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Clinical respiratory illness was compared in five homozygous chicken lines, originating from homozygous B2, B8, B12 and B19, and heterozygous B2/B12 birds after infection with either of two strains of the infectious bronchitis virus (IBV). All chickens used in these studies originated from White Leghorn and Ancona linages. IBV Gray strain infection of MHC homozygous B12 and B19 haplotype chicks resulted in severe respiratory disease compared to chicks with B2/B2 and B5/B5 haplotypes. Demonstrating a dominant B2 phenotype, B2/B12 birds were also more resistant to IBV. Respiratory clinical illness in B8/B8 chicks was severe early after infection, while illness resolved similar to the B5 and B2 homozygous birds. Following M41 strain infection, birds with B2/B2 and B8/B8 haplotypes were again more resistant to clinical illness than B19/B19 birds. Real time RT-PCR indicated that infection was cleared more efficiently in trachea, lungs and kidneys of B2/B2 and B8/B8 birds compared with B19/B19 birds. Furthermore, M41 infected B2/B2 and B8/B8 chicks performed better in terms of body weight gain than B19/B19 chicks. These studies suggest that genetics of B defined haplotypes might be exploited to produce chicks resistant to respiratory pathogens or with more effective immune responses., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

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.

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.

Infectious bronchitis virus (IBV), the first coronavirus described, has been a continuing problem in poultry for more than 70 years. IBV, causing a highly contagious respiratory disease in chickens, resembles the recently described severe acute respiratory syndrome virus in pathogenesis and genome organization. While previous studies demonstrated that effector and memory CD8(+) T lymphocytes are critical in controlling acute IBV infection and disease in chickens, here chicken anti-IBV antibody (IgG) secreting cells (ASC) in both peripheral blood mononuclear cells (PBMC) and spleens collected following IBV Gray infection were evaluated using an ELISPOT assay. The ASC in peripheral blood and spleens can be detected from 3 to 7 days post-infection (p.i.), which is 3-7 days earlier than anti-IBV IgG detected in the serum. The ASC frequency reached a maximum at 7-10 days p.i., and decreased more than 90% in the spleen and 70% in PBMC by 14 days p.i. The ASC levels in the PBMC then decreased gradually to 0.5 ASC/10(6) over the next 8 weeks. The higher concentration of about 20 ASC/10(6) cells in spleens may, at least partially, account for the presence of antibody in the serum although bone marrow ASC were not determined. In vitro stimulation of PBMC and splenocytes with IBV antigen demonstrated that memory B cells can be activated to secrete antibody by 3 weeks p.i. ELISPOT detection of primary B cells could be useful in the early detection of infection following infection with respiratory coronaviruses.

A 1.78 kb sequence, including the E, M, 5a and 5b genes, and the intergenic region between the M and 5a genes, of six US strains of infectious bronchitis (corona)virus (IBV) were sequenced and compared to the published sequences for two additional strains. The overall identities as determined through pairwise analyses of nucleotide sequences of the entire 1.78kb region ranged from 90 to 99%, with the 5b open reading frame (ORF) having the greatest identity (94-99%) while the identities of the E, 5a and M ORFs ranged from 87 to 100%. Nucleotide sequencing of recent field isolates from Alabama (Ala1) and California (Cal3) revealed distinct shifts in homology in the M gene, indicating two apparent recombination events between the Holland 52/Mass41-like strain and an Ark-like strain, both origins of commonly used vaccine strains. Putative sites of recombination could also be identified in both the E and M ORFs of laboratory strains of IBV.

