Jun Cao

GM-CSF fused with GP3 and GP5 of porcine reproductive and respiratory syndrome virus increased the immune responses and protective efficacy against virulent PRRSV challenge.

Porcine reproductive and respiratory syndrome virus (PRRSV) has recently caused catastrophic losses in swine industry worldwide. Current vaccination strategies only provide a limited protection against PRRSV infection. This study was aimed to construct the recombinant adenovirus co-expressing GP3 and GP5 of highly pathogenic PRRSV fused with swine granulocyte-macrophage colony stimulating factor (GM-CSF) (rAd-GF35), and to detect the immune response in mice and pigs. The results showed that the rAd-GF35 could induce significantly higher PRRSV-specific neutralizing antibodies than the recombinant adenovirus only expressing GP3 and GP5 (rAd-GP35). Moreover, the fusion of GM-CSF markedly increased the secretion of IFN-gamma and IL-4 in PRRSV-stimulated mice lymphocytes culture and pigs sera. Following challenge with PRRSV, piglets inoculated with recombinant rAd-GF35 had lighter clinical signs, lower viremia and less gross lesion of lungs, as compared to that of rAd-GP35 immunized group. It demonstrated that GM-CSF fused with GP3 and GP5 of PRRSV could significantly enhance the humoral and cellular immune responses and provide protection against PRRSV challenge in pigs. The recombinant adenovirus rAd-GF35 might be an attractive candidate vaccine for the prevention and control of highly pathogenic PRRSV infection.

HSP70 fused with GP3 and GP5 of porcine reproductive and respiratory syndrome virus enhanced the immune responses and protective efficacy against virulent PRRSV challenge in pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been mainly responsible for the heavy economic losses in pig industry all over the world. Current vaccination strategies provide only a limited protection. In this study recombinant adenoviruses expressing GP3/GP5 of highly pathogenic PRRSV and heat shock protein 70 (HSP70) gene of Heamophilus parasuis were constructed, and the immune responses and protective efficacy against homologous challenge were examined in pigs. The results showed that all animals vaccinated with rAd-GP35 (co-expressing GP3-GP5), rAd-HS35 and rAd-HSA35 (co-expressing GP3-GP5 fused with HSP70 using different linkers), developed specific anti-PRRSV ELISA antibody and neutralizing antibody. The humoral immune responses of rAd-HS35, especially rAd-HSA35 containing 2A of FMDV between HSP70 and GP3 gene, were significantly higher than that of rAd-GP35. Moreover, the fusion of HSP70 markedly induced both IFN-gamma and IL-4 in pigs sera. Following challenge with PRRSV, pigs inoculated with recombinant rAd-HS35 and rAd-HSA35 showed lighter clinical signs, lower viremia and less pathological lesion of lungs, as compared to those in rAd-GP35 group. Moreover, the protective efficiency induced by rAd-HSA35 was higher than that of rAd-HS35. It indicated that HSP70 fused with GP3 and GP5 of PRRSV could induce enhanced immune responses and provide protection against virulent PRRSV challenge in pigs. The recombinant adenovirus rAd-HSA35 might be an attractive candidate vaccine for the prevention and control of highly pathogenic PRRSV infections.

Replication characteristics of porcine reproductive and respiratory syndrome virus (PRRSV) European subtype 1 (Lelystad) and subtype 3 (Lena) strains in nasal mucosa and cells of the monocytic lineage: indications for the use of new receptors of PRRSV (Lena).

