Differentiation of avian infectious bronchitis virus serotypes

Abstract

The present invention relates, in general, to the genetics of viruses. In particular, the present invention provides methods of distinguishing between serotypes of avian infectious bronchitis virus based on restriction fragment length polymorphisms derived from the region of the S1 gene of IBV, peptides derived from the restriction fragments, related primers for polymerase chain reaction, and certain restriction length polymorphism patterns.

What is claimed is:  
     
         1 . A method of distinguishing between serotypes and/or detecting variants of infectious bronchitis virus comprising: 
 a. amplifying the S1 gene region of infectious bronchitis virus;    b. digesting the amplified S1 gene region with HaeIII, or a restriction endonuclease which cleaves at the HaeIII restriction site, to form restriction fragments;    c. separating the restriction fragment length polymorphisms by electrophoresis; and    d. analyzing restriction fragment length polymorphisms to distinguish between serotypes or detect variants of infectious bronchitis virus.    
     
     
         2 . A method of distinguishing between serotypes and/or detecting variants of infectious bronchitis virus comprising: 
 a. amplifying the S1 gene region of infectious bronchitis virus;    b. digesting the amplified S1 gene region with XcmI, or a restriction endonuclease which cleaves at the XcmI restriction site, to form restriction fragments;    c. separating the restriction fragments by electrophoresis; and    d. analyzing restriction fragment length polymorphisms to distinguish between serotypes or detect variants of infectious bronchitis virus.    
     
     
         3 . A method of distinguishing between serotypes and/or detecting variants of infectious bronchitis virus comprising: 
 a. amplifying the S1 gene region of infectious bronchitis virus;    b. digesting the amplified S1 gene region with BstYI, or a restriction endonuclease which cleaves at the BstYI restriction site, to form restriction fragments;    c. separating the restriction fragments by electrophoresis; and    d. analyzing restriction fragment length polymorphisms to distinguish between serotypes or detect variants of infectious bronchitis virus.    
     
     
         4 . A method of distinguishing between serotypes and/or detecting variants of infectious bronchitis virus comprising: 
 a. amplifying the S1 gene region of infectious bronchitis virus;    b. digesting the amplified S1 gene region with HaeIII, or a restriction endonuclease which cleaves at the HaeIII restriction site, to form a first set of restriction fragments;    c. separately digesting the amplified S1 gene region with XcmI, or a restriction endonuclease which cleaves at the XcmI restriction site, to form a second set of restriction fragments;    d. separating the restriction fragments within the first and second sets by electrophoresis; and    e. analyzing restriction fragment length polymorphisms to distinguish between serotypes or detect variants of infectious bronchitis virus.    
     
     
         5 . A method of distinguishing between serotypes and/or detecting variants of infectious bronchitis virus comprising: 
 a. amplifying the S1 gene region of infectious bronchitis virus;    b. digesting the amplified S1 gene region with HaeIII, or a restriction endonuclease which cleaves at the HaeIII restriction site, to form a first set of restriction fragments;    c. separately digesting the amplified S1 gene region with BstYI, or a restriction endonuclease which cleaves at the BstYI restriction site, to form a second set of restriction fragments;    d. separating the restriction fragments within the first and second sets by electrophoresis; and    e. analyzing restriction fragment length polymorphisms to distinguish between serotypes or detect variants of infectious bronchitis virus.    
     
     
         6 . A method of distinguishing between serotypes and/or detecting variants of infectious bronchitis virus comprising: 
 a. amplifying the S1 gene region of infectious bronchitis virus;    b. digesting the amplified S1 gene region with XcmI, or a restriction endonuclease which cleaves at the XcmI restriction site, to form a first set of restriction fragments;    c. digesting the amplified S1 gene region with BstYI, or a restriction endonuclease which cleaves at the BstYI restriction site, to form a second set of restriction fragments;    d. separating the restriction fragments within the first and second sets by electrophoresis; and    e. analyzing restriction fragment length polymorphisms to distinguish between serotypes or detect variants of infectious bronchitis virus.    
     
     
         7 . A method of distinguishing between serotypes and/or detecting variants of infectious bronchitis virus comprising: 
 a. amplifying the S1 gene region of infectious bronchitis virus;    b. digesting the amplified S1 gene region with HaeIII, or a restriction endonuclease which cleaves at the HaeIII restriction site, to form a first set of restriction fragments;    c. digesting the amplified S1 gene region with XcmI, or a restriction endonuclease which cleaves at the XcmI restriction site, to form a second set of restriction fragments;    d. digesting the amplified S1 gene region with BstYI, or a restriction endonuclease which cleaves at the BstYI restriction site, to form a third set of restriction fragments;    e. separating the restriction fragments within the first, second and third sets by electrophoresis; and    f. analyzing restriction fragment length polymorphisms to distinguish between serotypes or detect variants of infectious bronchitis virus.    
     
