Sequence Comparison of Avian Infectious Bronchitis Virus S1 Glycoproteins of the Florida Serotype and Five Variant Isolates from Georgia and California

The infectious bronchitis virus (IBV) spike glycoprotein S1 subunit is required to initiate infection and contains virus-neutralizing and serotype-specific epitope(s). Reported are the S1 gene nucleotide and predicted amino acid sequences for the Florida 18288 strain and isolates GA-92, CV-56b, CV-9437, CV-1686, and 1013. These sequences were compared with previously published gene sequences of IBV strains, and phylogenetic relationships are reported. The S1 amino acid sequence of Florida 18288 was 94.9% similar to the Connecticut strain, and GA-92 was 92.8% similar to the Arkansas 99 strain. S1 amino acid sequences of the California variants, CV-56b, CV-9437, and CV-1686, were 97.6–99.3% similar to one another and only 76.6%–76.8% similar to the Arkansas-type strains. Isolate 1013, also from California, was 84.0% similar to Ark DPI and 77.9% similar to CV-56b. When comparing 19 viruses isolated from the United States, sequence variations were observed between amino acids 55–96, 115–149, 255–309, and 378–395. Similar regions are reported to be involved in virus-neutralizing and/or serotype-specific epitopes. These data demonstrate that variant IBV strains continue to emerge, and unique variants may circulate among poultry in geographically isolated areas. This revised version was published online in July 2006 with corrections to the Cover Date.     

Detection of infectious bursal disease viruses by using cloned cDNA probes.

A molecular clone representing 445 base pairs at the 3' end of infectious bursal disease virus (IBDV) genome segment B was used in a dot blot hybridization assay to detect viral RNA from cell culture and from chicken bursa and spleen tissue specimens. The cloned nucleotide sequence represents approximately 14% of the virus-encoded polymerase (VP-1) gene. The lower detection limit of radiolabeled probes prepared from this clone was 0.1 ng of IBDV double-stranded RNA. The probe had broad specificity and was used to detect four serotype 1 IBDV strains and one serotype 2 IBDV strain. This probe, however, did not cross-react with nucleic acid extracted from nine unrelated poultry viruses. A rapid procedure for isolation of IBDV genomic RNA from bursa and spleen tissue specimens was developed and used with the dot blot hybridization assay to detect IBDV strains in tissue samples from experimentally infected and commercially reared chickens.     

Evaluation of VP 2 epitopes of infectious bursal disease virus using in vitro expression and radioimmunoprecipitation

Summary A clone (pV 17-7) spanning a portion of the VP2 gene of infectious bursal disease virus (IBDV) was selected from a cDNA library produced using the variant A virus strain. This clone was expressed in vitro and the protein products were immunoprecipitated with various virus-neutralizing antisera made against 6 different strains of IBDV. The antisera made against 4 variant strains immunoprecipitated the translation products from the pV 17-7 clone, but the antisera to the classic STC virus and the serotype 2 OH virus did not immunoprecipitate the pV 17-7 translation products.     

Molecular cloning and sequence comparison of the S1 glycoprotein of the Gray and JMK strains of avian infectious bronchitis virus

The nucleotide sequences of S1 glycoprotein genes of the Gray and JMK strains of avian infectious bronchitis virus (IBV) were determined and compared with published sequences for IBV. The IBV Gray and JMK strains had 99% nucleotide sequence similarity. The overall nucleotide sequence similarity of the Gray and JMK strains compared with other IBV strains was between 82.0% and 87.4%. The similarity of the predicted amino acid sequence for the S1 glycoproteins of the Gray and JMK strains was 98.8%. Six of the 10 differences in the amino acid sequence were found between residues 99 and 127, suggesting a possible role for that region in the tissue trophisms of the viruses. The S1 glycoprotein of the Gray and JMK strains had 79.5%–84.6% amino acid similarity with the published sequence of other IBV strains. Serine instead of phenylalanine was observed in the protease cleavage site between the S1 and S2 glycoprotein subunits for the Gray and JMK strains, which was similar to the published sequence for the Ark99 and SE17 strains. The significance of that amino acid change is not known. Based on the nucleotide sequence of the Gray and JMK strains, theBsmAI restriction enzyme was selected by computer analysis and was used in restriction fragment length polymorphism analysis to differentiate the two strains.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers GRAYS1=L14069 and JMKS1=L14070.     

Detection of Mycoplasma meleagridis and M. iozvae from dead-in-shell turkey embryos by polymerase chain reaction and culture.

This work reports the development of a 16S rRNA-based polymerase chain reaction (PCR) test for the detection of Mycoplasma meleagridis, and the application, together with a previously described multi-species PCR, for the diagnosis of M. meleagridis and M. iowae in dead-in-shell turkey embryos. Forty-six allantoic fluid samples from dead-in-shell turkey embryos from hatcheries in Israel and the United States were tested by PCR and cultured for the detection of M. meleagridis and M. iowae. Three methods of DNA extraction for PCR assay were compared in allantoic fluid samples from turkey embryos experimentally infected with M. iowae. The two PCR assays provided more rapid detection than culture and were more effective in detecting the components of a mixed infection. Furthermore, PCR has the ability to recognize all M. iowae strains irrespective of antigenic variation.     

