Progress 09/01/05 to 08/31/08
Outputs
OUTPUTS: A full-length cDNA infectious clone (pSD01-08) was generated from a low-virulence North American Type II PRRSV isolate, SD01-08. A green fluorescent protein (GFP) was inserted into the nonstructural protein 2 region of the virus as a positive marker. The availability of this infectious clone provides an important research tool for further study of the basic viral biology and pathogenic mechanisms of PRRSV. The pSD01-08-GFP clone is currently available to all PRRSV researchers, and we have sent the plasmid DNA to several universities, including Virginia Polytechnic Institute and State University, University of Nebraska, Lincoln, Cornell University and Leiden University Medical Center (The Netherlands). PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
With a PRRSV elimination program, the international trade in pigs and pork will require a marker vaccine in the future. Furthermore, as the world is progressively moving toward elimination of PRRSV, serosurveillance is an essential tool to verify disease status. The currently available conventional vaccines are unable to allow differentiation between wild-type infection and vaccination. Thus, serosurveillance is impossible in the face of ongoing vaccination or for several months after vaccination has ceased. Clearly, a marked vaccine would be of great benefit. In this study, we have constructed two genetic markers in the nsp2 region of the virus, a positive marker-GFP insertion, and a negative marker-ES4 epitope deletion. Antibody response to the positive marker can serve as an indicator of acquired immunity after vaccination and of vaccine compliance. In our case, one potential advantage of the GFP-tagged vaccine virus would be to follow the vaccine strain in the trial studies before its licensing. However, in disease control/eradication programs, it is essential to include a negative marker because if only the positive marker was present, animals dually exposed to wild-type virus and vaccine could not be differentiated from those animals that were vaccinated only. Our results showed that it is possible to develop such "double marker" PRRSV vaccines, so that vaccinated pigs could be differentiated from those exposed to wild-type virus or those that had mixed infections (vaccine virus and wild-type virus exposure).
Publications
- Fang, Y., Christopher-Hennings, J., Brown, E., Liu, H., Chen, Z., Lawson, S., Breen, R., Clement, T., Gao, X. Bao, J. Knudsen, D., Daly, R., and Nelson, E.A. 2008. Development of genetic markers in the nonstructural protein 2 region of a US type 1 porcine reproductive and respiratory syndrome virus: implications for future recombinant marker vaccine development. J. Gen. Virol. 89:3086-3096.
- Fang, Y., Rowland, R. R., Roof, M., Lunney, J. K., Christopher-Hennings, J., and Nelson, E. A. 2006. A full-length cDNA infectious clone of North American type 1 porcine reproductive and respiratory syndrome virus: expression of green fluorescent protein in the nsp2 region. J. Virology. 80(23):11447-11455.
- Fang, Y., Faaberg, K. S., Christopher-Hennings, J., Rowland, R. R., Pattnaik, A. K., Osorio F. A., Nelson, E. A. 2006. Construction of a European-like Type 1 PRRSV full-length cDNA infectious clone identified in the United States. Adv. Exp. Med. Biol. 581: 605-608.
