Pestivirus spillover effect: molecular detection of bovine viral diarrhea virus in domestic and feral pigs / Efeito spillover de pestivírus: detecção molecular do vírus da diarreia viral bovina em suínos domésticos e javalis

Pestivirus infections are important in the livestock industries, with infection occurring in cattle, sheep and pigs. The Pestivirus genus of the family Flaviviridae, includes four recognized species: bovine viral diarrhea virus 1 (BVDV-1), bovine viral diarrhea virus 2 (BVDV-2), border disease virus (BDV), and classical swine fever virus (CSFV). All pestivirus species can infect pigs, therefore accurate and specific pestivirus detection and differentiation is of great importance to assure control measures in swine populations. The aim of the study was the molecular detection of different pestiviruses in domestic and feral pigs. A total of 527 samples (92 pigs and 435 wild boars) were tested for pestiviruses detection using molecular assays. Eleven positive samples (6 wild boars and 5 domestic pigs) were identified using panpestivirus primers targeting the 5'- UTR region of the pestivirus RNA genome. Further all the positive samples were sequentially tested for detection of CSFV, BVDV-1 and BVDV-2 using specific primers. All RNAs were identified as positives for BVDV-1 and no amplification signals were obtained from BVDV-2 and CSFV. The current detection of BVDV-1 in clinical swine specimens highlights the important risk factor of swine population as reservoir and consequently carrier for BVDV.(AU)

As infecções por pestivírus são importantes nas indústrias pecuárias, com infecções em bovinos, ovinos e suínos. O gênero Pestivirus da família Flaviviridae inclui quatro espécies reconhecidas: vírus da diarreia viral bovina 1 (BVDV-1), vírus da diarreia viral bovina 2 (BVDV-2), vírus da doença de fronteira (VDF) e vírus da peste suína clássica (VPSC). Todas as espécies de pestivírus podem infectar porcos, portanto a detecção e diferenciação precisas e específicas de pestivírus são de grande importância para garantir medidas de controle nas populações suínas. O objetivo do estudo foi a detecção molecular de diferentes pestivírus em suínos domésticos e javali. Um total de 527 amostras (92 porcos e 435 javalis) foram testados para detecção de pestivírus usando ensaios moleculares. Onze amostras positivas (6 javalis e 5 porcos domésticos) foram identificadas usando iniciadores de panpestivírus visando a região 5'-UTR do genoma do RNA do pestivírus. Além disso, todas as amostras positivas foram testadas sequencialmente para detecção de VPSC, BVDV-1 e BVDV-2 usando iniciadores específicos. Todos os RNAs foram identificados como positivos para BVDV-1 e nenhum sinal de amplificação foi obtido do BVDV-2 e CSFV. A detecção atual do BVDV-1 em amostras clínicas de suínos destaca o importante fator de risco da população suína como reservatório e consequentemente portador do BVDV.(AU)

The potential efficacy of the E2-subunit vaccine to protect pigs against different genotypes of classical swine fever virus circulating in Vietnam

Histopathological Evaluation of the Efficacy for Plant-produced E2 Protein Vaccine against Classical Swine Fever Virus (CSFV) in Piglets

Classical swine fever (CSF), previously known as hog cholera, remains one of the most important swine-related contagious diseases worldwide. In order to eradicate classical swine fever virus (CSFV), it is commonly used in LOM-850 strain as a live attenuated CSF vaccine. However, there are symptoms of vaccination, such as the depression of feed intake, and difficulty of differentiation between infected and vaccinated hosts is impossible based on the antibodies induced. Nicotiana benthamiana were considered as an alternative to the production of recombinant vaccines on account of higher yields and levels of soluble protein than other models and crops in protein recombinant products. This study was conducted to evaluate histopathological validation of the plant-produced E2 fusion protein (ppE2) in piglets. The piglets were challenged by an injection of YC11WB strain in 7 days, 11 days and 14 days after one shot of the vaccination. The histopathological examination indicated that ppE2 can protect against lethal CSFV challenge at least 11 days of vaccination in piglets. These data suggest that the ppE2 can be an effective vaccine against CSFV in piglets.

