Inhibition of porcine reproductive and respiratory syndrome virus replication by short hairpin RNA in MARC-145 cells

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important contagious agents of swine in the world. The current vaccines cannot provide highly effective protection. In this study, the ability of specific short hairpin RNA directed against different genomic regions of PRRSV to inhibit virus replication in MARC-145 cells was examined. Seven plasmids expressing shRNA targeted to GP5 and nucleocapsid (N) protein coding region of PRRSV S1 strain RNA were constructed and delivered into MARC-145 cells. After infection, these cells, transfected with plasmids pSUPER-N3 or pSUPER-G1, showed a significant decrease in virus yield when compared to control cells, by detection using virus titers (TCID50), indirect immunofluorescence assay and real-time RT-PCR. The antiviral effect was sequence-specific and dose-dependent and could sustain for 96 h. Furthermore, by combination of treatment with plasmid pSUPER-N3 and pSUPER-G1, the viral inhibition cloud be significantly increased. In addition, the viral suppression efficiency by shRNA in previously infected cells was not significant different from that induced by shRNA before viral infection. It indicated that administration of the two different shRNA could have a synergistic effect. RNA interference targeting to the various regions of PRRSV might be a potential alternative virus control strategy.     

猪繁殖与呼吸综合征病毒M蛋白在重组牛痘病毒中的表达与鉴定

为重组表达猪繁殖与呼吸综合征病毒(PRRSV)M蛋白,本研究将RT-PCR获得的PRRSV CH-1a株M蛋白基因,克隆于pMD18-T载体中,经测序鉴定正确后,亚克隆至牛痘病毒重组转移质粒pSC11中,构建了转移重组质粒pSC11-PRRSV-M。将pSC11-PRRSV-M在脂质体的介导下转染WR株牛痘病毒感染的TK-143细胞,在含有X-gal的琼脂培养基上通过蓝斑筛选含有PRRSV M基因的重组病毒rWR-PRRSV-M。Western blot与IFA检测表明,重组病毒成功表达了PRRSV M蛋白,而且所表达的蛋白保持了良好的免疫原性。动物实验表明,rWR-PRRSV-M所表达的PRRSV M蛋白在免疫小鼠体内诱生了抗PRRSV的抗体。rWR-PRRSV-M的构建,为进一步探讨PRRSV M蛋白免疫原性提供了基础数据,也为PRRS重组活载体疫苗的研究奠定了基础。     

Acute phase response in porcine reproductive and respiratory syndrome virus infection

This study was focused on the changes observed in the serum concentration of haptoglobin (Hp), C-reactive protein (CRP), serum amyloid A (SAA) and Pig-major acute protein (Pig-MAP), during experimental porcine reproductive and respiratory syndrome virus (PRRSV) infection and in their relationship with the expression of interleukin 1β (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α). Hp and Pig-MAP serum levels were increased at 10 dpi, but CRP and SAA showed a delayed and highly variable increase. All three proinflammatory cytokines were poorly expressed, and only a mild increase in IL-1β was observed at 7 dpi. The increased expression of Hp coincided with the light enhancement observed in both IL-6 and TNF-α, and might be related with an increased expression of IL-10. The low expression of TNF-α might point to a possible mechanism of viral evasion of host-immune response. This issue and the delayed expression of CRP and SAA should be taken into account in future studies about modulation of the immune response by PRRSV infection.     

表达猪繁殖与呼吸综合征病毒CH-1a株核衣壳蛋白重组鸡痘病毒的构建

从含有猪繁殖与呼吸综合征病毒（PRRSV）核衣壳蛋白（N）的质粒扩增出N基因，构建禽痘病毒转移载体。该载体含有禽痘病毒早晚期启动子LP2EP2控制之下的PRRSVN基因、P11启动下的报告基因lacZ以及用于同源重组的禽痘病毒基因组的片段。在转移载体转染亲本病毒S—FPV-017感染的鸡胚成纤维细胞（CEF）之后，采用蓝色表型筛选的方法，筛选到表达N基因的重组病毒，并对其进行了6轮蚀斑纯化。PCR方法鉴定证明重组病毒的基因组中含有完整PRRSVN基因，间接免疫荧光试验证明了PRRSVN蛋白在重组病毒感染的CEF细胞中获得表达，本研究为猪繁殖与呼吸综合征非复制型疫苗的研制打下了基础。     

