共查询到20条相似文献,搜索用时 140 毫秒
1.
Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically significant viral diseases in
the swine industry. Infection with PRRSV following vaccination is common, since protection is incomplete. Persistent infection
may be one of the biggest obstacles to control of the disease. “Glycan shielding” was postulated to be a primary mechanism
to explain evasion from neutralizing immune response, ensuring in vivo persistence of virus, such as HIV, SIV, and HBV. The
objective of this study was to construct recombinant adenoviruses expressing single or multiple N-linked glycosylation site
(NGS) mutant GP5 of PRRSV, and evaluate the expression in cell culture, and potential to induce immune responses in BALB/c
mice. Six recombinant adenoviruses were constructed each expressing wild-type GP5 and 1-4 NGS mutants: N44S, N44/51S, N30/44/51S,
N30/33/44/51S and N30/33S. Inoculation of BALB/c mice with all five recombinants expressing NGS mutant GP5 resulted in a significant
neutralizing antibody responses which were significantly higher than that of recombinant adenovirus expressing wild-type GP5.
But there were no significant difference in lymphocyte proliferation responses induced by wild type and NGS mutant GP5. It
indicated that glycosylations of GP5 at residues N30, N33, N44 and N51 are critical for induction of neutralizing antibodies.
These NGS mutant PRRSV GP5 will be useful to characterize the effects of glycosylation on immunogenicity in the natural host,
and may lead to a new approach for the generation of PRRSV vaccines. 相似文献
2.
Enhanced immunogenicity of the modified GP5 of porcine reproductive and respiratory syndrome virus 总被引:9,自引:0,他引:9
The ORF5-encoded major envelope glycoprotein (GP5) is one of the key immunogenic proteins of the porcine reproductive and
respiratory syndrome virus (PRRSV) and is the leading target for the development of the new generation of vaccines against
PRRS. However, weak and tardy neutralizing antibodies have been elicited in several developed experimental vaccines expressing
PRRSV GP5. More recent evidence has demonstrated a non-neutralizing decoy epitope upstream of the neutralizing epitope of
GP5, which might prevent the development of a strong neutralizing antibody response against PRRSV. In the present study, we
modified the ORF5 gene by inserting a Pan DR T-helper cell epitope (PADRE) between the neutralizing epitope and the decoy
epitope to minimize or eliminate the decoy effect of the non-neutralizing epitope. The immunogenicity of the modified GP5
was further evaluated using DNA vaccination. The results showed that significantly enhanced neutralizing antibodies were elicited
in mice immunized with the DNA construct expressing the modified GP5 compared with the native GP5. Slightly increased levels
of GP5-specific ELISA antibodies and T-cell proliferative activities were also observed. These results indicate that the high
immunogenicity of the modified GP5 might facilitate the development of improved PRRS vaccines in the future. 相似文献
3.
Enhanced immune responses of mice inoculated recombinant adenoviruses expressing GP5 by fusion with GP3 and/or GP4 of PRRS virus 总被引:1,自引:0,他引:1
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. 相似文献
4.
Lu Z Zhang J Huang CM Go YY Faaberg KS Rowland RR Timoney PJ Balasuriya UB 《Virology》2012,432(1):99-109
Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) are members of family Arteriviridae; they are highly species specific and differ significantly in cellular tropism in cultured cells. In this study we examined the role of the two major envelope proteins (GP5 and M) of EAV and PRRSV in determining their cellular tropism. We generated three viable EAV/PRRSV chimeric viruses by swapping the N-terminal ectodomains of these two proteins from PRRSV IA1107 strain into an infectious cDNA clone of EAV (rMLVB4/5 GP5ecto, rMLVB4/5/6 Mecto and rMLVB4/5/6 GP5&Mecto). The three chimeric viruses could only infect EAV susceptible cell lines but not PRRSV susceptible cells in culture. Therefore, these data unequivocally demonstrate that the ectodomains of GP5 and M are not the major determinants of cellular tropism, further supporting the recent findings that the minor envelope proteins are the critical proteins in mediating cellular tropism (Tian et al., 2012). 相似文献
5.