Infectious bronchitis has remained one of the most difficult to control diseases in poultry since it was first described in 1931. Previous studies demonstrated that primary CD8(+) T lymphocytes collected at 10 days post-infection (p.i.) are important in controlling acute infection. To further investigate the role of memory T cells in protection, T lymphocytes collected from B19/B19 chicken spleens at 2, 3, 4, and 6 weeks p.i. were transferred to six-day-old syngeneic chicks one day prior to challenging with 10(6) EID(50) of the IBV Gray strain. Memory immune T cells collected at 3 to 6 weeks p.i. provided dose responsive protection from clinical illness. The greatest protection was observed after the transfer of 10(7) T cells collected at 6 weeks p.i., whereas T cells collected at 2 weeks p.i. did not protect. Annexin-V staining of the spleen cells demonstrated that the cells collected at 2 weeks p.i. were undergoing significantly more apoptosis than cells collected at 10 days p.i. Specific antibody production in sera collected at 7 days p.i. did not correlate with protection. T cell subtype depletion demonstrated that CD8(+), not CD4(+), T cells were critical. Memory T cells can be detected in peripheral blood mononuclear cells up to at least 10 weeks p.i. These results demonstrated that IBV specific CD8(+) memory T cells generated at 3 to 6 weeks p.i. can protect syngeneic chicks from acute IBV infection.

Infectious bronchitis virus (IBV) causes an economically important respiratory disease in poultry worldwide. Previous studies have shown that CD8(+) cytotoxic T lymphocytes (CTL) are critical in controlling acute IBV infection, but the role of innate immunity is unknown. This study describes the in vitro and in vivo anti-IBV activity of natural spleen cell-derived and recombinant chicken interferon type I (rChIFN-alpha). Both natural and rChIFN-alpha inhibited replication of the Beaudette strain of IBV in chicken kidney cells (CKC) in a dose-dependent manner, with the antiviral activity of the former accounted for entirely by its content of type I IFN. IFN at 100 U/ml reduced viral replication by 50% as measured by syncytia formation. In addition, the spleen cell-derived supernatants (natural IFN) inhibited tracheal ring ciliostasis mediated by the Gray strain of IBV. Optimal protection against IBV-induced respiratory disease was obtained after intravenous or oral administration of ChIFN given 1 day before virus challenge and each of 5 days thereafter. ChIFN-I protected chicks from clinical illness by delaying the onset of the disease and decreasing the severity of illness, demonstrating its potential as an immune enhancer.

Infectious bronchitis virus (IBV) infection and associated illness may be dramatically modified by passive transfer of immune T lymphocytes. Lymphocytes collected 10 days postinfection were transferred to naive chicks before challenge with virus. As determined by respiratory illness and viral load, transfer of syngeneic immune T lymphocytes protected chicks from challenge infection, whereas no protection was observed in the chicks receiving the MHC compatible lymphocytes from uninfected chicks. Protection following administration of T lymphocytes could be observed in chicks with three distinct MHC haplotypes: B(8)/B(8), B(12)/B(12), and B(19)/B(19). Nearly complete elimination of viral infection and illness was observed in chicks receiving cells enriched in alphabeta lymphocytes. In contrast, removal of gammadelta T lymphocytes had only a small effect on their potential to protect chicks. The adoptive transfer of enriched CD8(+) or CD4(+) T lymphocytes indicated that protection was also a function primarily of CD8-bearing cells. These results indicated that alphabeta T lymphocytes bearing CD8(+) antigens are critical in protecting chicks from IBV infection., (Copyright 2000 Academic Press.)

Various strains of infectious bronchitis virus (IBV) cause respiratory, kidney, enteric and reproductive illnesses in chickens, especially in newly hatched chicks. Assays have been developed to identify Gray strain IBV-specific cytotoxic T lymphocyte (CTL) responses using viral infected antigen presenting cells (APC) and using the Semliki Forest virus vector infected APC expressing individual viral polypeptides. It was shown that major histocompatibility complex restricted CTL are responsible for early control of IBV infection. The kinetics of viral load observed in the lungs and kidneys correlated with the level of IBV-specific CTL activity of effector cells prepared from spleens of infected chicks. Adoptive transfer of immune T cells to chicks prior to infection demonstrated that IBV primed CD8(+), alphabeta T lymphocytes could protect chicks from acute infection. CTL determinants in the viral particle can be mapped to the spike and nucleocapsid proteins but not to the membrane protein. The carboxyl terminus of the nucleocapsid protein houses an epitope(s) responsible for induction of CTL responses to IBV N protein. Inoculation of DNA plasmid expressing the carboxyl terminus of Gray strain N resulted in induction of CTL that cross-react with two distinct IBV strains. In addition, this potential DNA vaccine resulted in protection of chicks against acute infection.

