Identification of priority pathogens for aetiological diagnosis in adults with community-acquired pneumonia in China: a multicentre prospective study.

BACKGROUND: Community-acquired pneumonia (CAP) is a major public health challenge worldwide. However, the aetiological and disease severity-related pathogens associated with CAP in adults in China are not well established based on the detection of both viral and bacterial agents. METHODS: A multicentre, prospective study was conducted involving 10 hospitals located in nine geographical regions in China from 2014 to 2019. Sputum or bronchoalveolar lavage fluid (BALF) samples were collected from each recruited CAP patient. Multiplex real-time PCR and bacteria culture methods were used to detect respiratory pathogens. The association between detected pathogens and CAP severity was evaluated. RESULTS: Among the 3,403 recruited eligible patients, 462 (13.58%) had severe CAP, and the in-hospital mortality rate was 1.94% (66/3,403). At least one pathogen was detected in 2,054 (60.36%) patients, with two or more pathogens were co-detected in 725 patients. The ten major pathogens detected were Mycoplasma pneumoniae (11.05%), Haemophilus influenzae (10.67%), Klebsiella pneumoniae (10.43%), influenza A virus (9.49%), human rhinovirus (9.02%), Streptococcus pneumoniae (7.43%), Staphylococcus aureus (4.50%), adenovirus (2.94%), respiratory syncytial viruses (2.35%), and Legionella pneumophila (1.03%), which accounted for 76.06-92.52% of all positive detection results across sampling sites. Klebsiella pneumoniae (p < 0.001) and influenza viruses (p = 0.005) were more frequently detected in older patients, whereas Mycoplasma pneumoniae was more frequently detected in younger patients (p < 0.001). Infections with Klebsiella pneumoniae, Staphylococcus aureus, influenza viruses and respiratory syncytial viruses were risk factors for severe CAP. CONCLUSIONS: The major respiratory pathogens causing CAP in adults in China were different from those in USA and European countries, which were consistent across different geographical regions over study years. Given the detection rate of pathogens and their association with severe CAP, we propose to include the ten major pathogens as priorities for clinical pathogen screening in China.

The Environmental Stability of SARS-CoV-2 Variants Omicron BA.1 and BA.5 on the Surfaces of Widely Used Transport Packaging Materials.

The increased transmissibility of SARS-CoV-2 variants of concern (VOCs) has raised questions regarding the environmental stability of these viruses. Although a prolonged survival time has been reported for SARS-CoV-2, how long new variants can persist on contaminated surfaces and how environmental factors affect the persistence time are not fully characterized. The present study provides a comprehensive assessment of the stability of Omicron variants BA.1 and BA.5, which are currently circulating strains, on the surfaces of widely used transport packaging materials. By monitoring viable virus detection over a 7-day period under different environmental conditions, it was found that the environmental stability of SARS-CoV-2 Omicron variants depended heavily on the surface type, temperature, and virus concentration. In addition, virus nucleic acid exhibited high stability on the material surface independent of whether viable virus was detected. These findings provide useful information for logistics practitioners and the general public to appropriately deal with transport items under different conditions to minimize the risk of epidemic transmission. IMPORTANCE This study shows the environmental stability of SARS-CoV-2 Variants Omicron BA.1 and BA.5 on surfaces of widely used transport packaging materials. The findings demonstrate that the environmental stability of the SARS-CoV-2 Omicron variants varies based on material type. The viability of SARS-CoV-2 on material surfaces depends heavily on temperature and viral titer. Low temperatures and high viral titers promote virus survival. Moreover, in contrast to virus viability, virus nucleic acid exhibits high stability on the surfaces of widely used materials, making the detection of virus nucleic acid unsuitable for evaluating the risk of epidemic transmission.

Omicron BA.1 breakthrough infections in inactivated COVID-19 vaccine recipients induced distinct pattern of antibody and T cell responses to different Omicron sublineages.

