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1.
J Infect Dis ; 2022 Jun 27.
Article in English | MEDLINE | ID: covidwho-1908838

ABSTRACT

Using multiple cell types and isolates of Delta and Omicron variants of SARS-CoV-2, we report differences in virus production, replication, and infectivity in vitro. Ancestral and Delta SARS-CoV-2 variant exhibit reduced virus production and replication at 34°C compared to 37°C while Omicron replication is balanced between temperatures.

2.
J Med Virol ; 94(6): 2833-2836, 2022 06.
Article in English | MEDLINE | ID: covidwho-1669584

ABSTRACT

COVID-19 vaccines provide high levels of protection against severe disease and hospitalization due to severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infection. Vaccination may be less effective in preventing shedding of infectious viruses from otherwise immune patients. In this study, we describe breakthrough infections and shedding of infectious viruses in convalescent hamsters without significant replication in the lower respiratory tract following reinfection by Alpha and Delta variants despite high levels of circulating antibodies in sera. Using convalescent hamsters with long-term immunity (up to 1 year) following infection by ancestral SARS-CoV-2, we can model aspects of recurring COVID-19 in the context of preexisting immunity.


Subject(s)
COVID-19 , Animals , COVID-19/prevention & control , COVID-19 Vaccines , Cricetinae , Humans , Mesocricetus , SARS-CoV-2 , Trachea
3.
NPJ Vaccines ; 6(1): 145, 2021 Dec 03.
Article in English | MEDLINE | ID: covidwho-1550285

ABSTRACT

Numerous vaccine candidates against SARS-CoV-2, the causative agent of the COVID-19 pandemic, are under development. The majority of vaccine candidates to date are designed to induce immune responses against the viral spike (S) protein, although different forms of S antigen have been incorporated. To evaluate the yield and immunogenicity of different forms of S, we constructed modified vaccinia virus Ankara (MVA) vectors expressing full-length S (MVA-S), the RBD, and soluble S ectodomain and tested their immunogenicity in dose-ranging studies in mice. All three MVA vectors induced spike-specific immunoglobulin G after one subcutaneous immunization and serum titers were boosted following a second immunization. The MVA-S and MVA-ssM elicited the strongest neutralizing antibody responses. In assessing protective efficacy, MVA-S-immunized adult Syrian hamsters were challenged with SARS-CoV-2 (USA/WA1/2020). MVA-S-vaccinated hamsters exhibited less severe manifestations of atypical pneumocyte hyperplasia, hemorrhage, vasculitis, and especially consolidation, compared to control animals. They also displayed significant reductions in gross pathology scores and weight loss, and a moderate reduction in virus shedding was observed post challenge in nasal washes. There was evidence of reduced viral replication by in situ hybridization, although the reduction in viral RNA levels in lungs and nasal turbinates did not reach significance. Taken together, the data indicate that immunization with two doses of an MVA vector expressing SARS-CoV-2 S provides protection against a stringent SARS-CoV-2 challenge of adult Syrian hamsters, reaffirm the utility of this animal model for evaluating candidate SARS-CoV-2 vaccines, and demonstrate the value of an MVA platform in facilitating vaccine development against SARS-CoV-2.