Infection with Porcine reproductive and respiratory syndrome virus stimulates an early gamma interferon response in the serum of pigs

The early release of cytokines by cells involved in innate immunity is an important host response to intracellular pathogens. Gamma interferon (IFN-gamma) is an important cytokine produced during the early stages of an infection by macrophages, natural killer (NK) cells, and other cell types, and it is also a central cytokine mediator for the induction of cellular or Th1 immunity. To better understand innate and adaptive immune responses after infection with Porcine reproductive and respiratory syndrome virus (PRRSV), we investigated serum IFN-gamma concentrations and the duration of viremia. For 2 strains of atypical PRRSV, IFN-gamma was detectable in swine serum soon after infection and lasted for approximately 3 wk. Serum concentrations of IFN-gamma peaked at about 10 d after inoculation and returned to approximately baseline levels by day 22. However, individual pigs manifested short, sporadic increases in the serum concentration of IFN-gamma from 18 to 50 d after inoculation. Prior vaccination blocked the serum IFN-gamma response associated with homologous virus challenge and altered the kinetics of the response after heterologous challenge. Two other respiratory viruses of pigs, Porcine respiratory coronavirus and Swine influenza virus, do not appear to induce serum IFN-gamma. The early production of IFN-gamma in PRRSV-infected pigs might result from activation of NK cells, a response that is more characteristic of immune pathways stimulated by intracellular bacterial and protozoan infections.     

Development and characterization of stable cell lines constitutively expressing the porcine reproductive and respiratory syndrome virus nucleocapsid protein

Despite global efforts to control porcine reproductive and respiratory syndrome virus (PRRSV) infection, the virus continues to cause economic problems in the swine industry worldwide. In this study, we attempted to generate and characterize a panel of stable BHK cell lines that constitutively express the nucleocapsid (N) protein of type 1 or type 2 PRRSV. The established BHK cell lines were found to react well with N-specific antibodies as well as the hyperimmune serum of pigs raised against each genotype of PRRSV. Taken together, the data implicate a potential usefulness for the newly generated stable cell lines as a diagnostic reagent for PRRSV serology.     

Cytokines and synthetic double-stranded RNA augment the T helper 1 immune response of swine to porcine reproductive and respiratory syndrome virus

Immunization of pigs with a modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine initially elicits a weak interferon (IFN)-gamma response. To improve the immune response, an adjuvant consisting of plasmid encoding either porcine interleukin (IL)-12 or IFN-alpha was co-administered during vaccination. In the presence of either adjuvant, at least a three-fold increase in the primary virus-specific IFN-gamma response was observed. While this enhancement was only transient (1 week) when the IL-12 expressing plasmid was used, the effect was not only still apparent at 6 weeks after vaccination in the presence of the IFN-alpha expressing plasmid but even after challenge with a virulent genetically divergent PRRSV. In contrast, no effect of either adjuvant on the production of anti-virus antibodies was noticed throughout the study. Despite the apparent augmentation of a T helper (Th) 1 type response by the inclusion of IFN-alpha or IL-12 during vaccination, this modulation did not necessarily correlate with a reduction in viremia. Since a similar increase in the degree of the IFN-gamma response to the PRRSV vaccine could be achieved by substituting polyinosinic-polycytidylic acid in lieu of either cytokine, exposure to PRRSV in the presence of a variety of Th 1 polarizing molecules can positively influence the development of the cell-mediated immune response of swine to this pathogen. Conceivably, such intervention could be applied to improve the formulation of anti-PRRSV vaccines.     