Antigenic characterization of classical swine fever virus YC11WB isolates from wild boar

Classical swine fever (CSF), a highly contagious disease that affects domestic pigs and wild boar, has serious economic implications. The present study examined the virulence and transmission of CSF virus strain YC11WB (isolated from a wild boar in 2011) in breeding wild boar. Virulence of strain YC11WB in domestic pigs was also examined. Based on the severe clinical signs and high mortality observed among breeding wild boar, the pathogenicity of strain YC11WB resembled that of typical acute CSF. Surprisingly, in contrast to strain SW03 (isolated from breeding pigs in 2003), strain YC11WB showed both acute and strong virulence in breeding pigs. None of three specific monoclonal antibodies (7F2, 7F83, and 6F65) raised against the B/C domain of the SW03 E2 protein bound to the B/C domain of strain YC11WB due to amino acid mutations (⁷²⁰K→R and ⁷²³N→S) in the YC11WB E2 protein. Although strains YC11WB and SW03 belong to subgroup 2.1b, they had different mortality rates in breeding pigs. Thus, if breeding pigs have not developed protective immunity against CSF virus, they may be susceptible to strain YC11WB transmitted by wild boar, resulting in severe economic losses for the pig industry.

Hsp70 Fused with the Envelope Glycoprotein E0 of Classical Swine Fever Virus Enhances Immune Responses in Balb/c Mice / 病毒学报

Heat-shock protein (Hsp) 70 potentiates specific immune responses to some antigenic peptides fused to it. Here, the prokaryotic plasmids harboring the envelope glycoprotein E0 gene of classical swine fever virus (CSFV) and/or the Hsp70 gene of Haemophilus parasuis were constructed and expressed in Escherichia coli Rosseta 2(R2). The fusion proteins were then purified. Groups of Balb/c mice were immunized with these fusion proteins, respectively, and sera collected 7 days after the third immunization. Immune effects were determined via an enzyme-linked immunosorbent assay and flow cytometric analyses. E0-Hsp70 fusion protein and E0+Hsp70 mixture significantly improved the titer of E-specific antibody, levels of CD4+ and CD8+ T cells, and release of interferon-γ. These findings suggested that Hsp70 can significantly enhance the immune effects of the envelope glycoprotein E0 of CSFV, thereby laying the foundation of further application in pigs.

Research Progress in the Core Proteins of the Classical Swine Fever Virus / 病毒学报

The core protein (CP) of the classical swine fever virus (CSFV) is one of its structural proteins. Apart from forming the nucleocapsid to protect internal viral genomic RNA, this protein is involved in transcriptional regulation. Also, during viral infection, the CP is involved in interactions with many host proteins. In this review, we combine study of this protein with its disorders, structural/functional characteristics, as well as its interactions with the non-structural proteins NS3, NS5B and host proteins such as SUMO-1, UBC9, OS9 and IQGAP1. We also summarize the important part played by the CP in CSFV pathogenicity, virulence and replication of genomic RNA. We also provide guidelines for further studies in the CP of the CSFV.

Progress in new-type vaccines against classical swine fever / 生物工程学报

Classical swine fever (CSF), an acute and highly contagious disease of swine, is caused by classical swine fever virus. CSF is one of the most devastating diseases to the pig industry worldwide and results in serious economic losses. Currently prophylactic vaccination is still an important strategy for the control of CSF. Live attenuated vaccines (such as C-strain) are safe and effective. However, there are significant changes in the clinical features of CSF, displaying concurrent typical and atypical CSF, and simultaneous inapparent and persistent infections. Immunization failure has been reported frequently and it is difficult to distinguish between wild-type infected and vaccinated animals (DIVA). So there is an urgent need to develop more effective and safer DIVA or marker vaccines for the control of CSF. In this review, some of the most recent advances in new-type vaccines against CSF, including DNA vaccines, live virus-vectored vaccines, protein or peptide-based vaccines, gene-deleted vaccines and chimeric pestivirus-based vaccines, are reviewed and discussed.

