Recipient Organization
UNIV OF CONNECTICUT
438 WHITNEY RD EXTENSION UNIT 1133
STORRS,CT 06269
Performing Department
PATHOBIOLOGY & VETERINARY SCIENCE
Non Technical Summary
Infectious bronchitis virus (IBV) causes an acute highly contagious respiratory and urogenital disease of chickens, which results in significant economic losses in commercial broilers, layers, and breeders. Infectious bronchitis virus infection is mainly controlled by vaccination with attenuated and inactivated virus strains. The use of attenuated vaccine strains have reduced the economic losses due to IBV infections, but they might also be responsible for the emergence of new serotypes or variant strains by point mutation or recombination. The overall goal of this investigation is to develop a safe and effective recombinant subunit vaccine for infectious bronchitis virus (IBV).
Animal Health Component
20%
Research Effort Categories
Basic
50%
Applied
20%
Developmental
30%
Goals / Objectives
I. Clone and express the E, M, and S genes of IBV in a baculovirus expression system. We have already cloned and determined the nucleotide sequence of the S gene of IBV strain Mass41 (Current research). Therefore, we propose to clone the E and M genes of IBV-Mass41 that encode the envelope and matrix proteins. First, the S gene (without the transmembrane domain) will be subcloned into a baculovirus transfer vector, which will be used to generate a recombinant virus expressing spike protein in insect cells. Secondly, we will subclone the E and M genes into a baculovirus dual vector containing two different promoters to generate the recombinant baculovirus, which can coexpress the E and M proteins simultaneously in insect cells. II. Characterize the baculovirus-expressed IBV proteins and produce Virus Like Particles (VLP) of IBV. We will synthesize the E, M, and S proteins in insect cells and characterize the recombinant proteins by immunoblotting. We will coinfect the
cells with both recombinant viruses, optimize the conditions for VLP formation, and scale up the production of IBV recombinant proteins. III. Evaluate the efficacy of recombinant proteins in protection against IBV challenge. We will evaluate the efficacy of recombinant E + M proteins or a combination of both E + M, and S antigens in protection against IBV infection in chickens.
Project Methods
Objective I. Mass41 strain of IBV will be propagated in chicken eggs.. Total genomic RNA will be isolated from the purified IBV by TRIzol reagent (Invitrogen) according to the manufacturers protocol. Complementary DNA (cDNA) segments of IBV will be synthesized using standard cloning methods. Amplified fragments will be cloned into the EcoRI site of pCR2.1 vector using TOPO TA cloning kit (Invitrogen Corp). The major ORF of S gene will be cloned after the polyhedrin promoter of the pFastBacDUAL vector individually. In another construct, the M and E genes will be cloned behind the polyhedrin and p10 promoters, respectively in the same vector. The resulting donor plasmid will be then propagated in E. coli DH5a cells and transposed into E. coli DH10Bac cells to generate recombinant bacmid, as described in the Gibco BRL manual. Objective II. In this phase, we will monitor the expression of recombinant protein for production, characterize the expression products in different
baculoviruses, and optimize the conditions for Virus Like Particle (VLP) formations, and scale up production of these recombinant proteins for vaccine preparations. We will produce the IBV-specific proteins in insect cell cultures. Two separate lots of baculovirus-expressed IBV antigens will be prepared, one of S protein and the other of E+M proteins, and the third one with a combination of both. Objective III. We plan to use crude lysates containing the IBV spike, and envelope plus matrix proteins from baculovirus infected insect cells. Chickens will be immunized by intramuscular injection, using an emulsion prepared by mixing an equal volume of Freunds incomplete adjuvant and sonicated cell lysates. Four week old SPF chickens (n=180) will be divided into six groups and then immunized using Freunds incomplete adjuvant by intramuscular routes. Blood samples will be obtained weekly for up to 4 weeks. The sera will be tested for the presence of anti-IBV (Mass) antibodies in ELISA. Virus
neutralization test will be conducted according to the standard method to examine the virus neutralization antibodies. II) Challenge with virulent virus: At 6 weeks of age, each group of chickens from the above immunization studies will be challenged by eye drop with 104 EID50 of Mass 41 for group1-6. Deaths and characteristic clinical signs including tracheal rales, coughing and sneezing will be recorded each day. Tracheal swabs will be obtained for virus isolation from each chicken on day 5 after challenge. Birds will be euthanized by a USDA recommended procedure and tracheal and lung tissues will be collected for histopathology. Characteristic clinical signs (coughing and sneezing), microscopic lesion such as tracheal deciliation and inflammation and virus isolation will be used as criteria for IBV infection.