Analysis of immunogenicity of minor envelope protein GP3 of porcine reproductive and respiratory syndrome virus in mice 总被引:1,自引:0,他引:1
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. 相似文献
6.
Xu XG Wang ZS Zhang Q Li ZC Ding L Li W Wu HY Chang CD Lee LH Tong DW Liu HJ 《Journal of virological methods》2012,179(2):359-366
The GP5 glycoprotein of PRRSV is the main target for inducing neutralizing antibodies and protective immunity in the natural host. The capsid (Cap) protein is the major immunogenic protein and associated with the production of PCV2-specific neutralizing antibodies. In the present study, one genetic recombinant baculovirus BacSC-Dual-GP5-Cap was constructed. This virus displays simultaneously histidine-tagged GP5 and Cap proteins with the baculovirus glycoprotein gp64 TM and CTD on the virion surface as well as the surface of the virus-infected cells. After infection, the GP5 and Cap proteins were expressed and anchored simultaneously on the plasma membrane of Sf-9 cells, as revealed by Western blot and confocal microscopy. This report demonstrated first that both GP5 and Cap proteins were displayed successfully on the viral surface, revealed by immunogold electron microscopy. Vaccination of swine with recombinant baculovirus BacSC-Dual-GP5-Cap elicited significantly higher GP5 and Cap ELISA antibody titers in swine than the control groups. Virus neutralization test also showed that serum from the BacSC-Dual-GP5-Cap treated swine had significant levels of virus neutralization titers. Lymphocyte proliferation responses could be induced in swine immunized with BacSC-Dual-GP5-Cap than the control groups. These findings demonstrate that the BacSC-Dual-GP5-Cap bivalent subunit vaccine can be a potential vaccine against PRRSV and PCV2 infections. 相似文献
7.
We analyzed the complete genomic sequences of 17 porcine reproductive and respiratory syndrome virus (PRRSV) isolates from Southern China obtained between 2010 and 2011 and found that four of seven isolates from 2011 were closely related to the JXA1-R strain (vaccine virus of JXA1). This close relationship between field isolates and China domestic vaccine viruses has not been reported to date. The occurrence of vaccine-like viruses potentially creates a threat for the pig breeding industry and brings difficulties for control of this disease. 相似文献
8.
目的 探讨PRRSV M基因在哺乳动物细胞NIH/3T3细胞中的表达,建立一种非放射性、简便易行的检测特异性细胞毒T淋巴细胞的方法.方法 应用RT-PCR扩增得到M基因并克隆入真核表达载体pcDNA3,构建成重组表达载体pcDNA3-M;将重组质粒转染至NIH/3T3细胞,G418筛选克隆;IFA检测M蛋白在转基因NIH/3T3细胞中的表达.用表达M蛋白的重组腺病毒rAd-M免疫小鼠,用表达M蛋白的NIH/3T3细胞和乳酸脱氢酶法检测PRRSV特异性细胞毒T淋巴细胞的杀伤效应. 结果 获得有G418抗性的NIH/3T3/M细胞克隆;IFA检测到有PRRSV M蛋白表达.重组腺病毒rAd-M免疫组可以诱发CTL应答,并明显高于wtAd和PBS对照组. 结论 培育成功一株能表达PRRSV M蛋白的细胞株NIH/3T3/M,可作为PRRSV CTL检测方法中的靶细胞,证明PRRSV M基因能够诱发特异性的细胞免疫. 相似文献
9.
Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that has caused huge economic losses in the global swine industry. The advent of molecular farming has provided a cost-effective strategy for the development of transgenic plants as bioreactors to produce recombinant proteins. In this study, transgenic potato expressing GP5 protein of PRRSV was produced by Agrobacterium-mediated transformation, and confirmed using Southern blot and RT-PCR analyses. Recombinant GP5 protein was detected by ELISA and Western blot analyses. Mice immunized with transgenic potato extracts generated both serum and gut mucosal-specific antibodies, although low levels of neutralizing antibodies were elicited. This study provides a new approach for the production of vaccines against PRRSV. 相似文献
10.
11.
