IntroductionClostridium perfringens (C. perfringens), a ubiquitous Gram-positive bacterium in the environment and mammalian gut flora, is a leading cause of enterotoxemia in animals, necrotizing enteritis in humans and animals, and gas gangrene in both, attributed to its diverse exotoxin profile. Alpha-toxin, a pivotal virulence factor produced by all C. perfringens serotypes, plays a central role in the pathogenicity of these diseases.MethodsHere, we engineered a lipid nanoparticle encapsulated CPA-CTD mRNA vaccine targeting the conserved C-terminal domain of C. perfringens alpha-toxin and rigorously assessed its immunogenicity and protective efficacy in mouse and bovine models.ResultsThe CPA-CTD mRNA vaccine induced strong humoral and cellular immune responses in mice, particularly in promoting the rapid production of specific IgG and mucosal IgA antibodies, as well as enhancing T cell immune responses, surpassing conventional subunit vaccines. Protection was confirmed in dual challenge models --enterotoxemia and gas gangrene --where the vaccine provided complete immunity against lethal doses of alpha-toxin and C. perfringens infection. In cattle, the CPA-CTD mRNA vaccine induced high-titer IgG antibodies and toxin-neutralizing antibodies. Notably, immunization of pregnant cows led to efficient transfer of these antibodies via colostrum to newborn calves, providing passive protection.DiscussionThese results demonstrate that the CPA-CTD mRNA vaccine provides rapid and robust immune protection against C. perfringens alpha-toxin-associated diseases, with promising potential for applications in both veterinary and human health.

The segmented genome of live-attenuated rotavirus (RV) vaccines provides an ideal platform for developing multivalent vaccines against enteric pathogens. Here, we engineered the commercial human RV vaccine strain LLR as a vector to deliver Clostridium perfringens alpha-toxin (CPA), a critical virulence factor associated with gastroenteritis. A screen of four 2A peptide sequences identified that porcine teschovirus-1 2A (P2A) preceded by an N-terminal GSG spacer enabled optimal cleavage and antigen expression. In mice, the resulting recombinant virus elicited potent systemic antibody responses against both RV and CPA following oral administration. When tested in a maternal immunization model, intramuscular immunization of cows generated high titers of neutralizing antibodies in colostrum, demonstrating its potential utility for passive immunization. By integrating a key bacterial antigen into a licensed RV vaccine backbone, we establish a versatile strategy to combat viral-bacterial co-infections and polymicrobial enteric diseases, offering a promising new approach to reduce the global burden of diarrheal mortality.IMPORTANCEThe lack of vaccines targeting polymicrobial enteric infections represents a critical gap in the fight against diarrheal diseases, a leading cause of infant mortality worldwide. To bridge this gap, we engineered a novel bivalent vaccine designed to provide dual protection against rotavirus and Clostridium perfringens alpha-toxin (CPA)-mediated disease. Leveraging an enhanced reverse genetics system, we successfully utilized the commercialized rotavirus vaccine strain LLR as a viral vector to express the key C. perfringens virulence factor, CPA. This strategy not only offers a path to broader protection against diarrheal disease but also establishes a versatile platform for developing vaccines against other viral-bacterial co-infections.

Mucosal immunization represents a promising strategy for preventing enteric infections. Rotavirus (RV), a leading gastrointestinal pathogen distinguished by its remarkable stability and segmented double-stranded RNA genome, has been engineered into a versatile oral vaccine vector through advanced reverse genetics systems. The clinical efficacy of live-attenuated RV vaccines highlights their unique capacity to concurrently induce mucosal IgA responses and systemic neutralizing antibodies, positioning them as a multiple action vector for multiple immune protection. In this review, we summarize the RV colonization of the intestine and stimulation of intestinal immunity, as well as recent advancements in RV reverse genetics, and focus on their application in the rational design of a multivalent mucosal vaccine vector targeting enteric pathogens considering the advantages and challenges of RV as a vector. We further propose molecular strategies to overcome genetic instability in recombinant RV vectors, including the codon optimization of heterologous inserts. These insights provide a theoretical foundation for developing next-generation mucosal immunization platforms with enhanced safety, stability, and cross-protective efficacy.

