PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation.

Viruses exploit the host cell's energy metabolism system to support their replication. Mitochondria, known as the powerhouse of the cell, play a critical role in regulating cell survival and virus replication. Our prior research indicated that the classical swine fever virus (CSFV) alters mitochondrial dynamics and triggers glycolytic metabolic reprogramming. However, the role and mechanism of PKM2, a key regulatory enzyme of glycolytic metabolism, in CSFV replication remain unclear. In this study, we discovered that CSFV enhances PKM2 expression and utilizes PKM2 to inhibit pyruvate production. Using an affinity purification coupled mass spectrometry system, we successfully identified PKM as a novel interaction partner of the CSFV non-structural protein NS4A. Furthermore, we validated the interaction between PKM2 and both CSFV NS4A and NS5A through co-immunoprecipitation and confocal analysis. PKM2 was found to promote the expression of both NS4A and NS5A. Moreover, we observed that PKM2 induces mitophagy by activating the AMPK-mTOR signaling pathway, thereby facilitating CSFV proliferation. In summary, our data reveal a novel mechanism whereby PKM2, a metabolic enzyme, promotes CSFV proliferation by inducing mitophagy. These findings offer a new avenue for developing antiviral strategies. IMPORTANCE: Viruses rely on the host cell's material-energy metabolic system for replication, inducing host metabolic disorders and subsequent immunosuppression-a major contributor to persistent viral infections. Classical swine fever virus (CSFV) is no exception. Classical swine fever is a severe acute infectious disease caused by CSFV, resulting in significant economic losses to the global pig industry. While the role of the metabolic enzyme PKM2 (pyruvate dehydrogenase) in the glycolytic pathway of tumor cells has been extensively studied, its involvement in viral infection remains relatively unknown. Our data unveil a new mechanism by which the metabolic enzyme PKM2 mediates CSFV infection, offering novel avenues for the development of antiviral strategies.

Characteristics of Classical Swine Fever Virus Variants Derived from Live Attenuated GPE- Vaccine Seed.

The GPE- strain is a live attenuated vaccine for classical swine fever (CSF) developed in Japan. In the context of increasing attention for the differentiating infected from vaccinated animals (DIVA) concept, the achievement of CSF eradication with the GPE- proposes it as a preferable backbone for a recombinant CSF marker vaccine. While its infectious cDNA clone, vGPE-, is well characterized, 10 amino acid substitutions were recognized in the genome, compared to the original GPE- vaccine seed. To clarify the GPE- seed availability, this study aimed to generate and characterize a clone possessing the identical amino acid sequence to the GPE- seed. The attempt resulted in the loss of the infectious GPE- seed clone production due to the impaired replication by an amino acid substitution in the viral polymerase NS5B. Accordingly, replication-competent GPE- seed variant clones were produced. Although they were mostly restricted to propagate in the tonsils of pigs, similarly to vGPE-, their type I interferon-inducing capacity was significantly lower than that of vGPE-. Taken together, vGPE- mainly retains ideal properties for the CSF vaccine, compared with the seed variants, and is probably useful in the development of a CSF marker vaccine.

Classical swine fever virus C-strain with eight mutation sites shows enhanced cell adaptation and protects pigs from lethal challenge.

