Classical Swine Fever Virus Shimen Research Articles

Generation and epitope mapping of novel neutralizing monoclonal antibodies against glycoprotein E2 of CSFV

Classical swine fever virus (CSFV) is a highly contagious and economically important pathogen threatening pig industry worldwide, the envelope glycoprotein E2 of CSFV is the dominant antigen inducing strong antiviral neutralizing immunity. In this study, 7 monoclonal antibodies (mAbs) with neutralizing potency were generated using E2 protein of CSFV Shimen strain (SM) expressed by eukaryotic cells. Their reactivity with 116 CSFV strains in cell cultures and E2 proteins of 10 subgenotypes in western blots showed different CSFV spectrums they recognized. Of them, three (HCL-001, HCL-005 and HCL-010) reacted with all CSFV subgenotypes, while HCL-014 and HCL-002 reacted with most CSFV strains, except for some variants in genotype 2.3. In contrast, mAb HCL-009 reacted only with a few subgenotype 1.1 strains including SM, field strains and some vaccine strains. Interestingly, mAb HCL-018 reacted only with SM and field subgenotype 1.1 strains, not with any vaccine strains. Further epitope mapping using chimeric and site-directed mutated E2 proteins showed that HCL-001, HCL-005 and HCL-010 recognized a conservative epitope motif 143SPT145,L147, and HCL-002 recognized a conformational epitope with key aa motifs of 95GDD97,157RX(D/E)K(R)XFXXR164. HCL-014 recognized a new conservative epitope with key aa motifs of 41D,58XNVVXRR64. HCL-009 and HCL-018 recognized the epitope with key aa motifs of 36D,40ND41,45KXI47 and 69LHXGXLLT76, respectively. Taken together, present study has provided not only new insights into the antigenic structure of E2 protein, but also key reagents for antigenic characterization of CSFV strains and development of antibody assay for evaluation of the vaccination efficacy.

Generation of a DNA-launched classical swine fever virus infectious clone packaged in bacterial artificial chromosome

Reverse genetics system offers powerful tool for the research of RNA viruses. The infectious clones of classical swine fever virus (CSFV) were commonly constructed either in high- or low-copy number plasmids and transcribed to infectious RNA using phage RNA-polymerases. Herein, the full-length genome of CSFV Shimen strain, flanked by cytomegalovirus immediate-early (CMV) promoter (a eukaryotic RNA polymerase II promoter) sequence at the 5′-end and the hepatitis delta virus ribozyme along with the bovine growth hormone termination and polyadenylation signal sequences at the 3′-end, was packaged in bacterial artificial chromosome vector to establish a CSFV infectious clone pBAC-smCSFV. This infectious cDNA clone maintained stability after passaged 20 times in bacteria. Transfection of PK15 cells with this cDNA clone facilitated recovery of infectious progeny virus which was identical to parent virus as characterized by RT-qPCR, western blotting, indirect immunofluorescence assay, one-step growth kinetics analysis and nucleotide sequencing. Based on this CSFV infectious cDNA clone, the mCherry was inserted between viral Npro and C protein to develop reporter virus CSFV-mCherry. The mCherry was stably expressed after CSFV-mCherry was passaged 10 times in PK15 cells. Taken together, this present study develops a concise and efficient CSFV infectious cDNA clone and a reporter virus CSFV-mCherry. To the best of our knowledge, this is the first combination of CMV promoter and BAC system in construction of CSFV reverse genetics system. The CSFV infectious cDNA clone and the reporter virus will be useful in the study of CSFV virus biology, virulence determinants, molecular pathogenesis, vaccine development and virus-host interaction.

Proline to Threonine Mutation at Position 162 of NS5B of Classical Swine Fever Virus Vaccine C Strain Promoted Genome Replication and Infectious Virus Production by Facilitating Initiation of RNA Synthesis.

