Enhanced antibody responses in fully vaccinated individuals against pan-SARS-CoV-2 variants following Omicron breakthrough infection.

Omicron has become the globally dominant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant, creating additional challenges due to its ability to evade neutralization. Here, we report that neutralizing antibodies against Omicron variants are undetected following COVID-19 infection with ancestral or past SARS-CoV-2 variant viruses or after two-dose mRNA vaccination. Compared with two-dose vaccination, a three-dose vaccination course induces broad neutralizing antibody responses with improved durability against different SARS-CoV-2 variants, although neutralizing antibody titers against Omicron remain low. Intriguingly, among individuals with three-dose vaccination, Omicron breakthrough infection substantially augments serum neutralizing activity against a broad spectrum of SARS-CoV-2 variants, including Omicron variants BA.1, BA.2, and BA.5. Additionally, after Omicron breakthrough infection, memory T cells respond to the spike proteins of both ancestral and Omicron SARS-CoV-2 by producing cytokines with polyfunctionality. These results suggest that Omicron breakthrough infection following three-dose mRNA vaccination induces pan-SARS-CoV-2 immunity that may protect against emerging SARS-CoV-2 variants of concern.

Surgical safety in the COVID-19 era: present and future considerations.

The impacts of coronavirus disease 2019 (COVID-19) have been globally paradigm shifting in all aspects. Surgeons have experienced unprecedented changes regarding operation schedules, preparations before surgery, and the precautions needed both during and after surgery. Many medical centers simultaneously reported a decrease in their numbers of surgeries, whether they were elective or emergent, or for cancerous or benign resections. However, accumulated surgical outcomes from the last 2 years of experience presented postoperative morbidity and mortality data that were comparable to the pre-pandemic era, whether in elective or urgent settings. Although COVID-19 showed a significant association with postoperative morbidity and mortality, the majority of noninfected patients could be treated successfully with stringent mitigation protocols. Initially recommended to be avoided at the start of the pandemic, minimally invasive surgery seems to be safe and feasible according to reported surgical outcomes. Numerous sets of guidelines have now been produced from medical societies and adhering to the basic precautions has been found to be practicable. It is crucial that health care systems and surgical staff remain vigilant and attentive to the ever-changing situation in this pandemic in order to provide optimal medical support to their patients.

Attitudes toward telemedicine among urban and rural residents.

INTRODUCTION: Adoption of telemedicine by healthcare facilities has dramatically increased since the start of coronavirus pandemic; yet, major differences exist in universal acceptance of telemedicine across different population groups. The goal of this study was to examine population-based factors associated with current and/or future use of telemedicine in Alabama. METHODS: A cross-sectional survey was administered to 532 participants online or by phone, in four urban and eight rural counties in Alabama. Data were collected on: demographics, health insurance coverage, medical history, access to technology, and its use in accessing healthcare services. Generalized logit regression models were used to estimate the odds of choosing "virtual visit" and "phone communication" compared to "in-person visit" for the preferred choice of visit with the healthcare provider; as well as odds for willingness to participate in "virtual visit" in the future. RESULTS: Our study sample had a mean age of 43 (±15) years, 72.9% women, 45.9% Black or African American; 59.4% population living in an urban county. The odds of "phone communication" were higher compared to the odds of "in-person visit", with a unit increase in age (odds ratio: 1.02, 95% confidence interval: 1.00-1.03), after adjusting for other covariates. Among participants with past experience of virtual communications, the odds for choosing "virtual visit" were significantly higher compared to choice of in-person visit (odds ratio for virtual visit: 3.23, 95% confidence interval: 2.01-5.18), adjusted for other covariates. Further, people with college or more education were 71% less likely to choose "No" compared to those with high school or lower general education development education for future virtual visit [odds ratio for college or more: 0.29, 95% confodence interval: 0.10-0.87). Likewise, participants residing in rural counties were 57% less likely to choose "No" compared to urban counties for future virtual visit (odds ratio for rural participants: 0.43, 95% confidence interval:0.19-0.97). DISCUSSION: Our study found notable differences in age, education, and rurality for use and/or preference for telemedicine. Medical institutions and healthcare providers will need to account for these differences to ensure that the implementation of telemedicine does not exacerbate existing health disparities.

