A multiplex real-time PCR assay for simultaneous detection of classical swine fever virus,African swine fever virus,and atypical porcine pestivirus

With the implementation of the C-strain vaccine,classical swine fever(CSF) has been under control in China,which is currently in a chronic atypical epidemic situation.African swine fever(ASF) emerged in China in 2018 and spread quickly across the country.It is presently occurring sporadically due to the lack of commercial vaccines and farmers’ increased awareness of biosafety.Atypical porcine pestivirus(APPV) was first detected in Guangdong Province,China,in 2016,which mainly harms piglets and has a local epidemic situation in southern China.These three diseases have similar clinical symptoms in pig herds,which cause considerable losses to the pig industry.They are difficult to be distinguished only by clinical diagnosis.Therefore,developing an early and accurate simultaneous detection and differential diagnosis of the diseases induced by these viruses is essential.In this study,three pairs of specific primers and Taq-man probes were designed from highly conserved genomic regions of CSFV(5’ UTR),African swine fever virus(ASFV)(B646L),and APPV(5’ UTR),followed by the optimization of reaction conditions to establish a multiplex real-time PCR detection assay.The results showed that the method did not cross-react with other swine pathogens(porcine circovirus type 2(PCV2),porcine reproductive and respiratory syndrome virus(PRRSV),foot-and-mouth disease virus(FMDV),pseudorabies virus(PRV),porcine parvovirus(PPV),and bovine viral diarrhea virus BVDV).The sensitivity results showed that CSFV,ASFV,and APPV could be detected as low as 1 copy μL–1;the repeatability results showed that the intra-assay and interassay coefficient of variation of ASFV,CSFV,and APPV was less than 1%.Twenty-two virus samples were detected by the multiplex real-time PCR,compared with national standard diagnostic and patented method assay for CSF(GB/T 27540–2011),ASF(GB/T 18648–2020),and APPV(CN108611442A),respectively.The sensitivity of this triple real-time PCR for CSFV,ASFV,and APPV was almost the same,and the compliance results were the same(100%).A total of 451 clinical samples were detected,and the results showed that the positive rates of CSFV,ASFV,and APPV were 0.22% (1/451),1.3%(6/451),and 0%(0/451),respectively.This assay provides a valuale tool for rapid detection and accurate diagnosis of CSFV,ASFV,and APPV.

The Lapinized Chinese Strain Vaccine Against Classical Swine Fever Virus:A Retrospective Review Spanning Half A Century

Classical swine fever （CSF）, a list A disease of Office International des Epizooties, is caused by classical swine fever virus （CSFV） belonging to the Flaviviridae family. The well-known lapinized Chinese strain of CSFV, also known as C-strain, was developed in China in the mid-1950s. In the past half a century, the vaccine has been proved to be safe and immunogenic in pigs of essentially any age. It is of high efficacy, providing immunized animals with broad-spectrum, sometimes lifelong, protection, which is contributed by both cell-mediated immunity and humoral immunity, against essentially all genotypes or subgenotypes of the virus. The maternal antibodies derived from immunized sows can confer solid protection of their offspring from disease; however, they have been proved to inhibit the successful active immunization of C-strain vaccine. The complete genomes of C-strain and dozens of established or field strains have been sequenced and annotated. Recently, the reverse genetics system of C-strain has been developed, resulting in several C- strain-derived candidate marker vaccines. Many countries manage to control or even eradicate CSF with the aid of mass vaccination with C-strain. in spite of these efforts, the eradication of the disease worldwide remains a big challenge and needs to go a long way, and provably still resorts to genetically modified C-strain vaccine. The authors present an overview of the characteristics of the vaccine, which has stood the test of half a century.

Genomic Sequence Determination of Classical Swine Fever Virus Persistent Infection Strain

Full genomic sequence of a newly isolated persistent infection strain of classical swine fever virus was firstly determined. It was demonstrated by sequence analyses that nucleotides homologies of this strain compared with virulent Shimen and vaccine HCLV were 89.7%and 87.7%, and homologies of amino acids were 94.8%and 93.3%, respectively. The sequencing results primarily suggest a tighter relationship between this persistent infection strain and virulent Shimen strain than vaccine HCLV strain.

