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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.
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
3.
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
4.
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
5.
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.

6.
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
7.
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
8.
J Gen Virol ; 99(12): 1681-1685, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30355423

RESUMO

The spike glycoprotein (S) of infectious bronchitis virus (IBV) comprises two subunits, S1 and S2. We have previously demonstrated that the S2 subunit of the avirulent Beau-R strain is responsible for its extended cellular tropism for Vero cells. Two recombinant infectious bronchitis viruses (rIBVs) have been generated; the immunogenic S1 subunit is derived from the IBV vaccine strain, H120, or the virulent field strain, QX, within the genetic background of Beau-R. The rIBVs BeauR-H120(S1) and BeauR-QX(S1) are capable of replicating in primary chicken kidney cell cultures and in Vero cells. These results demonstrate that rIBVs are able to express S1 subunits from genetically diverse strains of IBV, which will enable the rational design of a future generation of IBV vaccines that may be grown in Vero cells.


Assuntos
Infecções por Coronavirus/prevenção & controle , Vírus da Bronquite Infecciosa/crescimento & desenvolvimento , Doenças das Aves Domésticas/prevenção & controle , Proteínas Recombinantes/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Vacinas Virais/imunologia , Replicação Viral , Animais , Células Cultivadas , Galinhas , Chlorocebus aethiops , Infecções por Coronavirus/veterinária , Vírus da Bronquite Infecciosa/genética , Vírus da Bronquite Infecciosa/imunologia , Proteínas Recombinantes/genética , Glicoproteína da Espícula de Coronavírus/genética , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/isolamento & purificação , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificação , Células Vero , Vacinas Virais/genética , Vacinas Virais/isolamento & purificação
9.
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
10.
J Virol ; 92(19)2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30021894

RESUMO

The spike (S) glycoprotein of the avian gammacoronavirus infectious bronchitis virus (IBV) is comprised of two subunits (S1 and S2), has a role in virulence in vivo, and is responsible for cellular tropism in vitro We have previously demonstrated that replacement of the S glycoprotein ectodomain from the avirulent Beaudette strain of IBV with the corresponding region from the virulent M41-CK strain resulted in a recombinant virus, BeauR-M41(S), with the in vitro cell tropism of M41-CK. The IBV Beaudette strain is able to replicate in both primary chick kidney cells and Vero cells, whereas the IBV M41-CK strain replicates in primary cells only. In order to investigate the region of the IBV S responsible for growth in Vero cells, we generated a series of recombinant IBVs expressing chimeric S glycoproteins, consisting of regions from the Beaudette and M41-CK S gene sequences, within the genomic background of Beaudette. The S2, but not the S1, subunit of the Beaudette S was found to confer the ability to grow in Vero cells. Various combinations of Beaudette-specific amino acids were introduced into the S2 subunit of M41 to determine the minimum requirement to confer tropism for growth in Vero cells. The ability of IBV to grow and produce infectious progeny virus in Vero cells was subsequently narrowed down to just 3 amino acids surrounding the S2' cleavage site. Conversely, swapping of the 3 Beaudette-associated amino acids with the corresponding ones from M41 was sufficient to abolish Beaudette growth in Vero cells.IMPORTANCE Infectious bronchitis remains a major problem in the global poultry industry, despite the existence of many different vaccines. IBV vaccines, both live attenuated and inactivated, are currently grown on embryonated hen's eggs, a cumbersome and expensive process due to the fact that most IBV strains do not grow in cultured cells. The reverse genetics system for IBV creates the opportunity for generating rationally designed and more effective vaccines. The observation that IBV Beaudette has the additional tropism for growth on Vero cells also invokes the possibility of generating IBV vaccines produced from cultured cells rather than by the use of embryonated eggs. The regions of the IBV Beaudette S glycoprotein involved in the determination of extended cellular tropism were identified in this study. This information will enable the rational design of a future generation of IBV vaccines that may be grown on Vero cells.


Assuntos
Infecções por Coronavirus , Vírus da Bronquite Infecciosa/fisiologia , Subunidades Proteicas , Glicoproteína da Espícula de Coronavírus , Tropismo Viral/fisiologia , Replicação Viral/fisiologia , Animais , Galinhas , Chlorocebus aethiops , Infecções por Coronavirus/genética , Infecções por Coronavirus/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero
11.
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
12.
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
13.
J Virol ; 91(5)2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-27974565

