Detection of latent pseudorabies virus in porcine tissue, using a DNA hybridization dot-blot assay

A DNA-hybridization dot-blot technique was developed to detect the presence of pseudorabies virus (PRV) DNA in porcine tissue. Seven 32P-nick translated probes of high specific activity were prepared from transformed Escherichia coli plasmids into which Bacillus amyloliquefaciens H (Bam HI) restriction fragments of PRV-DNA had been inserted. Samples of DNA that had been extracted from porcine tissue or from PRV grown in tissue culture were transferred to nitrocellulose paper, using a microsample filtration manifold and were hybridized to the probes under high-stringency conditions. Under optimal hybridization conditions, the minimum detection amount of PRV-DNA was 10(-11) g, which is equivalent to 1 copy of the PRV genome/80 host cells. Four probes did not show cross hybridization with DNA extracted from tissues of known PRV-negative swine, and these were subsequently used to detect PRV-DNA in infected porcine tissues. Generally, correlation between virus isolation and hybridization data was good for tissues from swine that had died of acute PRV infection. Furthermore, PRV-DNA was present in specific tissues of all 4 seropositive swine that had recovered from pseudorabies and in which no infective virus or viral products were detected at necropsy. Pseudorabies virus DNA was present in the rostralis cerebral cortex (n = 2) or in the medulla oblongata (n = 1) and trigeminal ganglion (n = 1). This probably indicated the portal of entry of the virus into the CNS. In another seropositive pig, there was evidence of a productive infection in the tonsils, although virus was not isolated in a tissue culture system.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA hybridization procedure to detect pseudorabies virus DNA in swine tissue

A DNA hybridization technique was developed to detect the presence of pseudorabies virus (PRV) DNA. P Nick translated probes of high specific activity were prepared from transformed Escherichia coli plasmids into which Bacillus amyloliquefaciens H (Bam H1) restriction fragments of PRV DNA had been inserted. Swine cellular DNA and tissue culture PRV DNA were digested with Bam H1, separated by agarosegel electrophoresis, transferred onto nitrocellulose paper, hybridized to the radioactive probes, and washed under high stringency conditions; autoradiographs were then prepared. Under the optimal hybridization conditions described, the detection limit of these probes was 10(-11)g of PRV DNA. In reconstruction experiments, 3 of the selected probes cross hybridized with digested swine cellular DNA, and 4 probes did not. The addition of polyuridylic acid and polyguanylic acid to the hybridization reactions did not alter the amount of hybridization. The results indicated that this procedure may be useful for studying the latency of pseudorabies viral infection.     

Detection of fowlpox virus DNA by in situ hybridization using a biotinylated probe

In situ hybridization was applied to detect fowlpox virus (FPV) DNA in formalin-fixed paraffin-embedded sections of the skin from infected chickens by using a biotinylated probe and a streptavidin-alkalinephosphatase conjugate. The immunohistochemical examination was applied to compare the distribution of the FPV DNA to that of related antigenic protein in serial sections. In the infected epithelial cells, FPV DNA was detected in cytoplasmic inclusion bodies and in the rest of cytoplasm. Likewise, immunohistochemical examination revealed the virus antigen in cytoplasm. Ultrastructurally, virions were observed in the cytoplasmic inclusion bodies, and immature virus particles were in the rest of the cytoplasm. The study proved restricted distribution of FPV DNA in the cytoplasm.     

应用原位杂交检测禽脑脊髓炎病毒

禽脑脊髓炎病毒(AEV)是属于小RNA病毒科、肠病毒属的一种正链RNA病毒，病毒粒子具有六边形轮廓，无囊膜，大小为22～25nm，病毒基因组全长为7055nt，具有PloyA尾，它主要侵害幼鸡中枢神经系统，从而引起以非化脓性脑炎为主要病理特征的一种病毒性传染病。由于AEV致病性     

