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1.
J Virol ; 96(6): e0205921, 2022 03 23.
Article in English | MEDLINE | ID: covidwho-1788916

ABSTRACT

The Gammacoronavirus infectious bronchitis virus (IBV) is a highly contagious global pathogen prevalent in all types of poultry flocks. IBV is responsible for economic losses and welfare issues in domestic poultry, resulting in a significant risk to food security. IBV vaccines are currently generated by serial passage of virulent IBV field isolates through embryonated hens' eggs. The different patterns of genomic variation accumulated during this process means that the exact mechanism of attenuation is unknown and presents a risk of reversion to virulence. Additionally, the passaging process adapts the virus to replicate in chicken embryos, increasing embryo lethality. Vaccines produced in this manner are therefore unsuitable for in ovo application. We have developed a reverse genetics system, based on the pathogenic IBV strain M41, to identify genes which can be targeted for rational attenuation. During the development of this reverse genetics system, we identified four amino acids, located in nonstructural proteins (nsps) 10, 14, 15, and 16, which resulted in attenuation both in vivo and in ovo. Further investigation highlighted a role of amino acid changes, Pro85Leu in nsp 10 and Val393Leu in nsp 14, in the attenuated in vivo phenotype observed. This study provides evidence that mutations in nsps offer a promising mechanism for the development of rationally attenuated live vaccines against IBV, which have the potential for in ovo application. IMPORTANCE The Gammacoronavirus infectious bronchitis virus (IBV) is the etiological agent of infectious bronchitis, an acute, highly contagious, economically important disease of poultry. Vaccination is achieved using a mixture of live attenuated vaccines for young chicks and inactivated vaccines as boosters for laying hens. Live attenuated vaccines are generated through serial passage in embryonated hens' eggs, an empirical process which achieves attenuation but retains immunogenicity. However, these vaccines have a risk of reversion to virulence, and they are lethal to the embryo. In this study, we identified amino acids in the replicase gene which attenuated IBV strain M41, both in vivo and in ovo. Stability assays indicate that the attenuating amino acids are stable and unlikely to revert. The data in this study provide evidence that specific modifications in the replicase gene offer a promising direction for IBV live attenuated vaccine development, with the potential for in ovo application.


Subject(s)
Amino Acids , Coronavirus Infections , Infectious bronchitis virus , Poultry Diseases , Viral Nonstructural Proteins , Viral Vaccines , Amino Acids/chemistry , Amino Acids/genetics , Animals , Chick Embryo , Chickens , Coronavirus Infections/prevention & control , Coronavirus Infections/veterinary , Coronavirus Infections/virology , Female , Infectious bronchitis virus/genetics , Poultry Diseases/prevention & control , Poultry Diseases/virology , Vaccines, Attenuated/genetics , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Vaccines/genetics
2.
Viruses ; 14(2)2022 02 08.
Article in English | MEDLINE | ID: covidwho-1674831

ABSTRACT

This article aims to review all currently known interactions between animal and human coronaviruses and their cellular receptors. Over the past 20 years, three novel coronaviruses have emerged that have caused severe disease in humans, including SARS-CoV-2 (severe acute respiratory syndrome virus 2); therefore, a deeper understanding of coronavirus host-cell interactions is essential. Receptor-binding is the first stage in coronavirus entry prior to replication and can be altered by minor changes within the spike protein-the coronavirus surface glycoprotein responsible for the recognition of cell-surface receptors. The recognition of receptors by coronaviruses is also a major determinant in infection, tropism, and pathogenesis and acts as a key target for host-immune surveillance and other potential intervention strategies. We aim to highlight the need for a continued in-depth understanding of this subject area following on from the SARS-CoV-2 pandemic, with the possibility for more zoonotic transmission events. We also acknowledge the need for more targeted research towards glycan-coronavirus interactions as zoonotic spillover events from animals to humans, following an alteration in glycan-binding capability, have been well-documented for other viruses such as Influenza A.


