Progress 03/11/15 to 09/30/19
Outputs
Target Audience:Target audiences include layer and broiler farmers in Indiana, layer and broiler farmers in the U.S., poultry and allied industries in the U.S., and poultry health and disease researchers in the U.S. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A post-doctoral scientist who worked with another faculty on immunology research after obtaining a PhD under my supervision has been guided by me for improving scientific writings in these couple of years. Her manuscripts related to DNA or vector vaccine research on infectious bursal disease virus conducted during her tenure as a PhD student has been further edited or revised by me for submission to professional journals. All the manuscripts submitted have been successfully peer-reviewed and published in professional journals. How have the results been disseminated to communities of interest?Research progress and up-to-date findings regarding infectious bursal disease virus pathogenesis, DNA vaccine optimization, and vector vaccine development have been presented to and communicated with poultry health disease researchers and allied scientists in the related fields from Indiana State Poultry Association, North Central Avian Diseases Conference, American Association of Avian Pathologists, and USDA National Committee 1180 on control of emerging and re-emerging avian respiratory diseases. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Accomplishment 1: Study was conducted to determine if innate immunity induced by chicken MDA5 (chMDA5) that sensed infectious bursal disease virus (IBDV) infection bridged to activate adaptive immune response by activating adaptive immunity modulating genes. Chicken HD11 macrophages were infected with IBDV LP1 at multiplicity of infection (MOI) of 0.5 or 10. In addition, HD11 cells with chMDA5 knockdown or overexpression by transfecting cells with chMDA5-targeting siRNA or chMDA5-expressing DNA were infected with IBDV LP1 at MOI of 10. Cell culture supernatants and lysates were collected at 2, 8, 16 and 24 hours post infection (hpi). IBDV-infected HD11 cells had IBDV titers and RNA loads increased up to 3.4x107 plaque forming unit (PFU)/mL and 1113.67 ng/µL at 24 hpi, respectively. IBDV infection in HD11 cells induced significantly upregulated (p<0.05) expression levels of chicken MDA5, interferon-β (IFN-β), dsRNA dependent protein kinase (PKR), 2', 5'-oligoadenylate synthetase (OAS), myxovirus resistance gene (Mx), interleukin-1β (IL-1β), IL-6, IL-8, IL-10, inducible nitric oxide (NO) synthase (iNOS) and major histocompatibility complex class I (MHC class I) up to 59.30, 692.98, 3.80, 286.03, 22.26, 4.95, 145.68, 4.33, 4.33, 15.17 and 4.41 folds, respectively. NO productions in the culture supernatants increased significantly (p<0.05) up to 6.53 μM. The expressed chMDA5 and IBDV-derived dsRNA were localized or co-localized in the cytoplasm of HD11 cells. ChMDA5-knockdown HD11 cells had higher or significantly higher (p<0.05) IBDV RNA loads at 16 and 24 hpi. HD11 cells with chMDA5 knockdown had lower or significantly lower (p<0.05) NO productions and expression levels of chicken MDA5, IPS-1, IRF-3, IFN-β, PKR, OAS, Mx, IL-1β, IL-6, IL-8, IL-12(p40), IL-18, IL-10, iNOS, MHC class I and CD86 at 24 hpi. The results indicated that chMDA5 in chicken macrophages sensed IBDV infection, initiated chMDA5-related innate immune response and activated adaptive immunity modulating genes, which bridged innate and adaptive immunity. Accomplishment 2: An infectious bursal disease virus (IBDV) polyprotein gene-based DNA (VP243/pcDNA) fused with avian influenza virus (AIV) hemagglutinin (HA) gene, VP243-HA/pcDNA, was constructed. One-day-old specific pathogen free (SPF) chickens were intramuscularly injected with 500 μg of constructed DNA weekly for three times, followed by a two-week interval for the fourth injection. Chickens inoculated with VP243/pcDNA had significantly higher (p<0.05) enzyme-linked immunosorbent assay (ELISA) titer to IBDV than those with VP243-HA/pcDNA 3 to 6 weeks after the first inoculation. The hemagglutination inhibition (HI) titers to AIV were significantly higher (p<0.05) in chickens inoculated with HA/pcDNA than those with VP243-HA/pcDNA 2 to 6 weeks after the first inoculation. The results indicated that a single plasmid construct carrying IBDV VP243 gene-based DNA fused with AIV HA gene can elicit specific antibody responses to both IBDV and AIV by DNA vaccination. Accomplishment 3: Infectious bursal disease virus (IBDV) has been established as a replication-competent viral vector capable of carrying an epitope at multiple loci in the genome. To enhance the safety and increase the insertion capacity of IBDV as a vector, a replication-incompetent IBDV vector was developed in the present study. The feasibility of replacing one of the viral gene loci, including pvp2, vp3, vp1, or the polyprotein vp243, with the sequence of green fluorescent protein (GFP) was