RESUMEN
Coronaviruses infect a wide variety of host species, resulting in a range of diseases in both humans and animals. The coronavirus genome consists of a large positive-sense single-stranded molecule of RNA containing many RNA structures. One structure, denoted s2m and consisting of 41 nucleotides, is located within the 3' untranslated region (3' UTR) and is shared between some coronavirus species, including infectious bronchitis virus (IBV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2, as well as other pathogens, including human astrovirus. Using a reverse genetic system to generate recombinant viruses, we investigated the requirement of the s2m structure in the replication of IBV, a globally distributed economically important Gammacoronavirus that infects poultry causing respiratory disease. Deletion of three nucleotides predicted to destabilize the canonical structure of the s2m or the deletion of the nucleotides corresponding to s2m impacted viral replication in vitro. In vitro passaging of the recombinant IBV with the s2m sequence deleted resulted in a 36-nucleotide insertion in place of the deletion, which was identified to be composed of a duplication of flanking sequences. A similar result was observed following serial passage of human astrovirus with a deleted s2m sequence. RNA modeling indicated that deletion of the nucleotides corresponding to the s2m impacted other RNA structures present in the IBV 3' UTR. Our results indicated for both IBV and human astrovirus a preference for nucleotide occupation in the genome location corresponding to the s2m, which is independent of the specific s2m sequence. IMPORTANCE Coronaviruses infect many species, including humans and animals, with substantial effects on livestock, particularly with respect to poultry. The coronavirus RNA genome consists of structural elements involved in viral replication whose roles are poorly understood. We investigated the requirement of the RNA structural element s2m in the replication of the Gammacoronavirus infectious bronchitis virus, an economically important viral pathogen of poultry. Using reverse genetics to generate recombinant IBVs with either a disrupted or deleted s2m, we showed that the s2m is not required for viral replication in cell culture; however, replication is decreased in tracheal tissue, suggesting a role for the s2m in the natural host. Passaging of these viruses as well as human astrovirus lacking the s2m sequence demonstrated a preference for nucleotide occupation, independent of the s2m sequence. RNA modeling suggested deletion of the s2m may negatively impact other essential RNA structures.
Asunto(s)
Virus de la Bronquitis Infecciosa , Mamastrovirus , Mutagénesis Insercional , Animales , Humanos , Regiones no Traducidas 3'/genética , Pollos/virología , Virus de la Bronquitis Infecciosa/genética , Mamastrovirus/genética , Mutagénesis Insercional/genética , Enfermedades de las Aves de Corral/virología , ARN Viral/genética , Replicación Viral/genética , Estabilidad del ARN/genética , Eliminación de Secuencia/genéticaRESUMEN
Avian coronavirus infectious bronchitis virus (IBV) is the etiological agent of infectious bronchitis, an acute highly contagious economically relevant respiratory disease of poultry. Vaccination is used to control IBV infections, with live-attenuated vaccines generated via serial passage of a virulent field isolate through embryonated hens' eggs. A fine balance must be achieved between attenuation and the retention of immunogenicity. The exact molecular mechanism of attenuation is unknown, and vaccines produced in this manner present a risk of reversion to virulence as few consensus level changes are acquired. Our previous research resulted in the generation of a recombinant IBV (rIBV) known as M41-R, based on a pathogenic strain M41-CK. M41-R was attenuated in vivo by two amino acid changes, Nsp10-Pro85Leu and Nsp14-Val393Leu; however, the mechanism of attenuation was not determined. Pro85 and Val393 were found to be conserved among not only IBV strains but members of the wider coronavirus family. This study demonstrates that the same changes are associated with a temperature-sensitive (ts) replication phenotype at 41°C in vitro, suggesting that the two phenotypes may be linked. Vaccination of specific-pathogen-free chickens with M41-R induced 100% protection against clinical disease, tracheal ciliary damage, and challenge virus replication following homologous challenge with virulent M41-CK. Temperature sensitivity has been used to rationally attenuate other viral pathogens, including influenza, and the identification of amino acid changes that impart both a ts and an attenuated phenotype may therefore offer an avenue for future coronavirus vaccine development. IMPORTANCE Infectious bronchitis virus is a pathogen of economic and welfare concern for the global poultry industry. Live-attenuated vaccines against are generated by serial passage of a virulent isolate in embryonated eggs until attenuation is achieved. The exact mechanisms of attenuation are unknown, and vaccines produced have a risk of reversion to virulence. Reverse genetics provides a method to generate vaccines that are rationally attenuated and are more stable with respect to back selection due to their clonal origin. Genetic populations resulting from molecular clones are more homogeneous and lack the presence of parental pathogenic viruses, which generation by multiple passage does not. In this study, we identified two amino acids that impart a temperature-sensitive replication phenotype. Immunogenicity is retained and vaccination results in 100% protection against homologous challenge. Temperature sensitivity, used for the development of vaccines against other viruses, presents a method for the development of coronavirus vaccines.
