Some characteristics of four attenuated vaccine virus strains and a virulent strain of Aujeszky's disease virus

In the present study four attenuated virus strains, used as vaccines, and a virulent strain of Aujeszky's disease virus (ADV) were compared with respect to their virulence in mice, their ability to induce virus-specified thymidine kinase (TK) in infected cells, and their cleavage profiles of viral DNA's after treatment with the restriction endonuclease KpnI. The survival time of mice inoculated with the B-KAL or the virulent NIA-3 strain was comparable, whereas the Bartha and BUK strains required significantly longer periods to kill mice. Mice were resistant to the MK-25 strain of ADV. The strains were assayed for TK phenotype by plaque autoradiography after 3H-thymidine labelling of infected cells. MK-25 proved to be the only strain defective in induction of TK in pig kidney cells. Restriction endonuclease analysis of viral DNA's revealed that each vaccine strain showed a characteristic fragment pattern that could easily be differentiated from that of other vaccine and field strains of ADV. The present results demonstrate that the mouse virulence test and the TK assay detect differences in biological properties of ADV strains, but that restriction endonuclease analysis is required for unambiguous identification of vaccine and field strains of ADV.     

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.     

Characterization of virulent and attenuated strains of pseudorabies virus for thymidine kinase activity, virulence and restriction patterns.

A pseudorabies virus mutant lacking thymidine kinase activity (TK-) was isolated and characterized. The mutant replicated as well in cell culture as the parental TK+ strain, was not temperature sensitive at 38.5 degrees C, and did not revert to TK+. Two pseudorabies virus field isolates and three commercial modified live virus vaccine strains were compared for TK activity and virulence for the mouse; all strains expressed TK: Km values for thymidine of the viral TKs ranged from 2.9 to 3.9 microns; the commercial modified live virus vaccine strains were reduced in virulence for the mouse two to ten-fold. The TK- mutant was avirulent for the mouse. Restriction enzyme analysis of the pseudorabies virus DNA from the strains under study revealed that two of the modified live virus vaccine strains are closely related and that all three modified live virus vaccine strains lack the largest PstI fragment characteristic of the other strains included in the study.     

Analysis of Korean strains of infectious laryngotracheitis virus by nucleotide sequences and restriction fragment length polymorphism

Korean field strains of infectious laryngotracheitis virus (ILTV) were analyzed by comparison of nucleotide sequences of thymidine kinase (TK) and glycoprotein G (gG) genes and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) patterns. Main differences among TK gene sequence were found in both amino acid at 252 and mRNA polyadenylation signals. In virulent strains, amino acid 252 of TK gene was methionine but was threonine in low virulence and vaccine strains. The mRNA polyadenylation signals of TK gene were identified at 24bp downstream from the stop codon in virulent strains, but not in low virulence and vaccine strains. The gG gene of all virulent strains showed the same nucleotide sequence except for N87278 which had a gG gene sequence identical to that of vaccine strains. The virulent ILTV strains differed from low virulence and vaccine strains in PCR-RFLP patterns of TK and gG genes. The RFLP patterns of TK and gG genes of low virulence ILTV strains were identical to those of vaccine strains. In the case of N87278, the PCR-RFLP patterns of TK and gG genes were identical to those of virulent and vaccine strains of ILTV, respectively. From these results, ILTV field strains were classified into three groups according to sequences of TK and gG genes and PCR-RFLP, and the virulent ILTV strains could be discriminated from low virulence and vaccine strains by PCR-RFLP of TK gene. And it was suspected that N87278 might be produced by in vivo recombination between virulent and vaccine strains of ILTV.     

Characterization of Japanese isolates of Aujeszky's disease virus by restriction endonuclease cleavage patterns, virulence in mice and thymidine kinase activity.