Development of CO2 Concentration Prediction Tool for Improving Office Indoor Air Quality Considering Economic Cost

Ventilation is becoming increasingly important to improve indoor air quality and prevent the spread of COVID-19. This study analyzed the indoor air quality of office spaces, where occupants remain for extended periods, among multi-use facilities with an increasing need for ventilation system application. A 'tool for office space CO2 prediction and indoor air quality improvement recommendation';was developed. The research method was divided into four steps. Step 1: Analysis of indoor air quality characteristics in office spaces was carried out with a questionnaire survey and indoor air quality experiment. Based on the CO2 concentration, which was found to be a problem in the indoor air quality experiment in the office space, Step 2: CO2 concentration prediction tool for office spaces, which requires inputs of regional and spatial factors and architectural and equipment elements, was developed. In Step 3: Development and verification of prediction tool considering economic feasibility, the cost of energy recovery ventilation systems based on the invoices of the energy recovery ventilation manufacturers was analyzed. In Step 4: Energy recovery ventilation proposal and indoor CO2 forecast, Office Space B, which can accommodate up to 15 people, was derived as an example of the proposed tool. As a result of the prediction, the optimal air volume of the energy recovery ventilation was determined according to the 'office CO2 prediction and indoor air quality improvement recommendations';. This study introduced simple tools, which can be used by non-experts, that are capable of showing changes in indoor air quality, CO2 concentration and cost according to activities.

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.

Antiviral effects of human placenta hydrolysate (Laennec®) against SARS-CoV-2 in vitro and in the ferret model.

The COVID-19 pandemic has caused unprecedented health, social, and economic crises worldwide. However, to date, there is an only a limited effective treatment for this disease. Human placenta hydrolysate (hPH) has previously been shown to be safe and to improve the health condition in patients with hyperferritinemia and COVID-19. In this study, we aimed to determine the antiviral effects of hPH against SARS-CoV-2 in vitro and in vivo models and compared with Remdesivir, an FDA-approved drug for COVID-19 treatment. To assess whether hPH inhibited SARS-CoV-2 replication, we determined the CC50, EC50, and selective index (SI) in Vero cells by infection with a SARS-CoV-2 at an MOI of 0.01. Further, groups of ferrets infected with 105.8 TCID50/ml of SARS-CoV-2 and treated with hPH at 2, 4, 6 dpi, and compared their clinical manifestation and virus titers in respiratory tracts with PBS control-treated group. The mRNA expression of immune-related cytokines was determined by qRT-PCR. hPH treatment attenuated virus replication in a dose-dependent manner in vitro. In a ferret infection study, treatment with hPH resulted in minimal bodyweight loss and attenuated virus replication in the nasal wash, turbinates, and lungs of infected ferrets. In addition, qRT-PCR results revealed that the hPH treatment remarkably upregulated the gene expression of type I (IFN-α and IFN-ß) and II (IFN-γ) IFNs in SARS-CoV-2 infected ferrets. Our data collectively suggest that hPH has antiviral efficacy against SARS-CoV-2 and might be a promising therapeutic agent for the treatment of SARS-CoV-2 infection.

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.

Therapeutic effect of CT-P59 against SARS-CoV-2 South African variant.

The global circulation of newly emerging variants of SARS-CoV-2 is a new threat to public health due to their increased transmissibility and immune evasion. Moreover, currently available vaccines and therapeutic antibodies were shown to be less effective against new variants, in particular, the South African (SA) variant, termed 501Y.V2 or B.1.351. To assess the efficacy of the CT-P59 monoclonal antibody against the SA variant, we sought to perform as in vitro binding and neutralization assays, and in vivo animal studies. CT-P59 neutralized B.1.1.7 variant to a similar extent as to wild type virus. CT-P59 showed reduced binding affinity against a RBD (receptor binding domain) triple mutant containing mutations defining B.1.351 (K417N/E484K/N501Y) also showed reduced potency against the SA variant in live virus and pseudovirus neutralization assay systems. However, in vivo ferret challenge studies demonstrated that a therapeutic dosage of CT-P59 was able to decrease B.1.351 viral load in the upper and lower respiratory tracts, comparable to that observed for the wild type virus. Overall, although CT-P59 showed reduced in vitro neutralizing activity against the SA variant, sufficient antiviral effect in B.1.351-infected animals was confirmed with a clinical dosage of CT-P59, suggesting that CT-P59 has therapeutic potential for COVID-19 patients infected with SA variant.

Impact of the COVID-19 Pandemic on Surgical Treatment Patterns for Colorectal Cancer in a Tertiary Medical Facility in Korea.

