Effects of interferon on immediate-early mRNA and protein levels in sensory neuronal cells infected with herpes simplex virus type 1 or pseudorabies virus

Most alphaherpesviruses are able to establish latency in sensory neurons and reactivate upon specific stimuli to cause recurrent symptoms. We have previously shown that interferon (IFN) is capable of inducing a quiescent HSV-1 and PRV infection that strongly resembles in vivo latency in primary cultures of TG neurons. This IFN-induced latency-like quiescence was found to correlate with suppression of the immediate-early protein ICP4 in HSV-1 and its ortholog IE180 in PRV. Here, we mechanistically investigated the IFN-mediated suppression of ICP4 and IE180 in sensory neuronal cells. RT-qPCR showed that mRNA levels of either HSV ICP4 or PRV IE180 at 4 hpi were mildly but not significantly different in IFN-treated samples versus control samples, whereas a strong reduction was observed at 8 hpi and 12 hpi. However, at 4 hpi, HSV ICP4 but not PRV IE180 protein expression was already markedly reduced in IFN-treated samples. In line with this difference in IFN-mediated suppression of HSV ICP4 versus PRV IE180 protein levels, we found that IFN resulted in an increase in phosphorylation of the translation initiation factor eIF2α in HSV-infected but not in PRV-infected cells. The latter finding indicates that PRV efficiently circumvents IFN-mediated translation inhibition by interfering with phosphorylation of eIF2α.     

近期部分规模化猪场猪伪狂犬病野毒抗体监测情况调查

猪伪狂犬病仍然是我国猪群的重要威胁性传染病，通常造成母猪繁殖障碍以及仔猪的神经症状和高死亡率，同时伪狂犬病病毒也是猪呼吸系统疾病综合征的重要原发性病原。用gE-ELISA野毒鉴别诊断方法共检测了来自8个省、市46个猪场1940份血清样品，其中阳性样品657份，样品总阳性率33.87%，阳性猪场25个。在检测的各场中，阳性率最高猪场达到75.76%(125/165)。而部分进行了科学的疫苗免疫和实施了严格的生物安全措施的猪场始终保持猪伪狂犬病阴性(0/135)。利用基因缺失疫苗科学免疫配合伪狂犬抗体鉴别诊断技术是控制和净化伪狂犬病的重要手段。     

Factors associated with circulation of pseudorabies virus within swine herds

Data were collected from 104 Minnesota swine farms quarantined for pseudorabies virus (PRV) infection. Each herd was serologically evaluated for the presence of antibodies to PRV in finishing pigs. Herd management practices, swine housing design, and disease profiles were described for each farm. Multiple logistic regression analysis was used to determine which factors were associated with circulation of PRV in the finishing pigs of farrow-to-finish farms. Sixty-seven (64%) of the herds had no serologic evidence of PRV circulation in the finishing section, whereas 37 herds (36%) contained at least one PRV seropositive finishing pig. The odds of a given finishing herd being seropositive for PRV were 2.85 times higher if the finishing pigs were housed in confinement (P = 0.01), 2 times higher if Actinobacillus (Haemophilus) pleuropneumoniae was a clinical problem in the herd (P = 0.03), 1.36 times less for each year that passed since the herd quarantine was issued (P = 0.01), 1.74 times higher if clinical signs of PRV were reported (P = 0.04), and 1.52 times higher if animal protein was included in at least one of the rations (P = 0.08).     

