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The exacerbating effect of infectious bronchitis virus infection on the infectious bursal disease virus-induced suppression of opsonization by Escherichia coil antibody in chickens.

Chickens infected with infectious bronchitis virus (IBV) and infectious bursal disease virus (IBDV) commonly develop secondary infection of the respiratory tract with Escherichia coli, resulting in significant economic losses. To understand the host factors that may contribute to the E. coli infection, we investigated macrophage-mediated E. coli phagocytosis, intracellular bacterial killing, and development of opsonizing antibody in previously uninfected chickens and in those infected with IBV, IBDV, and IBDV plus IBV. Macrophages from the peripheral blood and the respiratory tracts of chickens infected with IBV or IBDV plus IBV efficiently performed in vitro phagocytosis of E. coli in the presence of positive-control serum (i.e., E. coli antiserum produced in normal chickens). Those macrophages also had adequate bactericidal activity, indicating that IBV and IBDV infections had not affected their phagocytic activity or bactericidal function. The phagocytic activity of macrophages remained unaffected (P < 0.05) when the positive-control serum was replaced with E. coli antiserum produced in chickens infected with IBV alone. However, when E. coli antisera raised in IBDV-infected and, especially, that produced in IBDV plus IBV-infected chickens were supplemented, the percentage of phagocytosis and number of bacteria ingested per phagocyte were significantly (P < 0.05) less. These results indicate that although IBDV alone has the potential to markedly reduce opsonizing ability of antibody, this effect is significantly (P < 0.05) exacerbated by IBV infection.

Animals↗

Protective effect of vaccination in chicks with local infectious bronchitis viruses against field virus challenge.

Avian infectious bronchitis virus (IBV) causes tremendous economic losses to the poultry industry worldwide. Different serotypes of this virus show little cross-protection. The present study investigated the relationship between differences in the genotype based on the N-terminus of the spike protein and the protection provided by vaccination with IBVs. Cross-immunization tests were performed using both killed and live viruses in specific-pathogen-free chicks. One-day-old chicks were immunized with killed or high [10(5) 50% embryo infectious dose (EID50)] or low (10(3) EID50) doses of live IBV viruses and challenged with homologous or heterologous IBV strains. The immunization efficacy was evaluated by virus isolation from the challenged chicks. In the killed-virus and high-dose live-virus test groups, IBV vaccination protected against challenge by homologous but not heterologous IBV strains. However, a low dose of live IBV showed no protection against virulent IBV challenge. These results indicate that both the genotype based on the N-terminus of the spike protein and the virus dose are essential to IBV protection from immunization. Thus, development of vaccines from different local strains is necessary to control infectious bronchitis in poultry.

Animals↗

Increased incidence of airsacculitis in broilers infected with mycoplasma synoviae and chicken-passaged infectious bronchitis vaccine virus.

Infectious bronchitis vaccine virus is thought to cycle (i.e., pass from vaccine-virus-infected to susceptible chickens) in commercial broiler and pullet flocks. To simulate the effect of this cycling, mild infectious bronchitis vaccine virus was passaged in chickens serially six times. This sixth passage virus was used to infect chickens, which were then exposed to a moderately cold environment of 10 +/- 2 C and to Mycoplasma synoviae. In two separate experiments, the chicken-passaged vaccine virus resulted in a marked increase in the incidence of airsacculitis compared with nonpassaged vaccine virus. Intermediate passage levels also increased airsacculitis incidence but not as much as the sixth passage virus. In another experiment, virus chicken-passaged by contact transmission also caused increased airsacculitis incidence.

Air Sacs↗

Presence of an encephalomyocarditis virus internal ribosome entry site sequence in avian infectious bronchitis virus defective RNAs abolishes rescue by helper virus.