Protective immunity induced by a recombinant pseudorabies virus expressing the GP5 of porcine reproductive and respiratory syndrome virus in piglets

Pseudorabies virus (PRV) has been developed as a vaccine vector for expressing foreign immunogens. Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), continues to be a major problem to the pork industry worldwide. Many vaccine strategies have been developed to control the disease but most of them turn out to be unsuccessful. The objective of this research was to explore the feasibility of PRV-based vector vaccine in protection against PRRSV. A live attenuated vaccine-based PRV recombinant expressing the envelope protein GP5 of PRRSV was generated using recombinant DNA techniques. The Bartha-K61-derived recombinant virus, named rPRV-GP5, was shown to express PRRSV GP5 efficiently. Sixteen healthy piglets were assigned to one of four groups (one to four, four pigs per group). Animals in Groups 1 and 2 were each inoculated intramuscularly and intranasally with 10(7.0) PFU of rPRV-GP5 and its parent Bartha-K61, respectively; Group 3 were vaccinated intramuscularly with one-dose of PRRS inactivated vaccine; Group 4 was served as non-vaccinated control. One month later, all animals were all challenged with 10(6.5) TCID(50) of virulent PRRSV CH-1a. All animals in Groups 1 and 3 remained clinically healthy before and after challenge, with only a short period of fever (no more than 41 degrees C and 3 days), mild and gradually improving lung and kidney lesions, and short-term viremia (2 and 3 week, respectively) in spite of no detectable anti-PRRSV antibody before challenge. On the other hand, all animals in the other two groups showed evident clinical signs with higher temperatures (more than 41 degrees C) after challenge, and severe lung, kidney and spleen lesions and extended viremia (4 weeks). The results indicate that the rPRV-GP5 is safe for vaccinates and able to confer significant protection against clinical disease and reduce pathogenic lesions induced by PRRSV challenge in vaccinated pigs.     

Antibody production and blastogenic response in pigs experimentally infected with porcine reproductive and respiratory syndrome virus.

Seven five-week piglets were infected intranasally with 10(5) TCID50 of porcine reproductive and respiratory syndrome (PRRS) virus strain IAF.exp91. All virus-exposed pigs developed fever, labored abdominal breathing, conjunctivitis, and lymph node enlargement within the first 96 h postexposure (PE), which continued to d 10 to 14 PE. Two pigs that were necropsied at d 7 and 10 PE had diffuse interstitial pneumonitis, cardiopathy and lymphadenopathy. All 5 remaining pigs produced serum IgM and IgG antibodies against PRRS virus by 7 or 14 days PE, as demonstrated by indirect immunofluorescence. This corresponded with the capability of isolating the virus from serum d 7 to d 49 or d 63 PE. Low serum neutralizing antibody titers were detected in 3 of the virus-exposed pigs by 35 days PE. A transient episode of diminished proliferative response of peripheral blood lymphocytes to mitogens phytohemagglutinin (PHA) and concanavalin A (Con A) was observed in the virus-exposed pigs at d 3 PE. However, in vitro spontaneous uptake of [3H]-thymidine was significantly increased in lymphocyte cultures of the same pigs at d 7 or d 14 PE. These results suggest polyclonal activation of peripheral blood lymphocytes.     

Evidence of long distance airborne transport of porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae

The ability of porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae to be transported over long distances via the airborne route was evaluated. A source population of 300 grow-finish pigs was experimentally inoculated with PRRSV MN-184 and M. hyopneumoniae 232 and over a 50-day period, air samples were collected at designated distances from the source herd using a liquid cyclonic collector. Samples were tested for the presence of PRRSV RNA and M. hyopneumoniae DNA by PCR and if positive, further characterized. Of the 306 samples collected, 4 (1.3%) were positive for PRRSV RNA and 6 (1.9%) were positive for M. hyopneumoniae DNA. The PRRSV-positive samples were recovered 4.7 km to the northwest (NW) of the source population. Four of the M. hyopneumoniae-positive samples were obtained at the NW sampling point; 2 samples at approximately 2.3 km and the other 2 samples approximately 4.7 km from the source population. Of the remaining 2 samples, one sample was obtained at the southeast sampling point and the other at the southwest sampling point, with both locations being approximately 4.7 km from the source. The four PRRSV-positive samples contained infectious virus and were ≥ 98.8% homologous to the MN-184 isolate used to inoculate the source population. All 6 of the M. hyopneumoniae-positive samples were 99.9% homologous to M. hyopneumoniae 232. These results support the hypothesis that long distance airborne transport of these important swine pathogens can occur.     