Development of real-time polymerase chain reaction assays for rapid detection and differentiation of wild-type pseudorabies and gene-deleted vaccine viruses

The successful eradication of pseudorabies in U.S. domestic swine was accomplished through the use of glycoprotein E (gE) deleted modified live virus vaccines and an accompanying gE differential enzyme-linked immunosorbent assay (ELISA). Yet, pseudorabies virus (PRV) was established in feral swine in the United States, becoming a potential reservoir of PRV for infection of domestic swine and other native wildlife. A critical need for the current PRV surveillance program in the United States is the rapid detection of PRV infection. For this reason, a set of 2 real-time polymerase chain reaction (PCR) assays by using TaqMan chemistry was developed and evaluated for their capability in the detection and differentiation of field and vaccine strains of PRV. PCR primers and probes were designed for gB and gE genes of PRV, respectively. The newly developed PRV-specific real-time PCR assays could detect all wild-type PRV isolates from diagnostic submissions and differentiate them from vaccine strains. The analytical sensitivity of the assays was approximately 0.1 plaque-forming units per reaction. The assays were highly specific for PRV, because no positive results were obtained from testing other common swine viral pathogens and other animal herpesviruses. The results of testing samples from domestic and feral swine and from bovine showed that the real-time PCR assays are more sensitive than gel-based PCR. These results demonstrated the potential application of the developed real-time PCR assays as a differential test for rapid and specific detection of PRV in domestic and feral swine, as well as nonporcine species that can be infected with PRV and serve as carriers.     

Factors affecting the geographic distribution of pseudorabies (Aujeszky's disease) virus infection among swine herds in Illinois

Data on the geographic distribution of swine herds tested for pseudorabies virus (PRV) in the state of Illinois (USA) were analyzed to determine whether the prevalence of PRV-infected herds was clustered geographically at the county level. Second-order analysis of spatial dependence indicated there was a spatial clustering of counties of high PRV prevalence rates and that this clustering was greater than the observed clustering of counties with a large number of swine herds. The clustering of county PRV prevalence rates was most apparent within a radius of 120 km (on the average, approximately two couties away). The association of county PRV prevalence rates with average herd size, geographic density of swine herds in the country and regional (within 120 km) density of PRV-infected herds was analyzed using multiple linear regression. The primary factor affecting county PRV prevalence rates was the regional PRV density, which interacted with the other model variables. For counties with a low regional density of PRV infection, county PRV prevalence rates charged little with a change in county herd density or average herd size. In contrast, for counties with a high regional density of PRV infection, PRV prevalence within a county increased with increasing average herd size and increasing geographic density of swine herds in the county. The results of the current and previous studies implicate an important role for the geographic proximity of infected herds in the spread of PRV among swine herds.     

Tn5转座子介导表达猪瘟E2蛋白和EGFP重组伪狂犬病毒的构建

为构建猪瘟重组伪狂犬疫苗,以Tn5转座子为介导,采用体外转座的方法将猪瘟病毒（Classical swine fever virus,CSFV）E2基因和增强绿色荧光蛋白（enhanced green fluoresce protein,EGFP）的表达盒随机插入伪狂犬病毒（Pseudorabies virus,PRV）基因组中,随后将转座产物转染BHK-21细胞,获得表达E2蛋白和EGFP的病毒库,并对E2蛋白和EGFP在细胞上的表达效果进行PCR鉴定和间接免疫荧光分析。试验结果表明,运用体外转座的方法可以将外源基因插入PRV基因组中,并且外源基因可以在细胞中表达。本研究为重组病毒的研究提供了新思路,并为新型重组猪瘟疫苗的研制进一步奠定了基础。     

Estimating sample sizes for a two‐stage sampling survey of seroprevalence of pseudorabies virus (PRV)‐infected swine at a regional level in the Netherlands

Summary

In the European Union, vaccination campaigns against Pseudorabies virus (PRV) in swine have been started to eradicate PRV. Specific sampling designs are needed to monitor PRV seroprevalence at a regional level. This paper demonstrates how sampling theory can be applied to design a disease seroprevalence survey, using PRV as an example. In the spring of 1994, the four regions in the Netherlands covered by the regional Animal Health Services were monitored with respect to PRV seroprevalence. Per region, blood samples from approximately 1400 herds, with two animals per herd, were collected. The sampling design accounted for stratification by fattening pig and sow population within each region. The regional PRV seroprevalence of swine in the Southern region was the highest (24.9%), closely followed by the PRV seroprevalence of swine in the Eastern region (20.5%). These regions have the highest density of swine in the Netherlands. The PRV seroprevalence in the Western and Central region (11.7%) was about half of the seroprevalence in the Southern and Eastern regions; the lowest regional PRV seroprevalence was observed in the Northern region (3.5%). The Northern part also has the lowest pig density. The PRV seroprevalence was approximately two times higher in sows than in fattening pigs.     

