Construction Of Pseudotyped Virus Bearing Classical Swine Fever Virus Glycoprotein And Inhibition Of Replication Of CSFV By Capsid-Targeted Viral Inactivation

Classical swine fever(CSF) is a kind of acute, febrile and highly contagious disease that caused by the pathogen Pestivirus suis. CSF can cause great damages to the domestic and international pig breeding industry as it is characterized by high-contagious, high morbidity and mortality and wide range of transmission. The clinical symptoms including acute fever, hyperpyrexia, degeneration in micrangium wall, extensive dot-sized hemorrhage within the skin and infarction in the spleen. Office International Des Epizooties(OIE) has categorized CSF as A class infectious diseases according to related laws, and also specified CSF as one of the international quarantine item. According to the Implementating regultions for quarantine and prevention of livestock and poultry disease ordinance, CSF was classified as the Class A infectious disease.The retroviral envelope protein can be exchanged for envelope proteins from non-related viruses, a process called pseudotyping. Pseudotyped viruses with heterogenic glycoprotein incorporated into retroviral particles were proved to be a safe viral entry model, which only go through a single cycle infection(replication-deficient) and acquired the host range of the parent viruses where the glycoprotein were derived, thus they could facilitate the research on viral entry mechanism, viral tropism, neutralization antibody analysis, and receptor identification. In addition, One of the potential strategies, referred to as capsid-targeted viral inactivation (CTVI), is a conceptually powerful antiviral approach. In this strategy, the viral capsid protein is designed as the carrier of a deleterious enzyme, such as a nuclease, a proteinase, or even a single-chain antibody to bind to a native viral protein. These recombinant proteins are targeted specifically to progeny virions during their assembly to prevent the production of infectious viral particles and the subsequent spread of de novo infection. CTVI has been investigated extensively and shown to be a promising antiviral strategy against several important viruses.Therefore, in this study, the pseudotype system of MuLV particles with CSFV E012 was set up and it can be used to study the entry of CSFV. The results shown SK6、PK15 and ST infected were Lac Z positive, indicating viral entry, and revealed the pseudtype virions of MuLV-E012 were infectious. The pseudotyped particles were used to develop an in vitro micro neutralization assay that was both sensitive and specific for CSFV neutralizing antibody. In addition, serum samples from piglets immunized with E2 subunit vaccine were detected by this micro neutralization assay. To explore the feasibility of using capsid-targeted viral inactivation (CTVI) as an antiviral strategy against CSF infection, a stable cell line was constructed for expressing a fusion protein of CSFV capsid (Cap) and Staphylococcus aureus nuclease (SNase). Then we apply it into the study of anti-CSF infections.The contents of the paper contain five parts as following:1. Cloning and eukaryotic expression of the glycoprotein genes of CSFVTwo basic requirements are essential for successful pseudotyping:(1) the glycoprotein has to be incorporated onto the virions, and (2) the pseudotyped virions have to be infectious. Or the system is not useful. The generation of high titer retro viral stocks for the efficient transduction is an important technical for a pseudotype system.In this study, the glycoprotein E0, E2 and E012 genes of CSFV were amplified by RT-PCR and cloned into pMD-18T vector and sequenced. Then three genes were cloned into eukaryotic expression vector pcDNA3.0, designated pcDNA-E0, pcDNA-E2 and pcDNA-E012 respectively. HEK293T cell were transfected with these three plasmid using calcium phosphate method. The cells were collected cells after 48 h, and incubated for 1 h at 4℃with 1:100 CSFV anti-serum. After three washes with PBS/FCS, the cells were incubated with fluorescein-labelled staphylococcal protein A for 1h at 4℃, subsequently subjected to flow cytometry using a FACSCalibur. Meanwhile, protein expression of recombinant plasmids were also determined by Western blot assay. The results showed that the recombinant plasmids pcDNA-E0, pcDNA-E2 and pcDNA-E012, could be effective expressed in HEK293T cells. It provided a foundation to