Progress 10/01/08 to 08/31/12
Outputs
OUTPUTS: The project had two specific aims. 1. Analyze gN (UL49.5 homolog) mutants that lack TAP binding or gM binding domains with respect to their impact on TAP inhibition, gM processing and gN/gM envelope incorporation. 2. Characterize in vivo pathogenic and immunogenic properties of gN (UL 49.5) mutants lacking the gN-TAP binding domain or gM deleted mutants relative to their revertants. With respect to aim 1, we have completed analyzing gN mutants lacking the TAP binding domains and some of the results have been reported in PLOS Pathogen (Ref. # 1 below) and in PLOS ONE (Ref. #2 below). Further, we have characterized gN mutants that affect gN/gM interaction and gM processing. In addition, we have constructed and partially characterized a gM mutant lacking the cytoplasmic tail. These are important studies to fully understand gN/gM functions. Results of these studies were presented recently in the 37th Annual Herpesvirus Workshop (Calgary, CANADA; ref. 4 below) and a manuscript describing the results is in the final stage of preparation. With respect to specific aim 2, we have already characterized the in vivo pathogenicity and immunogenicity in calves of a gN mutant that lacked the TAP inhibition domain. Additionally, we performed a vaccine efficacy/challenge study. A manuscript reporting the results of the study has been published (Ref. # 3 below). PARTICIPANTS: Shafiqul Chowdhury Huiyong Wei Jiming Feng TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
BHV-1 gN (UL49.5) is a virulence factor and is important in the pathogenesis of IBR and BRD because it causes immune suppression while allowing extensive virus replication in the upper respiratory tract that promotes secondary bacterial infections. We have identified the viral sequences important for TAP inhibition and MHC-I down regulation and determined that animals immunized with a virus lacking the MHC-I down-regulation domain induced superior cellular and humoral immune response. These results are significant and will allow development of an effective BHV-1 vaccine because, i) it will be of reduced virulence, and ii) it will not be immunosuppressive and will induce effective immune protection.
Publications
- Wei H., He J., Paulsen D.B., Chowdhury, S.I. (2012). Bovine herpesvirus type 1 (BHV-1) UL49.5 luminal domain residues 30-32 and cytoplasmic tail residues 80-96 induce better immune responses in claves than the wild-type strain Cooper. Vet. Immunol. and Immunopathol. 147: 223-229.
- Wei H., He J., and Chowdhury S.I. (2012). BHV-1 envelope protein UL 49.5 residue C (cysteine) 42 is essential for covalently bonded UL49.5/gM complex formation and gM processing. (Abstract 8.26). 37th IHW (August 4-9), Calgary, Canada.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: BHV-1 envelope protein UL49.5 (gN homolog) and gM form a protein complex. UL49.5 is important for immune evasion and along with envelope protein gE, gM is important for secondary viral envelopment. Our long term goals are to determine the mechanism of UL49.5 mediated BHV-1 immune evasion, to determine the function(s) of UL49.5 /gM complex, and to determine the effect(s) of UL49.5 mutations on gM maturation and gM functions. Earlier we have reported that BHV-1 envelope protein UL49.5 residues 30-32 and 80 -96 are important for MHC-I down regulation. In addition, we determined that in cells infected with BHV-1 UL49.5 ∆ 30-32-CT null virus, there is no MHC I down regulation and more importantly, calves infected with the BHV-1 UL49.5∆30-32 CT- null virus had better serum neutralizing and cellular immune response. UL49.5 /gM complex formation is believed to be mediated by cysteine residues. The predicted UL49.5 ORF sequences contain two cysteine residues (C42 and C78) whereas predicted gM ORF sequences contain only one cysteine residue (C45). To determine which of the two UL49.5 cysteine residues is important for UL49.5/gM complex formation, two BHV-1 UL49.5 mutants were generated by exchanging C42 and C78, respectively with serine. Based on the mutant UL49.5/ wt gM co-immunoprecipitation results, C42 is critical for UL49.5/gM complex formation and gM processing in the Golgi (maturation). The results also showed that in the case of BHV-1 UL49.5 C42S exchange mutant, the gM incorporation in the virion envelope is defective. To determine whether UL49.5 C42S mutation has any lethal effect in the background of BHV-1 gE CT null virus, UL49.5 C42S mutation was introduced in the BHV-1gE Am453 (CT-null) mutant virus. Preliminary results showed that relative to parental BHV-1 gE CT-null and BHV-1 wt, BHV-1 UL49.5 C42S/gE CT- null virus produced significantly smaller plaques and had significantly reduced virus yields in infected cells. Further characterization of BHV-1 UL49.5 C42S/gE CT-null virus by confocal and electron microscopy is currently in progress. PARTICIPANTS: Shafiqul Chowdhury (PI- 15% effort) Huiyong Wei - Research Assistant Professor (50% effort) Zhongjian Guo- Post doctoral research associate (50%) Jiming Feng - Associate Professor (collaborator- 5% effort) TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
BHV-1 UL49.5 is a virulence factor and is important in the pathogenesis of IBR and BRD because it causes immune suppression while allowing extensive virus replication in the upper respiratory tract that promotes secondary bacterial infections. Results of this study will be useful for the development of a superior genetically engineered vaccine candidate against BHV-1.
Publications
- Wei H., Wang, Y.,and Chowdhury, S.I.(2011). Bovine herpesvirus type 1 (BHV-1) UL49.5 luminal domain residues 30-32 are critical for MHC-I down-regulation in virus-infected cells. PLoS one 6(10) e25742.