explored. A method combining TCID50 and immunoperoxidase monolayer assay (IPMA) determined the most feasible locus for gene replacement to be pvp2. The genomic segment containing gfp at the pvp2 locus was able to be encapsidated into IBDV particles. Furthermore, the expression of GFP in GFP-IBDV infected cells was confirmed by Western blotting and GFP IBDV particles showed similar morphology and size to that of wildtype IBDV by electron microscopy. By providing the deleted protein in trans in a packaging cell line (pVP2-DF1), replication-incompetent GFP-IBDV particles were successfully plaque-quantified. The gfp sequence from the plaque-forming GFP-IBDV in pVP2-DF1 was confirmed by RT-PCR and sequencing. GFP-IBDV developed in the present study is a replication-incompetent IBDV vector which expresses a foreign protein in infected cells without the capability to produce viral progeny. Additionally, such replication-incompetent IBDV vectors could serve as bivalent vaccine vectors for conferring protection against infections with IBDV and other economically important, or zoonotic, avian pathogens. Accomplishment 4: Replication incompetent IBDV vector was explored by replacing one of the viral genes, including vp2, vp3, vp1, or the polyprotein vp243 gene sequence with the green fluorescent protein (gfp) gene and providing the deleted viral protein in trans. Generation of a replication competent IBDV vector was conducted by insertion of the gfp gene into vp243 open reading frame with a linker sequence between vp3 and gfp. To create another open reading frame in segment A, adding 1, 2, 3, 10, 20, 26, 40 or 56 nucleotide (s) after the vp243 stop codon was investigated. Expression of GFP protein from all the gfp-containing segment A/B constructs was confirmed by direct observation and immunofluorescence antibody assay (IFA) for GFP protein in transfected cells. Both replication-incompetent and replication-competent strategies did not produce IBDV that can express the GFP protein. Transfection of each of the gfp containing viral cDNA plasmids in IBDV-infected cells was performed and the collected IBDV did not contain the gfp-containing viral mRNA, indicating that the in cis packaging signals on the viral mRNA was not present in all the gfp-containing viral cDNAs. Replication-competent IBDVs were generated when there was extra 1, 2, 3, 10, 20, 26 or 40 nucleotides inserted between the vp243 open reading frame and the original 3'noncoding sequence but the plaque size reduced as the insertion number increased. Replication-incompetent IBDV particles were obtained when 56 nucleotides were inserted as the third open reading frame after vp243 sequence. Accomplishment 5: Green fluorescent protein (GFP) has been successfully incorporated into the viral-like particles of infectious bursal disease virus (IBDV) with a linker at the C-terminus of VP3 in a baculovirus system. However, when the same locus in segment A was used to express GFP by a reverse genetic (RG) system, no viable GFP-expressing IBDV was recovered. To elucidate the underlying mechanism, cDNA construct of segment A with only the linker sequence (9 amino acids) was applied to generate RG IBDV virus (rIBDV). Similarly, no rIBDV was recovered. Moreover, when the incubation after transfection was extended, wildtype rIBDV without the linker was recovered suggesting a free C-terminus of VP3 might be necessary for IBDV replication. On the other hand, rIBDV could be recovered when additional sequence (up to 40 nucleotides) were inserted at the 3' noncoding region (NCR) adjacent to the stop codon of VP3, suggesting that the burden of the linker sequence was not in the stretched genome size but the disruption of the VP3 function. Finally, when the stop codon of VP3 was deleted in segment A to extend the translation into the 3' NCR without introducing additional genomic sequence, no rIBDV was recovered. Our data suggest that a free VP3 C-terminus is essential for IBDV replication.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Chen, Y.N., Wu, C.C., Bryan, T., Hooper, T., Schrader, D., and Lin, T.L. Pathogenicity, immunogenicity, protection efficacy, and spike protein gene sequence of a high-passage turkey coronavirus serially passages in embryonated turkey eggs. Taiwan Veterinary Journal, 44 (2): 1-14, 2018.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Mosley, Y-Y.C., Wu. C.C., and Lin, T.L. Infectious bursal disease virus as a replication-incompetent viral vector expressing green fluorescent protein. Archives of Virology, 162 (1): 23-32, 2017.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Mosley, Y-Y. C., Wu, C.C., and Lin, T.L. A free VP3 C-terminus is essential for the replication of infectious bursal disease virus. Virus Research, 232: 77ÿ¢ÿ¿ÿ¿79, 2017.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Mosley, Y-Y. C., Wu, C.C., and Lin, T.L. IBDV particles packaged with only
