During the co-evolution of viruses and their hosts, viruses have developed various strategies for overcoming host immunological defenses so that they can proliferate efficiently. Porcine reproductive and respiratory syndrome virus (PRRSV), a significant virus to the swine industry across the world, typically establishes prolonged infection via diverse and complicated mechanisms, which is one of the biggest obstacles for controlling the associated disease, porcine reproductive and respiratory syndrome (PRRS). In this review, we summarize the latest research on how PRRSV circumvents host antiviral responses from both the innate and adaptive immune systems and how this virus utilizes other evasion mechanisms, such as the manipulation of host apoptosis and microRNA. A thorough understanding of the exact mechanisms of PRRSV immune evasion will help with the development of novel antiviral strategies against PRRSV.

Background Porcine epidemic diarrhea (PED), caused by PED virus (PEDV), is a severe enteric disease burdening the global swine industry in recent years. Especially, the mortality of PED in neonatal piglets approaches 100%. Maternal antibodies in milk, particularly immunoglobulin A (IgA) antibodies, are of great importance for protection neonatal suckling piglets against PEDV infection as passive lactogenic immunity. Therefore, appropriate detection methods are required for detecting PEDV IgA antibodies in milk. In the current study, we prepared monoclonal antibodies (mAbs) against PEDV spike (S) glycoprotein. An enzyme-linked immunosorbent assay (ELISA) was subsequently developed based on PEDV antigen capture by a specific anti-S mAb. Results The developed ELISA showed high sensitivity (the maximum dilution of milk samples up to 1:1280) and repeatability (coefficient of variation values < 10%) in detecting PEDV IgA antibody positive and negative milk samples. More importantly, the developed ELISA showed a high coincidence rate with a commercial ELISA kit for PEDV IgA antibody detection in clinical milk samples. Conclusions The developed ELISA in the current study is applicable for PEDV IgA antibody detection in milk samples, which is beneficial for evaluating vaccination efficacies and neonate immune status against the virus.

Screening peptides with good affinity is an important step in peptide-drug discovery. Recent advancement in computer and data science have made machine learning a useful tool in accurately affinitive-peptide screening. In current study, four different tree-based algorithms, including Classification and regression trees (CART), C5.0 decision tree (C50), Bagged CART (BAG) and Random Forest (RF), were employed to explore the relationship between experimental peptide affinities and virtual docking data, and the performance of each model was also compared in parallel. All four algorithms showed better performances on dataset pre-scaled, -centered and -PCA than other pre-processed dataset. After model re-built and hyperparameter optimization, the optimal C50 model (C50O) showed the best performances in terms of Accuracy, Kappa, Sensitivity, Specificity, F1, MCC and AUC when validated on test data and an unknown PEDV datasets evaluation (Accuracy=80.4 %). BAG and RFO (the optimal RF), as two best models during training process, did not performed as expecting during in testing and unknown dataset validations. Furthermore, the high correlation of the predictions of RFO and BAG to C50O implied the high stability and robustness of their prediction. Whereas although the good performance on unknown dataset, the poor performance in test data validation and correlation analysis indicated CARTO could not be used for future data prediction. To accurately evaluate the peptide affinity, the current study firstly gave a tree-model competition on affinitive peptide prediction by using virtual docking data, which would expand the application of machine learning algorithms in studying PepPIs and benefit the development of peptide therapeutics.

With the continuous development of China's economy and society, people and the government have higher and higher requirements for food safety. Testing for food dopants and toxins can prevent the occurrence of various adverse health phenomena in the world's population. By deploying new and powerful sensors that enable rapid sensing processes, the food industry can help detect trace adulteration and toxic substances. At present, as a common food safety detection method, lateral flow immunochromatography (LFI) is widely used in food safety testing, environmental testing and clinical medical treatment because of its advantages of simplicity, speed, specificity and low cost, and plays a pivotal role in ensuring food safety. This paper mainly focuses on the application of lateral flow immunochromatography and new technologies combined with test strips in food safety detection, such as aptamers, surface-enhanced Raman spectroscopy, quantum dots, electrochemical test strip detection technology, biosensor test strip detection, etc. In addition, sensing principles such as fluorescence resonance energy transfer can also more effective. Different methods have different characteristics. The following is a review of the application of these technologies in food safety detection.