Virus isolation and immune responses in susceptible swine exposed with pseudorabies virus (Shope strain).

Eighteen seronegative swine weighing from 9 to 11 kg were exposed intranasally with the Shope strain of pseudorabies virus (PRV) and were observed for 21 days in an experiment to detect virus shedding and immune responses. All swine had PRV in their nasal passages at 7 days after exposure; they also had precipitating antibodies to PRV as determined by the microimmunodiffusion test (MIDT) and very low levels of virus-neutralizing (VN) antibodies. The PRV was isolated from only 2 swine at postexposure day 14; all swine were MIDT positive, and VN titers ranged from 4 to 128. Virus was not isolated from the swine at 21 days after exposure, but all were MIDT positive; VN titers ranged between 8 and greater than or equal to 256.     

Preferential sexual transmission of pseudorabies virus in feral swine populations may not account for observed seroprevalence in the USA

This paper compares the behavior of two competing models for the transmission of pseudorabies virus in feral swine in the USA. In first model, horizontal (non-sexual) density dependent transmission is the only transmission modality. In the second model, the only transmission modality is sexual transmission between mature males and females. The comparison of model behavior was carried out to test the hypothesis that preferential sexual transmission of PRV in feral swine can account for the seroprevalence observed in the field. The observed range of seroprevalence of PRV in mature feral swine in the USA is consistent with a preferential sexual transmission only if the feral swine mating system is a random mating system or a polygynous system in which there is a relatively large rate of acquisition of new mates. The observed range of seroprevalence of PRV in mature feral swine in the USA is not consistent with a preferential sexual transmission if there is mate guarding. This is important because the National Pseudorabies Surveillance Plan deems monitoring the risk of PRV introduction from feral swine to be a "minor objective" both in terms of the scope of the plan and with respect to the resources allocated. The rationale for this statement was derived from experimental studies, which suggested that the PRV indigenous to feral swine in the USA is preferentially sexually transmitted.     

Antiviral effectiveness of butylated hydroxytoluene against pseudorabies (Aujeszky's disease) virus in cell culture, mice, and swine

Butylated hydroxytoluene (BHT) was evaluated for antiviral effectiveness on pseudorabies virus (PRV) in cell culture, mice, and swine. When relatively small amounts of BHT were mixed with PRV and incubated at 37 C for 30 or 60 minutes before inoculation into cell cultures, the cell cultures did not become infected with virus. The PRV was not infectious when the virus was treated with BHT and then inoculated intraperitoneally into mice, but was infectious when BHT and PRV were inoculated simultaneously or when BHT was inoculated either 30 or 60 minutes before PRV. Swine fed BHT-medicated feed for 10 days before they were intranasally exposed with virulent PRV did not have overt signs of pseudorabies, had a lower concentration of PRV in nasal mucus than did control swine, and had acceptable blood enzyme and cholesterol concentrations during the experiment. The BHT was detected in tissues of 2 swine after they were fed BHT-medicated feed for 10 days, and higher concentrations of BHT were detected in tissues of 3 swine given BHT feed for 29 days.     

Production of a baculovirus-derived gp50 protein and utilization in a competitive enzyme-linked immunosorbent assay for the serodiagnosis of pseudorabies virus.