PRRSV GP5蛋白及猪IL-18基因共表达核酸疫苗免疫原性试验

本试验通过分子克隆技术分别构建了单独表达猪繁殖与呼吸综合征病毒（PRRSV）GP5基因以及PRRSVGP5基因和猪IL-18基因共表达的重组核酸疫苗质粒（pEGFP—GP5和pEGFP—ILl8-GP5）,并进行仔猪免疫原性研究,对构建的PRRSV核酸疫苗所诱导的体液免疫和细胞免疫水平进行检测,进一步研究了PRRSV核酸疫苗免疫效果以及猪IL-18基因对PRRSV核酸疫苗免疫调节作用的影响。同时,调查了商业上应用不同类型的PRRSV疫苗诱导的免疫效果,并与核酸疫苗免疫效果进行比较。结果表明,IL-18作为免疫佐剂在疫苗免疫猪后诱导的病毒特异性细胞免疫反应方面具有很好的调节作用,共表达IL18-GP5蛋白能够明显的改善DNA疫苗的免疫效力,增强抗PRRSV的免疫保护。因此,DNA疫苗做为一种新一代疫苗可用于对抗高致病性PRRSV感染。     

Evidence for a major QTL associated with host response to porcine reproductive and respiratory syndrome virus challenge

Porcine reproductive and respiratory syndrome (PRRS) causes decreased reproductive performance in breeding animals and increased respiratory problems in growing animals, which result in significant economic losses in the swine industry. Vaccination has generally not been effective in the prevention of PRRS, partially because of the rapid mutation rate and evolution of the virus. The objective of the current study was to discover the genetic basis of host resistance or susceptibility to the PRRS virus through a genome-wide association study using data from the PRRS Host Genetics Consortium PRRS-CAP project. Three groups of approximately 190 commercial crossbred pigs from 1 breeding company were infected with PRRS virus between 18 and 28 d of age. Blood samples and BW were collected up to 42 d post infection (DPI). Pigs were genotyped with the Illumina Porcine 60k Beadchip. Whole-genome analysis focused on viremia at each day blood was collected and BW gains from 0 to 21 DPI (WG21) or 42 DPI (WG42). Viral load (VL) was quantified as area under the curve from 0 to 21 DPI. Heritabilities for WG42 and VL were moderate at 0.30 and litter accounted for an additional 14% of phenotypic variation. Genomic regions associated with VL were found on chromosomes 4 and X and on 1, 4, 7, and 17 for WG42. The 1-Mb region identified on chromosome 4 influenced both WG and VL, exhibited strong linkage disequilibrium, and explained 15.7% of the genetic variance for VL and 11.2% for WG42. Despite a genetic correlation of -0.46 between VL and WG42, genomic EBV for this region were favorably and nearly perfectly correlated. The favorable allele for the most significant SNP in this region had a frequency of 0.16 and estimated allele substitution effects were significant (P < 0.01) for each group when the SNP was fitted as a fixed covariate in a model that included random polygenic effects with overall estimates of -4.1 units for VL (phenotypic SD = 6.9) and 2.0 kg (phenotypic SD = 3 kg) for WG42. Candidate genes in this region on SSC4 include the interferon induced guanylate-binding protein gene family. In conclusion, host response to experimental PRRS virus challenge has a strong genetic component, and a QTL on chromosome 4 explains a substantial proportion of the genetic variance in the studied population. These results could have a major impact in the swine industry by enabling marker-assisted selection to reduce the impact of PRRS but need to be validated in additional populations.     

Effects of a porcine reproductive and respiratory syndrome virus infection on the development of the immune response against pseudorabies virus