study the function relationship of the EO and E2 glycoproteins.2. Construction of pseudotype CSFV and study on characteristic and application of pseudotype virusThree plasmids, namely pcDNA-EO/2/012, pHIT60 (including the structural genes of MuLV) and pHIT111 (including the retroviral genome, containing LacZ as a reporter) were co-transfected into HEK293T cells for the production of pseudotyped virions with EO/2/012 glycoproteins of CSFV Shimen strain. The retroviral supernatants were harvested at 48 hours post-transfection, filtered through a 0.45μM filter, and used in western blot and infection assays. Parallel transfections were carried out with supernatants produced in absence of a viral envelope and with the vesicular stomatitis virus (VSV) G proteins, which is known to efficiently pseudotype MuLV. Western-blotting revealed only E012 could be expressed on the virions, indicated the glycoprotein E012 was incorporated onto the retroviral virions. Infection test were performed on SK6、PK15、ST、BHK21、Vero、COS7、HEK293T and CEF cells. The results shown SK6、PK15 and ST infected were Lac Z positive, indicating viral entry, and revealed the pseudtype virions of MuLV-E012 were infectious. The pseudotype system of MuLV particles with CSFV E012 was set up and it could be used to study the entry of CSFV. To assess whether the CSFV pseudotyped virus entry is pH-dependent, PK15 cells were treated with well-characterized lysosomotropic agent NH4Cl. The pH dependency of infection was evaluated by pretreating PK15 cells for 1 h with serum-free 1640 containing ammonium chloride at various concentrations (0-30 mM) at 37℃; this was followed by incubation with supernatants containing the pseudotyped viruses in the presence of ammonium chloride at the consistent concentration as in the pretreating procedure. After 2 h, the supernatants were replaced with 1640 containing 10% FBS. The luciferase activity was determined 48 h later as described above. Treatment with 30 mM NH4C1 caused >90% inhibition of infection by MuLV-E012 or MuLV-VSV G. These data indicated the MuLV-E012’s entry may be pH-dependent. The pseudotyped MuLV-E012 particles were used to develop an in vitro microneutralization assay that was both sensitive and specific for CSFV neutralizing antibody. Neutralization titers measured by this assay were highly parallel with those measured by the assay using live csfv high virulence strain. Because the pseudotype assay does not require handling live CSFV virus, it is a useful tool to determine serum neutralizing titers during natural infection and the preclinical evaluation of candidate vaccines.3. B-cell epitopes of classical swine fever virus glycoprotein E2 expressed in Escherichia coli as subunit vaccine induces protection against CSFVBased on sequence analysis, three B-cell epitopes were chosen to be expressed in prokaryotic express system. One of them was multiple epitopes, the others were mono-epitope. Three recombinant expression plasmids expressing thess epitopes were constructed into pET32a vector, designated pET-rE2-a, pET-rE2-b and pET-rE2-ba. They were transformed into host bacterium BL21(DE3). Single colony was chosen to incubate in LB at 37℃, the protein were induced with 0.1mM IPTG when OD600 was 0.6, the cell grown 3 hours in 37℃. The cell were harvested and lysed with ultrasonication, and then clarified by centrifuge at 12000rpm. The supernatant and pellet were analyzed with SDS-PAGE. In all these cases, most of the proteins were found predominantly in inclusion bodies. The proteins were purified on a His·Bind chelation affinity column. The SDS-PAGE analysis indicated three proteins were purified, and displayed a single band with a molecular weight of 22 kDa,22 kDa and 25 kDa, respectively. Western blotting indicated that purified rE2-a, rE2-b and rE2-ba were recognized by CSFV positive sera specifically and reacted strongly. This suggested that the three linear peptides all possessed immunogenicity.Twenty-five 6-week-old piglets with negative CSFV antibody titers, which were purchased for animal expriments and equally divided into five groups. The pigs were acclimatized for 2 weeks, the body temperatures were measured once daily. Three groups were inoculated with purified rE2-a, rE2-b, and rE2-ba. The fourth group was immunized with a commercial vaccine (HCLV) to serve as a positive control. The fifth group was immunized with PBS as a negative control. All three proteins, rE2-a, rE2-b and