RESUMO

Autophagy functions as an intrinsic antiviral defense. However, some viruses can subvert or even enhance host autophagic machinery to increase viral replication and pathogenesis. The role of autophagy during avibirnavirus infection, especially late stage infection, remains unclear. In this study, infectious bursal disease virus (IBDV) was used to investigate the role of autophagy in avibirnavirus replication. We demonstrated IBDV induction of autophagy as a significant increase in puncta of LC3+ autophagosomes, endogenous levels of LC3-II, and ultrastructural characteristics typical of autophagosomes during the late stage of infection. Induction of autophagy enhances IBDV replication, whereas inhibition of autophagy impairs viral replication. We also demonstrated that IBDV infection induced autophagosome-lysosome fusion, but without active degradation of their contents. Moreover, inhibition of fusion or of lysosomal hydrolysis activity significantly reduced viral replication, indicating that virions utilized the low-pH environment of acidic organelles to facilitate viral maturation. Using immuno-transmission electron microscopy (TEM), we observed that a large number of intact IBDV virions were arranged in a lattice surrounded by p62 proteins, some of which lay between virions. Additionally, many virions were encapsulated within the vesicular membranes, with an obvious release stage observed by TEM. The autophagic endosomal pathway facilitates low-pH-mediated maturation of viral proteins and membrane-mediated release of progeny virions.IMPORTANCE IBDV is the most extensively studied virus in terms of molecular characteristics and pathogenesis; however, mechanisms underlying the IBDV life cycle require further exploration. The present study demonstrated that autophagy enhances viral replication at the late stage of infection, and the autophagy pathway facilitates IBDV replication complex function and virus assembly, which is critical to completion of the virus life cycle. Moreover, the virus hijacks the autophagic vacuoles to mature in an acidic environment and release progeny virions in a membrane-mediated cell-to-cell manner. This autophagic endosomal pathway is proposed as a new mechanism that facilitates IBDV maturation, release, and reinternalization. This report presents a concordance in exit strategies among some RNA and DNA viruses, which exploit autophagy pathway for their release from cells.


Assuntos
Autofagia , Infecções por Birnaviridae/veterinária , Vírus da Doença Infecciosa da Bursa/fisiologia , Doenças das Aves Domésticas/virologia , Vacúolos/virologia , Animais , Infecções por Birnaviridae/virologia , Linhagem Celular , Embrião de Galinha , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Imunidade Inata , Transdução de Sinais , Vacúolos/fisiologia , Internalização do Vírus , Liberação de Vírus , Replicação Viral
14.
Avian Dis ; 60(3): 596-602, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27610718

RESUMO

The avian coronavirus infectious bronchitis virus (AvCoV-IBV) is recognized as an important global pathogen because new variants are a continuous threat to the poultry industry worldwide. This study investigates the genetic origin and diversity of AvCoV-IBV by analysis of the S1 sequence derived from 49 broiler flocks and 14 layer flocks in different regions of Turkey. AvCoV-IBV RNA was detected in 41 (83.6%) broiler flocks and nine (64.2%) of the layer flocks by TaqMan real-time RT-PCR. In addition, AvCoV-IBV RNA was detected in the tracheas 27/30 (90%), lungs 31/49 (62.2%), caecal tonsils 7/22 (31.8%), and kidneys 4/49 (8.1%) of broiler flocks examined. Pathologic lesions, hemorrhages, and mononuclear infiltrations were predominantly observed in tracheas and to a lesser extent in the lungs and a few in kidneys. A phylogenetic tree based on partial S1 sequences of the detected AvCoV-IBVs (including isolates) revealed that 1) viruses detected in five broiler flocks were similar to the IBV vaccines Ma5, H120, M41; 2) viruses detected in 24 broiler flocks were similar to those previously reported from Turkey and to Israel variant-2 strains; 3) viruses detected in seven layer flocks were different from those found in any of the broiler flocks but similar to viruses previously reported from Iran, India, and China (similar to Israel variant-1 and 4/91 serotypes); and 4) that the AVCoV-IBV, Israeli variant-2 strain, found to be circulating in Turkey appears to be undergoing molecular evolution. In conclusion, genetically different AvCoV-IBV strains, including vaccine-like strains, based on their partial S1 sequence, are circulating in broiler and layer chicken flocks in Turkey and the Israeli variant-2 strain is undergoing evolution.


Assuntos
Proteínas do Capsídeo/genética , Galinhas , Infecções por Coronavirus/veterinária , Variação Genética , Vírus da Bronquite Infecciosa/genética , Filogenia , Doenças das Aves Domésticas/virologia , Animais , Proteínas do Capsídeo/metabolismo , Infecções por Coronavirus/virologia , Vírus da Bronquite Infecciosa/classificação , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Análise de Sequência de RNA/veterinária , Turquia
15.
J Virol ; 90(16): 7519-7528, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27279618