Avian infectious bronchitis virus (IBV) defective RNAs (D-RNAs) have been used for the expression of heterologous genes in a helper-virus-dependent expression system. The heterologous genes were expressed under the control of an IBV transcription-associated sequence (TAS) derived from gene 5 of IBV Beaudette. However, coronavirus D-RNA expression vectors display an inherent instability following serial passage with helper virus, resulting in the eventual loss of the heterologous genes. The use of the picornavirus encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) sequence to initiate gene translation was investigated as an alternative method to the coronavirus-mediated TAS-controlled heterologous gene expression system. IBV D-RNAs containing the chloramphenicol acetyltransferase (CAT) reporter gene, under EMCV IRES control, were assessed for IRES-mediated CAT protein translation. CAT protein was detected from T7-derived IBV D-RNA transcripts in a cell-free protein synthesis system and in situ in avian chick kidney (CK) cells following T7-derived D-RNA synthesis from a recombinant fowlpox virus expressing the bacteriophage T7 DNA-dependent RNA polymerase. However, CAT protein was not detected in CK cells from IRES-containing IBV D-RNAs, in which the IRES-CAT construct was inserted at two different positions within the D-RNA, in the presence of helper IBV. Northern blot analysis demonstrated that the IRES-containing D-RNAs were not rescued on serial passage with helper virus, indicating that the EMCV IRES sequence had a detrimental effect on IBV D-RNA rescue.

Animals↗

Electron microscope observations on the entry of avian infectious bronchitis virus into susceptible cells.

Infectious bronchitis virus was observed to enter cells of chicken chorioallantoic membrane by viropexis. There was no support for the suggestion that entry took place by fusion of viral and plasma membranes. The results of electron microscopy showed that virus attachment occurred both at 4 degrees and at 37 degrees C. Viropexis was not observed until the preparations were warmed. Similar results were obtained using chicken kidney cells. Quantitative data obtained from a plaque counting system employing chicken kidney cells indicated that attachment was the same at both temperatures and that some virus particles were taken up at 4 degrees C. Virus uptake was triggered by attachment of the virus to the cell membrane and the subsequent process of virus entry visualised by E. M. appeared to proceed without the involvement of lysosomal enzymes. No intracellular virus was located by electron microscopy in warmed preparations when virus was treated with specific antiserum, either before or after adsorption to the cells.

Adsorption↗

Nephropathogenicity of infectious bronchitis virus.

Four strains of infectious bronchitis virus (IBV) were compared in chickens as to nephropathogenicity. Two of the viruses were from the United States, one was from Australia, and one from Italy. Each has been described as nephropathogenic. The Australian strain proved to be most pathogenic and was then followed by the Italian strain and, finally, the two viruses from the United States. With an increase in the age of the chickens there was an apparent decrease in pathogenic effect. All of the viruses were highly pathogenic in the respiratory tract regardless of the age of the test birds. Kidney damage, or nephritis, was detected more readily on histological study rather than by gross examination. Each of the viruses was unrelated to the others when the sera were assayed by virus neutralization. Massachusetts and Connecticut IBV vaccines gave erratic, or only, partial protection against challenge form the respective viruses which confirmed their aberrant nature. Lesions of IBV infection in the respiratory tract did not necessarily correlate with the results from challenge virus recovery attempts. One Massachusetts-type IB vaccine strain, Holland IBV, proved capable of inciting nephritis under the conditions of this study and from inoculating specific-pathogen-free chickens at 2 days of age. The lower embryo passage of this strain proved more nephropathogenic than the higher ones. Other Massachusetts-type vaccine viruses and the Connecticut-type did not induce nephritis. The nephropathogenic potential for certain vaccine strains is discussed.

Animals↗

Isolation and characterization of a novel antigenic subtype of infectious bronchitis virus serotype DE072.

Three infectious bronchitis virus (IBV) isolates, CU82792, CU82805, and CU82808, were recovered from sentinel chickens placed with three different layer flocks of a large commercial poultry farm in New York State. The three isolates were classified as members of the DE072 serotype on the basis of 1) their S1 genes could be amplified with only a modified primer designed for the DE072 serotype and 2) their restriction fragment length polymorphism patterns, after digestion with endonucleases HaeIII, BstyI and XcmI, were indistinguishable from that of DE072 virus. Additional characterization of one of the isolates, CU82805, revealed that its S1 gene bears approximately 96% identity at the nucleotide level and 94% identity at the amino acid level with DE072. Yet, in an in vitro reciprocal virus neutralization test, only a one-way neutralization was observed, i.e., antiserum to CU82805 neutralized DE072, whereas CU82805 was not neutralized by DE072 antiserum. Implications of these findings with regard to IBV diagnosis and immunization are discussed.

