The response of pigs inoculated with a thymidine kinase-negative (TK-) pseudorabies virus to challenge infection with virulent virus

12 Large-White-Landrace piglets were subdivided in four groups of 3 and housed in separate units. The piglets of three groups were inoculated with the 86/27V 6C2 thymidine kinase negative (TK-) mutant of pseudorabies virus (PRV), by different routes. A second inoculation with the same mutant was given to the pigs 21 days later. The animals of a fourth group were left as uninoculated controls. 21 days following the second inoculation with the TK- mutant all pigs were challenge infected with the virulent PRV. On post challenge day (PCD) 30 all pigs were killed and samples for virus detection and histology were taken from several organs. The inoculated TK- mutant of PRV did not induce any ill effects in the pigs except a transient febrile reaction in some animals. Virus was recovered from nasal swabbings from one pig 2 days after the first inoculation of the mutant. After challenge exposure with virulent PRV, the TK- mutant-inoculated pigs were apparently protected, whereas the control pigs all were severely affected and recovered very slowly over 3 weeks. Virus was isolated from the nasal swabbings from the TK- mutant-inoculated pigs on PCDs 2 and 4, whereas the nasal swabbings from the control piglets were all positive for virus from PCD 2 through PCD 10. DNA analysis of the virus recovered showed a pattern identical to that of the virulent PRV. Histologic lesions were found in the respiratory and the central nervous systems, however, the lesions in the TK- mutant-inoculated pigs were much milder compared to those registered for the control pigs. Virus was not isolated from any of the tissue samples that were tested, but viral DNA with sequences typical of PRV genome was detected by PCR in all samples of trigeminal ganglia from either the TK- mutant-inoculated pigs or from the controls.     

Determination of ability of a thymidine kinase-negative deletion mutant of bovine herpesvirus-1 to cause abortion in cattle.

The Cooper isolate of bovine herpesvirus-1, which causes abortion in cattle, was used to construct a thymidine kinase-negative (TK-) deletion mutant virus. Twelve heifers were inoculated IV at 25 to 29 weeks of pregnancy with either TK- or thymidine kinase-positive (TK+) Cooper virus. All heifers developed fevers of 1 to 2 C during the first week after inoculation. Temperatures of TK+ inoculates were slightly higher and remained above normal a few days longer than in TK- inoculates. Viremia was detected in 5 of 6 TK+ inoculates and in all 6 TK- inoculates. More virus isolations were made from nasal and vaginal swab specimens of TK+ inoculates than from swab specimens of TK- inoculates. All heifers developed virus neutralizing antibody within 14 days after inoculation and antibody titers were similar between the 2 groups. None of the TK- inoculated heifers aborted and their calves did not have neutralizing antibody at birth. Abortion occurred in 5 of 6 heifers given TK+ virus. All aborted fetuses were infected with bovine herpesvirus-1, as demonstrated by virus isolation or detection of viral antigen in fetal tissues. These results indicate that inactivation of the TK gene reduces abortifacient activity of bovine herpesvirus-1.     

Vaccination of swine with thymidine kinase-deficient mutants of pseudorabies virus

A mutant of pseudorabies virus (PRV) deficient in thymidine kinase (TK-) activity was isolated and characterized. The mutant grew well in cell culture and did not revert to the thymidine kinase-positive phenotype. The PRV-TK- was not virulent when inoculated intranasally into 3-to 4-week-old pigs and could not be reactivated from the ganglia of these pigs by explantation and cocultivation with susceptible cells several weeks after virus inoculation. Pigs that had been exposed to PRV-TK- were immune to challenge exposure with a virulent strain of PRV. Furthermore, the challenge virus was not recovered from the ganglia of most of these pigs, indicating that colonization of the ganglia by a super-infecting virulent PRV strain was considerably reduced by vaccination.     

Physicochemical properties of pseudorabies virus hemagglutinin.

