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1.
Front Cell Infect Microbiol ; 12: 880915, 2022.
Article in English | MEDLINE | ID: covidwho-1847156

ABSTRACT

With the epidemic of betacoronavirus increasing frequently, it poses a great threat to human public health. Therefore, the research on the pathogenic mechanism of betacoronavirus is becoming greatly important. Murine hepatitis virus strain-3 (MHV-3) is a strain of betacoronavirus which cause tissue damage especially fulminant hepatic failure (FHF) in mice, and is commonly used to establish models of acute liver injury. Recently, MHV-3-infected mice have also been introduced to a mouse model of COVID-19 that does not require a Biosafety Level 3 (BSL-3) facility. FHF induced by MHV-3 is a type of severe liver damage imbalanced by regenerative hepatocellular activity, which is related to numerous factors. The complement system plays an important role in host defense and inflammation and is involved in first-line immunity and/or pathogenesis of severe organ disorders. In this study, we investigated the role of aberrant complement activation in MHV-3 infection-induced FHF by strategies that use C3-deficient mice and intervene in the complement system. Our results showed that mice deficient in C3 had more severe liver damage, a higher viral load in the liver and higher serum concentrations of inflammatory cytokines than wild-type controls. Treatment of C57BL/6 mice with C3aR antagonist or anti-C5aR antibody reduced liver damage, viral load, and serum IFN-γ concentration compared with the control group. These findings indicated that complement system acts as a double-edged sword during acute MHV-3 infection. However, its dysregulated activation leads to sustained inflammatory responses and induces extensive liver damage. Collectively, by investigating the role of complement activation in MHV-3 infection, we can further understand the pathogenic mechanism of betacoronavirus, and appropriate regulation of immune responses by fine-tuning complement activation may be an intervention for the treatment of diseases induced by betacoronavirus infection.


Subject(s)
COVID-19 , Liver Failure, Acute , Murine hepatitis virus , Animals , Complement Activation , Liver Failure, Acute/pathology , Mice , Mice, Inbred C57BL
2.
Innovation (Camb) ; 3(2): 100221, 2022 Mar 29.
Article in English | MEDLINE | ID: covidwho-1713028

ABSTRACT

The highly pathogenic and readily transmissible SARS-CoV-2 has caused a global coronavirus pandemic, urgently requiring effective countermeasures against its rapid expansion. All available vaccine platforms are being used to generate safe and effective COVID-19 vaccines. Here, we generated a live-attenuated candidate vaccine strain by serial passaging of a SARS-CoV-2 clinical isolate in Vero cells. Deep sequencing revealed the dynamic adaptation of SARS-CoV-2 in Vero cells, resulting in a stable clone with a deletion of seven amino acids (N679SPRRAR685) at the S1/S2 junction of the S protein (named VAS5). VAS5 showed significant attenuation of replication in multiple human cell lines, human airway epithelium organoids, and hACE2 mice. Viral fitness competition assays demonstrated that VAS5 showed specific tropism to Vero cells but decreased fitness in human cells compared with the parental virus. More importantly, a single intranasal injection of VAS5 elicited a high level of neutralizing antibodies and prevented SARS-CoV-2 infection in mice as well as close-contact transmission in golden Syrian hamsters. Structural and biochemical analysis revealed a stable and locked prefusion conformation of the S trimer of VAS5, which most resembles SARS-CoV-2-3Q-2P, an advanced vaccine immunogen (NVAX-CoV2373). Further systematic antigenic profiling and immunogenicity validation confirmed that the VAS5 S trimer presents an enhanced antigenic mimic of the wild-type S trimer. Our results not only provide a potent live-attenuated vaccine candidate against COVID-19 but also clarify the molecular and structural basis for the highly attenuated and super immunogenic phenotype of VAS5.