Control of classical swine fever (CSF) in developing countries is achieved by immunization with attenuated vaccines, such as the lapinized C-strain vaccine that has been widely used in China. However, C-strain has relatively low growth rate in cell cultures, thus affecting productivity of the vaccine for the industry. In this study, eight amino acid residues were mutated on the C-strain backbone, resulting in a cell-adapted strain Cmut8. The mutant strain exhibited rapid growth with titer of about 100 fold higher than its parental C-strain. The mutation sites located at structural proteins Erns and E2 contributed more to cell adaptation than those located in non-structural proteins. Sera collected from pigs inoculated with Cmut8 and C-strain at the same dose showed similar antibody levels and neutralization titers. Pigs inoculated with different doses of Cmut8 (low, medium and high) and with C-strain offered full protection against challenge with a virulent strain, shown as absence of fever and other symptoms, marginal low levels of viral load, and no obvious gross pathological changes in major organs. Unvaccinated control pigs challenged with the virulent strain showed high fever from day 2 post-challenge and apparent clinical symptoms with two deaths. Viral load were markedly elevated in these control pigs after challenge. The pigs inoculated with high dose of Cmut8 did not show fever or other typical CSF symptoms, and no apparent pathological changes were observed in major organs. Besides, the Cmut8 strain did not induce typical fever response in rabbits. These results demonstrate that the cell-adapted Cmut8 strain remains non-pathogenic to the weaned pigs, provides full protection and could be a good candidate vaccine strain for improved yield at lower cost.

Interaction of Structural Glycoprotein E2 of Classical Swine Fever Virus with Protein Phosphatase 1 Catalytic Subunit Beta (PPP1CB).

Classical swine fever virus (CSFV) E2 protein, the major virus structural glycoprotein, is an essential component of the viral envelope. E2 is involved in virus absorption, induction of a protective immune response and is critical for virulence in swine. Using the yeast two-hybrid system, we identified protein phosphatase 1 catalytic subunit beta (PPP1CB), which is part of the Protein Phosphatase 1 (PP1) complex, as a specific binding host partner for E2. We further confirmed the occurrence of this interaction in CSFV-infected swine cells by using two independent methodologies: Co-immunoprecipitation and Proximity Ligation Assay. In addition, we demonstrated that pharmacological activation of the PP1 pathway has a negative effect on CSFV replication while inhibition of the PP1 pathway or knockdown of PPP1CB by siRNA had no observed effect. Overall, our data suggests that the CSFV E2 and PPP1CB protein interact in infected cells, and that activation of the PP1 pathway decreases virus replication.

Engagement of cellular cholesterol in the life cycle of classical swine fever virus: its potential as an antiviral target.

Classical swine fever virus (CSFV), the etiological agent of classical swine fever in pigs, is a member of the Pestivirus genus within the Flaviviridae family. It has been proposed that CSFV infection is significantly inhibited by methyl-ß-cyclodextrin (MßCD) treatment. However, the exact engagement of cellular cholesterol in the life cycle of CSFV remains unclear. Here, we demonstrated that pretreatment of PK-15 cells with MßCD significantly decreased the cellular cholesterol level and resulted in the inhibition of CSFV infection, while replenishment of exogenous cholesterol in MßCD-treated cells recovered the cellular cholesterol level and restored the viral infection. Moreover, we found that depletion of cholesterol acted on the early stage of CSFV infection and blocked its internalization into the host cells. Furthermore, we showed that 25-hydroxycholesterol, a regulator of cellular cholesterol biosynthesis, exhibited a potent anti-CSFV activity by reducing cellular cholesterol level. Taken together, our findings highlight the engagement of cholesterol in the life cycle of CSFV and its potential use as an antiviral target.

TRAF6 is a novel NS3-interacting protein that inhibits classical swine fever virus replication.

Classical swine fever virus (CSFV) non-structural protein 3 (NS3) is a multifunctional non-structural protein that plays a major role in viral replication. However, how exactly NS3 exerts these functions remains unknown. Here, we identified tumour necrosis factor receptor-associated factor 6 (TRAF6) as a novel NS3-interacting protein via yeast two-hybrid analysis, co-immunoprecipitation, and glutathione S-transferase pull-down assays. Furthermore, we observed that TRAF6 overexpression significantly inhibited CSFV replication, and TRAF6 knockdown promoted CSFV replication in porcine alveolar macrophages. Additionally, TRAF6 was degraded during CSFV infection or NS3 expression exclusively, indicating that CSFV and TRAF6 were mutually antagonistic and that TRAF6 degradation might contribute to persistent CSFV replication. Moreover, nuclear factor-kappa B (NF-κB) activity and interferon (IFN)-ß and interleukin (IL)-6 expression were increased in TRAF6-overexpressing cells, whereas TRAF6-knockdown cells exhibited decreased NF-κB activity and IFN-ß and IL-6 levels. Notably, TRAF6 overexpression did not reduce CSFV replication following inhibition of NF-κB activation by p65 knockdown. Our findings revealed that TRAF6 inhibits CSFV replication via activation of NF-κB-signalling pathways along with increases in the expression of its targets IFN-ß and IL-6. This work addresses a novel aspect concerning the regulation of innate antiviral immune response during CSFV infection.