Recently, it has been demonstrated that subtype 3 strains of European type porcine reproductive and respiratory syndrome virus (PRRSV) are more virulent/pathogenic than subtype 1 strains. This points to differences in the pathogenesis. In the present study, a new polarized nasal mucosa explant system was used to study the invasion of the low virulent subtype 1 PRRSV strain Lelystad (LV) and the highly virulent subtype 3 PRRSV strain Lena at the portal of entry. Different cell types of the monocytic lineage (alveolar macrophages (PAM), cultured blood monocytes and monocyte-derived dendritic cells (moDC)) were enclosed to examine replication kinetics of both strains in their putative target cells. At 0, 12, 24, 48 and 72xa0hours post inoculation (hpi), virus production was analyzed and the infected cells were quantified and identified. Lena replicated much more efficiently than LV in the nasal mucosa explants and to a lesser extent in PAM. Differences in replication were not found in monocytes and moDC. Confocal microscopy demonstrated that for LV, almost all viral antigen positive cells were CD163+Sialoadhesin (Sn)+, which were mainly located in the lamina propria of the respiratory mucosa. In Lena-infected nasal mucosa, CD163+Sn+, CD163+Sn- and to a lesser extent CD163-Sn- monocytic subtypes were involved in infection. CD163+Sn- cells were mostly located within or in the proximity of the epithelium. Our results show that, whereas LV replicates in a restricted subpopulation of CD163+Sn+ monocytic cells in the upper respiratory tract, Lena hijacks a broader range of subpopulations to spread within the mucosa. Replication in CD163+Sn- cells suggests that an alternative entry receptor may contribute to the wider tropism of Lena.

Inhibition of porcine reproductive and respiratory syndrome virus replication by adenovirus-mediated RNA interference both in porcine alveolar macrophages and swine

Porcine reproductive and respiratory syndrome virus (PRRSV) has been mainly responsible for the heavy economic losses in many swine-producing regions. Current vaccination strategies and antiviral drugs provide only limited protection. Consequently, there is a need to develop a new antiviral strategy. In this study, two recombinant adenoviruses expressing short-hairpin RNAs (shRNAs) directed against ORF1b of PRRSV S1 strain were constructed and the inhibition of PRRSV replication was determined. The results showed that pretreatment with these shRNAs delivered by recombinant adenovirus could induce a significant inhibition of viral RNA and protein level in Marc-145 cells infected with PRRSV S1 strains. One recombinant adenovirus (rAd-P2) was found to be also effective in inhibiting the replication of highly virulent PRRSV SY0608 strain in Marc-145 cells and porcine alveolar macrophages at both the protein and ORF1b mRNA level. The antiviral effect was dose-dependent and sustained for at least 96h. Twenty 6-week old piglets were assigned to four groups each with five piglets. Groups 1 and 2 were inoculated intramuscularly with rAd-P2 and mock construct rAd-mP2 individually. After 24h, groups 1, 2 and 3 were challenged intramuscularly with the SY0608 strain. Group 4 remained unchallenged but with PBS as mock. The results showed that the viral load of PRRSV in serum and lung tissue of swine was suppressed effectively by rAd-P2. The clinical signs and pathological lesions in the pigs inoculated with rAd-P2 were milder than those in rAd-mP2 negative and PRRSV control. These results indicated that shRNAs mediated by the adenovirus could inhibit PRRSV infection sufficiently in vitro as well as in vivo. RNAi mediated by recombinant adenovirus might be a potential new tool for controlling PRRSV infection. Of course, the protective efficiency of rAd-P2 should be made by using a large number of pigs in future.

CD40 ligand expressed in adenovirus can improve the immunogenicity of the GP3 and GP5 of porcine reproductive and respiratory syndrome virus in swine

Porcine reproductive and respiratory syndrome virus (PRRSV) has recently caused heavy economic losses in swine industry worldwide. Current vaccination strategies only provide a limited protective efficacy, thus immune modulators are being considered to enhance the effectiveness of PRRSV vaccines. In this study, the recombinant adenoviruses expressing porcine CD40 ligand (CD40L) and GP3/GP5 of PRRSV were constructed and the immune responses were examined in pigs. The results showed that rAd-CD40L-GP35 (co-expressing CD40L and GP3-GP5) or rAd-GP35 (expressing GP3-GP5) plus rAd-CD40L (expressing CD40L) could provide significant higher specific anti-PRRSV ELISA antibody and neutralizing antibody. And the levels of proliferative responses of peripheral blood mononuclear cells (PBMC), IFN-γ and IL-4 were markedly increased in rAd-CD40L-GP35 and rAd-CD40L plus rAd-GP35 groups than those in rAd-GP35 group. Following homologous challenge with Chinese isolate of the North-American genotype of PRRSV, pigs inoculated with recombinant rAd-CD40L-GP35 and rAd-CD40L plus rAd-GP35 showed lighter clinical signs and lower viremia, as compared to those in rAd-GP35 group. It indicated that porcine CD40L could effectively increase humoral and cell-mediated immune responses of GP3 and GP5 of PRRSV. Porcine CD40L might be used as an attractive adjuvant or immunotargeting strategies to enhance the PRRSV subunit vaccine responses in swine.