The 3′untranslated region (3′UTR) and NS5B of classical swine fever virus (CSFV) play vital roles in viral genome replication. In this study, two chimeric viruses, vC/SM3′UTR and vC/b3′UTR, with 3′UTR substitution of CSFV Shimen strain or bovine viral diarrhea virus (BVDV) NADL strain, were constructed based on the infectious cDNA clone of CSFV vaccine C strain, respectively. After virus rescue, each recombinant chimeric virus was subjected to continuous passages in PK-15 cells. The representative passaged viruses were characterized and sequenced. Serial passages resulted in generation of mutations and the passaged viruses exhibited significantly increased genomic replication efficiency and infectious virus production compared to parent viruses. A proline to threonine mutation at position 162 of NS5B was identified in both passaged vC/SM3′UTR and vC/b3′UTR. We generated P162T mutants of two chimeras using the reverse genetics system, separately. The single P162T mutation in NS5B of vC/SM3′UTR or vC/b3′UTR played a key role in increased viral genome replication and infectious virus production. The P162T mutation increased vC/SM3′UTRP162T replication in rabbits. From RNA-dependent RNA polymerase (RdRp) assays in vitro, the NS5B containing P162T mutation (NS5BP162T) exhibited enhanced RdRp activity for different RNA templates. We further identified that the enhanced RdRp activity originated from increased initiation efficiency of RNA synthesis. These findings revealed a novel function for the NS5B residue 162 in modulating pestivirus replication.

A self-assembling nanoparticle: Implications for the development of thermostable vaccine candidates

Effective controls on viral infections rely on the continuous development in vaccine technology. Nanoparticle (NP) antigens are highly immunogenic based on their unique physicochemical properties, making them molecular scaffolds to present soluble vaccine antigens. Here, viral targets (113–354 aas) were genetically fused to N terminal of mi3, a protein that self-assembles into nanoparticles composed of 60 subunits. With transmission electron microscopy, it was confirmed that target-mi3 fusion proteins which have insertions of up to 354 aas in N terminal form intact NPs. Moreover, viral targets are surface-displayed on NPs as indicated in dynamic light scattering. NPs exhibit perfect stability after long-term storage at room temperature. Moreover, SP-E2-mi3 NPs enhance antigen uptake and maturation in dendritic cells (DCs) via up-regulating marker molecules and immunostimulatory cytokines. Importantly, in a mouse model, SP-E2-mi3 nanovaccines against Classical swine fever virus (CSFV) remarkably improved CSFV-specific neutralizing antibodies (NAbs) and cellular immunity related cytokines (IFN-γ and IL-4) as compared to monomeric E2. Specially, improved NAb response with more than tenfold increase in NAb titer against both CSFV Shimen and HZ-08 strains indicated better cross-protection against different genotypes. Collectively, this structure-based, self-assembling NP provides an attractive platform to improve the potency of subunit vaccine for emerging pathogens.

Coordinated expression of vascular endothelial growth factor A and urokinase-type plasminogen activator contributes to classical swine fever virus Shimen infection in macrophages

BackgroundThe Shimen strain of classical swine fever (CSF) virus (CSFV) causes CSF, which is mainly characterised by disseminated intravascular haemorrhage. Macrophages are an essential component of innate immunity against pathogenic microorganisms; however, the role of macrophages in CSF pathogenesis remains unclear. To illuminate the infective mechanism of CSFV, we used gene co-expression networks derived from macrophages infected with CSFV Shimen and CSFV C as well as uninfected macrophages to screen key regulatory genes, and their contributions to the pathogenesis of CSF were discussed.ResultsVascular endothelial growth factor A (VEGFA) and plasminogen activator, urokinase (PLAU, which encodes urokinase-type plasminogen activator [uPA]) were identified as coordinated genes expressed in macrophages by gene co-expression networks. Quantitative polymerase chain reaction and western blot analysis confirmed that VEGFA and PLAU were significantly up-regulated at both the transcription and translation levels after infection. Further, confocal microscopy analysis proposed that the VEGFA and uPA proteins were temporally co-localised with the CSFV protein E2.ConclusionsOur findings suggest that co-expression of VEGFA and PLAU in macrophages contributes to CSFV Shimen infection and serves as a significant avenue for the strain to form an inflammatory microenvironment, providing new insight into the mechanisms of CSF caused by a virulent strain.

