Xianwei Wang

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.

Enhanced immune responses of mice inoculated recombinant adenoviruses expressing GP5 by fusion with GP3 and/or GP4 of PRRS virus

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important causes of economic losses of the swine industry. PRRS virus (PRRSV) infection poses a challenge to current vaccination strategies. In this study, three replication-defective adenovirus recombinants expressing fusion protein GP3-GP5, GP4-GP5, or GP3-GP4-GP5 were developed as potential vaccine against PRRSV in a mouse model. Six groups of BALB/c mice (24mice per group) were inoculated subcutaneously twice at 2-week intervals with above mentioned recombinants and other adenoviruses expressing single GP3, GP4, or GP5 protein. The results showed that the mice inoculated with recombinant adenoviruses developed PRRSV-specific antibodies, cellular immune response by 2 weeks post-boost-immunization. However, mice immunized with recombinant adenoviruses rAd-GP3-GP5, rAd-GP4-GP5, and rAd-GP3-GP4-GP5 developed significantly higher titers of neutralizing antibodies to PRRSV and produced stronger lymphocyte proliferation responses compared to mice immunized with rAd-GP3, rAd-GP4 or rAd-GP5 alone. It was also found that mice immunized with rAd-GP3-GP5 and rAd-GP3-GP4-GP5 were primed for significant higher levels of anti-PRRSV CTL responses than mice immunized with rAd-GP3 and rAd-GP5. These findings suggested that the recombinant adenoviruses expressing fusion proteins GP3-GP5 or GP3-GP4-GP5 might be an attractive candidate vaccine for preventing PRRSV infection.

Genetic analysis of porcine circovirus type 2 (PCV2) strains isolated between 2001 and 2009: genotype PCV2b predominate in postweaning multisystemic wasting syndrome occurrences in eastern China

Porcine circovirus type 2 (PCV2) is the essential infectious agent of postweaning multisystemic wasting syndrome (PMWS). In the present study, we obtained sequences of 31 PCV2 isolates from different farms of 11 provinces of eastern China and analyzed the genetic characterization of 136 eastern China-derivate PCV2 isolated during 2001–2009. The results showed that these PCV2 isolates could be divided into two groups, PCV2b (108 of 1A/1B, 19 of 1C) and PCV2a (1 of 2A, 2 of 2D, 6 of 2E). Among the 9 PCV2a isolates, eight were found before the year 2005. Meanwhile, three major heterogenic regions were observed in amino acid positions 53–91, 121–151, and 190–210; a few specific substitution patterns were found in each subgroup and several variant or conserved epitopes were also observed in the Cap protein. It indicated that PCV2b predominated in PMWS occurrences in eastern China and a few recombinants of different genotypes existed in nature.

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.

Genetic variation analysis of porcine reproductive and respiratory syndrome virus isolated in China from 2002 to 2007 based on ORF5

The complete open reading frame 5 (ORF5) sequences of 34 field porcine reproductive and respiratory syndrome virus (PRRSV) isolates from China in 2002-2007 were detected and compared with the different variable Chinese isolates S1, CH-1a, HB-1, HB-2 and JXA1. The results showed that all isolates were of type 2 PRRSV and could be assigned to two clusters. The isolates in cluster sg1 was high similar with the highly pathogenic PRRSV strain JXA1, while sg2 clustered with type 2 PRRSV isolate VR2332. It was interesting that the isolate SH02 which was isolated from Shanghai in 2002 has 98.8% identity with JXA1 emerged in 2006. And the ZJJ07 isolate was found to be a natural recombinant between a Chinese highly pathogenic SY0608 isolate and a VR-2332 derivative NH04 isolate. Analysis of the potential glycosylation sites indicated that they were frequently mutated and formed five putative N-linked glycosylation (NGS) sites patterns based on N30, 33-35, 44 and 51 in those isolates. It indicated that the highly variable PRRSV strain with different NGS patterns spread widely in China. The great genetic diversity could be taken into consideration for the control and prevention of this disease.

