SARS Coronavirus 2 (SARS-CoV-2) emerged in late 2019, leading to the Coronavirus Disease 2019 (COVID-19) pandemic that continues to cause significant global mortality in human populations. Given its ...sequence similarity to SARS-CoV, as well as related coronaviruses circulating in bats, SARS-CoV-2 is thought to have originated in Chiroptera species in China. However, whether the virus spread directly to humans or through an intermediate host is currently unclear, as is the potential for this virus to infect companion animals, livestock, and wildlife that could act as viral reservoirs. Using a combination of surrogate entry assays and live virus, we demonstrate that, in addition to human angiotensin-converting enzyme 2 (ACE2), the Spike glycoprotein of SARS-CoV-2 has a broad host tropism for mammalian ACE2 receptors, despite divergence in the amino acids at the Spike receptor binding site on these proteins. Of the 22 different hosts we investigated, ACE2 proteins from dog, cat, and cattle were the most permissive to SARS-CoV-2, while bat and bird ACE2 proteins were the least efficiently used receptors. The absence of a significant tropism for any of the 3 genetically distinct bat ACE2 proteins we examined indicates that SARS-CoV-2 receptor usage likely shifted during zoonotic transmission from bats into people, possibly in an intermediate reservoir. Comparison of SARS-CoV-2 receptor usage to the related coronaviruses SARS-CoV and RaTG13 identified distinct tropisms, with the 2 human viruses being more closely aligned. Finally, using bioinformatics, structural data, and targeted mutagenesis, we identified amino acid residues within the Spike-ACE2 interface, which may have played a pivotal role in the emergence of SARS-CoV-2 in humans. The apparently broad tropism of SARS-CoV-2 at the point of viral entry confirms the potential risk of infection to a wide range of companion animals, livestock, and wildlife.
The severity of disease following infection with SARS-CoV-2 is determined by viral replication kinetics and host immunity, with early T cell responses and/or suppression of viraemia driving a ...favourable outcome. Recent studies uncovered a role for cholesterol metabolism in the SARS-CoV-2 life cycle and in T cell function. Here we show that blockade of the enzyme Acyl-CoA:cholesterol acyltransferase (ACAT) with Avasimibe inhibits SARS-CoV-2 pseudoparticle infection and disrupts the association of ACE2 and GM1 lipid rafts on the cell membrane, perturbing viral attachment. Imaging SARS-CoV-2 RNAs at the single cell level using a viral replicon model identifies the capacity of Avasimibe to limit the establishment of replication complexes required for RNA replication. Genetic studies to transiently silence or overexpress ACAT isoforms confirmed a role for ACAT in SARS-CoV-2 infection. Furthermore, Avasimibe boosts the expansion of functional SARS-CoV-2-specific T cells from the blood of patients sampled during the acute phase of infection. Thus, re-purposing of ACAT inhibitors provides a compelling therapeutic strategy for the treatment of COVID-19 to achieve both antiviral and immunomodulatory effects. Trial registration: NCT04318314.
ChAdOx1 nCoV-19 (AZD1222) is a replication-deficient simian adenovirus-vectored vaccine encoding the spike (S) protein of SARS-CoV-2, based on the first published full-length sequence (Wuhan-1). ...AZD1222 has been shown to have 74% vaccine efficacy against symptomatic disease in clinical trials. However, variants of concern (VoCs) have been detected, with substitutions that are associated with a reduction in virus neutralizing antibody titer. Updating vaccines to include S proteins of VoCs may be beneficial, even though current real-world data is suggesting good efficacy following boosting with vaccines encoding the ancestral S protein. Using the Syrian hamster model, we evaluate the effect of a single dose of AZD2816, encoding the S protein of the Beta VoC, and efficacy of AZD1222/AZD2816 as a heterologous primary series against challenge with the Beta or Delta variant. Minimal to no viral sgRNA could be detected in lungs of vaccinated animals obtained at 3- or 5- days post inoculation, in contrast to lungs of control animals. In Omicron-challenged hamsters, a single dose of AZD2816 or AZD1222 reduced virus shedding. Thus, these vaccination regimens are protective against the Beta, Delta, and Omicron VoCs in the hamster model.
SARS-CoV-2 has a broad mammalian species tropism infecting humans, cats, dogs, and farmed mink. Since the start of the 2019 pandemic, several reverse zoonotic outbreaks of SARS-CoV-2 have occurred in ...mink, one of which reinfected humans and caused a cluster of infections in Denmark. Here we investigate the molecular basis of mink and ferret adaptation and demonstrate the spike mutations Y453F, F486L, and N501T all specifically adapt SARS-CoV-2 to use mustelid ACE2. Furthermore, we risk assess these mutations and conclude mink-adapted viruses are unlikely to pose an increased threat to humans, as Y453F attenuates the virus replication in human cells and all three mink adaptations have minimal antigenic impact. Finally, we show that certain SARS-CoV-2 variants emerging from circulation in humans may naturally have a greater propensity to infect mustelid hosts and therefore these species should continue to be surveyed for reverse zoonotic infections.
