| Protection of Chickens from Infectious Laryngotracheitis with aRecombinant Fowl Poxvirus Expressing Glycoprotein B ofInfectious Laryngotracheitis VirusPh.D Candidate: ZHANG Shaojie Supervisor: Prof. TONG GuangzhiAbstractInfectious laryngotracheitis(ILT) is an economically important viral respiratory tract infection of chickens characterized by signs of respiratory depression, gasping, expectoration of bloody mucus, and high mortality. The disease may cause severe production losses and/or decreased egg production. The causative agent of the disease is an alphaherpesvirus, infectious laryngotracheitis virus (ILTV), classified as gallid herpesvirus I. Present vaccines for ILT are all modified live viruses derived either by sequential passage in cell cultures or embryonated chicken eggs. As immunogenicity of ILTV is usually correlated with its virulence, almost all modified live ILT vaccines remained insufficient attenuation and have shown a variety of side effects including spread of vaccine virus to nonvaccinates, occurrence of long term "carrier" birds, and increasing virulence during in vivo passage. Therefore, vaccination has generally been used only in areas where the disease is endemic.There has been proved that vaccination with herpesvirus glycoproteins can induce protective immunity. For ILT, a subunit vaccine made of a 205 ku complex containing Glycoprotein B (gB) protected 100% of chickens against clinical disease and also against viral replication. This could prove that gB is a major protective immunogen of ILTV, and therefore a prime candidate for constructing recombinant vaccines.In the study, a gene coding for glycoprotein B (gB) of ILTV strain WG (a virulent strain isolated in Wanggang, Harbin.China) was amplified using a pair of primers based on the published sequence of ILTV strain SA2. The gB gene was then cloned into pUC19 at EcoRI site. The sequence analysis showed that the gB gene contains a complete open reading frame of 2619bp. The sequence comparison indicated that the gB gene of ILTV strain WG is identical to those of strain Throne 882 (England) and strain 632 (US), and shares homologies of 98.8% in nucleotide and 98.4% in amino acid with a vaccine strain SA2 (Australia).The cloned ILTVgB gene and LacZ gene were inserted individually into a transfer vector with promoters in frame of each gene. FPV infected chicken embryo fibroblast (CEF) cells were transfected with the transfer vector pSY781 taking lipofectin transfection procedure.Recombinant FPV was selected by blue plague purification. A recombinant FPV stably expressing 1LTV gB (designated as rFPV-ILTVgB) was obtained by six passages of plague purification. Expression of gB gene in rFPV-ILTVgB infected CEF cells was determined by Western blotting.The gB gene of strain WG was subcloned into the Baculovirus transfer vector pVL1393 and a recombinant vector rpVLgB was generated. Co-transfection of SF9 insect cells was performed with rpVLgB and Baculovirus linear DNA(BAC-N-Blue DNA). After three times purification, a recombinant baculovirus, designated as rpVL-ILTVgB was obtained. Expression of ILTV gB in rpVL-ILTVgB infected SF9 cells was detected by direct immuno-fluorescence and Dot-ELISA. The expressed glycoprotein B will be used as subunit vaccine for later immunization in combination with rFPV-ILTVgB.Protection study was performed in both SPF and commercial chickens. SPF chickens were divided into seven groups: commercial ILTV vaccine; rFPV-ILTVgB; S-FPV-017 (parental FPV of rFPV-ILTVgB); rFPV-ILTVgB+rFPV-ILTVgB (two weeks interval); rFPV-ILTV+ rpVL-ILTVgB (two weeks interval); ILTV-gB gene+rFPV-ILTVgB; unvaccinated (control). Commercial chickens were divided into four groups: commercial ILTV vaccine; S-FPV-017; rFPV-ILTVgB; unvaccinated (control group). The number of chickens in each group is 10 for SPF chicken and 20 for commercial chicken. At 5-6 weeks of age, chickens were eye-drop immunized with the recommended dose of commercial ILTV vaccine, or vaccinated intradermally via wing with 5><106PFU...
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