Zhiyuan Peng

HMG-CoA reductase is negatively associated with PCV2 infection and PCV2-induced apoptotic cell death.

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.

Pseudorabies virus can escape from CRISPR-Cas9-mediated inhibition.

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.

Expression, purification and antibody preparation using different constructs of PCV2 capsid protein.

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.

Expression, purification and antibody preparation of PCV2 Rep and ORF3 proteins

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.

Effect of atovastatin treatment on porcine circovirus 2 infection in BALB/c mice

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.

A dark-to-bright reporter cell for classical swine fever virus infection

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.

Inhibition of 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase increases the expression of interferon‐responsive genes

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.

Porcine circovirus 2 proliferation can be enhanced by stably expressing porcine IL-2 gene in PK-15 cell

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.

Live Cell Reporter Systems for Positive-Sense Single Strand RNA Viruses

Cell-based reporter systems have facilitated studies of viral replication and pathogenesis, virus detection, and drug susceptibility testing. There are three types of cell-based reporter systems that express certain reporter protein for positive-sense single strand RNA virus infections. The first type is classical reporter system, which relies on recombinant virus, reporter virus particle, or subgenomic replicon. During infection with the recombinant virus or reporter virus particle, the reporter protein is expressed and can be detected in real time in a dose-dependent manner. Using subgenomic replicon, which are genetically engineered viral RNA molecules that are capable of replication but incapable of producing virions, the translation and replication of the replicon could be tracked by the accumulation of reporter protein. The second type of reporter system involves genetically engineered cells bearing virus-specific protease cleavage sequences, which can sense the incoming viral protease. The third type is based on viral replicase, which can report the specific virus infection via detection of the incoming viral replicase. This review specifically focuses on the major technical breakthroughs in the design of cell-based reporter systems and the application of these systems to the further understanding and control of viruses over the past few decades.

Subculturing cells have no effect on CRISPR/Cas9-mediated cleavage of UL30 gene in pseudorabies virus

CRISPR/Cas9‐mediated genome editing can inhibit virus infection by targeting the conserved regions of the viral genomic DNA. Unexpectedly, we found previously that pseudorabies virus (PRV) could escape from CRISPR/Cas9‐mediated inhibition. In order to elucidate whether the escape of PRV from Cas9‐mediated inhibition was due to cell deficiencies, such as genetic instability of sgRNA or Cas9 protein, the positive cells were passaged ten times, and PRV infection in the sgRNA‐expressing cells was evaluated in the present study. The results showed that subculturing cells has no effect on Cas9‐mediated cleavage of PRV. Different passages of PX459‐PRV cells can stably express sgRNA to facilitate Cas9/sgRNA cleavage on the UL30 gene of PRV, resulting in a pronounced inhibition of PRV infection. Studies to elucidate the mechanism of PRV escape are currently in progress.