4.
Sci Rep ; 11(1): 22195, 2021 11 12.
Article in English | MEDLINE | ID: covidwho-1514424

ABSTRACT

To initiate SARS-CoV-2 infection, the Receptor Binding Domain (RBD) on the viral spike protein must first bind to the host receptor ACE2 protein on pulmonary and other ACE2-expressing cells. We hypothesized that cardiac glycoside drugs might block the binding reaction between ACE2 and the Spike (S) protein, and thus block viral penetration into target cells. To test this hypothesis we developed a biochemical assay for ACE2:Spike binding, and tested cardiac glycosides as inhibitors of binding. Here we report that ouabain, digitoxin, and digoxin, as well as sugar-free derivatives digitoxigenin and digoxigenin, are high-affinity competitive inhibitors of ACE2 binding to the Original [D614] S1 and the α/ß/γ [D614G] S1 proteins. These drugs also inhibit ACE2 binding to the Original RBD, as well as to RBD proteins containing the ß [E484K], Mink [Y453F] and α/ß/γ [N501Y] mutations. As hypothesized, we also found that ouabain, digitoxin and digoxin blocked penetration by SARS-CoV-2 Spike-pseudotyped virus into human lung cells, and infectivity by native SARS-CoV-2. These data indicate that cardiac glycosides may block viral penetration into the target cell by first inhibiting ACE2:RBD binding. Clinical concentrations of ouabain and digitoxin are relatively safe for short term use for subjects with normal hearts. It has therefore not escaped our attention that these common cardiac medications could be deployed worldwide as inexpensive repurposed drugs for anti-COVID-19 therapy.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/drug therapy , Cardiotonic Agents/pharmacology , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization/drug effects , A549 Cells , Animals , COVID-19/metabolism , Chlorocebus aethiops , Digitoxin/pharmacology , Digoxin/pharmacology , Humans , Lung/drug effects , Lung/metabolism , Ouabain/pharmacology , Protein Binding/drug effects , SARS-CoV-2/physiology , Vero Cells
5.
J Chem Inf Model ; 61(10): 5133-5140, 2021 10 25.
Article in English | MEDLINE | ID: covidwho-1467038

ABSTRACT

The newly emerging Kappa, Delta, and Lambda SARS-CoV-2 variants are worrisome, characterized with the double mutations E484Q/L452R, T478K/L452R, and F490S/L452Q, respectively, in their receptor binding domains (RBDs) of the spike proteins. As revealed in crystal structures, most of these residues (e.g., 452 and 484 in RBDs) are not in direct contact with interfacial residues in the angiotensin-converting enzyme 2 (ACE2). This suggests that albeit there are some possibly nonlocal effects, these mutations might not significantly affect RBD's binding with ACE2, which is an important step for viral entry into host cells. Thus, without knowing the molecular mechanism, these successful mutations (from the point of view of SARS-CoV-2) may be hypothesized to evade human antibodies. Using all-atom molecular dynamics (MD) simulation, here, we show that the E484Q/L452R mutations significantly reduce the binding affinity between the RBD of the Kappa variant and the antibody LY-CoV555 (also named as Bamlanivimab), which was efficacious for neutralizing the wild-type SARS-CoV-2. To verify simulation results, we further carried out experiments with both pseudovirions- and live virus-based neutralization assays and demonstrated that LY-CoV555 completely lost neutralizing activity against the L452R/E484Q mutant. Similarly, we show that mutations in the Delta and Lambda variants can also destabilize the RBD's binding with LY-CoV555. With the revealed molecular mechanism on how these variants evade LY-CoV555, we expect that more specific therapeutic antibodies can be accordingly designed and/or a precise mixing of antibodies can be achieved as a cocktail treatment for patients infected with these variants.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Mutation , Protein Binding , Spike Glycoprotein, Coronavirus/metabolism
6.
Life Sci Alliance ; 4(4)2021 04.
Article in English | MEDLINE | ID: covidwho-1389962

ABSTRACT

A critical question in understanding the immunity to SARS-COV-2 is whether recovered patients are protected against re-challenge and transmission upon second exposure. We developed a Syrian hamster model in which intranasal inoculation of just 100 TCID50 virus caused viral pneumonia. Aged hamsters developed more severe disease and even succumbed to SARS-CoV-2 infection, representing the first lethal model using genetically unmodified laboratory animals. After initial viral clearance, the hamsters were re-challenged with 105 TCID50 SARS-CoV-2 and displayed more than 4 log reduction in median viral loads in both nasal washes and lungs in comparison to primary infections. Most importantly, re-challenged hamsters were unable to transmit virus to naïve hamsters, and this was accompanied by the presence of neutralizing antibodies. Altogether, these results show that SARS-CoV-2 infection induces protective immunity that not only prevents re-exposure but also limits transmission in hamsters. These findings may help guide public health policies and vaccine development and aid evaluation of effective vaccines against SARS-CoV-2.