Chicken recombinant antibodies specific for very virulent infectious bursal disease virus

Summary. A phage-displayed single chain variable fragment (scFv) antibody library was constructed from the immune spleen cells of chickens immunized with very virulent infectious bursal disease virus (vvIBDV) strain CS89. A library consisting of around 9.2 × 10⁷ clones was subjected to 3 rounds of panning against captured CS89 virus. Analysis of individual clones by nucleotide sequencing revealed at least 22 unique scFv antibodies binding to vvIBDV in ELISA. Testing of the scFv antibody panel in ELISA against classical, variant or vaccine strains and a wide variety of vvIBDV isolates from the UK, China, France, Belgium, Africa, Brazil, Indonesia and the Netherlands identified one antibody, termed chicken recombinant antibody 88 (CRAb 88) that was specific for vvIBDV. CRAb 88 was capable of recognizing all vvIBDV strains tested regardless of their country of origin and showed no reactivity with classical, variant or vaccine strains, lending support to the use of this scFv as a powerful diagnostic tool for the differentiation of vvIBDV strains. Immunoprecipitation studies revealed that CRAb 88 was directed towards a highly conformational epitope located within the major neutralizing protein VP2. Sequence analysis of the hypervariable region of VP2 of the IBDV strains tested indicate that Ile(256) and Ile(294) may play roles in binding of CRAb 88. This is the first reagent of its type capable of positively distinguishing vvIBDV from other IBDV strains.     

Real-time RT-PCR analysis of two epitope regions encoded by the VP2 gene of infectious bursal disease viruses

Infectious bursal disease virus (IBDV) causes an immunosuppressive disease in chickens and leads to severe economic losses in the poultry industry. Vaccination may not be effective if there is exposure of the vaccinated flock to a different antigenic subtype, which reinforces the importance of identification of new IBDV variants. The virus outer capsid is constituted of VP2, in which the major neutralizing epitopes are located. Forty-eight bursa samples collected from IBDV infected commercial broiler flocks in the US were analyzed by real-time RT-PCR using probes designed for two epitope regions of VP2 denominated minor peak 1 and peak B. It was observed that 23, 48 and 44 samples tested with the minor peak probes Del-E, STC and F15, respectively, had a lower melting temperature (Tm) than expected. Furthermore, 44, 41 and 48 samples tested with the Del-E, STC and F15 peak B probes, respectively, had a lower Tm compared to the control, which indicates the presence of one or more nucleotide mutations in the samples. This fact was confirmed by nucleotide sequencing which also demonstrated that most mutations resulted in amino acid substitutions. Real-time RT-PCR can be a useful tool to assist in the development of more effective vaccination strategies.     

Differentiation of transmissible gastroenteritis virus from porcine respiratory coronavirus and other antigenically related coronaviruses by using cDNA probes specific for the 5'' region of the S glycoprotein gene.

Two cDNA clones prepared from the virulent Miller strain of transmissible gastroenteritis virus (TGEV) were identified, and their nucleotide sequences were determined. The clones were nonoverlapping and located in the 5' region of the S glycoprotein gene. Their nucleotide and predicted amino acid sequences were compared with published sequences of the attenuated Purdue strain of TGEV and feline infectious peritonitis virus (FIPV). TGEV clone pE21 contained 381 bp of the S glycoprotein gene and had greater than 98% nucleotide and amino acid sequence homology with Purdue TGEV and over 87% nucleotide and amino acid sequence homology with FIPV. TGEV clone pD24 contained 267 bp of the S glycoprotein gene. It had greater than 98% nucleotide and amino acid sequence homology with Purdue TGEV but only 54% nucleotide sequence homology and 24% amino acid sequence homology with FIPV. A probe prepared from pD24 could differentiate TGEV from porcine respiratory coronavirus and other antigenically related coronaviruses, FIPV, feline enteric coronavirus, and canine coronavirus in a dot blot hybridization assay.     

Spike Gene Analysis of the DE072 Strain of Infectious Bronchitis Virus: Origin and Evolution

The entire S2 gene of the DE072 strain of infectious bronchitis virus (IBV) was sequenced. The nucleotide and amino acid sequence was most similar to the D1466 strain and was 84.8% and 89.9% identity, respectively. The nucleotide and amino acid sequence similarity among the DE072 strain and other IBV strains was less than 71.9% and 76.6%, respectively. Phylogenetic analysis, based on both nucleotide and amino acid sequence, showed that IBV isolates were divided into two distinct groups. The DE072 strain clustered only with the D1466 strain, and all of the other strains were distinct from those two viruses. Further the nucleotide sequence analysis of the entire spike glycoprotein gene of the DE072 strain demonstrated that most of the gene contained a D1466-like sequence, and five putative cross-over sites were identified. Based on cross-over site, phylogenetic trees were constructed for different regions of the spike gene, and a difference in topology between these trees was observed. Considering the difference in S2 gene sequence identity and tree topology, we assume that DE072 and D1466 viruses share a different origin from other isolates of IBV. Furthermore, entire spike gene analysis indicates that the DE072 strain has undergone recombination event as well as extensive antigenic variation.