Analysis of in vitro apoptosis induced by virulent Korean isolate of classical swine fever virus in peripheral blood B cell line / 대한수의학회지

Classical swine fever (CSF) is a highly contagious disease among swine that has an important economic impact on worldwide. One clinical symptom of CSF is leukopenia, in particular lymphopenia, which is a characteristic event that occurs early in the course of CSF. Though lymphopenia associated with apoptosis, the pathogenic mechanism underlying the lymphopenia has not been well studied. To understand these mechanisms, we investigated the response of porcine B cell lines to infection with SW03, virulent strain isolated from swine tissue in Korea. This study demonstrated that SW03-infected L35 cell were induced apoptosis through the detection of activated caspase-3. In addition, SW03 infection leaded to alterations in pro-apoptotic, Bax, and anti-apoptotic, Bcl-xL proteins of Bcl-2 family. Our results would suggest that SW03-infected L35 cells induced apoptosis via intrinsic mitochondrial pathway.

Quantitative detection of anti-classical swine fever virus siRNA expression in cells by stem-loop RT-qPCR / 生物工程学报

RNA interference (RNAi) is a promising technology in development of specific antiviral therapy, but the quantitative detection of small interfering RNA (siRNA) expressed in vivo is the main challenge to assess its antiviral effect. In order to detect the siRNA molecules (siN1 and SiN2) particularly expressed in cells to inhibit the replication of classical swine fever virus (CSFV), serial specific stem-loop primers were designed and synthesized. Two of them (SLP-N1-6 and SLP-N2-8) were selected by screening in cross combination and successfully used in establishment of an optimal stem-loop RT-qPCR, which showed high specificity and sensitivity in detection of anti-CSFV siRNA expressed in PK-15 cells. The method was capable of detecting 10(2) to 10(8) copies of siRNA molecule with good parallel relationship (R(sq) = 0.999) and high amplification efficiency (Eff. = 98.2%). Therefore, the established stem-loop RT-qPCR can be used as an ideal tool in quantitative assessment of the anti-CSFV effects of RNAi in combination with detection of viral antigens using indirect immunofluorescent assay and TCID50, providing a novel technique for evaluating the antiviral effects of the siRNA expressed in anti-CSFV transgenic pigs to be established in future.

Establishment and characterization of an infectious cDNA clone of a classical swine fever virus LOM strain

Classical swine fever virus (CSFV) causes a highly contagious disease among swine that has an important economic impact worldwide. CSFV strain LOM is an attenuated virus of low virulent strain of Miyagi isolated from Japan in 1956. Eight DNA fragments representing the genome of the CSFV strain LOM were obtained by RT-PCR. These were used to determine the complete nucleotide sequence and construct a full-length cDNA clone which was called Flc-LOM. Sequence analysis of the recombinant clone (Flc-LOM) revealed the presence of eight mutations, resulting in two amino acid substitutions, when compared to the parental sequence. RNA transcripts of both LOM and Flc-LOM were directly infectious in PK-15 cells. The rescued Flc-LOM virus grew more slowly than the parental virus, LOM, in the cells. Intramuscular immunization with Flc-LOM was safe and highly immunogenic in pigs; no clinical signs or virus transmission to sentinel animals were observed after 35 days. CSFV-specific neutralizing antibodies were detected 14 days post-infection. After challenge with the virulent CSFV strain SW03, pigs immunized with Flc-LOM were shown to be fully protected. Thus, our newly established infectious clone of CSFV, Flc-LOM, could serve as a vaccine candidate.

Construction and immunogenicity of a recombinant adenovirus co-expressing the E2 protein of classical swine fever virus and the porcine interleukin 2 in rabbits / 病毒学报