Asunto(s)
Infecciones por Coronavirus , Virus de la Bronquitis Infecciosa , Enfermedades de las Aves de Corral , Vacunas Virales , Aminoácidos , Animales , Pollos , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/veterinaria , Aves de Corral , Enfermedades de las Aves de Corral/prevención & control , Enfermedades de las Aves de Corral/virología , Temperatura , Vacunas Atenuadas , Vacunas Virales/genéticaRESUMEN
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.
Asunto(s)
Aminoácidos , Infecciones por Coronavirus , Virus de la Bronquitis Infecciosa , Enfermedades de las Aves de Corral , Proteínas no Estructurales Virales , Vacunas Virales , Aminoácidos/química , Aminoácidos/genética , Animales , Embrión de Pollo , Pollos , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Femenino , Virus de la Bronquitis Infecciosa/genética , Enfermedades de las Aves de Corral/prevención & control , Enfermedades de las Aves de Corral/virología , Vacunas Atenuadas/genética , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Vacunas Virales/genéticaRESUMEN
Coronavirus infection induces the unfolded protein response (UPR), a cellular signalling pathway composed of three branches, triggered by unfolded proteins in the endoplasmic reticulum (ER) due to high ER load. We have used RNA sequencing and ribosome profiling to investigate holistically the transcriptional and translational response to cellular infection by murine hepatitis virus (MHV), often used as a model for the Betacoronavirus genus to which the recently emerged SARS-CoV-2 also belongs. We found the UPR to be amongst the most significantly up-regulated pathways in response to MHV infection. To confirm and extend these observations, we show experimentally the induction of all three branches of the UPR in both MHV- and SARS-CoV-2-infected cells. Over-expression of the SARS-CoV-2 ORF8 or S proteins alone is itself sufficient to induce the UPR. Remarkably, pharmacological inhibition of the UPR greatly reduced the replication of both MHV and SARS-CoV-2, revealing the importance of this pathway for successful coronavirus replication. This was particularly striking when both IRE1α and ATF6 branches of the UPR were inhibited, reducing SARS-CoV-2 virion release (~1,000-fold). Together, these data highlight the UPR as a promising antiviral target to combat coronavirus infection.
Asunto(s)
Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Virus de la Hepatitis Murina/efectos de los fármacos , Respuesta de Proteína Desplegada/efectos de los fármacos , Factor de Transcripción Activador 6/metabolismo , Animales , Antivirales/uso terapéutico , Línea Celular , Chlorocebus aethiops , Sistemas de Liberación de Medicamentos , Endorribonucleasas/metabolismo , Células HEK293 , Humanos , Ratones , Proteínas Serina-Treonina Quinasas/metabolismo , RNA-Seq , Células Vero , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
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.
Asunto(s)
Coronavirus/fisiología , Polisacáridos/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Alcaloides/química , Alcaloides/farmacología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células Cultivadas , Cromatografía Liquida , Biología Computacional/métodos , Coronavirus/efectos de los fármacos , Infecciones por Coronavirus/veterinaria , Regulación Viral de la Expresión Génica , Glicosilación/efectos de los fármacos , Virus de la Bronquitis Infecciosa/fisiología , Modelos Moleculares , Conformación Molecular , Peso Molecular , Pruebas de Neutralización , Oligosacáridos/química , Oligosacáridos/metabolismo , Polisacáridos/química , Enfermedades de las Aves de Corral/virología , Transporte de Proteínas , Espectrometría de Masa por Ionización de Electrospray , Glicoproteína de la Espiga del Coronavirus/genética , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
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.