The adaptive immunity against SARS-CoV-2 prototype strain and Omicron sublineages induced by BA.1 breakthrough infection in vaccinees of inactivated COVID-19 vaccines have not been well characterized. Here, we report that BA.1 breakthrough infection induced mucosal sIgA and resulted in higher IgG titers against prototype strain and Omicron sublineages in vaccinees than in vaccine naïve-infected individuals. BA.1 breakthrough infection boosted antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis to prototype strain and BA.1, BA.1.1, BA.2, BA.2.12.1, and BA.2.75 but not BA.4/5 and induced neutralization against prototype strain and BA.1, BA.1.1, BA.2, BA.2.12.1, BA.2.75, and BA.4/5 but not BF.7, BQ.1, and XBB. In total, BA.1 breakthrough infection individuals produced less extensive sIgA, plasma IgG and NAb responses against Omicron sublineages compared with those against prototype strain. Further, BA.1 breakthrough infection induced recall B cell response to prototype strain and Omicron variant, primarily targeting memory B cells producing conserved epitopes. Memory T cell responses against Omicron is largely preserved. Individuals with vaccine booster did not induce more beneficial immune responses to Omicron sublineages upon BA.1 breakthrough infection than those with primary vaccine dose only. The breakthrough infection individuals produced stronger adaptive immunity than those of inactivated vaccine-healthy individuals. These data have important implications for understanding the vaccine effectiveness and adaptive immunity to breakthrough infection in individuals fully immunized with inactivated vaccines. Omicron sublineages, especially for those emerged after BA.4/5 strain, evade NAb responses induced by BA.1 breakthrough infection. It is urgent to optimize the vaccine immunogen design and formulations to SARS-CoV-2 variants.

Deep Learning of Dual Plasma Fingerprints for High-Performance Infection Classification.

Infection classification is the key for choosing the proper treatment plans. Early determination of the causative agents is critical for disease control. Host responses analysis can detect variform and sensitive host inflammatory responses to ascertain the presence and type of the infection. However, traditional host-derived inflammatory indicators are insufficient for clinical infection classification. Fingerprints-based omic analysis has attracted increasing attention globally for analyzing the complex host systemic immune response. A single type of fingerprints is not applicable for infection classification (area under curve (AUC) of 0.550-0.617). Herein, an infection classification platform based on deep learning of dual plasma fingerprints (DPFs-DL) is developed. The DPFs with high reproducibility (coefficient of variation <15%) are obtained at low sample consumption (550 nL native plasma) using inorganic nanoparticle and organic matrix assisted laser desorption/ionization mass spectrometry. A classifier (DPFs-DL) for viral versus bacterial infection discrimination (AUC of 0.775) and coronavirus disease 2019 (COVID-2019) diagnosis (AUC of 0.917) is also built. Furthermore, a metabolic biomarker panel of two differentially regulated metabolites, which may serve as potential biomarkers for COVID-19 management (AUC of 0.677-0.883), is constructed. This study will contribute to the development of precision clinical care for infectious diseases.

SARS-CoV-2-specific antibody and T-cell responses 1 year after infection in people recovered from COVID-19: a longitudinal cohort study.

