Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1992-07-30
2001-04-10
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C536S023100, C536S024330
Reexamination Certificate
active
06214538
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates, in general, to the genetics of viruses. In particular, the present invention relates to methods of distinguishing between serotypes of avian infectious bronchitis virus based on restriction fragment length polymorphisms, peptides derived from the restriction fragments, related primers for polymerase chain reaction, and certain restriction fragment length polymorphism patterns.
BACKGROUND INFORMATION
Infectious bronchitis virus (IBV), the prototype of the family Coronaviridae, is the etiological agent of infectious bronchitis (IB), an acute, highly contagious disease of the respiratory and urogenital tracts of chickens (21). In spite of the use of vaccines for the control of IB, the disease continues to be a problem in commercial poultry because some serotypes do not cross protect against antigenically unrelated serotypes including variant strains of the virus (12,20).
Both the isolation and serotype identification of IBV field isolates are important because vaccines are selected based on the serotypes of field isolates in a given area. Identification of newly introduced IBV serotypes or variant strains of IBV in a geographical area can be used to modify vaccination programs in order to provide greater protection against the endemic serotypes.
Traditionally, IBV serotypes are identified by the virus neutralization (VN) test, or by the hemagglutination-inhibition (HI) test (9,11,19). Other methods such as, monoclonal antibodies (22), and oligonucleotide fingerprinting (6) have also been used to determine the serotype of IBV isolates. Traditional methods for identifying IBV serotypes are labor intensive and time-consuming. In addition, the use of antibodies, which is somewhat less labor-intensive, cannot detect variants.
IBV contains three structural proteins; the spike (S) glycoprotein, the membrane glycoprotein, and the nucleocapsid protein (30). The S glycoprotein comprises two glycopolypeptides, S1 and S2 (4). Neutralizing and serotype specific antibodies are directed against the S1 glycoprotein (5,23). Thus, unique epitopes on that glycoprotein determine serotype, and are reflected in the gene that codes for the S1 glycoprotein.
IBV has multiple serotypes and new variant serotypes continue to be recognized. When designing a vaccination program for IBV, it is important to identify the serotype of field strains since there may be limited cross-protection between different serotypes of the virus. Although several methods (6,7,16,22) have been used to group IBV isolates, those methods are time consuming and have produced conflicting results. The VN test is the traditional method of serotyping IBV isolates. Thus, any new method must agree with the results of the VN test to be of practical value.
It is well known that neutralizing and serotype specific epitopes are located on the S1 glycoprotein of IBV, but their exact location is not known (5,23).
Lin et al. (26,27) used a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for grouping IBV isolates. In that study, they amplified 400 bases of the N terminal region of the S2 glycoprotein gene, and categorized 12 IBV strains into several groups. In addition, the data set forth in Lin et al. does not adequately support distinguishing serotypes of IBV. That method required 9 different restriction endonuclease (RE) and utilized restriction fragment length polymorphism (RFLP) analysis as well as complicated RE digestion profiles. Thus, despite a great need for a simple and efficient method to distinguish serotypes, Lin et al. fell far short with this inadequate and complicated RE procedure. Further, the utility of that analysis was not verified with additional IBV isolates.
Thus, despite the efforts of many, there remains a great need for a simple, accurate method to distinguish IBV serotypes. The present invention satisfies this need by providing three restriction enzymes which, when used to cleave amplified S1 gene regions, are individually able to distinguish many serotypes of IBV. Further, when separately used on a test sample, the three restriction enzymes were able to distinguish virtually all available serotypes.
SUMMARY OF THE INVENTION
In one embodiment, the present invention provides 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.
In another embodiment, the present invention provides 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.
In another embodiment, the present invention provides 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.
In a further embodiment, the present invention provides 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.
In another embodiment, the present invention provides 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.
Another embodiment of the present invention is 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) digestin
Jackwood Mark W.
Kwon Hyuk Moo
Guzo David
Needle & Rosenberg PC
University of Georgia Research Foundation
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