Molecular Cloning and NucleotideSequence of Classical SwineFever Virus Strain Shimen

According to the previously published CSFV sequences, 18 paris of partially overlapping primers which span the entire genome of CSFV strain Shimen were designed and synthesized. Each cDNA fragment of strain Shimen was amplified by RT-PCR method from the anticoagulant blood of strain Shimen infected pig. The PCR fragments were cloned into pGEM-T vector respectively and sequenced. The results show that we have obtained the nucleotide sequence of strain Shimen. The viral RNA consists of 12 297 nucleotides including noncoding regions of 373 and 227 bases at the 5′ and 3′ end, respectively, and a single large open reading frame spanning 11 697 nucleotides in the middle, which encodes an amino acid sequence of 3 989 residues with a calculated molecular weight of 437.6×103. The precisely sequencing of 5′ and 3′ termini is undertaking.

A Novel RT-LAMP Assay for Rapid and Simple Detection of Classical Swine Fever Virus

A simple and rapid assay for the detection of Classical swine fever virus(CSFV)was established using reverse transcription loop-mediated isothermal amplification(RT-LAMP).This study describes the amplification of the genomic RNA of CSFV under isothermal conditions(63℃)within one hour,using a set of six primers(two outer primers,two inner primers and two loop primers).This RT-LAMP assay showed 100-fold higher sensitivity than the standard RT-PCR method and identified eighteen additional positive cases that were negative when tested by RT-PCR.This RT-LAMP was able to detect all the 13 strains of CSFV but not the BVDV.PRRSV.SIV. PRV-PCV,thus showed a good specificity.Products amplified by RT-LAMP can be visualized by agarose gel electrophoresis and in addition,either as a white precipitate at the bottom of the tube after a pulse spin or as a color change when dyed with SYBR Green I which are visible to the naked eye.Because RT-LAMP is low-cost and produces rapid results,it has the potential to be an excellent tool for CSFV surveillance in the field,especially in developing countries.

An Indirect ELISA of Classical Swine Fever Virus Based on Quadruple Antigenic Epitope Peptide Expressed in E.coli

In this study,a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV)E2 glycoprotein was expressed in E.coli to a obtain target protein.This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs.The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve(ROC)analysis based on 30 negative sera and 80 positive samples.The test gave 97.5%sensitivity and 96.7%specificity compared with the indirect hemagglutination(IHA)test.The inter-assay and intra-assay coefficients of variation (CVs)for 16 sera were both≤6.8%.No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus(BVDV)antibodies was observed.

Discrepancy of Classical Swine Fever Virus in Different Tissues by One-Step RT-PCR and Nested RT-PCR

Reverse transcription polymerase chain reaction (RT-PCR) was used for the detection of classical swine fever virus (CSFV) in blood and tissue samples of field cases and experimentally inoculated pigs. The distribution of CSFV in different organ samples showed some discrepancies in infected pigs. Four weaner pigs were inoculated with C-strain vaccine virus, then samples of spleen, tonsil, lung, mesenteric lymph node, kidney and brain were collected after slaughter and tested for E2 and NS5B genes using one-step RT-PCR and nested RT-PCR. Using the same method, 12 field cases were simultaneously studied. A discrepancy of CSFV in different samples was found upon detecting the target gene. The most reliable diagnostic organs were spleen and tonsil, and the nested RT-PCR assay provided a highly sensitive and specific method with comparable performance to the one-step RT-PCR assay.

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-RzTФ. Direct transfection of pOKShimen-RzTqb 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.

Generation and Immunogenicity of a Recombinant Adenovirus Co-Expressing the E2 Protein of Classical Swine Fever Virus and the GP5 Protein of Porcine Reproduction and Respiratory Syndrome Virus

Classical swine fever （CSF） and porcine reproduction and respiratory syndrome （PRRS） are both economically important, highly contagious diseases of swine worldwide. To develop an effective vaccine to control these two diseases, we constructed a recombinant adenovirus rAdV-GP52AE2, using a replication-defective human adenovirus serotype 5 as a delivery vector, to co-express the GP5 protein of highly pathogenic porcine reproduction and respiratory syndrome virus （PRRSV） and the E2 protein of classical swine fever virus （CSFV）. Foot-and-mouth disease virus （FMDV） 2A peptide was used as a linker between the GP5 and E2 proteins to allow automatic self-cleavage of the polyprotein. The GP5 and E2 genes were expressed as demonstrated by immunofluorescence assay and Western blotting. Immunization of mice resulted in a CSFV-neutralizing antibody titer of 1：128 and a PRRSV-neutralizing antibody titer of 1：16. The lymphoproliferative responses were detected by Cell Counting Kit-8 assay and the stimulation index of CFSV-specific and PRRSV-specific lymphocytes in the rAdV-GP52AE2 group was significantly higher than that in the negative control group. The results show that rAdV-GP52AE2 can induce both effective humoral and cell-mediated immune responses in mice. The protective efficacy of the recombinant virus against CSF was evaluated in immunized rabbits, which were protected from fever induced by challenge with C-strain. Our study provides supporting evidence for the use of FMDV 2A to develop a bivalent genetically-engineered vaccine.