Background: The memory immune response is crucial for preventing reinfection or reducing disease severity. However, the robustness and functionality of the humoral and T-cell response to SARS-CoV-2 remains unknown 12 months after initial infection. The aim of this study is to investigate the durability and functionality of the humoral and T-cell response to the original SARS-CoV-2 strain and variants in recovered patients 12 months after infection. Methods: In this longitudinal cohort study, we recruited participants who had recovered from COVID-19 and who were discharged from the Wuhan Research Center for Communicable Disease Diagnosis and Treatment at the Chinese Academy of Medical Sciences, Wuhan, China, between Jan 7 and May 29, 2020. Patients received a follow-up visit between Dec 16, 2020, and Jan 27, 2021. We evaluated the presence of IgM, IgA, and IgG antibodies against the SARS-CoV-2 nucleoprotein, Spike protein, and the receptor-binding domain 12 months after initial infection, using ELISA. Neutralising antibodies against the original SARS-CoV-2 strain, and the D614G, beta (B.1.351), and delta (B.1.617.2) variants were analysed using a microneutralisation assay in a subset of plasma samples. We analysed the magnitude and breadth of the SARS-CoV-2-specific memory T-cell responses using the interferon γ (IFNγ) enzyme-linked immune absorbent spot (ELISpot) assay and intracellular cytokine staining (ICS) assay. The antibody response and T-cell response (ie, IFN-γ, interleukin-2 [IL-2], and tumour necrosis factor α [TNFα]) were analysed by age and disease severity. Antibody titres were also analysed according to sequelae symptoms. Findings: We enrolled 1096 patients, including 289 (26·4%) patients with moderate initial disease, 734 (67·0%) with severe initial disease, and 73 (6·7%) with critical initial disease. Paired plasma samples were collected from 141 patients during the follow-up visits for the microneutralisation assay. PBMCs were collected from 92 of 141 individuals at the 12-month follow-up visit, of which 80 were analysed by ELISpot and 92 by ICS assay to detect the SARS-CoV-2-specific memory T-cell responses. N-IgG (899 [82·0%]), S-IgG (1043 [95·2%]), RBD-IgG (1032 [94·2%]), and neutralising (115 [81·6%] of 141) antibodies were detectable 12 months after initial infection in most individuals. Neutralising antibodies remained stable 6 and 12 months after initial infection in most individuals younger than 60 years. Multifunctional T-cell responses were detected for all SARS-CoV-2 viral proteins tested. There was no difference in the magnitude of T-cell responses or cytokine profiles in individuals with different symptom severity. Moreover, we evaluated both antibody and T-cell responses to the D614G, beta, and delta viral strains. The degree of reduced in-vitro neutralising antibody responses to the D614G and delta variants, but not to the beta variant, was associated with the neutralising antibody titres after SARS-CoV-2 infection. We also found poor neutralising antibody responses to the beta variant; 83 (72·2%) of 115 patients showed no response at all. Moreover, the neutralising antibody titre reduction of the recovered patient plasma against the delta variant was similar to that of the D614G variant and lower than that of the beta variant. By contrast, T-cell responses were cross-reactive to the beta variant in most individuals. Importantly, T-cell responses could be detected in all individuals who had lost the neutralising antibody response to SARS-CoV-2 12 months after the initial infection. Interpretation: SARS-CoV-2-specific neutralising antibody and T-cell responses were retained 12 months after initial infection. Neutralising antibodies to the D614G, beta, and delta viral strains were reduced compared with those for the original strain, and were diminished in general. Memory T-cell responses to the original strain were not disrupted by new variants. This study suggests that cross-reactive SARS-CoV-2-specific T-cell responses could be particularly important in the protection against severe disease caused by variants of concern whereas neutralising antibody responses seem to reduce over time. Funding: Chinese Academy of Medical Sciences, National Natural Science Foundation, and UK Medical Research Council.

Humoral responses after inactivated COVID-19 vaccination in individuals with and without prior SARS-CoV-2 infection: A prospective cohort study.

We evaluated and compared humoral immune responses after inactivated coronavirus disease 2019 (COVID-19) vaccination among naïve individuals, asymptomatically infected individuals, and recovered patients with varying severity. In this multicenter, prospective cohort study, blood samples from 666 participants were collected before and after 2 doses of inactivated COVID-19 vaccination. Among 392 severe acute respiratory syndrome coronavirus 2-naïve individuals, the seroconversion rate increased significantly from 51.8% (median antispike protein pan-immunoglobulins [S-Igs] titer: 0.8 U/ml) after the first dose to 96% (median S-Igs titer: 79.5 U/ml) after the second dose. Thirty-two percent of naïve individuals had detectable neutralizing antibodies (NAbs) against the original strain but all of them lost neutralizing activity against the Omicron variant. In 274 individuals with natural infection, humoral immunity was significantly improved after a single vaccine dose, with median S-Igs titers of 596.7, 1176, 1086.5, and 1828 U/ml for asymptomatic infections, mild cases, moderate cases, and severe/critical cases, respectively. NAb titers also improved significantly. However, the second dose did not substantially increase antibody levels. Although a booster dose is needed for those without infection, our findings indicate that recovered patients should receive only a single dose of the vaccine, regardless of the clinical severity, until there is sufficient evidence to confirm the benefits of a second dose.

