Utilizing fowlpox virus recombinants to generate defective RNAs of the coronavirus infectious bronchitis virus.
Coronavirus defective RNAs (D-RNAs) have been used as RNA vectors for the expression of heterologous genes and as vehicles for reverse genetics by modifying coronavirus genomes by targetted recombination. D-RNAs based on the avian coronavirus infectious bronchitis virus (IBV) D-RNA CD-61 have been rescued (replicated and packaged into virions) in a helper virus-dependent manner following electroporation of in vitro-generated T7 transcripts into IBV-infected cells. In order to increase the efficiency of rescue of IBV D-RNAs, cDNAs based on CD-61, under the control of a T7 promoter, were integrated into the fowlpox virus (FPV) genome. The 3'-UTR of the D-RNAs was flanked by a hepatitis delta antigenomic ribozyme and T7 terminator sequence to generate suitable 3' ends for rescue by helper IBV. Cells were co-infected simultaneously with IBV, the recombinant FPV (rFPV) containing the D-RNA sequence and a second rFPV expressing T7 RNA polymerase for the initial expression of the D-RNA transcript, subsequently rescued by helper IBV. Rescue of rFPV-derived CD-61 occurred earlier and with higher efficiency than demonstrated previously for electroporation of in vitro T7-generated RNA transcripts in avian cells. Rescue of CD-61 was also demonstrated for the first time in mammalian cells. The rescue of rFPV-derived CD-61 by M41 helper IBV resulted in leader switching, in which the Beaudette-type leader sequence on CD-61 was replaced with the M41 leader sequence, confirming that helper IBV virus replicated the rFPV-derived D-RNA. An rFPV-derived D-RNA containing the luciferase gene under the control of an IBV transcription-associated sequence was also rescued and expressed luciferase on serial passage. (+info)
cis-acting sequences required for coronavirus infectious bronchitis virus defective-RNA replication and packaging.
The parts of the RNA genome of infectious bronchitis virus (IBV) required for replication and packaging of the RNA were investigated using deletion mutagenesis of a defective RNA (D-RNA) CD-61 (6.1 kb) containing a chloramphenicol acetyltransferase reporter gene. A D-RNA with the first 544, but not as few as 338, nucleotides (nt) of the 5' terminus was replicated; the 5' untranslated region (UTR) comprises 528 nt. Region I of the 3' UTR, adjacent to the nucleocapsid protein gene, comprised 212 nt and could be removed without impairment of replication or packaging of D-RNAs. A D-RNA with the final 338 nt, including the 293 nt in the highly conserved region II of the 3' UTR, was replicated. Thus, the 5'-terminal 544 nt and 3'-terminal 338 nt contained the necessary signals for RNA replication. Phylogenetic analysis of 19 strains of IBV and 3 strains of turkey coronavirus predicted a conserved stem-loop structure at the 5' end of region II of the 3' UTR. Removal of the predicted stem-loop structure abolished replication of the D-RNAs. D-RNAs in which replicase gene 1b-derived sequences had been removed or replaced with all the downstream genes were replicated well but were rescued poorly, suggesting inefficient packaging. However, no specific part of the 1b gene was required for efficient packaging. (+info)
The coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus.
The coronavirus nucleoprotein (N) has been reported to be involved in various aspects of virus replication. We examined by confocal microscopy the subcellular localization of the avian infectious bronchitis virus N protein both in the absence and in the context of an infected cell and found that N protein localizes both to the cytoplasmic and nucleolar compartments. (+info)
Effect of liquid paraffin on antibody responses and local adverse reactions of bivalent oil adjuvanted vaccines containing newcastle disease virus and infectious bronchitis virus.
Effects of liquid paraffin on antibody responses and local adverse reactions after intramuscular injection of oil adjuvanted vaccines containing Newcastle disease (ND) and infectious bronchitis (IB) virus were investigated in chickens. Each vaccine was prepared with a liquid paraffin such as Carnation, Crystol 52 and Lytol. These vaccines induced sustained antibody responses against ND and IB. Among local adverse reactions, Lytol induced granulomatous reactions and abscesses, but Carnation and Crystol 52 did not. The residual weight of liquid paraffin at the injection site decreased in the order Carnation, Crystol 52, Lytol. Crystol 52 was composed of relatively few short-chain hydrocarbons (i.e., n-C20H42). The vaccine with liquid paraffin mainly composed of n-C16H34-n-C20H42 was suggested to induce fewer adverse reactions. (+info)
Serological monitoring on layer farms with specific pathogen-free chickens.
