Performing Department
Veterinary Medicine
Non Technical Summary
Infectious laryngotracheitis (ILT) is a highly contagious disease of chickens, which is caused by infectious laryngotracheitis virus (ILTV), a member of the family Herpesviridae. The disease is characterized by signs of respiratory depression, gasping, expectoration of blood,mucus, and high mortality. The disease occurs worldwide and varies in severity from mild respiratory disease to severe outbreaks with mortality as high as 70%. The disease causes significant economic losses due to morbidity, mortality, decreased egg production, weight loss, and predisposition to other avian pathogens . Although the exact economic losses due to ILT is not known, the poultry industry of the United States experiences multi‐million dollar losses each year as a result of ILTV‐induced mortality, delayed growth, and decreased egg production. Traditionally, ILTV is controlled by the use of modified‐live vaccines. However, current vaccines have a number of disadvantages including insufficient attenuation, production of latently infected carriers, and increased virulence as a result of bird‐to‐bird passage. Therefore, there is a need for safer and more effective ILTV vaccines which can be used in both layers and broilers. We believe that a NDV‐vectored ILT vaccine will be highly effective in controlling ILT in the poultry industry. A NDV‐vectored ILTV vaccine can be used as early as one day of age; therefore, it can be used in broiler chickens. NDV‐vectored vaccines can be administered by spray or in drinking water, which will allow vaccination of a large number of birds in a short time. Chickens in the United States are routinely vaccinated with live‐attenuated NDV vaccine. Therefore, NDV‐vectored ILTV vaccine will be a bivalent vaccine and will not require introduction of a new vaccine. Another advantage of the NDV‐vectored ILTV vaccine is that vaccinated chickens can be distinguished from naturally ILTV infected birds by serological tests. We also strongly believe that use of an effective NDV‐vectored ILTV vaccine will eventually eradicate ILT. Therefore, development of a NDV‐vectored ILTV vaccine will be highly beneficial to the poultry industry in the United States. General methods used in the study includes (A) Isolation of ILTV DNA (B) Cloning of ILTV gB, gC and gD genes (C) Construction of rNDV cDNAs containing gB, gC and gD genes of ILTV individually and in all possible combinations (D) Recovery of rNDVs containing the glycoprotein genes of ILTV (E) Characterization of the recovered rNDV containing ILTV genes (F)Immunogenicity and protective efficacy of rNDVs expressing the envelope glycoproteins of ILTV for chickens.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The goal of this proposal is to develop an efficacious and safe vaccine for Infectious laryngotracheitis. Hence,we propose to use the Newcastle disease virus (NDV) vaccine strain LaSota as a vector to express the immunogenic protein(s) of Infectious laryngotracheitis virus (ILTV). We hypothesize that recombinant NDV (rNDV) expressing the envelope glycoprotein(s) of ILTV would provide a complete protection to chickens from virulent ILTV challenge. We have developed a reverse genetics system to produce infectious rNDV strain LaSota from cloned cDNAs. We propose to evaluate the individual contributions of three important envelope glycoproteins of ILTV (gB, gC, and gD) in protection and immunity.In closely related human and animal herpes viruses, gB, gC, and gD have been shown to be the major protective immunogens. Therefore, we have decided to evaluate the roles of these three ILTV glycoproteins in our study. The immunogenicity and protective efficacy of glycoproteins gB, gC and gD will be evaluated individually and also in all possible combinations. Our specific aims are: 1. Construction of recombinant NDV strain LaSota cDNAs containing gB, gC, and gD genes of ILTV individually and in all possible combinations. Anticipated results:We do not anticipate any problems in completing the proposed work because we have previously cloned the gD gene of bovine herpes virus‐1 in NDV cDNA. 2. Recovery and characterization of recombinant NDVs containing gB, gC and gD genes of ILTV individually and in all possible combinations. Anticipated results: We are quite conversant with techniques of recovering recombinant NDVs expressing foreign genes. Therefore, we do not foresee any problem in recovering rNDV expressing ILTV glycoproteins. However, we recognize that we may encounter a problem in expressing multiple ILTV genes using a single rNDV. We do not expect any problem in expressing two ILTV genes simultaneously because we have previously expressed two foreign genes using a single rNDV. If problems arise, we will mix one rNDV expressing two different ILTV glycoproteins and a second rNDV expressing the third ILTV glycoprotein for our triple combination (rNDV‐gB+gC+gD) vaccination group. 3. Evaluation of immunogenicity and protective efficacy of recombinant NDVs expressing ILTV glycoproteins in chickens following challenges with virulent NDV and ILTV. Anticipated results: The experiments proposed will enhance our current knowledge concerning the role of ILTV gB, gC, and gD proteins in protection and immunity. This study will provide the critical information required for the rational design of safe vaccines to protect against ILTV infection.
Project Methods
(A)Isolation of ILTV DNA: The ILTV vaccine Trachivax will be propagated in chick embryo kidney cells. Virus cultures will be harvested at moderate level of cytopathic effect and cellular debris will be removed by slow centrifugation. Virus will be harvested by pelleting the clarified supernatant through 30% sucrose. Viral DNA will be purified according to the standard procedure. (B)Cloning of ILTV gB, gC and gD genes: Three sets of PCR primers will be used to amplify the ORF of gB, gC and gD genes of ILTV. All the primers contain PmeI sites, the NDV gene end and gene start transcriptional signals, and a six‐nucleotide Kozak sequence for efficient translation. After amplification the products (ORF of gB, gC and gD genes respectively) will be cloned into cloning vector. (C)Construction of rNDV cDNAs containing gB, gC and gD genes of ILTV individually and in all possible combinations: The TOPO vectors bearing the glycoproteins (gB, gC and gD) gene of ILTV will be digested with PmeI and cloned at the unique PmeI site between P and M genes of full‐length NDV plasmid to yield pNDV‐gB, pNDV‐gC and pNDV‐gD. NDV cDNAs containing various combinations of ILTV glycoprotein genes will also be constructed by sequential cloning. All inserted ILTV genes of the resulting plasmids will be sequenced to confirm the orientation and the absence of adventitious mutations. (D)Recovery of rNDVs containing the glycoprotein genes of ILTV: The recombinant viruses will be recovered using the reverse genetics techniques established in our laboratory. For confirmation of recovered virus, the virus plaques will be visualized by immunological staining using a monoclonal antibody specific to NDV HN protein. To confirm that the recovered virus contains the ILTV sequences, RT‐PCR will be performed on RNA extracted from purified recovered rNDV followed by sequencing. (E)Characterization of the recovered rNDV containing ILTV genes: The recovered viruses will be characterized in vitro for their ability to replicate, their levels of ILTV glycoprotein expression, pathogenicity and stability. We will analyze the kinetics and the virus titers of the recovered virus under multi‐cycle growth condition in chicken embryo fibroblast (DF1) cells. The expression of ILTV glycoproteins by the recombinant viruses will be examined by immunofluorescence and western blot assays using ILTV gene specific polyclonal sera. (F)Immunogenicity and protective efficacy of rNDVs expressing the envelope glycoproteins of ILTV for chickens. Four‐week‐old SPF white leghorn chickens will be immunized with different combinations of rNDVs expressing the envelope glycoproteins of ILTV. At 4 weeks post vaccination, chickens in each group will be divided into two subgroups, one subgroup will be transferred to BSL3 facility for NDV challenge. The remaining chickens will be kept in BSL‐2 facility for virulent ILTV challenge. Blood from all vaccinated birds will be collected on the day of challenge and 10 days post challenge from different groups of chickens. Serum will be evaluated for NDV and ILTV antibodies using ELISA and VN tests.