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1.
J Gen Virol ; 102(8)2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34424155

RESUMO

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.


Assuntos
Coronavirus/fisiologia , Polissacarídeos/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Alcaloides/química , Alcaloides/farmacologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células Cultivadas , Cromatografia Líquida , Biologia Computacional/métodos , Coronavirus/efeitos dos fármacos , Infecções por Coronavirus/veterinária , Regulação Viral da Expressão Gênica , Glicosilação/efeitos dos fármacos , Vírus da Bronquite Infecciosa/fisiologia , Modelos Moleculares , Conformação Molecular , Peso Molecular , Testes de Neutralização , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Polissacarídeos/química , Doenças das Aves Domésticas/virologia , Transporte Proteico , Espectrometria de Massas por Ionização por Electrospray , Glicoproteína da Espícula de Coronavírus/genética , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos
2.
PLoS Pathog ; 17(6): e1009644, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34138976

RESUMO

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.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Vírus da Hepatite Murina/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Fator 6 Ativador da Transcrição/metabolismo , Animais , Antivirais/uso terapêutico , Linhagem Celular , Chlorocebus aethiops , Sistemas de Liberação de Medicamentos , Endorribonucleases/metabolismo , Células HEK293 , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , RNA-Seq , Células Vero , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos
3.
Viruses ; 12(10)2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-33003350

RESUMO

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.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Vírus da Bronquite Infecciosa/efeitos dos fármacos , Vírus da Bronquite Infecciosa/fisiologia , Tripsina/uso terapêutico , Tropismo Viral/efeitos dos fármacos , Animais , Linhagem Celular , Chlorocebus aethiops , Gammacoronavirus/efeitos dos fármacos , Vírus da Bronquite Infecciosa/metabolismo , Cinética , Inoculações Seriadas , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Proteínas do Envelope Viral/metabolismo , Vírion/efeitos dos fármacos , Vírion/metabolismo , Replicação Viral/efeitos dos fármacos
4.
Methods Mol Biol ; 2203: 147-165, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833211

RESUMO

We have developed a reverse genetics system for the avian coronavirus infectious bronchitis virus (IBV) in which a full-length cDNA corresponding to the IBV genome is inserted into the vaccinia virus genome under the control of a T7 promoter sequence. Vaccinia virus as a vector for the full-length IBV cDNA has the advantage that modifications can be introduced into the IBV cDNA using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. Here, we describe the use of transient dominant selection as a method for introducing modifications into the IBV cDNA that has been successfully used for the substitution of specific nucleotides, deletion of genomic regions, and the exchange of complete genes. Infectious recombinant IBVs are generated in situ following the transfection of vaccinia virus DNA, containing the modified IBV cDNA, into cells infected with a recombinant fowlpox virus expressing T7 DNA-dependent RNA polymerase.


Assuntos
Vírus da Bronquite Infecciosa/genética , Transfecção/métodos , Vírus Vaccinia/genética , Animais , Bacteriófagos/genética , Chlorocebus aethiops , DNA Polimerase Dirigida por DNA/metabolismo , Vírus da Varíola das Aves Domésticas/genética , Recombinação Homóloga , Microrganismos Geneticamente Modificados , Vírus Vaccinia/isolamento & purificação , Células Vero
5.
Genes (Basel) ; 11(8)2020 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-32785186

RESUMO

The coronaviruses are a large family of enveloped RNA viruses that commonly cause gastrointestinal or respiratory illnesses in the infected host. Avian coronavirus infectious bronchitis virus (IBV) is a highly contagious respiratory pathogen of chickens that can affect the kidneys and reproductive systems resulting in bird mortality and decreased reproductivity. The interferon-inducible transmembrane (IFITM) proteins are activated in response to viral infections and represent a class of cellular restriction factors that restrict the replication of many viral pathogens. Here, we characterize the relative mRNA expression of the chicken IFITM genes in response to IBV infection, in vivo, ex vivo and in vitro using the pathogenic M41-CK strain, the nephropathogenic QX strain and the nonpathogenic Beaudette strain. In vivo we demonstrate a significant upregulation of chIFITM1, 2, 3 and 5 in M41-CK- and QX-infected trachea two days post-infection. In vitro infection with Beaudette, M41-CK and QX results in a significant upregulation of chIFITM1, 2 and 3 at 24 h post-infection. We confirmed a differential innate response following infection with distinct IBV strains and believe that our data provide new insights into the possible role of chIFITMs in early IBV infection.


