Currently, vaccination with influenza vaccines is still an effective strategy to prevent infection by seasonal influenza virus. However, seasonal influenza vaccines frequently fail to induce effective immune protection against rapidly changing seasonal influenza viruses and emerging zoonotic influenza viruses. In addition, seasonal influenza vaccines may not confer potent protection in elderly and immunocompromised individuals. There is an urgent need to develop potent broad-spectrum influenza vaccines to address this problem. Herein, we designed an mRNA-based broad-spectrum influenza vaccine candidate encoding cholera toxin subunit B and conserved antigens of influenza viruses. In both adult and aged mice, this universal influenza mRNA vaccine candidate stimulated robust T-cell and humoral immune responses and conferred effective protection against broad-spectrum influenza viruses in both adult and aged mice.

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 mL−1; the repeatability results showed that the intra-assay and inter-assay 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.

Classical swine fever (CSF) and porcine epidemic diarrhea (PED) are highly contagious viral diseases that pose a significant threat to piglets and cause substantial economic losses in the global swine industry. Therefore, the development of a bivalent vaccine capable of targeting both CSF and PED simultaneously is crucial. In this study, we genetically engineered a recombinant classical swine fever virus (rCSFV) expressing the antigenic domains of the porcine epidemic diarrhea virus (PEDV) based on the modified infectious cDNA clone of the vaccine strain C-strain. The S1N and COE domains of PEDV were inserted into C-strain cDNA clone harboring the mutated 136th residue of Npro and substituted 3′UTR to generate the recombinant chimeric virus vC/SM3′UTRN-S1NCOE. To improve the efficacy of the vaccine, we introduced the tissue plasminogen activator signal (tPAs) and CARD domain of the signaling molecule VISA into vC/SM3′UTRN-S1NCOE to obtain vC/SM3′UTRN-tPAsS1NCOE and vC/SM3′UTRN-CARD/tPAsS1NCOE, respectively. We characterized three vaccine candidates in vitro and investigated their immune responses in rabbits and pigs. The NproD136N mutant exhibited normal autoprotease activity and mitigated the inhibition of IFN-β induction. The introduction of tPAs and the CARD domain led to the secretory expression of the S1NCOE protein and upregulated IFN-β induction in infected cells. Immunization with recombinant CSFVs expressing secretory S1NCOE resulted in a significantly increased in PEDV-specific antibody production, and coexpression of the CARD domain of VISA upregulated the PEDV-specific IFN-γ level in the serum of vaccinated animals. Notably, vaccination with vC/SM3′UTRN-CARD/tPAsS1NCOE conferred protection against virulent CSFV and PEDV challenge in pigs. Collectively, these findings demonstrate that the engineered vC/SM3′UTRN-CARD/tPAsS1NCOE is a promising bivalent vaccine candidate against both CSFV and PEDV infections.

To develop the new classical swine fever (CSF) vaccine candidate with differentiating infected vaccinated animals (DIVA) characteristics, a chimeric CSF virus (CSFV) was constructed based on an infectious cDNA clone of the CSF vaccine C-strain. The 5’- and 3’-untranslated regions (UTRs) and partial E2 region (residues 690-860) of the C-strain were substituted with the corresponding regions of bovine viral diarrhoea virus (BVDV) to construct the chimeric cDNA clone pC/bUTRs-tE2. The chimeric virus rC/bUTRs-tE2 was generated by several passages of pC/bUTRs-tE2-transfected PK15 cells. Stable growth and genetic properties of rC/bUTRs-tE2 were obtained after 30 serial passages. Compared to parental rC/bUTRs-tE2 (1st passage), two residue mutations (M834K and M979K) located in E2 in rC/bUTRs-tE2 P30 were observed. Compared to the C-strain, rC/bUTRs-tE2 exhibited unchanged cell tropism and decreased plaque-forming ability. Substituting the C-strain UTRs with the BVDV UTRs resulted in significantly increased viral replication in PK15 cells. Compared to CSFV Erns-positive and BVDV tE2-negative antibody responses induced by the CSF vaccine C-strain, immunization of rabbits and piglets with rC/bUTRs-tE2 resulted in serological profiles of CSFV Erns- and BVDV tE2-positive antibodies, which are used to serologically discriminate pigs that are clinically infected and vaccinated. Vaccination of piglets with rC/bUTRs-tE2 conferred complete protection against lethal CSFV challenge. Our results suggest that rC/bUTRs-tE2 is a promising new CSF marker vaccine candidate.