Development of functionalized gold nanoparticles as nanoflare probes for rapid detection of classical swine fever virus.

Classical swine fever (CSF) is a devastating viral disease affecting pigs that causes major economic losses worldwide. Conventional assays to identify classical swine fever virus (CSFV) face challenges, such as the required molecular amplification of the target molecules via polymerase chain reaction (PCR). We designed a gold nanoflare probe to directly detect CSFV. Gold nanoparticles (AuNPs) were conjugated with a pair of complementary DNA sequences that specifically recognized and captured CSFV RNA, resulting in a fluorescence signal to indicate the existence of CSFV. The constructed nanocomposite was then utilized in a quantitative analysis to recognize the virus sequence present at amounts as low as 50 pg/μL. The CSFV-AuNP probe enabled real-time, quantitative detection of native CSFV in response to doses of the specific RNA sequence (CSFV NS2) that indicated active viral replication of CSFV Shimen in macrophages after 12, 24, and 48 h. The potential diagnostic applications of the probe were demonstrated by measuring CSFV without nucleic acid amplification in samples from seven types of tissue samples, specifically heart, spleen, kidney, liver, lymph, intestine, and muscle samples obtained from one pig confirmed to suffer CSF. The speed, sensitivity, and versatility of this CSFV-AuNP biosensor make it an ideal candidate for further application in the prevention and control of animal epidemic diseases.

Comprehensive evaluation of the host responses to infection with differentially virulent classical swine fever virus strains in pigs

Classical swine fever virus (CSFV) infection causes most variable clinical syndromes from chronic or latent infection to acute death, and it is generally acknowledged that the course of disease is affected by both virus and host factors. To compare host immune responses to differentially virulent CSFV strains in pigs, fifteen 8-week-old specific-pathogen-free pigs were randomly divided into four groups and inoculated with the CSFV Shimen strain (a highly virulent strain), the HLJZZ2014 strain (a moderately virulent strains), C-strain (an avirulent strain), and DMEM (mock control), respectively. Infection with the Shimen or HLJZZ2014 strain resulted in fever, clinical signs and histopathological lesions, which were not observed in the C-strain-inoculated pigs, though low viral genome copies were detected in the peripheral blood and tissue samples. The data showed that the virulence of the strains affected the outcome of duration and intensity of the disease rather than the tissue tropism of the virus. Furthermore, leukopenia, lymphocytopenia, differentiation of T-cells, and the secretion of cytokines associated with inflammation or apoptosis such as interferon alpha (IFN-α), tumor necrosis factor alpha (TNF-α), interleukin 2 (IL-2), IL-4, IL-6, and IL-10 were induced by the virulent CSFV infection, the differences reflected in onset and extent of the regulation. Taken together, our results revealed that the major differences among the three strains resided in the kinetics of host response to the infection: severe and immediate with the highly virulent strain, while progressive and delayed with the moderately virulent one. This comparative study will help to dissect the pathogenesis of CSFV.

Tissue expression of Toll-like receptors 2, 3, 4 and 7 in swine in response to the Shimen strain of classical swine fever virus.

The Toll-like receptors (TLRs) of the innate immune system provide the host with the ability to detect and respond to viral infections. The present study aimed to investigate the mRNA and protein expression levels of TLR2, 3, 4 and 7 in porcine tissues upon infection with the highly virulent Shimen strain of classical swine fever virus (CSFV). Reverse transcription-quantitative polymerase chain reaction was used to detect the mRNA expression levels of CSFV and TLR, whereas western blotting was used to detect the expression levels of TLR proteins. In addition, tissues underwent histological examination and immunohistochemistry to reveal the histopathological alterations associated with highly virulent CSFV infection and to detect TLR antigens. Furthermore, porcine monocyte-derived macrophages (pMDMs) were prestimulated with peptidoglycan from Staphylococcus aureus (PGN-SA), polyinosinic-polycytidylic acid [poly (I:C)], lipopolysaccharide from Escherichia coli 055:B5 (LPS-B5) or imiquimod (R837) in order to analyze the association between TLR expression and CSFV replication. Following stimulation for 12 h (with TLR-specific ligands), cells were infected with CSFV Shimen strain. The results revealed that the expression levels of TLR2 and TLR4 were increased in the lung and kidney, but were decreased in the spleen and lymph nodes in response to CSFV. TLR3 was strongly expressed in the heart and slightly upregulated in the spleen in response to CSFV Shimen strain infection, and TLR7 was increased in all examined tissues in the presence of CSFV. Furthermore, R837 and LPS-B5 exerted inhibitory effects on CSFV replication in pMDMs, whereas PGN-SA and poly(I:C) had no significant effect. These findings highlight the potential role of TLR expression in the context of CSFV infection.