To construct a recombinant adenovirus co-expressing the E2 protein of classical swine fever virus (CSFV) and the porcine interleukin 2 (pIL-2), the CSFV E2 gene and pIL-2 gene were amplified respectively from the plasmids pMD19-T-E2 and pMD19-T-pIL-2 by PCR. E2-pIL-2 fusion gene was obtained by using 5 consecutive glycine codons as a linker and cloned into the adenoviral shuttle plasmid AdTrack. The AdTrack-E2-pIL-2 was linearized and transformed into E. coli BJ5183 with the backbone plasmid AdEasy1. The resultant recombinant plasmid AdEasy-E2-pIL-2 was transfected into the 293 cells where the recombinant adenovirus rAd-E2-pIL-2 was produced. The immunogenicity of rAd-E2-pIL-2 was evaluated in rabbits. The results of RT-PCR and Western-blotting showed that rAd-E2-pIL-2 could carry and express E2 and pIL-2 proteins. The titer of the rAd-E2-pIL-2 was 10(8.12) PFU/mL. After immunized with rAd-E2pIL-2, The injected rabbits developed a high level of CSFV specific antibodies. Regular fever was not detected in the rAd-E2-pIL-2-immunized rabbits upon challenge with CSFV C stain, and specific lymphoproliferative responses to the CSFV was detected in the lymphocytes from the immunized rabbits. In conclusion, rAd-E2-pIL-2 was constructed successfully and it could be an attractive vaccine candidate against CSFV.

Selection of a Less Pathogenic BVDV Strain for the Construction of Avirulent Chimeric Pestivirus

To select a less pathogenic bovine viral diarrhea virus (BVDV) strain for the construction of chimeric pestivirus harboring classical swine fever virus (CSFV) E2 gene, five Korean BVDV isolates (four type 1 isolates and a type 2 isolate) were evaluated for their pathological and biological properties with respect to porcine infection. Each of five groups of 100-day-old pigs was inoculated intranasally with one of the five BVDV isolates. No clinical sign or leukopenia was observed in any pig throughout the duration of the experiment, but viruses were detected in blood, nasal discharges and postmortem samples using RT-PCR. These results indicated that although the five BVD viruses could infect pigs, they did not cause clinically apparent symptoms. Because of its proper infection dynamics shown in this preliminary animal study and its fast growth rate and quick CPE in cell culture, one isolate (KD26-1) was chosen among the five isolates to test its virulence and immunogenic properties in 40-day-old piglets. Neither clinical sign nor pathological lesion was observed in 40-day-old piglets during the course of infection of isolate KD26-1. The first neutralizing antibodies were detectable 14 days post-inoculation (PI) and increased to 1:128~1:256 28 days PI. A BVDV specific gene was detectable by RT-PCR in tonsil, spleen, inguinal lymph node and brain until 14 days PI. According to this study, it can be concluded that isolate KD26-1 has little pathological effect in pigs and is a candidate for construction of chimeric pestivirus harboring CSFV E2 gene.

Selection of a Less Pathogenic BVDV Strain for the Construction of Avirulent Chimeric Pestivirus

To select a less pathogenic bovine viral diarrhea virus (BVDV) strain for the construction of chimeric pestivirus harboring classical swine fever virus (CSFV) E2 gene, five Korean BVDV isolates (four type 1 isolates and a type 2 isolate) were evaluated for their pathological and biological properties with respect to porcine infection. Each of five groups of 100-day-old pigs was inoculated intranasally with one of the five BVDV isolates. No clinical sign or leukopenia was observed in any pig throughout the duration of the experiment, but viruses were detected in blood, nasal discharges and postmortem samples using RT-PCR. These results indicated that although the five BVD viruses could infect pigs, they did not cause clinically apparent symptoms. Because of its proper infection dynamics shown in this preliminary animal study and its fast growth rate and quick CPE in cell culture, one isolate (KD26-1) was chosen among the five isolates to test its virulence and immunogenic properties in 40-day-old piglets. Neither clinical sign nor pathological lesion was observed in 40-day-old piglets during the course of infection of isolate KD26-1. The first neutralizing antibodies were detectable 14 days post-inoculation (PI) and increased to 1:128~1:256 28 days PI. A BVDV specific gene was detectable by RT-PCR in tonsil, spleen, inguinal lymph node and brain until 14 days PI. According to this study, it can be concluded that isolate KD26-1 has little pathological effect in pigs and is a candidate for construction of chimeric pestivirus harboring CSFV E2 gene.