Asunto(s)
Virus de la Bronquitis Infecciosa/genética , ARN Mensajero/genética , ARN Viral/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Transcripción Genética/genética , Regiones no Traducidas 5'/genética , Animales , Secuencia de Bases , Línea Celular , Pollos , Chlorocebus aethiops , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Sistemas de Lectura Abierta/genética , Enfermedades de las Aves de Corral/virología , Células Vero , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral/genéticaRESUMEN
Like all coronaviruses, avian infectious bronchitis virus (IBV) possesses a long, single-stranded, positive-sense RNA genome (â¼27 kb) and has a complex replication strategy that includes the production of a nested set of subgenomic mRNAs (sgmRNAs). Here, we used whole-transcriptome sequencing (RNASeq) and ribosome profiling (RiboSeq) to delineate gene expression in the IBV M41-CK and Beau-R strains at subcodon resolution. RNASeq facilitated a comparative analysis of viral RNA synthesis and revealed two novel transcription junction sites in the attenuated Beau-R strain, one of which would generate a sgmRNA encoding a ribosomally occupied open reading frame (dORF) located downstream of the nucleocapsid coding region. RiboSeq permitted quantification of the translational efficiency of virus gene expression and identified, for the first time, sites of ribosomal pausing on the genome. Quantification of reads flanking the programmed ribosomal frameshifting (PRF) signal at the genomic RNA ORF1a/ORF1b junction revealed that PRF in IBV is highly efficient (33 to 40%). Triplet phasing of RiboSeq data allowed precise determination of reading frames and revealed the translation of two ORFs (ORF4b and ORF4c on sgmRNA IR), which are widely conserved across IBV isolates. Analysis of differential gene expression in infected primary chick kidney cells indicated that the host cell response to IBV occurs primarily at the level of transcription, with global upregulation of immune-related mRNA transcripts following infection and comparatively modest changes in the translation efficiencies of host genes. Cellular genes and gene networks differentially expressed during virus infection were also identified, giving insights into the host cell response to IBV infection.IMPORTANCE IBV is a major avian pathogen and presents a substantial economic burden to the poultry industry. Improved vaccination strategies are urgently needed to curb the global spread of this virus, and the development of suitable vaccine candidates will be aided by an improved understanding of IBV molecular biology. Our high-resolution data have enabled a precise study of transcription and translation in cells infected with both pathogenic and attenuated forms of IBV and expand our understanding of gammacoronaviral gene expression. We demonstrate that gene expression shows considerable intraspecies variation, with single nucleotide polymorphisms being associated with altered production of sgmRNA transcripts, and our RiboSeq data sets enabled us to uncover novel ribosomally occupied ORFs in both strains. The numerous cellular genes and gene networks found to be differentially expressed during virus infection provide insights into the host cell response to IBV infection.
Asunto(s)
Virus de la Bronquitis Infecciosa/genética , Virulencia/genética , Animales , Pollos/genética , Codón/genética , Infecciones por Coronavirus/virología , Sistema de Lectura Ribosómico , Expresión Génica/genética , Perfilación de la Expresión Génica/métodos , Regulación Viral de la Expresión Génica/genética , Virus de la Bronquitis Infecciosa/metabolismo , Sistemas de Lectura Abierta , Enfermedades de las Aves de Corral/virología , ARN Mensajero/genética , ARN Viral/genética , Ribosomas/metabolismo , Transcriptoma/genética , Secuenciación del Exoma/métodosRESUMEN
The gammacoronavirus infectious bronchitis virus (IBV) causes an acute, highly contagious respiratory disease of poultry. Live attenuated vaccines are traditionally generated by serial passage of a virulent strain in embryonated chicken eggs; however, the molecular mechanism of attenuation is unknown. M41-CK, a virulent lab-adapted strain of IBV, was egg passaged over 100 times in four parallel independent replicates. All four final egg-passaged viruses were attenuated in vivo and exhibited similar growth phenotypes in adult chicken kidney cells and ex vivo tracheal organ cultures. The virus populations were sequenced by 454 pyrosequencing at the end of passaging, and the results showed that overall sequence diversity in the IBV population increased but the four replicates only had between 11 and 17 consensus-level single nucleotide polymorphisms (SNPs). Although hot spots of variation were identified in spike and nucleocapsid structural proteins as well as the 3' untranslated region, each attenuated virus possessed a different pattern of genomic variation. Overall, only a small number of consensus-level SNPs were acquired during egg passage, leaving a potentially short route back to virulence. These results highlight the unpredictable nature of attenuation by serial egg passage and the need to develop mechanisms to rationally attenuate IBV for the next generation of effective vaccines.IMPORTANCE Infectious bronchitis remains a major problem in the global poultry industry, despite the existence of many different vaccines. IBV vaccines are currently developed by serial passage of a virulent strain on embryonated hen's eggs until attenuation; however, little is known about the evolution of the viral population during the process of attenuation. High-throughput sequencing of four replicates of a serially egg-passaged IBV revealed a different pattern of genomic variation in each attenuated replicate and few consensus-level SNPs. This raises concerns that only a small number of genomic mutations are required to revert to a virulent phenotype, which may result in vaccine breakdown in the field. The observed hot spots of variation in the attenuated viruses have the potential to be used in the rational attenuation of virulent IBV for next-generation vaccine design.