Tendency Cluster Analysis Based on the Deep Sequencing Database to Explore the Response Mechanism of Swine Umbilical Vein Endothelial Cells to Classical Swine Fever Virus Infection

Endothelial cell is one of main targets of classical swine fever virus (CSFV) infection that causes classical swine fever (CSF). Although the pathogenesis of CSF has been studied for a long time, the new method is still needed to elucidate the underlying mechanism. Genomics analysis based on the deep sequencing provides a possibility for further studies of CSFV infection mechanism. Tendency cluster analysis and bioinformatics analysis were developed for interpreting the significant gene responses of endothelial cells to CSFV in this work. Key genes contributed to CSFV Shimen were identified differing from CSFV C infection and the control. GO (gene ontology) and KEGG analysis suggested that CSFV Shimen infection caused changes in biological processes such as cytoplasmic sequestering of NF-kappaB, regulation of apoptotic process and toll-like receptor 4 signaling pathway. And there was a significant response from PI3K-Akt signaling pathway, viral myocarditis, prolactin signaling pathway and so on during the Shimen infestation. Trend analysis based on series-cluster for deep sequencing database obtained better interpretation to CSFV Shimen, which provided more valuable information to understand the development of CSF.

Classical swine fever virus Shimen infection increases p53 signaling to promote cell cycle arrest in porcine alveolar macrophages.

Classical swine fever virus (CSFV) replicates in macrophages and causes persistent infection. Despite its role in disastrous economic losses in swine industries, the molecular mechanisms underlying its pathogenesis are poorly understood. The virus evades the neutralizing immune response, subverting the immune system to ensure its own survival and persistence. Our genome-wide analysis of porcine alveolar macrophage transcriptional responses to CSFV Shimen infection using the Solexa/Illumina digital gene expression system revealed that p53 pathway components and cell cycle molecules were differentially regulated during infection compared to controls. Further, we investigated the molecular changes in macrophages infected with CSFV Shimen, focusing on the genes involved in the p53 pathway. CSFV Shimen infection led to phosphorylation and accumulation of p53 in a time-dependent manner. Furthermore, CSFV Shimen infection upregulated cyclin-dependent kinase inhibitor 1A (p21) mRNA and protein. In addition, CSFV Shimen infection induced cell cycle arrest at the G1 phase, as well as downregulation of cyclin E1 and cyclin-dependent kinase 2 (CDK2). The expression of genes in the p53 pathway did not change significantly after p53 knockdown by pifithrin-α during CSFV Shimen infection. Our data suggest that CSFV Shimen infection increases expression of host p53 and p21, and inhibits expression of cyclin E1 and CDK2, leading to cell cycle arrest at the G1 phase. CSFV may utilize this strategy to subvert the innate immune response and proliferate in host cells.