Amino Acid Sequence↗

S1 gene sequence analysis of a nephropathogenic strain of avian infectious bronchitis virus in Egypt.

BACKGROUND: Infectious bronchitis is highly contagious and constitutes one of the most common and difficult poultry diseases to control. IBV is endemic in probably all countries that raise chickens. It exists as dozens of serotypes/genotypes. Only a few amino acid differences in the S1 protein of vaccine and challenge strains of IBV may result in poor protection. Tropism of IBV includes the respiratory tract tissues, proventriculus and caecal tonsils of the alimentary tract, the oviduct and the kidney. RESULTS: Infectious bronchitis virus (IBV) strain closely related to Massachusetts (Mass) serotype was isolated from broiler chickens suffering from severe renal and respiratory distresses. The isolate was serologically identified by Dot-ELISA and further characterized by RT-PCR then genotyped using S1 gene sequence analysis. Alignment of the S1 sequence of the isolate with 16 IBV strains revealed high homology to isolates related to Mass serotype. Inoculation with the strain reproduced the disease in experimental 1-day-old chickens and resulted in 20% mortality, severe renal and moderate respiratory distresses. Marked histopathological changes in both kidney and trachea were observed in experimentally infected chickens. A protection study using the H120 live attenuated vaccine showed low protection rate in spite of high S1 sequence homology (97%). Protection based criteria were: virus re-isolation attempts from trachea, tracheal and renal histopathology as well as IBV antigens detection by immunofluorescent antibody technique in kidney sections. CONCLUSION: Periodical evaluation of cross-protective capabilities of IBV vaccine(s) versus recently recovered field isolates should be performed to ensure optimum control of IBV.

Animals↗

The polypeptide composition of isolated surface projections of avian infectious bronchitis virus.

Disruption of avian infectious bronchitis virus (IBV) particles with 4% Triton X-100 and 1.0 M-KCl and centrifugation through a sucrose gradient containing 0.1% Triton X-100 and 1.0 M-KCl enabled separation of the petal-shaped surface projections. By negative-contrast electron microscopy the separated projections appeared mainly as rosettes containing 3 to 12 projections radiating from a central core, although single projections and rosettes containing up to 16 projections were seen. SDS-PAGE of these preparations revealed two polypeptides of 86,000 and 66,000 mol. wt. The larger polypeptide was glycosylated.

Coronaviridae↗

Antigenic and S-1 genomic characterization of the Delaware variant serotype of infectious bronchitis virus.

A previously unrecognized infectious bronchitis virus (IBV) serotype, referred to hereafter as the Delaware variant (DE var), was isolated from commercial broiler chickens during a severe, widespread respiratory disease epornitic in the Delmarva peninsula region of the United States in January-March 1992. The DE var serotype was found to be antigenically unrelated by virus-neutralization (VN) test to nine reference IBV serotypes from North America. Additional VN tests indicated that the DE var isolates (DE/072/92, DE/121/ 92, DE/152/92, and DE/174/92) from broilers were fully or partially neutralized by monospecific antisera prepared against themselves and against two IBV field isolates (DE/492/90 and DE/903/90) recovered from a Delmarva commercial layer flock experiencing egg production losses in 1990. Antigenic relatedness values determined by VN indicated layer isolate DE/492/90 was more closely related to the broiler DE var isolates than was layer isolate DE/903/90. Cross-challenge tests performed in specific-pathogen-free chickens also demonstrated the antigenic similarity of the broiler (DE/072/92 and DE/174/92) and the layer isolates (DE/492/90 and DE/903/90), with heterologous strain protection values ranging from 55% to 100%. Protection values of DE var isolates vs. Massachusetts 41 and Arkansas DPI were considerably lower (0-60%). The S-1 gene of the US/DE/072/92 isolate of the DE var serotype was amplified by reverse transcription polymerase chain reaction, cloned, and sequenced. The DE var S-1 gene sequence was compared with the S-1 gene sequences of IBV serotypes from North America, Europe, and Australia. A dendrogram based on this analysis supported the conclusion that the DE var serotype is highly novel among IBV. A high degree of similarity (> 88%) was observed between the S-1 genes of the DE var broiler isolates (DE/072/92 and DE/174/92) and layer isolates (DE/492/90 and DE/903/90). These data, taken with the VN and cross-challenge results, establish a genetic as well as an antigenic link between the isolates from layers and broilers and indicate the DE var serotype was responsible for both infectious bronchitis outbreaks.