Subject(s)
COVID-19/immunology , COVID-19/transmission , Immunity , Reinfection/immunology , Reinfection/transmission , SARS-CoV-2/immunology , Age Factors , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/virology , Chlorocebus aethiops , Cricetinae , Disease Models, Animal , Female , HEK293 Cells , Humans , Male , RNA, Viral/genetics , Reinfection/virology , SARS-CoV-2/genetics , Transfection , Vero Cells , Viral Load
7.
mSphere ; : e0050721, 2021 Jun 16.
Article in English | MEDLINE | ID: covidwho-1270880

ABSTRACT

Epidemiological studies have revealed the emergence of multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC), including the lineage B.1.1.7 that is rapidly replacing old variants. The B.1.1.7 variant has been linked to increased morbidity rates, transmissibility, and potentially mortality. To assess viral fitness in vivo and to address whether the B.1.1.7 variant is capable of immune escape, we conducted infection and reinfection studies in naive and convalescent Syrian hamsters (>10 months old). Nasal wash samples from hamsters infected by a B.1.1.7 variant exhibited slightly higher viral RNA levels but lower infectious titers than those from B.1 (G614) variant-infected hamsters, and the two variants induced comparable lung pathologies in hamsters. Despite a sporadic and transient low-level infection in the nasal cavity, convalescent hamsters that had recovered from a previous USA-WA1 isolate (D614) infection displayed no observable clinical signs or lung pathology following B.1.1.7 rechallenge. Altogether, our study did not find that the B.1.1.7 variant significantly differs from the B.1 variant in pathogenicity in Syrian hamsters and that a heterologous natural infection-induced immunity confers protection against a secondary challenge by the B1.1.7 variant. IMPORTANCE The rapid emergence of several variants of concern of SARS-CoV-2 calls for evaluations of viral fitness and pathogenicity in animal models in order to understand the mechanism of enhanced transmission and the possible increases in morbidity and mortality rates. Here, we demonstrated that immunity naturally acquired through a prior infection with the first-wave variant does confer nearly complete protection against the B.1.1.7 variant in Syrian hamsters upon reexposure. Strikingly, although the B.1.1.7 variant appears to replicate to a higher level in the nose than the ancestral B.1 variant, it does not induce more severe lung pathology in hamsters.

8.
Lancet Microbe ; 2(6): e259-e266, 2021 06.
Article in English | MEDLINE | ID: covidwho-1164728

ABSTRACT

BACKGROUND: Faecal shedding of SARS-CoV-2 has raised concerns about transmission through faecal microbiota transplantation procedures. Validation parameters of authorised tests for SARS-CoV-2 RNA detection in respiratory samples are described in product labelling, whereas the published methods for SARS-CoV-2 detection from faecal samples have not permitted a robust description of the assay parameters. We aimed to develop and validate a test specifically for detection of SARS-CoV-2 in human stool. METHODS: In this validation study, we evaluated performance characteristics of a reverse transcriptase real-time PCR (RT-rtPCR) test for detection of SARS-CoV-2 in human stool specimens by spiking stool with inactivated SARS-CoV-2 material. A modified version of the US Centers for Disease Control and Prevention RT-rtPCR SARS-CoV-2 test was used for detection of viral RNA. Analytical sensitivity was evaluated in freshly spiked stool by testing two-fold dilutions in replicates of 20. Masked samples were tested by a second laboratory to evaluate interlaboratory reproducibility. Short-term (7-day) stability of viral RNA in stool samples was assessed with four different stool storage buffers (phosphate-buffered saline, Cary-Blair medium, Stool Transport and Recovery [STAR] buffer, and DNA/RNA Shield) kept at -80°C, 4°C, and ambient temperature (approximately 21°C). We also tested clinical stool and anal swab specimens from patients who were SARS-CoV-2 positive by nasopharyngeal testing. FINDINGS: The lower limit of detection of the assay was found to be 3000 viral RNA copies per g of original stool sample, with 100% detection across 20 replicates assessed at this concentration. Analytical sensitivity was diminished by approximately two times after a single freeze-thaw cycle at -80°C. At 100 times the limit of detection, spiked samples were generally stable in all four stool storage buffers tested for up to 7 days, with maximum changes in mean threshold cycle values observed at -80°C storage in Cary-Blair medium (from 29·4 [SD 0·27] at baseline to 30·8 [0·17] at day 7; p<0·0001), at 4°C storage in DNA/RNA Shield (from 28·5 [0·15] to 29·8 [0·09]; p=0·0019), and at ambient temperature in STAR buffer (from 30·4 [0·24] to 32·4 [0·62]; p=0·0083). 30 contrived SARS-CoV-2 samples were tested by a second laboratory and were correctly identified as positive or negative in at least one of two rounds of testing. Additionally, SARS-CoV-2 RNA was detected using this assay in the stool and anal swab specimens of 11 of 23 individuals known to be positive for SARS-CoV-2. INTERPRETATION: This is a sensitive and reproducible assay for detection of SARS-CoV-2 RNA in human stool, with potential uses in faecal microbiota transplantation donor screening, sewage monitoring, and further research into the effects of faecal shedding on the epidemiology of the COVID-19 pandemic. FUNDING: National Institute of Allergy and Infectious Diseases, US National Institutes of Health; Center for Biologics Evaluation and Research, US Food and Drug Administration.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Humans , Pandemics , RNA, Viral/genetics , Reproducibility of Results , SARS-CoV-2/genetics
9.
J Virol ; 2021 Mar 08.
Article in English | MEDLINE | ID: covidwho-1123591