Efficacy of the marker vaccine rAdV-SFV-E2 against classical swine fever in the presence of maternally derived antibodies to rAdV-SFV-E2 or C-strain

Classical swine fever (CSF) is an economically important disease caused by Classical swine fever virus (CSFV). In order to eradicate CSF, many marker vaccines that allow differentiation of infected from vaccinated animals (DIVA) have been developed. In our previous studies, a DIVA CSF vaccine rAdV-SFV-E2 has been demonstrated to completely protect pigs against lethal CSFV challenge. In the context of risk assessments for an emergency vaccination scenario, the question has been raised whether preexisting maternally derived antibodies (MDAs) interfere with the efficacy of the vaccine. In this study, six groups of piglets (n=5), with or without anti-C-strain or anti-rAdV-SFV-E2 MDAs, were immunized twice with 106 TCID50 rAdV-SFV-E2 and challenged with the CSFV Shimen strain. Clinical signs, CSFV-specific antibodies, viremia and pathological and histopathological changes were monitored. The results showed that the vaccinated piglets, either with or without MDAs directed against C-strain (about 67% blocking rate) or rAdV-SFV-E2 (about 50% blocking rate) were completely protected; however, the mock-vaccinated piglets displayed severe CSF-typical clinical symptoms, viremia, pathological/histopathological changes and deaths (5/5). These findings demonstrate that the MDAs to either rAdV-SFV-E2 or C-strain do not interfere with the efficacy of rAdV-SFV-E2, which highlights the great potential of the vaccine for control and eradication of CSF.

Caveolin-1-mediated endocytic pathway is involved in classical swine fever virus Shimen infection of porcine alveolar macrophages

Macrophages are at the frontline of defense against pathogenic microorganisms. However, very little is known about the cell invasion mechanism of classical swine fever virus (CSFV) Shimen strain. To elucidate the infective mechanism of this important pathogen, we screened deep-sequencing data derived from macrophages infected with CSFV Shimen and uninfected macrophages, and identified a role of caveolin-1 (CAV1). Digital gene expression profiling showed that CAV1 was differentially expressed in CSFV Shimen-infected macrophages. Quantitative polymerase chain reaction and western blot analyses showed that the transcription and translation of CAV1 were significantly up-regulated in CSFV Shimen-infected macrophages. In addition, immunofluorescent confocal microscopy analysis suggested that CAV1 was temporally colocalized with CSFV E2 throughout the course of the infection. Through the overexpression of recombinant CAV1 or the silencing of CAV1 expression using small interfering RNA in macrophages, we demonstrated that CAV1 expression is beneficial for the replication of CSFV Shimen. However, RNA silencing of CAV1 did not prevent viral replication, which may indicate that CSFV can also enter macrophages by other mechanisms. Our findings suggest that CAV1-mediated endocytosis is advantageous for productive CSFV Shimen infection in macrophages, providing a new insight into the mechanisms of evasion of host immunity for successful viral infection.

Different RNA splicing mechanisms contribute to diverse infective outcome of classical swine fever viruses of differing virulence: insights from the deep sequencing data in swine umbilical vein endothelial cells.

Molecular mechanisms underlying RNA splicing regulation in response to viral infection are poorly understood. Classical swine fever (CSF), one of the most economically important and highly contagious swine diseases worldwide, is caused by classical swine fever virus (CSFV). Here, we used high-throughput sequencing to obtain the digital gene expression (DGE) profile in swine umbilical vein endothelial cells (SUVEC) to identify different response genes for CSFV by using both Shimen and C strains. The numbers of clean tags obtained from the libraries of the control and both CSFV-infected libraries were 3,473,370, 3,498,355, and 3,327,493 respectively. In the comparison among the control, CSFV-C, and CSFV-Shimen groups, 644, 158, and 677 differentially expressed genes (DEGs) were confirmed in the three groups. Pathway enrichment analysis showed that many of these DEGs were enriched in spliceosome, ribosome, proteasome, ubiquitin-mediated proteolysis, cell cycle, focal adhesion, Wnt signalling pathway, etc., where the processes differ between CSFV strains of differing virulence. To further elucidate important mechanisms related to the differential infection by the CSFV Shimen and C strains, we identified four possible profiles to assess the significantly expressed genes only by CSFV Shimen or CSFV C strain. GO analysis showed that infection with CSFV Shimen and C strains disturbed ‘RNA splicing’ of SUVEC, resulting in differential ‘gene expression’ in SUVEC. Mammalian target of rapamycin (mTOR) was identified as a significant response regulator contributed to impact on SUVEC function for CSFV Shimen. This computational study suggests that CSFV of differing virulence could induce alterations in RNA splicing regulation in the host cell to change cell metabolism, resulting in acute haemorrhage and pathological damage or infectious tolerance.