Continuous Passaging of a Recombinant C-Strain Virus in PK-15 Cells Selects Culture-Adapted Variants that Showed Enhanced Replication but Failed to Induce Fever in Rabbits.

Classical swine fever virus (CSFV) is the etiologic agent of classical swine fever, a highly contagious disease that causes significant economic losses to the swine industry. The lapinized C-strain, a widely used vaccine strain against CSFV, has low growth efficiency in cell culture, which limits the productivity in the vaccine industry. In this study, a recombinant virus derived from C-strain was constructed and subjected to continuous passaging in PK-15 cells with the goal of acquiring a high progeny virus yield. A cell-adapted virus variant, RecCpp80, had nearly 1,000-fold higher titer than its parent C-strain but lost the ability to induce fever in rabbits. Sequence analysis of cell-adapted RecC variants indicated that at least six nucleotide changes were fixed in RecCpp80. Further adaption of RecCpp80 variant in swine testicle cells led to a higher virus yield without additional mutations. Introduction of each of these residues into the wild-type RecC backbone showed that one mutation, M979R (T3310G), located in the C-terminal region of E2 might be closely related to the cell-adapted phenotype. Rabbit inoculation revealed that RecCpp80+10 failed to induce fever in rabbits, whereas RecCpp40+10 caused a fever response similar to the commercial C-strain vaccine. In conclusion, the C-strain can be adapted to cell culture by introducing specific mutations in its E2 protein. The mutations in RecCpp80 that led to the loss of fever response in rabbits require further investigation. Continuous passaging of the C-strain-based recombinant viruses in PK-15 cells could enhance its in vitro adaption. The non-synonymous mutations at 3310 and 3531 might play major roles in the enhanced capacity of general virus reproduction. Such findings may help design a modified C-strain for improved productivity of commercial vaccines at reduced production cost.

A novel ViewRNA in situ hybridization method for the detection of the dynamic distribution of Classical Swine Fever Virus RNA in PK15 cells.

BACKGROUND: Classical swine fever (CSF) is a highly contagious fatal infectious disease caused by classical swine fever virus (CSFV). A better understanding of CSFV replication is important for the study of pathogenic mechanism of CSF. With the development of novel RNA in situ Hybridization method, quantitatively localization and visualization of the virus RNA molecular in cultured cell or tissue section becomes very important tool to address these pivotal pathogenic questions. In this study, we established ViewRNA ISH method to reveal the dynamic distribution of CSFV RNA in PK15 cells. METHODS: We designed several specific probes of CSFV RNA and reference gene ß-actin for host PK15 cells to monitor the relative location of CSFV RNA and house-keeping gene in the infected cells. After determining the titer of reference strain CSFV (HeBHH1/95) with the 50% tissue culture infective dose (TCID50), we optimized the protease K concentration and formalin fixation time to analyze the hybridization efficiency, fluorescence intensity and repeatability. In order to measure the sensitivity of this assay, we compared it with the fluorescent antibody test (FAT) and immunohistochemical(IHC) method. Specificity of the ViewRNA ISH was tested by detecting several sub genotypes of CSFV (sub genotype 1.1, 2.1, 2.2 and 2.3) which are present in China and other normal pig infectious virus (bovine viral diarrhea virus (BVDV), porcine parvovirus (PPV), porcine pseudorabies virus (PRV) and porcine circovirusII(PCV-2). RESULTS: The lowest detection threshold of the ViewRNA ISH method was 10-8, while the sensitivity of FAT and IHC were 10-5 and 10-4, respectively. The ViewRNA ISH was specific for CSFV RNA including 1.1, 2.1, 2.2 and 2.3 subtypes, meanwhile, there was no cross-reaction with negative control and other viruses including BVDV, PPV, PRV and PCV-2. Our results showed that after infection at 0.5 hpi (hours post inoculation, hpi), the CSFV RNA can be detected in nucleus and cytoplasm; during 3-9 hpi, RNA was mainly distributed in nucleus and reached a maximum at 12hpi, then RNA copy number was gradually increased around the cell nucleus during 24-48 hpi and reached the peak at 72hpi. CONCLUSIONS: To our knowledge, this is the first to reveal the dynamic distribution of medium virulence CSFV RNA in PK15 cells using the ViewRNA ISH method. The sensitivity of the ViewRNA ISH was three to four orders of magnitude higher than that of FAT and IHC methods. The specificity experiment showed that the ViewRNA ISH was highly specific for CSFV and no cross-reaction occurred to negative control and other pig infectious virus. This assay is more suitable for studying the CSFV RNA life cycle in cell nucleus. The results proved that CSFV RNA enters into PK15 cells earlier than 0.5hpi, relative to the eclipse period of cytoplasm is 6-9 hpi and CSFV RNA has ever existed in nucleus.

