Porcine circovirus 2 (PCV2) is the causative agent of porcine circovirus diseases and porcine circovirus-associated diseases (PCVD/PCVAD), which are widely present in every major swine farm. However, lower propagation rate of PCV2 in vitro seriously hindered the production of PCV2 vaccine. Previously, we found that interleukin-2 (IL-2) can increase PCV2 yield in vitro. In the present study, porcine IL-2 gene was amplified and stably transfected into PK-15 cells. The results demonstrated that PCV2 proliferation can be significantly enhanced in cells stably expressing porcine IL-2 gene, suggesting that porcine IL-2 contributes to proliferation of PCV2. These results indicated that cells overexpressing porcine IL-2 gene can be used as a promising cell line for vaccine development of PCV2.

The denitrifying bacterium Acinetobacter johnsonii strain DBP-3 which was capable of removing phosphate, nitrate, and ammoniacal salt is psychrotolerant, whereas, the cold shock response mechanisms or the cold shock proteins (Csps) was unclear. In this article, the optimal growth temperature (25 °C) and cold shock temperature (7.5 °C) were determined firstly by an Arrhenius plot of the growth of the strain DBP-3. Then, among the seven cold shock-like protein genes which were cloned and identified referenced by A. johnsonii SH046 genome, qRT-PCR and shotgun-LTQ mass spectrometry showed that Csp3 and Csp4 were overexpressed under cold shock condition. Furthermore, Western blotting confirmed the result with the antibodies against Csp3 and Csp4 prepared by ourselves. Finally, the phylogenetic analysis showed that the similarity percent between Csp3 and Csp4 was 76.85 %, and Csp3 and Csp4 belonged to CspE family. The results indicated that CspE is overproduced by temperature downshift and may play an important role in the psychrotolerant process of strain DBP-3.

Porcine circovirus type 2 (PCV2) is the smallest DNA virus, which causes porcine circovirus diseases and porcine circovirus-associated diseases (PCVD/PCVAD). Due the small size of viral genomic DNA, PCV2 replication predominantly relies on the host factors. In this study, effects of PKC and HMGCR on PCV2 infection were evaluated using real time PCR and western blot. We found that PKC and HMGCR participated in different stages of PCV2 infection. HMGCR works on the early stage of the infection to inhibit the virus infection, while PKC enhances the infection at the late stage. Furthermore, PKC enhances PCV2 replication by activating JNK1/2 and inactivating HMGCR via regulating phosphorylation of these two proteins, while HMGCR can suppress phosphorylation of JNK1/2. The results in the present study will provide new sights in the pathogenesis of PCV2 infection, as well as interactions between host factors during PCV2 infection.

The CRISPR-Cas9 system is a newly developed genome-engineering tool used to inhibit virus infection by targeting the conserved regions of the viral genomic DNA. In the present study, we constructed a cell line stably expressing Cas9 endonuclease and sgRNA targeting the conserved UL30 gene of pseudorabies virus (PRV). During the PRV infection, the CRISPR-Cas9 system was efficient in cleaving the UL30 gene in each passage. However, deletions and insertions occurred at low passages, while substitutions were frequently observed at high passages. Furthermore, copy numbers and virus titers of PRV were significantly increased in a passage-dependent manner, indicating that viral genomic replication and assembly were more effective at the high passages than at low passages. These results demonstrated that PRV could escape from CRISPR-Cas9-mediated inhibition. Therefore, whether the CRISPR-Cas9 system is suitable for antiviral application should be considered and carefully verified.

Rep and ORF3 proteins are important functional proteins of porcine circovirus 2 (PCV2). Here, Rep and ORF3 genes were cloned, expressed and used to raise polyclonal antibodies. The result showed the recombinant plasmids of Rep and ORF3 genes constructed in this study were expressed efficiently in the prokaryotic system, and the recombinant proteins had antigenicity and immunogenicity. Furthermore, reactivity and specificity of the antiserums were characterized by western blot and indirect immunofluorescent assays. The results elucidated that polyclonal antiserum prepared with Rep or ORF3 had good reactivity and specificity against PCV2, or the Rep and ORF3 expressed in PK-15 cells, respectively. The Rep protein is promising for PCV2 antibody and vaccine development. These results will be helpful for further studies focusing on pathogenesis of PCV2 and serology diagnostic test or vaccine development against PCV2.

The HMG-CoA reductase (HMGCR) pathway is an important metabolic route, which is not only present in almost every organism, but also involves virus infection. It has recently been shown that expression levels of IFN-responsive genes were significantly increased in HMGCR-downregulated cells and HMGCR inhibitor-treated cells. The aim of this study was to determine whether inhibition of HMGCR by atovastatin would significantly affect Porcine circovirus type 2 (PCV2) infection and immunological reaction in BALB/c mice. The results showed atovastatin significantly stimulated PCV2 replication in vivo. Immunological reaction in atovastatin-treated mice was also significantly enhanced during PCV2 infection. Atovastatin also enhanced PCV2-induced illness in mice. The results of this study will provide new insight into the role of atovastatin in PCV2 infection.

