Recipient Organization
UNIV OF MARYLAND
(N/A)
COLLEGE PARK,MD 20742
Performing Department
Veterinary Medicine
Non Technical Summary
Porcine reproductive and respiratory syndrome (PRRS) causes significant economic losses to the swine industry. The current strategies to control PRRS are inadequate due to the limited efficacy of the current vaccines. An improved vaccine is one of the top priorities in the PRRS virus (PRRSV) research. PRRSV-infected pigs develop delayed and low titer of virus-neutralizing antibodies and weak cell-mediated immune response, which indicates that PRRSV elicits a poor protective immune response. Innate immunity is the first line of defense against viral infection and plays a crucial role in activating adaptive immunity. PRRSV inhibits innate immunity, especially interferons and their downstream signaling. Suppression of interferon signaling is believed to be an essential contributing factor to the PRRSV modulation of host immune responses. Fortunately, we have discovered an atypical PRRSV strain that induces type I interferons in infected cells. We have also identified the critical residues in the PRRSV proteins responsible for inhibiting interferon signaling. This project aims to generate infectious mutant clones with a weakened effect on interferon-activated signaling while maintaining the capacity of interferon induction with reverse genetics technology. Completing this project will facilitate the development of an improved vaccine against PRRS.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Goals / Objectives
We discovered that PRRSV infection inhibits the interferon-activated STAT1 and STAT2 signaling by reducing the protein level of STAT2 and blocking the nuclear translocation of STAT1 (5, 6). Reverse genetics will be used to generate a mutant virus for an improved vaccine. The mutant virus that loses the ability to reduce STAT1 and STAT2 signaling will be characterized by growth properties.Construct a cDNA clone of A2MC2-P75 and compare the recovered virus with its parental strain. PRRSV inhibits the production and signaling of type I interferons (IFNs) (5, 12). Fortunately, we have discovered an atypical PRRSV strain, A2MC2, that evokes the production of type I IFNs in cultured cells (10). This strain induces earlier production and a higher titer of neutralizing antibodies than a commercial vaccine strain (11). Serial passaging of A2MC2 for 90 passages in cultured cells attenuates the virus but does not affect its capacity to induce IFNs (9). Genome sequencing of A2MC2-P90 shows there is a deletion of 543 nucleotides in comparison with the wild-type strain. A pig vaccination and challenge study showed that A2MC2-P90 was attenuated without detectable shedding, improved weight gain, and offered protection to the pigs challenged with field strain VR-2385 by reduction of virus load and macroscopic lung lesions (8). The data indicate that A2MC2-P90 has limited proliferation (8). A2MC2-P75 was shown to have better replication in pigs (9) and is expected to provide improved protection. Under this objective, an infectious cDNA clone for A2MC2-P75 will be constructed, which will enable the manipulation of the A2MC2-P75 genome for functional studies and future vaccine development.Mutate the cDNA clone of A2MC2-P75 by site-directed mutagenesis to minimize interference of the STAT1 and STAT2 signaling. We discovered that PRRSV nsp1β and nsp11 inhibit STAT1 and STAT2 signaling, respectively (6, 7). The amino acid residue valine 19 of nsp1β is critical for the inhibition of STAT1 signaling and residue lysine 59 of nsp11 is critical for the inhibition of STAT2 signaling. In this experiment, these two residues in nsp1β and nsp11 in the cDNA infectious clone of A2MC2-P75 will be mutated to minimize the capacity of A2MC2-P75 in antagonizing STAT1 and STAT2 signaling. Site-directed mutagenesis of A2MC2-P75 cDNA clone in nsp1β and nsp11 will be conducted. The experiment will generate three infectious clones: pIR-A2MC2-p75-nsp1β-V19I, pIR-A2MC2-p75-nsp11-K59A, and pIR-A2MC2-p75-nsp1β-V19I&nsp11-K59A. Virus recovery from the infectious mutant clones will be performed. The recovered mutant viruses will be tested if the feature of IFN induction is maintained. The effect of the mutant viruses on STAT1 and STAT2 signaling will be assessed in comparison with the wild-type and parental strains. Growth properties of the mutant viruses in both MARC-145 and porcine primary pulmonary alveolar macrophages (PAM) cells will be determined.
