Development of a multiplex qRT-PCR assay for the detection of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine Deltacoronavirus.

Currently, porcine coronaviruses are prevalent in pigs, and due to the outbreak of COVID-19, porcine coronaviruses have become a research hotspot. porcine epidemic diarrhea virus (PEDV), Transmissible Gastroenteritis Virus (TGEV), and Porcine Deltacoronavirus (PDCoV) mentioned in this study mainly cause diarrhea in pigs. These viruses cause significant economic losses and pose a potential public health threat. In this study, specific primers and probes were designed according to the M gene of PEDV, the S gene of TGEV, and the M gene of PDCoV, respectively, and TaqMan probe-based multiplex real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was developed for the simultaneous detection of PEDV, TGEV, and PDCoV. This method has high sensitivity and specificity, and the detection limit of each virus can reach 2.95 × 100 copies/µl. An assay of 160 clinical samples from pigs with diarrhea showed that the positive rates of PEDV, TGEV, and PDCoV were 38.13, 1.88, and 5.00%; the coinfection rates of PEDV+TGEV, PEDV+PDCoV, TGEV+PDCoV, PEDV+TGEV+PDCoV were 1.25, 1.25, 0, 0.63%, respectively. The positive coincidence rates of the multiplex qRT-PCR and single-reaction qRT-PCR were 100%. This method is of great significance for clinical monitoring of the porcine enteric diarrhea virus and helps reduce the loss of the breeding industry and control the spread of the disease.

Higher SARS-CoV-2 shedding in exhaled aerosol probably contributed to the enhanced transmissibility of Omicron BA.5 subvariant.

The global pandemic of the BA.5 subvariant had moved from prediction to reality. In this study, we compared SARS-CoV-2 aerosol emissions from patients with BA.2 or BA.5 subvariant infection. First, patients with BA.2 subvariant infection had higher upper respiratory viral loads than patients with BA.5 subvariant infection. However, the average breath emission rate (BER) of patients with BA.5 subvariant infection, which represented the concentration of exhaled SARS-CoV-2 aerosols, was nearly 40 times higher than that of patients with BA.2 subvariant. Second, aerosols exhaled by patients with BA.5 subvariant infection exhibited SARS-CoV-2 RNA detection positive rate than patients with BA.1 or BA.2 subvariant infection. Meanwhile, for BA.5 subvariant infection, patients that exhaled infectious SARS-CoV-2 aerosols accounted for 14.8% of all patients. Third, since the onset of COVID-19, the SARS-CoV-2 RNA detection signals of throat swabs showed a gradual decline trend, although the decline process was accompanied by fluctuations. Overall, the monitoring of infectious SARS-CoV-2 aerosols may provide the data support for the transmissibility evaluation of the Omicron BA.5 subvariant. This article is protected by copyright. All rights reserved.

Emerging Omicron subvariants evade neutralizing immunity elicited by vaccine or BA.1/BA.2 infection.

The newly emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.2.75 and BA.2.76 subvariants contained 35 and 29 additional mutations in its spike (S) protein compared with the reference SARS-CoV-2 genome, respectively. Here, we measured the evasion degree of the BA.1, BA.2, BA.4, BA.5, BA.2.75, and BA.2.76 subvariants from neutralizing immunity in people previously infected with the Omicron BA.1 and BA.2, determined the effect of vaccination on immune evasion, and compared the titers of neutralizing antibodies in serums between acute infection and convalescence. Results showed that the neutralization effect of serums from patients with different vaccination statuses and BA.1/BA.2 breakthrough infection decreased with the Omicron evolution from BA.1 to BA.2, BA.4, BA.5, BA.2.75, and BA.2.76. This study also indicated that the existing vaccines could no longer provide effective protection, especially for the emerging BA.2.75 and BA.2.76 subvariants. Therefore, vaccines against emerging epidemic strains should be designed specifically. In the future, we can not only focus on the current strains, but also predict and design new vaccines against potential mutant strains. At the same time, we can combine the virus strains' infection characteristics to develop protective measures for virus colonization areas, such as nasal protection spray. Besides, further studies on the Y248N mutation of BA.2.76 subvariant were also necessary to explore its contribution to the enhanced immune evasion ability.

Randomized, Double-Blinded, Placebo-Controlled Phase 2 Trial of an Inactivated Severe Acute Respiratory Syndrome Coronavirus 2 Vaccine in Healthy Adults.