rE2-ba, were combined with ISA 206 VG adjuvant in immunization. The pigs were inoculated with 50μg of each peptide for the first and second immunization at an interval of 2 weeks. Pigs immunized were inoculated intranasally (mimicking natural infection) with a lethal dose(200 TCID50) of CSFV on day 21 after the second immunization. Serum samples of immunized pigs were collected by jugular venipuncture every week before and after immunization and every week post-challenge. Each sample was heated to inactivate at 56℃for 30 min, and then was subjected to detection by a safe MuLV-E012 neutralization assay. In vivo, all these epitope-based proteins induced an antibody response and protected pigs against lethal challenge with virulent CSFV strain Shimen. The multiple epitope protein rE2-ba showed better protective effect (similar to that of HCLV vaccine) than that of mono-epitope peptide (rE2-a or rE2-b). The results demonstrated that the reactogenic and immunogenic proteins were produced by the prokaryotic system, and CSFV B-cell linear epitope peptides induced immunoregulation, similar to that of attenuated virus. Therefore, CSFV B-cell linear epitope based peptides expressed in a prokaryotic system can be used as immunogens in pigs, and may be used to develop more effective subunit vaccines. 4. Establishment and identification of a stable cell line by CSFV capsid and Staphylococcus aureus nuclease fusion proteinFor construction of the vector expressing the fusion protein, a pair of specific primers were designed and used to amplify the coding region of the Cap. Then the gene Cap was ligated into the expression plasmid pcDNA-SNase and resulted in pcDNA-Cap-SNase plasmid. The positive recombinant products were confirmed by restriction enzyme digestion and DNA sequencing. Then pcDNA-Cap-SNase was transfected into the PK-15 cells. The cell line was passaged continuously for 15 generations or more under G418 selection, which was named as PK-15/Cap-SNase cells. In order to confirm whether the screened PK-15/Cap-SNase cells expressed stably Cap gene (317 bp) and SNase gene (469 bp), PCR was performed using specific primers to amplify the products from the isolated total RNA; however, the products were not detected in Rnase-treated total RNA (negative control), indicating that fusion gene Cap-SNase was transcribed in the PK-15/Cap-SNase cells. To identify further the expression products of the recombinant plasmid, Western blot was carried out using an anti-Cap antibody. A protein band of 31 KDa (Cap=14 KDa and SNase=17 KDa) was detected in the transfected cell lysis. The expressed fusion protein was detected by indirect immunofluorescent signals in the cytoplasm.An in vitro DNA digestion assay was designed to determine nuclease activity. The activity of 5μL of cell lysate containing Cap-SNase was similar to that of 0.5 pg of a standard preparation of SNase, while the linearized plasmid DNA could not be digested when removing Ca+ by EDTA treatment, indicating that the expressed Cap-SNase retained a good Ca2+-dependent nuclease activity.5. Inhibition of replication of CSFV by capsid-targeted virus inactivationPK-15/Cap-SNase and normal PK-15 cells were infected with the CSFV Shimen strain, and then titers of the progeny virus in cell supernatants were detected. The results showed that after infection for 5 d, virus titer produced by PK-15/Cap-SNase cells was 102 lower compared with that of normal PK-15 cells. After 6 d, a greater antiviral effect was observed in the PK-15/Cap-SNase which produced a virus titer that was 103.5 lower than the control (P<0.01). The results of Real-time PCR showed that genomic copy numbers of CSFV in PK-15 cells and PK-15/Cap-SNase cells both increased with time post-infection. The virus in PK-15 cells reached peak at 6dpi then gradually decreased. However, the viral genomic copy number in PK-15/Cap-SNase cells began to decrease at 3dpi, reaching a stable low level at 6 dpi, and the inhibitory rate was 70.8% at 8 dpi. Compared with the parental PK-15 cells, the CSFV genome replication was significantly inhibited in PK-15/Cap-SNase cells (P<0.01). CSFV was positive in the supernatant of PK-15 cell culture but weakly positive in the supernatant of PK-15/Cap-SNase cell culture, further indicating fusion Cap-SNase protein mediated inhibition of CSFV replication. The ELISA results showed stable expression of Cap-SNase could inhibit the proliferation of CSFV virions in PK-15 cells.