Pseudorabies virus hemagglutinin was readily adsorbed on mouse erythrocytes at 4, 22, or 37 degrees C, but not on cattle erythrocytes. The adsorbed hemagglutinin could not be eluted from the cells by resuspending in phosphate-buffered saline (PBS), by incubating at 37 or 50 degrees C, or by incubating in the presence of neuraminidase. The receptor on mouse erythrocytes for the hemagglutinin was inactivated by trypsin, but not by neuraminidase, sodium deoxycholate (DOC), potassium periodate (KIO4), dithiothreitol (DTT), 2-mercaptoethanol (2-ME) and formalin. The hemagglutinin was inactivated by trypsin, alpha-amylase, pepsin, DOC, KIO4, and ethylendiamine-tetraacetic acid (EDTA), but not by papain, beta-glucosidase, phospholipase C, neuraminidase, DTT, 2-ME, Tween-80, ethylether, chloroform, trichloro-trifluoroethane, beta-propiolactone and formalin, suggesting that the hemagglutinin active component involved glycoproteins. The hemagglutinin was stable at 37 degrees C for lower temperatures but not at 60 degrees C or higher. The hemagglutinin activity was resistant to ultraviolet irradiation, while the infectivity was very susceptible. The hemagglutinin and the infectivity were readily sedimented by ultracentrifugation at 48,000 x g for 3 hr. In rate zonal centrifugation of the preparation on a sucrose density gradient, the hemagglutination (HA) activity showed a sharp peak at 1.22 g/ml coinciding with the peak of infectivity. The HA activity in the peak fraction seemed to be structually associated with virus particles. After fractionation of the virus by Nonidet P-40, the HA activity was found only in the fraction of the envelope material, indicating that the hemagglutinin is situated in the viral envelop.     

Avirulent ts and thymidine kinase-deficient mutant of Aujeszky's disease virus.

The temperature-sensitive (ts), thymidine kinase-deficient (TK-) mutant designated ZHtsTK- strain, of Aujeszky's disease virus (ADV) was isolated from a virulent strain with the treatments using 5-bromodeoxyuridine and arabinosylthymine. The ZHtsTK- strain was easily distinguished from the other virulent ADV strains by plaque size on HmLu-1 and chicken embryo fibroblast cells and by restriction endonuclease analyses using Bam HI, Sal I and Kpn I. The ZHtsTK- strain was avirulent for mice, guinea pigs and rabbits, and produced neutralizing antibodies to ADV in these animals. The rabbits inoculated with the ZHtsTK- strain did not shed detectable amounts of virus after dexamethasone treatment. The ZHtsTK- strain was also avirulent for 5-day-old piglets and did not cause disease. No virus was detected from the piglets inoculated intramuscularly in the nasal swabs or the tissues examined on postinoculation day 9. These findings presented here suggested that there is a significant correlation between pathogenicity and properties such as ts and TK-, and the combination of ts and TK- properties plays a much larger role in reducing virulence for animals.     

A vaccine strain of pseudorabies virus with deletions in the thymidine kinase and glycoprotein X genes

A pseudorabies virus (PRV) mutant with deletions in genes for glycoprotein X (gX) and thymidine kinase, designated delta GX delta TK, was constructed and evaluated as a vaccine for protecting swine against PRV-induced mortality. Doses greater than or equal to 10(3) plaque-forming units (PFU) of this strain given to mice provided protection from challenge exposure with virulent PRV. Sera tested from mice inoculated with delta GX delta TK had high titers of neutralizing antibody to PRV, but reactivity in the same sera was not significantly different from that in sera from noninoculated mice (controls) when sera from both groups were evaluated by use of an ELISA with gX antigen produced in Escherichia coli. Compared with noninoculated pigs (controls), those given delta GX delta TK (greater than or equal to 10(2) PFU) were protected completely from lethal challenge exposure, without experiencing adverse effects on weight gain and with reduction of shedding of virulent challenge virus. Serotest results indicated that, although inoculated pigs responded with strong neutralizing antibody titers, the response of delta GX delta TK-inoculated pigs to gX, as determined by ELISA before challenge exposure, was not significantly greater than the ELISA values obtained from control pigs. The ELISA values from a group of pigs inoculated with a commercially available vaccine were significantly (P less than 0.05) higher than those of control pigs. The experimental vaccine, delta GX delta TK, was avirulent for mice, swine, and sheep, but was mildly virulent for calves (mortality, 1 of 12) and more virulent for dogs (mortality, 3 of 6) and cats (mortality, 2 of 6).(ABSTRACT TRUNCATED AT 250 WORDS)     