BACKGROUND: We evaluated an inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine for immunogenicity and safety in adults aged 18-59 years. METHODS: In this randomized, double-blinded, controlled trial, healthy adults received a medium dose (MD) or a high dose (HD) of the vaccine at an interval of either 14 days or 28 days. Neutralizing antibody (NAb) and anti-S and anti-N antibodies were detected at different times, and adverse reactions were monitored for 28 days after full immunization. RESULTS: A total of 742 adults were enrolled in the immunogenicity and safety analysis. Among subjects in the 0, 14 procedure, the seroconversion rates of NAb in MD and HD groups were 89% and 96% with geometric mean titers (GMTs) of 23 and 30, respectively, at day 14 and 92% and 96% with GMTs of 19 and 21, respectively, at day 28 after immunization. Anti-S antibodies had GMTs of 1883 and 2370 in the MD group and 2295 and 2432 in the HD group. Anti-N antibodies had GMTs of 387 and 434 in the MD group and 342 and 380 in the HD group. Among subjects in the 0, 28 procedure, seroconversion rates for NAb at both doses were both 95% with GMTs of 19 at day 28 after immunization. Anti-S antibodies had GMTs of 937 and 929 for the MD and HD groups, and anti-N antibodies had GMTs of 570 and 494 for the MD and HD groups, respectively. No serious adverse events were observed during the study period. CONCLUSIONS: Adults vaccinated with inactivated SARS-CoV-2 vaccine had NAb as well as anti-S/N antibody and had a low rate of adverse reactions. CLINICAL TRIALS REGISTRATION: NCT04412538.

Common mtDNA variations at C5178a and A249d/T6392C/G10310A decrease the risk of severe COVID-19 in a Han Chinese population from Central China.

BACKGROUND: Mitochondria have been shown to play vital roles during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and coronavirus disease 2019 (COVID-19) development. Currently, it is unclear whether mitochondrial DNA (mtDNA) variants, which define mtDNA haplogroups and determine oxidative phosphorylation performance and reactive oxygen species production, are associated with COVID-19 risk. METHODS: A population-based case-control study was conducted to compare the distribution of mtDNA variations defining mtDNA haplogroups between healthy controls (n = 615) and COVID-19 patients (n = 536). COVID-19 patients were diagnosed based on molecular diagnostics of the viral genome by qPCR and chest X-ray or computed tomography scanning. The exclusion criteria for the healthy controls were any history of disease in the month preceding the study assessment. MtDNA variants defining mtDNA haplogroups were identified by PCR-RFLPs and HVS-I sequencing and determined based on mtDNA phylogenetic analysis using Mitomap Phylogeny. Student's t-test was used for continuous variables, and Pearson's chi-squared test or Fisher's exact test was used for categorical variables. To assess the independent effect of each mtDNA variant defining mtDNA haplogroups, multivariate logistic regression analyses were performed to calculate the odds ratios (ORs) and 95% confidence intervals (CIs) with adjustments for possible confounding factors of age, sex, smoking and diseases (including cardiopulmonary diseases, diabetes, obesity and hypertension) as determined through clinical and radiographic examinations. RESULTS: Multivariate logistic regression analyses revealed that the most common investigated mtDNA variations (> 10% in the control population) at C5178a (in NADH dehydrogenase subunit 2 gene, ND2) and A249d (in the displacement loop region, D-loop)/T6392C (in cytochrome c oxidase I gene, CO1)/G10310A (in ND3) were associated with a reduced risk of severe COVID-19 (OR = 0.590, 95% CI 0.428-0.814, P = 0.001; and OR = 0.654, 95% CI 0.457-0.936, P = 0.020, respectively), while A4833G (ND2), A4715G (ND2), T3394C (ND1) and G5417A (ND2)/C16257a (D-loop)/C16261T (D-loop) were related to an increased risk of severe COVID-19 (OR = 2.336, 95% CI 1.179-4.608, P = 0.015; OR = 2.033, 95% CI 1.242-3.322, P = 0.005; OR = 3.040, 95% CI 1.522-6.061, P = 0.002; and OR = 2.890, 95% CI 1.199-6.993, P = 0.018, respectively). CONCLUSIONS: This is the first study to explore the association of mtDNA variants with individual's risk of developing severe COVID-19. Based on the case-control study, we concluded that the common mtDNA variants at C5178a and A249d/T6392C/G10310A might contribute to an individual's resistance to developing severe COVID-19, whereas A4833G, A4715G, T3394C and G5417A/C16257a/C16261T might increase an individual's risk of developing severe COVID-19.

Immunological evaluation of an inactivated SARS-CoV-2 vaccine in rhesus macaques.

Because of the relatively limited understanding of coronavirus disease 2019 (COVID-19) pathogenesis, immunological analysis for vaccine development is needed. Mice and macaques were immunized with an inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine prepared by two inactivators. Various immunological indexes were tested, and viral challenges were performed on day 7 or 150 after booster immunization in monkeys. This inactivated SARS-CoV-2 vaccine was produced by sequential inactivation with formaldehyde followed by propiolactone. The various antibody responses and specific T cell responses to different viral antigens elicited in immunized animals were maintained for longer than 150 days. This comprehensive immune response could effectively protect vaccinated macaques by inhibiting viral replication in macaques and substantially alleviating immunopathological damage, and no clinical manifestation of immunopathogenicity was observed in immunized individuals during viral challenge. This candidate inactivated vaccine was identified as being effective against SARS-CoV-2 challenge in rhesus macaques.

The safety and immunogenicity of an inactivated SARS-CoV-2 vaccine in Chinese adults aged 18-59 years: A phase I randomized, double-blinded, controlled trial.