Identification of a novel coronavirus causing severe pneumonia in human: a descriptive study.

BACKGROUND: Human infections with zoonotic coronaviruses (CoVs), including severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV, have raised great public health concern globally. Here, we report a novel bat-origin CoV causing severe and fatal pneumonia in humans. METHODS: We collected clinical data and bronchoalveolar lavage (BAL) specimens from five patients with severe pneumonia from Wuhan Jinyintan Hospital, Hubei province, China. Nucleic acids of the BAL were extracted and subjected to next-generation sequencing. Virus isolation was carried out, and maximum-likelihood phylogenetic trees were constructed. RESULTS: Five patients hospitalized from December 18 to December 29, 2019 presented with fever, cough, and dyspnea accompanied by complications of acute respiratory distress syndrome. Chest radiography revealed diffuse opacities and consolidation. One of these patients died. Sequence results revealed the presence of a previously unknown ß-CoV strain in all five patients, with 99.8% to 99.9% nucleotide identities among the isolates. These isolates showed 79.0% nucleotide identity with the sequence of SARS-CoV (GenBank NC_004718) and 51.8% identity with the sequence of MERS-CoV (GenBank NC_019843). The virus is phylogenetically closest to a bat SARS-like CoV (SL-ZC45, GenBank MG772933) with 87.6% to 87.7% nucleotide identity, but is in a separate clade. Moreover, these viruses have a single intact open reading frame gene 8, as a further indicator of bat-origin CoVs. However, the amino acid sequence of the tentative receptor-binding domain resembles that of SARS-CoV, indicating that these viruses might use the same receptor. CONCLUSION: A novel bat-borne CoV was identified that is associated with severe and fatal respiratory disease in humans.

Serum 8-Hydroxydeoxyguanosine Is a Potential Indicator for the Severity and Prognosis in Patients with Community-Acquired Pneumonia: A Prospective Cohort Study.

Previous studies have demonstrated that 8-hydroxydeoxyguanosine (8-OHdG) exerted key roles in various pulmonary diseases, but the evidence for its role in community-acquired pneumonia (CAP) was lacking. The goal of this research was to evaluate the correlations of serum 8-OHdG with the severity and prognosis among patients with CAP through a prospective cohort study. A total of 239 patients with CAP and 239 healthy participants were enrolled. Fasting blood samples were collected. 8-OHdG and inflammatory cytokines were measured by ELISA. On admission, serum 8-OHdG was significantly increased in patients with CAP compared with control subjects. Besides, serum 8-OHdG was incrementally increased in line with CAP severity scores. Pearson correlative analysis found that serum 8-OHdG was correlated with clinical characteristics and inflammatory cytokines in patients with CAP. Linear and logistic regression analysis showed that serum 8-OHdG was positively associated with CAP severity scores. Furthermore, the prognostic outcomes were tracked. Higher serum 8-OHdG on admission increased the risks for intensive care unit admission, mechanical ventilation, vasoactive agent usage, death, and longer hospital stay among patients with CAP. Serum 8-OHdG combination with confusion, respiratory rate, blood pressure, and age ≥65 y or pneumonia severity index had stronger predictive powers for death than single 8-OHdG, CAP severity scores, or several inflammatory cytokines in patients with CAP. These results indicated that serum 8-OHdG is positively associated with the severity and poor prognosis in patients with CAP, demonstrating that 8-OHdG may be involved in the pathophysiology process of CAP.

