Classical Swine Fever Virus Shimen Strain Research Articles

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.

P108 and T109 on E2 Glycoprotein Domain I Are Critical for the Adaptation of Classical Swine Fever Virus to Rabbits but Not for Virulence in Pigs.

The classical swine fever virus (CSFV) live attenuated vaccine C-strain is adaptive to rabbits and attenuated in pigs, in contrast with the highly virulent CSFV Shimen strain. Previously, we demonstrated that P108 and T109 on the E2 glycoprotein (E2P108-T109) in domain I (E2DomainI) rather than R132, S133, and D191 in domain II (E2DomainII) determine C-strain's adaptation to rabbits (ATR) (Y. Li, L. Xie, L. Zhang, X. Wang, C. Li, et al., Virology 519:197-206, 2018). However, it remains elusive whether these critical amino acids affect the ATR of the Shimen strain and virulence in pigs. In this study, three chimeric viruses harboring E2P108-T109, E2DomainI, or E2DomainII of C-strain based on the non-rabbit-adaptive Shimen mutant vSM-HCLVErns carrying the Erns glycoprotein of C-strain were generated and evaluated. We found that E2P108-T109 or E2DomainI but not E2DomainII of C-strain renders vSM-HCLVErns adaptive to rabbits, suggesting that E2P108-T109 in combination with the Erns glycoprotein (E2P108-T109-Erns) confers ATR on the Shimen strain, creating new rabbit-adaptive CSFVs. Mechanistically, E2P108-T109-Erns of C-strain mediates viral entry during infection in rabbit spleen lymphocytes, which are target cells of C-strain. Notably, pig experiments showed that E2P108-T109-Erns of C-strain does not affect virulence compared with the Shimen strain. Conversely, the substitution of E2DomainII and Erns of C-strain attenuates the Shimen strain in pigs, indicating that the molecular basis of the CSFV ATR and that of virulence in pigs do not overlap. Our findings provide new insights into the mechanism of adaptation of CSFV to rabbits and the molecular basis of CSFV adaptation and attenuation.IMPORTANCE Historically, live attenuated vaccines produced by blind passage usually undergo adaptation in cell cultures or nonsusceptible hosts and attenuation in natural hosts, with a classical example being the classical swine fever virus (CSFV) lapinized vaccine C-strain, which was developed by hundreds of passages in rabbits. However, the mechanism of viral adaptation to nonsusceptible hosts and the molecular basis for viral adaptation and attenuation remain largely unknown. In this study, we demonstrated that P108 and T109 on the E2 glycoprotein together with the Erns glycoprotein of the rabbit-adaptive C-strain confer adaptation to rabbits on the highly virulent CSFV Shimen strain by affecting viral entry during infection but do not attenuate the Shimen strain in pigs. Our results provide vital information on the different molecular bases of CSFV adaptation to rabbits and attenuation in pigs.

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.

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.

Enhanced protective immunity of the chimeric vector-based vaccine rAdV-SFV-E2 against classical swine fever in pigs by a Salmonella bacterial ghost adjuvant.

Classical swine fever (CSF) is a highly contagious swine disease caused by classical swine fever virus (CSFV). Previously, we demonstrated that rAdV-SFV-E2, an adenovirus-delivered, Semliki Forest virus replicon-vectored marker vaccine against CSF, is able to protect pigs against lethal CSFV challenge. From an economical point of view, it will be beneficial to reduce the minimum effective dose of the vaccine. This study was designed to test the adjuvant effects of Salmonella enteritidis-derived bacterial ghosts (BG) to enhance the protective immunity of rAdV-SFV-E2 in pigs. Groups of 5-week-old pigs (n = 4) were immunized intramuscularly twice with 105 median tissue culture infective doses (TCID50) rAdV-SFV-E2 combined with 1010 colony forming units (CFU) BG, 106 or 105 TCID50 rAdV-SFV-E2 alone or 1010 CFU BG alone at an interval of 3 weeks, and challenged with the highly virulent CSFV Shimen strain at 1 week post-booster immunization. The results show that the pigs inoculated with 105 TCID50 rAdV-SFV-E2 plus BG or 106 TCID50 rAdV-SFV-E2 alone were completely protected from lethal CSFV challenge, in contrast with the pigs vaccinated with 105 TCID50 rAdV-SFV-E2 or BG alone, which displayed partial or no protection following virulent challenge. The data indicate that BG are a promising adjuvant to enhance the efficacy of rAdV-SFV-E2 and possibly other vaccines.