3.
Front Microbiol ; 12: 799150, 2021.
Article in English | MEDLINE | ID: covidwho-1643526

ABSTRACT

Purpose: To investigate and characterize the putative Elizabethkingia anophelis contaminant isolated from throat and anal swab samples of patients from three fever epidemic clusters, which were not COVID-19 related, in Shenzhen, China, during COVID-19 pandemic. Methods: Bacteria were cultured from throat (n = 28) and anal (n = 3) swab samples from 28 fever adolescent patients. The isolated bacterial strains were identified using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) and the VITEK2 automated identification system. Nucleic acids were extracted from the patient samples (n = 31), unopened virus collection kits from the same manufacturer as the patient samples (n = 35, blank samples) and from unopened throat swab collection kits of two other manufacturers (n = 22, control samples). Metagenomic sequencing and quantitative real-time PCR (qPCR) detection were performed. Blood serum collected from patients (n = 13) was assessed for the presence of antibodies to E. anophelis. The genomic characteristics, antibiotic susceptibility, and heat resistance of E. anophelis isolates (n = 31) were analyzed. Results: The isolates were identified by MALDI-TOF/MS and VITEK2 as Elizabethkingia meningoseptica. DNA sequence analysis confirmed isolates to be E. anophelis. The patients' samples and blank samples were positive for E. anophelis. Control samples were negative for E. anophelis. The sera from a sub-sample of 13 patients were antibody-negative for isolated E. anophelis. Most of the isolates were highly homologous and carried multiple ß-lactamase genes (bla B, bla GOB, and bla CME). The isolates displayed resistance to nitrofurans, penicillins, and most ß-lactam drugs. The bacteria survived heating at 56°C for 30 min. Conclusion: The unopened commercial virus collection kits from the same manufacturer as those used to swab patients were contaminated with E. anophelis. Patients were not infected with E. anophelis and the causative agent for the fevers remains unidentified. The relevant authorities were swiftly notified of this discovery and subsequent collection kits were not contaminated. DNA sequence-based techniques are the definitive method for Elizabethkingia species identification. The E. anophelis isolates were multidrug-resistant, with partial heat resistance, making them difficult to eradicate from contaminated surfaces. Such resistance indicates that more attention should be paid to disinfection protocols, especially in hospitals, to avoid outbreaks of E. anophelis infection.

4.
Nat Commun ; 12(1): 5654, 2021 09 27.
Article in English | MEDLINE | ID: covidwho-1440471

ABSTRACT

There is an urgent need for animal models to study SARS-CoV-2 pathogenicity. Here, we generate and characterize a novel mouse-adapted SARS-CoV-2 strain, MASCp36, that causes severe respiratory symptoms, and mortality. Our model exhibits age- and gender-related mortality akin to severe COVID-19. Deep sequencing identified three amino acid substitutions, N501Y, Q493H, and K417N, at the receptor binding domain (RBD) of MASCp36, during in vivo passaging. All three RBD mutations significantly enhance binding affinity to its endogenous receptor, ACE2. Cryo-electron microscopy analysis of human ACE2 (hACE2), or mouse ACE2 (mACE2), in complex with the RBD of MASCp36, at 3.1 to 3.7 Å resolution, reveals the molecular basis for the receptor-binding switch. N501Y and Q493H enhance the binding affinity to hACE2, whereas triple mutations at N501Y/Q493H/K417N decrease affinity and reduce infectivity of MASCp36. Our study provides a platform for studying SARS-CoV-2 pathogenesis, and unveils the molecular mechanism for its rapid adaptation and evolution.


Subject(s)
COVID-19/diagnosis , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/metabolism , Animals , Binding Sites/genetics , COVID-19/mortality , COVID-19/virology , Disease Models, Animal , Female , Humans , Male , Mice , Protein Binding/genetics , Protein Domains/genetics , SARS-CoV-2/genetics , Severity of Illness Index , Spike Glycoprotein, Coronavirus/genetics
6.
Emerg Microbes Infect ; 9(1): 2368-2378, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-910382