Decrease in ambient volatile organic compounds during the COVID-19 lockdown period in the Pearl River Delta region, south China.

During the COVID-19 lockdown, ambient ozone levels are widely reported to show much smaller decreases or even dramatical increases under substantially reduced precursor NOx levels, yet changes in ambient precursor volatile organic compounds (VOCs) have been scarcely reported during the COVID-19 lockdown, which is an opportunity to examine the impacts of dramatically changing anthropogenic emissions on ambient VOC levels in megacities where ozone formation is largely VOC-limited. In this study, ambient VOCs were monitored online at an urban site in Guangzhou in the Pearl River Delta region before, during, and after the COVID-19 lockdown. The average total mixing ratios of VOCs became 19.1% lower during the lockdown than before, and those of alkanes, alkenes and aromatics decreased by 19.0%, 24.8% and 38.2%, respectively. The levels of light alkanes (C < 6) decreased by only 13.0%, while those of higher alkanes (C ≥ 6) decreased by 67.8% during the lockdown. Disappeared peak VOC levels in morning rush hours and the drop in toluene to benzene ratios during the lockdown suggested significant reductions in vehicle exhaust and industrial solvent emissions. Source apportioning by positive matrix factorization model revealed that reductions in industrial emissions, diesel exhaust (on-road diesel vehicles and off-road diesel engines) and gasoline-related emissions could account for 48.9%, 42.2% and 8.8%, respectively, of the decreased VOC levels during the lockdown. Moreover, the reduction in industrial emissions could explain 56.0% and 70.0% of the reductions in ambient levels of reactive alkenes and aromatics, respectively. An average increase in O3-1 h by 17% and a decrease in the daily maximum 8-h average ozone by 11% under an average decrease in NOx by 57.0% and a decrease in VOCs by 19.1% during the lockdown demonstrated that controlling emissions of precursors VOCs and NOx to prevent ambient O3 pollution in megacities such as Guangzhou remains a highly challenging task.

Dynamics of the Upper Respiratory Tract Microbiota and Its Association with Mortality in COVID-19.

Rationale: Alteration of human respiratory microbiota had been observed in coronavirus disease (COVID-19). How the microbiota is associated with the prognosis in COVID-19 is unclear. Objectives: To characterize the feature and dynamics of the respiratory microbiota and its associations with clinical features in patients with COVID-19. Methods: We conducted metatranscriptome sequencing on 588 longitudinal oropharyngeal swab specimens collected from 192 patients with COVID-19 (including 39 deceased patients) and 95 healthy controls from the same geographic area. Meanwhile, the concentration of 27 cytokines and chemokines in plasma was measured for patients with COVID-19. Measurements and Main Results: The upper respiratory tract (URT) microbiota in patients with COVID-19 differed from that in healthy controls, whereas deceased patients possessed a more distinct microbiota, both on admission and before discharge/death. The alteration of URT microbiota showed a significant correlation with the concentration of proinflammatory cytokines and mortality. Specifically, Streptococcus-dominated microbiota was enriched in recovered patients, and showed high temporal stability and resistance against pathogens. In contrast, the microbiota in deceased patients was more susceptible to secondary infections and became more deviated from the norm after admission. Moreover, the abundance of S. parasanguinis on admission was significantly correlated with prognosis in nonsevere patients (lower vs. higher abundance, odds ratio, 7.80; 95% CI, 1.70-42.05). Conclusions: URT microbiota dysbiosis is a remarkable manifestation of COVID-19; its association with mortality suggests it may reflect the interplay between pathogens, symbionts, and the host immune status. Whether URT microbiota could be used as a biomarker for diagnosis and prognosis of respiratory diseases merits further investigation.

Substantial changes of chemical composition and sources of fine particles during the period of COVID-19 pandemic in Taiyuan, Northern China.