Background Classical swine fever (CSF), African swine fever (ASF), and atypical porcine pestivirus (APPV) are acute, virulent, and contagious viral diseases currently hampering the pig industry in China, which result in mummification or stillbirths in piglets and mortality in pigs. Diagnostic assays for the differentiation of infection and vaccination of CSFV, in addition to the detection of ASFV and APPV, are urgently required for better prevention, control, and elimination of these viral diseases in China. Methods A quadruple PCR-based gene microarray assay was developed in this study to simultaneously detect wild-type and vaccine CSFV strains, ASFV and APPV according to their conserved regions. Forty-two laboratory-confirmed samples, including positive samples of 10 other swine viral diseases, were tested using this assay to confirm its high specificity. Results This assay's limit of detections (LODs) for the wild-type and vaccine CSFV were 6.98 and 6.92 copies/µL. LODs for ASFV and APPV were 2.56 × 10 and 1.80 × 10 copies/µL, respectively. When compared with standard RT-PCR or qPCR for CSFV (GB/T 26875–2018), ASFV (MARR issue No.172), or APPV (CN108611442A) using 219 clinical samples, the coincidence was 100%. The results showed that this assay with high sensitivity could specifically distinguish ASFV, APPV, and CSFV, including CSFV infection and immunization. Conclusion This assay provides a practical, simple, economic, and reliable test for the rapid detection and accurate diagnosis of the three viruses and may have good prospects for application in an epidemiological investigation, prevention, and control and elimination of these three diseases.

利用转录组测序技术(RNA-sequencing,RNA-seq)进行转录组分析是了解病原体入侵宿主分子变化的重要工具,在同时分析病原体与宿主转录组时,RNA-seq技术需要分别构建病原体及宿主的cDNA文库,再将其各自映射到病原体及宿主参考基因组中,而互作转录组测序技术(Dual RNA-seq)无需分离两物种,只需构建一个转录组文库,便能同时对两个(或多个)研究对象进行测序和分析,可以直观地揭示病原体和宿主相互作用过程中转录组学动态变化,因此Dual RNA-seq技术被广泛应用到人类疾病和生物感染模型的相互作用研究中.为了解Dual RNA-seq技术及其在宿主-病原体相互作用研究中的前景,就Dual RNA-seq技术概述以及近年来该技术在原核生物、真核生物以及病毒研究中的应用现状及发展前景进行综述.Dual RNA-seq技术可为病原体与宿主相互作用的研究提供新视角,有助于更好地识别和理解感染过程中病原体和宿主的转录组学变化,从而揭示潜在的新靶点或生物标记物.

Classical swine fever (CSF) is a highly contagious and important swine disease in China. Sporadic outbreaks with mild clinical signs are still being reported despite massive vaccination with the CSF C-strain vaccine. One possible reason for vaccine failure could be interference from maternally derived antibodies (MDAs) during vaccination in the field. The aim of this study was to evaluate the efficacy of different CSF vaccines in the presence of MDAs and to assess the different vaccination schemes in the field. The results demonstrated that vaccination with a single dose of C-strain-PK vaccine protected pigs against severe clinical signs and significantly reduced viremia. The impact of MDAs was negligible. The interference was also mild during a prime and boost vaccination scheme using the C-strain-ST vaccine. In contrast, a significant influence of MDAs on the efficacy of the subunit E2 vaccine in a one-dose vaccination scheme was observed, with pigs showing severe clinical signs, CSF-associated death, typical pathological lesions and a high level of viremia after challenge, despite robust E2 antibody induction. A field vaccination and challenge study further confirmed the superior effectiveness of a single dose of C-strain-PK vaccine in the presence of MDAs in comparison to a routine prime and boost vaccination scheme applied in the field, with pigs having fever, chronic signs, significant viremia and shedding after challenge. Delaying the vaccination time from the age of 28 days to 45 days, when MDA was low, was beneficial for improving the clinical protection and immunity induced by vaccines. Altogether, the results presented here emphasize that a high-quality vaccine and a scientific design of the vaccination scheme based on serological surveillance are essential pillars to control and eliminate CSF in China.