Asunto(s)
Huevos/virología , Virus de la Bronquitis Infecciosa , Polimorfismo de Nucleótido Simple , Vacunas Virales , Animales , Línea Celular , Pollos , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/inmunología , Virus de la Bronquitis Infecciosa/genética , Virus de la Bronquitis Infecciosa/inmunología , Enfermedades de las Aves de Corral/genética , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/virología , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Virales/genética , Vacunas Virales/inmunologíaRESUMEN
Vaccination regimes against Infectious bronchitis virus (IBV), which are based on a single virus serotype, often induce insufficient levels of cross-protection against serotypes and two or more antigenically diverse vaccines are used in attempt to provide broader protection. Amino acid differences in the surface protein, spike (S), in particular the S1 subunit, are associated with poor cross-protection. Here, homologous vaccination trials with recombinant IBVs (rIBVs), based on the apathogenic strain, BeauR, were conducted to elucidate the role of S1 in protection. A single vaccination of specific-pathogen-free chickens with rIBV expressing S1 of virulent strains M41 or QX, BeauR-M41(S1) and BeauR-QX(S1), gave incomplete protection against homologous challenge, based on ciliary activity and clinical signs. There could be conformational issues with the spike if heterologous S1 and S2 are linked, suggesting a homologous S2 might be essential. To address this, a homologous vaccination-challenge trial incorporating rIBVs expressing full spike from M41, BeauR-M41(S), and S2 subunit from M41, BeauR-M41(S2) was conducted. All chimeric viruses grew to similar titers in vitro, induced virus-specific partial protective immunity, evident by cellular infiltrations, reductions in viral RNA load in the trachea and conjunctiva and higher serum anti-IBV titers. Collectively, these findings show that vaccination with rIBVs primed the birds for challenge but the viruses were cleared rapidly from the mucosal tissues in the head. Chimeric S1 and S2 viruses did not protect as effectively as BeauR-M41(S) based on ciliary activity and clinical signs. Booster vaccinations and an rIBV with improved in vivo replication may improve the levels of protection.IMPORTANCE Infectious bronchitis virus causes an acute, highly contagious respiratory disease, responsible for significant economic losses to the poultry industry. Amino acid differences in the surface protein, spike (S), in particular the S1 subunit, have been associated with poor cross-protection. Available vaccines give poor cross-protection and rationally designed live attenuated vaccines, based on apathogenic BeauR, could address these. Here, to determine the role of S1 in protection, a series of homologous vaccination trials with rIBVs were conducted. Single vaccinations with chimeric rIBVs induced virus-specific partial protective immunity, characterized by reduction in viral load and serum antibody titers. However, BeauR-M41(S) was the only vaccination to improve the level of protection against clinical signs and the loss of tracheal ciliary activity. Growth characteristics show that all of the rIBVs replicated in vitro to similar levels. Booster vaccinations and an rIBV with improved in vivo replication may improve the levels of protection.
Asunto(s)
Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/veterinaria , Virus de la Bronquitis Infecciosa/inmunología , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/prevención & control , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas Virales/inmunología , Replicación Viral , Animales , Anticuerpos Antivirales/inmunología , Pollos , Infecciones por Coronavirus/virología , ADN Recombinante , Virus de la Bronquitis Infecciosa/genética , Virus de la Bronquitis Infecciosa/crecimiento & desarrollo , Enfermedades de las Aves de Corral/virología , Organismos Libres de Patógenos Específicos , Glicoproteína de la Espiga del Coronavirus/genética , Vacunación , Carga Viral , Vacunas Virales/administración & dosificaciónRESUMEN
The replicase gene of the coronavirus infectious bronchitis virus (IBV) encodes 15 non-structural proteins (nsps). Nsp 3 is a multi-functional protein containing a conserved ADP-ribose-1â³-phosphatase (ADRP) domain. The crystal structures of the domain from two strains of IBV, M41 (virulent) and Beaudette (avirulent), identified a key difference; M41 contains a conserved triple-glycine motif, whilst Beaudette contains a glycine-to-serine mutation that is predicted to abolish ADRP activity. Although ADRP activity has not been formally demonstrated for IBV nsp 3, Beaudette fails to bind ADP-ribose. The role of ADRP in virulence was investigated by generating rIBVs, based on Beaudette, containing either a restored triple-glycine motif or the complete M41 ADRP domain. Replication in vitro was unaffected by the ADRP modifications and the in vivo phenotype of the rIBVs was found to be apathogenic, indicating that restoration of the triple-glycine motif is not sufficient to restore virulence to the apathogenic Beaudette strain.