To better understand the effects of COVID-19 on air quality in Taiyuan, hourly in situ measurements of PM2.5(particulate matter with an aerodynamic diameter less than 2.5 mm) and chemical components (water-soluble ions, organic carbon (OC), elemental carbon (EC), and trace elements) were conducted before (P1: 1 January-23 January 2020) and during (P2: 24 January-15 February 2020) the coronavirus disease 2019 (COVID-19) outbreak. The average concentrations of PM2.5 dropped from 122.0 µg/m3 during P1 to 83.3 µg/m3 during P2. Compared with P1, except for fireworks burning-related chemical components (K+, Mg2+, K, Cu, Ba), the concentrations of other chemical components of PM2.5 decreased by14.9-69.8%. Although the large decrease of some emission sources, fireworks burning still resulted in the occurrence of pollution events during P2. The analysis results of positive matrix factorization model suggested that six PM2.5 sources changed significantly before and during the outbreak of the epidemic. The contributions of vehicle emission, industrial process, and dust to PM2.5 decreased from 23.1%, 3.5%, and 4.0% during P1 to 7.7%, 3.4%, and 2.3% during P2, respectively, whereas the contributions of secondary inorganic aerosol, fireworks burning, and coal combustion to PM2.5 increased from 62.0%, 1.8%, and 5.5% to 71.5%, 9.0%, and 6.2%, respectively. The source apportionment results were also affected by air mass transport. The largest reductions of vehicle emission, industrial process, and dust source were distinctly seen for the air masses from northwest. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11869-021-01082-y.

SARS-CoV-2-specific immune response in COVID-19 convalescent individuals.

We collected blood from coronavirus disease 2019 (COVID-19) convalescent individuals and investigated SARS-CoV-2-specific humoral and cellular immunity in these discharged patients. Follow-up analysis in a cohort of 171 patients at 4-11 months after the onset revealed high levels of IgG antibodies. A total of 78.1% (164/210) of the specimens tested positive for neutralizing antibody (NAb). SARS-CoV-2 antigen peptide pools-stimulated-IL-2 and -IFN-γ response can distinguish COVID-19 convalescent individuals from healthy donors. Interestingly, NAb survival was significantly affected by the antigen peptide pools-stimulated-IL-2 response, -IL-8 response, and -IFN-γ response. The antigen peptide pools-activated CD8+ T cell counts were correlated with NAb. The antigen peptide pools-activated natural killer (NK) cell counts in convalescent individuals were correlated with NAb and disease severity. Our data suggested that the development of NAb is associated with the activation of T cells and NK cells. Our work provides a basis for further analysis of the protective immunity to SARS-CoV-2 and for understanding the pathogenesis of COVID-19. It also has implications for the development of an effective vaccine for SARS-CoV-2 infection.

High anal swab viral load predisposes adverse clinical outcomes in severe COVID-19 patients.

To identify the association between the kinetics of viral load and clinical outcome in severe coronavirus disease 2019 (COVID-19) patients, a retrospective study was performed by involved 188 hospitalized severe COVID-19 patients in the LOTUS China trial. Among the collected 578 paired throat swab (TS) and anal swab (AS) samples, viral RNA was detected in 193 (33.4%) TS and 121 (20.9%) AS. A higher viral RNA load was found in TS than that of AS, with means of 1.0 × 106 and 2.3 × 105 copies/ml, respectively. In non-survivors, the viral RNA in AS was detected earlier than that in survivors (median of 14 days vs 19 days, P = 0.007). The positivity and viral load in AS were higher in non-survivors than that of survivors at week 2 post symptom onset (P = 0.006). A high initial viral load in AS was associated with death (OR 1.368, 95% CI 1.076-1.741, P = 0.011), admission to the intensive care unit (OR 1.237, 95% CI 1.001-1.528, P = 0.049) and need for invasive mechanical ventilation (OR 1.340, 95% CI 1.076-1.669, P = 0.009). Our findings indicated viral replication in extrapulmonary sites should be monitored intensively during antiviral therapy.