Interferon-Inducible Oligoadenylate Synthetase-Like Protein Acts as an Antiviral Effector against Classical Swine Fever Virus via the MDA5-Mediated Type I Interferon-Signaling Pathway.

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), which poses a serious threat to the global pig industry. Interferons (IFNs) and IFN-stimulated genes (ISGs) play a key role in host antiviral defense. We have previously screened the porcine 2'-5'-oligoadenylate synthetase-like protein (pOASL) as a potential anti-CSFV ISG using a reporter CSFV. This study aimed to clarify the underlying antiviral mechanism of pOASL against CSFV. We confirmed that CSFV replication was significantly suppressed in lentivirus-delivered, pOASL-overexpressing PK-15 cells, whereas silencing the expression of endogenous pOASL by small interfering RNAs markedly enhanced CSFV growth. In addition, the transcriptional level of pOASL was upregulated both in vitro and in vivo upon CSFV infection. Interestingly, the anti-CSFV effects of pOASL are independent of the canonical RNase L pathway but depend on the activation of the type I IFN response. Glutathione S-transferase pulldown and coimmunoprecipitation assays revealed that pOASL interacts with MDA5, a double-stranded RNA sensor, and further enhances MDA5-mediated type I IFN signaling. Moreover, we showed that pOASL exerts anti-CSFV effects in an MDA5-dependent manner. In conclusion, pOASL suppresses CSFV replication via the MDA5-mediated type I IFN-signaling pathway.IMPORTANCE The host innate immune response plays an important role in mounting the initial resistance to viral infection. Here, we identify the porcine 2'-5'-oligoadenylate synthetase-like protein (pOASL) as an interferon (IFN)-stimulated gene (ISG) against classical swine fever virus (CSFV). We demonstrate that the anti-CSFV effects of pOASL depend on the activation of type I IFN response. In addition, we show that pOASL, as an MDA5-interacting protein, is a coactivator of MDA5-mediated IFN induction to exert anti-CSFV actions. This work will be beneficial to the development of novel anti-CSFV strategies by targeting pOASL.

Establishment and characterization of a chimeric infectious cDNA clone of classical swine fever virus.

