Progress 10/01/99 to 09/30/04
Outputs
We have produced a monoclonal antibody (MAb) (7G10) that has high neutralizing activity against porcine reproductive and respiratory syndrome virus (PRRSV). In this study, we identified the 7G10 MAb-binding target(s) by 2-D electrophoresis, followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), as the members of cytoskeletal filaments: vimentin, cytokeratin 8, keratin 18, actin and hair type II basic keratin. In many viruses, it has already been shown that cytoskeletal filaments play an important role in the virus infection. Vimentin bound to PRRSV nucleocapsid protein N and polyclonal antivimentin antibody showed PRRSV neutralizing activity. Vimentin was also expressed on the surface of MARC-145 cells (20 percent of the cells). These results suggest that vimentin is part of PRRSV receptor complex and plays an important role in PRRSV infection, together with the other cytoskeletal filaments (U.S. Patent filed November
10, 2004).
Impacts
We are applying these findings for improved swine breeding for PRRSV resistance.
Publications
- Kim, J.K., Fahad, Al-Majhdi, Shamukhappa, K., and S. Kapil. 2004. Defining the cellular target of PRRSV neutralizing monoclonal antibody (7G10). Submitted.
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Progress 01/01/03 to 12/31/03
Outputs
Porcine reproductive and respiratory syndrome is the most economically devastating disease of swine industry world wide. There is currently no published information on the molecular pathogenesis of PRRS virus. We found that a tetrespanin, CD151 confers susceptibility to baby hamster kidney 21 cells. The recombinant BHK21 become susceptible to PRRSV compared to parent BHK21 cell line. We studied the genomic structure of CD151. The genomic structure of porcine CD151 is thus similar to human and murine CD151.
Impacts
The development of non simian cell lines for PRRSV will help develop better vaccines for PRRSV.
Publications
- Kapil, S and Shanmukhappa, K. 2003. Identification and applications of PRRSV host susceptibility factor(s) for improved swine breeding. 09/772,044.
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Progress 01/01/02 to 12/31/02
Outputs
We are trying to understand the biochemical mechanisms of PRRS virus replication, specially the role of host proteins in PRRS RNA replication. Because 3' untranslated RNA (+) of PRRS virus is critical in virus replication, we studied its interaction with host cell proteins. We previously found out that about 11 proteins specifically bind to PRRS virus 3' UTR (+) RNA (Fahad and Kapil, 2002). By screening the MARC protein expression library with 3' UTR (+) riboprobe, one of the RNA binding proteins identified was CD151, a tetraspan (Shammukappa and Kapil, 2002). The role of CD151 was proven by transfecting CD151 negative cell line (BHK21) with the simiam CD151. After transfecting with CD151, BHK21 cells became susceptible to PRRS virus infection. We have further expanded our investigation of identification and role of MARC cell proteins by performing 2D-SDS-PAGE followed by North Western assay. The identity of the proteins has been confirmed by MALDI-mass spectroscopy.
The roles of these newly identified proteins are under investigation.
Impacts
Porcine reproductive and respiratory syndrome virus is the number one economically significant problem faced by the swine industry worldwide. However, there is no effective treatment for the virus and no effective vaccine to prevent the disease. Thus, we are focusing on the project to evenutally develop PRRS resistant pigs.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
Porcine Reproductive and Respiratory Syndrome (PRRS) is an RNA virus which makes a nested set of transcripts having a common 3'UTR. This 3'UTR is extremely important in the replication of the virus. This region was cloned by RT-PCR and was used as a riboprobe for screening a Meat Animal Research Center (MARC) cell library. On the basis of multiple screenings we identified an RNA-binding protein, CD151. This tetraspanin plays an important role in PRRS replication. A PRRS nonsuscepible Baby Hamster Kidney (BHK-21) cell line allows the attachment of PRRSV but does not allow PRRSV infection. However, we found that when BHK-21 cell line is transfected with CD151 it becomes susceptible to PRRSV infection. CD151 therefore facilitates the entry of PRRS virus inside the cell. We are further studying the mechanism and role of CD151 in PRRS infection. Understanding the mechanism of PRRS virus penetration into the cell as well as PRRS replication will allow us to control this
economically significant disease of pigs worldwide.
Impacts
Porcine reproductive and respiratory syndrome (PRRS) is the number one economically significant disease of pigs in the world. It causes about $236-502/sow losses in the United States (Polson et al., 1994). We believe we have discovered an essential component, CD151, in PRRS entry and replication. Because CD 151 specifically binds to the common 3' UTR of PRRS RNA, it could function as an RNA delivery molecule inside the cell and might also facilitate the transport of viral mRNAs. Through understanding of the entry mechanisms and replication of PRRSV, we can develop significant control of this disease.
Publications
- Fahad Al-Majhdi, Shanmukhappa, S., and Kapil, S. 2001. Interactions of Arteriviral Positive Strand Untranslated Region RNA with Host Cell Proteins. Virus Research. Submitted.
- Shanmukhappa, S. and Kapil, S. 2002. CD151/PETA-3, a Tetraspanin Molecule Interacts with 3'Untranslated Region of Porcine Reproductive and Respiratory Syndrome Virus RNA. J. Virol. Under Revision.
- Polson, D.D., Gorcyca, D., and Morrison, R.B. 1994. An evaluation of the finanicial impact of porcine reproductive and repiratory syndrome (PRRS) in nursery pigs. Proc. 13th Intl. Pigt. Vet. Congress P. 436.
- Daginakatte, Girish and Kapil, S. 2001. Mapping of the RNA-Binding Domain of Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein. Arch. Virol. Submitted.
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Progress 01/01/00 to 12/31/00
Outputs
Porcine reproductive and respiratory syndrome virus (PRRSV) is the most important disease problem facing the swine industry worldwide. The nucleoprotein of PRRS virus is an immunogenic protein and is one of the most predominant proteins of the virus. Pigs elicit a strong immune response to the protein. We expressed the full-length nucleoprotein of PRRSV in E. coli and used the purified recombinant nucleoprotein in an ELISA to detect the antibodies. The sensitivity (85.3%) and specificity (81.7%) of the Kansas State University ELISA were good, correlating well (82.4%) with the IDEXX HerdChek ELISA, currently a more expensive method of herd screening. Previous studies in our laboratory of interactions between the 3' untranslated region of porcine reproductive and respiratory syndrome virus (PRRSV) and MARC 145 cell cytoplasmic proteins have identified 11 RNA binding proteins. We have developed 11 monoclonal antibodies against these proteins.
Impacts
We have developed a rapid, sensitive, and inexpensive alternative method for detecting antibodies to PRRSV. Monoclonal antibodies against proteins reacting with the 3'untranslated region of PRRSV will enhance our understanding of RNA-protein interactions.
Publications
- Steven B. Witte, Cindy Chard-Bergstrom, Thomas A. Loughin, and Sanjay Kapil. 2000. Development of a Recombinant Nucleoprotein-Based Enzyme-Linked Immunosorbent Assay for Quantification of Antibodies against Porcine Reproductive and Respiratory Syndrome Virus. Clinical and Diagnostic Laboratory Immunology, p. 700-702.
- Kumar Shanmukhappa, Fahad Majhdi, and Sanjay Kapil. 2000. Production, Characterization, and Uses of Monoclonal antibodies Against Porcine Reproductive and Respiratory Syndrome Virus 3' Untranslated Region and Nucleoprotein RNA Binding Proteins. Hybridoma, p. 263-267.
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