Comparison of three TaqMan real-time reverse transcription-PCR assays in detecting SARS-CoV-2.

Quick and accurate detection of SARS-CoV-2 is critical for COVID-19 control. Dozens of real-time reverse transcription PCR (qRT-PCR) assays have been developed to meet the urgent need of COVID-19 control. However, methodological comparisons among the developed qRT-PCR assays are limited. In the present study, we evaluated the sensitivity, specificity, amplification efficiency, and linear detection ranges of three qRT-PCR assays, including the assays developed by our group (IPBCAMS), and the assays recommended by WHO and China CDC (CCDC). The three qRT-PCR assays exhibited similar sensitivities, with the limit of detection (LoD) at about 10 copies per reaction (except the ORF 1b gene assay in CCDC assays with a LoD at about 100 copies per reaction). No cross reaction with other respiratory viruses were observed in all of the three qRT-PCR assays. Wide linear detection ranges from 106 to 101 copies per reaction and acceptable reproducibility were obtained. By using 25 clinical specimens, the N gene assay of IPBCAMS assays and CCDC assays performed better (with detection rates of 92 % and 100 %, respectively) than that of the WHO assays (with a detection rate of 60 %), and the ORF 1b gene assay in IPBCAMS assays performed better (with a detection rate of 64 %) than those of the WHO assays and the CCDC assays (with detection rates of 48 % and 20 %, respectively). In conclusion, the N gene assays of CCDC assays and IPBCAMS assays and the ORF 1b gene assay of IPBCAMS assays were recommended for qRT-PCR screening of SARS-CoV-2.

Development of two TaqMan real-time reverse transcription-PCR assays for the detection of severe acute respiratory syndrome coronavirus-2.

The outbreak of coronavirus disease 2019 (COVID-19) in Wuhan, China, was caused by a novel coronavirus (CoV), named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The rapid detection of viral nucleic acids is critical for the early identification of infected cases. We have developed two TaqMan real-time reverse transcription-PCR assays to detect SARS-CoV-2. The designed primers target the nucleocapsid (N) and open reading frame (ORF) 1b gene regions, where the probes discriminate SARS-CoV-2 from other human and animal CoVs. The sensitivities are one genomic copy per reaction for the N gene assay and ten copies for the ORF 1b gene assay. The overall linear detection ranges are 1-106 and 10-106 copies per reaction for the N gene assay and the ORF 1b gene assay, respectively. Surveillance of 23 suspected COVID-19 patients demonstrated that SARS-CoV-2 could be detected from 100% (23/23) and 62.5% (16/23) of clinical specimens by the N gene assay and the ORF 1b gene assay, respectively. All of the samples not detected by the ORF 1b gene assay were throat swabs, indicating a lower viral load in the upper respiratory tract and the relatively lower sensitivity of the ORF 1b gene assay. The assays developed in the present study offer alternative diagnostic tests for COVID-19.

The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19), which has become a public health emergency of international concern1. Angiotensin-converting enzyme 2 (ACE2) is the cell-entry receptor for severe acute respiratory syndrome coronavirus (SARS-CoV)2. Here we infected transgenic mice that express human ACE2 (hereafter, hACE2 mice) with SARS-CoV-2 and studied the pathogenicity of the virus. We observed weight loss as well as virus replication in the lungs of hACE2 mice infected with SARS-CoV-2. The typical histopathology was interstitial pneumonia with infiltration of considerable numbers of macrophages and lymphocytes into the alveolar interstitium, and the accumulation of macrophages in alveolar cavities. We observed viral antigens in bronchial epithelial cells, macrophages and alveolar epithelia. These phenomena were not found in wild-type mice infected with SARS-CoV-2. Notably, we have confirmed the pathogenicity of SARS-CoV-2 in hACE2 mice. This mouse model of SARS-CoV-2 infection will be valuable for evaluating antiviral therapeutic agents and vaccines, as well as understanding the pathogenesis of COVID-19.