To monitor the existence of avian pathogens in laying chicken flocks, specific pathogen-free (SPF) chickens were introduced into two layer farms and reared with laying hens for 12 months. SPF chickens were bled several times after their introduction and examined for their sero-conversion to avian pathogens. As a result, antibodies to eight or ten kinds of pathogens were detected in SPF chickens on each farm. Antibodies to infectious bronchitis virus (IBV), avian nephritis virus, Mycoplasma gallisepticum and M. synoviae were detected early within the first month. Antibody titer to IBV suggested that the laying chickens were infected with IBV repeatedly during the experiment on both farms. However, antibodies to infectious bursal disease virus and 6 pathogens were not detected. (+info)
The missing link in coronavirus assembly. Retention of the avian coronavirus infectious bronchitis virus envelope protein in the pre-Golgi compartments and physical interaction between the envelope and membrane proteins.
One missing link in the coronavirus assembly is the physical interaction between two crucial structural proteins, the membrane (M) and envelope (E) proteins. In this study, we demonstrate that the coronavirus infectious bronchitis virus E can physically interact, via a putative peripheral domain, with M. Deletion of this domain resulted in a drastic reduction in the incorporation of M into virus-like particles. Immunofluorescent staining of cells coexpressing M and E supports that E interacts with M and relocates M to the same subcellular compartments that E resides in. E was retained in the pre-Golgi membranes, prior to being translocated to the Golgi apparatus and the secretory vesicles; M was observed to exhibit similar localization and translocation profiles as E when coexpressed with E. Deletion studies identified the C-terminal 6-residue RDKLYS as the endoplasmic reticulum retention signal of E, and site-directed mutagenesis of the -4 lysine residue to glutamine resulted in the accumulation of E in the Golgi apparatus. The third domain of E that plays a crucial role in virus budding is a putative transmembrane domain present at the N-terminal region, because deletion of the domain resulted in a free distribution of the mutant protein and in dysfunctional viral assembly. (+info)
Induction of caspase-dependent apoptosis in cultured cells by the avian coronavirus infectious bronchitis virus.
Avian coronavirus infectious bronchitis virus (IBV) is the causative agent of chicken infectious bronchitis, an acute, highly contagious viral respiratory disease. Replication of IBV in Vero cells causes extensive cytopathic effects (CPE), leading to destruction of the entire monolayer and the death of infected cells. In this study, we investigated the cell death processes during acute IBV infection and the underlying mechanisms. The results show that both necrosis and apoptosis may contribute to the death of infected cells in lytic IBV infection. Caspase-dependent apoptosis, as characterized by chromosomal condensation, DNA fragmentation, caspase-3 activation, and poly(ADP-ribose) polymerase degradation, was detected in IBV-infected Vero cells. Addition of the general caspase inhibitor z-VAD-FMK to the culture media showed inhibition of the hallmarks of apoptosis and increase of the release of virus to the culture media at 16 h postinfection. However, neither the necrotic process nor the productive replication of IBV in Vero cells was severely affected by the inhibition of apoptosis. Screening of 11 IBV-encoded proteins suggested that a 58-kDa mature cleavage product could induce apoptotic changes in cells transiently expressing the protein. This study adds one more example to the growing list of animal viruses that induce apoptosis during their replication cycles. (+info)
The autocatalytic release of a putative RNA virus transcription factor from its polyprotein precursor involves two paralogous papain-like proteases that cleave the same peptide bond.
The largest replicative protein of coronaviruses is known as p195 in the avian infectious bronchitis virus (IBV) and p210 (p240) in the mouse hepatitis virus. It is autocatalytically released from the precursors pp1a and pp1ab by one zinc finger-containing papain-like protease (PLpro) in IBV and by two paralogous PLpros, PL1pro and PL2pro, in mouse hepatitis virus. The PLpro-containing proteins have been recently implicated in the control of coronavirus subgenomic mRNA synthesis (transcription). By using comparative sequence analysis, we now show that the respective proteins of all sequenced coronaviruses are flanked by two conserved PLpro cleavage sites and share a complex (multi)domain organization with PL1pro being inactivated in IBV. Based upon these predictions, the processing of the human coronavirus 229E p195/p210 N terminus was studied in detail. First, an 87-kDa protein (p87), which is derived from a pp1a/pp1ab region immediately upstream of p195/p210, was identified in human coronavirus 229E-infected cells. Second, in vitro synthesized proteins representing different parts of pp1a were autocatalytically processed at the predicted site. Surprisingly, both PL1pro and PL2pro cleaved between p87 and p195/p210. The PL1pro-mediated cleavage was slow and significantly suppressed by a non-proteolytic activity of PL2pro. In contrast, PL2pro, whose proteolytic activity and specificity were established in this study, cleaved the same site efficiently in the presence of the upstream domains. Third, a correlation was observed between the overlapping substrate specificities and the parallel evolution of PL1pro and PL2pro. Collectively, our results imply that the p195/p210 autoprocessing mechanisms may be conserved among coronaviruses to an extent not appreciated previously, with PL2pro playing a major role. A large subset of coronaviruses may employ two proteases to cleave the same site(s) and thus regulate the expression of the viral genome in a unique way. (+info)