Classical swine fever virus (CSFV) causes a highly contagious disease among swine that has an important economic impact worldwide. There are two important CSFV strains in China, Shimen and hog cholera lapinized virus (HCLV). Shimen strain is highly virulent while HCLV, also referred to as C-strain, is a live attenuated vaccine strain considered to be one of the most effective and safest live vaccines. In this study, a chimeric infectious cDNA clone of CSFV named pT7SM-c was engineered by replacing the E(rns) genomic region of an infectious clone of CSFV Shimen strain, pT7SM, with the same region obtained from HCLV. RNA transcripts of pT7SM-c containing an engineered EcoRI site that served as a genetic marker were directly infectious in PK15 cells. The rescued virus vT7SM-c showed similar growth kinetics and cytopathic effect with the parental virus vT7SM in the cells. The chimeric infectious cDNA clone can be used as a practical tool for further studying of the virulence, protein function and pathogenesis of CSFV through genetic manipulation.

Generation and evaluation of a chimeric classical swine fever virus expressing a visible marker gene.

Classical swine fever virus (CSFV) is a noncytopathogenic virus, and the incorporation of an enhanced green fluorescent protein (EGFP) tag into the viral genome provides a means of direct monitoring of viral infection without immunostaining. It is well established that the 3' untranslated region (3'-UTR) of the CSFV plays an important role in viral RNA replication. Although CSFV carrying a reporter gene and chimeric CSFV have been generated and evaluated, a chimeric CSFV with a visible marker has not yet been reported. Here, we generated and evaluated a chimeric virus containing the EGFP tag and the 3'-UTR from vaccine strain HCLV (C-strain) in the genetic background of the highly virulent CSFV Shimen strain. The chimeric marker CSFV was fluorescent and had an approximately 100-fold lower viral titer, lower replication level of viral genome, and weaker fluorescence intensity than the recombinant CSFV with only the EGFP tag or the parental virus. Furthermore, the marker chimera was avirulent and displayed no viremia in inoculated pigs, which were completely protected from lethal CSFV challenge as early as 15 days post-inoculation. The chimeric marker virus was visible in vitro and attenuated in vitro and in vivo, which suggests that CSFV can be engineered to produce attenuated variants with a visible marker to facilitate in vitro studies of CSFV infection and replication and to develop of novel vaccines against CSF.

N pro of Bungowannah virus exhibits the same antagonistic function in the IFN induction pathway than that of other classical pestiviruses.

Bungowannah virus is the most divergent atypical pestivirus that had been detected up to now, and does not fit into any of the four approved species: Bovine viral diarrhea virus type 1 (BVDV-1) and type 2 (BVDV-2), Classical swine fever virus (CSFV) and Border disease virus (BDV). However, the presence of N(pro) and E(rns) coding regions, which are unique to pestiviruses, provides clear evidence of a pestivirus. Nevertheless, the amino acid identity of Bungowannah virus N(pro) and BVDV-1 N(pro) (strain CP7) is only 51.5%. By using a BVDV-1 backbone, a novel chimeric construct was generated, in which the genomic region encoding the non-structural protein N(pro) was replaced by that of Bungowannah virus (CP7_N(pro)-Bungo). In vitro studies of CP7_N(pro)-Bungo revealed autonomous replication with the same efficacy as the BVDV backbone CP7 and infectious high-titer virus could be collected. In order to compare the ability of interferon (IFN) suppression, two reporter gene assays, specific for type-I IFN, were carried out. In virus-infected cells, no significant difference in blocking of IFN expression between the parental virus CP7, Bungowannah virus and the chimeric construct CP7_N(pro)-Bungo could be detected. In contrast, an N(pro) deletion mutant showed an impaired replication in bovine cells and a marked type-I IFN response. Taken together, our findings reveal the compatibility of non-structural protein N(pro) of atypical Bungowannah virus with a BVDV type 1 backbone and its characteristic feature as an inhibitor of type-I IFN induction with an inhibitor-activity comparable to other pestiviruses.

Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA.

BACKGROUND: Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. RESULTS: A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable rescue of chimeric pestiviruses (vR26_E2gif and vR26_TAV) with potential as new marker vaccine candidates. Sequencing of the BACs revealed a high genetic stability during passages within bacteria. The complete genome sequences of rescued viruses, after extensive passages in mammalian cells showed that modifications in the E2 protein coding sequence were stably maintained. A single amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence. CONCLUSIONS: These results show that targeted recombination-mediated mutagenesis provides a powerful tool for expediting the construction of novel RNA genomes and should be applicable to the manipulation of other RNA viruses.