为了建立猪瘟病毒(CSFV)血清抗体检测方法,将CSFV E rns和E2蛋白主要抗原区串联表达的编码核酸序列克隆至原核表达载体pET-32a中,转化大肠杆菌Rosetta TM2(DE3)构建重组表达菌.SDS-PAGE、Western blot鉴定及蛋白质可溶性分析结果显示,表达的E rns/E2蛋白分子质量约为43 ku,能够被CSFV阳性血清识别,主要以包涵体形式存在.通过快速稀释法复性了纯化的包涵体蛋白,复性效率大于40％.以纯化复性的E rns/E2蛋白为包被抗原,经优化各反应条件,建立了间接ELISA检测CSFV抗体方法(E rns/E2-ELISA).特异性、敏感性和重复性试验结果表明,建立的检测方法特异性强、敏感性高、重复性好.利用建立的E rns/E2-ELISA检测方法对121份随机采取的临床血清样本进行检测,与IDEXX CSFV抗体检测试剂盒相比,E rns/E2-ELISA检测方法符合率为86.78％,敏感性为92.11％,特异性为77.78％.利用建立的E rns/E2-ELISA检测方法和IDEXX CSFV抗体检测试剂盒同时评价CSFV C株疫苗免疫的4只30日龄健康仔猪抗体消长规律,可分别于免疫后7 d和14 d开始检出阳性,35～42 d抗体水平达到高峰,49～56 d抗体趋于稳定.

Vesicular stomatitis (VS), characterized by vesicular lesions, produces significant economic losses in livestock industry. Infection by its causative agent, VS virus (VSV), has been previously shown to be mediated by the glycoprotein (G) during attachment, endocytosis and membrane fusion. In the current study, we revealed a novel role of VSV G protein in negative regulation of host cell pro-inflammatory responses. We determined that VSV G protein inhibited lipopolysaccharide (LPS)-induced pro-inflammatory responses as naïve VSV virions in murine peritoneal macrophage-like cell line RAW 264.7. Furthermore, we identified that VSV G protein suppressed nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK)-mediated pro-inflammatory pathways in a dose-dependent manner. Moreover, we demonstrated that α2-3-linked sialic acids on VSV G protein were involved in antagonizing NF-κB- and MAPK-mediated pro-inflammatory responses. All these results expand the knowledge of VSV pathogenesis and strengthen the importance of VSV G protein in host innate immunity, which support implications for the development of VSV-based vaccination and oncolysis.