Han K Seo HW Shin JH Oh Y Kang I Park C Chae C 《Clinical and Vaccine Immunology : CVI》2011,18(10):1600-1607
The objective of the present study was to compare the effects of the modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine (Ingelvac PRRS MLV; Boehringer Ingelheim Animal Health, St. Joseph, MO) on European and North American PRRSV shedding in the semen of experimentally infected boars. The boars were randomly divided into six groups. Vaccinated boars shed the North American PRRSV at the rate of 10(0.1) to 10(1.0) viral genome copies per ml and 3.63 to 10(1.1) 50% tissue culture infective doses (TCID(50))/ml, respectively, in semen, whereas nonvaccinated boars shed the North American PRRSV at the rate of 10(0.2) to 10(4.7) viral genome copies per ml and 1.14 to 10(3.07) TCID(50)/ml, respectively, in semen. Vaccinated boars shed the European PRRSV at the rate of 10(0.1) to 10(4.57) viral genome copies per ml and 1.66 to 10(3.10) TCID(50)/ml, respectively, in semen, whereas nonvaccinated boars shed the European PRRSV at the rate of 10(0.3) to 10(5.14) viral genome copies per ml and 1.69 to 10(3.17) TCID(50)/ml, respectively, in semen. The number of genomic copies of the European PRRSV in semen samples was not significantly different between vaccinated and nonvaccinated challenged European PRRSV boars. The present study demonstrated that boar vaccination using commercial modified live PRRSV vaccine was able to decrease subsequent shedding of North American PRRSV in semen after challenge but was unable to decrease shedding of European PRRSV in semen after challenge. 相似文献
12.
Sipos W Duvigneau C Pietschmann P Höller K Hartl R Wahl K Steinborn R Gemeiner M Willheim M Schmoll F 《Viral immunology》2003,16(3):335-346
Parameters of humoral and cellular immunity were investigated in pigs experimentally infected with a modified-live European porcine reproductive and respiratory syndrome virus (PRRSV, strain DV). PRRSV was detected by real-time RT-PCR and PRRSV-specific antibodies by a commercial ELISA test-kit, respectively. Interleukins IL-1alpha, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF-alpha) and interferon-gamma (IFN-gamma) as well as IL-2 receptor (IL-2R) were quantified at mRNA level using RT-PCR. Subpopulations of blood lymphocytes were assayed using flow cytometry. No significant changes neither in cytokine expression nor in shifts of CD4 and CD8 markers could be found, but similar curve diagrams concerning CD8 single positive T cells could be observed in all vaccinated animals with an initial decrease and an increase between post-infection days (PIDs) 7 and 14. In the vaccination group, TNF-alpha and IL-6 tended to be increased at PIDs 22 and 40, whereas no increase could be seen in IFN-gamma. When comparing the in vivo immune response to that being seen in in vitro experiments, similar shifts of CD4/CD8 lymphocyte subpopulations may be seen. Cytokine curve diagrams, however, do not reflect the in vitro findings to that extent. 相似文献
13.
14.
Porcine reproductive and respiratory syndrome virus (PRRSV) is the cause of an economically important swine disease that has been devastating the global swine industry since the early 1990s. The current PRRSV vaccines are not very effective largely due to heterogeneic nature of the virus. The major envelope protein, GP5, exposes outside the virion, induces neutralizing antibodies, and thus is a primary target for developing a subunit vaccine. In this study, we report a process for purification of GP5 protein from native virions of PRRSV propagated in MARC-145 cells. PRRSV virions were first purified and concentrated through sucrose cushion ultracentrifugation. GP5 protein was subsequently solubilized with Triton X-100 detergent for further processing. Cation exchange chromatography (CEX) was utilized for partial fractionation of GP5, although the viral nucleocapsid protein (N) was a major impurity in CEX elution fractions. During a second chromatographic step, hydrophobic interaction chromatography (HIC) further purified GP5 protein by means of a two-stage elution scheme. Pure GP5 protein was eluted from the HIC resin in the second HIC elution stage by Triton X-100 displacement; however the protein is present as a homodimeric/tetrameric aggregate. This process may be useful in PRRSV subunit vaccine development. 相似文献
15.
