Xinrong Chen

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.

Interactions of porcine circovirus 2 with its hosts

Porcine circovirus 2 (PCV2) can cause porcine circovirus diseases and porcine circovirus-associated diseases (PCVD/PCVAD), which are widely presented in swine-producing countries. Since the discovery of this virus, considerable efforts have been devoted to understanding this pathogen and its interactions with its host. Here, we review the current state of knowledge on interactions between host cell factors and PCV2 with respect to viral proliferation, virus-induced cell apoptosis and autophagy, and host antiviral defenses during PCV2 infection. We also review mouse model systems for PCV2 infection.

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.

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.

Genetically modified pigs are protected from classical swine fever virus

Classical swine fever (CSF) caused by classical swine fever virus (CSFV) is among the most detrimental diseases, and leads to significant economic losses in the swine industry. Despite efforts by many government authorities try to stamp out the disease from national pig populations, the disease remains widespread. Here, antiviral small hairpin RNAs (shRNAs) were selected and then inserted at the porcine ROSA26 (pROSA26) locus via a CRISPR/Cas9-mediated knock-in strategy. Finally, anti-CSFV transgenic (TG) pigs were produced by somatic nuclear transfer (SCNT). Importantly, in vitro and in vivo viral challenge assays demonstrated that these TG pigs could effectively limit the growth of CSFV and reduced CSFV-associated clinical signs and mortality, and the disease resistance was stably transmitted to F1-generation. The use of these TG pigs can improve the well-being of livestock and substantially reduce virus-related economic losses. Additionally, this antiviral approach may provide a reference for future antiviral research. Author summary Classical swine fever (CSF), caused by classical swine fever virus (CSFV), and is a highly contagious, often fatal porcine disease with significant economic losses. Due to its economic importance to the pig industry, the biology and pathogenesis of CSFV have been investigated extensively. Despite efforts by many government authorities to stamp out the disease from national pig populations, the disease remains widespread in some regions and seems to be waiting for the reintroduction and the next round of disease outbreaks. These highlight the necessity and urgency of developing more effective approaches to eradicate the challenging CSFV. In this study, we successfully produced anti-CSFV transgenic pigs and confirmed that these transgenic pigs could effectively limit the growth of CSFV in vivo and in vitro and that the disease resistance traits in the TG founders can be stably transmitted to their F1-generation offspring. This study suggests that these TG pigs can improve the well-being of livestock and contribute to offer potential benefits over commercial vaccination. The use of these TG pigs can improve the well-being of livestock and substantially reduce CSFV-related economic losses.

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.

HMGCR inhibits the early stage of PCV2 infection, while PKC enhances the infection at the late stage.

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.

Detection of Kobe-type and Otsu-type Babesia microti in wild rodents in China's Yunnan province

Babesiosis is an emerging tick-transmitted zoonosis prevalent in large parts of the world. This study was designed to determine the rates of Babesia microti infection among small rodents in Yunnan province, where human cases of babesiosis have been reported. Currently, distribution of Babesia in its endemic regions is largely unknown. In this study, we cataloged 1672 small wild rodents, comprising 4 orders, from nine areas in western Yunnan province between 2009 and 2011. Babesia microti DNA was detected by polymerase chain reaction in 4·3% (72/1672) of the rodents analyzed. The most frequently infected rodent species included Apodemus chevrieri and Niviventer fulvescens. Rodents from forests and shrublands had significantly higher Babesia infection rates. Genetic comparisons revealed that Babesia was most similar to the Kobe- and Otsu-type strains identified in Japan. A variety of rodent species might be involved in the enzootic maintenance and transmission of B. microti, supporting the need for further serological investigations in humans.