PRRSV REGULATION FOR INFLAMMATORY MEDIATORS AND CLINICAL ATTENUATION DURING BACTERIAL CO-INFECTION

Project Methods
Five specific experimental approaches are illustrated below to complete the project.1.Elucidation of the viral strategies for immune dysregulation and host mRNA nuclear retention: We will use the PA8 strain as a model virus. We also use P129 and NVSL 97-7895 (FL12)for which infectious clones are available.Genes for nsp1α, nspβ, and N are availableas a fusion with a FLAG tag and a Myc tag so that these proteins can be detected by anti-FLAG or anti-Myc antibodies.Signaling proteins will be detected byimmunoblotand immunofluorescence. Protein-protein interactions will be determined by GST-pull down and co-immunoprecipitation assays. The porcine PIAS is in possession.Nuclear translocation ofIRF3, PIAS, NF-kB will be determined will be examined by confocal microscopy and cell fractionation followed by Western blot.Phosphorylationwill be determined by Western blot from cells expressing respective proteins.Promoter activation will be determined by luciferase reporter assays. Reporter assays are routine in my laboratory, and the IRF3-, NF-kB-, IFNβ-, TNF-α-, and ISRE- promoter constructs are in possesion.2. Generation of nsp1β-mediated IFN suppression-negative, reversion-resistant infectious PRRSV and phenotype characterization in cells: Substitution of leucine (L)at 126 or 135 to alanine (A) results in FN suppression-negative phenotype in cells and pigs. Since reversion occurs in these sites,amino-acid deletions will be introduced to PRRSV. A single-deletion or double-deletion will be introduced in the nsp1β gene by PCR-based site-directed mutagenesis. The mutant nsp1β proteins will be expressed in cells and their IFN-suppression activity will be examined for their negative-phenotype confirmation.To make deletion mutants in the current project, infectious clones will be used to make mutant PRRSV. We have developed a technique for extra-long PCR-based mutagenesis and have used this technique to introduce specific mutations in the PRRSV full-length genomic clone. DNA manipulation and gene cloning are routine in my laboratory and will be performed according to standard procedures.MARC-145 cells will be transfected with a full-length DNA clone.The culture supernatants will be collected and designated 'passage-1'. The 'passage-1' virus will be used to inoculate fresh MARC-145 cells, and the 5-day harvest will be designated 'passage-2'. The 'passage-3' virus will be prepared in the same way as for passage-2. Mutants will be examined for their growth kinetics, viral titers, and plaque morphology in cell culture. Each passage virus will be titrated and stored at -80 C. Viral RNA will be extracted from 'passage 3' and sequenced to ensure the presence of deletions.3. Construction of N-mediated NF-κB activation-negative, reversion-resistant, infectious PRRSV by reverse genetics: PRRSV is known to upregulate proinflammatory cytokines in pigs, and NF-κB is the major player for such responses. Wewill confirm N as the PRRSV protein upregulating proinflammatory cytokines.Immunofluorescence and NF-κB promoter-based reporter assays will be conducted in N-gene transfected cells. The PIAS binding region of N overlaps NLS, and thus NLS-null N protein is hypothesized as NF-κB activation-negative.Proinflammatory cytokine productions by these mutants will be assessed by RT-qPCR in cells expressing NLS-null mutant. Deletions will be introduced to our full-length genomic clones, and infectious PRRSV will be generated.4.Rescue of NF-κB:IFN double-mutant PRRSV by reverse genetics:PRRSV infectious clones will be used to make double-deletion mutant PRRSV. Mutant PRRSV will be examined for stability of mutations, growth characteristics, and viral titers. IFN phenotype and NF-κB activity will be initially determined in the IFNβ, -IRF3-, NF-kB-, and IFN-reporter systems. RT-qPCR and cytokine ELISA will also be used to determine the production of proinflammatory cytokines and type I IFNs in cells. Their phenotypes will be confirmed in PAMs from supernatants following infection. Besides luciferase reporters, VSV-GFP bioassays will be conducted. We have done such experiments, and VSV-GFP is in our possession.5.Infection studies for clinical attenuation and immunological consequence in pigs during co-infection of double-mutant PRRSV with a bacterial pathogen:Infection studieswill be conducted in pigs using six groups of piglets;1) placebo, 2) wild-type PRRSV, 3) Bacterial pathogen, 4) NF-κB-negative and IFN-negative double mutant PRRSV, 5) co-infection with a bacterial pathogen and wild-type PRRSV, 6) co-infection with a bacterial pathogen and NF-κB-negative and IFN-positive double mutant PRRSV.Clinical signs, collection of serum, and tissue samples: Clinical signs will be monitored daily for general conditions, depression, appetite, coughing, sneezing, and respiratory distress. Blood samples will be taken on days weekly post-infection for virus isolation and serology. We will determine IFN levels and proinflammatory cytokines.At necropsy,lung lesion will be scored.Tonsil samples will be collected for persistence and evaluation of viral RNA by RT-PCR. PAMs will be collected at necropsy from BAL RT-PCR will be carried out to determine the expression of proinflammatory.Viremia, ELISA, serum neutralization, and RT-PCR: Viremia will be measured by a standard plaque assay in MARC-145 cells in duplicate. PRRSV-specific antibody titers will be determined using IDEXX antibody detection kit.Serum neutralization tests will be performed by a standard plaque reduction assay. RT-PCR for detection of viral RNA is a routine procedure in my laboratory and will be performed using viral RNA extracted from sera, tonsils, and lymph nodes for detection of the N and nsp1β genes. IFNs and proinflammatory cytokines will be determined by ELISA.