猪瘟病毒(CSFV)基因编码的结构和非结构蛋白参与病毒的吸附、复制或影响宿主细胞的免疫应答等.CSFV在感染宿主细胞的过程中,会利用与宿主细胞蛋白的相互作用完成其增殖等生命周期.本文对CSFV的结构和非结构蛋白在复制、增殖和毒力方面的相关研究进展进行了总结,旨在为进一步研究CSFV致病机制,建立新的防控方法提供参考.

Classical swine fever is an important swine disease in China, and sporadic outbreaks with mild clinical signs despite compulsory vaccination have raised questions about the virulence and pathogenicity of prevalent subgenotype 2.1 strains, and the ability of C-strain vaccines to cross-protect against them. To investigate this, three field isolates were evaluated in experimentally infected piglets and compared with the highly virulent reference Shimen strain. Clinical signs for the field strains ranged from mild to severe, and mortality ranged from 0 to 80 %. These data show differences in virulence among the subgenotype 2.1 field isolates and support the use of field strain GD191 as a genotype 2 challenge virus to assess efficacy of C-strain vaccines. In contrast to the historical genotype 1 strain, which caused acute infection with significant virus shedding in non-vaccinated animals, the subgenotype 2.1 GD191 strain produced different clinical manifestations in weaned piglets and adults. Adult pigs showed subclinical infection with viral shedding, whereas weaned piglets showed overt signs of infection. Efficacy of, and duration of immunity conferred by a C-strain vaccine were assessed using the reference Shimen strain and field isolate GD191 at 12 and 15 months after vaccination. A robust antibody response and sterilising protection were seen in all vaccinated animals and lasted up to 15 months post-vaccination. This study confirms that C-strain vaccines confer both clinical and virological protection against the historical genotype 1 Shimen strain and cross-protection against the prevalent genotype 2 field strain.

为建立一种能够快速鉴别猪瘟病毒野毒和疫苗弱毒株的临床检测方法,本实验针对猪瘟病毒野毒株及疫苗弱毒株分别设计了特异性鉴别引物及探针,利用RT-PCR方法特异性扩增目的基因后进行芯片杂交反应并显色,直观判定检测结果,经反应条件优化建立了一种鉴别检测猪瘟病毒的基因芯片方法,并对该方法的特异性、敏感性及重复性进行了试验.结果显示,该芯片检测方法仅能鉴别检测出猪瘟病毒野毒株与疫苗弱毒株,不能检测猪的其他12种主要传染病病原,特异性较强.对猪瘟病毒野毒株和疫苗弱毒株重组质粒标准品的最低检出限分别为6.98拷贝/μL和6.92×101拷贝/μL,敏感性较高.选择不同批次基因芯片对同一质粒标准品的检测结果均为阳性,重复性良好.利用该方法检测177份临床样本,结果与国标猪瘟病毒RT-nPCR检测方法(GB/T 26875-2018)相比较,总体符合率为99.43％(176/177).可视化猪瘟病毒鉴别诊断基因芯片方法可在2 h内完成对猪瘟野毒感染和疫苗免疫的临床样本的鉴别检测,且检测结果可用肉眼直观判定,在基层猪瘟流行病学监测及我国猪瘟的防控和净化方面具有良好的应用前景.

规律成簇的间隔短回文重复序列系统(clustered regularly interspaced short palindromic repeat,CRISPR)是一种广泛存在于古细菌和细菌中,由RNA介导在Cas蛋白协助下发挥作用的获得性免疫系统,目前,已发现的CRISPR系统中以CRISPR/Cas9应用最为广泛,本文主要对CRISPR/Cas9系统的基本原理和研究进展进行概述,着重介绍其在重要猪病毒病防控中的应用,包括改造宿主和改造病毒两方面,该技术为研究病毒致病机制、新型疫苗研发以及抗病育种研究等提供了强有力的工具,对疫病的控制有着深远的影响.

