Juan Bai

Two-Dimensional Liquid Chromatography–Tandem Mass Spectrometry Coupled with Isobaric Tags for Relative and Absolute Quantification (iTRAQ) Labeling Approach Revealed First Proteome Profiles of Pulmonary Alveolar Macrophages Infected with Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) has devastated the pig industry worldwide for almost 25 years, and its virus (PRRSV) preferentially infects and replicates in pulmonary alveolar macrophages (PAMs). To discover cellular protein responses in PRRSV-infected PAMs, two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the differentially expressed proteins between the PRRSV-infected groups and the controls. A total of 160 cellular proteins in PAMs that were significantly altered post-infection were identified. These differentially expressed proteins are related to the biological processes of virus binding, cell structure, signal transduction, cell adhesion, etc., and their interactions. This is the first report that analyzed the cellular protein profile of PRRSV-infected PAMs using iTRAQ technology, and this data provides important information to help understand the host response to PRRSV and to define the cellular requirements for the underlying mechanism of PRRSV replication and pathogenesis.

A novel inactivated gE/gI deleted pseudorabies virus (PRV) vaccine completely protects pigs from an emerged variant PRV challenge.

A highly virulent and antigenic variant of pseudorabies virus (PRV) broke out in China at the end of 2011 and caused great economic loss in the pig industry. In this study, an infectious bacterial artificial chromosome (BAC) clone containing the full-length genome of the emerged variant PRV ZJ01 strain was generated. The BAC-derived viruses, vZJ01-GFPΔgE/gI (gE/gI deleted strain, and exhibiting green autofluorescence), vZJ01ΔgE/gI (gE/gI deleted strain), and vZJ01gE/gI-R (gE/gI revertant strain), showed similar in vitro growth to their parent strain. In pigs, inactivated vZJ01ΔgE/gI vaccine generated significantly high levels of neutralizing antibodies against ZJ01 compared with Bartha-K61 live vaccine (p<0.05). After fatal ZJ01 challenge, all five animals in the inactivated vZJ01ΔgE/gI vaccine group survived without exhibiting any clinical sings, but two of five animals exhibited central nervous signs in the Bartha-K61 group. Meanwhile, all the non-vaccinated control animals died at 7 days post-challenge. This indicates that the inactivated vZJ01ΔgE/gI vaccine is a promising vaccine candidate for controlling the variant strains of PRV now circulating in China.

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.

Two-dimensional liquid chromatography–tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling approach revealed first proteome profiles of pulmonary alveolar macrophages infected with porcine circovirus type 2

Porcine circovirus type 2 (PCV2) has been identified as the essential causal agent of postweaning multisystemic wasting syndrome, which has spread worldwide. Monocyte/macrophage lineage cells are the major target cells of PCV2. To discover cellular protein responses of pulmonary alveolar macrophages (PAMs) to PCV2 infection, two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the proteins that were differentially expressed in PAMs from the PCV2-infected group compared to the uninfected control group. A total of 145 cellular proteins in PAMs that were significantly altered at different time periods post-infection were identified. These differentially expressed proteins were related to the biological processes of binding, cell structure, signal transduction, cell adhesion, etc., and their interactions. The high number of differentially expressed proteins identified should be very useful to elucidate the mechanism of replication and pathogenesis of PCV2 in the future.

Pathogenicity and antigenicity of a novel NADC30-like strain of porcine reproductive and respiratory syndrome virus emerged in China

Porcine reproductive and respiratory syndrome virus (PRRSV) has spread globally and caused huge economic loss. In recent years, a new kind of highly pathogenic NADC30-like strain has emerged in China. However, the pathogenicity and antigenicity of the virus are not well understood. In this study, PRRSV strain FJ1402 was isolated from piglets with clinical signs in Fujian Province in China in 2014. The complete genomic sequence analysis showed that it arose from recombination of North America NADC30 strain and highly pathogenic PRRSV (HP-PRRSV) in China. Experiment in piglets showed that FJ1402 had similar virulence to HP-PRRSV strain BB0907. The commercial PRRSV modified live vaccines TJM-F92 and R98 could partly provide protective efficacy against FJ1402 challenge in piglets. This should be helpful for preventing and controlling this disease in the future.

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.