Development of Multiple ELISAs for the Detection of Antibodies against Classical Swine Fever Virus in Pig Sera

The major immunogenic proteins （Ems, E2 and NS3） of classical swine fever virus （CSFV） （Shimen strain） were expressed in E. coli and purified by affinity chromatography. The recombinant antigens were applied to develop multiple enzyme-linked immunosorbent assays （ELISAs） for the detection of specific antibodies in pig sera. Optimum cut-off values were determined by receiver operating characteristic （ROC） analysis after testing 201 sera of vaccinated pigs and 64 negative sera of unvaccinated piglets. The multiple ELISAs were validated with 265 pig sera yielding high sensitivity and specificity in comparison with the virus neutralization results. The results demonstrated that multiple ELISAs can be a valuable tool for the detection of CSFV infection and serological surveys in CSFV-free countries or for the evaluation of the antibody responses in pigs induced by a live attenuated C-strain vaccination

Heterogeneity and Secondary Structure Analysis of 3' Untranslated Region in Classical Swine Fever Viruses

The attenuated vaccine strains of CSFV have a 12-nucleotides （nt） insertion in the 3＇-UTR of genome as compared to that of CSFV virulent strains. In this study, we found a distinct heterogeneity in the 3＇-UTR of attenuated Thiverval and HCLV strains. The longest 3＇-UTR of Thiverval strain was 259 base pairs （bp） with a 32-nt insertion, the shortest 3＇-UTR had only 233 bp with a 6-nt insertion. The longest 3＇-UTR of HCLV strain was 244 bp with a 17-nt insertion and the shortest 3＇ UTR was 235 bp with a 8-nt insertion. Compared with the published sequences of 3＇-UTR of vaccine and virulent strains, the 3＇-UTR of CSFV vaccine strains have two variable regions where insertion among the different vaccine strains were frequently found. The first is located between the second conservative TALk codon and the start of T-rich region where we found the variable length insertion in the same vaccine strain Thiveral or HCLV and the second is located between the end of T-rich region and the front of GAA eodon, however, a 4-nt deletion was found in this region in the virulent Shimen strain. These two regions may represent the ＂hot spot＂ for mutation. Modeling the secondary structures of the 3＇-UTR suggests that the T-rich insertion could result in the change of structure and free energy, thus affecting the stability of the 3＇-UTR structure. These findings will help to understand the mechanism of attenuated vaccines and improve vaccine safety, stability, and efficacy.

Detection of nucleic acid of classical swine fever virus by reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Classical swine fever virus (CSFV) is the causative agent of Classical swine fever which is a highly contagious disease affecting swine and resulting in severe economic losses. In this study, we developed reverse transcription loopmediated isothermal amplification (RT-LAMP) assay targeting the 5’UTR gene for the detection of CSFV. This amplification method can be obtained in 1 h under isothermal conditions (65°C) employing a set of six specific primers mixtures. Amplification product was visualized by using hydroxynaphthol blue (HNB) dye and agarose gel electrophoresis. The sensitivity was 100 copy numbers. No cross-reactivity related to Japanese encephalitis virus (JEV) and porcine reproductive and respiratory syndrome virus (PRRSV) was demonstrated. The results demonstrated that the RT-LAMP assay is a useful tool for the rapid and sensitive for CSFV detection in swine.

Effect of Attenuated Highly Pathogenic Pig Reproductive and Respiratory Syndrome(HP-PRRS) TJM-F92 Strain Vaccine on Immune Antibody Levels against Classical Swine Fever(CSF) and Foot-and-Mouth Disease(FMD)

Effects of attenuated highly pathogenic pig reproductive and respiratory syndrome（HP-PRRS）TJM-F92 strain vaccine on immune antibody level against classical swine fever（CSF）and foot-and-mouth disease（FMD）were studied from October 8 to November 12 in 2014,in order to optimize vaccination program of CSF,HP-PRRS and FMD and to provide scientific guidance for animal disease control and prevention work.The results showed that attenuated HP-PRRS（TJMF92 strain）vaccine had no significant effect on immune antibody level of hog cholera lapinized virus（HCLV,ST passage cell vaccine）attenuated vaccine and FMD-O inactivated vaccines（OZK/93 strain）,and single or combined use of three vaccines received good immunization effects.

