Taxonomic studies on strains of avian infectious bronchitis virus using neutralisation tests in tracheal organ cultures

Summary The antigenic relationships of 24 strains of avian infectious bronchitis virus (IBV) were investigated by serum neutralisation tests performed in chick embryo tracheal organ cultures.The serum dilution that neutralised 100 median ciliostatic doses (CD₅₀) of virus was estimated from the linear relationship between varying concentrations of each virus strain and the neutralisation titre of homologous antiserum; this dilution defined 1 antibody unit. Antisera diluted to contain 20 antibody units were then tested by neutralisation against 1.5–2.5 log₁₀ CD₅₀ of each strain.Clusters of both strains and antisera in turn were established by methods of numerical taxonomy using as measures of resemblance Euclidean distance and correlation coefficient, and by analysis by principal components. These analyses identified a group of 8 similar strains; neutralisation of the remaining 16 strains was slight.Similar results were obtained by classifying antisera, except that a further group of 3 antisera was demonstrated, each having a neutralising capacity for most strains. Implications for vaccine formulation are discussed.With 3 Figures     

Growth comparisons of avian infectious bronchitis virus strains in organ cultures of chicken tissues

Summary Six strains of avian infectious bronchitis virus (IBV) were used to inoculate explants of a range of 15 chicken tissues and virus growth kinetics observed over a period of 96 hours thereafter.Similar patterns of virus production were given by all 6 strains from explants of any particular tissue such as the nasal turbinates, trachea, lung, air sacs and oviduct. Nevertheless, significant differences in behaviour between strains were noted for different tissues and in the efficiency with which certain tissues produced virus.It is suggested that the method has a potential value in determining the virulence of different strains of IBV by comparing their pathogenesis for chicken tissuesin vitro.With 3 Figures     

The use of chicken tracheal organ cultures for the isolation and assay of avian infectious bronchitis virus

Summary A study has been made of the use of chicken tracheal organ cultures for the isolation and assay of avian infectious bronchitis (AIB) virus from both naturally and experimentally infected chickens. Six strains of AIB virus were investigated, 3 of which had been isolated from natural outbreaks of disease. Two of the virus isolations from the outbreaks of AIB were made directly into tracheal organ cultures without passage in embryonated eggs.Organ cultures prepared from 20-day-old embryos were used since they were found to be somewhat more sensitive in virus assay than those derived from chickens of up to 31 days of age. Ciliostasis, which was used as the marker of infectivity, was complete by 3 days after inoculation with each strain of virus examined.Virus could be isolated from both respiratory and non-respiratory tissue in tracheal organ cultures and these cultures were found to be at least as sensitive as 9-day-old embryonated eggs in detecting AIB virus either in pathological material or in serial dilutions. When virus was assayed in both systems, the titres were very similar. It is considered, therefore, that chicken embryo tracheal organ cultures offer a reliable alternative system to embryonated eggs for studying AIB virus.With 1 Figure     

An in vitro comparison of the virulence of seven strains of infectious bronchitis virus using tracheal and oviduct organ cultures.

The virulence of seven infectious bronchitis virus (IBV) strains for tracheal and oviduct ciliated epithelium was assessed using an in vitro tracheal organ culture (TOC) and oviduct organ culture (OOC) system. The OOC were prepared using oviducts obtained from oestrogen-treated chicks. All strains tested stopped ciliary beating in the oviduct by day 5 post-inoculation (p.i.) and in the trachea by day 3 p.i. This corresponded with the absence of immunofluorescent-stained cells in the epithelium at that time. The time taken for a 50% reduction of relative ciliary activity (RCA) of oviduct cilia was shortest for strain 6 (serotype D207) and longest for strain G (enterotropic variant). Strains 7 (serotype D3896) and M41 were the most pathogenic for tracheal cilia, while strains 6 and G were less pathogenic. A calmod-ulin (CAM) assay was standardized to quantify the epithelial cell damage to oviducts caused by IBV. It was found that strains 6 and M41 were the most pathogenic. The use of time taken to achieve a 50% reduction in RCA and the CAM assay for in vitro pathotyping of IBV isolates is discussed. The susceptibility of OOC or TOC for five IBV strains was compared. It was found that strains 25 and 793B had equal predilection for both, while for 25, G and M41, TOC was more susceptible.     