为了得到一种能够高通量检测细胞培养物的方法,并将其用于筛选特异性强、敏感度高的抗猪繁殖与呼吸综合征病毒(PRRSV)单克隆抗体.用每孔能感染约100个细胞的病毒量接种单层覆盖96孔板的Marc-145细胞,12 h后用含3％H2 O2的甲醇固定细胞,以制备免疫过氧化物酶单层细胞试验(IPMA)反应板.以100μL/孔的量将融合后继续培养10 d的杂交瘤细胞的培养上清加入至IPMA反应板,以辣根过氧化物酶标记的羊抗鼠IgG-HRP作为二抗,以3-氨基-9-乙基咔唑(AEC)作为显色底物,于倒置显微镜下进行观察.结果表明,共筛选出1D1、7G8等39份PRRSV单克隆抗体,这39份单抗能够使PRRSV中高致病毒株HN07-1和经典毒株BJ-4感染的Marc-145细胞被特异性染色,而对猪瘟病毒、猪伪狂犬病毒、猪流行性腹泻病毒感染的Marc-145细胞无交叉染色.因此,构建的IPMA方法能够敏感、准确地捕捉到PRRSV单克隆抗体.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a single-stranded positive-sense RNA virus, and the current strategies for controlling PRRSV are limited. Interferon gamma-inducible protein 16 (IFI16) has been reported to have a broader role in the regulation of the type I interferons (IFNs) response to RNA and DNA viruses. However, the function of IFI16 in PRRSV infection is unclear. Here, we revealed that IFI16 acts as a novel antiviral protein against PRRSV-2. IFI16 could be induced by interferon-beta (IFN-β). Overexpression of IFI16 could significantly suppress PRRSV-2 replication, and silencing the expression of endogenous IFI16 by small interfering RNAs led to the promotion of PRRSV-2 replication in MARC-145 cells. Additionally, IFI16 could promote mitochondrial antiviral signaling protein (MAVS)-mediated production of type I interferon and interact with MAVS. More importantly, IFI16 exerted anti-PRRSV effects in a MAVS-dependent manner. In conclusion, our data demonstrated that IFI16 has an inhibitory effect on PRRSV-2, and these findings contribute to understanding the role of cellular proteins in regulating PRRSV replication and may have implications for the future antiviral strategies.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection which caused severe reproductive failure and respiratory disorders in swine is accompanied with severe nervous symptoms. Our previous studies demonstrated that microglia, the resident innate immune cells in central nervous system (CNS), could support PRRSV infection and replication in vitro. And PRRSV infection led to the increased expressions of large amounts of proinflammatory cytokines and chemokines which contributed to neuropathogenesis of PRRSV. Interleukin-1 beta (IL-1 beta) is one of the increased proinflammatory cytokines, which possesses diverse functions in immune response upon virus infection, including activation of innate immune and modulation of adaptive immune responses. Importantly, considerable evidences indicated that IL-1 beta is involved in neuronal injury. Here, we demonstrated that PRRSV infection up-regulated IL-1 beta expression at both the mRNA and protein levels in microglia in a dose-dependent manner. Myeloid differentiation primary response gene 88 (MyD88), extracellular signal-regulated kinase1/2 (ERK) and activator protein 1 (AP-1) were involved in PRRSV induced IL-1 beta production in microglia. Moreover, NOD-like receptor protein 3 (NLRP3) inflammasome is activated by PRRSV in microglia, which is required for IL-1 beta secretion. Taken together, our data indicated that PRRSV infection could induce IL-1 beta up-regulation, which was likely mediated by MyD88/ERK/AP-1 and NLRP3 inflammasome. These findings will provide new insights into the molecular mechanisms of IL-1 beta production and some implications for neuropathogenesis of PRRSV.

Porcine epidemic diarrhea (PED) has caused huge economic losses to the global pork industry. Infection by its causative agent PED virus (PEDV), an Alpha-coronavirus, was previously proven to be mediated by its spike (S) glycoprotein and a cellular receptor porcine aminopeptidase N (pAPN). Interestingly, some recent studies have indicated that pAPN is not a functional receptor for PEDV. To date, there is a lack of a direct evidence for the interaction between pAPN and PEDV S protein in vitro. Here, we prepared pAPN ectodomain and the truncated variants of PEDV S protein in Drosophila S2 cells. These recombinant proteins were homogeneous after purification by metal-affinity and size-exclusion chromatography. We then assayed the purified target proteins through immunogenicity tests, PEDV binding interference assays, circular dichroism (CD) measurements, pAPN activity assay and structural determination, demonstrating that they were biologically functional. Finally, we characterized their interactions by gel filtration chromatography, native-polyacrylamide gel electrophoresis (PAGE) and surface plasmon resonance (SPR) analyses. The results showed that their affinities were too low to form complexes, which suggest that pAPN may be controversial as the genuine receptor for PEDV. Therefore, further research needs to be carried out to elucidate the interaction between PEDV and its genuine receptor.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection which caused severe reproductive failure and respiratory disorders in swine is accompanied with severe nervous symptoms. Our previous studies demonstrated that microglia, the resident innate immune cells in central nervous system (CNS), could support PRRSV infection and replication in vitro. And PRRSV infection led to the increased expressions of large amounts of proinflammatory cytokines and chemokines which contributed to neuropathogenesis of PRRSV. Interleukin-1β (IL-1β) is one of the increased proinflammatory cytokines, which possesses diverse functions in immune response upon virus infection, including activation of innate immune and modulation of adaptive immune responses. Importantly, considerable evidences indicated that 1L-1β is involved in neuronal injury. Here, we demonstrated that PRRSV infection up-regulated IL-1β expression at both the mRNA and protein levels in microglia in a dose-dependent manner. Myeloid differentiation primary response gene 88 (MyD88), extracellular signal-regulated kinase1/2 (ERK) and activator protein 1 (AP-1) were involved in PRRSV induced IL-1β production in microglia. Moreover, NOD-like receptor protein 3 (NLRP3) inflammasome is activated by PRRSV in microglia, which is required for IL-1β secretion. Taken together, our data indicated that PRRSV infection could induce IL-1β up-regulation, which was likely mediated by MyD88/ERK/AP-1 and NLRP3 inflammasome. These findings will provide new insights into the molecular mechanisms of IL-1β production and some implications for neuropathogenesis of PRRSV.