Porcine reproductive and respiratory syndrome virus (PRRSV) causes apoptosis during its replication in fetal implantation sites 总被引:1,自引:0,他引:1
Karniychuk UU Saha D Geldhof M Vanhee M Cornillie P Van den Broeck W Nauwynck HJ 《Microbial pathogenesis》2011,51(3):194-202
Reproductive failure due to porcine reproductive and respiratory syndrome virus (PRRSV) is characterized by late-term abortions, early farrowing and an increase of dead and mummified fetuses and weak-born piglets. The mechanism of PRRSV-induced reproductive failure is poorly understood. Human pregnancies, complicated by some pathogens leading to reproductive disorders exhibit increased apoptosis in the fetal membranes. Because PRRSV-target cells are present in endometrium/fetal placentas from healthy sows and PRRSV-infected macrophages in other organs die by apoptosis, we hypothesized that PRRSV can replicate and induce apoptosis in the fetal implantation sites at the last stage of gestation. In the present study, identification, localization and quantification of the PRRSV-positive and apoptotic cells were performed in the fetal implantation sites. Three dams were inoculated intranasally with 105 TCID50 PRRSV 07V063 at 90 days of gestation and sampled at 10 days post-inoculation. Two non-inoculated dams that were euthanized at 100 days of gestation served as control animals. Inoculation of the dams resulted in a viremia that lasted until the end of the study. Transplacental PRRSV spread was detected in all inoculated dams. Using immunofluorescence staining, single PRRSV-positive cells were found in the endometrial connective tissues adjacent to both PRRSV-positive and PRRSV-negative fetuses. In the fetal placental mesenchyme of the PRRSV-positive fetuses, infected cells were more abundant and spread focally. Double staining showed that all PRRSV-positive cells in the fetal implantation sites were positive for sialoadhesin and CD163. Apoptotic cells (TUNEL+) were detected in endometrium and fetal placentas of both non- and PRRSV-inoculated dams. The number of apoptotic cells was significantly higher in PRRSV-positive endometrium/fetal placentas. PRRSV caused apoptosis in infected cells since 20-61% of PRRSV-positive cells were apoptotic and in surrounding cells since 43-91% of the apoptotic cells were virus-negative. The main conclusion obtained from the present study is that PRRSV replicates in the fetal implantation sites and causes apoptosis in infected macrophages and surrounding cells at the last stage of gestation. The possible mode of PRRSV replication in the fetal implantation sites and the events that might contribute to the reproductive disorders are discussed. 相似文献
16.
Oleksiewicz MB Stadejek T Maćkiewicz Z Porowski M Pejsak Z 《Journal of virological methods》2005,129(2):134-144
A peptide ELISA was developed based on an immunodominant and hypervariable epitope in the ORF4 envelope glycoprotein of porcine reproductive and respiratory syndrome virus (PRRSV). The peptide sequence was derived from the Porcilis live-attenuated PRRSV vaccine strain (genotype 1, European). Antibodies induced by the field PRRSVs currently circulating in Poland were not detected by the Porcilis ORF4 peptide ELISA. In contrast, Porcilis-vaccinated animals seroconverted in the ORF4 peptide ELISA at 21 days post-vaccination. Maximal titers were seen 30-92 days post-vaccination; most sera had endpoint titers between 1:1000 and 1:100,000. In a paired format, where sera were assayed in two separate ELISAs using ORF4 peptides derived from the genetically very closely related Porcilis and Lelystad PRRSV strains, it was possible to differentiate between antibodies induced by these two viruses. The Porcilis and Lelystad ORF4 peptide ELISAs had sensitivities of 89 and 100%, respectively. Thus, ORF4 peptide ELISA afforded specific detection of antibodies induced by an European-genotype live-attenuated vaccine PRRSV strain (Porcilis). The results suggest that specific ORF4 peptide ELISAs can be custom-made for European-genotype PRRSV strains, using general peptide design criteria described in this work. Thus, ORF4 ELISAs may be generally useful, to monitor safety and operational aspects of European-genotype live-attenuated PRRSV vaccine virus use in populations with circulating field European-genotype PRRSVs. 相似文献
17.