Pathogenicity of Australian strains of avian infectious bronchitis virus

The pathogenicity of 25 strains of infectious bronchitis virus (IBV) isolated in Australia between 1961 and 1994 was compared in white leghorn specific pathogen-free chicks. Twelve strains were nephropathogenic and 10 respiratory, the other three being of mixed pathogenicity. The IBV strains identified as nephropathogenic induced clinical nephritis, gross and histological kidney lesions, and mortality of 5-90%. According to the severity of these features, the nephropathogenic strains could be further subdivided into strains of high, moderate or low pathogenicity. The three strains of mixed pathogenicity induced tracheitis, mild clinical nephritis and kidney lesions but no mortality. The 10 respiratory strains caused histological lesions in the trachea but not in the kidney, and did not induce clinical nephritis or mortality. Of 12 IBV strains isolated between 1961 and 1976, nine were nephropathogenic, inducing mortality of 15-90%. In contrast, of 13 strains isolated between 1981 and 1994, only three were nephropathogenic, inducing mortality of 5-37%, whereas nine were respiratory. Seven of these nine strains, unlike other respiratory strains, failed completely to replicate in the kidney. The results indicated a change in the prevalent IBV strains from highly nephropathogenic (1960s to 1970s) to respiratory (1980s to early 1990s); moreover, the late 1980s saw the emergence of respiratory strains with altered tissue tropism.     

Antigenic variation in strains of avian infectious bronchitis virus

Summary Fifteen british field strains of IBV were compared using cross serum neutralization tests in embryonated eggs with seven standard reference strains of IBV. While the British field strains were considered to form a relatively homogeneous group considerable antigenic variation did occur. It was considered that it was not feasible at this time to describe accurately a serotype classification for IBV, similar to that described for other virus groups.     

Plastic multiwell plates to assay avian infectious bronchitis virus in organ cultures of chicken embryo trachea.

Simple assay systems for infectivity titrations of avian infectious bronchitis virus (IBV) in chicken embryo trachea organ cultures (OC) were developed using plastic multiplate wells with one tracheal ring per well; these assays appeared to be much more satisfactory than the conventional rolled-tube method. The medium, 0.05 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-buffered Eagle minimal essential medium was not changed during observation. A medium containing 0.4% bovine serum albumin did not influence the virus yield, but did stabilize virus viability during storage. Reproducibility of results obtained in the OC system was confirmed by performing replicate titrations of the Beaudette strain with three different passage histories. The mean virus titers in the OC were lower than those in chicken embryos, depending on the IBV passage histories. The time required for ciliostasis was related not only to the concentration of virus, but also to the IBV passage history. Application of OC techniques for the constant serum-variable virus neutralization test gave low neutralization indexes with excellent reproducibility as compared with those obtained in the chicken embryo assay system. Also, the slopes of neutralization curves obtained by assays in OC were less steep than those seen in the chicken embryo system.     

Comparison of the haemagglutination inhibition test and the serum neutralisation test in tracheal organ cultures for typing infectious bronchitis virus strains

Five strains of infectious bronchitis (IB) virus, which had been compared antigenically by the serum neutralisation (SN) test in tracheal organ cultures (OC), were arbitarily coded and then compared by the haemagglutination inhibition (HI) test. Their antigenic relationships were found to be similar by the two methods but, because of the high and variable cross reactions found in the HI test, the differences between the strains were less clear by that method. It was concluded that the HI test, in its present state of development, is considerably less type-specific than the SN test in OC, and cannot be recommended for defining antigenic relationships between strains of IB virus. However, it retains its value for diagnosing IB or for monitoring the vaccinal status of flocks.     

Interferon induction by several strains of avian infectious bronchitis virus, a coronavirus, in chickens

Eight U.S. and Japanese strains of avian infectious bronchitis virus (IBV) were tested for their interferon-inducing ability in chickens. All strains induced interferon (IFN) in several organs of six-week-old SPF chickens. However, the extent of IFN formation in these chickens was not necessarily the same from one strain to another.     