segment A dsRNA. Virology, 488: 68-72, 2016.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Mosley, Y-Y. C., Wu, C.C., and Lin, T.L. Avian viral vector vaccines for infectious bursal disease. Taiwan Veterinary Journal, 41 (3): 153ÿ¢ÿ¿ÿ¿163, 2015.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Lee, C.C., Wu, C.C., and Lin, T.L. Bursal transcriptome of chickens protected by DNA vaccination versus those challenged with infectious bursal disease virus Archives of Virology, 160: 69-80, 2015.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Mosley, Y-Y. C., Wu, C.C., and Lin, T.L. Eliciting specific humoral immunity
from a plasmid DNA encoding infectious bursal disease virus polyprotein gene fused with avian influenza virus hemagglutinin gene. Journal of Virological Methods, 211: 36ÿ¢ÿ¿ÿ¿42, 2015.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Lee, C.C., Wu, C.C., and Lin, T.L. Role of chicken melanoma differentiation-
associated gene 5 in induction and activation of innate and adaptive immune responses to infectious bursal disease virus in cultured macrophages. Archives of Virology, 160:3021ÿ¢ÿ¿ÿ¿3035, 2015.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Lee, C.C., Tung, C.Y., Wu, C.C., and Lin, T.L. Avian innate immunity with an emphasis on chicken melanoma differentiation-association gene 5 (MDA5). Taiwan Veterinary Journal, 45 (3): 43-55, 2019.
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Target audiences include layer and broiler farmers in Indiana, layer and broiler farmers in the U.S., poultry and allied industries in the U.S., and poultry health researchers in the U.S. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Research progress and up-to-date findings have been presented to and communicated with Indiana State Poultry Association, North Central Avian Diseases Conference, American Association of Avian Pathologists, and USDA National Committee 1180 on control of emerging and re-emerging avian respiratory diseases. What do you plan to do during the next reporting period to accomplish the goals?Work will be planned to continue to study mechanisms of DNA or vector vaccination against infectious bursal disease virus (IBDV) infection, investigate the pathogenesis of IBDV infection, and determine immunogenicity and protection efficacy of various reverse genetics based IBDV vectors generated.
Impacts
What was accomplished under these goals?
Replication incompetent IBDV vector was explored by replacing one of the viral genes, including vp2, vp3, vp1, or the polyprotein vp243 gene sequence with the green fluorescent protein (gfp) gene and providing the deleted viral protein in trans. Generation of a replication-competent IBDV vector was conducted by insertion of the gfp gene into vp243 open reading frame with a linker sequence between vp3 and gfp. To create another open reading frame in segment A, adding 1, 2, 3, 10, 20, 26, 40 or 56 nucleotide (s) after the vp243 stop codon was investigated. Expression of GFP protein from all the gfp-containing segment A/B constructs was confirmed by direct observation and immunofluorescence antibody assay (IFA) for GFP protein in transfected cells. Both replication-incompetent and replication-competent strategies did not produce IBDV that can express the GFP protein. Transfection of each of the gfp containing viral cDNA plasmids in IBDV-infected cells was performed and the collected IBDV did not contain the gfp-containing viral mRNA, indicating that the in cis packaging signals on the viral mRNA was not present in all the gfp-containing viral cDNAs. Replication-competent IBDVs were generated when there was extra 1, 2, 3, 10, 20, 26 or 40 nucleotides inserted between the vp243 open reading frame and the original 3' noncoding sequence but the plaque size reduced as the insertion number increased. Replication-incompetent IBDV particles were obtained when 56 nucleotides were inserted as the third open reading frame after vp243 sequence.
Publications
|
Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Target audiences include layer and broiler farmers in Indiana, layer and broiler farmers in the U.S., poultry and allied industries in the U.S., and poultry health researchers in the U.S. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Research progress and up-to-date findings have been presented to and communicated with Indiana State Poultry Association, North Central Avian Diseases Conference, American Association of Avian Pathologists, and USDA National Committee 1180 on control of emerging and re-emerging avian respiratory diseases. What do you plan to do during the next reporting period to accomplish the goals?Work will be planned to continue to study mechanisms of DNA or vector vaccination against infectious bursal disease virus (IBDV) infection, study the pathogenesis of IBDV infection, generate various reverse genetics based VP3-deleted IBDV mutants, and study the effects of molecular adjuvants on IBDV-based DNA or vector vaccination.
Impacts
What was accomplished under these goals?