Schmallenberg virus (SBV) is an emerging orthobunyavirus transmitted by Culicoides midges. It poses a serious global health threat to ruminants, especially during pregnancy, causing abortion, stillbirths, and congenital malformations. Since its first outbreak in 2011, SBV has spread across Europe and other regions. Its transmission has expanded due to global climate change and increased animal trade, resulting in recurrent outbreaks in endemic regions and a growing risk of introduction into non-endemic areas. This situation highlights the urgent need for improved control strategies. This review summarizes the pathogenic and epidemiological characteristics of SBV and provides an overview of recent advancements in diagnostic approaches, vaccine development, and vector control. Diagnostic approaches, such as serological assays and nucleic acid-based tests, have become the primary tools for SBV detection. However, their applicability in clinical settings still requires further optimization. In terms of vaccine development, existing inactivated vaccines have limitations, including the inability to distinguish between vaccinated and infected animals. This has driven the development of next-generation vaccines, such as recombinant protein, viral vector, and mRNA-based platforms. For vector control, integrated approaches combining chemical, ecological, and biological strategies have been proposed to interrupt the transmission of the virus by Culicoides midges. Additionally, this review emphasizes the necessity of region-specific control strategies tailored to the differing epidemiological contexts. In endemic regions, comprehensive measures, including pathogen surveillance, vaccination programs, and Culicoides control, are critical. In non-endemic regions, the focus should be on enhancing border biosecurity, monitoring international trade, and establishing early warning systems. These strategies not only provide a scientific foundation for SBV control but also offer practical guidance for managing the spread of similar vector-borne viruses globally.

Bovine viral diarrhoea virus (BVDV) imposes significant economic burdens and biosecurity risks on the global animal trade and biological product industries. Addressing the challenges posed by the virus's complex subgenotypes requires the development of cross-protective vaccines. In this study, we evaluated the immunogenicity of two lipid nanoparticle-encapsulated mRNA (mRNA-LNP) vaccines: a full-length, membrane-anchored E2 (mE2) mRNA-LNP vaccine and a secretory, truncated E2 (tE2) mRNA-LNP vaccine, in both mice and calves. mE2 and tE2 mRNA-LNP induced robust E2-specific IgG antibodies and neutralising antibodies in mice. Neutralising antibody data further demonstrated that the mE2 mRNA-LNP exhibited superior immunogenicity compared with the tE2 mRNA-LNP in calves. Notably, administering two doses of the 100 μg mE2 mRNA-LNP effectively protected calves against BVDV challenge. Furthermore, the mE2 mRNA-LNP elicited robust cross-neutralising antibodies against multiple BVDV-1 and BVDV-2 strains for up to six months and induced promising cross-reactive cellular immune responses. The safety of mE2 mRNA-LNP was confirmed through the administration of two high doses (500 μg) in calves without adverse effects. Importantly, the rate of BVDV infection within the mE2 mRNA-LNP-vaccinated herd declined significantly, demonstrating that this vaccine effectively reduces BVDV transmission in field. In conclusion, this study comprehensively highlights the safety, efficacy, and broad cross-immune responses of the mE2 mRNA-LNP vaccine platform for preventing and controlling BVDV in calves.

>Bovine viral diarrhea virus(BVDV) is a positive-sense single-stranded RNA virus, belonging to the genus Pestivirus in the Flaviviridae family(Riitho et al. 2020). Currently, BVDV is divided into 3 main genotypes, BVDV-1,BVDV-2, and BVDV-3, based on the genetic differences in the 5′untranslated region(5′UTR)(Muasya et al. 2022). BVDV-1 is the dominant genotype circulating in China(Deng et al. 2020), while BVDV-2 is mainly identified in North America and occasionally in Europe and China(Liu et al. 2012). The BVDV-3 Hobby-like strain has recently been described in Brazil, but the global prevalence has not been investigated yet(Stalder et al. 2005). In addition, the BVDV exists as 2 biotypes: the non-cytopathogenic(NCP) and the cytopathogenic(CP) biotypes.

Unveiling the molecular mechanisms underlying rotavirus replication and pathogenesis has been hampered by the lack of a reverse genetics (RG) system in the past. Since 2017, multiple plasmid-based RG systems for simian, human, and murine-like rotaviruses have been established. However, none of the described methods have supported the recovery of bovine rotaviruses (BRVs). Here, we established an optimized plasmid-based RG system for BRV culture-adapted strain (BRV G10P [15] BLR) and clinical isolates (BRV G6P [1] C73, G10P [11] HM26) based on a BHK-T7 cell clone stably expressing T7 polymerase. Furthermore, using this optimized RG system, we successfully rescued the reporter virus BRV rC73/Zs, rHM26/Zs and rBLR/Zs, harboring a genetically modified 1.8-kb segment 7 encoding full-length nonstructural protein 3 (NSP3) fused to ZsGreen, a 232-amino acid green fluorescent protein. Analysis of the stability of genomic insertions showed that the rC73/Zs and rBLR/Zs replicated efficiently and were genetically stable in seven rounds of serial passaging, while rHM26/Zs can be stabilized only up to the third generation, indicating that the BRV segment composition may influence the viral fitness. In addition, we adopted the recombinant reporter viruses for high-throughput screening application and discovered 12 candidates out of 1440 compounds with potential antiviral activities against rotavirus. In summary, this improved RG system of BRVs represents an important tool with great potential for understanding the molecular biology of BRV and facilitates the development of novel therapeutics and vaccines for BRV.