Red homologous recombination has been extensively used in recombineering. Because foreign sequences, such as antibiotic resistance genes, FRT-sites, or loxP-sites, are often unwanted in mutant Escherichia coli, we established a markerless deletion system containing short homologous sequences, a positive-selectable marker (kan), and a negative-selectable marker (sacB) for E. coli. For markerless deletion of a specific region of the E. coli genome, a two-step recombination procedure using two different PCR fragments, which were amplified from pUC57-kan-sacB and pUC57-298, was performed. The generation of a pheA-tyrA deficient mutant demonstrated that this markerless deletion system was a simple and efficient method to generate markerless chromosomal deletions in E. coli.

Current methods to quantitate classical swine fever virus (CSFV) infectivity in cell culture are time-consuming and labor-intensive. This study described the generation of a dark-to-bright fluorescent reporter cells to facilitate in vitro studies of CSFV infection and replication. This assay was based on a novel reporter cell stably expressing the enhanced green fluorescent protein (EGFP) fused in-frame to a quenching peptide via a special recognition sequence of the CSFV NS3 protease. Chromophore maturation of EGFP can be prevented by quenching peptide until the quenching peptide was specifically cleaved by NS3 protease during CSFV infection, making it a dark-to-bright reporter of CSFV infection. The result demonstrated that the CSFV-infected cells were clearly distinguishable from mock-infected cells and cells infected with other viruses. There was a strong correlation between the fluorescence intensity and viral RNA replication in CSFV-infected cells. The cell enabled rapid and sensitive detection of CSFV infection and viral replication in cell culture. The best time to examine the fluorescence in CSFV-infected cells was at 48h post-inoculation. These data suggested that the cells can be used as a reporter cell in CSFV infection assays. This reporter cell provides a sensitive method for the detection and isolation of CSFV and it will be useful for the screening of antiviral drugs or neutralizing antibody assays.

In the present study, 89 porcine reproductive and respiratory syndrome virus (PRRSV) isolates in China during 2007 to 2012 were randomly selected from the GenBank genetic sequence database. Evolutionary characteristics of these isolates were analyzed based on the sequences of non-structural protein 2 (Nsp2) and glycoprotein 5 (GP5). The genetic variations of the isolates were also compared with six representative strains. The results showed that a high degree of genetic diversity exists among the PRRSV population in China. Highly pathogenic PRRSV isolates, with a discontinuous deletion of a 30 amino acid residue in the Nsp2 region, remained the most dominant virus throughout 2007–2012 in China. Owing to the extensive use of representative vaccine strains, natural recombination events occurred between strains. Three isolates — HH08, DY, and YN-2011 — were more closely related to vaccine strains than the other isolates. Both YN-2011 and DY were the evolutionary products of recombination events between strains SP and CH-1R. The results of the present study provide useful information for the epidemiology of PRRSV as well as for vaccine development.

We examined the role of HMG-CoA reductase (HMGCR) during porcine circovirus 2 (PCV2) infection. The results demonstrated that levels of endogenous HMGCR were not significantly different in PCV2-infected cells and mock-infected cells. However, the level of phosphorylated HMGCR, an inactivated form of HMGCR, was increased in PCV2-infected cells. Furthermore, HMGCR was upregulated by overexpression, silenced by siRNA or inactivated using its dominant-negative form in PK-15 cells. The results showed that PCV2 infection was inhibited by HMGCR overexpression, whereas it was significantly increased in HMGCR-silenced cells and HMGCR inhibitor-treated cells. Moreover, there was a robust apoptotic response at 48 h post-infection (p.i.) in HMGCR-inactivated cells, and this response was significantly greater than that observed in PK-15 cells. A modest apoptotic response was also observed in HMGCR-silenced cells. Caspase-3 activity was also analysed in PCV2-infected cells at 48 h p.i. As expected, caspase-3 activity was significantly increased in HMGCR-inactivated and -silenced cells compared with PK-15 cells. PCV2 replication was dose-dependently increased in HMGCR-inactivated cells when treated with increasing amounts of caspase-3 inhibitor. Altogether, HMGCR was negatively associated with PCV2 infection and PCV2-induced apoptotic cell death. These data demonstrated that HMGCR can be used as a candidate target for PCV2 disease control and antivirus research. Furthermore, the cells generated in this study can be used to evaluate the potential effects of HMGCR on PCV2 replication.

The 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) pathway is an important metabolic route that is present in almost every organism. However, whether HMGCR affects the expression of interferon (IFN)-responsive genes is unclear. In the present study, expression levels of IFN-responsive genes were monitored by real time polymerase chain reaction and enzyme-linked immunosorbent assay. The results showed that expression levels of IFN-responsive genes were significantly increased in HMGCR-downregulated cells and HMGCR inhibitor-treated cells, indicating that inhibition of HMGCR activates the expression of IFN-responsive genes. The result in this study will provide new insight into the role of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in antiviral research.