Cytotoxic T lymphocyte (CTL) activity to infectious bronchitis virus (IBV) was examined at regular intervals between 3 and 30 days post infection (p.i.). The maximal CTL lysis of target cells infected with IBV with 82% was detected at 10 days p.i. The specific CTL activity began to decrease only after viral loads, which peaked at day 8 p.i. in both kidneys and lungs, started to decline. Therefore, the CTL response correlated with elimination of acute infection. IgM antibody did not appear until day 10 and levels peaked at day 12 p.i. whereas IgG antibody titers were detectable only by day 15 p.i., but continued to increase exponentially until day 30 p.i., the last day examined. IBV specific CTL epitope(s) were mapped within the carboxyl terminal 120 amino acids of nucleocapsid protein. In vivo inoculation of this fragment, as cDNA, induced protection against acute infection. The absence of viral neutralizing epitopes on the nucleocapsid protein would suggest that protection with known CTL eptiope(s) can be induced in the absence of neutralizing antibody.

The nucleocapsid protein of the Gray strain of infectious bronchitis virus (IBV) is highly immunogenic and cross-reactive among various distinct serotypes. Recombinant nucleocapsid polypeptide expressed in bacteria with a histidine tag at the amino terminus has been used as antigen for developing an assay to detect IBV-specific antibody. This fusion protein was produced readily in bacteria and easily purified with a nickel column which bound to the histidine tag. Conditions were optimized for using these preparations for an IBV-specific ELISA. Although differences in optical densities could be detected between pre-immune and positive sera for the Ark, Mass, and Gray strains with antigen concentrations between 50 and 0.1 microg per well, the greatest differences could be detected with 3 and 1.5 microg of protein per well. Maximum differences in optical densities between pre-immune and positive sera were obtained using 2.4 microg per well of protein and sera diluted between 1:80 and 1:160. In addition, as little as 30 ng/dot of recombinant nucleocapsid consistently detected IBV-specific sera in immunoblot assays which have convenient field applications.

Specific cytotoxic T-lymphocyte (CTL) responses to nucleocapsid of infectious bronchitis virus (IBV) were identified by using target cells infected with a Semliki Forest virus (SFV) vector. Effector cells for CTL assays were collected from chickens infected with the Gray strain of IBV or inoculated with a DNA plasmid encoding nucleocapsid proteins. IBV-specific CTL epitopes were mapped within the carboxyl-terminal 120 amino acids of the nucleocapsid protein. CTL lysis of target cells infected with SFV encoding nucleocapsid was major histocompatibility complex restricted and mediated by CD8+ T cells. In addition, splenic T cells collected from chickens inoculated in the breast muscle with a DNA plasmid encoding this CTL epitope(s) recognized target cells infected with wild-type virus or an SFV vector encoding nucleocapsid proteins. CTL activity of splenic T cells collected from chicks immunized with a DNA plasmid encoding CTL epitopes was cross-reactive, in that lysis of target cells infected with serologically distinct strains of IBV was dose responsive in a manner similar to that for lysis of target cells infected with the homologous strain of IBV. Furthermore, chickens immunized with a DNA plasmid encoding a CTL epitope(s) were protected from acute viral infection.

Cytotoxic T-lymphocyte (CTL) responses to infectious bronchitis virus (IBV) were determined at regular intervals between 3 and 30 days postinfection (p.i.). The maximum response with 82% lysis of labeled target cells was detected at 10 days p.i. The specific CTL response did not begin to decline until the amount of virus, which peaked at day 8 p.i. in both the kidneys and lungs, started to decrease. Clinical respiratory signs of illness also correlated with amount of virus. CTL activity was shown to be major histocompatibility complex (MHC) class restricted because the lysis of MHC-mismatched targets was negligible, and lysis was mediated by CD8+ CD4- T cells, as the CTL response could be abolished with removal of CD8+ CD4- but not CD4+ CD8- lymphocytes. In contrast, immunoglobulin M (IgM) antibody was not detected until day 10 p.i., and levels peaked at day 12 p.i.; IgG antibody levels were minimal until day 15 p.i. but continued to increase exponentially until day 30 p.i., the last day examined. In summary, CTL responses correlated with initial decreases in infection and illness.