Phylogeny and Homologous Recombination Occurring in Classical Swine Fever Viruses

Classical swine fever virus(CSFV) is the causative agent of classical swine fever, a highly contagious disease of pigs. But there is little information on the recombination in natural populations of CSFVs. Therefore, a phylogenetic analysis of 62 fulllength genome CSFV strains, isolated from all over the world, was performed to detect potential recombination events, with the recombinant sequences being analyzed with the SimPlot and RDP programs. The results identified a mosaic virus, Chinese CSFV HCLV(2)(AF091507.1), which is the one naturally emerged recombinant CSFV with two recombination breakpoints at 2 484 and 2 900 bp of the genome alignment. Its two putative parental-like strains were CSFV Shimen(AF092448.2) and CSFV strain C/HVRI(AY805221.1). This work demonstrated that homologous recombination did occur in natural CSFV populations. It had significant implications for understanding the molecular epidemiology of CSFV, and revealed that recombination was an important factor for high genetic diversities of CSFV.

Development of Reverse Transcriptase Loop-Mediated Isothermal Amplification for Rapid and Visualized Detection of Classical Swine Fever Virus

[ Objective] To develop a rapid and visualized detection method of classical swine fever virus （CSFV） using reverse transcriptase loopmediated isothermal amplification （RT-LAMP）. [ Method ] A total of six special primers were designed based on the conserved sequences of CSFV gene. After optimizing, the reaction of RT-LAMP was carded out at 63℃ for 45 rain. The RT-LAMP products were analyzed by agarose gel electro- phoresis. The sensitivity, specificity and repeatability were verified, respectively. [ Result] The RT-LAMP method could be used for detecting CSFV rather than six generic viruses. The sensitivity of RT-LAMP was 100 times higher than that of RT-PCR. The detection of 27 clinical samples by RT- LAMP and RT-PCR showed that RT-LAMP is more reliable and convenient. [ Conclusion] The RT-LAMP method is sensitive and reliable for the detection of CSFV.

Primary Selection of MicroRNA Acting on Classical Swine Fever Virus in Swine Umbilical Vein Endothelial Cells

The aim of the study was to identify the microRNAs that act on classical swine fever virus (CSFV) in swine umbilical vein endothelial cells (SUVECs). The 3'-and 5'-untranslated regions ( UTR) of CSFV were cloned and then inserted into psiCHECH TM-2 plasmids carrying Firefly and Renilla luciferases reporter genes; microRNAs that acted with CSFV were predicted by bio-informatics analysis; then the recombinant plasmids and inhibitors of the predicted microRNA were co-transfected into SUVECs. The activities of Firefly and Renilla luciferases were detected by luminometry. The PCR products of the CSFV 5'-UTR (373 bp) and 3'-UTR (252 bp) were detected by electrophoresis on 1% agarose gels. Four microRNAs (ssc-miR-let7c, ssc-miR-106a, ssc-miR-18, ssc-miR-139) were screened out and evaluated. The CSFV 3'-UTR is the important target site of microRNA in SUVECs. The four microRNAs mentioned above had different inhibitory effects on the CSFV 3'-UTR, of which the ssc-miR-18 played the most important role.

A Universal Real-Time Fluorescence qPCR Method for Identifying Epidemic Strains of African Swine Fever Virus

Objective Establishing a highly sensitive real-time fluorescence quantitative PCR (qPCR) method for universal testing of epidemic African swine fever virus (ASFV) strains. Methods The ASFV p72 gene was targeted to design primer probes covering 24 p72 genotypes. The optimal amount of dimethylsulphoxide (DMSO) for qPCR amplification was determined, Various sensitivity and limit of detection (LOD) tests were performed, and clinical samples from China and imported goods were tested. Results The optimal primer-probe combination could specifically detect ASFV, 1.5% DMSO was optimal for qPCR, and LOD reached 3.2 copies/μL with good reproducibility (n = 20, p = 0.369). The method was employed to test 142 clinically suspected samples, of which 30 pig blood and 37 pig tissue samples were ASFV-positive. Moreover, the positive testing rate for ASFV was higher than for the standard qPCR method recommended by the Office International Des Epizooties (OIE), and for the commercially available kit. Thus, our method is superior for testing weakly positive samples with low virus titre, and epidemic strains present in imported goods. Conclusion Our method could be employed for universal testing of epidemic ASFV strains worldwide, ensuring wider coverage of hosts and ASFV strains/endemic strains, reducing false negatives, and benefitting early diagnosis.