RESUMO

UNLABELLED: During infection of their host cells, viruses often inhibit the production of host proteins, a process that is referred to as host shutoff. By doing this, viruses limit the production of antiviral proteins and increase production capacity for viral proteins. Coronaviruses from the genera Alphacoronavirus and Betacoronavirus, such as severe acute respiratory syndrome coronavirus (SARS-CoV), establish host shutoff via their nonstructural protein 1 (nsp1). The Gammacoronavirus and Deltacoronavirus genomes, however, do not encode nsp1, and it has been suggested that these viruses do not induce host shutoff. Here, we show that the Gammacoronavirus infectious bronchitis virus (IBV) does induce host shutoff, and we find that its accessory protein 5b is indispensable for this function. Importantly, we found that 5b-null viruses, unlike wild-type viruses, induce production of high concentrations of type I interferon protein in vitro, indicating that host shutoff by IBV plays an important role in antagonizing the host's innate immune response. Altogether, we demonstrate that 5b is a functional equivalent of nsp1, thereby answering the longstanding question of whether lack of nsp1 in gammacoronaviruses is compensated for by another viral protein. As such, our study is a significant step forward in the understanding of coronavirus biology and closes a gap in the understanding of some IBV virulence strategies. IMPORTANCE: Many viruses inhibit protein synthesis by their host cell to enhance virus replication and to antagonize antiviral defense mechanisms. This process is referred to as host shutoff. We studied gene expression and protein synthesis in chicken cells infected with the important poultry pathogen infectious bronchitis virus (IBV). We show that IBV inhibits synthesis of host proteins, including that of type I interferon, a key component of the antiviral response. The IBV-induced host shutoff, however, does not require degradation of host RNA. Furthermore, we demonstrate that accessory protein 5b of IBV plays a crucial role in the onset of host shutoff. Our findings suggest that inhibition of host protein synthesis is a common feature of coronaviruses and primarily serves to inhibit the antiviral response of the host.


Assuntos
Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Vírus da Bronquite Infecciosa/imunologia , Vírus da Bronquite Infecciosa/patogenicidade , Interferon Tipo I/antagonistas & inibidores , Proteínas Virais/metabolismo , Animais , Células Cultivadas , Galinhas , Técnicas de Inativação de Genes , Vírus da Bronquite Infecciosa/genética , Proteínas Virais/genética
16.
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
17.
Infect Genet Evol ; 39: 349-364, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26883378

RESUMO

Infectious bronchitis virus (IBV) is the causative agent of a highly contagious disease that results in severe economic losses to the global poultry industry. The virus exists in a wide variety of genetically distinct viral types, and both phylogenetic analysis and measures of pairwise similarity among nucleotide or amino acid sequences have been used to classify IBV strains. However, there is currently no consensus on the method by which IBV sequences should be compared, and heterogeneous genetic group designations that are inconsistent with phylogenetic history have been adopted, leading to the confusing coexistence of multiple genotyping schemes. Herein, we propose a simple and repeatable phylogeny-based classification system combined with an unambiguous and rationale lineage nomenclature for the assignment of IBV strains. By using complete nucleotide sequences of the S1 gene we determined the phylogenetic structure of IBV, which in turn allowed us to define 6 genotypes that together comprise 32 distinct viral lineages and a number of inter-lineage recombinants. Because of extensive rate variation among IBVs, we suggest that the inference of phylogenetic relationships alone represents a more appropriate criterion for sequence classification than pairwise sequence comparisons. The adoption of an internationally accepted viral nomenclature is crucial for future studies of IBV epidemiology and evolution, and the classification scheme presented here can be updated and revised novel S1 sequences should become available.


Assuntos
Infecções por Coronavirus/virologia , Vírus da Bronquite Infecciosa/classificação , Vírus da Bronquite Infecciosa/genética , Filogenia , Proteínas do Envelope Viral/genética , Animais , Galinhas , Biologia Computacional/métodos , Genótipo , Recombinação Genética , Análise de Sequência de DNA
18.
J Virol ; 89(23): 12047-57, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26401035

RESUMO

UNLABELLED: The innate immune response is the first line of defense against viruses, and type I interferon (IFN) is a critical component of this response. Similar to other viruses, the gammacoronavirus infectious bronchitis virus (IBV) has evolved under evolutionary pressure to evade and counteract the IFN response to enable its survival. Previously, we reported that IBV induces a delayed activation of the IFN response. In the present work, we describe the resistance of IBV to IFN and the potential role of accessory proteins herein. We show that IBV is fairly resistant to the antiviral state induced by IFN and identify that viral accessory protein 3a is involved in resistance to IFN, as its absence renders IBV less resistant to IFN treatment. In addition to this, we found that independently of its accessory proteins, IBV inhibits IFN-mediated phosphorylation and translocation of STAT1. In summary, we show that IBV uses multiple strategies to counteract the IFN response. IMPORTANCE: In the present study, we show that infectious bronchitis virus (IBV) is resistant to IFN treatment and identify a role for accessory protein 3a in the resistance against the type I IFN response. We also demonstrate that, in a time-dependent manner, IBV effectively interferes with IFN signaling and that its accessory proteins are dispensable for this activity. This study demonstrates that the gammacoronavirus IBV, similar to its mammalian counterparts, has evolved multiple strategies to efficiently counteract the IFN response of its avian host, and it identifies accessory protein 3a as multifaceted antagonist of the avian IFN system.


Assuntos
Vírus da Bronquite Infecciosa/imunologia , Vírus da Bronquite Infecciosa/metabolismo , Interferon Tipo I/imunologia , Fator de Transcrição STAT1/imunologia , Transdução de Sinais/imunologia , Proteínas Virais Reguladoras e Acessórias/metabolismo , Análise de Variância , Animais , Western Blotting , Células Cultivadas , Embrião de Galinha , Chlorocebus aethiops , Primers do DNA/genética , Células HEK293 , Humanos , Imuno-Histoquímica , Vírus da Bronquite Infecciosa/genética , Luciferases , Células Vero
20.
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
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