Linzhu Ren

Human MxA protein inhibits the replication of classical swine fever virus

Classical swine fever virus (CSFV) has a spherical enveloped particle with a single stranded RNA genome, the virus belonging to a pestivirus of the family Flaviviridae is the causative agent of an acute contagious disease classical swine fever (CSF). The interferon-induced MxA protein has been widely shown to inhibit the life cycle of certain RNA viruses as members of the Bunyaviridae family and others. Interestingly, it has been reported that expression of MxA in infected cells was blocked by CSFV and whether MxA has an inhibitory effect against CSFV remains unknown to date until present. Here, we report that CSFV replicated poorly in cells stably transfected with human MxA. The proliferation of progeny virus in both PK-15 cell lines and swine fetal fibroblasts (PEF) continuously expressing MxA was shown significantly inhibited as measured by virus titration, indirect immune fluorescence assay and real-time PCR.

Complete Genome Sequence of Porcine Circovirus 2b Strain CC1

ABSTRACT Porcine circovirus type 2 (PCV2) is the etiologic agent of porcine circovirus-associated disease, and it is mainly divided into five genotypes. Here, we report the complete genome sequence of PCV2 strain GDYX, which belongs to PCV2d and has a unique amino acid variation at position 169 (S to G).

Comparative analysis of different methods to enhance porcine circovirus 2 replication.

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 1.81×10(10) genome copy numbers per mL of cell lysate at 72 hpi, and the viral titer (-lgTCID50/100 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.

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.

Isolation and culture of embryonic stem-like cells from pig nuclear transfer blastocysts of different days.

This study was conducted to establish pig embryonic stem (ES)-like cell lines from nuclear transfer blastocysts. A green fluorescent protein (GFP)-expressing cell line was used as the source of donor cells injected into the enucleated oocytes. Blastocysts were collected at D5 (the fifth day), D7 (the seventh day) and D9 (the ninth day). Differential staining was used to assay the viability and development of blastocysts from the 3 days. The number of inner cell mass (ICM) cells increased from 1.83 ± 0.8 (D5) to 5.37 ± 1.2 (D7) to 7.56 ± 1.5 (D9). The expression profiles of embryonic stem (ES) cell factors (OCT4, SOX2, KLF4 and c-MYC) correlated best with the undifferentiated ES state and were identified by qPCR. The expression of the four factors was increased from D5 to D7, whereas the expression decreased from D7 to D9. We tried to isolate ES-like cells from these embryos. However, ES-like cells from the D7 blastocysts grew slowly and expressed alkaline phosphatase. The cells from the D9 blastocysts grew rapidly but did not express alkaline phosphatase. ES-like cells were not isolated from the D5 blastocysts. These results show that the cells from the D7 embryos are pluripotent but grow slowly. The cells from the D9 embryos grow rapidly but start to lose pluripotency.

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.

Construction of a recombinant human FGF1 expression vector for mammary gland-specific expression in human breast cancer cells

Human Fibroblast growth factor 1 (FGF1) has been recognized as a valuable protein drug for the treatment of many diseases because of its multiple functions in regulating a variety of biological processes involved in embryonic development, cell growth and differentiation, morphogenesis, tissue repair, and others. The aim of this study was to develop an FGF1 mammary gland-specific expression vector to produce FGF1 on a large scale from transgenic cows to meet the demand for FGF1 in medical use. In this study, we generated an FGF1 mammary gland-specific expression vector and validated its function in human MCF-7 cells. This vector was shown to successfully express functional FGF1, thus potentially enabling the generation of transgenic cows to be used as mammary gland bioreactors.

Development of a Rapid Method for the Visible Detection of Pork DNA in Halal Products by Loop-Mediated Isothermal Amplification

Disclosing the composition of meat products in detail is essential to consumers’ rights. In this study, we developed a rapid method based on loop-mediated isothermal amplification to visibly identify pork DNA in meat products. Four pig specific primers were designed according to the mitochondrial DN1 gene sequence. The analytical sensitivity of the LAMP assay for pork DNA detection is 0.5xa0pg in our experiment, and the results were not affected by processing temperature. We used the LAMP assay and the industry standard for Chinese entry-exit inspections and quarantines to analyze commercial halal products, and the results were comparable. In conclusion, the LAMP assay has excellent sensitivity and specificity and is a convenient method for pork DNA detection.

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.