Infectious bursal disease virus (IBDV) has been established as a replication-competent viral vector capable of carrying an epitope at multiple loci in the genome. To enhance the safety and increase the insertion capacity of IBDV as a vector, a replication-incompetent IBDV vector was developed in the present study. The feasibility of replacing one of the viral gene loci, includingpvp2, vp3,vp1,or the polyproteinvp243, with the sequence of green fluorescent protein (GFP) was explored. A method combining TCID50and immunoperoxidase monolayer assay (IPMA) determined the most feasible locus for gene replacement to bepvp2. The genomic segment containinggfpat thepvp2locus was able to be encapsidated into IBDV particles. Furthermore, the expression of GFP in GFP-IBDV infected cells was confirmed by Western blotting and GFP-IBDV particles showed similar morphology and size to that of wildtype IBDV by electron microscopy. By providing the deleted proteinin transin a packaging cell line (pVP2-DF1), replication-incompetent GFP-IBDV particles were successfully plaque-quantified. Thegfp sequence from the plaque-forming GFP-IBDV in pVP2-DF1 was confirmed by RT-PCR and sequencing. GFP-IBDV developed in the present study is a replication-incompetent IBDV vector which expresses a foreign protein in infected cells without the capability to produce viral progeny. Additionally, such replication-incompetent IBDV vectors could serve as bivalent vaccine vectors for conferring protection against infections with IBDV and other economically important, or zoonotic, avian pathogens. ?
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Mosley, Y-Y. C., Wu, C.C., and Lin, T.L. 2017. Infectious bursal disease virus as a replication-incompetent viral vector expressing green fluorescent protein. Archives of Virology, 162: 23-32.
|
Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Target audiences include layer and broiler farmers in Indiana, layer and broiler farmers in the U.S., poultry and allied industries in the U.S., and poultry health researchers in the U.S. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Research progress and up-to-date findings have been presented to and communicated with Indiana State Poultry Association, North Central Avian Diseases Conference, American Association of Avian Pathologists, and USDA National Committee 1180 on control of emerging and re-emerging avian respiratory diseases. What do you plan to do during the next reporting period to accomplish the goals?Work will be planned to continue to study mechanisms of DNA or vector vaccination against infectious bursal disease virus (IBDV) infection, study the pathogenesis of IBDV infection, generate various reverse genetics based VP3-deleted IBDV mutants, and study the effects of molecular adjuvants on IBDV-based DNA or vector vaccination.
Impacts
What was accomplished under these goals?
Studies were conducted to determine functional genomics of chicken innate immune cytosolic sensor nucleotide-binding oligomerization domain like receptor pyrin domain containing 3 (NLRP3) inflammasome. RNA was extracted from chicken macrophage cell line HD11 cells or bursa of Fabricius, reversely transcribed to cDNA, and subjected to PCR amplification of chicken NLRP3. The PCR amplicons were cloned and sequenced. The expression level of NLRP3 in various chicken tissues was determined by SyBr Green-based chicken NLRP3-specific real-time RT-PCR. Functional characterization of NLRP3 was determined by 12-hour lipopolysaccharide (LPS) and additional 15-minute ATP stimulation of HD11 cells or HD11 cells with knockdown of chicken NLRP3 by small interfering RNA (siRNA) targeting chicken NLRP3. Chicken IL-1β levels in the cell culture supernatants were analyzed and determined by Western Blotting. The chicken NLRP3 has an open reading frame encompassing 2778 base pairs of nucleotides and encoding a protein of 925 amino acids. There is one pyrin domain (PYD) in the N-terminal region and leucine-rich repeat domain (LRR) in C-terminal region. The domain architecture is conserved among NLRP3 paralogues. Chicken NLRP3 is phylogenetically different from mammalian counterpart. The highest mRNA levels of chicken NLRP3 were present in kidney, bursa and spleen at 9.4x1010, 9.33x1010 and 8.57x1010 (copy number/µl), respectively. Production of mature chicken IL-1β was detected on the Western blots from the supernatants of HD11 cells stimulated with LPS for 12 hours. Increased amounts of chicken IL-1β were detected in the supernatants of HD11 cells, following stimulation with LPS for 12 hours and exposure to ATP for additional 15 minutes. Production of mature chicken IL-1β was partially abrogated or decreased in the supernatants of chicken NLRP3-knockdown HD11 cells stimulated with LPS and ATP. The result indicated that chicken tissues possess NLRP3, encompassing 2778 base pairs of nucleotides and encoding for 925 amino acids. Chicken NLRP3 is phylogenetically different from mammalian NLRP3. Chicken NLRP3 is ubiquitously expressed in chicken tissues. In addition, chicken NLRP3 functions as a cytosolic sensor for LPS and ATP and production and activation of mature chicken IL-1β is chicken NLRP3 dependent.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Mosley, Y-Y. C., Wu, C.C., and Lin, T.L. 2016. IBDV particles packaged only segment A. Virology, 488: 68-72.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Mosley, Y-Y. C., Wu, C.C., and Lin, T.L. 2015. Avian viral vector vaccines for infectious bursal disease. Taiwan Veterinary Journal, 41: 153-163.