猪瘟疫苗免疫是我国猪瘟防控的核心,猪瘟病毒抗体ELISA检测是进行免疫效果评价的最常用手段.中国兽医药品监察所国家/OIE猪瘟参考实验室(以下简称:参考实验室)承担了国家认证认可监督管理委员会(以下简称“认监委”)组织的A类能力验证项目“猪瘟诊断检测技术”(CNCA-19-A01),全面考察和评价我国动物疫病检测机构对猪瘟病毒抗体的检测能力.参考实验室负责制备检测样品,并进行随机编号.向每个参试实验室发放4份待检盲样,由其采用ELISA方法对各盲样进行定性判断,并向参考实验室提供检验结果及原始检验报告.出现“不满意”结果的单位可参加补测1次,若补测结果仍为“不满意”,则表明该实验室暂不具备该参数的检测能力.参加本次能力验证猪瘟病毒抗体检测的实验室共有103家,初测满意率为92.23％,初测和补测的总体满意率为100％.此次能力验证结果表明,参加此次能力比对试验的猪瘟检测机构均能够提供正确的检测结果,可以满足猪瘟病毒抗体监测的需求.通过本次能力验证项目查找出了存在的问题,参试单位可以及时纠正错误,进一步提升猪瘟抗体的检测能力.

为了验证猪瘟病毒化学发光竞争ELISA抗体检测试剂盒的临床应用效果,对6个地区的2 200份血清样本进行检测,并采用进口试剂同步检测,比较2种试剂盒的一致性;田间筛选了 391份猪瘟抗体阴性和阳性血清,采用该试剂盒与荧光抗体病毒中和试验同时进行检测,比较试剂盒的一致性;为了进一步验证对免疫抗体的检出情况,采用本试剂盒定期对猪瘟疫苗免疫后抗体消长情况进行检测.结果显示:本试剂盒与进口试剂盒具有高度的一致性(Kappa值为0.78);与荧光抗体病毒中和试验的结果几乎完全一致(Kappa值为0.908);免疫后18 d,所有免疫猪的抗体全部转阳.

对猪瘟病毒核酸的检测是实验室检测猪瘟感染的主要手段.为全面考察动物疫病检测机构对猪瘟病毒核酸的检测能力,国家认证认可监督管理委员会(以下简称"认监委")组织了A类能力验证项目"猪瘟诊断检测技术"(CNCA-19-A01),该项目由中国兽医药品监察所国家/OIE猪瘟参考实验室(以下简称:参考实验室)承担.参考实验室负责制备了检测样品,并进行随机编号.向每个参试实验室发放4份待检盲样,不限定检测方法,参试实验室对各个盲样进行定性判断,并向参考实验室提供检验结果及原始检验报告.出现"不满意"结果的单位可参加补测一次,若补测结果仍为"不满意",则表明该实验室暂不具备该参数的检测能力.参加本次猪瘟病毒核酸检测的实验室共有97家,初测满意率为93.81％,初测和补测的总体满意率为98.97％.此次能力验证结果表明,我国90％以上猪瘟检测机构能够提供正确的检测结果,可以满足猪瘟诊断和监测的需求.对不达标机构,通过本次能力验证项目查找出了存在的问题,帮助该单位及时纠正错误,进一步提升猪瘟诊断能力.

对转录组测序(RNA-seq)的技术特点及其发展进行概述,对该技术在猪瘟感染研究中的应用进行详细综述,并对其存在的问题及未来发展做了进一步展望,以期为猪瘟的相关研究提供参考.