Serological relationship among ten strains of avian infectious bronchitis virus

Ten strains of avian infectious bronchitis virus (IBV) were studied serologically by cross-neutralization test using rabbit and chicken immune sera. With the chicken sera all 10 IBV strains were antigenically related. In particular, anti-KH serum neutralized all heterologous strains except of the Ishida strain; Nerima strain was neutralized by all antisera except of anti-Ishida serum. Most cross-reactions were less or more heterologous, thus all 10 IBV strains seemed to belong to one serological type. Using rabbit sera, all strains except of Connecticut A-5968, cross-reacted with certain other strains. Most cross-reactions were partially heterologous showing one-way-relationship; heterologous relations were observed less frequently than with chicken sera.     

A clearance test to assess protection in chickens vaccinated against avian infectious bronchitis virus

A clearance test is described that is designed as a model for the quantitative assessment of protection in chickens vaccinated against avian infectious bronchitis virus (IBV). This is based on the ability of a vaccinating strain to have induced within 3 weeks an immunity sufficient to inhibit the replication of a challenge strain in the upper respiratory tract by the 4th day subsequent to challenge. A median protective dose (PD(50)) was determined for each of 4 vaccinating strains (H120, H52, D41 and Doorn 274), and chickens were vaccinated with 20 PD(50) of one of these intranasally. Challenge strains were administered intratracheally 3 weeks later and assays of residual challenge virus in the trachea as well as the kidneys and oviducts were made after a further 4 days. Observations were also made on tracheal ciliary activity and histopathological changes. The H120 and H52 strains were efficient in clearing antigenically related challenge strains, but the H120 strain was less so in the case of the Doom 274 strain and D41 strain. The Doom 274 strain was effective against the heterologous strains examined with the exception of the T strain. The D41 strain was generally effective in protecting against all the strains selected for challenge, and its candidature as a broad-spectrum vaccine strain is endorsed accordingly. A small number (6%) of unvaccinated chickens had virus in low titre in the kidneys or oviducts after challenge with some strains. Virus was not detected in these organs of vaccinated birds.     

Phylogenetic analysis of avian infectious bronchitis virus strains isolated in Japan

Summary To define the origin and evolution of recent avian infectious bronchitis virus (IBV) in Japan, a genetic analysis was performed. By phylogenetic analysis based on the S1 gene including the sequence of the hypervariable regions, IBV isolates in Japan were classified into five genetic groups, which included two already-known groups (Mass and Gray). Among them, three major genetic groups were associated with the recent outbreaks of IB in Japan. One group is indigenous to Japan and could not be placed within the known existing groups in other countries. The remaining two groups, which have emerged recently, are related to isolates in China and Taiwan.     

A simple method for immunofluorescence staining of tracheal organ cultures for the rapid identification of infectious bronchitis virus

A method is described for the simple application of immunofluorescence (IF) staining for the identification of infectious bronchitis virus (IBV) in tracheal organ cultures (TOC). It involves the application of antiserum and fluorescent antiglobulin to unfixed TOC. In TOC inoculated with high titres of virus, specific fluorescence was observed in less than 24 h. IF staining almost always occurred before ciliostasis. Following experimental infection of chicks with IBV, virus could sometimes be detected in material from tracheal or cloacal swabs within 24 h of inoculation of TOC.     

Susceptibility of organ cultures from chicken tissues for strains of infectious bronchitis virus isolated from the intestine.

Organ cultures were prepared from various levels of intestine and kidney of 2 to 4-week old specific-pathogen-free chickens, and their susceptibility to ten strains of infectious bronchitis virus isolated from the chicken intestine and the Massachusetts strain M41 was investigated. The ability of a virus to grow depended on the strain of virus and size of the inoculum. While proventriculus, bursa and kidney were found to be universally susceptible to all viruses tested, some strains did not grow in caecal tonsils or rectum. Strain M41 showed little difference in pattern of tissue replication compared with several other strains isolated from the gut and actually grew in a wider range than some. Duodenum, jejunum and ileum were found to be non-permissive to all strains tested, even after a high input inoculum.     