伪狂犬病病毒gE/TK基因缺失突变株的构建

在伪狂犬病病毒转移载体pBdTK-Uni的多克隆位点中插入由SV40启动子控制下的IacZ基因表达盒,同时在右侧同源臂下游插入一个1.7kb的KpnI片段,构建成一个新的转移载体pUhi-LacZ.用该载体与Bartha-K61株基因组通过脂质体法共转染Vero细胞,经过10代蓝斑筛选纯化和PCR鉴定获得了一株稳定表达LacZ基因的伪狂犬病病毒gE/TK基因缺失突变株,命名为rPrV-LacZ.在不同的细胞(PK-15、IBRS-2、Vero和CEF)上,对该重组病毒与亲本病毒的增殖滴度和细胞病变进行比较,未见显著差异.结果表明转移载体pBdTK-Uni具有实用性,可用于构建伪狂犬病病毒基因工程活载体疫苗.     

Genotypic screening of pseudorabies virus strains for thymidine kinase deletions by use of the polymerase chain reaction.

Genetic recombination between field strains and vaccine strains of pseudorabies virus (PRV) has been suggested as a scenario that might arise from use of deletion-mutant modified-live vaccine strains, particularly those strains attenuated by deletions within the thymidine kinase (TK-) gene locus. To address this hypothesis experimentally, it is necessary to screen large numbers of PRV isolates for their TK genotype. Techniques to detect the native TK genotype are routinely used in molecular virology laboratories, but are time-consuming. We adapted the polymerase chain reaction to define the genotypic status of PRV isolates with regard to the presence or absence of deletions in the TK gene locus. Used in tandem with the existing glycoprotein-specific ELISA that discriminate between PRV-vaccinated and field strain-infected swine populations, the described technique may help to clarify whether vaccine-derived recombinants are generated under natural conditions and after normal vaccine usage.     

一例猪伪狂犬病病毒的诊断

2015年11月,当地某养殖户15日龄仔猪出现角弓反张、精神沉郁等疑似猪伪狂犬病病毒感染的典型神经症状,但查看免疫程序,已免疫猪伪狂犬病疫苗（Bartha-k61株）。经gE基因血清学调查和针对gE基因的PCR诊断,证实该病例为猪伪狂犬病病毒感染所致。     

猪伪狂犬病病毒的分离和鉴定

从病猪脑部分离到1株病毒.该病毒接种Balb/c小鼠出现神经症状,病死率为60% ,接种PK-15细胞出现拉网病变.伪狂犬病阳性血清能特异性的中和该分离毒.根据基因库(GenBank)的PRV gE基因设计的引物能扩增出特异性片段,证实该病毒为伪狂犬病病毒.     

Latent infection and subsequent reactivation of pseudorabies virus in swine exposed to pseudorabies virus while nursing immune dams

The ability of pseudorabies virus (PRV) to infect and establish latency in pigs with passively acquired (maternal) antibody for PRV was tested by exposing such pigs to the virus and subsequently attempting to reactivate latent virus by administering large doses of dexamethasone. Pigs of each of 4 litters that had nursed gilts with relatively high (512, gilts 1 and 2), moderate (32, gilt 3), and no (less than 2, gilt 4) serum titers of virus-neutralizing (VN) antibodies for PRV were allotted to 3 treatment groups (A, B, C) when they were 2 weeks old. Group-A pigs were separated from littermates and dam and thereafter kept in isolation; group-B pigs were experimentally exposed oronasally to PRV and 1 hour later returned to their dam; group-C pigs were kept with their dam and potentially exposed to PRV by contact with littermates of group B. Sera obtained from pigs at selected intervals until they were 17 weeks old were tested for VN activity and for precipitating activity for radiolabeled viral proteins. All group-A pigs remained clinically normal throughout the experiment. Depending on the initial amount of passively acquired antibody, little or no serum VN or precipitating activity remained by the time these pigs were 17 weeks old. Group-B and -C pigs, with relatively high amounts of passively acquired antibody when exposed to PRV, also remained clinically normal. However, most became latently infected as subsequently evidenced by either dexamethasone-induced or noninduced virus reactivation. Noninduced reactivation may have been initiated by weaning the pigs when they were about 8 weeks old.(ABSTRACT TRUNCATED AT 250 WORDS)     