The aim of this study was to investigate the effects of a porcine reproductive and respiratory syndrome virus (PRRSV) infection on the development of the immune response after pseudorabies virus (PRV) vaccination in pigs. Pigs were intranasally inoculated with the European PRRSV strain, Lelystad virus ter Huurne, and were vaccinated intramuscularly with PRV 2 weeks later (LV-PRV group). Control pigs were vaccinated with PRV only (PRV group). Eight weeks after PRV vaccination, pigs from both groups were challenged intranasally with wild-type PRV. We measured the lymphoproliferative, and the cytolytic responses to PRV of peripheral blood mononuclear cells (PBMC), isolated from blood samples. In addition, serum samples were examined for antibodies against PRV and LV. One week after PRV vaccination, PBMC proliferated abundantly to PRV in both groups. However, in the LV-PRV group the lymphoproliferative response declined after 1 week, whereas, in the PRV group, the lymphoproliferative response was high for 3 weeks and declined thereafter (P<0.05). After challenge, the lymphoproliferative response was 1 week earlier and was consistently and significantly higher in the PRV group than in the LV-PRV group. The PRV-specific killing was higher at 3 weeks after PRV vaccination and 5 weeks after PRV challenge 19+/-3 and 24+/-6%, respectively, in the PRV group, compared to 7+/-4 and 6+/-9%, respectively, in the LV-PRV group (P<0.05). However, later after vaccination and challenge the cytolytic response was identical in both groups. The antibody titre against PRV developed equally in both groups. After challenge, no PRV virus was isolated from both groups. From these results we conclude that, although PRRSV infection did cause changes in the time course of the T-lymphocyte response after PRV vaccination, PRRSV infection did not inhibit the development of vaccine-induced protection after PRV.     

PRRSV变异株ORF6基因与IL-18共表达真核质粒的构建及其表达

针对白细胞介素18(IL-18)基因和本实验室分离的PRRSVJL/07/SW毒株全基因序列,分别设计了2对特异性引物,扩增出IL-18基因和ORF6基因。将2个目的基因克隆于真核表达载体pEGFP-N1中,成功构建了重组真核质粒pEGFP-ORF6和pEGFP-IL18-ORF6,阳性质粒转染Marc-145细胞进行筛选。通过表达载体在转染细胞中的高效转染,增强型绿色荧光蛋白(EGFP)快速、准确的反映出阳性质粒在细胞质中的正确表达,再通过SDS-PAGE和Western blot鉴定构建的真核表达质粒pEGFP-IL18-ORF6的正确性。结果表明,ORF6基因和IL-18基因在Macr-145细胞中能有效表达。结论,所构建的真核质粒pEGFP-IL18-ORF6结构正确,能够在Marc-145细胞中高效表达,而且表达产物具有特异免疫学反应。     

表达猪繁殖与呼吸综合征病毒ORF5/ORF6基因重组鸡痘病毒的构建与小鼠免疫试验

本试验构建了1株表达猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)RF5/ORF6基因的重组鸡痘病毒,并进行了小鼠免疫实验,对其诱导BALB/c小鼠产生体液免疫和细胞免疫反应的能力进行了评价。结果表明,重组鸡痘病毒能够刺激免疫鼠产生特异性PRRSV ELISA抗体,促进特异性T淋巴细胞增殖。对免疫小鼠血清中细胞因子检测结果表明,IFN-γ的分泌显著提高。结果表明,所构建重组鸡痘病毒可以对小鼠起到良好的免疫效果,具有成为抗PRRSV感染新型疫苗的潜力。     

Systemic and mucosal immunity induced by attenuated Salmonella enterica serovar Typhimurium expressing ORF7 of porcine reproductive and respiratory syndrome virus

Oral administration of attenuated Salmonella vaccine may provide valuable advantages such as low cost, easy preparation, and safety. Attenuated Salmonella vaccines also serve as carriers of foreign antigens and immunomodulatory cytokines. Presently, an attenuated Salmonella enterica serovar Typhimurium strain was used as a carrier for open reading frame 7 (ORF7) protein of porcine reproductive and respiratory syndrome virus (PRRSV), a swine pathogen of significant global economic importance. Initially, an attenuated S. enterica serovar Typhimurium expressing ORF7 gene derived from PRRSV Korean isolate was constructed. Following oral administration of a single dose of the attenuated Salmonella vaccine expressing PRRSV ORF7, humoral and cell-mediated immune responses specific for ORF7 were induced at both systemic and mucosal sites including spleen, mesenteric lymph node, Peyer's patch, and laminar propria, as evaluated by determining serum ORF7-specific IgG and mucosal IgA responses, as well as Th1- and Th2-type cytokine production from antigen-stimulated T cells. The induced humoral responses were sustained for at least 12 weeks post-immunization. In particular, the immunized mice displayed immune responses to both the foreign ORF7 antigen and Salmonella itself. The results indicate the value of attenuated S. enterica serovar Typhimurium as an oral carrier of PRRSV antigenic proteins to induce effective systemic and mucosal immunity.     

