Recombinant Encephalomyocarditis Viruses Elicit Neutralizing Antibodies against PRRSV and CSFV in Mice

<div><p>Encephalomyocarditis virus (EMCV) is capable of infecting a wide range of species and the infection can cause myocarditis and reproductive failure in pigs as well as febrile illness in human beings. In this study, we introduced the entire ORF5 of the porcine reproductive and respiratory syndrome virus (PRRSV) or the neutralization epitope regions in the E2 gene of the classical swine fever virus (CSFV), into the genome of a stably attenuated EMCV strain, T1100I. The resultant viable recombinant viruses, CvBJC3m/I-ΔGP5 and CvBJC3m/I-E2, respectively expressed partial PRRSV envelope protein GP5 or CSFV neutralization epitope A1A2 along with EMCV proteins. These heterologous proteins fused to the N-terminal of the nonstructural leader protein could be recognized by anti-GP5 or anti-E2 antibody. We also tested the immunogenicity of these fusion proteins by immunizing BALB/c mice with the recombinant viruses. The immunized animals elicited neutralizing antibodies against PRRSV and CSFV. Our results suggest that EMCV can be engineered as an expression vector and serve as a tool in the development of novel live vaccines in various animal species.</p></div

Growth characteristics of the recombinant viruses in BHK-21 cells.

<p>^a Plaque size was expressed as mean diameters ± SD. The asterisk represents statistically significant differences between parental T1100I and CvBJC3m/I-ΔGP5.</p><p>^b Virus titer was determined as mean titer of three continuous passages in BHK-21 cells (P3 to P5).</p><p>Growth characteristics of the recombinant viruses in BHK-21 cells.</p

Determination of expressed fusion-proteins by IFA and western blot analysis.

<p>(A) IFA identification of fusion proteins. BHK-21 cells were infected with recombinant viruses or parental T1100I at MOI 5 and stained with anti-VP1, anti-GP5 or anti-E2 antibody at 16 hpi. The scale bars in the pictures represent 50 μm. (B) Western blot of fusion proteins. BHK-21 cell lysates harvested at 16 hpi were probed for fusion proteins with same antibodies. M refers to mock infected BHK-21 cell lysates. ΔGP5 and E2 are herein abbreviated for CvBJC3m/I-ΔGP5 and CvBJC3m/I-E2 infected cell lysates respectively.</p

Neutralizing antibodies induced by recombinant EMC viruses in mice.

<p>^a Eight-week-old male BALB/c mice (n = 5) were intraperitoneally immunized with 5×10⁴ PFU recombinant virus and T1100I in 0.2 ml 2% DMEM. Mice (n = 3) inoculated with 0.2 ml 2% DMEM in the same route were used as negative control.</p><p>^b Neutralization antibody titers were defined as the reciprocal of highest serum dilution that neutralized 100 TCID₅₀ of JXwn06 and were expressed as the geometric mean value of 5 mice.</p><p>^c Neutralization antibody titers were defined as the reciprocal of highest serum dilution that neutralized 200 TCID₅₀ of CSFV C-strain and were expressed as the geometric mean value of 5 mice.</p><p>^d Serum samples were collected at day 0 before immunization.</p><p>^e Mice were boosted with identical dose (5×10⁴ PFU) of either viruses or medium at 14 dpi.</p><p>Neutralizing antibodies induced by recombinant EMC viruses in mice.</p

Replication of parental and recombinant viruses in BHK-21 cells.

<p>(A) Plaque morphology of recombinant viruses. (B) One-step growth curves of recombinant viruses. BHK-21 cells were infected with parental T1100I or recombinant viruses CvBJC3m/I-ΔGP5 and CvBJC3m/I-E2 at MOI 5. Freeze-thawed samples collected from duplicate wells at indicated time points (hpi) and virus titers determined using plaque assay. SD bars are shown for triplicate independent experiments. Asterisks represent statistically significant differences between parental T1100I and respective recombinant viruses as described in Material and Methods. (C) VP1 expression of recombinant viruses by western blot. Infected cells from the same cultures used for panel B were lysed and capsid protein VP1 was detected using anti-VP1 monoclonal antibody. M refers to mock infected BHK-21 cell lysates. ΔGP5 and E2 are herein abbreviated for CvBJC3m/I-ΔGP5 and CvBJC3m/I-E2 infected cell lysates respectively.</p

Gram's staining and molecular identification of Staphylococcus isolate.

<p>A. Gram's staining of the clinical Staphylococcus isolate. B. Identification of Staphylococcus subspecies by ITS-PCR analysis of ribosomal DNA in the isolates from natural EE (Lane 1) and experimental EE (Lane 2). C. Examination of exfoliative toxin gene of the Staphylococcus isolate by PCR. Lanes 1 through 5 represent the PCR products of Exh-A, Exh-B, Exh-C, Exh-D and 23s ribosomal DNA, respectively.</p

Analysis of an apoptosis signaling crosstalk by Nsp10.

<p>(A) Anti-Bid siRNA was transfected into MARC-145 cells or cells that were expressing Nsp10 for 24 h, activation of procaspase-8, Bid expression, and PARP cleavage were examined by Western blot. Anti-HA antibody was used for the detection of Nsp10 expression. β-actin was used as a protein loading control. (B) MARC-145 cells were pretreated with 30 μM Z-IETD-FMK or DMSO for 30 min, and then transfected with the plasmid-expressing Nsp10 or empty vector pCMV-HA for 24 h. Procaspase-8 and PARP were examined by Western blot.</p

Clinical case of Exudative Epidermitis (EE) in piglets.

<p>A–B. Skin lesions (indicated by arrows) of five-day-old piglets with EE. C. Morbidity and mortality of suckling piglets from September of 2005 (n = 23), December of 2005 (n = 33) through March of 2006 (n = 95).</p

Induction of pro-apoptotic pathways by Nsp4 and Nsp10.

<p>(A) Procaspase-8, procaspase-9, and cleved-caspase-12 were examined in MARC-145 cells that were expressing Nsp3, Nsp4, and Nsp10, separately. (B) Protein levels of Bcl-2 family members were examined, including Bid, Bim, Bax, Bcl-xL, and Bcl-2. Black arrows represent each of the indicated isoforms. Empty vector (EV) pWPXL-GFP and Nsp3 were used as negative control. β-actin was used as a protein loading control. Relative proteins levels were calculated for each target protein. Green, blue, grey, and yellow bars represents the cells that were expressing GFP, Nsp3, Nsp4 and Nsp10, respectively.</p

Reproduction of EE in newborn piglets inoculated with <i>S. sciuri</i> HBXX06 via <i>i.m.</i> injection or oral feeding.

<p>A. Four out of six newborn piglets treated with <i>S. sciuri</i> HBXX06 via <i>i.m.</i> injection or oral feeding at the dose of 1×10¹⁰ CFU per pig succumbed to death within 24 hours post infection. B–D. The skin lesions (indicated by arrows) of the survivals of the piglets on days 2, 3 and 4 respectively following oral feeding with 1×10¹⁰ CFU per piglet.</p