efficient protection from SARS-CoV-2 challenge, showing no fever, body weight loss, or clinical symptoms. Furthermore, vaccinated ferrets showed rapid clearance of infectious virus in nasal washes and lungs as well as of viral RNA in respiratory organs. This study demonstrates that spike RBD-nanoparticles are an effective protein vaccine candidate against SARS-CoV-2.

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.

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.

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.