伪狂犬病病毒云南变异毒株的分离鉴定及gE、gD、gC、TK基因序列分析

2013-2016年期间,从云南陆良、富源、寻甸及西双版纳规模猪场经Bartha-K61疫苗免疫猪群中发生流产的胎儿内脏组织病料中成功分离获得4株伪狂犬病病毒流行毒株,分别命名为FY-YN-2014、LL-YN-2014、XSBN-YN-2015和XD-YN-2014株。经TCID_(50)测定、动物致病性试验、免疫保护试验及gE、gD、gC、TK基因序列分析,结果显示TCID_(50)分别为10~(-6.083)/100μL、10~(-5.75)/100μL、10~(-6.583)/100μL、10~(-6.5)/100μL。采用Bartha-K61疫苗免疫过的经产母猪血清样品对XSBN-YN-2015分离毒株进行微量病毒中和试验,其结果显示gB抗体阳性、gE抗体阴性的血清样品的中和效价在1∶16～1∶32之间,用gB、gE抗体均为阳性的血清样品时,其中和效价在1∶64～1∶128之间。4株分离毒株接种昆明小白鼠、家兔2～5 d后,均出现奇痒的典型伪狂犬病临床症状。对健康非免疫断奶仔猪进行攻毒,同样出现伪狂犬病的典型发病症状,且能从脑及内脏组织中扩增出伪狂犬病病毒gE基因并成功回收分离到病毒株。在昆明小白鼠上的LD_(50)分别是10~(2.625) TCID_(50)、10~(2.875) TCID_(50)、10~(2.625) TCID_(50)、10~(2.5) TCID_(50)。XSBN-YN-2015株在非免疫断奶仔猪(伪狂犬病病毒gE、gB抗体阴性)测得的LD_(50)=10~(5.33) TCID_(50)。XSBN分离毒株对免疫2次Bartha-K61疫苗的断奶仔猪的攻击试验结果显示,被攻击仔猪均出现高热、精神萎顿、厌食等临床表现,但1周后均能耐过并逐渐康复,提示Bartha-K61疫苗株免疫猪只对当前流行的伪狂犬病病毒云南变异毒株的攻击基本能够提供100%保护。4株伪狂犬病病毒云南流行毒株与近年来国内报道的TJ、HeN1、NH1201等变异毒株的gE、gC、gD及TK基因核苷酸及氨基酸序列均高度一致,同源性高达99%～100%,均分属于同一进化树分支,不过与早期的国内毒株SC、GDSH株,国外毒株Becker、Nia-1、CL15、Kaplan、Kolchis、Hercules、NIA3及疫苗株Bartha相比,均存在一定程度的变异,也未分属于同一进化分支,因此,4株伪狂犬病病毒云南流行毒株也属于变异毒株。     

重组伪狂犬病病毒(rPRV-GP5)对绵羊的安全和效力试验

为了验证表达猪繁殖与呼吸综合征病毒(PRRSV)GP5蛋白的重组伪狂犬病病毒(rPRV-GP5)的安全性和免疫原性,将10只5～6月龄健康绵羊分成三组,安检组(3只)肌注108.0PFU/只、效检组(4只)肌注106.0 PFU/只,3只作为不接种对照组.接种后14 d,效检组与对照组每只臀部肌注伪狂犬病病毒"双城系"猪源强毒S株103 LD50,结果效检组全部保护,对照组全部死亡,安检组未见异常.结果表明,重组病毒完全符合伪狂大病活疫苗制造及检验规程中对疫苗的安全和效力检验标准.     

伪狂犬病病毒IE180基因的研究进展

伪狂犬病病毒（Pseudorabies virus，PRV）是疱疹病毒科a疱疹病毒亚科的成员，临床上引起Aujeszky氏病（Aujeszky＇s disease，AD）。根据该病毒感染细胞后DNA转录、表达时间的先后可将PRV基因分为立即早期基因（immediate—early gene，IE），早期基因（earlygene）和晚期基因（late gene），这3种基因产物以瀑布方式调节。现已证实伪狂犬病病毒只含一个立即早期基因——IE180基因，该基因对病毒在动物体内的复制至关重要，只有IE180基因开放转录、并翻译成IE180蛋白，其下游的基因才能在IE180蛋白的激活作用下得以完全开放转录，否则病毒复制将不能完成。IE180基因的表达产物IE180蛋白是一个多功能蛋白，不仅能激活同源蛋白基因启动子，而且能激活某些异源蛋白基因启动子。此外，IE180蛋白还具有诱导机体产生免疫保护的功能。IE180的转基因小鼠能够抵抗PRV的攻击，但最近发现IE180在小鼠体内的表达会导致小鼠小脑发育异常，表现出共济失调、震颤等症状。     