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Progress 03/11/15 to 09/30/15
Outputs
Target Audience:Target audiences include layer and broiler farmers in Indiana, layer and broiler farmers in the U.S., poultry and allied industries in the U.S., and poultry health researchers in the U.S. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Research progress and up-to-date findings have been presented to and communicated with Indiana State Poultry Association, North Central Avian Diseases Conference, American Association of Avian Pathologists, and USDA National Committee 1180 on Control of Endemic, Emerging and Re-emerging Poultry Respiratory Diseases in the United States. What do you plan to do during the next reporting period to accomplish the goals?Work will be planned to continue to study mechanisms of DNA or vector vacciantion againstinfectious bursal disease virus (IBDV) infection andgenerate various reverse genetics basedVP3-deleted IBDV mutants.
Impacts
What was accomplished under these goals?
During infectious bursal disease virus (IBDV) infection in cultured macrophages (HD11 cells), IBDV infection induced significantly upregulated (p<0.05) expression levels of chicken (ch) melanoma differentiation-associated gene 5 (MDA5), interferon-β (IFN-β), dsRNA dependent protein kinase (PKR), 2', 5'-oligoadenylate synthetase (OAS), interleukin-1β (IL-1β), IL-6, IL-8, IL-10, inducible nitric oxide synthase (iNOS) and major histocompatibility complex class I (MHC class I) up to 59, 693, 4, 286, 5, 146, 4, 4, 15 and 4 folds, respectively. Nitric oxide productions in the culture supernatants increased significantly (p<0.05) up to 6.5 μM at 24 hpi. ChMDA5-knockdown HD11 cells had significantly higher (p<0.05) IBDV RNA loads at 24 hpi and significantly lower (p<0.05) nitric oxide productions and expression levels of chMDA5, IFN-β, PKR, OAS, IL-1β, IL-6, IL-8, IL-12(p40), IL-18, IL-10, iNOS, MHC class I and CD86 at 24 hpi. In addition, chMDA5-overexpressed HD11 cells had significantly reduced (p<0.05) IBDV titers and RNA loads and significantly enhanced (p<0.05) nitric oxide productions at 16 and 24 hpi. They also had significantly higher (p<0.05) expression levels of chMDA5, IFN-β, PKR, OAS, Mx, IL-1β, IL-6, IL-8, IL-12(p40), IL-10 and iNOS at 2 hpi. The results indicated that chMDA5 sensed IBDV infection in chicken macrophages and was associated with IBDV-induced expression of IFN-β and initiation of innate immune response, which bridged to activate adaptive immune response and limited IBDV replication. In another study, an infectious bursal disease virus (IBDV) polyprotein gene-based DNA (VP243/pcDNA) fused with avian influenza virus (AIV) hemagglutinin (HA) gene, VP243-HA/pcDNA, was constructed. One-day-old specific pathogen free (SPF) chickens were intramuscularly injected with 500 μg of constructed DNA weekly for three times, followed by a two-week interval for the fourth injection. Chickens inoculated with VP243/pcDNA had significantly higher (p<0.05) enzyme-linked immunosorbent assay (ELISA) titer to IBDV than those with VP243-HA/pcDNA 3 to 6 weeks after the first inoculation. The hemagglutination inhibition (HI) titers to AIV were significantly higher (p<0.05) in chickens inoculated with HA/pcDNA than those with VP243-HA/pcDNA 2 to 6 weeks after the first inoculation. The results indicated that a single plasmid construct carrying IBDV VP243 gene-based DNA fused with AIV HA gene can elicit specific antibody responses to both IBDV and AIV by DNA vaccination.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Lee, C.C., Wu, C.C., and Lin, T.L. 2015. Bursal transcriptome of chickens protected by DNA vaccination versus those challenged with infectious bursal disease virus Archives of Virology, 160: 69-80.
Mosley, Y-Y. C., Wu, C.C., and Lin, T.L. 2015. Eliciting specific humoral immunity from a plasmid DNA encoding infectious bursal disease virus polyprotein gene fused with avian influenza virus hemagglutinin gene. Journal of Virological Methods, 211: 3642.
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