In vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication.
Molecular clones of infectious bronchitis virus (IBV), derived from the Vero cell adapted Beaudette strain, were constructed, using an in vitro assembly method. In vitro transcribed RNA from a cDNA template that had been constructed from seven cDNA fragments, encompassing the entire genome of IBV, was electroporated into BHK-21 cells. The cells were overlaid onto the susceptible Vero cells and viable virus was recovered from the molecular clone. The molecularly cloned IBV (MIBV) demonstrated growth kinetics, and plaque size and morphology that resembled the parental Beaudette strain IBV. The recombinant virus was further manipulated to express enhanced green fluorescent protein (EGFP) by replacing an open reading frame (ORF) of the group-specific gene, ORF 5a, with the EGFP ORF. The rescued recombinant virus, expressing EGFP (GIBV), replicated to lower viral titers and formed smaller plaques compared to the parental virus and the MIBV. After six passages of GIBV, a minority of plaques were observed that had reverted to the larger plaque size and virus from these plaques no longer expressed EGFP. Direct sequencing of RT-PCR products derived from cells infected with the plaque-purified virus, which had lost expression of EGFP, confirmed loss of the EGFP ORF. The loss of EGFP expression (Delta5a IBV) was also accompanied by reversion to growth kinetics resembling the standard virus and intact recombinant virus. This study demonstrates that the 5a ORF is not essential for viral multiplication in Vero cells. (+info)
Emergence of a nephropathogenic avian infectious bronchitis virus with a novel genotype in India.
We describe the emergence of a nephropathogenic avian infectious bronchitis virus (IBV) with a novel genotype in India. The Indian IBV isolate exhibited a relatively high degree of sequence divergence with reference strains. The highest homology was observed with strain 6/82 (68%) and the least homology with strain Mex/1765/99 (34.3%). (+info)
Isolation of avian infectious bronchitis coronavirus from domestic peafowl (Pavo cristatus) and teal (Anas).
Coronavirus-like viruses, designated peafowl/China/LKQ3/2003 (pf/CH/LKQ3/03) and teal/China/LDT3/2003 (tl/CH/LDT3/03), were isolated from a peafowl and a teal during virological surveillance in Guangdong province, China. Partial genomic sequence analysis showed that these isolates had the S-3-M-5-N gene order that is typical of avian coronaviruses. The spike, membrane and nucleocapsid protein genes of pf/CH/LKQ3/03 had >99 % identity to those of the avian infectious bronchitis coronavirus H120 vaccine strain (Massachusetts serotype) and other Massachusetts serotype isolates. Furthermore, when pf/CH/LKQ3/03 was inoculated experimentally into chickens (specific-pathogen-free), no disease signs were apparent. tl/CH/LDT3/03 had a spike protein gene with 95 % identity to that of a Chinese infectious bronchitis virus (IBV) isolate, although more extensive sequencing revealed the possibility that this strain may have undergone recombination. When inoculated into chickens, tl/CH/LDT3/03 resulted in the death of birds from nephritis. Taken together, this information suggests that pf/CH/LKQ3/03 might be a revertant, attenuated vaccine IBV strain, whereas tl/CH/LDT3/03 is a nephropathogenic field IBV strain, generated through recombination. The replication and non-pathogenic nature of IBV in domestic peafowl and teal under field conditions raises questions as to the role of these hosts as carriers of IBV and the potential that they may have to transmit virus to susceptible chicken populations. (+info)
Development and application of a Saccharomyces cerevisiae-expressed nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibodies against infectious bronchitis virus.
A Saccharomyces cerevisiae-expressed nucleocapsid (N) polypeptide of the M41 strain of infectious bronchitis virus (IBV) was used as antigen in a recombinant yeast-expressed N protein-based enzyme-linked immunosorbent assay (Y-N-ELISA). The Y-N-ELISA was rapid, sensitive, and specific for detecting chicken serum antibodies to IBV, and it compared favorably with a commercial ELISA. (+info)
Infectious bronchitis virus 3a protein localizes to a novel domain of the smooth endoplasmic reticulum.