Assuntos
Galinhas/genética , Galinhas/virologia , Infecções por Coronavirus/veterinária , Interações Hospedeiro-Patógeno/genética , Proteínas de Membrana/genética , Animais , Infecções por Coronavirus/genética , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno/fisiologia , Vírus da Bronquite Infecciosa/patogenicidade , Vírus da Bronquite Infecciosa/fisiologia , Técnicas de Cultura de Órgãos , Doenças das Aves Domésticas/etiologia , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/virologia , Carga Viral , Tropismo Viral
6.
Viruses ; 12(7)2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32674326

RESUMO

The Gammacoronavirus infectious bronchitis virus (IBV) is a highly contagious economically important respiratory pathogen of domestic fowl. Reverse genetics allows for the molecular study of pathogenic determinants to enable rational vaccine design. The recombinant IBV (rIBV) Beau-R, a molecular clone of the apathogenic Beaudette strain, has previously been investigated as a vaccine platform. To determine tissues in which Beau-R could effectively deliver antigenic genes, an in vivo study in chickens, the natural host, was used to compare the pattern of viral dissemination of Beau-R to the pathogenic strain M41-CK. Replication of Beau-R was found to be restricted to soft tissue within the beak, whereas M41-CK was detected in beak tissue, trachea and eyelid up to seven days post infection. In vitro assays further identified that, unlike M41-CK, Beau-R could not replicate at 41 °C, the core body temperature of a chicken, but is able to replicate a 37 °C, a temperature relatable to the very upper respiratory tract. Using a panel of rIBVs with defined mutations in the structural and accessory genes, viral replication at permissive and non-permissive temperatures was investigated, identifying that the Beau-R replicase gene was a determinant of temperature sensitivity and that sub-genomic mRNA synthesis had been affected. The identification of temperature sensitive allelic lesions within the Beau-R replicase gene opens up the possibility of using this method of attenuation in other IBV strains for future vaccine development as well as a method to investigate the functions of the IBV replicase proteins.


Assuntos
Infecções por Coronavirus/prevenção & controle , Vírus da Bronquite Infecciosa/imunologia , Doenças das Aves Domésticas/prevenção & controle , Vacinação/veterinária , Vacinas Virais/imunologia , Animais , Linhagem Celular , Embrião de Galinha , Galinhas , Aves Domésticas/virologia , Doenças das Aves Domésticas/virologia , RNA Viral/genética , Temperatura , Vacinas Atenuadas/imunologia , Replicação Viral/genética , Replicação Viral/fisiologia
7.
J Gen Virol ; 101(10): 1103-1118, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32720890

RESUMO

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.


Assuntos
Vírus da Bronquite Infecciosa/genética , RNA Mensageiro/genética , RNA Viral/genética , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição Genética/genética , Regiões 5' não Traduzidas/genética , Animais , Sequência de Bases , Linhagem Celular , Galinhas , Chlorocebus aethiops , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Fases de Leitura Aberta/genética , Doenças das Aves Domésticas/virologia , Células Vero , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/genética
8.
Vaccines (Basel) ; 8(2)2020 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-32580371

RESUMO

Gammacoronavirus infectious bronchitis virus (IBV) causes an economically important respiratory disease of poultry. Protective immunity is associated with the major structural protein, spike (S) glycoprotein, which induces neutralising antibodies and defines the serotype. Cross-protective immunity between serotypes is limited and can be difficult to predict. In this study, the ability of two recombinant IBV vaccine candidates, BeauR-M41(S) and BeauR-4/91(S), to induce cross-protection against a third serotype, QX, was assessed. Both rIBVs are genetically based on the Beaudette genome with only the S gene derived from either M41 or 4/91, two unrelated serotypes. The use of these rIBVs allowed for the assessment of the potential of M41 and 4/91 S glycoproteins to induce cross-protective immunity against a heterologous QX challenge. The impact of the order of vaccination was also assessed. Homologous primary and secondary vaccination with BeauR-M41(S) or BeauR-4/91(S) resulted in a significant reduction of infectious QX load in the trachea at four days post-challenge, whereas heterologous primary and secondary vaccination with BeauR-M41(S) and BeauR-4/91(S) reduced viral RNA load in the conjunctiva-associated lymphoid tissue (CALT). Both homologous and heterologous vaccination regimes reduced clinical signs and birds recovered more rapidly as compared with an unvaccinated/challenge control group. Despite both rIBV BeauR-M41(S) and BeauR-4/91(S) displaying limited replication in vivo, serum titres in these vaccinated groups were higher as compared with the unvaccinated/challenge control group. This suggests that vaccination with rIBV primed the birds for a boosted humoral response to heterologous QX challenge. Collectively, vaccination with the rIBV elicited limited protection against challenge, with failure to protect against tracheal ciliostasis, clinical manifestations, and viral replication. The use of a less attenuated recombinant vector that replicates throughout the respiratory tract could be required to elicit a stronger and prolonged protective immune response.