Piglets with maternally derived antibodies from sows immunized with rAdV-SFV-E2 were completely protected against lethal CSFV challenge

Classical swine fever (CSF) is an economically important infectious disease of pigs caused by Classical swine fever virus (CSFV). To facilitate the eradication of CSF in endemic areas, a marker vaccine enabling differentiation of infected from vaccinated animals (DIVA) is urgently needed. Previously, we have demonstrated that the DIVA vaccine rAdV-SFV-E2, an adenovirus-vectored Semliki Forest virus replicon expressing the E2 glycoprotein of CSFV, induces complete protection from lethal CSFV challenge. The aim of this study was to investigate whether maternally derived antibodies (MDAs) from sows immunized with rAdV-SFV-E2 can effectively protect piglets against lethal CSFV challenge. Three groups of five-week-old piglets (n=4), with or without MDAs, were challenged with the highly virulent CSFV Shimen strain. Clinical signs, CSFV-specific antibodies, viremia and pathological and histopathological changes were monitored. The results showed that the piglets with MDAs from the sow immunized with rAdV-SFV-E2 were protected clinically, virologically and pathologically, while the piglets with undetectable MDAs from the rAdV-SFV-E2-immunized sow were partially protected (2/4 survival), in contrast with the piglets from the non-vaccinated sow, which displayed CSF-typical clinical signs, viremia, deaths (4/4) and pathological/histopathological lesions. These results indicate that MDAs from the sow immunized with rAdV-SFV-E2 are able to confer full passive immunity to newborn piglets.

Safety and immunogenicity of a gE/gI/TK gene-deleted pseudorabies virus variant expressing the E2 protein of classical swine fever virus in pigs

Classical swine fever (CSF) and pseudorabies (PR) are both major infectious diseases of pigs, causing enormous economic losses to the swine industry in many countries. A marker vaccine that enables differentiation of infected from vaccinated animals (DIVA) is highly desirable for control and eradication of these two diseases in endemic areas. Since late 2011, PR outbreaks have been frequently reported in many Bartha-K61-vaccinated pig farms in China. It has been demonstrated that a pseudorabies virus (PRV) variant with altered antigenicity and increased pathogenicity was responsible for the outbreaks. Previously, we showed that rPRVTJ-delgE/gI/TK, a gE/gI/TK-deleted PRV variant, was safe for susceptible animals and provided a complete protection against lethal PRV variant challenge, indicating that rPRVTJ-delgE/gI/TK can be used as an attractive vaccine vector. To develop a safe bivalent vaccine against CSF and PR, we generated a recombinant virus rPRVTJ-delgE/gI/TK-E2 expressing the E2 protein of classical swine fever virus (CSFV) based on rPRVTJ-delgE/gI/TK and evaluated its safety and immunogenicity in pigs. The results indicated that pigs (n=5) immunized with rPRVTJ-delgE/gI/TK-E2 of different doses did not exhibit clinical signs or viral shedding following immunization, the immunized pigs produced anti-PRV or anti-CSFV neutralizing antibodies and the pigs immunized with 106 or 105 TCID50 rPRVTJ-delgE/gI/TK-E2 were completely protected against the lethal challenge with either CSFV Shimen strain or variant PRV TJ strain. These findings suggest that rPRVTJ-delgE/gI/TK-E2 is a promising bivalent DIVA vaccine candidate against CSFV and PRV coinfections.

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.

A comparison of the impact of Shimen and C strains of classical swine fever virus on Toll-like receptor expression.

Classical swine fever is one of the most important swine diseases worldwide and has tremendous socioeconomic impact. In this study, we focused on the signalling pathways of Toll-like receptors (TLRs) because of their roles in the detection and response to viral infections. To this end, two classical swine fever virus (CSFV) strains, namely the highly virulent CSFV Shimen strain and the avirulent C strain (a vaccine strain), were employed, and the expression of 19 immune effector genes was analysed by real-time PCR, Western blot analyses, ELISA and flow cytometry analyses. In vitro experiments were conducted with porcine monocyte-derived macrophages (pMDMs). The results showed that the mRNA and protein levels of TLR2, TLR4 and TLR7 were upregulated in response to CSFV infection, but TLR3 remained unchanged, and was downregulated after infection with the C strain and the Shimen virus, respectively. Furthermore, TLR3-mediated innate immune responses were inhibited in Shimen-strain-infected pMDMs by stimulation with poly(I : C). Accordingly, comprehensive analyses were performed to detect TLR-dependent cytokine responses and the activation of TLR signalling elements. CSFV infection induced mitogen-activated protein kinase activation, but did not elicit NFκB activation, thereby affecting the production of pro-inflammatory cytokines. The Shimen strain infection resulted in a significant activation of IFN regulatory factor IRF7 and suppression of IRF3. These data provided clues for understanding the effect of CSFV infection on the TLR-mediated innate immune response and associated pathological changes.