All coronaviruses possess small open reading frames (ORFs) between structural genes that have been hypothesized to play important roles in pathogenesis. Infectious bronchitis virus (IBV) ORF 3a is one such gene. It is highly conserved among group 3 coronaviruses, suggesting that it has an important function in infection. IBV 3a protein is expressed in infected cells but is not detected in virions. Sequence analysis predicted that IBV 3a was a membrane protein; however, only a fraction behaved like an integral membrane protein. Microscopy and immunoprecipitation studies demonstrated that IBV 3a localized to the cytoplasm in a diffuse pattern as well as in sharp puncta in both infected and transfected cells. These puncta did not overlap cellular organelles or other punctate structures. Confocal microscopy demonstrated that IBV 3a puncta lined up along smooth endoplasmic reticulum (ER) tubules and, in a significant number of instances, were partially surrounded by these tubules. Our results suggest that IBV 3a is partially targeted to a novel domain of the smooth ER. (+info)
The virion N protein of infectious bronchitis virus is more phosphorylated than the N protein from infected cell lysates.
Because phosphorylation of the infectious bronchitis virus (IBV) nucleocapsid protein (N) may regulate its multiple roles in viral replication, the dynamics of N phosphorylation were examined. 32P-orthophosphate labeling and Western blot analyses confirmed that N was the only viral protein that was phosphorylated. Pulse labeling with 32P-orthophosphate indicated that the IBV N protein was phosphorylated in the virion, as well as at all times during infection in either chicken embryo kidney cells or Vero cells. Pulse-chase analyses followed by immunoprecipitation of IBV N proteins using rabbit anti-IBV N polyclonal antibody demonstrated that the phosphate on the N protein was stable for at least 1 h. Simultaneous labeling with 32P-orthophosphate and 3H-leucine identified a 3.5-fold increase in the 32P:3H counts per minute (cpm) ratio of N in the virion as compared to the 32P:3H cpm ratio of N in the cell lysates from chicken embryo kidney cells, whereas in Vero cells the 32P:3H cpm ratio of N from the virion was 10.5-fold greater than the 32P:3H cpm ratio of N from the cell lysates. These studies are consistent with the phosphorylation of the IBV N playing a role in assembly or maturation of the viral particle. (+info)
Evaluation of serum- and animal protein-free media for the production of infectious bronchitis virus (M41) strain in a continuous cell line.
Infectious bronchitis virus (IBV) is produced in vitro using specific pathogen free chicken embryos, primary chicken kidney cells (CKC) and/or tracheal organ culture (TOC). Regulatory authorities in Europe (EMEA) and in the United States (FDA) have encouraged biological manufactures to reduce or eliminate the use of live animals and/or products of animal origin in biological manufacturing processes. In this paper, a stable chicken embryo-related (CER) cell line was adapted and maintained in serum free (SF) or animal protein free (APF) medium after a direct switch of the medium. The most suitable media were Ex Cell 520 and Ex Cell 302. CER monolayers adapted to SF or APF were infected with IBV (M41 strain) in agitated suspended culture and the IBV titre obtained 48 h post infection was 2.8 x 104 PFU/ml in both cases. Thus, propagation of CER cells and culture of IBV can be performed without the use of animal serum or animal protein. (+info)
Contribution of trafficking signals in the cytoplasmic tail of the infectious bronchitis virus spike protein to virus infection.
Coronavirus spike (S) proteins are responsible for binding and fusion with target cells and thus play an essential role in virus infection. Recently, we identified a dilysine endoplasmic reticulum (ER) retrieval signal and a tyrosine-based endocytosis signal in the cytoplasmic tail of the S protein of infectious bronchitis virus (IBV). Here, an infectious cDNA clone of IBV was used to address the importance of the S protein trafficking signals to virus infection. We constructed infectious cDNA clones lacking the ER retrieval signal, the endocytosis signal, or both. The virus lacking the ER retrieval signal was viable. However, this virus had a growth defect at late times postinfection and produced larger plaques than IBV. Further analysis confirmed that the mutant S protein trafficked though the secretory pathway faster than wild-type S protein. A more dramatic phenotype was obtained when the endocytosis signal was mutated. Recombinant viruses lacking the endocytosis signal (in combination with a mutated dilysine signal or alone) could not be recovered, even though transient syncytia were formed in transfected cells. Our results suggest that the endocytosis signal of IBV S is essential for productive virus infection. (+info)