Specific primers were designed upon the conservative sequences of Marek's disease virus (MDV),infectious laryngotracheitis virus (ILTV),and fowlpox virus (FPV) genomic nucleotide sequences from registered in GenBank.Three pairs of specific primers for each virus were designed,a total of 9 pairs of primers for three viruses(MDV,ILTV,FPV) were screened by biological software,and three pairs of primers matched each other.After verification of three pair suitable primers with singleplex PCR,the reaction conditions of the multiplex PCR were optimized,including annealing temperature,concentrations of primers and Taq DNA polymerase.The simple multiplex PCR assay was successfully established after specificity,sensitivity and simplified test.The specific bands with lengths of 228 bp(MDV),400 bp (ILTV),499 bp(FPV) were amplified by the multiplex PCR,which were consistent with those expected.The results demonstrated that the method could simultaneously amplify these three viruses at a sensitivity of 100 fg when all three viruses were present.According to the principle of PCR technology,the classical PCR(a three step thermal cycle assay) was improved to be a two step thermal cycle assay,namely annealing and extension step were merged into one step (62 ℃),and the reaction time was shortened.This multiplex PCR for three DNA viruses was both highly specific and sensitive,and could be used as a rapid diagnostic assay for clinical samples.

According to the pseudorabies virus strain BJ/YT gD gene sequences in GenBank,a pair of primers were designed. The B-cell epitopes of gD was amplified,and the length of PCR product was 702 bp. The amplified fragment was cloned into the cloning vector pMD19-T successfully named pMD19-T-gD. The fragment was digested by BamH Ⅰ and Hind Ⅲ restriction enzymes and then cloned into the expression vector pET-28a named pET-28a-gD. The expression of protein was induced with IPTG,specific protein bands appeared at the site of 25 ku by SDS-PAGE electrophoresis. Western blot analysis showed that the expression product could be identified by pseudorabies virus positive serum. In this study, gD protein epitope was cloned and expressed successfully, and the expression product had good biological activity.

依据Gen Bank中注册的禽流感病毒（AIV）、鸡传染性支气管炎病毒（IBV）、新城疫病毒（NDV）和传染性法氏囊病毒（IBDV）基因组核苷酸序列,通过序列比对获得AIV、IBV、NDV、IBDV的相对保守序列。根据每种病毒的保守序列利用生物学软件分别设计3对特异性引物,4种病毒共设计12对引物,并对12对引物进行模拟筛选,获得匹配最佳的4对引物。对获得的最佳匹配引物进行单项PCR验证,优化复合PCR反应条件,确定最佳的退火温度、引物浓度和Taq DNA聚合酶使用量;经特异性、敏感性及简化PCR试验,最终建立了简易复合PCR检测方法。结果表明,建立的检测方法可同时扩增出4条大小与设计相符的特异性片段,即IBV（146 bp）、NDV（215 bp）、IBDV（345 bp）、AIV（435 bp）;建立的复合PCR方法具有较强的敏感性和特异性,能检测出100 pg AIV、IBV、IBDV的cDNA和100 fgNDV的cDNA;将三温热循环改进为二温热循环,即将退火和延伸过程合为一步（62℃）,大大缩短了反应时间。

The key goals of immunocontraception research are to obtain full contraceptive effects using vaccines administered to both males and females. Current research concerning human anti-sperm contraceptive vaccines is focused on delineating infertility-related epitopes to avoid autoimmune disease. We constructed phage-display peptide libraries to select epitope peptides derived from human posterior head 20 (hPH20) and homo sapiens sperm acrosome associated 1 (hSPACA1) using sera collected from infertile women harbouring anti-sperm antibodies. Following five rounds of selection, positive colonies were reconfirmed for reactivity with the immunoinfertile sera. We biopanned and analysed the chemical properties of four epitope peptides, named P82, Sa6, Sa37 and Sa76. Synthetic peptides were made and coupled to either bovine serum albumin (BSA) or ovalbumin. We used the BSA-conjugated peptides to immunise BALB/c mice and examined the effects on fertility in female and male mice. The synthetic peptides generated a sperm-specific antibody response in female and male mice that caused a contraceptive state. The immunocontraceptive effect was reversible and, with the disappearance of peptide-specific antibodies, there was complete restoration of fertility. Vaccinations using P82, Sa6 and Sa76 peptides resulted in no apparent side effects. Thus, it is efficient and practical to identify epitope peptide candidates by phage display. These peptides may find clinical application in the specific diagnosis and treatment of male and female infertility and contraceptive vaccine development.