Chimeric infectious bronchitis virus (IBV) genomes with cross-over sites in the S1 gene were generated by co-infection with two distinct IBV strains. Recombinant viruses were collected from chicken embryos, embryonic cultured cells and chickens co-infected with Ark99 and Mass41 strains and purified by differential centrifugation. The recombinant S1 genes were identified by reverse transcription polymerase chain reaction (RTPCR) using heterologous primers and confirmed by nucleotide sequencing. The recombinants with Ark99 5' and Mass41 3' sequences were identified following the in vitro, in ovo and in vivo co-infections. Mixed RNA extracted from Ark99 and Mass41 did not produce RTPCR products with these primers at the PCR conditions used. Cross-over sites within the amplified 580 (Mass41) or 604 (Ark99) bases of the 5' S1 gene could only be detected between nucleotides 50 and 155. While this region, lying upstream of the S1 hypervariable region, corresponded with sites commonly identified in naturally occurring isolates, recombination sites identified in these studies could not be detected within the HVR of S1 of the genomes of chimeric viruses.

The infectious bronchitis virus (IBV) nucleocapsid protein was expressed as a bacterial fusion protein which differed from the native protein only in the addition of six amino terminus histidine residues. Using RNA overlay protein blot assays, the recombinant protein was shown to bind to RNA fragments specific for the positive sense 3' noncoding end of the IBV genome. At greater concentrations of sodium chloride, the native and fusion nucleocapsid proteins similarly bound to G RNA, representing the terminal 1805 3' nt of the genome, whereas bovine serum albumin and allantoic fluid protein did not bind to labeled G RNA. Competitive gel shift assays with labeled G RNA indicated that the protein interacted with several unlabeled RNA representing sequences at the 3' noncoding end of the IBV genome. Cache Valley virus (a bunyavirus) mRNA transcribed from the small segment cDNA also inhibited the interaction with IBV G RNA to approximately the same extent as homologous unlabeled G RNA, whereas reactions with bovine liver RNA and yeast tRNA were considerably weaker. Whereas yeast tRNA did not inhibit the interaction with the labeled large G RNA, interactions of the fusion protein with EF, a region from 78 to 217 nt from the 3' terminus of the IBV genome, were also apparently weaker than interactions with fragment CD which consisted of the 3' terminal 155 nt. On a molar basis, the latter interacted in an identical nature to a RNA consisting of CD and an additional 1053 nt of plasmid sequences. Compared to bovine liver RNA, unlabeled G specifically inhibited binding to the two smaller labeled IBV fragments in gel shift assays. The binding of IBV nucleocapsid protein with RNA probably requires specific sequences and/or structures that are present on the genome, and may represent a common mechanism used by similar viral nucleoproteins whose functions depend on binding to RNA.

The predicted amino acid sequence and secondary structures of S1 of the spike protein (S) of infectious bronchitis viral (IBV) strains from Europe, the U.S.A., and Japan were compared. An antigenic determinant that was highly conserved in both the primary amino acid sequence and secondary structure of all strains was identified between amino acid positions 240 to 255. A synthesized peptide corresponding to this region was found to react with all polyclonal antisera examined from various IBV strains and with one monoclonal antibody (MAb), 9B1B6, out of nine known to react with the S of Gray. The specificity of the interaction with MAb 9B1B6 was confirmed by competitive ELISA using bound and unbound peptide. Interestingly, the previously described epitope for 9B1B6 had been characterized as cross-reactive with several strains of IBV, as conformation-independent but reacting only with intact whole S, and as associated with the functional integrity of other epitopes, including neutralizing epitopes on the S protein. The apparent critical functional and structural nature of this highly immunogenic determinant suggests a potential contribution in developing protective, cross-reactive subunit vaccines to IBV.