Asunto(s)
Virus de la Bronquitis Infecciosa/genética , Virus de la Bronquitis Infecciosa/patogenicidad , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Virales/genética , Adenosina Difosfato Ribosa , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Línea Celular , Pollos , Regulación Viral de la Expresión Génica , Mutación , Unión Proteica , ARN Polimerasa Dependiente del ARN/genética , Proteínas Virales/química , VirulenciaRESUMEN
Coronavirus subgenomic mRNA (sgmRNA) synthesis occurs via a process of discontinuous transcription involving transcription regulatory sequences (TRSs) located in the 5' leader sequence (TRS-L) and upstream of each structural and group-specific gene (TRS-B). Several gammacoronaviruses including infectious bronchitis virus (IBV) contain a putative open reading frame (ORF), localized between the M gene and gene 5, which is controversial due to the perceived absence of a TRS. We have studied the transcription of a novel sgmRNA associated with this potential ORF and found it to be transcribed via a previously unidentified noncanonical TRS-B. Using an IBV reverse genetics system, we demonstrated that the template-switching event during intergenic region (IR) sgmRNA synthesis occurs at the 5' end of the noncanonical TRS-B and recombines between nucleotides 5 and 6 of the 8-nucleotide consensus TRS-L. Introduction of a complete TRS-B showed that higher transcription levels are achieved by increasing the number of nucleotide matches between TRS-L and TRS-B. Translation of a protein from the sgmRNA was demonstrated using enhanced green fluorescent protein, suggesting the translation of a fifth, novel, group-specific protein for IBV. This study has resolved an issue concerning the number of ORFs expressed by members of the Gammacoronavirus genus and proposes the existence of a fifth IBV accessory protein. We confirmed previous reports that coronaviruses can produce sgmRNAs from noncanonical TRS-Bs, which may expand their repertoire of proteins. We also demonstrated that noncanonical TRS-Bs may provide a mechanism by which coronaviruses can control protein expression levels by reducing sgmRNA synthesis.
Asunto(s)
Coronaviridae/genética , Regulación Viral de la Expresión Génica , ARN Mensajero/genética , ARN Viral/genética , Transcripción Genética , Animales , Células Cultivadas , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , ARN Viral/metabolismoRESUMEN
The avian Gammacoronavirus infectious bronchitis virus (IBV) causes major economic losses in the poultry industry as the aetiological agent of infectious bronchitis, a highly contagious respiratory disease in chickens. IBV causes major economic losses to poultry industries across the globe and is a concern for global food security. IBV vaccines are currently produced by serial passage, typically 80 to 100 times in chicken embryonated eggs (CEE) to achieve attenuation by unknown molecular mechanisms. Vaccines produced in this manner present a risk of reversion as often few consensus level changes are acquired. The process of serial passage is cumbersome, time consuming, solely dependent on the supply of CEE and does not allow for rapid vaccine development in response to newly emerging IBV strains. Both alternative rational attenuation and cell culture-based propagation methods would therefore be highly beneficial. The majority of IBV strains are however unable to be propagated in cell culture proving a significant barrier to the development of cell-based vaccines. In this study we demonstrate the incorporation of a heterologous Spike (S) gene derived from the apathogenic Beaudette strain of IBV into a pathogenic M41 genomic backbone generated a recombinant IBV denoted M41K-Beau(S) that exhibits Beaudette's unique ability to replicate in Vero cells, a cell line licenced for vaccine production. The rIBV M41K-Beau(S) additionally exhibited an attenuated in vivo phenotype which was not the consequence of the presence of a large heterologous gene demonstrating that the Beaudette S not only offers a method for virus propagation in cell culture but also a mechanism for rational attenuation. Although historical research suggested that Beaudette, and by extension the Beaudette S protein was poorly immunogenic, vaccination of chickens with M41K-Beau(S) induced a complete cross protective immune response in terms of clinical disease and tracheal ciliary activity against challenge with a virulent IBV, M41-CK, belonging to the same serogroup as Beaudette. This implies that the amino acid sequence differences between the Beaudette and M41 S proteins have not distorted important protective epitopes. The Beaudette S protein therefore offers a significant avenue for vaccine development, with the advantage of a propagation platform less reliant on CEE.