     
         8 . A primer for amplifying the S1 gene region of infectious bronchitis virus comprising a nucleic acid, having at least 20 nucleotides, which selectively hybridizes between nucleotide positions −100 through +1, relative to the ATG start site of S1, of the negative strand of infectious bronchitis virus.  
     
     
         9 . The primer of  claim 8 , wherein the primer is SEQ ID NO:1 (S10LIG05′).  
     
     
         10 . A primer for amplifying the S1 gene region of infectious bronchitis virus comprising a nucleic acid, having at least 20 nucleotides, which selectively hybridizes between nucleotide positions 1600 through 1700, relative to the ATG start site of S1, of the positive strand of infectious bronchitis virus.  
     
     
         11 . The primer of  claim 10 , wherein the primer is SEQ ID NO:2 (S10LIG03′).  
     
     
         12 . A method of detecting the presence of a serotype of infectious bronchitis virus comprising ascertaining the presence of a nucleotide sequence associated with a restriction endonuclease cleavage site, created by digestion with HaeIII, by hybridizing the sequence with a selective nucleic acid probe.  
     
     
         13 . The method of  claim 12 , wherein the sequence is amplified prior to hybridization.  
     
     
         14 . A method of detecting the presence of a serotype of infectious bronchitis virus comprising ascertaining the presence of a nucleotide sequence associated with a restriction endonuclease cleavage site, created by digestion with HaeIII, by selective amplification.  
     
     
         15 . A method of detecting the presence of a serotype of infectious bronchitis virus comprising ascertaining the presence of a nucleotide sequence associated with a restriction endonuclease cleavage site, created by digestion with HaeIII, by hybridizing the sequence with a selective nucleic acid probe.  
     
     
         16 . The method of  claim 15 , wherein the sequence is amplified prior to hybridization.  
     
     
         17 . A method of detecting the presence of a serotype of infectious bronchitis virus comprising ascertaining the presence of a nucleotide sequence associated with a restriction endonuclease cleavage site, created by digestion with HaeIII, by determining the order of nucleotides by sequencing.  
     
     
         18 . A method of detecting the presence of a serotype of infectious bronchitis virus comprising ascertaining the presence of a nucleotide sequence associated with a restriction endonuclease cleavage site, created by digestion with XcmI, by selective amplification.  
     
     
         19 . A method of detecting the presence of a serotype of infectious bronchitis virus comprising ascertaining the presence of a nucleotide sequence associated with a restriction endonuclease cleavage site, created by digestion with XcmI, by hybridizing the sequence with a selective nucleic acid probe.  
     
     
         20 . The method of  claim 19 , wherein the sequence is amplified prior to hybridization.  
     
     
         21 . A method of detecting the presence of a serotype of infectious bronchitis virus comprising ascertaining the presence of a nucleotide sequence associated with a restriction endonuclease cleavage site, created by digestion with XcmI, by determining the order of nucleotides by sequencing.  
     
     
         22 . A method of detecting the presence of a serotype of infectious bronchitis virus comprising ascertaining the presence of a nucleotide sequence associated with a restriction endonuclease cleavage site, created by digestion with BstYI, by selective amplification.  
     
     
         23 . A method of detecting the presence of a serotype of infectious bronchitis virus comprising ascertaining the presence of a nucleotide sequence associated with a restriction endonuclease cleavage site, created by digestion with BstYI, by hybridizing the sequence with a selective nucleic acid probe.  
     
     
         24 . The method of  claim 23 , wherein the sequence is amplified prior to hybridization.  
     
     
         25 . A polypeptide portion of the S1 protein of infectious bronchitis virus comprising the amino acids encoded by the nucleotide sequence associated with the HaeIII restriction site.  
     
     
         26 . The polypeptide of  claim 25 , further comprising the amino acids encoded by the nucleotide sequence associated with the XcmI restriction site.  
     
     
         27 . The polypeptide of  claim 25 , further comprising the amino acids encoded by the nucleotide sequence associated with the BstYI restriction site.  
     
     
         28 . The polypeptide of  claim 25 , further comprising the amino acids encoded by the nucleotide sequence associated with the XcmI and BstYI restriction sites.  
     
     
         29 . A polypeptide portion of the S1 protein of infectious bronchitis virus comprising the amino acids encoded by the nucleotide sequence associated with the XcmI restriction site.  
     
     
         30 . The polypeptide of  claim 29 , further comprising the amino acids encoded by the nucleotide sequence associated with the BstYI restriction site.  
     
     
         31 . A polypeptide portion of the S1 protein of infectious bronchitis virus comprising the amino acids encoded by the nucleotide sequence associated with the BstYI restriction site.  
     
     
         32 . The restriction length polymorphism pattern shown in lanes 3 through 11 of FIG. 2.  
     
     
         33 . The restriction length polymorphism pattern shown in lanes 3 through 5 of FIG. 3.  
     
     
         34 . The restriction length polymorphism pattern shown in lanes 3 and 4 of FIG. 4.  
     
     
         35 . The restriction length polymorphism pattern shown in lanes 3 through 8 of FIG. 5.