Application of real-time quantitative polymerase chain reaction to monitoring infection of classic swine fever virus and determining optimal harvest time in large-scale production.

Due to the non-cytopathogenic replication of classical swine fever virus (CSFV) in cell culture, large-scale production of CSFV using bioreactor system remains the problem of monitoring the time of maximum virus production for optimal harvest. In this study, we proposed the application of real-time quantitative PCR assay to monitoring the progress of CSFV infection and yield determination in large scale. The region of NS5B of CSFV responsible for CSFV genome replication was used for the designation of primers and probe. Viral titers determined by the real-time quantitative PCR assay were compared with the conventional cell-culture based method of immunofluorescent staining. Results from large scale production show that a similar profile of CSFV production was successfully outlined by real-time quantitative PCR and virus yields were comparable to the results from immunofluorescent staining assay. By using this method, an optimal harvesting time of the production could be rapidly and precisely determined leading to an improvement in virus harvest.

The feasibility of a novel bioreactor for vaccine production of classical swine fever virus.

The performance of a new type of tide mode culture system was investigated in this study. This novel bioreactor provides two separated stages, liquid and gas, for cell growth requirements. The immobilized cells absorbed the nutrient from medium during the liquid stage and subsequently were exposed directly to fresh air to absorb oxygen during the gas stage. Operating with PK15 cells under optimal conditions, we obtained 2.3×10(9) cells in 500ml reactor. It is 30 times higher than the initial inoculum and about 11 times higher than the production by roller bottle. For the vaccine production of classical swine fever (CSFV), a high virus titer of 2.1×10(9) median tissue culture infective dose (TCID(50)) was yielded which provided exceed 300 doses per milliliter of CSFV solution. Therefore, this new cultural system performed well not only for cell production but also for virus yield. It should be a highly efficient production for the CSFV vaccine and have practical potential in other animal cell culture vaccine.

Poly(C)-binding protein 1, a novel N(pro)-interacting protein involved in classical swine fever virus growth.

N(pro) is a multifunctional autoprotease unique to pestiviruses. The interacting partners of the N(pro) protein of classical swine fever virus (CSFV), a swine pestivirus, have been insufficiently defined. Using a yeast two-hybrid screen, we identified poly(C)-binding protein 1 (PCBP1) as a novel interacting partner of the CSFV N(pro) protein and confirmed this by coimmunoprecipitation, glutathione S-transferase (GST) pulldown, and confocal assays. Knockdown of PCBP1 by small interfering RNA suppressed CSFV growth, while overexpression of PCBP1 promoted CSFV growth. Furthermore, we showed that type I interferon was downregulated by PCBP1, as well as N(pro). Our results suggest that cellular PCBP1 positively modulates CSFV growth.

Selection of classical swine fever virus with enhanced pathogenicity reveals synergistic virulence determinants in E2 and NS4B.

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious disease of pigs. There are numerous CSFV strains that differ in virulence, resulting in clinical disease with different degrees of severity. Low-virulent and moderately virulent isolates cause a mild and often chronic disease, while highly virulent isolates cause an acute and mostly lethal hemorrhagic fever. The live attenuated vaccine strain GPE(-) was produced by multiple passages of the virulent ALD strain in cells of swine, bovine, and guinea pig origin. With the aim of identifying the determinants responsible for the attenuation, the GPE(-) vaccine virus was readapted to pigs by serial passages of infected tonsil homogenates until prolonged viremia and typical signs of CSF were observed. The GPE(-)/P-11 virus isolated from the tonsils after the 11th passage in vivo had acquired 3 amino acid substitutions in E2 (T830A) and NS4B (V2475A and A2563V) compared with the virus before passages. Experimental infection of pigs with the mutants reconstructed by reverse genetics confirmed that these amino acid substitutions were responsible for the acquisition of pathogenicity. Studies in vitro indicated that the substitution in E2 influenced virus spreading and that the changes in NS4B enhanced the viral RNA replication. In conclusion, the present study identified residues in E2 and NS4B of CSFV that can act synergistically to influence virus replication efficiency in vitro and pathogenicity in pigs.