9.
J Virol ; 93(18)2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-31243124

RESUMO

Like all coronaviruses, avian infectious bronchitis virus (IBV) possesses a long, single-stranded, positive-sense RNA genome (∼27 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.


Assuntos
Vírus da Bronquite Infecciosa/genética , Virulência/genética , Animais , Galinhas/genética , Códon/genética , Infecções por Coronavirus/virologia , Mudança da Fase de Leitura do Gene Ribossômico , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação Viral da Expressão Gênica/genética , Vírus da Bronquite Infecciosa/metabolismo , Fases de Leitura Aberta , Doenças das Aves Domésticas/virologia , RNA Mensageiro/genética , RNA Viral/genética , Ribossomos/metabolismo , Transcriptoma/genética , Sequenciamento Completo do Exoma/métodos
10.
J Virol ; 93(14)2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31043525

RESUMO

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.


Assuntos
Ovos/virologia , Vírus da Bronquite Infecciosa , Polimorfismo de Nucleotídeo Único , Vacinas Virais , Animais , Linhagem Celular , Galinhas , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Vírus da Bronquite Infecciosa/genética , Vírus da Bronquite Infecciosa/imunologia , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/virologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Virais/genética , Vacinas Virais/imunologia
11.
J Virol ; 92(23)2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30209177

RESUMO

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.


Assuntos
Infecções por Coronavirus/imunologia , Infecções por Coronavirus/veterinária , Vírus da Bronquite Infecciosa/imunologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Replicação Viral , Animais , Anticorpos Antivirais/imunologia , Galinhas , Infecções por Coronavirus/virologia , DNA Recombinante , Vírus da Bronquite Infecciosa/genética , Vírus da Bronquite Infecciosa/crescimento & desenvolvimento , Doenças das Aves Domésticas/virologia , Organismos Livres de Patógenos Específicos , Glicoproteína da Espícula de Coronavírus/genética , Vacinação , Carga Viral , Vacinas Virais/administração & dosagem
12.
J Gen Virol ; 99(8): 1097-1102, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29893665

RESUMO

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-1″-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.


Assuntos
Vírus da Bronquite Infecciosa/genética , Vírus da Bronquite Infecciosa/patogenicidade , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/genética , Adenosina Difosfato Ribose , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Galinhas , Regulação Viral da Expressão Gênica , Mutação , Ligação Proteica , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/química , Virulência
13.
Methods Mol Biol ; 1602: 83-102, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28508215

RESUMO

We have developed a reverse genetics system for the avian coronavirus infectious bronchitis virus (IBV) in which a full-length cDNA corresponding to the IBV genome is inserted into the vaccinia virus genome under the control of a T7 promoter sequence. Vaccinia virus as a vector for the full-length IBV cDNA has the advantage that modifications can be introduced into the IBV cDNA using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. Here, we describe the use of transient dominant selection as a method for introducing modifications into the IBV cDNA that has been successfully used for the substitution of specific nucleotides, deletion of genomic regions, and exchange of complete genes. Infectious recombinant IBVs are generated in situ following the transfection of vaccinia virus DNA, containing the modified IBV cDNA, into cells infected with a recombinant fowlpox virus expressing T7 DNA-dependant RNA polymerase.