IntroductionNeonatal calf diarrhea (NCD) is one of the most common diseases in calves, causing huge economic and productivity losses to the bovine industry worldwide. The main pathogens include bovine rotavirus (BRV), bovine coronavirus (BCoV), and Enterotoxigenic Escherichia coli (ETEC) K99. Since multiple infectious agents can be involved in calf diarrhea, detecting each causative agent by traditional methods is laborious and expensive.MethodsIn this study, we developed a one-step multiplex Real-Time PCR assay to simultaneously detect BRV, BCoV, and E. coli K99+. The assay performance on field samples was evaluated on 1100 rectal swabs of diseased cattle with diarrhea symptoms and compared with the conventional gel-based RT-PCR assay detect BRV, BCoV, and E. coli K99+.ResultsThe established assay could specifically detect the target pathogens without cross-reactivity with other pathogens. A single real-time PCR can detect ~1 copy/µL for each pathogen, and multiplex real-time PCR has a detection limit of 10 copies/µL. Reproducibility as measured by standard deviation and coefficient of variation were desirable. The triple real-time PCR method established in this study was compared with gel-based PT-PCR. Both methods are reasonably consistent, while the real-time PCR assay was more sensitive and could rapidly distinguish these three pathogens in one tube. Analysis of surveillance data showed that BRV and BCoV are major enteric viral pathogens accounting for calves’ diarrhea in China. DiscussionThe established assay has excellent specificity and sensitivity and was suitable for clinical application. The robustness and high-throughput performance of the developed assay make it a powerful tool in diagnostic applications and calf diarrhea research. ​

IntroductionBovine viral diarrhea virus (BVDV), a positive-sense single-stranded RNA virus, causes significant economic losses in the cattle industry. Current diagnostic methods for BVDV exhibit variable sensitivity and specificity, underscoring the need for more rapid and accurate detection approaches. Here, we developed a novel competitive ELISA (cELISA) to detect antibodies against the BVDV E2 protein.Methods and resultsWe generated three monoclonal antibodies (mAbs)—3E6, 2D5, and 5B9—by immunizing mice with purified BVDV E2 protein expressed in Expi293F cells. Among these, mAb 3E6 displayed superior competitive binding abilities to the E2 protein, enabling effective differentiation between BVDV positive and negative sera. Remarkably, mAb 3E6 exhibited pan-genotypic recognition of various BVDV strains, including BVDV-1a, -1b, -1c, -1m, -1p, -1v, and -2a, while showing no cross-reactivity with the classical swine fever virus (CSFV). Computational modeling using AlphaFold 3 identified domain B of the E2 protein as the primary binding site for mAb 3E6. Building upon these findings, we established a cELISA employing mAb 3E6 and recombinant E2 protein. Receiver-operating characteristic (ROC) analysis revealed outstanding diagnostic performance, achieving a sensitivity of 99.26% and specificity of 98.99%. Further tests confirmed the cELISA's specificity for detecting BVDV-specific antibodies, with no cross-reactivity with antisera from animals infected or immunized against BCoV, BHV-1, BRV, AKAV, LSDV, BLV, and CSFV. Consistency was observed between results from the BVDV E2 cELISA and traditional virus neutralization test (VNT), demonstrating high sensitivity for monitoring antibody dynamics. In performance evaluations, the established cELISA exhibited high concordance with VNT in assessing 160 vaccinated sera and 190 clinical samples.DiscussionThe BVDV E2 cELISA, utilizing mAb 3E6 to target domain B of the BVDV E2 protein, represents a reliable and effective serological diagnostic tool for the detection of antibodies against both BVDV-1 and BVDV-2. This methodology holds significant promise for applications in clinical diagnosis and the evaluation of vaccine efficacy.