猪瘟病毒C株表位突变毒株的构建及拯救

本研究旨在突变猪瘟病毒C株WH303位点,构建感染性克隆,为猪瘟病毒C株标记疫苗的研究提供候选毒株。本试验以猪瘟病毒C株为研究材料,经重叠PCR方法突变WH303位点,再分别连接至C株和C-flag株全长感染性克隆。体外转录RNA,转染PK15细胞,细胞连续传代完成病毒的拯救和增殖。结果显示,经特异性限制内切酶酶切反应验证,感染性克隆构建成功。经RT-PCR、过氧化物酶单层细胞试验检测,表明病毒成功拯救。病毒细胞传代至17代,检测到病毒,表明病毒可以稳定传代。综上,本研究成功构建了C株WH303突变感染性克隆MC和MC-flag,并成功拯救MC株,得到可以稳定传代的病毒株。

基于真核CMV启动子构建猪瘟病毒感染性cDNA克隆与病毒拯救

为建立猪瘟病毒(CSFV)反向遗传操作系统,本研究将CSFV石门株(SM)全基因组克隆至pBAC11载体中,并采用重叠延伸PCR方法在病毒基因组5'端和3'端分别加入CMV真核启动子序列和RzBGH序列,并将其基因组nt2809的碱基C突变为T,将原有的AfeⅠ酶切位点突变消失作为拯救病毒的遗传标记。将构建的质粒转染PK-15细胞培养后,收集细胞上清盲传3代后通过RT-PCR扩增蛋白编码基因,并经间接免疫荧光试验(IFA)鉴定。RT-PCR鉴定结果显示扩增到CSFV特异性基因;IFA结果显示接种病毒的细胞出现绿色荧光。表明病毒拯救成功,将其命名为BAC-rSM。将BAC-rSM株在PK-15细胞中连续传18代,将第18代病毒经RT-PCR扩增E2基因,并对遗传标记的位点经Afe I酶切和鉴定,分析BAC-rSM病毒的遗传稳定性,结果显示第18代BAC-rSM株遗传标记稳定存在,表明拯救的病毒具有遗传稳定性。将BAC-rSM和SM株病毒分别以MOI 1感染PK-15细胞,在感染后不同时间点收获病毒并测定病毒滴度,结果显示BAC-rSM和SM株生长动力学特征基本一致。对第18代BAC-rSM株与SM株进行全基因组序列比对分析,利用在线软件npsa-prabi.ibcp.fr/对Erns和E2蛋白二级结构预测,采用荧光定量PCR(qPCR)对BAC-rSM和SM株感染PK-15细胞中IFN-βmRNA的转录水平。全基因测序比对分析结果显示,BAC-rSM株基因组共存在46个碱基突变,经预测其Erns和E2的突变导致各自的二级结构改变;qPCR检测结果显示,相对SM感染的PK-15细胞,BAC-rSM感染后8 h和24 h PK-15细胞中IFN-βmRNA的转录水平显著或极显著降低(P<0.05、P<0.01)。本研究建立了一种简单、操作方便的CSFV反向遗传操作系统的方法,为进一步开展CSFV的分子生物学、毒力决定因素、分子发病机制、疫苗开发和病毒与宿主相互作用的研究提供了新的技术方法。

种公猪精液中CSFV、PCV2和PRRSV多重PCR检测方法的建立及应用

旨在建立可同时检测猪精液中猪瘟病毒(CSFV)、猪圆环病毒2型(PCV2)和猪繁殖与呼吸综合征病毒(PRRSV)的多重PCR方法。根据GenBank数据库中的CSFV E2基因、PCV2 ORF2基因及PRRSV ORF5基因,设计3对特异性引物,通过优化反应条件和特异性、敏感性检验,建立可同时检测上述3种病原的多重PCR方法,用该方法对30份精液样品进行检测。结果表明,所建立的方法可同时扩增出734 bp(CSFV)、476 bp(PCV2)、305 bp(PRRSV)的目的条带,且对猪的其他病毒(PRV、PEDV、TEGV)扩增结果呈阴性,多重PCR最低检出限1×10^(7) copies/μL,具有良好的特异性和较高的灵敏度。用单一PCR与多重PCR方法对临床样品进行检测,两种方法的检测结果一致,表明该方法可同时对种公猪精液中CSFV、PCV2和PRRSV的检测。