ABSTRACT

Managing recovered COVID-19 patients with recurrent-positive SARS-CoV-2 RNA test results is challenging. We performed a population-based observational study to characterize the viral RNA level and serum antibody responses in recurrent-positive patients and evaluate their viral transmission risk. Of 479 recovered COVID-19 patients, 93 (19%) recurrent-positive patients were identified, characterized by younger age, with a median discharge-to-recurrent-positive length of 8 days. After readmission, recurrent-positive patients exhibited mild (28%) or absent (72%) symptoms, with no disease progression. The viral RNA level in recurrent-positive patients ranged from 1.8 to 5.7 log10 copies/mL (median: 3.2), which was significantly lower than the corresponding values at disease onset. There are generally no significant differences in antibody levels between recurrent-positive and non-recurrent-positive patients, or in recurrent-positive patients over time (before, during, or after recurrent-positive detection). Virus isolation of nine representative specimens returned negative results. Whole genome sequencing of six specimens yielded only genomic fragments. 96 close contacts and 1,200 candidate contacts of 23 recurrent-positive patients showed no clinical symptoms; their viral RNA (1,296/1,296) and antibody (20/20) tests were negative. After full recovery (no longer/never recurrent-positive), 60% (98/162) patients had neutralizing antibody titers of ≥1:32. Our findings suggested that an intermittent, non-stable excretion of low-level viral RNA may result in recurrent-positive occurrence, rather than re-infection. Recurrent-positive patients pose a low transmission risk, a relatively relaxed management of recovered COVID-19 patients is recommended.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , RNA, Viral/analysis , Adult , Betacoronavirus/genetics , Betacoronavirus/immunology , COVID-19 , COVID-19 Testing , Coronavirus Infections/therapy , Coronavirus Infections/transmission , Female , Genome, Viral/genetics , Humans , Male , Middle Aged , Pandemics , Pneumonia, Viral/therapy , Pneumonia, Viral/transmission , Recurrence , SARS-CoV-2 , Whole Genome Sequencing , Young Adult
7.
Science ; 369(6511): 1603-1607, 2020 09 25.
Article in English | MEDLINE | ID: covidwho-690532

ABSTRACT

The ongoing coronavirus disease 2019 (COVID-19) pandemic has prioritized the development of small-animal models for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We adapted a clinical isolate of SARS-CoV-2 by serial passaging in the respiratory tract of aged BALB/c mice. The resulting mouse-adapted strain at passage 6 (called MASCp6) showed increased infectivity in mouse lung and led to interstitial pneumonia and inflammatory responses in both young and aged mice after intranasal inoculation. Deep sequencing revealed a panel of adaptive mutations potentially associated with the increased virulence. In particular, the N501Y mutation is located at the receptor binding domain (RBD) of the spike protein. The protective efficacy of a recombinant RBD vaccine candidate was validated by using this model. Thus, this mouse-adapted strain and associated challenge model should be of value in evaluating vaccines and antivirals against SARS-CoV-2.


Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/prevention & control , Disease Models, Animal , Mice , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Viral Vaccines/immunology , Administration, Intranasal , Angiotensin-Converting Enzyme 2 , Animals , Betacoronavirus/genetics , Betacoronavirus/pathogenicity , COVID-19 , COVID-19 Vaccines , Coronavirus Infections/immunology , Female , High-Throughput Nucleotide Sequencing , Humans , Immunogenicity, Vaccine , Lung/virology , Lung Diseases, Interstitial/virology , Mice, Inbred BALB C , Mice, Transgenic , Mutation , Peptidyl-Dipeptidase A/genetics , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunology , Viral Vaccines/administration & dosage , Virulence/genetics
8.
Science ; 368(6491): 638-642, 2020 05 08.
Article in English | MEDLINE | ID: covidwho-20742

ABSTRACT

Responding to an outbreak of a novel coronavirus [agent of coronavirus disease 2019 (COVID-19)] in December 2019, China banned travel to and from Wuhan city on 23 January 2020 and implemented a national emergency response. We investigated the spread and control of COVID-19 using a data set that included case reports, human movement, and public health interventions. The Wuhan shutdown was associated with the delayed arrival of COVID-19 in other cities by 2.91 days. Cities that implemented control measures preemptively reported fewer cases on average (13.0) in the first week of their outbreaks compared with cities that started control later (20.6). Suspending intracity public transport, closing entertainment venues, and banning public gatherings were associated with reductions in case incidence. The national emergency response appears to have delayed the growth and limited the size of the COVID-19 epidemic in China, averting hundreds of thousands of cases by 19 February (day 50).


Subject(s)
Betacoronavirus , Coronavirus Infections/prevention & control , Coronavirus Infections/transmission , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Pneumonia, Viral/transmission , Travel , COVID-19 , China/epidemiology , Communicable Disease Control , Coronavirus Infections/epidemiology , Epidemics , Humans , Incidence , Models, Statistical , Pneumonia, Viral/epidemiology , Public Health Practice , Regression Analysis , SARS-CoV-2
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