Classical swine fever virus NS5A protein interacts with 3'-untranslated region and regulates viral RNA synthesis.

To investigate the function of classical swine fever virus (CSFV) NS5A protein, the experiments for viral RNA synthesis and viral replication were performed in the co-presence of NS5A and NS5B. Results showed that small concentrations of NS5A stimulated, large concentrations of NS5A inhibited, viral RNA synthesis and viral replication. Affinity chromatography experiments and UV-crosslinking assays revealed that CSFV NS5A and NS5B bound its cognate 3'UTR and that NS5A had higher affinity than NS5B protein in binding to 3'UTR. 200 ng of NS5A inhibited NS5B-3'UTR complex formation by about 95%. CSFV 3'UTR was found to contain two NS5A-binding sites, located in 3'UTRSL-1 (nt 161-231) and 3'UTRSL-2 (nt 90-160), respectively, a NS5B-binding site, also located in 3'UTRSL-1. The 3'UTRSL-1 is the common binding site for NS5A and NS5B. Furthermore, competitive electrophoretic mobility shift assays indicated that binding of CSFV NS5A to 3'UTRSL-1 is more efficiently than to 3'UTRSL-2. These results suggested that the different concentrations of NS5A, the different binding activities of NS5A and NS5B to 3'UTR and binding of NS5A to different regions of 3'UTR might contribute at least partially to modulation of CSFV replication.

Inhibition of replication of classical swine fever virus in a stable cell line by the viral capsid and Staphylococcus aureus nuclease fusion protein.

Classical swine fever (CSF) is one of the major diseases causing serious economic losses to the swine industry. To explore the feasibility of using capsid-targeted viral inactivation (CTVI) as an antiviral strategy against CSF infection, a plasmid pcDNA-Cap-SNase was constructed for expressing a fusion protein of CSFV capsid (Cap) and Staphylococcus aureus nuclease (SNase). Under G418 selection, a mammalian cell line PK-15 expressing stably the fusion protein Cap-SNase(PK-15/Cap-SNase) could be detected by rabbit antiserum against CSFV capsid protein and had good nuclease activity in cleaving linearized plasmid DNA. The CSFV titer produced from infection of this PK-15/Cap-SNase stable cell line was reduced by an order of 10(2)-10(3.5) or 70.8% compared to that produced in control PK-15 cells. Detection of the virus by ELISA indicated that CSFV propagation was inhibited in the PK-15/Cap-SNase cell line. It was demonstrated clearly that the fusion protein Cap-SNase could inhibit effectively the production of CSFV, resulting in a reduction in infectious titers. Therefore, CTVI may be valuable therapeutic approach against CSFV.

Inactivation of classical swine fever virus in porcine casing preserved in salt.

Pig intestines used for the production of natural sausage casings may carry classical swine fever (CSF) virus. Feeding pigs with human food waste that contains pig casings may then spread the virus to CSF-free animals. Casings derived from a pig experimentally infected with CSF by dosing with 10(6) tissue culture infectious doses (TCID50) of the highly virulent CSF virus strain "Koslov", were treated with phosphate supplemented or citrate supplemented NaCl, instead of with NaCl alone, which is the standard preservation treatment for casings. Treated casings were stored for 30 days at either 4 degrees C or 20 degrees C. After storage the casings were fed to 16 susceptible pigs. CSF infection was confirmed in the four animals that had been fed casings treated with citrate supplemented salt and stored at 4 degrees C. All other animals remained healthy. It is therefore possible to avoid the inadvertent spread of CSF virus via porcine sausage casings by treating casings with phosphate supplemented salt and storing them for 30 days at temperatures over 4 degrees C.