Enhanced immunogenicity of multiple-epitopes of foot-and-mouth disease virus fused with porcine interferon α in mice and protective efficacy in guinea pigs and swine

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating vesicular disease of cloven-hoofed animals. In this study, three amino acid residues 21-60, 141-160 and 200-213 from VP1 protein of FMDV were selected as multiple-epitopes (VPe), and a recombinant adenovirus expressing the multiple-epitopes fused with porcine interferon alpha (rAd-pIFN alpha-VPe) was constructed. Six groups of female BALB/c mice (18 mice per group) were inoculated subcutaneously (s.c.) twice at 2-week intervals with the recombinant adenoviruses and the immune responses were examined. Following this the protective efficacy of rAd-pIFN alpha-VPe was examined in guinea pigs and swine. The results showed that both FMDV-specific humoral and cell-mediated immune responses could be induced by rAd-VPe and increased when rAd-pIFN alpha is included in this regime in mice model. Moreover, the levels of the immune responses in the group inoculated with rAd-pIFN alpha-VPe were significantly higher than the group inoculated with rAd-VPe plus rAd-pIFN alpha. All guinea pigs and swine vaccinated with rAd-pIFN alpha-VPe were completely protected from viral challenge. It demonstrated that recombinant adenovirus rAd-pIFN alpha-VPe might be an attractive candidate vaccine for preventing FMDV infection.

Suppression of NK cell-mediated cytotoxicity against PRRSV-infected porcine alveolar macrophages in vitro

The adaptive immunity against PRRSV has already been studied in depth, but only limited data are available on the innate immune responses against this pathogen. In the present study, we analyzed the interaction between porcine natural killer (NK) cells and PRRSV-infected primary porcine alveolar macrophages (PAMs), since NK cells are one of the most important components of innate immunity and PAMs are primary target cells of PRRSV infection. NK cytotoxicity assays were performed using enriched NK cells as effector cells and virus-infected or mock-inoculated PAMs as target cells. The NK cytotoxicity against PRRSV-infected PAMs was decreased starting from 6h post inoculation (hpi) till the end of the experiment (12 hpi) and was significantly lower than that against pseudorabies virus (PrV)-infected PAMs. UV-inactivated PRRSV also suppressed NK activity, but much less than infectious PRRSV. Furthermore, co-incubation with PRRSV-infected PAMs inhibited degranulation of NK cells. Finally, using the supernatant of PRRSV-infected PAMs collected at 12 hpi showed that the suppressive effect of PRRSV on NK cytotoxicity was not mediated by soluble factors. In conclusion, PRRSV-infected PAMs showed a reduced susceptibility toward NK cytotoxicity, which may represent one of the multiple evasion strategies of PRRSV.

Identification of B-cell epitopes in the NSP1 protein of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) was divided into North American and European genotypes. NSP1 was an important non-structural protein of PRRSV, which was auto-cleaved from the replicase polyprotein into NSP1α and NSP1β subunits and played an important role in the immune suppression. In this study, six monoclonal antibodies (MAbs) against the recombinant PRRSV NSP1, expressed in Escherichia coli system, were screened out and identified. Western blot and IFA results indicated that 4 out of 6 MAbs recognized the recombinant NSP1α and 2 MAbs recognized NSP1β. Epitope mapping results indicated that MAb 4H2 recognized the linear epitopes E(54)EPLRW(59) in NSP1α, MAbs (2G5, 3E11 and 4D4) recognized the epitopes H(157)VLTNLP(163) in NSP1α, and MAbs 3C7 and 1H7 reacted with the epitopes 185aa to 232aa in NSP1β. Protein sequence alignment of NSP1 indicated E(54)EPLRW(59) was conserved in all North American PRRSV strains, whereas European type strains has variable amino acids in this region. The epitope H(157)VLTNLP(163) was relatively conserved among all PRRSV strains, except for a L162→S162 change in European type strains. The epitope 185-232aa was variable among North American PRRSV strains. These results may facilitate future investigations into the function of NSP1 of PRRSV and diagnostic methods for PRRSV infection.