Asunto(s)
Gammacoronavirus , Virus de la Bronquitis Infecciosa , Vacunas , Animales , Chlorocebus aethiops , Glicoproteína de la Espiga del Coronavirus/genética , Células Vero , Pollos , Virus de la Bronquitis Infecciosa/genéticaRESUMEN
Avian infectious bronchitis is an acute respiratory disease of poultry of particular concern for global food security. Investigation of infectious bronchitis virus (IBV), the causative agent of avian infectious bronchitis, via reverse genetics enables deeper understanding of virus biology and a rapid response to emerging variants. Classic methods of reverse genetics for IBV can be time consuming, rely on recombination for the introduction of mutations, and, depending on the system, can be subject to genome instability and unreliable success rates. In this study, we have applied data-optimized Golden Gate Assembly design to create a rapidly executable, flexible, and faithful reverse genetics system for IBV. The IBV genome was divided into 12 fragments at high-fidelity fusion site breakpoints. All fragments were synthetically produced and propagated in E. coli plasmids, amenable to standard molecular biology techniques for DNA manipulation. The assembly can be carried out in a single reaction, with the products used directly in subsequent viral rescue steps. We demonstrate the use of this system for generation of point mutants and gene replacements. This Golden Gate Assembly-based reverse genetics system will enable rapid response to emerging variants of IBV, particularly important to vaccine development for controlling spread within poultry populations.
Asunto(s)
Virus de la Bronquitis Infecciosa , Genética Inversa , Virus de la Bronquitis Infecciosa/genética , Genética Inversa/métodos , Animales , Genoma Viral , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/veterinaria , Plásmidos/genética , Enfermedades de las Aves de Corral/virología , Escherichia coli/genéticaRESUMEN
The avian coronavirus, infectious bronchitis virus (IBV), is an economically important infectious disease affecting chickens, with a diverse range of serotypes found globally. The major surface protein, spike (S), has high diversity between serotypes, and amino acid differences in the S1 sub-unit are thought to be responsible for poor cross-protection afforded by vaccination. Here, we attempt to address this, by using epitope mapping technology to identify shared and serotype-specific immunogenic epitopes of the S glycoprotein of three major circulating strains of IBV, M41, QX, and 4/91, via CLIPS peptide arrays based on peptides from the S1 sub-units. The arrays were screened with sera from chickens immunised with recombinant IBV, based on Beau-R backbone expressing heterologous S, generated in two independent vaccination/challenge trials. The screening of sera from rIBV vaccination experiments led to the identification of 52 immunogenic epitopes on the S1 of M41, QX, and 4/91. The epitopes were assigned into six overlapping epitope binding regions. Based on accessibility and location in the hypervariable regions of S, three sequences, 25YVYYYQSAFRPPNGWHLQGGAYAVVNSTN54, 67TVGVIKDVYNQSVASI82, and 83AMTVPPAGMSWSVS96, were selected for further investigation, and synthetic peptide mimics were recognised by polyclonal sera. These epitopes may have the potential to contribute towards a broader cross-protective IBV vaccine.
Asunto(s)
Infecciones por Coronavirus , Virus de la Bronquitis Infecciosa , Enfermedades de las Aves de Corral , Vacunas Virales , Animales , Epítopos , Mapeo Epitopo , Pollos , Péptidos , Glicoproteínas , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/veterinaria , Glicoproteína de la Espiga del CoronavirusRESUMEN
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.
Asunto(s)
Interacciones Microbiota-Huesped , Polisacáridos/metabolismo , Receptores Virales/metabolismo , SARS-CoV-2/metabolismo , Tropismo Viral , Animales , COVID-19/transmisión , COVID-19/virología , Humanos , Unión Proteica , SARS-CoV-2/inmunología , Internalización del VirusRESUMEN
The envelope (E) protein of the avian coronavirus infectious bronchitis virus (IBV) is a small-membrane protein present in two forms during infection: a monomer and a pentameric ion channel. Each form has an independent role during replication; the monomer disrupts the secretory pathway, and the pentamer facilitates virion production. The presence of a T16A or A26F mutation within E exclusively generates the pentameric or monomeric form, respectively. We generated two recombinant IBVs (rIBVs) based on the apathogenic molecular clone Beau-R, containing either a T16A or A26F mutation, denoted as BeauR-T16A and BeauR-A26F. The replication and genetic stability of the rIBVs were assessed in several different cell types, including primary and continuous cells, ex vivo tracheal organ cultures (TOCs) and in ovo. Different replication profiles were observed between cell cultures of different origins. BeauR-A26F replicated to a lower level than Beau-R in Vero cells and in ovo but not in DF1, primary chicken kidney (CK) cells or TOCs. Genetic stability and cytopathic effects were found to differ depending on the cell system. The effect of the T16A and A26F mutations appear to be cell-type dependent, which, therefore, highlights the importance of cell type in the investigation of the IBV E protein.