ABSTRACT

Biochemical and structural analyses suggest that SARS-CoV-2 is well-adapted to infecting humans and the presence of four residues (PRRA) at the S1/S2 site within the spike (S) protein, which may lead to unexpected tissue or host tropism. Here we report that SARS-CoV-2 efficiently utilized ACE2 of 9 species to infect 293T cells. Similarly, pseudoviruses bearing S protein derived from either the bat RaTG13 or pangolin GX, two closely related animal coronaviruses, utilized ACE2 of a diverse range of animal species to gain entry. Removal of PRRA from SARS-CoV-2 S protein displayed distinct effects on pseudoviral entry into different cell types. Unexpectedly, insertion of PRRA into the RaTG13 S protein selectively abrogated the usage of horseshoe bat and pangolin ACE2 but enhanced the usage of mouse ACE2 by the relevant pseudovirus to enter cells. Together, our findings identified a previously unrecognized effect of the PRRA insert on SARS-CoV-2 and RaTG13 S proteins.ImportanceThe four-residue insert (PRRA) at the boundary between the S1and S2 subunits of SARS-CoV-2 has been widely recognized since day 1 for its role in SARS-CoV-2 S protein processing and activation. As this PRRA insert is unique to SARS-CoV-2 among group b betacoronaviruses, it is thought to affect the tissue and species tropism of SARS-CoV-2. We compared the usage of 10 ACE2 orthologs and found that the presence of PRRA not only affects the cellular tropism of SARS-CoV-2 but also modulates the usage of ACE2 orthologs by the closely related bat RaTG13 S protein. The binding of pseudovirions carrying RaTG13 S with a PRRA insert to mouse ACE2 was nearly 2-fold higher than that of pseudovirions carrying RaTG13 S.

10.
Virology ; 556: 96-100, 2021 04.
Article in English | MEDLINE | ID: covidwho-1046110

ABSTRACT

Dynamic tracking of variant frequencies among viruses circulating in the global pandemic has revealed the emergence and dominance of a D614G mutation in the SARS-CoV-2 spike protein. To address whether pandemic SARS-CoV-2 G614 variant has evolved to become more pathogenic, we infected adult hamsters (>10 months old) with two natural SARS-CoV-2 variants carrying either D614 or G614 spike protein to mimic infection of the adult/elderly human population. Hamsters infected by the two variants exhibited comparable viral loads and pathology in lung tissues as well as similar amounts of virus shed in nasal washes. Altogether, our study does not find that naturally circulating D614 and G614 SARS-CoV-2 variants differ significantly in pathogenicity in hamsters.


Subject(s)
COVID-19/virology , SARS-CoV-2/pathogenicity , Animals , Antibodies, Neutralizing/blood , COVID-19/pathology , Chlorocebus aethiops , Disease Models, Animal , Female , Lung/pathology , Lung/virology , Male , Mesocricetus , Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Vero Cells , Viral Load , Weight Loss
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