猪流行伪狂犬病毒gE基因检测及生物信息学分析

为了解当前伪狂犬病毒(PrV)地方流行毒株gE基因的变异特点,根据Guizhou DY株设计引物序列扩增gE基因,并对扩增产物进行序列分析和蛋白特性预测。成功获得1 654bp目的片段,提交GenBank获得登录号KM079613。核酸序列分析在142~144和1 480~1 482位发现2处特征性CGA插入,并导致氨基酸序列在48和494位天冬氨酸插入。相对Ea株编码蛋白O-GlcNAc糖基化位点在563和571位出现偏移,抗原倾向性在420~460位区域明显下降。进化分析显示与2012年以来国内流行毒株WY、ZM、HuXT2012、HBBD、HBLF、XiangA和ZJNB2012同源性较高,达99.8%,而与欧洲、美洲毒株及2012年以前国内毒株同源性较低。表明所感染的伪狂犬病病毒已发生一定的变异,并且gE基因编码蛋白与国内早期分离的Ea株相比在O-GlcNAc糖基化位点和抗原倾向性上也发生了改变。     

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。     

Reactivation of latent pseudorabies virus infection in vaccinated commercial sows

Pseudorabies virus (PRV) was isolated from 9 of 44 PRV-vaccinated seropositive sows on 5 of 11 farms. Although serum-neutralization antibody titers were 1:16 to 1:256, 28 virus isolates were obtained from tonsil, nasal, or buccal swab samples from 9 sows given 2 ml of dexamethasone/kg of body weight IM for 5 days. Pseudorabies virus was isolated from 6 of 20 sows (3 of 5 farms) given a killed-virus vaccination. Virus was obtained from 3 of 24 sows (2 of 6 farms) given modified-live virus and killed-virus vaccination. Evaluation of the 9 PRV with 5 restriction endonucleases revealed 4 PRV existing genotypes. The 9 isolated types of PRV appeared to be indistinguishable by Kpn I and BamHI restriction endonuclease analysis; however, when analyzed with Sal I, HinfI, and Pst I, isolates 7 (farm D), 8 (farm C), and 9 (farm B) had numerous differences. Isolates 1, 2, 3, and 4 (farm F) and 5 and 6 (farm G) appeared to be the same genotype when further analyzed with Pst I, HinfI, and Sal I.     

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)     

Shedding patterns in swine of virulent and attenuated pseudorabies virus

Shedding patterns of 2 virulent (P-2208 and KC-152-D) and 1 attenuated (BUK) strains of pseudorabies virus (PRV) were determined in groups of intranasally inoculated feeder pigs. Clinical signs observed following inoculation with the P-2208 and KC-152-D strains included increase in rectal temperatures up to 42.2 C, anorexia, severe respiratory disturbance, and fatal CNS signs in 2 cases. Clinical signs in pigs inoculated with 7.2 X 10(7) median tissue culture infective dose (TCID50) of the BUK strain were limited to depression and a rise in rectal temperatures to 40.5 C for 3 to 4 days. Evaluation of the efficacy of the virus isolation method used showed that the presence on swabs of only 12.5 TCID50 of the P-2208 strain or 8.4 TCID50 of the BUK strain resulted in a 50% chance of virus recovery. Intranasal inoculations with 500 TCID50 of the P-2208 or KC-152-D strain did not result in synchronous infection of the whole group. Intranasal inoculations with 5,000 TCID50 of the KC-152-D strain or 50,000 TCID50 of the P-2208 strain resulted in continuous virus shedding in all pigs between postinoculation days (PID) 4 and 13 (KC-152-D strain) or 14 (P-2208 strain). Some of the pigs in these 2 groups further shed the P-2208 or KC-152-D strain in a continuous or discontinuous pattern up to PID 19 (P-2208 strain) or 20 (KC-152-D strain). The time of onset or the level of virus neutralizing antibody production in individual pigs was not found to have an influence on their shedding patterns.(ABSTRACT TRUNCATED AT 250 WORDS)     

A skin test for pseudorabies virus infection in swine.

Heat-inactivated pseudorabies virus caused cutaneous delayed-type hypersensitive reactions when injected intradermally on the dorsolateral aspect of the thorax and tips of the ears of pigs previously exposed to the homologous virus. The reaction induced by subcutaneous injection in the lower eyelid was more easily administered and evaluated. Nonexposed control pigs did not react to the antigen and exposed and control pigs did not react when injected with a cell control antigen prepared in a similar manner. A positive response was detectable as early as seven days after exposure, reached near maximal levels by 28 days, and remained at similar levels for at least 90 days.     