Project Methods
Objective 1. Construct an infectious clone of A2MC2-P75 and compare the recovered virus with its parental strain. Construction of the full-length cDNA clone of A2MC2-P75 will facilitate the development of an improved vaccine against PRRS. Under this objective, an infectious clone of A2MC2-P75 will be constructed in a similar manner as done for wild-type A2MC2 (13). The P75 virus is avirulent to pigs and keeps the feature of IFN induction (9). Thus we select this passage for the construction of an infectious cDNA clone and exploration of it for vaccine development. First, the genomic RNA of the A2MC2-P75 virus will be isolated and converted into cDNA. Next, four cDNA fragments will be amplified and cloned into a pIR-A2MC2 plasmid by fragment swapping. The pIR-A2MC2 is a cDNA clone for the wild-type A2MC2. The full-length cDNA cloned into the pIR plasmid will be re-sequenced to confirm the correct P75 sequence. Thirdly, the newly constructed pIR-A2MC2-P75 will be used for virus recovery. This pIR-A2MC2-P75 plasmid is a DNA-launched system and is expected to initiate virus replication once mRNA is made in the cells. The recovered virus will be tested in MARC-145 and PAM cells for growth properties and induction of type I IFNs.Research methodsRNA isolation from the A2MC2-P75 virus and cDNA preparation. RNA isolation and cDNA synthesis will be performed as described previously (10). The PRRSV virions in the culture supernatant will be concentrated and purified by sucrose gradient centrifugation. Viral RNA will be extracted from purified virions with QIAmp Viral RNA Mini Kit (Qiagen). Primers will be designed to amplify and clone the A2MC2-P75 genomic sequence as we did for cloning the wild-type A2MC2. The cDNA synthesis will be conducted with Fermentas Maxima Universal First Strand cDNA Synthesis Kit (ThermoFisher) with gene-specific primers.Construction of cDNA clone of A2MC2-P75. Phusion High-Fidelity DNA Polymerase (ThermoFisher) will be used in PCR amplification of the PRRSV cDNA fragments for cloning. Specific restriction sites will be introduced into the beginning and end of the cDNA fragments for later identification. Four cDNA fragments covering the whole PRRSV genome will be cloned into the pIR-A2MC2 plasmid. Sequencing of clones containing P75 cDNA fragments will be conducted to confirm the correct sequence being cloned. Q5® Site-Directed Mutagenesis Kit (New England Biolabs) will be used to modify any nucleotide variations introduced during cloning.The full-length cDNA clone of A2MC2-P75 will be obtained after all four fragments spanning the whole genome are replaced. The full-length cDNA in the pIR-A2MC2-P75 plasmid will be re-sequenced to confirm the correct full-length sequence.Recovery of A2MC2-P75 virus from the infectious clone. The pIR-A2MC2-P75 is a DNA-launch system and will be used to transfect MARC-145 cells. The pIR-A2MC2 of the wild-type virus will be used as a positive control. To amplify any low titer virus, we will use the culture supernatant of the transfected cells to inoculate fresh cells on the fifth day after transfection and passage the virus three times. To detect the virus replication, we will conduct an immunofluorescence assay (IFA) with a monoclonal antibody against PRRSV N-protein as previously described (14). The culture supernatant from cells with apparent PRRSV replication will be used to inoculate fresh MARC-145 cells to propagate the rescued virus.Determine the growth properties of the recovered A2MC2-P75 virus. To discriminate the recovered virus from the parental virus, we will isolate RNA and conduct RT-PCR with primers covering one restriction site engineered into the cloned virus. The recovered virus will be tested in MARC-145 cells for growth properties as previously described (10). A plaque assay will be performed to compare the plaque sizes of the recovered virus with its parental strain. Multi-step growth curve will be done to determine the propagation property of the recovered virus (13).Determine the capacity of the recovered virus to induce IFNs. The recovered A2MC2-P75 virus will be used to inoculate MARC-145 and PAM cells. Supernatant samples will be harvested at 24 and 48 h post-infection (hpi). IFN bioassay will be conducted in Vero (for supernatant from MARC-145 cells) and CRL2843 (for supernatant from PAMs) (a cell line derived from porcine alveolar macrophages with a loss of CD163, the PRRSV receptor; so it is not susceptible to PRRSV) cells as described (10). The rescued virus is expected to retain the feature of IFN induction as its parental virus. We will also isolate RNA and detect the expression of the interferon-stimulated genes ISG15 and ISG56 in the treated cells by RT-qPCR, as done in Figure 1. Objective 2. Mutate the cDNA clone of A2MC2-P75 by site-directed mutagenesis to minimize interference of the STAT1 and STAT2 signaling. An infectious cDNA clone for A2MC2-P75 will enable the manipulation of the A2MC2-P75 genome for functional studies and future vaccine development. Mutation of the cDNA infectious clone of A2MC2-P75 will be conducted. The critical residues in nsp1β and nsp11 will be subjected to mutagenesis.Conduct site-directed mutagenesis of the A2MC2-P75 cDNA clone. Nsp1β and nsp11 inhibit STAT1 and STAT2 signaling, respectively (6, 7). A2MC2-P75 was found to be similar to wild-type PRRSV strains in the reduction of STAT1 and STAT2 signaling. We will conduct site-directed mutagenesis to minimize the inhibition by nsp1β and nsp11 of rA2MC2-P75.Interferon bioassay. To exclude the possibility that the mutations in nsp1β and nsp11 affect interferon induction of the mutant virus, we will first conduct an interferon bioassay. The rescued mutant and parent viruses will be used to inoculate MARC-145 and PAM cells. Supernatant samples will be harvested at 24 and 48 hpi. An interferon bioassay will be conducted as described (10). It is expected that the recovered mutant viruses will retain the property of interferon induction, as the mutations in nsp1β and nsp11 lead to a loss of inhibition of STAT1 and STAT2, which are downstream of interferon induction.Determine the effect on the STAT1 and STAT2 protein levels and signaling by the recovered mutant viruses. The effects of the recovered mutant viruses on the STAT1 and STAT2 protein levels and signaling will be tested in comparison with the A2MC2-P75 and recovered virus from the pA2MC2-P75. To test their effects on IFN-activated JAK/STAT signaling, we will treat the infected MARC-145 cells with IFN-α and detect the phosphorylation levels and nuclear translocation of STAT1 and STAT2 in comparison cells infected with the parental virus and mock-infected cells. We will also isolate RNA and detect the expression of the interferon-stimulated genes ISG15 and ISG56 in the treated cells by RT-qPCR, as done in Figure 1. An IFN-stimulated response element (ISRE) luciferase reporter assay will also be conducted to confirm the observations.