Genome Characteristics and Evolution of Pseudorabies Virus Strains in Eastern China from 2017 to 2019

Since late 2011, outbreaks of pseudorabies virus(PRV) have occurred in southern China causing major economic losses to the pig industry. We previously reported that variant PRV forms and recombination in China could be the source of continued epidemics. Here, we analyzed samples from intensive pig farms in eastern China between 2017 and 2019, and sequenced the main glycoproteins(gB, gC, gD, and gE) to study the evolution characteristics of PRV. Based on the g C gene, we found that PRV variants belong to clade 2 and detected a founder effect during by the PRV epidemic. In addition,we detected inter-and intra-clade recombination; in particular, inter-clade recombination in the g B genes of strains FJ-ZXF and FJ-W2, which were recombinant with clade 1 strains. We also found specific amino-acid changes and positively selected sites, possibly associated with functional changes. This analysis of the emergence of PRV in China illustrates the need for continuous monitoring and the development of vaccines against specific variants of PRV.     

Pseudorabies virus DNA-binding protein stimulates the exonuclease activity and regulates the processivity of pseudorabies virus DNase

The pseudorabies virus (PRV) DNase is an alkaline exonuclease and endonuclease, which exhibits an Escherichia coli RecBCD-like catalytic function. The PRV DNA-binding protein (DBP) promotes the renaturation of complementary single strands of DNA, which is an essential function for recombinase. To investigate the functional and physical interactions between PRV DBP and DNase, these proteins were purified to homogeneity. PRV DBP stimulated the DNase activity, especially the exonuclease activity, in a dose-dependent fashion. Acetylation of DBP by acetic anhydride resulted in a loss of DNA-binding ability and a 60% inhibition of the DNase activity, suggesting that DNA-binding ability of PRV DBP was required for stimulating the DNase activity. PRV DNase behaved in a processive mode; however, it was converted into a distributive mode in the presence of DBP, implying that PRV DBP stimulated the dissociation of DNase from DNA substrates. The physical interaction between DBP and DNase was further analyzed by enzyme-linked immunosorbent assay, and a significant interaction was observed. Thus, these results suggested that PRV DBP interacted with PRV DNase and regulated the DNase activity in vitro.     

Antiviral Activity of Antimicrobial Lipopeptide from Bacillus subtilis fmbj Against Pseudorabies Virus, Porcine Parvovirus, Newcastle Disease Virus and Infectious Bursal Disease Virus in Vitro

Bacillus subtilis fmbj can produce lipopeptide antimicrobial substance, whose main components were surfactin and fengycin. In the study, the antiviral activity of antimicrobial lipopeptides (AMLs) from B. subtilis fmbj (CGMCC No. 0934) against Pseudorabies Virus (PRV), Porcine Parvovirus (PPV), Newcastle Disease Virus (NDV) and Infectious Bursal Disease Virus (IBDV) was evaluated in vitro. The AMLs represented a direct inactivation effect on cell-free virus stocks of PRV, PPV, NDV and IBDV, and it could effectively inhibit infection and replication of the NDV and IBDV, but failed to affect PRV and PPV. The AMLs were represented higher toxicity for the Porcine Kidney (PK-15) cells (50% cytotoxic concentration (CC₅₀) value was 32.87 μM) and lower for the Chicken Embryo Fibroblasts (CEF) cells (CC₅₀ value was 89.16 μM). The Selectivity index of AMLs on PRV, PPV, NDV and IBDV was 1.44, 2.23, 8.40 and 12.19, respectively.     

表达猪繁殖与呼吸综合征病毒修饰型GP5m蛋白的重组伪狂犬病毒的构建及其在小鼠的免疫反应

猪伪狂犬病毒（PRV）是一种良好的兽用活病毒疫苗载体。但以PRV基因缺失疫苗株TK^-/gE^-/LacZ⁺为载体表达PRRSV GP5的重组病毒TK^-/gE^-/GP5⁺免疫实验动物后难以激发抗PRRSV的中和抗体。为了进一步增强这种重组病毒的免疫效力,用具有更好免疫原性的修饰的ORF5基因（ORF5m）代替天然ORF5基因,构建了表达PRRSV的修饰型GP5m蛋白的重组伪狂犬病毒TK^-/gE^-/GP5m⁺。经PCR、Southern blot、Western blot 证实构建正确,并能表达具有活性的GP5m蛋白。将TK^-/gE^-/GP5m⁺与TK^-/gE^-/GP5⁺分别免疫Balb/c小鼠,结果TK^-/gE^-/GP5m⁺免疫小鼠不仅产生了较高水平的抗PRRSV的中和抗体(3/6只达到了1∶16),而且在诱导PRRSV特异性细胞免疫方面也显著优于TK^-/gE^-/GP5⁺,表明TK^-/gE^-/GP5m⁺是一种极有希望的PRRSV和PRV二价基因工程候选疫苗。     