Intracloacal infection with avian infectious bronchitis virus

An infectious bronchitis virus (IBV) strain HS-91 isolated from kidneys of chicks which died of nephrosis was inoculated via the cloaca of specific pathogen-free (SPF) chicks. These chicks showed more severe clinical signs, grosser kidney lesions and higher mortality than chicks inoculated with the same IBV strain via the trachea.     

The polypeptide composition of avian infectious bronchitis virus

Summary Avian infectious bronchitis virus grownin ovo was purified by differential centrifugation and isopycnic sedimentation in density gradients. The purified virus was analysed by SDS polyacrylamide gel electrophoresis and found to comprise up to sixteen polypeptides, four of which were glycopeptides. Bromelain treatment of the particles removed three polypeptides and two glycopeptides.With 3 Figures     

Two particle types of avian infectious bronchitis virus

Two distinct types of avian infectious bronchitis virus (IBV) particles were isolated on sucrose density gradients. The higher density particles banded at 1.18 g/ml, had typical coronavirus morphology and contained all the structural polypeptides and a complete genome. The less dense particles of density 1.13 g/ml appeared to have typical coronavirus morphology, although they were much more flattened than the more dense particles. Furthermore, these particles lacked the ribonucleoprotein polypeptide and the genome, although all the other polypeptides were present in the same amounts as in the denser particles.     

Comparison of the susceptibility to avian infectious bronchitis virus infection of two inbred lines of white leghorn chickens

Two inbred lines of White Leghorn chickens, line C which are highly resistant to infection with avian infectious bronchitis virus (IBV), and line 151, which are highly susceptible, were challenged at 11 days of age with IBV Massachusetts-41 (M41) strain. Line 151 chickens showed more severe respiratory signs than line C and although similar amounts of virus were recovered from the tracheas of both lines for the first four days after inoculation, overall more virus was recovered from the respiratory tract organs and from kidneys of line 151 chicks. There was however, no difference in the rate of virus production or in the amount produced by explants of tissues from each line following inoculation with IBV in vitro. When tracheal ciliary movement of lines C and 151 chickens was examined following inoculation with M41. ciliostasis was observed earlier in line 151 than in line C and lasted for a longer time.     

Immune responses to structural proteins of avian infectious bronchitis virus.

Chickens were vaccinated with live and inactivated infectious bronchitis virus (IBV), and antibody responses to the individual structural proteins, S1, S2, M and N, followed by ELISA and western blotting. All four structural proteins elicited an antibody response in chicks vaccinated with either live or inactivated IBV. The S1, S2 and N proteins elicited similar titres of antibodies following vaccination with live IBV, whereas the M glycoprotein elicited significantly lower titres. Time of appearance and the course of development of the S1, S2 and N ELISA antibodies were similar, being first detected 2 weeks after vaccination and coincided with appearance of virus neutralizing antibodies. The M antibodies were first detected 4 weeks after vaccination. S1, S2, and N antibody titres were significantly higher in chicks vaccinated at 14 days of age than in chicks vaccinated at either 1 or 7 days of age, and reached maximum levels 4 weeks after the second vaccination. The S1, S2 and N proteins induced cross-reactive antibodies, whereas the M glycoprotein induced antibodies of limited cross-reactivity. Titres of cross-reactive N antibodies were higher than titres of cross-reactive S1 and S2 antibodies, which were similar. Epitopes on the N and S2 proteins that gave rise to cross-reactive antibodies showed the same degree of conservation, whereas the cross-reactive S1 epitopes were marginally less conserved. Vaccination with inactivated virus induced significantly lower antibody titres and at least three vaccinations were necessary for induction of S1, S2, N and M antibodies in all chicks. The S2 glycoprotein was the most immunogenic structural protein following vaccination with inactivated virus. All four proteins induced cell-mediated immune responses in chicks vaccinated with live IBV as determined by a delayed type hypersensitivity response.