Localization of classical swine fever virus from chronically infected pigs by in situ hybridization and immunohistochemistry

Classical swine fever (CSF) virus (CSFV) nucleic acid and antigen were detected in 15 pigs with naturally occurring chronic CSF by in situ hybridization and immunohistochemistry. The most consistent and prominent microscopic lesions were perivascular mononuclear cell infiltration and gliosis in the central nervous system of pigs with chronic CSF. Positive cells typically exhibited a dark brown (in situ hybridization) or red (immunohistochemistry) reaction product in the cytoplasm without background staining. A positive signal for both in situ hybridization and immunohistochemistry was detected in mononuclear cells and lymphocytes of lymphoid tissues. Viral nucleic acid was detected in some tissue sections in the absence of viral antigen. The in situ hybridization technique developed in this study was useful for the detection of CSFV RNA in tissues taken from chronically infected pigs and may be a valuable technique for studying the pathogenesis of chronic CSFV infection.     

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).     

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.     

Spread of pseudorabies virus among breeding swine in quarantined herds.

In theory, pseudorabies virus (PRV) may be eliminated from any size of breeding herd by phased test and removal if replacement gilts are not infected with PRV, culling decisions are partially based on PRV status, and the cull rate is higher than the incidence rate of PRV. Annual cull rates are commonly at least 50%, but little information exists on the incidence of PRV within enzootically infected swine herds. The purpose of this study was to develop a method by which spread of PRV could be detected among breeding swine within enzootically infected herds and to determine the incidence of PRV infection in these herds. Data were collected from 17 herds that were quarantined for PRV and ranged in size from 120 to 1,100 sows. At each herd, within the first 5 days of introduction, a group of approximately 30 replacement gilts was identified, vaccinated with a glycoprotein X-deleted PRV vaccine, and blood sample was collected. The owner of 1 herd had a nonvaccinated breeding herd and elected to leave incoming gilts nonvaccinated. After vaccination, blood samples were collected every 1 to 2 months for an average of 13.6 months. Serum samples from vaccinated gilts were tested for antiglycoprotein X antibodies by a specific differential ELISA. Samples from nonvaccinated gilts were evaluated by serum neutralization test. Product-limit method was used to estimate the probability of not becoming infected with PRV. Spread was detected in 7 of 8 herds that had more than 400 sows and in 2 of 9 herds that had less than 400 sows.(ABSTRACT TRUNCATED AT 250 WORDS)     

猪伪狂犬病病毒的分离鉴定与防制

从福清海口某猪场病猪淋巴结分离到1株病毒。该病毒接种Balb/c小鼠出现神经症状,死亡率为100%;接种Vero细胞出现拉网病变;PRV阳性血清能特异性中和该分离毒。以上结果证实该病毒为猪伪狂犬病毒。确诊后采取隔离、消毒及注射疫苗等措施,取得了一定的效果。     

Detection of infectious bursal disease virus in experimentally infected chickens by in situ hybridization

In situ hybridization was used in a pathogenesis study of three vaccine pathotypes (Delaware variant A, D78, and BursaVac) of infectious bursal disease virus (IBDV). Tissues were excised (bursa, thymus, spleen, proventriculus, and cecal tonsils), fixed in formalin, and paraffin embedded at 12, 24, 48, 72, and 120 hr postinoculation (HPI). With an antisense VP2 gene probe, viral nucleic acid was detected in bursas from both D78- and BursaVac-infected chickens at 24, 48, 72, and 120 HPI. However, viral RNA was detected only in the Delaware variant A-infected birds at 72 HPI. Thymus and spleen were positive in the D78-infected birds at 48 HPI and in the BursaVac-inoculated group at 72 HPI. Viral nucleic acid was not present in detectable levels among any of the tissues tested at 12 HPI. However, by 24 hr, scattered positive lymphoid cells were visualized in the bursal follicles of chickens infected with D78 and BursaVac. In addition, low levels of viral nucleic acids were detected in the thymus and spleen among the D78- and BursaVac-infected birds. The sites of viral replication were consistent between the two vaccine-infected groups (D78 and BursaVac), whereas the chickens infected with Delaware variant A had limited IBDV replication in the bursa.     

猪伪狂犬病调查

这几年 ,猪伪狂犬病在我国不少地区呈流行或暴发流行 ,主要引起母猪的繁殖障碍 ,新生仔猪大量发病并死亡 ,给养猪业造成重大的经济损失。现将广西某瘦肉型猪场发生猪伪狂犬病的情况报告如下。1　基本情况该猪场为一家新办的小型瘦肉型猪场 ,场址选择在山边 ,不设围墙 ,鼠患相当