Animals↗

The susceptibility of chicken kidney and oviduct organ cultures to a vaccine strain of avian infectious bronchitis virus.

The minimal infectious dose of the H52 strain of infectious bronchitis virus for organ cultures of oviduct and kidney was compared in chickens of different ages. Organ cultures of oviduct were found to be highly susceptible to infection regardless of the age of chicken and no difference in susceptibility could be demonstrated between cultures of the magnum and uterus regions of the mature oviduct. Kidney organ cultures were less susceptible and resistance to infection increased significantly (P less than 0.001) with the age of the chicken from which cultures were prepared.

Age Factors↗

Protection of chickens after live and inactivated virus vaccination against challenge with nephropathogenic infectious bronchitis virus PA/Wolgemuth/98.

Protection provided by live and inactivated virus vaccination against challenge with the virulent nephropathogenic infectious bronchitis virus (NIBV) strain PA/Wolgemuth/98 was assessed. Vaccinations with combinations of live attenuated strains Massachusetts (Mass) + Connecticut (Conn) or Mass + Arkansas (Ark) were given by eyedrop to 2-wk-old specific-pathogen-free leghorn chickens. After live infectious bronchitis virus (IBV) vaccination, some chickens at 6 wk of age received an injection of either an oil emulsion vaccine containing inactivated IBV strains Mass + Ark or an autogenous vaccine prepared from NIBV PA/Wolgemuth/98. Challenge with PA/Wolgemuth/98 was given via eyedrop at 10 wk of age. Serum IBV enzyme-linked immunosorbent assay antibody geometric mean titers (GMTs) after vaccination with the combinations of live attenuated strains were low, ranging from 184 to 1,354, prior to NIBV challenge at 10 wk of age. Both inactivated vaccines induced an anamnestic response of similar magnitudes with serum GMTs of 6,232-12,241. Assessment of protection following NIBV challenge was based on several criteria virus reisolation from trachea and kidney and renal microscopic pathology and IBV-specific antigen immunohistochemistry (IHC). Live attenuated virus vaccination alone with combinations of strains Mass + Conn or Mass + Ark did not protect the respiratory tract and kidney of chickens after PA/Wolgemuth/98 challenge. Chickens given a live combination vaccination of Mass + Conn and boosted with an inactivated Mass + Ark vaccine were also susceptible to NIBV challenge on the basis of virus isolation from trachea and kidney butshowed protection on the basis of renal microscopic pathology and IHC. Live IBV-primed chickens vaccinated with an autogenous inactivated PA/Wolgemuth/98 vaccine had the highest protection against homologous virulent NIBV challenge on the basis of virus isolation.

Animals↗

One-pot Golden Gate Assembly of an avian infectious bronchitis virus reverse genetics system.

Avian infectious bronchitis is an acute respiratory disease of poultry of particular concern for global food security. Investigation of infectious bronchitis virus (IBV), the causative agent of avian infectious bronchitis, via reverse genetics enables deeper understanding of virus biology and a rapid response to emerging variants. Classic methods of reverse genetics for IBV can be time consuming, rely on recombination for the introduction of mutations, and, depending on the system, can be subject to genome instability and unreliable success rates. In this study, we have applied data-optimized Golden Gate Assembly design to create a rapidly executable, flexible, and faithful reverse genetics system for IBV. The IBV genome was divided into 12 fragments at high-fidelity fusion site breakpoints. All fragments were synthetically produced and propagated in E. coli plasmids, amenable to standard molecular biology techniques for DNA manipulation. The assembly can be carried out in a single reaction, with the products used directly in subsequent viral rescue steps. We demonstrate the use of this system for generation of point mutants and gene replacements. This Golden Gate Assembly-based reverse genetics system will enable rapid response to emerging variants of IBV, particularly important to vaccine development for controlling spread within poultry populations.