The pseudorabies virus (PRV) gp50 envelope glycoprotein gene was cloned and expressed in a recombinant baculovirus. An anti-gp50 Mab (1842) recognized a protein of approximately 40 kDa in immunoblotting assays from infected insect cell lysates, while this product was not present in cells infected with wild-type baculovirus. The recombinant protein was purified by lectin affinity chromatography, utilizing lectins specific for O-linked oligosaccharides (Artocarpus integrifolia and Glycine max). Competitive (c) ELISAs, using either crude or lectin-purified antigen, were devised for the detection of antibodies to PRV in sera, and were capable of monitoring sero-conversion by day 14 post-infection. Furthermore, a specificity of 100% and sensitivity of 98% (crude lysate antigen) or 96% (lectin-purified antigen) was found for a panel of 80 swine sera, using the cELISA, as compared to a serum neutralization (SN) test. These studies demonstrated that recombinant PRV gp50 protein shows promise as a cELISA antigen, for serodetection of PRV.     

Pseudorabies virus propagated in rabbit kidney-derived RK13 cells is neutralized by natural IgM antibodies in normal swine serum which specifically lyse host cells

Pseudorabies virus (PRV) propagated in rabbit kidney-derived RK-13 cells (PRV-RK) was neutralized by serum obtained from specific pathogen-free pigs through the activation of complement. The virus-neutralizing activity of swine serum was lost after treatment with ethylene glycol-bis-aminoethylether-N,N,N',N'-tetraacetic acid (EGTA) or ethylenediaminetetraacetic acid (EDTA). Anti-C1q and anti-IgM antibodies also inhibited virus-neutralizing activity. Though IgG-depleted swine serum neutralized PRV, IgM and IgG-free swine serum lost virus-neutralizing activity. Pre-incubation of swine serum with RK-13 cells, but not with swine kidney-derived CPK cells, at 4 degrees C eliminated the virus-neutralizing activity to PRV-RK. Results indicated that swine serum contained natural IgM against an antigen(s) on the RK-13 cell surface and that this surface antigen was integrated into the PRV envelope during the budding process. Thus the natural IgM in swine serum reacted with the RK-13 antigen on the viral envelope, activated the complement cascade and neutralized the PRV-RK.     

我国猪群伪狂犬病病毒变异株感染人的警示

2018年以来国内报道人感染伪狂犬病病毒23例,其中20例患者与生猪养殖及加工相关。我国是养猪大国,庞大的从业人数遇上猪群伪狂犬病病毒变异株流行是巨大的公共卫生隐患。科学认识伪狂犬病感染人的风险,坚定地落实猪群伪狂犬病净化在Covid-19背景下意义重大。     

伪狂犬病病毒立即早期180基因的克隆及序列分析简

为了研究伪狂犬病病毒（pseudorabies virus,PRV）立即早期180基因（immediate early 180,IE180）及其功能,试验采用设计的特异性引物通过PCR法对IE180基因进行扩增,并成功构建了IE180克隆载体,经序列测定后,用DNAStar软件对该序列和已发表的7个PRV参考株的IE180基因核苷酸序列及推导的氨基酸序列进行比对分析。结果表明,HB-98株IE180基因编码区大小为4425 bp,编码1474个氨基酸,与GenBank中登录的参考株的IE180基因核苷酸同源性为98.4%～99.1%,氨基酸同源性为97.8%～98.4%。系统进化树分析结果表明,该毒株与毒株TNL(登录号：AF352564.2）的亲缘关系较近。     

检测猪伪狂犬病病毒gE抗体红细胞凝集试验的建立及应用

以纯化的抗人红细胞单链抗体(ScFv)—猪伪狂犬病病毒(PRV)gE蛋白双功能融合蛋白为抗原,建立了检测猪伪狂犬病毒gE抗体的红细胞凝集试验。利用方阵滴定试验筛选出最佳抗原工作浓度为55μg/mL,血清最佳稀释度为1∶20,作用时间15 min,与猪瘟(CSF)、猪细小病毒(PPV)、猪繁殖与呼吸综合征(PRRS)、猪乙型脑炎(JE)、猪布氏杆菌病(Brucellosis)阳性血清和PRV gE缺失疫苗接种的猪免疫血清均不出现红细胞凝集现象,与PRV标准阳性血清反应出现肉眼可见的凝集圈。与美国进口的PRV抗体检测gE-ELISA诊断试剂盒检测结果比较,50份猪血清的阴、阳性检出符合率均为100%。红细胞凝集试验检测方法具有操作简便、敏感性和特异性较高的特点,可用于PRV野毒感染的快速筛查。     

Replication and immunosuppressive effects of Pseudorabies virus on swine peripheral blood mononuclear cells.