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•Y453F, F486L, and N501T often arise in SARS-CoV-2 spike during ferret/mink adaptation•These mutations specifically adapt SARS-CoV-2 to use ferret ACE2•Common ferret or mink adaptations attenuate the virus in human airway cells•SARS-CoV-2 variants can use ferret ACE2 with any adaptation
Zhou et al. show that common mink/ferret adaptations of SARS-CoV-2 enhance the use of the otherwise poorly used ferret ACE2 receptor, while weakening virus replication in human airway cells. However, many SARS-CoV-2 variants can intrinsically enter cells via ferret ACE2 implying these hosts may be more susceptible to novel strains.
Nipah virus (NiV) poses a significant threat to human and livestock populations across South and Southeast Asia. Vaccines are required to reduce the risk and impact of spillover infection events. ...Pigs can act as an intermediate amplifying host for NiV and, separately, provide a preclinical model for evaluating human vaccine candidate immunogenicity. The aim of this study was therefore to evaluate the immunogenicity of an mRNA vectored NiV vaccine candidate in pigs. Pigs were immunized twice with 100 μg nucleoside-modified mRNA vaccine encoding soluble G glycoprotein from the Malaysia strain of NiV, formulated in lipid nanoparticles. Potent antigen-binding and virus neutralizing antibodies were detected in serum following the booster immunization. Antibody responses effectively neutralized both the Malaysia and Bangladesh strains of NiV but showed limited neutralization of the related (about 80% amino acid sequence identity for G) Hendra virus. Antibodies were also capable of neutralizing NiV glycoprotein mediated cell-cell fusion. NiV G-specific T cell cytokine responses were also measurable following the booster immunization with evidence for induction of both CD4 and CD8 T cell responses. These data support the further evaluation of mRNA vectored NiV G as a vaccine for both pigs and humans.
There has been limited characterisation of bat-borne coronaviruses in Europe. Here, we screened for coronaviruses in 48 faecal samples from 16 of the 17 bat species breeding in the UK, collected ...through a bat rehabilitation and conservationist network. We recovered nine complete genomes, including two novel coronavirus species, across six bat species: four alphacoronaviruses, a MERS-related betacoronavirus, and four closely related sarbecoviruses. We demonstrate that at least one of these sarbecoviruses can bind and use the human ACE2 receptor for infecting human cells, albeit suboptimally. Additionally, the spike proteins of these sarbecoviruses possess an R-A-K-Q motif, which lies only one nucleotide mutation away from a furin cleavage site (FCS) that enhances infectivity in other coronaviruses, including SARS-CoV-2. However, mutating this motif to an FCS does not enable spike cleavage. Overall, while UK sarbecoviruses would require further molecular adaptations to infect humans, their zoonotic risk warrants closer surveillance.
The spread of SARS-CoV-2 has caused a worldwide pandemic that has affected almost every aspect of human life. The development of an effective COVID-19 vaccine could limit the morbidity and mortality ...caused by infection and may enable the relaxation of social-distancing measures. Age is one of the most significant risk factors for poor health outcomes after SARS-CoV-2 infection; therefore, it is desirable that any new vaccine candidates elicit a robust immune response in older adults.
Here, we use in-depth immunophenotyping to characterize the innate and adaptive immune response induced upon intramuscular administration of the adenoviral vectored ChAdOx1 nCoV-19 (AZD-1222) COVID-19 vaccine candidate in mice.
A single vaccination generates spike-specific Th1 cells, Th1-like Foxp3+ regulatory T cells, polyfunctional spike-specific CD8+ T cells. and granzyme-B-producing CD8 effectors. Spike-specific IgG and IgM are generated from both the early extrafollicular antibody response and the T follicular helper cell-supported germinal center reaction, which is associated with the production of virus-neutralizing antibodies. A single dose of this vaccine generated a similar type of immune response in aged mice but of a reduced magnitude than in younger mice. We report that a second dose enhances the immune response to this vaccine in aged mice.
This study shows that ChAdOx1 nCoV-19 induces both cellular and humoral immunity in adult and aged mice and suggests a prime-boost strategy is a rational approach to enhance immunogenicity in older persons.
This study was supported by BBSRC, Lister institute of Preventative Medicine, EPSRC VaxHub, and Innovate UK.