Immune responses in pigs induced by recombinant canine adenovirus 2 expressing the glycoprotein 5 of porcine reproductive and respiratory syndrome virus

To develop a new type vaccine for porcine reproductive and respiratory syndrome (PRRS) prevention by using canine adenovirus 2(CAV-2) as vector, the Glycoprotein 5(GP5) gene from PRRSV strain JL was amplified by RT-PCR, and the expression cassette of GP5 was constructed using the human cytomegalovirus (HCMV) promoter and the simian virus 40 (SV40) early mRNA polyadenylation signal. The expression cassette of Glycoprotein 5 was cloned into the CAV-2 genome in which E3 region had been partly deleted, and the recombinant virus (CAV-2-GP5) was obtained by transfecting the recombinant CAV-2-GP5 genome into MDCK cells together with Lipofectamine™ 2000. Immunization trial in pigs with the recombinant virus CAV-2-GP5 showed that CAV-2-GP5 could stimulate a specific immune response to PRRSV. Immune response to the GP5 and PRRSV was confirmed by ELISA, neutralization test and lymphocyte proliferative responses, and western blotting confirmed expression of GP5 by the vector in cells. These results indicated that CAV-2 may serve as a vector for development of PRRSV vaccine in pigs, and the CAV-2-GP5 might be a candidate vaccine to be tested for preventing PRRSV infection.     

Interleukin-12 (IL-12) ameliorates the effects of porcine respiratory and reproductive syndrome virus (PRRSV) infection

Porcine respiratory and reproductive syndrome virus (PRRSV) disease, one of the most economically significant viral diseases in the swine industry, is characterized by miscarriages, premature farrowing, stillborn pigs, and respiratory disease associated with death and chronic poor performance of nursing and weaned pigs. Interleukin-12 (IL-12) is a key component in driving the development of cell-mediated immunity as well as stimulating interferon-gamma (IFN-gamma) production from T cells and natural killer cells. Although some studies have investigated the use of IL-12 as a vaccine adjuvant in swine, little is known about its effectiveness as a treatment against viral diseases in swine. The present study investigated whether recombinant porcine IL-12 (rpIL-12) enhances the immune response and thereby diminishes the effects of PRRSV infection in young pigs. Interestingly, in vitro experiments demonstrated that rpIL-12 is capable of inducing swine pulmonary alveolar macrophages (PAMs), the target cells of PRRSV, to produce IFN-gamma in a dose and time dependent manner. In addition, in vitro studies also revealed that rpIL-12 treatment was capable of significantly reducing PRRSV viral titers in PAMs. In vivo administration of rpIL-12 significantly decreased PRRSV titers in the lungs and blood of infected animals. Furthermore, treatment with rpIL-12 prevented significant growth retardation in PRRSV-infected animals. Finally, in response to viral antigen recall challenge, PAMs isolated from rpIL-12-treated/PRRSV-infected animals produced greater amounts of IFN-gamma and lesser amounts of interleukin-10 than PAMs isolated from non-rpIL-12-treated/PRRSV-infected animals. Taken together our data indicate that treatment with rpIL-12 may provide an effective approach to control or ameliorate PRRSV-induced disease in swine.     

猪繁殖与呼吸综合征病毒GP5基因及猪IL-18基因共表达真核表达系统的建立

采用RT-PCR方法扩增了JL/07/SW毒株GP5蛋白和猪IL-18基因。将该基因克隆入真核表达载体pEG-FP-N1,获得重组质粒pEGFP-GP5和pEGFP-IL18-GP5。将重组质粒转染Marc-145细胞,通过Western blotting和green fluorescent检测产物的表达情况。结果显示,所构建的2个重组质粒在Marc-145细胞中能进行有效的转录。结果表明,所构建的重组质粒pEGFP-GP5和pEGFP-IL18-GP5在Marc-145细胞中得到表达,为进一步研究PRRSV基因工程疫苗奠定了基础。