Because of their reluctance to visit the hospital due to concerns about contracting coronavirus disease 2019 (COVID-19), patients with colorectal cancer have been affected by delays in care during the pandemic. This study assessed the effects of the pandemic on the clinical characteristics and surgical treatment patterns of colorectal cancer patients at a tertiary medical facility in Korea. Patients who underwent colorectal cancer surgery at our institution between March and September 2020 were analyzed. Clinicopathological and treatment characteristics were compared with those of patients who underwent surgery in 2018 and 2019. The patients who did not undergo tumor resection (4.1% vs. 1.8%, p < 0.001) and who received neoadjuvant treatment (16.7% vs. 14.7%, p = 0.039) were significantly higher during the COVID period. The minimally invasive approach was performed less during the COVID period (81.2% vs. 88%, p < 0.001). More patients in the COVID period required combined resection of organs adjacent to the tumor (4.8% vs. 2.8%, p = 0.017). Surgical aggressiveness, as shown by the proportion of patients undergoing minimally invasive surgery and adjacent organ resection, was significantly influenced by the pandemic. In addition, resectability decreased during the COVID period. These characteristics will likely influence long-term oncological outcomes, indicating the need for long-term monitoring of this cohort.

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.

Factors Associated with Perceived Susceptibility to COVID-19 Among Urban and Rural Adults in Alabama.

We examined factors associated with and reasons for perceived susceptibility to COVID-19 among urban and rural adults in Alabama. We surveyed 575 eligible participants' engagement in preventive behaviors, concern about COVID-19 in their communities, perceived susceptibility to the virus, and reasons for susceptibility across three response options (Yes, No, and Don't Know/Not Sure). Bivariate analyses compared characteristics by level of perceived susceptibility to COVID-19. A multinomial logistic regression model evaluated the association of demographics, health insurance coverage, and chronic illness status with perceived susceptibility. Participants' race, gender, and educational attainment were significantly associated with perceived susceptibility to COVID-19. African Americans and males had higher odds of responding 'No', compared to 'Yes' and 'Don't Know/Not Sure' than Whites and females. Participants with a high school education and lower had higher odds of responding 'Don't Know/Not Sure' versus 'Yes' compared to those with college or higher education. Those unconcerned about COVID-19 in their community had higher odds of responding 'No' (OR = 2.51, CI 1.35-4.68) and 'Don't Know/Not Sure' (OR = 2.51, CI 1.26-4.99) versus 'Yes', as compared to those who were concerned. Possibility of exposure at work was the most frequent reasons for perceiving themselves susceptible to COVID-19, engagement in recommended preventive measures was the most frequent reason among respondents who indicated 'No', and uncertainty/perception that everyone is at risk was the most frequent reason among the ones who indicated 'Don't Know/Not Sure'. Results indicate that tailored efforts to heighten perceived susceptibility to COVID-19 among specific demographics are needed.

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.

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.

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.

Development of a reverse transcription-loop-mediated isothermal amplification as a rapid early-detection method for novel SARS-CoV-2.

The previous outbreaks of SARS-CoV and MERS-CoV have led researchers to study the role of diagnostics in impediment of further spread and transmission. With the recent emergence of the novel SARS-CoV-2, the availability of rapid, sensitive, and reliable diagnostic methods is essential for disease control. Hence, we have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the specific detection of SARS-CoV-2. The primer sets for RT-LAMP assay were designed to target the nucleocapsid gene of the viral RNA, and displayed a detection limit of 102 RNA copies close to that of qRT-PCR. Notably, the assay has exhibited a rapid detection span of 30 min combined with the colorimetric visualization. This test can detect specifically viral RNAs of the SARS-CoV-2 with no cross-reactivity to related coronaviruses, such as HCoV-229E, HCoV-NL63, HCoV-OC43, and MERS-CoV as well as human infectious influenza viruses (type B, H1N1pdm, H3N2, H5N1, H5N6, H5N8, and H7N9), and other respiratory disease-causing viruses (RSVA, RSVB, ADV, PIV, MPV, and HRV). Furthermore, the developed RT-LAMP assay has been evaluated using specimens collected from COVID-19 patients that exhibited high agreement to the qRT-PCR. Our RT-LAMP assay is simple to perform, less expensive, time-efficient, and can be used in clinical laboratories for preliminary detection of SARS-CoV-2 in suspected patients. In addition to the high sensitivity and specificity, this isothermal amplification conjugated with a single-tube colorimetric detection method may contribute to the public health responses and disease control, especially in the areas with limited laboratory capacities.