Assuntos
Vírus da Bronquite Infecciosa/genética , Plasmídeos/genética , Genética Reversa , Animais , Técnicas de Cultura de Células , Chlorocebus aethiops , Infecções por Coronavirus/veterinária , DNA Complementar , Regulação Viral da Expressão Gênica , Genoma Viral , Recombinação Homóloga , RNA Viral , Genética Reversa/métodos , Transfecção , Vírus Vaccinia/genética , Células Vero , Ensaio de Placa Viral , Vírion/genética , Vírion/crescimento & desenvolvimento , Vírion/isolamento & purificação , Replicação Viral
14.
Sci Rep ; 6: 27126, 2016 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-27255716

RESUMO

Positive-strand RNA (+RNA) viruses rearrange cellular membranes during replication, possibly in order to concentrate and arrange viral replication machinery for efficient viral RNA synthesis. Our previous work showed that in addition to the conserved coronavirus double membrane vesicles (DMVs), Beau-R, an apathogenic strain of avian Gammacoronavirus infectious bronchitis virus (IBV), induces regions of ER that are zippered together and tethered open-necked double membrane spherules that resemble replication organelles induced by other +RNA viruses. Here we compared structures induced by Beau-R with the pathogenic lab strain M41 to determine whether membrane rearrangements are strain dependent. Interestingly, M41 was found to have a low spherule phenotype. We then compared a panel of pathogenic, mild and attenuated IBV strains in ex vivo tracheal organ culture (TOC). Although the low spherule phenotype of M41 was conserved in TOCs, each of the other tested IBV strains produced DMVs, zippered ER and spherules. Furthermore, there was a significant correlation for the presence of DMVs with spherules, suggesting that these structures are spatially and temporally linked. Our data indicate that virus induced membrane rearrangements are fundamentally linked to the viral replicative machinery. However, coronavirus replicative apparatus clearly has the plasticity to function in different structural contexts.


Assuntos
Membrana Celular/virologia , Galinhas/virologia , Retículo Endoplasmático/virologia , Gammacoronavirus/patogenicidade , Animais , Células Cultivadas , Retículo Endoplasmático/química , Gammacoronavirus/classificação , Gammacoronavirus/fisiologia , Técnicas de Cultura de Órgãos , Fenótipo , Traqueia/virologia , Virulência , Replicação Viral
15.
Methods Mol Biol ; 1282: 109-12, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25720476

RESUMO

RNA viruses are known for a high mutation rate and rapid genomic evolution. As such an RNA virus population does not consist of a single genotype but is rather a collection of individual viruses with closely related genotypes-a quasispecies, which can be analyzed by next-generation sequencing (NGS). This diversity of genotypes provides a mechanism in which a virus population can evolve and adapt to a changing environment. Sample preparation is vital for successful sequencing. The following protocol describes the process of generating a high-quality RNA preparation from IBV grown in embryonated eggs and then partially purified and concentrated through a 30% sucrose cushion for NGS.


Assuntos
Vírus da Bronquite Infecciosa/isolamento & purificação , RNA Viral/isolamento & purificação , Animais , Embrião de Galinha , Sequenciamento de Nucleotídeos em Larga Escala , Vírus da Bronquite Infecciosa/genética , RNA Viral/genética , Análise de Sequência de RNA
16.
Methods Mol Biol ; 1282: 115-33, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25720477

RESUMO

We have developed a reverse genetics system for the avian coronavirus infectious bronchitis virus (IBV) in which a full-length cDNA corresponding to the IBV genome is inserted into the vaccinia virus genome under the control of a T7 promoter sequence. Vaccinia virus as a vector for the full-length IBV cDNA has the advantage that modifications can be introduced into the IBV cDNA using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. Here, we describe the use of transient dominant selection as a method for introducing modifications into the IBV cDNA; this has been successfully used for the substitution of specific nucleotides, deletion of genomic regions, and the exchange of complete genes. Infectious recombinant IBVs are generated in situ following the transfection of vaccinia virus DNA, containing the modified IBV cDNA, into cells infected with a recombinant fowlpox virus expressing T7 DNA-dependent RNA polymerase.


Assuntos
Vírus da Bronquite Infecciosa/genética , Animais , Técnicas de Cultura de Células , Galinhas , Chlorocebus aethiops , DNA Viral/genética , DNA Viral/isolamento & purificação , Eletroforese em Gel de Campo Pulsado , Genes Dominantes , Genes Virais , Engenharia Genética , Recombinação Homóloga , Vírus da Bronquite Infecciosa/isolamento & purificação , Genética Reversa , Seleção Genética , Transfecção , Vírus Vaccinia/isolamento & purificação , Vírus Vaccinia/fisiologia , Células Vero , Cultura de Vírus
17.
Bioengineered ; 5(5): 288-92, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25482229