Asunto(s)
Infecciones por Coronavirus , Gammacoronavirus , Virus de la Bronquitis Infecciosa , Animales , Pollos , Chlorocebus aethiops , Infecciones por Coronavirus/veterinaria , Virus de la Bronquitis Infecciosa/genética , Mutación , Células VeroRESUMEN
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 in vitro and in the upper respiratory tract in vivo, PRCV strains causing more severe lung pathology were also able to replicate in ex vivo tracheal organ cultures as well as in vivo 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 vitro 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 in vitro organ cultures, will enable key events in coronavirus infection and disease pathogenesis to be identified.
Asunto(s)
COVID-19 , Coronavirus Respiratorio Porcino , Enfermedades de los Porcinos , Virus de la Gastroenteritis Transmisible , Animales , SARS-CoV-2 , PorcinosRESUMEN
BACKGROUND: Endometrial cancer is the most common genital tract carcinoma among women in developed countries, with most women presenting with stage 1 disease. Adjuvant progestagen therapy has been advocated following primary surgery to reduce the risk of recurrence of disease. OBJECTIVES: To evaluate the effectiveness and safety of adjuvant progestagen therapy for the treatment of endometrial cancer. SEARCH STRATEGY: We searched the Cochrane Gynaecological Cancer Group Trials Specilaised Register, Cochrane Central Register of Controlled Trials (CENTRAL) Issue 2, 2009. MEDLINE and EMBASE up to April 2009. SELECTION CRITERIA: Randomised controlled trials (RCTs) of progestagen therapy in women who have had surgery for endometrial cancer. DATA COLLECTION AND ANALYSIS: Two review authors independently abstracted data and assessed risk of bias. Risk ratios (RRs) comparing survival in women who did and did not receive progestagen were pooled in random effects meta-analyses. . MAIN RESULTS: Seven trials assessing 4556 women were identified. Three trials included women with stage one disease only, whereas four included women with more advanced disease. Meta-analysis of four trials showed that there was no significant difference in the risk of death at five years between adjuvant progestagen therapy and no further treatment (RR = 1.00, 95% CI 0.85 to 1.18). This conclusion is also robust to single trial analyses at 4 and 7 years and in one trial across all points in time using a hazard ratio (HR). There was also no significant difference between progestagen therapy and control in terms of the risk of death from endometrial cancer, cardiovascular disease and intercurrent disease. Relapse of disease appeared to be reduced by progestagen therapy in one trial (HR = 0.71, 95% CI 0.52 to 0.97 and 5 year RR = 0.74, 95% CI 0.58 to 0.96), but there was no evidence of a difference in disease recurrence in another trial at 7 years (RR = 1.34, 95% CI 0.79 to 2.27). AUTHORS' CONCLUSIONS: There is no evidence to support the use of adjuvant progestagen therapy in the primary treatment of endometrial cancer.
Asunto(s)
Antineoplásicos Hormonales/uso terapéutico , Neoplasias Endometriales/tratamiento farmacológico , Hidroxiprogesteronas/uso terapéutico , Acetato de Medroxiprogesterona/uso terapéutico , Progestinas/uso terapéutico , Causas de Muerte , Quimioterapia Adyuvante , Neoplasias Endometriales/mortalidad , Neoplasias Endometriales/patología , Neoplasias Endometriales/terapia , Femenino , Humanos , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
BACKGROUND: Cervical intraepithelial neoplasia (CIN) is the most common pre-malignant lesion. Surgical treatments for CIN are commonly associated with blood loss. OBJECTIVES: To assess the effectiveness and safety of interventions for preventing blood loss during the treatment of CIN. SEARCH STRATEGY: We searched the Cochrane Gynaecological Cancer Group Trials Register, MEDLINE, EMBASE and CENTRAL up to April 2009. We also searched registers of clinical trials, abstracts of scientific meetings and reference lists of included studies. SELECTION CRITERIA: Randomised controlled trials (RCTs) of vasopressin, tranexamic acid, haemostatic sutures, Amino-Cerv or Monsel's solution in women undergoing surgery for CIN. DATA COLLECTION AND ANALYSIS: Two reviewers independently abstracted data and assessed risk of bias. Risk ratios comparing adverse events in women who received one of the interventions were pooled in a random-effects meta-analyses or included in single trial analyses. MAIN RESULTS: Twelve RCTs (N = 1602, of whom 1512 were