|
Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: Objective 1: This objective is completed and the result is published (J. Virology, 80:11447-11455). The full-length cDNA infectious clone pSD01-08 has been sent to two other PRRSV research laboratories, including laboratories at University of Nebraska, LinColn (Drs. F. Osorio and A. Pattnaik) and Virginia Tech (Dr. X. J. Meng). Objective 2: This objective is in progress. Initially, we inserted a green fluorescent protein (GFP) into the unique deletion site of nsp2 (at amino acids 733/734 of SD01-08 ORF1a). The GFP virus was recovered from the MARC-145 cells. However, the GFP gene is not stable. Sequence analysis showed that the N-terminal amino acids 1-159 of GFP were deleted by the seventh passage of the GFP virus on the cell culture. To overcome this problem, we deleted the 32 amino acids (ES3) upstream and 55 amino acids (ES4) down-stream of the GFP. This ES3+4 epitope-deletion GFP virus is viable, and GFP appears stable for the first seven passage in the
cell culture. We are continuing passage this GFP virus in cell culture to determine the stability of the GFP gene in the viral genome. In addition, in vivo stability of this GFP virus is also under the investigation in experimental pigs. Objective 3: This objective is completed. Among B cell epitopes of Nsp2, ES2-ES7, the ES2 epitope is located in the most conserved region. However, deletion of ES2 epitope generated non-viable virus. We further performed site-directed mutagenesis on each of the amino acid of ES2 epitope. The amino acid D458 mutation is the only mutation generated the viable virus. Under the support of SD Ag experimental station, we performed the in vivo characterization of the D459 mutant virus. The results showed that the synthetic peptide with the D458 mutation can not differentiate the antibody response to the D458 mutated virus from those of wild type virus. We further investigated the potential of using the other epitopes of Nsp2 as the genetic marker. Each of
the B-cell epitopes ES3-ES7 in the nsp2 region was deleted from the full-length cDNA infectious clone pSD01-08. Viable viruses were recovered from ∆ES3, ∆ES4, ∆ES7, ∆ES3+4+7 epitope deletion mutants. In MARC-145 cells, the ∆ES3 mutant reached a similar viral titer as that of original cloned virus (106 TCID50/ml), but ∆ES4, ∆ES7, ∆ES3+4+7 mutants had lower viral titer compared to that of parental virus, the peak viral titer for ∆ES3+4+7 mutant has two logs difference in comparison to that of parental virus. As a companion test for the marker identification, we have evaluated the ES3, ES4 and ES7 epitope-based ELISA tests for detecting the antibody specific to the deleted epitopes. Three hundred and sixty serum samples, as sequential bleeds from 0 dpi to 62 dpi, were tested. Antibody specific to ES4 recombinant protein can be detected as early as 14 dpi from the pigs infected with the parental viruses, but lacked the antibody
response to the deleted epitope from the pigs infected with corresponding mutant viruses (ΔES4 and ΔES3+4+7). The ES3 or ES7 epitope based- ELISA was not as sensitive as ES4 epitope-based ELISA. PARTICIPANTS: Collaborators: Dr. Eric Nelson. eric.nelson@sdstate.edu; Dr. Jane Christopher-Hennings. jane.hennings@sdstate.edu; Dr. Ruse Daly. Ruse.daly@sdstate.edu; Dr. David Knudson. david.knudson@sdstate.edu; Postdoc, graduate and undergraduate students: Elizabeth Brown, Haixia Liu, Rachael Breen, Xiaofei Gao, Jingjing Bao, Zhenghai Chen, Travis Clement, Lindsey Reister, Brandon Berkenpas, Craig Welbon, Steve Lawson, Scott Vanderpoel, Xiaoxin Zhou and Brett Tostenson TARGET AUDIENCES: Swine research workers, swine veterinarian, and swine producer. PROJECT MODIFICATIONS: None
Impacts
Based on the in vitro characterization of our marker-engineered virus, we received funding from National Pork Board, and SD Ag experimental station to conduct the in vivo study to evaluate the genetic markers in experimental pigs. Our data showed that the ES4 epitope deletion marked-PRRSV recombinants, in conjunction with an ES4 epitope-based ELISA diagnostic test, enable serological differentiation of vaccinated animals from infected animals, which would be a useful tool in PRRSV eradication programs.