BACKGROUND: This study examined the safety and immunogenicity of an inactivated SARS-CoV-2 vaccine. METHOD: In a phase I randomized, double-blinded, placebo-controlled trial involving 192 healthy adults 18-59 years old, two injections of three doses (50 EU, 100 EU, 150 EU) of an inactivated SARS-CoV-2 vaccine or placebo were administered intramuscularly at a 2- or 4-week interval. The safety and immunogenicity of the vaccine were evaluated. RESULTS: Vaccination was completed in 191 subjects. Forty-four adverse reactions occurred within 28 days, most commonly mild pain and redness at the injection site or slight fatigue. At days 14 and 28, the seroconversion rates were 87.5% and 79.2% (50 EU), 100% and 95.8% (100 EU), and 95.8% and 87.5% (150 EU), respectively, with geometric mean titers (GMTs) of 18.1 and 10.6, 54.5 and 15.4, and 37.1 and 18.5, respectively, for the schedules with 2-week and 4-week intervals. Seroconversion was associated with synchronous upregulation of antibodies against the S protein, N protein and virion and a cytotoxic T lymphocyte (CTL) response. No cytokines and immune cells related to immunopathology were observed. Transcriptome analysis revealed the genetic diversity of immune responses induced by the vaccine. INTERPRETATION: In a population aged 18-59 years in this trial, this inactivated SARS-CoV-2 vaccine was safe and immunogenic. TRIAL REGISTRATION: CTR20200943 and NCT04412538.

High resolution computed tomography for the diagnosis of 2019 novel coronavirus (2019-nCoV) pneumonia: a study from multiple medical centers in western China.

BACKGROUND: To evaluate the role of high-resolution computed tomography (HRCT) in the diagnosis of 2019 novel coronavirus (2019-nCoV) pneumonia and to provide experience in the early detection and diagnosis of 2019-nCoV pneumonia. METHODS: Seventy-two patients confirmed to be infected with 2019-nCoV from multiple medical centers in western China were retrospectively analyzed, including epidemiologic characteristics, clinical manifestations, laboratory findings and HRCT chest features. RESULTS: All patients had lung parenchymal abnormalities on HRCT scans, which were mostly multifocal in both lungs and asymmetric in all patients, and were mostly in the peripheral or subpleural lung regions in 52 patients (72.22%), in the central lung regions in 16 patients (22.22%), and in both lungs with "white lung" manifestations in 4 patients (5.56%). Subpleural multifocal consolidation was a predominant abnormality in 38 patients (52.78%). Ground-glass opacity was seen in 34 patients (47.22%). Interlobular septal thickening was found in 18 patients, 8 of whom had only generally mild thickening with no zonal predominance. Reticulation was seen in 8 patients (11.11%), and was mild and randomly distributed. In addition, both lungs of 28 patients had 2 or 3 CT imaging features. Out of these 72 patients, 36 were diagnosed as early stage, 32 patients as progressive stage, and 4 patient as severe stage pneumonia. Moreover, the diagnostic accuracy of HRCT features combined with epidemiological history was not significantly different from the detection of viral nucleic acid (all P >0.05). CONCLUSIONS: The HRCT features of 2019-nCoV pneumonia are characteristic to a certain degree, which when combined with epidemiological history yield high clinical value in the early detection and diagnosis of 2019-nCoV pneumonia.

High resolution computed tomography for the diagnosis of 2019 novel coronavirus (2019-nCoV) pneumonia: a study from multiple medical centers in western China (preprint)

Objective: To evaluate the role of high-resolution computed tomography (HRCT) in the diagnosis of 2019 novel coronavirus (2019-nCoV) pneumonia and to provide experience in the early detection and diagnosis of 2019-nCoV pneumonia. Methods: 72 patients confirmed to be infected with 2019-nCoV from multiple medical centers in western China were retrospectively analyzed, including epidemiologic characteristics, clinical manifestations, laboratory findings and HRCT chest features. Results: All patients had lung parenchymal abnormalities on HRCT scans, which were mostly multifocal in both lungs and asymmetric in all patients, and were mostly in the peripheral or subpleural lung regions in 52 patients (72.22%), in the central lung regions in sixteen (22.22%), and in both lungs, with "white lung "manifestations in four (5.56%). Subpleural multifocal consolidation was predominant abnormality in 38 patients (52.78%). Ground-glass opacity was seen in 34 patients (47.22%). Interlobular septal thickening was found in 18patients, of which eight had only generally mild thickening with no zonal predominance. Reticulation was seen in 8 patients (11.11%), in all of whom it was mild and randomly distributed. In addition, both lungs of 28 patients had two or three CT imaging features. Out of these 72 patients, 36 were diagnosed as early stage, 32 patients as progressive stage and 4 patient as severe stage pneumonia. Moreover, the diagnostic accuracy of HRCT features combined with epidemiological history was not significantly different from the detection of viral nucleic acid (all P >0.05). Conclusion: The HRCT features of 2019-nCoV pneumonia are characteristic to a certain degree, which when combined with epidemiological history yield high clinical value in the early detection and diagnosis of 2019-nCoV pneumonia.Authors Hong-Wei Li, Li-Hua Zhuo, Gao-Wu Yan contributed equally to this work.