Capsid protein (Cap) of porcine circovirus 2 (PCV2) contained critical epitopes for inducing a protective immune response. Here, different fragments of PCV2 Cap protein were cloned, expressed, purified and used to raise polyclonal antibodies. The result showed the recombinant plasmids expressed efficiently in the prokaryotic system. Western blot and ELISA showed the recombinant protein had antigenicity and immunogenicity. Furthermore, efficiency of different constructs to produce antibody against PCV2 was compared. Reactivity and specificity of the polyclonal antibody were characterized by Western blot and indirect immunofluorescent assays. The results indicated that polyclonal antiserum prepared from protein ΔCap17-233 had better reactivity and specificity against PCV2 in comparison to that of protein ΔCap51-233 and the inactivated vaccine. These results will contribute to further studies focusing on the gene and vaccine development against PCV2.

Porcine CD4 proximal promoter and enhancer sequences were cloned and aligned with the corresponding human and murine sequences. The alignment showed nucleotide homology between porcine and human sequences was 62.4 % for the CD4 promoter and 56.6 % for the CD4 enhancer. The nucleotide homology between porcine and murine sequences was 42.5 % for the CD4 promoter and 25.4 % for the CD4 enhancer. The proximal enhancer and promoter regions of the CD4 gene from porcine, murine and human cells were compared for their ability to direct foreign gene expression in transiently transfected human cell lines. The results indicated the porcine CD4 promoters and enhancers could effectively direct expression of a foreign gene in human cells. The porcine promoter was equally efficient as CMV and EF-1α in directing gene expression.

<正>猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)俗称猪蓝耳病,是由猪繁殖与呼吸综合征病毒(PRRSV)引起的一种以母猪繁殖障碍、仔猪呼吸道症状和高病死率为特征的传染病,是一种世界范围内广泛流行的猪传染性疾病。自1987年首次在美国发现以来,相继在各国呈流行性感染,给世界养猪业造成了严重的经济损失,被世界动物卫生组织

A porcine circovirus 2 (PCV2) strain, designated CC1, was isolated and purified from tissue samples from pigs with wasting syndromes in China. We report the complete genome sequence of PCV2b strain CC1 with a deletion of C at position 1053 resulting in elongation of open reading frame 2 (ORF2) and formation of ORF5. There were 11 ORFs in the genome.

<正>热休克蛋白(Heat shock protein,HSP)是1962年由Ritossa等在制备果蝇唾液腺细胞染色体的过程中发现的一类应激蛋白。HSPs广泛存在于原核生物和真核生物中,在应激情况下由热休克基因编码合成,是序列高度保守的伴随细胞蛋白。热休克蛋白一般在细胞处于各种不利于自

In order to construct cell lines without CD163gene,agene of porcine reproductive and respiratory syndrome virus receptor,and find out role of CD163in PRRSV infection,5'and 3'homologous arms of CD163gene of porcine reproductive and respiratory syndrome virus receptor were amplified and cloned into pGEM-T vector.Results of sequencing showed that 5'and 3'homologous arms of CD163gene were 4.5kb and 1.1kb,respectively.Then,the homologous arms of CD163gene were subcloned into pSSC-9to construct a targeting vector pSSC-Larm-Sarm for CD163gene knock-out.The vector will be used to construct fibroblast cells with CD163gene knockout.

In order to develop effective detection methods and prepare monoclonal antibody of porcine circovirus type Ⅱ , Cap and Rep genes of porcine circovirus type Ⅱ were amplified and cloned into pMD18-Tsimple vector. And the genes were sequenced. Then Cap and Rep genes were sub-cloned into pACYCDuet-1 vector to generate three prokaryotic expression plasmids named pACYCDuet-1-Cap, pACYCDuet-1-Rep, and pACYCDuet-1-Rep -Cap. The plasmids were transformed into E.coli BL21 (DE3) and expressed. The results of Western-blotting showed that the two proteins were expressed successfully in E. coli BL21 (DE3). Furthermore, plasmid for coexpression of two proteins expressed better than the other two plasmids.

Porcine circovirus 2 (PCV2) is an extremely slow-growing virus, and PCV2 infection and replication in cell culture yield very low viral titers. The effects of different methods of PCV2 cultivation in vitro were compared with the purpose of increasing viral yield. The results showed that treatment with IL-2, ConA, and d-glucosamine increased PCV2 yield more effectively than other treatments. Additionally, treatment with IL-2, ConA, d-glucosamine and MβCD consistently increased PCV2 infection in PK-15 cells during consecutive viral passages. A combinatorial treatment with ConA, MβCD and d-glucosamine increased PCV2 yield significantly in PK-15 cells, to 1.81 × 1010 genome copy numbers per mL of cell lysate at 72 hpi, and the viral titer (−lg TCID50/100 μL) was 8.6. The results of this study may be helpful for the investigation of PCV2 replication and the production of a PCV2 vaccine.

<正>目前位点特异性重组酶系统在基因打靶中获得广泛应用,是条件性基因打靶、诱导性基因打靶、时空特异性基因打靶策略的技术核心。该系统目前主包括Cre/LoxP系统,FLP/FRT系统,该系统可以使某个基因在特定的细胞类型、特定时间表达或被沉默,进而研究该基因的特定功能。本研究应用FLP/FRT重组酶系统将FLP基因连接到可以游离于染色体外的载体PCEP4上获