Effects of the nuclear localization of the Npro protein of classical swine fever virus on its virulence in pigs

The Npro protein of classical swine fever virus (CSFV) is localized in the cytoplasm and nucleus. However, it is unknown whether the nuclear localization of Npro correlates with the virulence of CSFV in the host. Previously, we showed that the Npro protein fused with interferon regulatory factor 3 (IRF3) was present only in the cytoplasm. Here, we generated and evaluated a recombinant CSFV vSM-IRF3 harboring the IRF3 gene inserted into the Npro gene of the highly virulent CSFV Shimen strain. Compared to the even nuclear and cytoplasmic distribution of the enhanced green fluorescent protein (EGFP)-Npro fusion expressed by the recombinant CSFV EGFP-CSFV, vSM-IRF3 expressed an IRF3-Npro fusion protein that only was localized in the cytoplasm. vSM-IRF3 was markedly attenuated in vitro and in vivo, and the inoculated pigs were completely protected from lethal CSFV challenge, whereas the parental virus as well as EGFP-CSFV exhibited a typical virulent phenotype. Taken together, the nuclear localization of Npro plays a significant role in the CSFV replication and virulence.

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.

Experimental infection of Bama miniature pigs with a highly virulent classical swine fever virus

BackgroundCurrently, larger domestic pigs are only animals widely used in vaccine evaluation and pathogenicity study of classical swine fever virus (CSFV). This study was aimed to create an alternative animal experimental infection model of CSFV.ResultsTwenty specific-pathogen-free Bama miniature pigs were randomly divided into two groups and rooms, infected and non-infected, and the pigs in the infected group were inoculated intramuscularly with 104, 105 or 106 TCID50 (median tissue culture infective dose) CSFV Shimen strain (n = 5 × 3) or left uninoculated to serve as in-contact pigs (n = 3). The uninfected control pigs (n = 2) were housed in a separate room. Clinical signs, body temperature, viraemia, tissue antigen distribution, pathological changes and seroconversion were monitored. Clinical signs were observed as early as 2 days post-inoculation (dpi) in all infected pigs (though mild in contact pigs), but not non-infected control pigs. All inoculated pigs showed viraemia by 6 dpi. The in-contact pigs showed lower levels of viraemia. At 10 dpi, seroconversion was noted in five of the 15 inoculated pigs. All inoculated or one in-contact pigs died by 15 dpi.ConclusionsThese results show that Bama miniature pigs support productive CSFV infection and display clinical signs and pathological changes consistent with CSFV infections observed in larger domestic pigs.

Proteomic analysis of swine serum following highly virulent classical swine fever virus infection

BackgroundClassical swine fever virus (CSFV) belongs to the genus Pestivirus within the family Flaviviridae. Virulent strains of classical swine fever virus (CSFV) cause severe disease in pigs characterized by immunosuppression, thrombocytopenia and disseminated intravascular coagulation, which causes significant economic losses to the pig industry worldwide.MethodsTo reveal proteomic changes in swine serum during the acute stage of lethal CSFV infection, 5 of 10 pigs were inoculated with the virulent CSFV Shimen strain, the remainder serving as uninfected controls. A serum sample was taken at 3 days post-infection from each swine, at a stage when there were no clinical symptoms other than increased rectal temperatures (≥40°C). The samples were treated to remove serum albumin and immunoglobulin (IgG), and then subjected to two-dimension differential gel electrophoresis.ResultsQuantitative intensity analysis revealed 17 protein spots showing at least 1.5-fold quantitative alteration in expression. Ten spots were successfully identified by MALDI-TOF MS or LTQ MS. Expression of 4 proteins was increased and 6 decreased in CSFV-infected pigs. Functions of these proteins included blood coagulation, anti-inflammatory activity and angiogenesis.ConclusionThese proteins with altered expression may have important implications in the pathogenesis of classical swine fever and provide a clue for identification of biomarkers for classical swine fever early diagnosis.

Enhanced immunity against classical swine fever in pigs induced by prime-boost immunization using an alphavirus replicon-vectored DNA vaccine and a recombinant adenovirus

Classical swine fever (CSF) – caused by the classical swine fever virus (CSFV) – is a fatal disease of pigs that is responsible for extensive losses to the swine industry worldwide. We had demonstrated previously that a prime-boost vaccination strategy using an alphavirus (Semliki Forest virus, SFV) replicon-vectored DNA vaccine (pSFV1CS-E2) and a recombinant adenovirus (rAdV-E2) expressing the E2 glycoprotein of CSFV induced enhanced immune responses in a mouse model. In this study, we evaluated further the efficacy of the heterologous prime-boost immunization approach in pigs, the natural host of CSFV. The results showed that the pigs ( n = 5) receiving pSFV1CS-E2/rAdV-E2 heterologous prime-boost immunization developed significantly higher titers of CSFV-specific neutralizing antibodies and comparable CD4 + and CD8 + T-cell proliferation, compared to the pigs receiving double immunizations with rAdV-E2 alone. When challenged with virulent CSFV Shimen strain, the pigs of the heterologous prime-boost group did not show clinical symptoms or viremia, which were observed in one of the 5 pigs immunized with rAdV-E2 alone and all the 5 control pigs immunized with an empty adenovirus. The results demonstrate that the heterologous DNA prime and recombinant adenovirus boost strategy can induce solid protective immunity.

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.