IntroductionAkabane virus (AKAV) is a worldwide epidemic arbovirus belonging to the Bunyavirales order that predominantly infects livestock and causes severe congenital malformations. Reporter-expressing recombinant virus represents a powerful tool to characterize the viral biology in vitro and in vivo.MethodsIn this study, we have successfully established a reverse genetics system for AKAV. The recued virus possessed similar growth characteristics to the parental virus in vitro. Moreover, the recombinant AKAV reporter viruses expressing nanoluciferase (Nluc) or mWasabi were constructed by inserting into S segment, named rAKAV-Nluc and rAKAV-mWasabi, respectively.ResultsWe investigated the virological characteristics of rAKAV-Nluc and rAKAV-mWasabi and found that rAKAV-Nluc displayed similar growth kinetics as the parental virus and could stably produce the nano-luciferase even after 10 rounds of serial passages. rAKAV-mWasabi also exhibited comparable growth kinetics and genetic stability as the parental virus. We further used the two reporter viruses to test the susceptibility of different cell lines to AKAV and found that cell lines derived from various host species, including human, swine, cattle, and monkey enables AKAV replication efficiently, accelerating our understanding of the AKAV cell tropism range.DiscussionTaken together, our established reverse genetics system for AKAV provides more convenient screening tools and can be used to study AKAV virulence and tropism, and to elucidate the molecular biology of AKAV.

Infection with bovine leukemia virus (BLV) leads to enzootic bovine leukosis, the most prevalent neoplastic disease in cattle. Due to the lack of commercially available vaccines, reliable eradication of the disease can be achieved through the testing and elimination of BLV antibody-positive animals. In this study, we developed a novel competitive ELISA (cELISA) to detect antibodies against BLV capsid protein p24. Recombinant p24 protein expressed by Escherichia coli, in combination with the monoclonal antibody 2G11 exhibiting exceptional performance, was used for the establishment of the cELISA. Receiver-operating characteristic curve analysis showed that the sensitivity and specificity of the assay were 98.85 % and 98.13 %, respectively. Furthermore, the established cELISA was specific for detecting BLV-specific antibodies, without cross-reactivity to antisera for six other bovine viruses. Significantly, experimental infection of cattle and sheep with BLV revealed that the cELISA accurately monitors seroconversion. In a performance evaluation, the established cELISA displayed a high agreement with Western blotting and the commercial BLV gp51 cELISA kit in the detection of 242 clinical samples, respectively. In conclusion, the novel p24 cELISA exhibited the potential to be a reliable and efficient diagnostic tool for BLV serological detection with a broad application prospect.

本研究采集内蒙古自治区呼和浩特市某牛场腹泻犊牛粪样,对其进行牛轮状病毒分离鉴定和遗传进化分析.将RT-PCR检测阳性的腹泻粪样接种于MA104细胞,盲传4代后分离得到一株出现明显细胞病变的毒株,命名为HSX-21;对分离的毒株进行VP7多重半套式PCR鉴定基因型,将分离毒株测序得到的VP6、VP7和VP4基因进行遗传进化分析.结果表明分离到的毒株基因型为A群G6P[1]型,VP6基因与美国分离株NCDV同源性高达99.7％,VP7基因与英国分离株RF同源性高达99.9％;VP4基因与中国分离株NMG17044同源性高达98.8％.通过对牛轮状病毒的分离和基因型的确定,可为有效的防控本地区牛轮状病毒感染提供前期基础和理论依据.

Bluetongue virus (BTV), a member of the genus Orbivirus in the family Reoviridae, is transmitted by biting midges and causes severe disease in domestic and wild ruminants. In the present study, a BTV strain, BTV-20/GX015/China/2013 (GX015), was isolated from sentinel cattle in Guangxi, China. Virus neutralization tests and phylogenetic analyses based on genomic segments 2 (S2) and 6 (S6) indicated that GX015 belongs to BTV serotype 20 (BTV-20) and represents a new topotype within BTV-20 strains, which makes GX015 the first BTV-20 strain isolated in China. Genomic analyses suggested that the 10 genomic segments of GX015 originated from a reassortment event, in which S2 and S6 are derived from exotic BTV-20 strains (South Africa or Australia), whereas the remaining eight genomic segments are apparently of Chinese origin and most likely share the same ancestor with a Taiwanese BTV-12 strain. Importantly, we evaluated the infectivity and pathogenicity of the BTV-20 strain in mice lacking the interferon receptor (IFNAR-/- mice, a good animal model for studying the pathogenesis, virulence and transmission of BTVs) and sheep for the first time, and found that GX015 causes severe disease and death in IFNAR-/- mice and clinical signs and viraemia in the natural host sheep. These results improve our understanding of the genetic characteristics, diversity and pathogenicity of BTVs, which is important for developing diagnostic methods and vaccines for the surveillance and prevention of bluetongue disease.