RESUMO

Infectious bronchitis virus (IBV) is an economically important virus infecting chickens, causing large losses to the poultry industry globally. While vaccines are available, there is a requirement for novel vaccine strategies due to high strain variation and poor cross-protection. This requires a more detailed understanding of virus-host cell interactions to identify candidates for targeted virus attenuation. One key area of research in the positive sense RNA virus field, due to its central role in virus replication, is the induction of cellular membrane rearrangements by this class of viruses for the assembly of virus replication complexes. In our recent work, we identified the structures induced by IBV during infection of cultured cells, as well as primary cells and ex vivo organ culture. We identified structures novel to the coronavirus family, which strongly resemble replication sites of other positive sense RNA viruses. We have begun to extend this work using recombinant IBVs, which are chimera of different virus strains to study the role of viral proteins in the induction of membrane rearrangements.


Assuntos
Infecções por Coronavirus/veterinária , Retículo Endoplasmático/virologia , Vírus da Bronquite Infecciosa/fisiologia , Membranas Intracelulares/química , Doenças das Aves Domésticas/virologia , Animais , Humanos
18.
J Virol ; 87(4): 2128-36, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23221558

RESUMO

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.


Assuntos
Coronaviridae/genética , Regulação Viral da Expressão Gênica , RNA Mensageiro/genética , RNA Viral/genética , Transcrição Genética , Animais , Células Cultivadas , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA Viral/metabolismo
19.
Eur J Emerg Med ; 19(3): 177-80, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21857228

RESUMO

OBJECTIVE: Digit preference bias has previously been described in a number of different clinical settings including the emergency department. This study aimed to assess whether the implementation of a computerised recording system affects the digit preference bias in recording of times of arrival, assessment and departure at an emergency department. METHODS: A preintervention/postintervention study was undertaken in a type 1 district general hospital emergency department that receives approximately 70 000 attendances per annum. Consecutive 8-week samples were taken before and after the introduction of an electronic whiteboard/patient tracking system. Timings of arrival, nursing and medical assessment and departure were compared. RESULTS: Twelve thousand four hundred and ninety-three patients presented during the 8-week control period and 11 758 patients presented in the 8-week period after the introduction of electronic data capturing. Within the control group, over 80% of the nursing assessment (82.7%), medical examination (92.5%) and departure times (92.7%) ended in '0' or '5', compared with just over 20% after electronic recordings (22.0, 21.7 and 21.8% respectively). CONCLUSION: The introduction of the patient tracking system eliminated the digit preference bias found in recording of the time of nursing assessment, examination and departure that was present in the preintervention data.


Assuntos
Viés , Computadores , Eficiência Organizacional/estatística & dados numéricos , Serviço Hospitalar de Emergência/estatística & dados numéricos , Dedos , Listas de Espera , Eficiência , Acesso aos Serviços de Saúde , Necessidades e Demandas de Serviços de Saúde , Humanos , Qualidade da Assistência à Saúde/estatística & dados numéricos , Fatores de Tempo , Reino Unido
20.
Bioeng Bugs ; 3(2): 114-9, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22179147

RESUMO

Infectious bronchitis virus (IBV) causes an infectious respiratory disease of domestic fowl that affects poultry of all ages causing economic problems for the poultry industry worldwide. Although IBV is controlled using live attenuated and inactivated vaccines it continues to be a major problem due to the existence of many serotypes, determined by the surface spike protein resulting in poor cross-protection, and loss of immunogenicity associated with vaccine production. Live attenuated IBV vaccines are produced by the repeated passage in embryonated eggs resulting in spontaneous mutations. As a consequence attenuated viruses have only a few mutations responsible for the loss of virulence, which will differ between vaccines affecting virulence and/or immunogenicity and can revert to virulence. A new generation of vaccines is called for and one means of controlling IBV involves the development of new and safer vaccines by precisely modifying the IBV genome using reverse genetics for the production of rationally attenuated IBVs in order to obtain an optimum balance between loss of virulence and capacity to induce immunity.


Assuntos
Vírus da Bronquite Infecciosa/imunologia , Vírus da Bronquite Infecciosa/patogenicidade , Doenças das Aves Domésticas/prevenção & controle , Doenças das Aves Domésticas/virologia , Vacinas Virais/imunologia , Animais , Galinhas/imunologia , Galinhas/virologia , Proteção Cruzada , Doenças das Aves Domésticas/imunologia , Genética Reversa/métodos , Vacinas Atenuadas/imunologia , Vacinas de Produtos Inativados/imunologia , Virulência
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