Publications
- J. Bao, X. Gao, Brown, E., J. Christopher-Hennings, H. Liu, R. Daly, E. Nelson, R. Breen, D. Knudson and Y. Fang. 2007. Genetic marker development in the Nsp2 region of a US Type1 PRRSV: Implications for future recombinant marker vaccine development. Xth International PRRSV Symposium & 88th Conference of Research Workers in Animal Disease. Chicago, IL.
|
Progress 09/01/06 to 08/31/07
Outputs
OUTPUTS: Objective 1: This objective is completed and the result is published (J. Virology, 80:11447-11455). The full-length cDNA infectious clone pSD01-08 has been sent to two other PRRSV research laboratories, including laboratories at University of Nebraska, LinColn (Drs. F. Osorio and A. Pattnaik) and Virginia Tech (Dr. X. J. Meng). Objective 2: This objective is in progress. Initially, we inserted a green fluorescent protein (GFP) into the unique deletion site of nsp2 (at amino acids 733/734 of SD01-08 ORF1a). The GFP virus was recovered from the MARC-145 cells. However, the GFP gene is not stable. Sequence analysis showed that the N-terminal amino acids 1-159 of GFP were deleted by the seventh passage of the GFP virus on the cell culture. To overcome this problem, we deleted the 32 amino acids (ES3) upstream and 55 amino acids (ES4) down-stream of the GFP. This ES3+4 epitope-deletion GFP virus is viable, and GFP appears stable for the first seven passage in the cell culture. We are continuing passage this GFP virus in cell culture to determine the stability of the GFP gene in the viral genome. In addition, in vivo stability of this GFP virus is also under the investigation in experimental pigs. Objective 3: This objective is completed. Among B cell epitopes of Nsp2, ES2-ES7, the ES2 epitope is located in the most conserved region. However, deletion of ES2 epitope generated non-viable virus. We further performed site-directed mutagenesis on each of the amino acid of ES2 epitope. The amino acid D458 mutation is the only mutation generated the viable virus. Under the support of SD Ag experimental station, we performed the in vivo characterization of the D459 mutant virus. The results showed that the synthetic peptide with the D458 mutation can not differentiate the antibody response to the D458 mutated virus from those of wild type virus. We further investigated the potential of using the other epitopes of Nsp2 as the genetic marker. Each of the B-cell epitopes ES3-ES7 in the nsp2 region was deleted from the full-length cDNA infectious clone pSD01-08. Viable viruses were recovered from ∆ES3, ∆ES4, ∆ES7, ∆ES3+4+7 epitope deletion mutants. In MARC-145 cells, the ∆ES3 mutant reached a similar viral titer as that of original cloned virus (~106 TCID50/ml), but ∆ES4, ∆ES7, ∆ES3+4+7 mutants had lower viral titer compared to that of parental virus, the peak viral titer for ∆ES3+4+7 mutant has two logs difference in comparison to that of parental virus. As a companion test for the marker identification, we have evaluated the ES3, ES4 and ES7 epitope-based ELISA tests for detecting the antibody specific to the deleted epitopes. Three hundred and sixty serum samples, as sequential bleeds from 0 dpi to 62 dpi, were tested. Antibody specific to ES4 recombinant protein can be detected as early as 14 dpi from the pigs infected with the parental viruses, but lacked the antibody response to the deleted epitope from the pigs infected with corresponding mutant viruses (ΔES4 and ΔES3+4+7). The ES3 or ES7 epitope based- ELISA was not as sensitive as ES4 epitope-based ELISA. PARTICIPANTS: Collaborators: Dr. Eric Nelson. eric.nelson@sdstate.edu; Dr. Jane Christopher-Hennings. jane.hennings@sdstate.edu; Dr. Ruse Daly. Ruse.daly@sdstate.edu; Dr. David Knudson. david.knudson@sdstate.edu; Postdoc, graduate and undergraduate students: Elizabeth Brown, Haixia Liu, Rachael Breen, Xiaofei Gao, Jingjing Bao, Zhenghai Chen, Travis Clement, Lindsey Reister, Brandon Berkenpas, Craig Welbon, Steve Lawson, Scott Vanderpoel, Xiaoxin Zhou and Brett Tostenson TARGET AUDIENCES: Swine research workers, swine veterinarian, and swine producer. PROJECT MODIFICATIONS: None
Impacts
Based on the in vitro characterization of our marker-engineered virus, we received funding from National Pork Board, and SD Ag experimental station to conduct the in vivo study to evaluate the genetic markers in experimental pigs. Our data showed that the ES4 epitope deletion marked-PRRSV recombinants, in conjunction with an ES4 epitope-based ELISA diagnostic test, enable serological differentiation of vaccinated animals from infected animals, which would be a useful tool in PRRSV eradication programs.