为了探究宿主蛋白核仁素(NCL)对牛肠道病毒(BEV)复制的影响及其作用机制,本研究将构建的重组质粒pHA-NCL和NCL shRNA分别转染BHK-21细胞,采用western blot检测细胞中NCL的表达.结果显示,NCL在BHK-21细胞中分别获得了过表达和下调表达.在此基础上,将BEV以MOI 1感染上述过表达和下调NCL表达的BHK-21细胞,于感染8h、10 h和12 h时收集上清液和细胞,采用qPCR、western blot(检细胞)分别检测病毒基因组RNA的复制水平、BEV结构蛋白VP1表达水平及通过上清液检测病毒滴度的变化.结果表明,过表达NCL明显促进BEV在BHK-21细胞中的复制,而下调NCL的表达则明显抑制BEV的复制.利用兔源NCL特异性抗体及同源IgG对感染BEV的BHK-21细胞进行RNA免疫共沉淀试验,并利用RT-PCR检测免疫复合物中BEV相应基因片段.结果显示,免疫复合物NCL抗体-NCL蛋白-病毒RNA免疫复合物中扩增出BEV目的基因片段,而IgG抗体对照组未扩增出任何条带,表明NCL与BEV基因组RNA存在相互作用.通过激光共聚焦试验观察BEV感染BHK-21细胞后不同时间(5 h和10 h)NCL蛋白的亚细胞定位,结果显示,BEV感染细胞后使NCL由核仁迁移至细胞质,并与病毒基因组在细胞质中共定位.利用同源重组试剂盒将线性化的pmirGLO及扩增的BEV内部核糖体进入位点(IRES)构建双荧光素酶报告质粒pmirGLO-BEV IRES,并经测序鉴定正确后分别转染NCL过表达和下调表达的BHK-21细胞,采用双荧光素酶试剂盒分别检测上述细胞中两种荧光素酶FLuc和RLuc(BEV IRES依赖性的)的活性.结果显示,与转染pHA的对照细胞相比,NCL过表达的BHK-21细胞中FLuc值无显著变化,而RLuc值显著升高(P＜0.01);与转染shRNA NC的对照细胞相比,NCL表达下调的BHK-21细胞中,FLuc值无显著变化,而RLuc值显著降低(P＜0.01).表明,NCL正调控BEV IRES的翻译起始活性.本研究首次证实NCL与BEV基因组间存在相互作用,该互作可促进BEV IRES介导的病毒蛋白翻译活性,从而提高BEV的复制水平,为BEV复制的分子调控机制提供了新的见解.

In the prevention and treatment of infectious diseases, mRNA vaccines hold great promise because of their low risk of insertional mutagenesis, high potency, accelerated development cycles, and potential for low-cost manufacture. In past years, several mRNA vaccines have entered clinical trials and have shown promise for offering solutions to combat emerging and re-emerging infectious diseases such as rabies, Zika, and influenza. Recently, the successful application of mRNA vaccines against COVID-19 has further validated the platform and opened the floodgates to mRNA vaccine's potential in infectious disease prevention, especially in the veterinary field. In this review, we describe our current understanding of the mRNA vaccines and the technologies used for mRNA vaccine development. We also provide an overview of mRNA vaccines developed for animal infectious diseases and discuss directions and challenges for the future applications of this promising vaccine platform in the veterinary field.