The infectivity and potential immunosuppressive effects of Pseudorabies virus (PRV) was evaluated in swine peripheral blood mononuclear cells (PBMC). Virus progeny titers and viral DNA synthesis at various intervals post-inoculation revealed the replication of PRV in both peripheral blood monocytes and lymphocytes; however, replication in lymphocytes was restricted compared with monocytes. PRV infection resulted in the damage and death of monocytes. Although PRV did not appear to affect the viability of the lymphocytes, PRV infection suppressed lymphocyte functions such as proliferation and interleukin-2 (IL-2) synthesis in response to Concanavalin A. This immunosuppression was dependent upon the multiplicity of infection (MOI) of infectious PRV. UV-inactivated PRV was not immunosuppressive. There was no effect of PRV on natural killer (NK) cell activity. The reduction of lymphocyte proliferation by PRV was not reversible by the addition of supernatant containing porcine IL-2 and non-infected monocytes to the infected cultures. The results from these in vitro studies demonstrate that PRV can infect and cause immunosuppressive effects on swine PBMC. These effects may explain the potential role of PRV in predisposing infected pigs to secondary infection and support the hypothesis that PRV can spread systemically by infected PBMC in blood and lymph.     

猪伪狂犬病病毒多克隆抗体的制备及免疫学活性研究

本研究旨在获得抗猪伪狂犬病病毒(PRV)闽A株的多克隆抗体,为PRV的治疗与检测提供理论基础.本研究在PK-15细胞上进行PRV的增殖,测定其TCID₅₀为10^-7.372,粗提蛋白后,测定PRV蛋白浓度为3.6 mg/mL.试验选用25只健康、雄性、体重为2.5 kg±0.2 kg的新西兰大白兔为试验动物,用获得的PRV为抗原免疫后,获得抗PRV多克隆抗体.测定其抗血清效价为1:32 000,抗原包被稀释度为1:40,最佳包被条件为4 ℃ 12 h,最佳封闭时间为1 h,酶标二抗最佳工作稀释度为1:8 000.细胞病变中和试验结果表明,本研究制备的PRV抗血清在1:16的稀释情况下能保护50%的PK-15细胞免受PRV的攻击,而阴性血清不能保护PK-15细胞免受PRV的感染.结果表明本研究成功制备了PRV多克隆抗体.     

Preparation of Polyclonal Antibodies against Swine Pseudorabies Virus and Study on the Immunological Activity

This study was aimed to obtain polyclonal antibody against swine pseudorabies virus (PRV) Min A strain,and provide a theoretical basis for the study of the treatment and detection of PRV.This study was performed on PK-15 cell and proliferation of PRV was measured as TCID₅₀ 10^-7.372,the protein concentration of PRV was measured as 3.6 mg/mL.Choosing five healthy male rabbits (2.5 kg±0.2 kg) as experimental animals and using PRV obtained as the antigen,we got polyclonal antibody against PRV.Antiserum titer was 1:32 000,antigen coating dilution was 1:40,the best coating conditions was 4 ℃ 12 h,the best blocking time was 1 h,the best working dilution of enzyme labled antibody was 1:8 000,the result of cell lesions neutralization test showed that PRV antiserum prepared in this assay at 1:16 dilution could protect 50% of PK-15 cells from being infected by PRV,and negative serum couldn't protect PK-15 cells from being infected by PRV.The study successfully prepared polyclonal antibodies against PRV.