- Wei H., Paulsen D.B., Chowdhury, S.I. Bovine herpesvirus type 1 (BHV-1) UL49.5 luminal domain residues 30-32 and cytoplasmic tail residues 80-96 induce better immune responses in claves than the wild-type strain Cooper. (submitted to Veterinary Immunology and Immunopathology in 2011).
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Earlier we have reported that BHV-1 envelope glycoprotein (g) N interferes with MHC class I antigen presentation by inhibiting and degrading the transporter associated with antigen processing (TAP) and that a gN cytoplasmic tail-deleted mutant (BHV-1 gN Am80) did not degrade the TAP yet blocked the TAP function. We have constructed a BHV-1 gN ∆ 30-32-CT null recombinant virus using a parental BHV-1 gN cytoplasmic tail-null virus in which the gN luminal domain residues 30-32 are deleted. In the BHV-1 gN ∆ 30-32-CT null virus, the TAP 1 degradation, and the TAP 1 mediated MHC I down regulation are abolished. Most notably, in a calf infection and challenge experiment, i) BHV-1 gN ∆ 30-32-CT null virus replicated efficiently in the nasal epithelium, ii) There were 2-fold increase in serum neutralization titers in BHV-1 gN ∆ 30-32-CT null virus- infected calves relative to the wild-type BHV-1 infected calves both at 21-days post infection and at 2 weeks post challenge (Fig. 5) and iii) There were 3 fold (270%) and 50% increases in CD8+ cell proliferation in BHV-1 gN ∆ 30-32-CT null virus-infected calves relative to the wild-type BHV-1 infected calves at 7 days post-infection and 7 days post wild type BHV-1 challenge, respectively. Based on these results incorporation of gN ∆ 30-32 mutation in a vaccine virus will improve immunogenicity of the vaccine virus. PARTICIPANTS: Shafiqul Chowdhury (PI- 15% effort) Huiyong Wei - Research Assistant Professor (100% effort) Jiming Feng - Associate Professor (collaborator- 5% effort) TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
BHV-1 gN is a virulence factor and is important in the pathogenesis of IBR and BRD because it causes immune suppression while allowing extensive virus replication in the upper respiratory tract that promotes secondary bacterial infections. Identification of viral sequences important for TAP inhibition and MHC-I down regulation will allow development of an effective BHV-1 vaccine because, i) it will be of reduced virulence, and ii) it will not be immunosuppressive and will induce effective immune protection
Publications
- Wei, H., Wang, Y. and Chowdhury S.I. Bovine herpesvirus type 1 (BHV-1) glycoprotein N (gN) residues 30-32 are critical for the gN-mediated TAP inhibition/MHC downregulation. Veterinary Herpesvirus Workshop/ International Herpesvirus Workshop (IHW). July 24-29, 2010, Salt Lake City, Utah. (manuscript submitted to JVI).
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Earlier we have reported that BHV-1 envelope glycoprotein (g) N interferes with MHC class I antigen presentation by inhibiting and degrading the transporter associated with antigen processing (TAP) and that a gN cytoplasmic tail-deleted mutant (BHV-1 gN Am80) did not degrade the TAP yet blocked the TAP function. We have now constructed several BHV-1 gN mutants with short sequence deletion in the gN luminal or transmembrane domains using a cytoplasmic tail truncated BHV-1 gNAm80 as a backbone. Plaque morphology and one step growth curve studies revealed that all four BHV-1 gN mutants (BHV-1gNΔ30-32Am80, gNΔ37-40Am80, gNΔ43-46Am80 and gNΔ76-79Am80) had very similar plaque sizes, growth kinetics and similar virus yields relative to the parental BHV-1 gNAm80 virus or BHV-1 wild-type. In addition, the mutants exhibited normal gM matuartion and gN /gM complex formation. However, two other BHV-1gN mutants (BHV-1 gNΔ41-42Am80 and gNΔ74-79Am80) produced ~50% smaller size plaques, reduced (10 fold) viral yield and had defective gM processing due to the lack of gN/gM complex formation and gN degradation, respectively. Among all the gN mutants tested, only one mutant BHV-1gN∆30-32Am80 did not inhibit TAP function and the mutant virus-infected cells displayed significantly higher MHC-I surface expression (90% versus 30% for the wild-type). Preliminary results showed that relative to BHV-1 wild-type-infected rabbits there is a two-fold increase of BHV-1-specific serum neutralizing antibody titer in rabbits infected with the BHV-1gN∆30-32Am80 virus. Taken together, we have determined that gN residues 30 to 32 are critical for gN mediated peptide transport inhibition /MHC I down-regulation, and that BHV-1gN∆30-32Am80 mutant may be useful as a BHV-1 live attenuated vaccine. In the very near future, this mutant will be tested for its pathogenecity and its ability to induce immune response in calves relative to BHV-1 wild-type. PARTICIPANTS: Shafiqul Chowdhury Huiyong Wei - Research Assistant Professor Jiming Feng - Associate Professor TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
BHV-1 gN is a virulence factor and is important in the pathogenesis of IBR and BRD because it causes immune suppression while allowing extensive virus replication in the upper respiratory tract that promotes secondary bacterial infections. Identification of viral sequences important for TAP inhibition and MHC-I down regulation will allow development of an effective BHV-1 vaccine because, i) it will be of reduced virulence, and ii) it will not be immunosuppressive and will induce effective immune protection.
Publications
- Wei, H., Wang, Y., and Chowdhury S.I. BHV-1 glycoprotein N (gN) transmembrane domain mutants-infected MDBK cells have significantly increased MHC class I molecules. Veterinary Herpesvirus Workshop/ International Herpesvirus Workshop (IHW). July 25-31, 2009, Cornell University, Ithaca.
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