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ChAdOx1 nCoV-19 induces spike-specific polyfunctional CD8+ and CD4+ T cellsChAdOx1 nCoV-19 stimulates extrafollicular plasma cell and germinal center formationA single dose of ChAdOx1 nCoV-19 induces cellular and humoral immunity in aged miceA booster dose of ChAdOx1 nCoV-19 enhances immunogenicity in aged mice
Effective COVID-19 vaccines will play a central role in the exit strategy from the worldwide pandemic. However, older persons often do not generate protective immunity upon vaccination due to age-dependent changes in their immune system. Because older people are more likely to have poor clinical outcomes after SARS-CoV-2 infection, vaccine strategies that elicit an optimal immune response in older bodies are urgently required. Researchers from the Babraham Institute , the Jenner Institute, and the Pirbright Institute in the UK performed pre-clinical testing of the COVID-19 vaccine candidate ChAdOx1 nCoV-19 in aged mice to assess how aging influences the immune response to this vaccine. The results show that a “prime-boost” vaccine regime enhances immunogenicity in aged mice, indicating that this approach is a rational strategy for vaccinating older persons.
Older persons are more likely to have poor health outcomes after SARS-CoV-2 infection; therefore, they are most in need of effective COVID-19 vaccines. Silva-Cayetano et al. show that a prime-boost strategy of the COVID-19 vaccine ChAdOx1 nCoV-19 enhances immunogenicity in aged mice, suggesting this as a strategy for immunizing older people.
In the light of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, we have developed a porcine respiratory coronavirus (PRCV) model for in depth mechanistic evaluation of the ...pathogenesis, virology and immune responses of this important family of viruses. Pigs are a large animal with similar physiology and immunology to humans and are a natural host for PRCV. Four PRCV strains were investigated and shown to induce different degrees of lung pathology. Importantly, although all four strains replicated equally well in porcine cell lines
and in the upper respiratory tract
, PRCV strains causing more severe lung pathology were also able to replicate in
tracheal organ cultures as well as
in the trachea and lung. The time course of infection of PRCV 135, which caused the most severe pulmonary pathology, was investigated. Virus was shed from the upper respiratory tract until day 10 post infection, with infection of the respiratory mucosa, as well as olfactory and sustentacular cells, providing an excellent model to study upper respiratory tract disease in addition to the commonly known lower respiratory tract disease from PRCV. Infected animals made antibody and T cell responses that cross reacted with the four PRCV strains and Transmissible Gastroenteritis Virus. The antibody response was reproduced
in organ cultures. Comparison of mechanisms of infection and immune control in pigs infected with PRCVs of differing pathogenicity with human data from SARS-CoV-2 infection and from our
organ cultures, will enable key events in coronavirus infection and disease pathogenesis to be identified.
For enveloped viruses, such as SARS-CoV-2, transmission relies on the binding of viral glycoproteins to cellular receptors. Conventionally, this process is recapitulated in the lab by infection of ...cells with isolated live virus. However, such studies can be restricted due to the availability of high quantities of replication-competent virus, biosafety precautions and associated trained staff. Here, we present a protocol based on pseudotyping to produce recombinant replication-defective lentiviruses bearing the SARS-CoV or SARS-CoV-2 attachment Spike glycoprotein, allowing the investigation of viral entry in a lower-containment facility. Pseudoparticles are produced by cells transiently transfected with plasmids encoding retroviral RNA packaging signals and
proteins, for the reconstitution of lentiviral particles, and a plasmid coding for the viral attachment protein of interest. This approach allows the investigation of different aspects of viral entry, such as the identification of receptor tropism, the prediction of virus host range, and zoonotic transmission potential, as well as the characterisation of antibodies (sera or monoclonal antibodies) and pharmacological inhibitors that can block entry. Graphic abstract: SARS-CoV and SARS-CoV-2 pseudoparticle generation and applications.
RaTG13 is a close relative of SARS-CoV-2, the virus responsible for the COVID-19 pandemic, sharing 96% sequence similarity at the genome-wide level. The spike receptor binding domain (RBD) of RaTG13 ...contains a number of amino acid substitutions when compared to SARS-CoV-2, likely impacting affinity for the ACE2 receptor. Antigenic differences between the viruses are less well understood, especially whether RaTG13 spike can be efficiently neutralised by antibodies generated from infection with, or vaccination against, SARS-CoV-2. Using RaTG13 and SARS-CoV-2 pseudotypes we compared neutralisation using convalescent sera from previously infected patients or vaccinated healthcare workers. Surprisingly, our results revealed that RaTG13 was more efficiently neutralised than SARS-CoV-2. In addition, neutralisation assays using spike mutants harbouring single and combinatorial amino acid substitutions within the RBD demonstrated that both spike proteins can tolerate multiple changes without dramatically reducing neutralisation. Moreover, introducing the 484 K mutation into RaTG13 resulted in increased neutralisation, in contrast to the same mutation in SARS-CoV-2 (E484K). This is despite E484K having a well-documented role in immune evasion in variants of concern (VOC) such as B.1.351 (Beta). These results indicate that the future spill-over of RaTG13 and/or related sarbecoviruses could be mitigated using current SARS-CoV-2-based vaccination strategies.
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