The changes of peripheral blood leucocytes subpopulation after challenge with CSFV virulent strain Shimen / 病毒学报

To investigate the effects of classical swine fever virus (CSFV) virulent strain Shimen (SM) infection on piglets peripheral blood leucocytes, the 60-days weanling piglets were infected with the shinen strain and the peripheral blood samples of the piglets were collected to analyze the kinetics of the CSEV nucleic acid, the peripheral blood leucocytes subpopulation and SLA molecule expression on the peripheral blood leukocytes. The results showed that the piglets rectal temperature increased 48 hours after intramuscular injection of CSFV SM strain, the CSFV nucleic acid was detected in the peripheral blood at 2DPI, the content of CSFV nucleic acid increased and up-regulated to a peak at 6DPI as 10 (4.84 +/- 0.98 times as 2DPI. The amount of WBC, LYM and PLT significantly decreased, where in the amount of WBC decreased to 65.87% at 1DPI and 50% at 2DPI respectively; the amount of LYM decreased to 70.68%, 47.88% and 23.29% at 1DPI, 2DPI, and 3DPI, respectively; the amount of PLT decreased day by day and to 34.59% at 6DPI; the amount of NK, gammadeltaT, Tc, Th, CD3+ CD4+ CD8+ and CD3- CD4- CD8- cells decreased after infection; 78.49% of NK cells decreased at 1DPI and then there was no significant change from 2DPI to 6DPI. The amount of gammadeltaT, Tc, CD4- CD8- CD3-,CD4+ CD8+ CD3+ cells decreased to 41.74%, 43.83%, 15.87%, and 32.96% at 3DPI, respectively, However, the amount of T helper cells decreased continually to 42.95% at 6DPI; the amount of SLA I positive lymphocytes decreased significantly and the amount of SLA I positive CD3 cells decreased to 23.07% and 15.38% at 1DPI and 2DPI respectively; the SLA I positive granulocytes increased continually from 92.20% at 1DPI to 98.30% at 3DPI; the amount of CD3 SLA II + cells in lymphocytes decreased from 1.38% at 1DPI to 0.22% at 2DPI, while the SLA II + granulocytes increased continually to a peak at 3DPI and 53.76% of granulocytes expressed the SLA II molecule, but the percentage of the granulocytes expressing SLA II molecules decreased to 12.54% and 4.06% at 4DPI and 5DPI respectively. The study indicated that the CSFV SM strain infection could escape the immune surveillance and cause immunosuppression through inhibiting the host's innate antiviral immunity and the SLA molecule expression to affect the antigen presentation.

A prime-boost vaccination strategy using a Semliki Forest virus replicon vectored DNA vaccine followed by a recombinant adenovirus protects pigs from classical swine fever / 生物工程学报

We have previously evaluated a Semliki Forest virus (SFV) replicon vectored DNA vaccine (pSFV1CS2-E2) and a recombinant adenovirus (rAdV-E2) expressing the E2 glycoprotein of classical swine fever virus (CSFV) in pigs. The results showed that the immunized pigs were protected from virulent challenge, but few pigs showed short-term fever and occasional pathological changes following virulent challenge. To enhance the immunogenecity of the vaccines, we tried a prime-boost vaccination strategy using a combination of prime with pSFV1CS2-E2 followed by boost with rAdV-E2. The results showed that all the immunized pigs developed high-level CSFV-specific antibodies following prime-boost immunization. When challenged with virulent CSFV, the immunized pigs (n = 5) from the heterologous boost group showed no clinical symptoms, and CSFV RNA was not detected following challenge, whereas one of five pigs from the homologous boost group developed short-term fever and CSFV RNA was detected. This demonstrates that the heterologous prime-boost vaccination regime has the potential to prevent against virulent challenge.

High expression of antimicrobial peptide Cecropin AD in Escherichia coli by fusion with EDDIE / 生物工程学报

In this study, we efficiently expressed the active antimicrobial peptide (CAD), which fused with the site-mutated coat protein (EDDIE) of the classical swine fever virus, in Escherichia coli. First, we obtained the e-cad fusion gene from the CAD gene and the EDDIE gene using overlapping PCR. Then to get the recombinant expression vector (pETED), the e-cad fusion gene was cloned into the pET30a vector by a site-directed homologous recombination technique. The EDDIE-CAD fusion protein expressed in E. coli as inclusion bodies, and its yield was more than 40% of total bacterial proteins. After renaturated in vitro and self-cleavage of the fusion protein, we obtained the antimicrobial peptide Cecropin AD. Antimicrobial experiments showed that the Cecropin AD efficiently inhibited the growth of G+ and G- bacteria, but it weakly inhibited the growth of Saccharomyces. This method provides an excellent way for high expression of antimicrobial peptides when fused with EDDIE.