assessed) were included.Vasopressin significantly reduced perioperative bleeding (mean difference (MD) = -100.80, 95% confidence interval (CI) -129.48 to -72.12) and was associated with a decreased risk of bleeding that required haemostatic sutures or further vasopressin, compared to placebo (risk ratio (RR) = 0.39, 95% CI 0.27 to 0.56).Tranexamic acid significantly reduced risk of secondary haemorrhage (RR = 0.23, 95% CI 0.11 to 0.50), but not primary haemorrhage (RR = 1.24, 95% CI 0.04 to 38.23) after knife and laser cone biopsy, compared with placebo. There was also a statistically significant reduction in postoperative blood loss compared with placebo (MD = -55.60, 95% CI -94.91 to -16.29).Packing with Monsel's solution resulted in less perioperative blood loss (MD = -22.00, 95% CI -23.09 to -20.91) and decreased the risk of dysmenorrhoea (RR = 0.37, 95% CI 0.16 to 0.84), unsatisfactory colposcopy (RR = 0.43, 95% CI 0.30 to 0.63) and cervical stenosis (RR = 0.35, 95% CI 0.25 to 0.49) compared to routine suturing, but was not statistically different to sutures for risk of primary and secondary haemorrhages.Amino-Cerv antibiotic gel failed to make a difference on secondary haemorrhage but was associated with significantly less vaginal discharge at 2 weeks compared with routine care (RR = 0.27, 95% CI 0.09 to 0.86).There was no significant difference in blood loss between women who received ball electrode diathermy and those who received Monsel's paste (MD = 4.82, 95% CI -3.45 to 13.09). AUTHORS' CONCLUSIONS: Bleeding associated with surgery of the cervix appears to be reduced by vasopressin, used in combination with local anaesthetic. Tranexamic acid appears to be beneficial after knife and laser cone biopsy. There are insufficient data to assess the effects on primary haemorrhage. There is some evidence that haemostatic suturing has an adverse effect on blood loss, cervical stenosis and satisfactory colposcopy.
Asunto(s)
Hemostasis Quirúrgica/métodos , Hemostáticos/uso terapéutico , Displasia del Cuello del Útero/cirugía , Neoplasias del Cuello Uterino/cirugía , Pérdida de Sangre Quirúrgica/prevención & control , Femenino , Compuestos Férricos/uso terapéutico , Humanos , Ensayos Clínicos Controlados Aleatorios como Asunto , Sulfatos/uso terapéutico , Suturas , Ácido Tranexámico/uso terapéutico , Vasopresinas/uso terapéuticoRESUMEN
BACKGROUND: Cervical intraepithelial neoplasia (CIN) is the most common pre-malignant lesion. Atypical squamous changes occur in the transformation zone of the cervix with mild, moderate or severe changes described by their depth (CIN 1, 2 or 3). Cervical intraepithelial neoplasia is treated by local ablation or lower morbidity excision techniques. Choice of treatment depends on the grade and extent of the disease. OBJECTIVES: To assess the effectiveness and safety of alternative surgical treatments for CIN. SEARCH STRATEGY: We searched the Cochrane Gynaecological Cancer Group Trials Register, Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library), MEDLINE and EMBASE (up to April 2009). We also searched registers of clinical trials, abstracts of scientific meetings and reference lists of included studies. SELECTION CRITERIA: Randomised controlled trials (RCTs) of alternative surgical treatments in women with cervical intraepithelial neoplasia. DATA COLLECTION AND ANALYSIS: Two review authors independently abstracted data and assessed risks of bias. Risk ratios that compared residual disease after the follow-up examination and adverse events in women who received one of either laser ablation, laser conisation, large loop excision of the transformation zone (LLETZ), knife conisation or cryotherapy were pooled in random-effects model meta-analyses. MAIN RESULTS: Twenty-nine trials were included. Seven surgical techniques were tested in various comparisons. No significant differences in treatment failures were demonstrated in terms of persistent disease after treatment. Large loop excision of the transformation zone appeared to provide the most reliable specimens for histology with the least morbidity. Morbidity was lower than with laser conisation, although the trials did not provide data for every outcome measure. There were not enough data to assess the effect on morbidity when compared with laser ablation. AUTHORS' CONCLUSIONS: The evidence suggests that there is no obvious superior surgical technique for treating cervical intraepithelial neoplasia in terms of treatment failures or operative morbidity.