Determination of the effectiveness of Pseudorabies marker vaccines in experiments and field trials

The aim of vaccination in an eradication campaign is not only to induce clinical protection, but primarily to stop transmission of infections within and between herds by inducing herd immunity. Consequently, vaccines should be evaluated for their capacity to reduce virus transmission in the population. Glycoprotein E (gE) negative marker vaccines against Pseudorabies virus (PRV) infections in pigs have been evaluated this way in experiments and field studies. PRV infection in groups of (vaccinated) pigs was determined by measuring antibodies against gE of PRV from regularly taken serum samples. For the statistical analysis of the experiments a stochastic susceptible-infectious-removed (SIR) model was used. A measure for the transmission of virus is the reproduction ratio R, which is defined as the average number of secondary cases caused by one typical infectious individual. This implies that an infection will always fade out in a population when R < 1, but the infection can spread massively when R > 1. From several experiments it was shown that R < 1. Field studies showed that the R within herds was still > 1, but by reducing further contacts the R could be reduced to a value below one. This would imply that PRV could be eradicated by means of vaccination. In The Netherlands, an eradication campaign was launched in 1993, and in 2002 the virus was eradicated, as shown by a negligible number of gE-positive pigs. Farmers' organizations have to decide whether or not to stop vaccination.     

伪狂犬病毒gI基因的克隆表达及其对病毒增殖的影响

从伪狂犬病毒(PRV)国内地方分离Ea株基因组DNA片段中克隆了完整的gI基因,序列分析结果表明,gI基因编码区全长1101bp,可编码366个氨基酸残基,二级结构预测具有典型I型膜蛋白特征。与GenBank中收录的国外Rice株的同源比较发现,Ea株gI在核苷酸和氨基酸水平上均存在多处突变,尤其是潜在胞浆区中连续两个碱基的缺失导致移码突变,致使gI基因的读码框架后移,从而导致Ea株gI较rice株长16个氨基酸残基。将gI基因克隆到真核表达载体pcDNA31+中的人巨细胞病毒早期启动子下游,构建的真核表达质粒转染PK15细胞,间接免疫荧光检测证实gI获得正确表达。进一步测定天然缺失gI的PRV弱毒Bartha株在表达gI细胞系和空白载体转染的对照细胞系中的蚀斑形成单位(pfu)和组织细胞培养半数感染量(TCID50),结果显示：Bartha株在表达gI细胞系中的pfu和TCID\-\{50\}分别为对照细胞系的164%和200%。说明gI具有促进病毒增殖的功能。     

大鼠孤束核中神经肽Y样神经元与咽肌运动神经元的联系——PRV和NPY免疫荧光双标记法研究

用PRV和NPY免疫荧光双标记法研究了大鼠孤束核中NPY样神经元对咽肌运动神经元的调控。PRV注射大鼠咽肌后,在孤束核的中介亚核和中间亚核中可见许多PRV和NPY双标记细胞。首次证明了大鼠孤束核中的NPY样神经元和咽肌运动神经元的联系。推测NPY可能对咽肌运动的精确调控有关。     

伪狂犬病病毒ul24基因表达蛋白的胞内定位研究

根据GenBank已发表的PrVul24基因序列(NC006151),设计并合成一对引物,PCR扩增出ul24基因编码区,克隆于pEGFP-N1载体,得到重组质粒pUL24-GFP。酶切鉴定,测序及WesternBlot验证重组质粒。ul24基因序列测定结果已提交GenBank,登录号DQ226544。Westernblot分析结果表明UL24-GFP融合蛋白为45KD。将pUL24-GFP转染真核细胞,激光共聚焦显微镜观察融合蛋白的细胞内定位,结果表明UL24-GFP融合蛋白定位于细胞核。     

检测PCV2、PPV、PRV疫苗株与野毒株的多重PCR方法

本文建立了一种同时检测猪圆环病毒2型(PCV2)、细小病毒(PPV)、及伪狂犬病毒(PRV)疫苗株与野毒株的多重PCR方法.根据GenBank上发表的PCV2、PPV和PRV gB、gE基因序列,针对各自保守区各设计一对特异性引物,用这四对引物对同一样品中的PCV2、PPV和PRV gB、gE进行检测,结果可同时扩增出269bp(PCV2)、581bp(PPV)、372bP(PRV gB)及147bp(PRV gE)四条特异性片段.对JEV、PRRSRV、大肠杆菌和双蒸水的PCR扩增结果均为阴性;敏感性测定结果表明,该多重PCR能检出10pg PCV2、PPV和PRV gB、gE检测敏感度分别为10-6.2、10-3.8、10-5.8TCID50的模板.该方法的建立对临床上进行这三种疾病的鉴别诊断和混合感染的检测具有重要意义.