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.

Discovering up-regulated VEGF-C expression in swine umbilical vein endothelial cells by classical swine fever virus Shimen.

Infection of domestic swine with the highly virulent Shimen strain of classical swine fever virus causes hemorrhagic lymphadenitis and diffuse hemorrhaging in infected swine. We analyzed patterns of gene expression for CSFV Shimen in swine umbilical vein endothelial cells (SUVECs). Transcription of the vascular endothelial growth factor (VEGF) C gene (VEGF-C) and translation of the corresponding protein were significantly up-regulated in SUVECs. Our findings suggest that VEGF-C is involved in mechanisms of acute infection caused by virulent strains of CSFV.

Rapid Recovery of Classical Swine Fever Virus Directly from Cloned cDNA

The reverse genetics for classical swine fever virus (CSFV) is currently based on the transfection of in vitro transcribed RNA from a viral genomic cDNA clone, which is inefficient and time-consuming. This study was aimed to develop an improved method for rapid recovery of CSFV directly from cloned cDNA. Full-length genomic cDNA from the CSFV Shimen strain, which was flanked by a T7 promoter, the hepatitis delta virus ribozyme and T7 terminator sequences, was cloned into the low-copy vector pOK12, producing pOKShimen-RzTΦ. Direct transfection of pOKShimen-RzTΦ into PK/T7 cells, a PK-15-derived cell line stably expressing bacteriophage T7 RNA polymerase, allowed CSFV to be rescued rapidly and efficiently, i.e., at least 12 h faster and 31.6-fold greater viral titer when compared with the in vitro transcription-based rescue system. Furthermore, the progeny virus rescued from PK/T7 cells was indistinguishable, both in vitro and in vivo, from its parent virus and the virus rescued from classical reverse genetics. The reverse genetics based on intracellular transcription is efficient, convenient and cost-effective. The PK/T7 cell line can be used to rescue CSFV directly from cloned cDNA and it can also be used as an intracellular transcription and expression system for studying the structure and function of viral genes.

Efficient and stable rescue of classical swine fever virus from cloned cDNA using an RNA polymerase II system

Conventional reverse genetics for classical swine fever virus (CSFV) is based on the transfection of permissive cells with either in vitro or intracellularly synthesized RNA transcripts from a viral genomic cDNA clone. These strategies are complicated, inefficient and time-consuming. This study is aimed to develop an improved reverse genetics method for the direct, rapid and efficient recovery of CSFV from cloned cDNA. The cDNA clone pBRCISM was constructed, which harbors the full-length genomic sequence from the CSFV Shimen strain flanked by the cytomegalovirus promoter (an RNA polymerase II promoter), a chimeric intron, and hammerhead ribozyme sequences at the 5'-end and the hepatitis delta virus ribozyme and SV40 polyadenylation signal sequences at the 3'-end. Infectious progeny virus was rescued from PK-15 cells directly transfected with pBRCISM, and its morphology, one-step growth characteristics and pathogenicity were indistinguishable from the parent virus and virus rescued from classical reverse genetics. The reverse genetics based on RNA polymerase II yielded a 120-fold increase in the titer of nascent virus in 12-h less time than a reverse genetics method based on in vitro transcription. The full-length cDNA clone remained stable and infectious after 20 passages in bacterial cells, in contrast to the instability of the full-length clone without the intron after 9 passages. The improved reverse genetics method developed in the present study is efficient, stable, convenient and cost-effective and will be valuable for the rapid recovery of CSFV mutants.