Analysis of immunogenicity of minor envelope protein GP3 of porcine reproductive and respiratory syndrome virus in mice

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically significant viral diseases in swine industry. Though the minor envelope protein GP3 is associated with protective immunity, its immunogenicity and protective mechanism are poorly known. In this study, two recombinant adenoviruses, rAd-GP3 expressing complete GP3 and rAd-tGP3 expressing truncated GP3 in which aa2-64 were deleted, were constructed and the immunogenicity were tested in a mouse model. Four groups of BALB/c mice were immunized subcutaneously twice at 2-week internals with the recombinants rAd-GP3 and rAd-tGP3 or with wild type adenovirus (wtAd) and PBS as control. The results showed that the mice immunized with recombinant adenoviruses developed PRRSV-specific neutralizing antibodies and cellular immune response, including T-cell proliferation responses and cytotoxic T responses, by 2 weeks post-primary immunization. Moreover, the levels of immune responses of mice immunized with rAd-tGP3 were significantly higher than that of mice with rAd-GP3. It indicated that the first 64aa fragment of GP3 might affect the conformation of the antigen structures of GP3 protein. GP3 protein should be one of candidate molecules for developing a new safer effective vaccine.

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.

Adenovirus-mediated shRNA interference against porcine circovirus type 2 replication both in vitro and in vivo.

Porcine circovirus type 2 (PCV2) is the primary causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome (PMWS), which is responsible for the heavy economic losses in stockbreeding. There are no specific antiviral drugs for treatment of the virus infection. We have now constructed two recombinant adenoviruses expressing short-hairpin RNAs (shRNAs) directed against either ORF1 (rAdS1) or ORF2 (rAdS2) of PCV2 and measured the inhibition of PCV2 replication. The results showed that delivery of these shRNAs by recombinant adenovirus into PK15 cells could induce a significant inhibition of viral RNA and DNA replication and protein synthesis level in cells subsequently infected with PCV2. The antiviral effect was dose-dependent and could sustain at least for 120h and the inhibition of virus replication could be significantly strengthened by combination of rAdS1 with rAdS2. Mice injected with shRNA before PCV2 infection showed substantial and low level of PCV2 DNA replication in the spleen during the period of 21-28 days post-PCV2 infection. These results indicated that shRNAs generated by adenovirus could sufficiently and continuously inhibit PCV2 infection in vitro as well as in vivo. The adenovirus based shRNA targeting ORF1 and ORF2 of PCV2 might be a new potential alternative strategy for controlling PCV2 infection.

Hsp70 positively regulates porcine circovirus type 2 replication in vitro.

The Hsp70 chaperone plays a central role in multiple processes within cells. Porcine circovirus type 2 (PCV2) is the essential causal agent of post-weaning multisystemic wasting syndrome (PMWS), which has spread worldwide. But the mechanism of PCV2 replication remains poorly understood. In this study, we firstly found the positive effect of heat stress on the replication of PCV2 in the continuous porcine monocytic cell line 3D4/31. Downregulation of Hsp70 by the specific chaperone inhibitor Quercetin or RNA interference and upregulation of Hsp70 by expression from a recombinant adenovirus showed that Hsp70 enhanced PCV2 genome replication and virion production. A specific interaction between Hsp70 and PCV2 Cap was confirmed by colocalization by confocal microscopy and co-immunoprecipitation. Furthermore, the NF-κB pathway was activated and caspase-3 activity was reduced when Hsp70 was overexpressed in PCV2-infected 3D4/31 cells. These data suggested that Hsp70 positively regulated PCV2 replication, which being helpful for understanding the molecular mechanism of PCV2 infection.

Genetic analysis of two porcine reproductive and respiratory syndrome viruses with different virulence isolated in China

The S1 and SY0608 strains of porcine reproductive and respiratory syndrome virus (PRRSV) were individually isolated and had different pathogenicity in pigs in 1997 and 2006. In order to understand their genomic characteristics, the full-length genome of S1 and SY0608 isolates were sequenced and analyzed. The results indicated that their genome composition differed significantly and shared only 88.5% nucleotide identity with each other. The genetic variation and amino acid substitutions were not randomly distributed in the genome, and mainly focused on ORF1a, ORF3 and ORF5. The SY0608 strain, with high pathogenicity, had a 30-amino-acid deletion at amino acid positions 480 and 532-560 in comparison with the S1 strain. The alignment of amino acid sequence of Nsp1-Nsp8, GP2-GP5, M and N of S1 and SY0608 with other PRRSV isolates demonstrated that variation was mainly found in the Nsp2, GP3 and GP5 proteins. In comparison with the S1 strain, the SY0608 strain showed some potential glycosylation site mutations in GP5 at amino acid positions between 26 and 39, which might be associated with viral antigenicity. Phylogenetic analysis showed that the two strains belonged to two different branches that do not indicate differences in pathogenicity. Interestingly, the deletion strains isolated recently in China formed a new minor branch, revealing the same evolutionary trend.