Studies On The Antiviral Activity Of Porcine Interferon-α Against PRRSV And Genetic Analysis Of PRRSV In Henan Province

Porcine interferon-α could inhibit viral propagation significantly. Porcine interferon-α(PoIFN-α) from eukaryotic expression possesses the bioactivity as natural PoIFN-α. It is ofimportant significance to study the antiviral of PoIFN-α from eukaryotic expression againstporcine reproductive and respiratory syndrome virus (PRRSV), which belongs to the RNA virus.This study focused on the antiviral activity of PoIFN-α against PRRSV at the cellular level.PoIFN-α was produced by transfecting eukaryotic expression plasmid pcDNA3.0-poIFNα intoPK-15cell. The genome of PRRSV is easy to be mutational, especially for glycoprotein5.Analyzing the genetic variation about the glycoprotein5gene could reveal the variation of thewhole genome. This article also studied on the PRRSV genetic variation and phylogeneticcharacteristic based on the93PRRSV isolates.To produce PoIFN-α, the eukaryotic expression plasmid pcDNA3.0-poIFNα wastransfected into PK-15cell and the cells were selected by G418sulphate. The polymerase chainreaction and enzyme-linked immunosorbent assay were employed to confirm the expression ofPoIFN-α, whose concentration was910.56pg/ml as measured by ELISA according to thestandard curve.The antiviral activity of PoIFN-α against PRRSV was detected by using fluorescentReal-time PCR to measure the mRNA level after the monocytes and macrophages, which werepretreated with different concentration PoIFN-α, were infected with PRRSV for24h,48h,72h.The results revealed that PoIFN-α could inhibit viral propagation in monocytes and macrophagesat the concentration28.46pg/ml to455.28pg/ml at24h and48h post infection. On the contrary,the viral mRNA level all increased in monocytes and macrophages at72h post infectioncompared with the virus control group. Namely, viral RNA copies in monocytes pretreated with56.91pg/ml IFN-α decreased8.8times significantly compared with the virus control group at24h post infection. And viral RNA copies in monocytes pretreated with455.28pg/ml,56.91pg/ml,14.23pg/ml IFN-α decreased4.7,3.4,4.8times，respectively, which were compared withthe virus control group at48h post infection. Meanwhile, viral RNA copies in macrophagespretreated with56.91pg/ml IFN-α decreased4.64and11.01times significantly at24h and48hpost infection compared with the virus control group. In conclusion, PoIFN-α expressed thehighest anti-PRRSV bioactivity at the concentration56.91pg/ml. The antiviral activity of PoIFN-α was associated with time and dose. PoIFN-α displayed the same function rules both inmonocytes and macrophages.A total of93sequences, consisting of60emanated from Henan from2004to2011, wereretrieved from GenBank and subjected to variable and phylogenetic analysis. The sequencehomology between the genome sequences of60Henan isolates was87.7%-100%. There werethree subgroups in Henan, subgroup Ⅰ (3/60), subgroup Ⅱ (1/60) and subgroup Ⅲ (56/60),respectively, all belonging to the North American Typs. The recombination analysis revealed thatJX0601in subgroupⅠand HuB0801in subgroup Ⅲ yielded the strain ZJJ07. No recombinationwas detected among Henan PRRSV isolates. Selective analysis revealed that there were morevariable residues in signal peptide, ectodomain1and C-terminal. Codon bias analysis revealedthat ORF5of Henan strains had preferences for codons AGA(R)、ACC(T)、AGC(S)、AUU(I)、UUG(L)、AAC(N)、UCC(S)、CCU(P)、GGU(G)、GUC(V)、CCC(P)、UAU(Y)、GUG(V).In conclusion, these results revealed that there were more amino acids undertaking geneticvariation and positive selection, whose mutations could enhance the virulence, decrease theantibody recognition and weaken the immune response. Wild type strain and vaccine strain couldyield additional strain by genetic recombination. It also indicated that viral evolution and geneticvariation were associated with the immunologic performance induced by PRRSV, such asimmune evasion and immunological suppression. Further more, the consequence of viralevolution may give rise to the enhancement of infectivity and virulence, which was possibly apattern of self-protection.