Dachrit Nilubol Thitima Tripipat Tawatchai Hoonsuwan Pavita Tipsombatboon Jittima Piriyapongsa 《Archives of virology》2014,159(1):17-27
The objective of this study was to investigate the dynamics and evolution of porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 following the use of a modified live PRRSV (MLV) vaccine. A PRRSV-positive farm with coexistence of types 1 and 2 and no history of MLV vaccination was investigated. Vaccination with a type 2 MLV (Ingelvac PRRS MLV, Boehringer Ingelheim, USA) was implemented. All sows were vaccinated at monthly intervals for two consecutive months and then every third month. Piglets were vaccinated once at 7-10 days of age and weaned to nursery facilities at 21-23 days of age. Serum samples were collected monthly before and after vaccination from four population groups, including replacement gilts and suckling, nursery and finishing pigs, and assayed by PCR. After a year of blood collection, amplified products were sequenced, resulting in 277 complete ORF5 gene sequences from 145 type 1 and 132 type 2 isolates. Prior to and following vaccination, both type 1 and type 2 PRRSV were isolated and found to coexist in an individual pig. Each genotype evolved separately without influencing the strain development of the other. Although the substitution rates of both genotypes were relatively similar, MLV vaccination appears to increase the heterogenicity of type 2 PRRSV, resulting in the emergence of three novel type 2 PRRSV clusters in the herd, including an MLV-like cluster, which disappeared within the month following whole-herd vaccination. Two additional clusters included one related to the MLV vaccine and one related to the endemic cluster of the herd. 相似文献
18.
Erika Silva-Campa Lorenzo Fraile Lilian Flores-Mendoza Jesús Hernández 《Virology》2010,396(2):264-8204
The aim of this study was to characterize the immune responses of DCs after infection with four different EU strains of PRRSV and whether they show any ability to immunomodulate T cells activation. Our results show that all EU strains can efficiently infect and replicate in DCs. Nevertheless, SLA-II levels remained unaltered in DC infected by all EU PRRSV strains, whereas SLA-I expression was only reduced when strain 2992 was used. IL-10 production was induced by three EU PRRSV strains, being strain 2992 the highest inducer. However, no induction of Treg cells, measured by CD25 and Foxp3 expression on lymphocytes co-cultured with infected DCs, was found. TGF-β induction was not detected in DC infected with any EU strain tested. In conclusion, DCs infected with EU PRRSV strains exhibited an unbalanced ability to stimulate T cell response and was strain dependent. However, Treg cells were not induced, at least in vitro. 相似文献
19.
To evaluate the effect of natural infection with TTSuV1 on the antibody response to vaccination with PRRS vaccine and clinical
signs when co-infected with virulent PRRSV, 15 4-week-old TTSuV1-positive piglets and 20 TTSuV1-negative piglets were selected
by PCR from two pig farms in Jiangsu province. TTSuV1-negative pigs were divided into four groups, and TTSuV1-positive pigs
were divided into three groups. Experimental pigs were vaccinated with a PRRSV modified live virus (MLV) at 6 weeks of age
and subsequently challenged with a virulent strain of PRRSV at 10 weeks of age. A TTSuV1-negative control group and an unvaccinated
PRRS MLV control group were tested at the same time. The levels of antibody/cytokine and protective efficiency against PRRS
MLV vaccine were evaluated. TTSuV1-infected/PRRSV-vaccinated pigs had lower levels of PRRSV antibody, as well as IFN-γ, IL-10
and T lymphocyte proliferation, than the TTSuV1-uninfected/PRRSV-vaccinated group (P < 0.05, except IL-10) after vaccination at only one time point. TTSuV1-infected/PRRS MLV-vaccinated/PRRSV-challenged pigs had more
severe clinical signs (P > 0.05), more macroscopic lung lesions (P < 0.05) and lower levels of PRRSV antibody (P < 0.05 at 7 to 14 days post-PRRSV-challenge) than TTSuV1-uninfected/PRRSV-vaccinated/PRRSV-challenged pigs. These data indicate
that TTSuV1 natural infection has an adverse effect on the development of host immune responses, suppresses immunization by
the PRRS MLV vaccine, and exacerbates PRRS to a certain extent in pigs. 相似文献