为了对患腹泻疾病的犊牛进行病原学鉴定,并进一步丰富我国牛轮状病毒(BRV)的流行病学数据,本研究采用RT-PCR方法对8份腹泻犊牛的粪便样品进行BRV VP7基因检测,结果显示有1份样品为阳性.将该阳性病料样品接种Marc-145细胞.对产生细胞病变(CPE)的阳性样品连续传5代后,经间接免疫荧光试验(IFA)和电镜观察,结果表明从粪便样品中分离的病毒为BRV,命名为DZ株.对DZ株的11个基因节段进行RT-PCR扩增、测序、拼接后对11个基因节段进行同源性及分型分析;构建分离病毒VP7、VP4基因进化树,分析其遗传进化关系及其氨基酸序列的变异情况.结果 显示,DZ株基因组10个基因节段分别与来源于牛(VP2、VP7、NSP1、NSP3)、犬(VP3、NSP5)、羔羊(NSP2)、马(VP6)、猕猴(VP1)、人(NSP4)以及人-牛基因重配(vP4)的轮状病毒株.其中VP1基因与猕猴轮状病毒(RRV)的同源性最高,为81.3％,但低于VP1基因分型的临界值83％,因此DZ株的VP1为新出现的基因型,命名为R17;分离株NSP3基因与法国RF株NSP3基因的同源性高达98％,DZ株与RF株的NSP3属于同一基因型,命名为T18型.所以,本研究分离的BRV DZ株的基因型为G6-P5-I2-R17-C2-M2-A3-N2-T18-E2-H5.VP7和VP4基因遗传进化分析结果显示,DZ株VP7基因来源于韩国KJ69-1株,VP4基因来源于美国疫苗株RotaTeq-SC2-9.与同源性最高的病毒株相比,DZ株VP4、VP7蛋白氨基酸序列分别发生了9处和8处突变.这些突变可能会引起VP7和VP4蛋白的抗原性和组织嗜性变化,进而影响病毒的免疫原性、宿主嗜性以及致病性.综上所述,DZ株是多宿主来源的基因重配病毒株,且主要抗原蛋白VP7和VP4发生了较大变异.本研究为我国BRV遗传进化及其分子流行病学和疫苗的研究奠定了实验基础.

为建立检测牛轮状病毒(BRV)快速特异的荧光定量RT-PCR方法,本研究针对BRV NSP5基因设计了一对特异性引物经PCR扩增目的片段,并克隆于pCI载体作为重组质粒标准品(pCI-NSP5),经优化反应条件,建立了基于NSP5基因的BRV荧光定量RT-PCR方法,并对其进行了特异性、敏感性及重复性试验.结果显示,建立的SYBR Green Ⅰ荧光定量RT-PCR方法最佳引物浓度为10 μmol/L,模板为2μL,退火温度为58℃;特异性试验结果显示,该方法除对BRV的检测结果为阳性外,对牛病毒性腹泻病毒、牛冠状病毒、牛副流感3型病毒、牛呼吸道合胞体病毒的检测结果均为阴性;敏感性试验结果显示,该方法对重组质粒标准品的最低检测限为12拷贝/μL,对BRV的最低检测限为1×102TCID50/反应;批内和批间重复性试验的变异系数均小于2％.利用该方法检测30份BRV接种犊牛的粪便样品,结果25份呈阳性,而利用VP7基因的常规RT-PCR和病毒分离方法检出的阳 性样品分别为20份和25份,表明本研究建立的检测方法的敏感性高于VP7基因的常规RT-PCR方法,而与病毒分离方法的符合率为100％.以上结果表明,本研究建立的BRVNSP5基因荧光定量RT-PCR检测方法具有良好的特异性、敏感性和重复性,可以用于BRV的快速检测、病原流行病学调查以及疫苗免疫攻毒试验的病毒载量定量等研究.

旨在建立一种SYBR GreenⅠ实时荧光定量PCR(RT-qPCR)检测牛轮状病毒(BRV)的方法,并采用针对BRV vp6基因序列保守区设计的引物,使用RT-qPCR方法进行目的基因扩增,优化反应条件,制备阳性标准品,建立标准曲线,确定重复性、敏感性和特异性,与常规PCR方法比较.结果显示,建立的RT-qPCR方法最佳引物浓度为5μmol/L,退火温度为58℃,40个循环,熔解温度为77℃±0.5℃,最小检出量8.13 copies/μL.两种方法检出率分别为30.4％和19.6％,RT-qPCR具有较高敏感性.建立的RT-qPCR可为BRV感染早期诊断、分子流行病学调查及定量检测分析等提供技术保障.

Picornaviruses, as a large family of human and animal pathogens, cause a bewildering array of disease syndromes. Many host factors are implicated in the pathogenesis of these viruses, and some proteins interact with the viral IRES elements to affect function. Here, we report for the first time that cellular hnRNP L specifically interacts with the IRES of the picornavirus FMDV and negatively regulates FMDV replication through inhibiting viral RNA synthesis. Further, our results showed that hnRNP L coimmunoprecipitates with FMDV 3D pol in a viral RNA-dependent manner, suggesting that it may remain in the replication complex to function. The data presented here would facilitate further understanding of virus-host interactions and the pathogenesis of picornavirus infections.