Recombinant porcine interferon to control PRRSV and select other viral pathogens

Interferons (IFNs), particularly IFN-α and IFN-β, provide one of the first lines of defense against
virus infections. Many viruses that affect swine, i.e. porcine reproductive and respiratory syndrome
virus (PRRSV), Seneca Valley virus (SVV), porcine epidemic diarrhea virus (PEDV) and swine
influenza virus (SIV), encode proteins that block IFN activity. These viruses have a devastating
economic impact on the swine industry. Restoring the anti-viral state in pigs by IFN administration
could greatly reduce the economic losses associated with viral disease outbreaks. The goals of this
project were to produce inexpensive and industrially scalable porcine IFN using recombinant
technology and to assess the ability of these IFNs to suppress the in vitro replication of PRRSV, SVV,
PEDV and SIV in porcine cells. Recombinant IFN-α and IFN-β were produced using a Bacillus
megaterium shuttle vector. First, however, it was necessary to modify the shuttle vector by cloning a
cleavable self-aggregating (cSAT) module downstream of the xylose promoter (PxylA). The module
consists of the coding sequence for a gyrA intein, a PT linker and a self-aggregating ELK16 tag.
Next, the coding sequences of porcine IFN-α and IFN-β were codon optimized and cloned into the
vector. The IFN coding sequences were introduced upstream of and in-frame to the cSAT module,
resulting in the production of an IFN fusion protein. The IFN genes were be under the control of the
inducible promoter PxylA, which could be activated by supplementation of the bacterial culture
media with xylose. The shuttle vector was constructed in E.coli then transformed directly into B.
megaterium by electroporation. Transformants were selected for tetracycline resistance. Expression
of the fusion proteins was induced by providing xylose to the culture media. Aliquots of culture
samples were collected at increasing optical densities until the optical density reached 1.5, at which
point the bacteria were pelleted for protein purification. The aliquots were tested for the fusion
protein by western blotting using tag-specific antibodies. These experiments revealed that the
proteins were successfully purified. Bacterial pellets were sonicated and centrifuged to separate the
soluble and insoluble fractions. Due to self-aggregating nature of the ELK16 tag in the cSAT
module, the IFN fusion proteins accumulated in the insoluble fractions. IFN was released from the
insoluble fractions by activating the self-cleaving ability of the GyrA intein using reducing buffer.
The insoluble, cleaved cSAT module was removed from the soluble IFN by centrifugation followed by
dialysis in a refolding buffer. The biological activity of purified porcine IFN-α and IFN-β was
measured in Vero cells using the lytic vesicular stomatitis virus cytopathic assay. We compared the
biological activity of our IFNs to commercially available recombinant human IFN-α and β (BPS
BioSciences). Cells were incubated with serial twofold dilutions of each IFN starting with 100 pg/ml
for 20 hr. Twenty-four hours after inoculation with VSV, cells were fixed and virus titers were calculated. VSV titers were approximately 1 x 108 pfu/ml in the absence of IFN. Virus titers were
greatly reduced in the presence of all four IFNs that were tested. These findings demonstrate that
the porcine IFNs that we produced are biologically active, although their ability to suppress VSV
replication was approximately tenfold less as that observed with the commercially available human
IFNs. Next, we assessed the ability of recombinant porcine IFNs to suppress the in vitro replication
of PRRSV, SVV, PEDV and SIV in swine alveolar macrophages (MARC-145 cells). The cells were
cultured in six-well plates then inoculated with virus in the presence or absence of IFN. All four
viruses (PRRSV, SVV, PEDV and SIV) were used at a multiplicity of infection of 1.0 and the IFNs
(porcine IFN-α and IFN-β) were tested both separately and together. Plaque assay experiments
revealed that the ability of each virus to replicate in MARC-145 did not significantly differ when
comparisons were performed between cultures with no IFNs, 2 units of rPor-IFN-α or rPor-IFN-β or
2 units of both rPor-IFN-α and rPor-IFN-β.