为开发一种操作简便、省时省力、特异性强、敏感度高的猪瘟抗体检测方法,使其更适合于我国猪瘟防控需要,本研究采用真核系统表达纯化的猪瘟病毒E2蛋白作为包被物,辣根过氧化物酶标记的E2蛋白单克隆抗体作为竞争抗体,化学发光底物作为显色体系,同时将猪瘟血清国家参考品作为溯源血清,绘制标准曲线,建立了猪瘟病毒化学发光竞争ELISA抗体检测方法.结果表明,该方法的敏感性为96.67％,特异性为96.68％;通过标准曲线的绘制,能够实现检测结果的相对定量;简化了操作步骤,检测时间大大缩短(＜1 h/96样本).猪瘟病毒化学发光竞争ELISA抗体检测方法适合于猪瘟的田间大规模普查,能够提供更高的准确度,缩短检测时间,降低工作强度,减少人工成本,具有较好的应用前景.

介绍了WHO 2004年公布的最新的国际及其他生物制品标准物质制备、鉴定和建立的指导原则,旨在引进科学的、具有指导性意义的国际生物制品标准物质建立程序和技术标准,为我国兽用生物制品标准物质制备程序的建立提供依据.

采用杆状病毒表达系统表达并纯化猪瘟病毒Erns蛋白,并对其反应原性进行鉴定.将猪瘟病毒的Erns基因克隆至pFastbac-HT-A载体上,构建了pFA-Erns重组质粒;经转座、转染后构建重组杆状病毒rBac-Erns;通过优化接毒时间和接毒量等参数,最终确定表达条件.将表达产物采用亲和层析法纯化后,采用Western Blotting鉴定Erns蛋白的反应性,并采用间接ELISA方法对其应用进行了初步探索.结果 显示,获得了重组杆状病毒rBac-Erns,经Western Blotting和间接免疫荧光(IFA)鉴定,该重组病毒能够与猪瘟阳性血清和Erns单抗发生特异性反应.纯化后的Erns蛋白纯度较好,浓度为0.2 mg/mL,经Western Blotting方法鉴定证明,纯化后的蛋白与Erns单抗和猪瘟阳性血清能够发生特异性反应;初步建立了间接ELISA方法检测猪瘟抗体,证明表达的蛋白可以区分猪瘟阳性血清和阴性血清.表明本研究利用杆状病毒表达系统成功表达并纯化了猪瘟病毒Erns蛋白,纯化后的Erns蛋白具有良好的反应性,可以作为开发鉴别诊断试剂的备选蛋白以及用于Erns蛋白的结构和生物学功能研究.

1株Asia 1型口蹄疫疫苗毒株在BHK21悬浮培养细胞中传代时与宿主BHK21细胞mRNA重组,导致VP1第206位与第207位氨基酸之间插入了VDLV序列,该插入突变使得疫苗免疫效果大幅下降,不能产生有效保护.比较发现,含12 nt插入序列的变异毒株(Vac-Asia1-VDLV)与亲本毒株(Asia 1/HN/06)在BHK21细胞上的生长曲线存在较大差异,变异毒株可感染CHO-K1,pgsA-745,pgsB-618,pgsD-677细胞,毒价可达到1×105 PFU/mL以上,而亲本毒不能感染CHO类细胞并产生蚀斑.动物试验结果显示,乳鼠半数致死量(LD50)与亲本毒株相比变化不明显.但牛感染试验表明,大剂量(1×107 LD50)接种牛舌面,Vac-Asia1-VDLV不引起体温反应和其它临床症状,荧光定量PCR检测鼻拭子和血液时未发现病毒,而Asia 1/HN/06引起典型的口蹄疫症状,鼻拭子和血液中检出病毒.上述试验结果表明,与亲本毒相比,变异毒株Vac-Asia 1-VDLV对牛的致病性明显降低.

一、我国猪瘟流行现状 根据OIE最新数据,截止2019年5月,全球有36个国家根除猪瘟. 1.中国猪瘟现状 从2011-2018年病原学监测结果来看,中国地区猪瘟发病率和死亡率一直处于下降状态,阳性带毒率从9.26％下降到0.70％,说明我国C株疫苗对我国现有的猪瘟流行毒株能提供有效保护,也为我国猪瘟的净化和根除提供了坚实的技术支持.