Hepatitis B virus envelope proteins can serve as therapeutic targets embedded in the host cell plasma membrane.

Hepatitis B virus (HBV) infection is a major health threat causing 880,000 deaths each year. Available therapies control viral replication but do not cure HBV, leaving patients at risk to develop hepatocellular carcinoma. Here, we show that HBV envelope proteins (HBs)-besides their integration into endosomal membranes-become embedded in the plasma membrane where they can be targeted by redirected T-cells. HBs was detected on the surface of HBV-infected cells, in livers of mice replicating HBV and in HBV-induced hepatocellular carcinoma. Staining with HBs-specific recombinant antibody MoMab recognising a conformational epitope indicated that membrane-associated HBs remains correctly folded in HBV-replicating cells in cell culture and in livers of HBV-transgenic mice in vivo. MoMab coated onto superparamagnetic iron oxide nanoparticles allowed to detect membrane-associated HBs after HBV infection by electron microscopy in distinct stretches of the hepatocyte plasma membrane. Last but not least, we demonstrate that HBs located on the cell surface allow therapeutic targeting of HBV-positive cells by T-cells either engrafted with a chimeric antigen receptor or redirected by bispecific, T-cell engager antibodies. TAKE AWAYS: HBs become translocated to the plasma membrane. Novel, recombinant antibody confirmed proper conformation of HBs on the membrane. HBs provide an interesting target by T-cell-based, potentially curative therapies.

Synchronised infection identifies early rate-limiting steps in the hepatitis B virus life cycle.

Hepatitis B virus (HBV) is an enveloped DNA virus that contains a partially double-stranded relaxed circular (rc) DNA. Upon infection, rcDNA is delivered to the nucleus where it is repaired to covalently closed circular (ccc) DNA that serves as the transcription template for all viral RNAs. Our understanding of HBV particle entry dynamics and host pathways regulating intracellular virus trafficking and cccDNA formation is limited. The discovery of sodium taurocholate co-transporting peptide (NTCP) as the primary receptor allows studies on these early steps in viral life cycle. We employed a synchronised infection protocol to quantify HBV entry kinetics. HBV attachment to cells at 4°C is independent of NTCP, however, subsequent particle uptake is NTCP-dependent and reaches saturation at 12 h post-infection. HBV uptake is clathrin- and dynamin dependent with actin and tubulin playing a role in the first 6 h of infection. Cellular fractionation studies demonstrate HBV DNA in the nucleus within 6 h of infection and cccDNA was first detected at 24 h post-infection. Our studies show the majority (83%) of cell bound particles enter HepG2-NTCP cells, however, only a minority (<1%) of intracellular rcDNA was converted to cccDNA, highlighting this as a rate-limiting in establishing infection in vitro. This knowledge highlights the deficiencies in our in vitro cell culture systems and will inform the design and evaluation of physiologically relevant models that support efficient HBV replication.

CspE is Overproduced by Temperature Downshift in the Acinetobacter johnsonii DBP-3.

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.

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.

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.

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.

Complete genome sequence of porcine circovirus 2b strain CC1.

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.

Porcine CD4 promoters and enhancers can direct foreign gene expression in human cells.

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.

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.

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.

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.

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.

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.

Markerless Deletion System for Escherichia coli Using Short Homologous Sequences and Positive-Negative Selectable Cassette.

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.

Analysis of molecular variation in porcine reproductive and respiratory syndrome virus in China between 2007 and 2012.

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.

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.

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 MßCD consistently increased PCV2 infection in PK-15 cells during consecutive viral passages. A combinatorial treatment with ConA, MßCD 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.