The infectious bronchitis virus (IBV) nucleocapsid protein was expressed as a fusion protein in bacteria. The coding sequence differed from the native protein only in the addition of six histidine residues at the amino terminus which were used for enrichment with a nickel affinity column. In gel shift assays, the mobility of labelled G RNA was decreased with increasing concentrations of the fusion protein. Competitive gel shift assays with labelled G RNA indicated that the protein interacted with relatively high avidities to several unlabelled RNAs representing sequences at the 3' noncoding end of the IBV genome. Cache Valley virus (a bunyavirus) mRNA transcribed from the smaller segment cDNA also inhibited the interaction with IBV G RNA to the same extent as homologous unlabelled G RNA. In contrast, interactions of the fusion proteins with a region from 99 to 249 bases from the 3' terminus of the IBV genome and bovine liver RNA were relatively weak. The binding of IBV nucleocapsid protein with RNA probably requires specific sequences and/or structures that are present at a number of sites on the genome, and may represent a common mechanism used by similar viral proteins whose functions depend on binding to RNA.

The large number of phenotypically distinct strains of infectious bronchitis virus (IBV) provide a broad genetic background for examining naturally occurring coronavirus variation. Comparisons of the published nucleotide sequence of S1 genes of strains isolated in Europe, Japan and the USA and four additional American strains described in this report identified 4 genetically distinct groups. The Dutch group was the most divergent sharing only about 60% identity with the American, Mass and European groups which were about 80% homologous with each other. Whereas the strains within the Mass, European and Dutch strains were at least 95% homologous, the strains within the American group were most variable, sharing about 80% identity. The hypervariable region (HVR) which tended to correlate with serotype extended from amino acid residue 53 to 148. In addition to the previously described putative recombination events in the S1 gene of PP14 and SE17, we have now described similar shifts in homology in the corresponding gene of the Gray, Holte, 6/82 (European strain), and Iowa strains. Although minor cross-over sites were identified in the more conserved 3' end at approximately nt 1000 and 1400, a frequently used hot-spot for recombination extended from nt 25 to a region immediately upstream of, but not including, the hypervariable region (HVR). In addition to point mutations, deletions, and insertions, recombination often involving Mass-like and Ark-like sequences, is a commonly used mechanism responsible for the evolution of IBV.

Previous studies on infectious bronchitis virus (IBV) cDNA have identified a region of about 184 bases in the 3' non-coding terminus of both the U.S. prototype strain (Beaudette) and a Japanese strain (KB8523), that was not present in an antigenically closely related U.S. strain, Massachusetts (Mass) 41 (Boursnell et al., 1985; Sutou et al., 1988). In order to investigate the origin and function of this region and its occurrence in nature, the cDNA sequences of the 3' non-coding regions of three additional strains of IBV, Gray, Arkansas (Ark) 99 and Holland (Holl) 52, were determined and compared to the sequences of the Beaudette, KB8523 and Mass41 strains. Not only was this Urich sequence absent from the 3' non-coding region of the Mass41 strain, it was also highly variable, especially in comparison to the highly conserved 3' non coding region downstream of this sequence. Computer analyses of the sequences adjacent to this hypervariable region (HVR) showed that the 3' end of the IBV genome was highly conserved downstream of this region, with 94.3 to 97.8% similarity. However, the similarities for the HVR ranged from 53.2% between Holl52 and Ark99, to 92.8% between Beaudette and Gray. The flanking sequences were not only conserved but these sequences upstream and downstream of the HVR also formed mirrored images.

During an outbreak of severe respiratory disease, a field strain of infectious bronchitis virus (IBV), PP14, was isolated from a bird in a Texas flock that had been previously vaccinated with an attenuated Mass serotype virus. After cloning and sequencing the S1 gene from several IBV strains, it was found that the 5' end of the cDNA was 96% identical to the published sequences of Mass41 and 77% identical with Ark99. The following 402 bases which included the hypervariable regions (HVR) of the S1 gene were 94% homologous with Ark99 and only 69% with Mass41. In addition, the HVR in the 3' noncoding region of the genome, which is totally absent in Mass41, was 99% homologous with the Ark99 strain. This abrupt shift in identity of PP14 in the S1 strongly indicated that a recombination event had occurred about 98 bases from the beginning of the S1 gene between an Ark-like and a Mass-like virus. Downstream, 33 bases from the PP14 recombination junction, a second putative "cross-over" site was identified in the S1 of the SE17 strain where the 5'131 bases of the S1 gene of the Ark99 and SE17 were found to be 95% identical and the following 368 base sequence was only 78% homologous. In addition, a second shift in homology in the S1 of SE17 was identified between nucleotide 1112 and 1460 which shared 95% identity with Mass41. The putative recombination junctions which were downstream of the signal sequence and upstream of the S1 HVR may represent a "hot spot," but not an exclusive region, for exchanging genetic material between IBV strains. Genetic shifts are apparently not only common mechanisms for variation in nature, but vaccine strains may actually play a critical role in these events in the evolution of virulent strains of IBV.