Subject(s)
Host Microbial Interactions , Polysaccharides/metabolism , Receptors, Virus/metabolism , SARS-CoV-2/metabolism , Viral Tropism , Animals , COVID-19/transmission , COVID-19/virology , Humans , Protein Binding , SARS-CoV-2/immunology , Virus Internalization
3.
J Gen Virol ; 102(8)2021 08.
Article in English | MEDLINE | ID: covidwho-1368372

ABSTRACT

Infectious bronchitis virus (IBV) is an economically important coronavirus, causing damaging losses to the poultry industry worldwide as the causative agent of infectious bronchitis. The coronavirus spike (S) glycoprotein is a large type I membrane protein protruding from the surface of the virion, which facilitates attachment and entry into host cells. The IBV S protein is cleaved into two subunits, S1 and S2, the latter of which has been identified as a determinant of cellular tropism. Recent studies expressing coronavirus S proteins in mammalian and insect cells have identified a high level of glycosylation on the protein's surface. Here we used IBV propagated in embryonated hens' eggs to explore the glycan profile of viruses derived from infection in cells of the natural host, chickens. We identified multiple glycan types on the surface of the protein and found a strain-specific dependence on complex glycans for recognition of the S2 subunit by a monoclonal antibody in vitro, with no effect on viral replication following the chemical inhibition of complex glycosylation. Virus neutralization by monoclonal or polyclonal antibodies was not affected. Following analysis of predicted glycosylation sites for the S protein of four IBV strains, we confirmed glycosylation at 18 sites by mass spectrometry for the pathogenic laboratory strain M41-CK. Further characterization revealed heterogeneity among the glycans present at six of these sites, indicating a difference in the glycan profile of individual S proteins on the IBV virion. These results demonstrate a non-specific role for complex glycans in IBV replication, with an indication of an involvement in antibody recognition but not neutralisation.


Subject(s)
Coronavirus/physiology , Polysaccharides/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Alkaloids/chemistry , Alkaloids/pharmacology , Amino Acid Sequence , Animals , Binding Sites , Cells, Cultured , Chromatography, Liquid , Computational Biology/methods , Coronavirus/drug effects , Coronavirus Infections/veterinary , Gene Expression Regulation, Viral , Glycosylation/drug effects , Infectious bronchitis virus/physiology , Models, Molecular , Molecular Conformation , Molecular Weight , Neutralization Tests , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Polysaccharides/chemistry , Poultry Diseases/virology , Protein Transport , Spectrometry, Mass, Electrospray Ionization , Spike Glycoprotein, Coronavirus/genetics , Structure-Activity Relationship , Virus Replication/drug effects
4.
Nat Commun ; 12(1): 542, 2021 01 22.
Article in English | MEDLINE | ID: covidwho-1044339

ABSTRACT

There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic.


Subject(s)
Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , COVID-19/prevention & control , Peptides/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/immunology , Animals , Antibodies, Blocking/immunology , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , COVID-19/immunology , Cell Line , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Protein Interaction Domains and Motifs , Protein Multimerization , Swine
5.
PLoS Biol ; 18(12): e3001016, 2020 12.
Article in English | MEDLINE | ID: covidwho-992640

ABSTRACT

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.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Viral Tropism , Virus Attachment , Amino Acid Substitution , Animals , Binding Sites , Cats , Cattle , Dogs , Guinea Pigs , HEK293 Cells , Host-Pathogen Interactions , Humans , Rabbits , Rats , Viral Zoonoses/virology
6.
Viruses ; 12(10)2020 09 29.
Article in English | MEDLINE | ID: covidwho-906373

ABSTRACT

The Gammacoronavirus infectious bronchitis virus (IBV) causes a highly contagious and economically important respiratory disease in poultry. In the laboratory, most IBV strains are restricted to replication in ex vivo organ cultures or in ovo and do not replicate in cell culture, making the study of their basic virology difficult. Entry of IBV into cells is facilitated by the large glycoprotein on the surface of the virion, the spike (S) protein, comprised of S1 and S2 subunits. Previous research showed that the S2' cleavage site is responsible for the extended tropism of the IBV Beaudette strain. This study aims to investigate whether protease treatment can extend the tropism of other IBV strains. Here we demonstrate that the addition of exogenous trypsin during IBV propagation in cell culture results in significantly increased viral titres. Using a panel of IBV strains, exhibiting varied tropisms, the effects of spike cleavage on entry and replication were assessed by serial passage cell culture in the presence of trypsin. Replication could be maintained over serial passages, indicating that the addition of exogenous protease is sufficient to overcome the barrier to infection. Mutations were identified in both S1 and S2 subunits following serial passage in cell culture. This work provides a proof of concept that exogenous proteases can remove the barrier to IBV replication in otherwise non-permissive cells, providing a platform for further study of elusive field strains and enabling sustainable vaccine production in vitro.