An immunochromatographic strip was developed for the serological detection of pseudorabies virus (PRV) in swine. In the strip, the expressed protein of gB, one of the glycoproteins of PRV, labeled with colloidal gold, was used as the detector; staphylococcal protein A and swine anti-pseudorabies virus antibody were blotted on nitrocellulose membrane for the test and control lines, respectively. The specificity of the strip was 98.1%, and the sensitivity of the strip with reference anti-PRV serum was 96.0%. Swine serum samples (296) were collected to evaluate the characteristics of the strip in comparison with an existing commercial kit. The agreement was 93.6%. Furthermore, the dipstick assay based on the strip is rapid (5 min) and easy to perform with no requirement of professional skills, reagents, or equipment. This suggests that the immunochromatographic strip is an acceptable alternative for use in clinical laboratories lacking specialized equipment and for field diagnosis.

In several parts of China, there have been a large number of pseudorabies (PR) outbreaks which have devastated many swine farms even though the herds had been previously immunized with gE-deleted vaccines (Bartha-K61). The emergence of these outbreak-associated PRV strains might indicate that Bartha-K61 vaccine could not provide effective protection and poses challenges for current serologic diagnostics of anti-PRV antibodies. Here, we performed phylogenetic analyses based on partial gE, gB, and gC genes to provide information about the molecular epidemiology, diagnostics, and immune protection in these outbreak-associated PRV strains. Our results indicated that the maximal nucleotide sequence divergence for gE, gB, and gC genes are 1.7, 0.4, and 2.7 % within the cluster where outbreak-associated PRV strains were located, and are 2.3, 2.7, and 7.6 % with other clusters in the phylogenetic trees, respectively. Phylogenetic analyses revealed that gE, gB, and gC genes of the twelve outbreak-associated PRV strains clustered to a relatively independent branch of the tree, and evolved from the same ancestor with strains Ea-China-1999, Fa-China-2001, and BJ-China-2008. The genetic relationship between these outbreak-associated PRV strains and strain Bartha is not close which may genetically explain the emergence of PR outbreaks in Bartha-K61-vaccinated swine farms. We suggest that these outbreak-associated PRV strains originate from earlier strains in local regions in China.

An isolate of pseudorabies virus(PRV) was obtained from the tissues of dead piglets from a farm with PRV-like disease in Henan province.This isolate of PRV could induce typical cytopathogenic effects after several passages on PK-15 cells and be neutralized by PRV positive serum in microneutralization test.This isolate was sensitive to chloroform and ether,and could be inactivated in 56 ℃ water bath after 30 min.After inoculation,both rabbits and mice showed typical clinical symptoms of pseudorabies.The cloning and sequencing of gp50 gene showed that the nucleotide homology of this isolate with other 5 PRV isolates previously published in GenBank was 99%,indicating that this isolate is PRV.

Three pairs of primers were designed according to conservative sequences on DBP genes on Pseudorabies virus(PRV).A method of loop-mediated isothermal amplification(LAMP) for rapid and visible detection of PRV has been established by optimizing reacting system using rhodamine B derivatives as the indicator.The results showed that the visible results can be observed at 40 min under the condition of 63 ℃ water bath.The blue color is regarded as positive while the purple is negative.The result of visible LAMP was in accordant with that of agarose gel electrophoresis.

MicroRNAs(miRNAs)are a class of non-coding small RNA in length of 22-25 nt and distribute extensively in human and other organism genomes.They regulate gene expression through sequence specific interactions with target mRNAs resulting in translational repression or mRNA degradation.Viral microRNAs are a class of newly discovered miRNA.This paper introduces the progress on the researches of the generation,functions and mechanisms of viral micro-RNAs and its function involved in animal diseas.