猪伪狂犬病病毒的分离与鉴定

猪伪狂犬病是由疱疹病毒科的猪疱疹病毒1型病毒引起的一种急性传染病，多数动物均可感染，猪是本病的主要宿主和带毒者。本病主要以成年妊娠母猪流产、死产、木乃伊胎、新生仔猪急性死亡及3周龄以内的仔猪表现神经症状为主要特征。近年来我国也相继有本病发生的报道。1997年10月黑龙江省牡丹江地区某养猪场从外地引进种母猪，回来后直接放入后备猪舍与其他后备母猪混养，5日后同舍母猪出现厌食、发热、精神沉郁，有的呕吐、咳嗽。     

猪伪狂犬病病毒河南株的分离及鉴定

采用PCR方法对河南不同地市采集的疑似猪伪狂犬病病毒(PRV)感染病料进行PCR检测,PCR阳性的病料经处理后,接种ST细胞,分离到15株PRV。分离PRV传至第3代后,在ST细胞上均能出现较稳定的细胞病变,传代至第7代时病毒滴度在10~(5.6) TCID_(50)/0.1 mL~10~9 TCID_(50)/0.1 mL,且均能扩增出特异性gE基因片段(429bp)。将分离PRV接种6周龄雌性昆明小鼠,引起皮炎及小鼠先后死亡。以NY株为例进行理化特性试验,结果表明,对分析纯氯仿、胰酶敏感;对酸、碱及热有较强抵抗力,pH3.0盐酸处理1h、pH11.0 NaOH处理1h、56℃水浴处理1h,才能使其失活;对紫外线处理30min,仍具有感染性。经电镜观察,可看到大小均一、近似圆形的病毒粒子,大小为110~150nm,囊膜表面有呈放射状排列的纤突,表明所分离病毒均为PRV野毒株。     

猪伪狂犬病活疫苗(Bartha K61株)对变异株的保护效力

本研究旨在检测仔猪免疫猪伪狂犬病活疫苗(Bartha K61株)后,抵抗伪狂犬病病毒(PRV)变异株攻击的保护效果。取4~6周龄PRV抗体阴性仔猪,接种猪伪狂犬病活疫苗,1周后用PRV变异株(AH02LA株)攻毒,检测攻毒后临床症状、直肠温度、鼻腔排毒和肺部病变。疫苗免疫组在免疫后7 d均可以检测到gB抗体。攻毒对照组攻毒后出现典型伪狂犬症状,发病率为100%,死亡率为60%,所有猪只鼻拭子均检出排毒,所有猪只肺部均有出血、淤血等病变。免疫组的猪只攻毒后,所有猪只均未出现明显临床症状,部分猪只鼻拭子检出排毒,排毒持续时间缩短,排毒量显著减少,所有免疫猪只肺部未见明显病变。结果表明:伪狂犬病活疫苗免疫猪后对PRV变异株的攻击具有良好的保护效果。     

Pathogenicity of a modified-live pseudorabies vaccine virus in lambs

Subcutaneous injections of modified-live pseudorabies virus (PRV) vaccine into lambs caused clinical signs and death within 1 week after injection in 4 of 5 inoculated lambs. The clinical signs included depression, high fever, muscle fasciculations and convulsions, occasionally followed by death within 48 hours of the initial clinical signs. Histologic examinations and virus isolation procedures demonstrated PRV in the CNS of infected lambs. Sera from sick lambs remained negative for PRV antibodies. Two subsequent serial passages of the vaccine virus in lambs resulted in similar clinical signs and death in 6 of 10 inoculated lambs. Again, PRV was isolated from tissues of sick lambs, and the histopathologic findings were characteristic of the disease. Affected lambs remained seronegative to PRV, as did lambs that remained clinically normal after inoculation. There was no evidence of PRV transmission to uninoculated lambs and pigs housed with the infected lambs.