Infectious bronchitis virus↗

Systemic and local antibody responses to infectious bronchitis virus in chickens inoculated with infectious bursal disease virus and control chickens.

Serum and local (respiratory) antibody responses to infectious bronchitis virus (IBV) were studied in 5-wk-old white leghorn-type control chickens and chickens inoculated with infectious bursal disease virus (IBDV) at 1 day of age. Of the chickens inoculated with IBV alone, 93% had detectable levels of IBV antibodies in the sera and 87% had detectable antibodies in the respiratory lavage fluids. Compared to this group, only 73% and 65% of IBDV-IBV inoculated chickens had serum and respiratory antibodies, respectively. In chickens inoculated with IBV alone, the IBV antibodies were evenly associated with immunoglobulin classes IgM, IgG, and IgA, whereas the IBDV-IBV inoculated chickens mainly produced IgM-associated antibodies with low to negligible levels of IgA- and IgG-associated antibodies. These results suggest that the lack of adequate IgA- and IgG-associated antibody production in IBDV-infected chickens may account for their increased susceptibility to IBV infection.

Animals↗

Variant infectious bronchitis virus isolated from Indiana chickens.

Infectious bronchitis virus (IBV) associated with a catarrhal tracheitis, sudden decline in egg production, and reduced shell quality was isolated from an Indiana White Leghorn breeder flock. It was found to be serologically different from Massachusetts, Connecticut, Iowa 97, Iowa 609, Florida, Arkansas 99, JMK, Holte, Gray and SE 17 IBV serotypes. Two different Massachusetts vaccine strains protected chickens from respiratory signs but not against virus infection using the isolant for challenge in laboratory trials. The isolant was passed through a 0.22 mu. filter. It was heat (56 C), acid pH (3.0), ether and chloroform labile. In embryos it produced deaths or lesions of infectious bronchitis in one to five days after inoculation. It is suggested that this IBV isolant be designated Indiana-type.

Animals↗

The polypeptide composition of avain infectious bronchitis virus particles.

Egg grown avian infectious bronchitis virus (IBV) centrifuged on sucrose density gradients was found to consist of a major virus peak of density 1.17 to 1.18 g/cm(3). Three different IBV strains were examined and no morphological differences were detected between virus particles of different densities or from different strains. The polypeptides of the different density virus particles from the three IBV strains were analysed on polyacrylamide gels. In all cases 7 polypeptides were observed, although there were differences in the proportions of these polypeptides in particles of different densities and those from the different strains. The polypeptides have been called VP1 (molecular weight 130,000), VP2 (105,000), VP3 (97,000), VP4 (81,000), VP5 (74,000), VP6 (51,000) and VP7 (33,000). Additional polypeptides were produced if slightly harsher treatments were used.

Animals↗

Identification and analysis of the Georgia 98 serotype, a new serotype of infectious bronchitis virus.

Twenty-five field infectious bronchitis viruses (IBVs) similar to, but genetically distinct from, the DE072 serotype were isolated from several states in the United States from 1990 through 1999 and were examined molecularly and antigenically. A 421-bp sequence in the hypervariable region of the S1 gene was examined, and phylogenetic analysis on that region indicated that these viruses are closely related but fall into unique groups. Cross-virus neutralization testing and entire S1 sequence analysis on selected isolates further confirmed that fact, and we divided the viruses into the DE072 serotype and two other unique groups. In a vaccine protection trial, the commercially available DE072 vaccine showed less than 50% protection against viruses in one of the groups. The majority of the recent isolates belong to that group and share very low antigenic relatedness to the DE072 strain as well as other serotypes of IBV. Consequently, we designated this group as a new serotype, Georgia 98. We developed a restriction fragment length polymorphism analysis that can differentiate this new serotype from all other serotypes of IBV.

Animals↗