Publications
- J. Bao, X. Gao, Brown, E., J. Christopher-Hennings, H. Liu, R. Daly, E. Nelson, R. Breen, D. Knudson and Y. Fang. 2007. Genetic marker development in the Nsp2 region of a US Type1 PRRSV: Implications for future recombinant marker vaccine development. Xth International PRRSV Symposium & 88th Conference of Research Workers in Animal Disease. Chicago, IL.
|
Progress 09/01/05 to 08/31/06
Outputs
We have completed objective (1); objectives (2) and (3) are in progress as described below: Objective (2) to insert a marker into the Nsp2 region of a European-like Type 1 PRRSV infectious clone as a means to identify vaccinated animals using routine serology. We inserted a green fluorescent protein (GFP) into the unique deletion site of Nsp2 (at amino acids 733/734 of SD01-08 ORF1a). The construct, pSD01-08-GFP was in vitro transcribed and transfected into BHK-21 cells. Cell culture supernatants from transfected BHK cells were passaged onto MARC-145 cells, resulting in GFP-expressing cells, which could be clearly visualized as early as 6 hr after infection. The GFP virus was serially passaged on MARC-145 cells. By the seventh passage, there appeared a subpopulation (~15%) of non-GFP expressing infected cells. The loss of GFP, analyzed by RT-PCR and sequencing, revealed that the N-terminal amino acids 1-159 of GFP were deleted. More interestingly, two amino acids,
methionine (M) and glutamic acid (E) were inserted by the virus before the GFP amino acid 160. Thus, selection of GFP deleted viral genome accounted for the decline in the percentage of GFP expressing cells. Current research is directed to identify a viral region that can stably express foreign antigens. Objective (3) to mutate an epitope in the NSP2 region as a means to detect the presence of an immune response to a wild-type virus in vaccinated pigs. Based on Oleksiewicz et al.(2001a), Type 1 PRRSV contains six B-cell epitopes, ES2-ES7, in the predicted Nsp2 region. Among these six epitopes, ES2 is located in the highly conserved cysteine protease region, and induces the highest antibody response. Using reverse genetics, the ES2 epitope deletion produced non-viable virus, but a mutation introduced in the ES2 epitope by replacing the amino acid D458 to A resulted in viable virus. This ES2 mutated virus may have potential for future construction of marker vaccines. An ES2 tandem
epitope based-ELISA was also developed in our laboratory for future use as a diagnostic assay to differentiate the serum antibody response of pigs infected with the ES2 marker vaccine from that of wild-type virus.
Impacts
This project addresses the development of tools to identify vaccinated pigs and naturally infected pigs. The results and assays derived from this study can be directly integrated into current or future MLV vaccine constructions.
Publications
- 1. Fang Y., Rowland, R. R., Roof, M., Lunney, J. K., Christopher-Hennings, J., and Nelson, E. A. 2006. A full-length cDNA infectious clone of North American type 1 porcine reproductive and respiratory syndrome virus: expression of green fluorescent protein in the nsp2 region. J. Virology, 80 (23):11447-11455.
- 2. Fang, Y., Faaberg, K. S., Rowland, R. R., Christopher-Hennings, J., Pattnaik, A. K., Osorio, F., and Nelson, E. A. 2006. Construction of a full-length cDNA infectious clone of a European-like Type 1 PRRSV isolated in the U. S. Adv Exp Med Biol. 581:605-608.
|
|