Applications of reverse genetics in studying classical swine fever virus / 生物工程学报

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), has been epidemic or endemic in many countries, and causes great economical losses to pig industry worldwide. Attenuated vaccines (such as C-strain) have played an important role in the control of CSF. Recently some new phenomena appear, such as atypical and persistent infections of CSF, immunization failure and so on. Meanwhile, eradication programs have been implemented in many countries, restricting the widespread applications of attenuated vaccines. Thus, currently the priority is to strengthen the research in pathogenesis and transmission mechanisms, as well as to develop marker vaccines. Recently, the applications of reverse genetics technology open up a new way for research of structure and function of CSFV proteins and development of novel vaccines against CSF. This review focuses on the progress of applications of reverse genetics in the functional analysis and marker vaccine development of CSFV, and also discusses the problems confronted now and prospective aspects in the study of CSFV.

Establishment and identification of classical swine fever virus (CSFV) capsid targeted nuclease expression system / 病毒学报

One pair of primers was designed based on the sequence encoding capsid protein C of classical swine fever virus (CSFV). The C gene fragment was amplified by RT-PCR and PCR products were inserted into eukaryotic expression vector pcDNA-SN containing staphylococcal nuclease (SN) gene resulting in recombinant plasmid pcDNA-C-SN. 48h after transfection of the recombinant into porcine kidney (PK)-15 cells using liposome, the expression of fusion protein was identified through RT-PCR, Western blot and indirect immunofluorescence, and nuclease activity was detected by in vitro DNA digestion assay. The results showed that fusion protein of C-SN was expressed stably in PK-15 cells, and could be identified by rabbit polyclonal antibody against CSFV capsid protein and had good nuclease activity to cleave DNA. Meanwhile, the expressed fusion protein of C-SN in the transfected cells could effectively inhibit the proliferation of CSFV, reducing the infection rate by 10(2)-10(3) times. Our findings laid a foundation for further application of capsid-targeted antiviral strategies for CSFV.

Expression profiles of apoptotic genes of pig peripheral blood leukocytes caused by classical swine fever virus infection / 病毒学报

Classical swine fever (CSF) is a contagious swine disease charactered by hemorrhagic fever and leukopenia,usually leading to substantial economic losses. To obtain a insight of leucopenia caused by CSFV infection, DNA microarray analyses of peripheral blood leucocytes (PBL) of the infected pigs was performed. Three health pigs were inoculated with a lethal dose of CSFV Shimen strain and their PBLs were isolated when the onset of typical clinical signs and then subjected to total RNA extraction followed by microarray analysis with Affymetrix Porcine Genome Array GeneChips. The results showed that the significant differences were observed in cellular apoptotic genes expression at 7 days post-infection (p. i.). The changes of the genes expression were confirmed by real time RT-PCR of some selected apoptosis-related genes. This study provided a valuable information for further investigating the molecular mechanism of apoptosis caused by CSFV infection.

Construction of recombinant fowlpox virus expressing E0 gene of classical swine fever virus shimen strain and the animal immunity experiment / 病毒学报

The CSFV E0 gene was amplified from the plasmid pMD18-T-E0 by PCR and cloned into the FPV-P11 and FPV-pSY. The identified recombinant DNA was transfected into chicken embryo fibroblasts (CEF) to package Fowlpox virus. E0 gene was confirmed to be integrated into the genome of recombinant Fowlpox virus by PCR, and Western blot was employed for detection of E0 expression in the chicken embryo fibroblasts infected with recombinant Fowlpox virus . The results of ELISA showed that systemic immune response to CSFV could be induced effectively after the mice were immunized three times with recombinant Fowlpox virus through celiac route, the titer of antibody was 1 : 4096. The protection experiment showed that 75% of piglets immunized three times with recombinant Fowlpox virus were survived, indicating that the recombinant Fowlpox virus was effective. This paper lays foundation for the study of CSFV live vector vaccine.