Poly(I:C) inhibits porcine reproductive and respiratory syndrome virus replication in MARC-145 cells via activation of IFIT3.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major cause of heavy economic losses in many swine-producing regions. Current vaccination strategies and antiviral drugs provide only limited protection. Interferon (IFN)-induced protein with tetratricopeptide repeats 3 (IFIT3) has been characterized as the product of a novel antiviral gene and as an important modulator in innate immunity. However, the role of IFIT3 in PRRSV infection is scarcely understood. In this study, polyinosinic-polycytidylic acid (poly(I:C)) inhibited PRRSV replication in MARC-145 cells, following the appearance of increased IFIT3. Overexpression of porcine IFIT3 resulted in a decrease of PRRSV. Knockdown of IFIT3 in MARC-145 cells increased PRRSV replication and impaired the antiviral activity mediated by poly(I:C). Moreover, in the presence or absence of IFIT3, poly(I:C)-induced IFN-β promoter activity was significantly boosted or crippled, respectively. IFIT3, TBK1 and phosphorylation of IRF3 were activated in poly(I:C)-transfected MARC-145 cells. It demonstrated that IFIT3 plays an important role in IFN-β induction in MARC-145 cells, and, when activated, it can inhibit PRRSV replication.

The amino acid residues at 102 and 104 in GP5 of porcine reproductive and respiratory syndrome virus regulate viral neutralization susceptibility to the porcine serum neutralizing antibody

Porcine reproductive and respiratory syndrome virus (PRRSV) is mainly responsible for the heavy economic losses in pig industry in the world. A number of neutralizing epitopes have been identified in the viral structural proteins GP3, GP4, GP5 and M. In this study, the important amino acid (aa) residues of HP-PRRSV strain BB affecting neutralization susceptibility of antibody were examined using resistant strains generated under neutralizing antibody (NAb) pressure in MARC-145 cells, reverse genetic technique and virus neutralization assay. HP-PRRSV strain BB was passaged under the pressure of porcine NAb serum in vitro. A resistant strain BB34s with 102 and 104 aa substitutions in GP5, which have been predicted to be the positive sites for pressure selection (Delisle et al., 2012), was cloned and identified. To determine the effect of the two aa residues on neutralization, eight recombinant PRRSV strains were generated, and neutralization assay results confirmed that the aa residues 102 and 104 in GP5 played an important role in NAbs against HP-PRRSV in MARC-145 cells and porcine alveolar macrophages. Alignment of GP5 sequences revealed that the variant aa residues at 102 and 104 were frequent among type 2 PRRSV strains. It may be helpful for understanding the mechanism regulating the neutralization susceptibility of PRRSV to the NAbs and monitoring the antigen variant strains in the field.

Development of a multiplex TaqMan probe-based real-time PCR for discrimination of variant and classical porcine epidemic diarrhea virus

Since October 2010, porcine diarrhea outbreaks have occurred widely, resulting in major losses in suckling piglets in China. A variant porcine epidemic diarrhea virus (PEDV), characterized by base deletion and insertion in the S gene, compared to classical PEDV CV777, was shown to be responsible for this outbreak. In this study, a multiplex TaqMan probe-based real-time PCR was developed for detecting PEDV and differentiating the variant from classical PEDV, by using two sets of primers and probes based on the S gene of PEDV. The limits of detection of both variant and classical PEDV were 5×10(2) DNA copies. Specificity was determined using eight other viral pathogens of swine. Reproducibility was evaluated using standard dilutions, with coefficients of variation <1.4%. Standard dilutions included in each test allowed quantification of the amount of PEDV. Among 42 intestinal samples from pigs with severe watery diarrhea, 36 variant PEDV and three classical PEDV samples were detected, with viral loads of 10(2)-10(8) copies/μl and 10(3)-10(5) copies/μl, respectively, which suggested that the variant PEDV was prevalent in China. The multiplex TaqMan probe-based real-time PCR should be a useful tool for quantifying viral load, detecting PEDV, and differentiating variant from classical PEDV.

The N-N non-covalent domain of the nucleocapsid protein of type 2 porcine reproductive and respiratory syndrome virus enhances induction of IL-10 expression.

Porcine reproductive and respiratory syndrome virus (PRRSV) usually establishes a prolonged infection and causes an immunosuppressive state. It has been proposed that IL-10 plays an important role in PRRSV-induced immunosuppression. However, this mechanism has not been completely elucidated. In this study, we found that transfection of 3D4/2 macrophages with the N protein gene of type 2 PRRSV significantly upregulated IL-10 expression at the transcriptional level. Moreover, alanine substitution mutation analysis revealed that the N protein residues 33-37, 65-68 and 112-123 were related to the upregulation of IL-10 promoter activity. Recombinant PRRSV with mutations at residues 33-37 in the N protein (rQ33-5A and rS36A) recovered from corresponding infectious cDNA clones and induced significantly lower levels of IL-10 production in infected monocyte-derived dendritic cells, as compared with their revertants rQ33-5A(R) and rS36A(R), and the wild-type recombinant PRRSV strain rNT/wt. These data indicate that the type 2 PRRSV N protein plays an important role in IL-10 induction and the N-N non-covalent domain is associated with this activity.