Influence of PPV, PRV and PRRSV on Efficacy of the Lapinized Hog Cholera Vaccine and Pathogenicity of Classical swine fever virus

Classical swine fever caused by Classical swine fever virus (CSFV) is a serious problem for swine industries in developing countries, which successful control of the disease have been relying on vaccination. However, classical swine fever still occurs in some immunized swine herds for various reasons. In this study, we conducted animal experiments to examine the influence of single or mixed infection with Porcine parvo virus (PPV), Pseudorabies virus (PRV) and Porcine reproductive and respiratory syndrome virus (PRRSV) on the protective immunity induced by the Lapinized hog cholera virus (HCLV) vaccine and the pathogenicity of CSFV. In experiment 1, pigs were first inoculated with PPV, PRV or PRRSV, then immunized with HCLV, and finally challenged with a highly virulent CSFV Shimen strain. All of the pigs immunized with HCLV survived after the challenge, while all of the pigs in the non-immunized control group died after the challenge. The pigs in the group immunized with HCLV did not show any clinical symptoms of classical swine fever and were negative with CSFV after the challenge. The pigs infected with the non-CSFV before HCLV immunization did not display any clinical symptoms after the challenge with CSFV Shiman strain, but 11 of the 12 pigs were positive with CSFV. In experiment 2, pre-infections with PPV, PRV, and PRRSV were followed by inoculation with a low-virulence CSFV strain (CSFV 39), and then the pigs were challenged with the CSFV Shimen strain. Infections by either PPV, PRV or PRRSV did not enhance the virulence of CSFV-39, but pigs infected by a mixture of the 3 viruses developed clinical symptoms after inoculation with CSFV-39. The mixed infection also increased mortality caused by the challenge with the CSFV Shimen strain. Together, these results showed PPV, PRV and PRRSV infections in pigs can reduce the efficacy of the HCLV vaccine and enhance the pathogenicity of CSFV, which may partly explain the immunization failure against CSFV in some swine herds.

Expression of major antigen domains of E2 gene of CSFV and analysis of its immunological activity

E2 is an envelope glycoprotein of Classical swine fever virus (CSFV) and contains sequential neutralizing epitopes to induce virus-neutralizing antibodies and mount protective immunity in the natural host. In this study, four antigen domains (ABCD) of the E2 gene was cloned from CSFV Shimen strain into the retroviral vector pBABE puro and expressed in eukaryotic cell (PK15) by an retroviral gene expression system, and the activity of recombinant E2 protein to induce immune responses was evaluated in rabbits. The results indicated that recombinant E2 protein can be recognized by fluorescence antibodies of CSFV and CSFV positive serum (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China) using Western blot, indirect immunofluorescence antibody test (IFAT) and ELISA, Furthermore, anti-CSFV specific antibodies and lymphocyte proliferation were elicited and increased by recombinant protein after vaccination. In the challenge test, all of rabbits vaccinated with recombinant protein and Chinese vaccine strain (C-strain) were fully protected from a rabbit spleen virus challenge. These results indicated that a retroviral-based epitope-vaccine carrying the major antigen domains of E2 is able to induce high level of epitope-specific antibodies and exhibits similar protective capability with that induced by the C-strain, and encourages further work towards the development of a vaccine against CSFV infection.

Influence of a 12-nt insertion present in the 3′ untranslated region of classical swine fever virus HCLV strain genome on RNA synthesis

The function of a 12-nt insertion ‘CTTTTTTCTTTT’ in the 3′ untranslated region (3′UTR) of the HCLV strain, a vaccine strain derived from the Shimen strain of classical swine fever virus (CSFV), was examined in vitro. RNA synthesis increased when the 12-nt insertion was deleted from the 3′UTR of the HCLV strain. RNA synthesis also decreased when the 12-nt insertion was introduced into the 3′UTR of the CSFV Shimen, virulent strain. Therefore, the 12-nt insertion present in the 3′UTR of the HCLV strain may be a marker for avirulence. It was also found that the predicted secondary structure of the 3′UTR of the HCLV strain was more stable when the 12-nt insertion was deleted, and that the 3′UTR of Shimen strain was more unstable when the 12-nt insertion was introduced. Replacement of the 12-nt insertion ‘CTTTTTTCTTTT’ by another 12-nt fragment ‘ATTATTATTTAT’ in the 3′UTR of HCLV strain facilitated RNA synthesis and stabilized the predicted secondary structure, which was also observed in the 3′UTR of the Shimen strain.