The natural sequence variations of the nucleocapsid genes of the Gray, Arkansas99 (Ark99), and Holland52 (Holl52) strains of infectious bronchitis virus (IBV) were determined. These were compared with previously published sequencing data of other IBV strains, as well as other coronaviruses, in order to correlate the serological and evolutionary relationship of coronaviruses. IBV nucleotide sequence alignment shows that overall the sequences are highly conserved, with homologies from 91.1 to 96.5%. However, there are also two regions (730 to 800 and 1138 to 1166) that appear to be even more highly conserved. Overall, the nucleocapsid protein is highly variable both in size and composition between coronavirus major antigenic groups but is conserved within these groups. A phylogenetic tree of the nucleocapsid protein of various coronaviruses indicates that the coronaviruses fall into distinct groups that correspond to the three major antigenic groups; however, a phylogenetic tree of the IBV nucleocapsid shows that this does not hold true for the type specific antigenic groups of IBV.

A recombinant DNA probe with specificity for the 3' end of genomic RNA from the Ark 99 strain of infectious bronchitis virus (IBV) was found to hybridize with extracted RNA of three strains with the Ark serotype, as well as the Mass41, Holl52, Gray, JMK, Conn, Fla and SE17 strains of IBV. Viral infection was detected in the cytoplasm of chicken embryo kidney cells inoculated with Mass41, Ark99, SE17 or two recent field isolates of IBV using in situ cytohybridization and a biotinylated probe. In vivo infections were detected in individual cells of tracheas and lungs 2,4, and 6 days after inoculation of chicks with Mass41 and Ark99. In situ hybridization of Ark99 infected tissue sections using 32P-dATP labelled probe indicated that more viral replication was present in the trachea on day 4 than either days 2 or 6; whereas more viral RNA was found in the lungs on day 6 than days 2 or 4 after inoculation.

The genomic ribonucleic acid (RNA) of Arkansas DPI at embryonic passages 10 and 100, Australian T, and Massachusetts 41 strains of infectious bronchitis virus (IBV) were compared using two-dimensional, 32PpCp-labeled oligonucleotide fingerprinting. The Arkansas DPI strain embryonic passage 10 was pathogenic for chickens and had one oligonucleotide not present in the attenuated passage 100. The remaining Arkansas DPI oligonucleotides had identical electrophoretic mobility. The fingerprint patterns of the Australian T, Massachusetts 41, and Arkansas DPI IBV genomes were dissimilar, thus indicating they were genetically distinct. The distinctive fingerprint patterns of these virus strains, all identified as IBV by other criteria, indicate considerable genomic variation. Fingerprinting is reliable and sensitive, and should be used in combination with other characterization methods for identifying and differentiating IBV strains.

The antigenic diversity of ten strains of avian infectious bronchitis virus (IBV) was examined by Western blot analyses using polyclonal antisera specific for the Massachusetts 41 (M41), Gray, Arkansas DPI (Ark DPI), Connecticut (Conn) and Australian T (Aust T) serotypes. Although antigenic variation was found in all three structural viral proteins, the matrix protein appeared to be antigenically the most highly variable. Four distinct antigenic groups, which did not correspond to virulence or pathotype, could be defined according to the variations observed in the matrix protein. Somewhat less variation was seen in the spike polypeptide. The only variation in the nucleocapsid protein was indicated by the lack of a detectable reaction between the Aust T antiserum and the Ark DPI nucleocapsid protein. Antisera made against M41 had the broadest reactivity while antisera against Aust T, the only strain tested which was exotic to the U.S.A., had the greatest specificity.