Subject(s)
Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Infectious bronchitis virus/drug effects , Infectious bronchitis virus/physiology , Trypsin/therapeutic use , Viral Tropism/drug effects , Animals , Cell Line , Chlorocebus aethiops , Gammacoronavirus/drug effects , Infectious bronchitis virus/metabolism , Kinetics , Serial Passage , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Viral Envelope Proteins/metabolism , Virion/drug effects , Virion/metabolism , Virus Replication/drug effects
7.
Viruses ; 12(10):1102, 2020.
Article | MDPI | ID: covidwho-803018

ABSTRACT

The Gammacoronavirus infectious bronchitis virus (IBV) causes a highly contagious and economically important respiratory disease in poultry. In the laboratory, most IBV strains are restricted to replication in ex vivo organ cultures or in ovo and do not replicate in cell culture, making the study of their basic virology difficult. Entry of IBV into cells is facilitated by the large glycoprotein on the surface of the virion, the spike (S) protein, comprised of S1 and S2 subunits. Previous research showed that the S2′cleavage site is responsible for the extended tropism of the IBV Beaudette strain. This study aims to investigate whether protease treatment can extend the tropism of other IBV strains. Here we demonstrate that the addition of exogenous trypsin during IBV propagation in cell culture results in significantly increased viral titres. Using a panel of IBV strains, exhibiting varied tropisms, the effects of spike cleavage on entry and replication were assessed by serial passage cell culture in the presence of trypsin. Replication could be maintained over serial passages, indicating that the addition of exogenous protease is sufficient to overcome the barrier to infection. Mutations were identified in both S1 and S2 subunits following serial passage in cell culture. This work provides a proof of concept that exogenous proteases can remove the barrier to IBV replication in otherwise non-permissive cells, providing a platform for further study of elusive field strains and enabling sustainable vaccine production in vitro.

8.
NPJ Vaccines ; 5(1): 69, 2020.
Article in English | MEDLINE | ID: covidwho-689622

ABSTRACT

Clinical development of the COVID-19 vaccine candidate ChAdOx1 nCoV-19, a replication-deficient simian adenoviral vector expressing the full-length SARS-CoV-2 spike (S) protein was initiated in April 2020 following non-human primate studies using a single immunisation. Here, we compared the immunogenicity of one or two doses of ChAdOx1 nCoV-19 in both mice and pigs. Whilst a single dose induced antigen-specific antibody and T cells responses, a booster immunisation enhanced antibody responses, particularly in pigs, with a significant increase in SARS-CoV-2 neutralising titres.

9.
J Gen Virol ; 101(10): 1103-1118, 2020 10.
Article in English | MEDLINE | ID: covidwho-688886

ABSTRACT

Coronavirus sub-genomic mRNA (sgmRNA) synthesis occurs via a process of discontinuous transcription involving complementary transcription regulatory sequences (TRSs), one (TRS-L) encompassing the leader sequence of the 5' untranslated region (UTR), and the other upstream of each structural and accessory gene (TRS-B). Several coronaviruses have an ORF located between the N gene and the 3'-UTR, an area previously thought to be non-coding in the Gammacoronavirus infectious bronchitis virus (IBV) due to a lack of a canonical TRS-B. Here, we identify a non-canonical TRS-B allowing for a novel sgmRNA relating to this ORF to be produced in several strains of IBV: Beaudette, CR88, H120, D1466, Italy-02 and QX. Interestingly, the potential protein produced by this ORF is prematurely truncated in the Beaudette strain. A single nucleotide deletion was made in the Beaudette strain allowing for the generation of a recombinant IBV (rIBV) that had the potential to express a full-length protein. Assessment of this rIBV in vitro demonstrated that restoration of the full-length potential protein had no effect on viral replication. Further assessment of the Beaudette-derived RNA identified a second non-canonically transcribed sgmRNA located within gene 2. Deep sequencing analysis of allantoic fluid from Beaudette-infected embryonated eggs confirmed the presence of both the newly identified non-canonically transcribed sgmRNAs and highlighted the potential for further yet unidentified sgmRNAs. This HiSeq data, alongside the confirmation of non-canonically transcribed sgmRNAs, indicates the potential of the coronavirus genome to encode a larger repertoire of genes than has currently been identified.


Subject(s)
Infectious bronchitis virus/genetics , RNA, Messenger/genetics , RNA, Viral/genetics , Regulatory Sequences, Nucleic Acid/genetics , Transcription, Genetic/genetics , 5' Untranslated Regions/genetics , Animals , Base Sequence , Cell Line , Chickens , Chlorocebus aethiops , Coronavirus Infections/veterinary , Coronavirus Infections/virology , Open Reading Frames/genetics , Poultry Diseases/virology , Vero Cells , Viral Proteins/genetics , Viral Proteins/metabolism , Virus Replication/genetics
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