Twenty four cloned isolates of Aujeszky's disease virus collected from outbreaks of Aujeszky's disease from 1981 through 1989 in Japan were characterized by their restriction endonuclease (RE) cleavage patterns, virulence for mice and thymidine kinase (TK) activity. All of the isolates belonged to Type II of the four types classified by Herrmann et al. (1984). Based on the number and migration rate of the restriction fragments, the isolates were divided into 7 groups with Bam HI, 9 groups with Kpn I, 3 groups with BstE II and 2 groups with Sal I. The results indicate that the RE analysis, especially with Bam HI and Kpn I, provides useful epidemiological information about field isolates of Aujeszky's disease virus. All of the isolates showed virulence for mice ranging from 6.9 to 63.0 (PFU/LD50). It was interesting that the Nagano S87 strain, which had the highest virulence for the mouse, showed unique RE cleavage patterns with four enzymes. On the other hand, ara-T-resistant, TK-negative strain, was avirulent for mice (greater than 10(6.4) PFU/LD50). All of the isolates investigated in this study showed TK activity by the thymidine plaque autoradiography.     

PCR扩增TK基因检测鸡传染性喉气管炎病毒的研究

根据已发表序列设计一对包含鸡传染性喉气管炎病毒（ILTV）TK基因全长核苷酸的1259bp引物，对2株ILTV强毒和1株ILTV疫苗毒进行PCR扩增，均能扩增出预期大小的目的片段，酶切分析证实了目的片段的特异性，而其它禽病原体的扩增均为阴性。PCR检测ILTV DNA的最小检测量为75pg。此方法检测人工接种鸡的气管棉拭样品，均能检测到ILTV，因此可用于临诊样品鸡传染性喉气管炎病的检测和诊断。     

Attenuated properties of thymidine kinase-negative deletion mutant of pseudorabies virus

A thymidine kinase (TK)-negative (TK-) deletion mutant of the Bucharest (BUK) strain of pseudorabies virus (PRV) was isolated. The mutant, designated as PRV (BUK d13), did not revert to TK-positive (TK+), even when propagated in medium that selected for TK+ viruses. The mutant also replicated equally well at 39.1 C and 34.5 C, and was easily distinguished from other PRV strains by molecular hybridization experiments, restriction nuclease fingerprints, and plaque autoradiography or other assays for the TK phenotype. The PRV (BUK d13) had greatly reduced virulence for mice and rabbits, compared with parental TK+ strains, PRV (BUK-5) and PRV (BUK-5A-R1), and provided mice with solid protection against the TK+ BUK and Aujeszky strains of PRV. Experiments were done in 5- to 6-week-old pigs to assess the safety and efficacy of PRV (BUK d13) in the natural host. In one experiment, pigs were vaccinated IM with 7.5 X 10(8) plaque-forming units of TK- PRV (BUK d13), and were then challenge exposed intranasally (IN) with 4.3 X 10(8) TCID50 of virulent PRV [Indiana-Funkhauser (IND-F)]. Vaccinated pigs did not have clinical signs of illness after vaccination or after challenge exposure. One nonvaccinated control pig died on postchallenge day 4; a 2nd nonvaccinated control pig became moribund, but eventually recovered. Pigs developed virus-neutralizing antibodies after vaccination, and had a secondary immunologic response after challenge exposure; however, PRV was not isolated from the tonsils or trigeminal ganglia of vaccinated pigs at postchallenge exposure day 11.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative study of two strains of Bovid herpesvirus-4

Two strains of Bovid herepesvirus-4 (BHV-4), i.e. the prototype strain DN-599, obtained from a steer suffering of a respiratory disease, and the strain 85/BH 16TV, originated from a cow with vulvovaginitis, were compared in studies which included restriction endonuclease analysis, experimental infection and reciprocal cross protection tests. The restriction endonuclease analysis revealed that the resultant DNA patterns of the isolates were generally similar with only a difference in one fragment. The two strains were capable of causing respiratory tract infection in calves, even if they displayed a different level of virulence: the strain 85/BH 16TV being the most virulent while the strain DN-599 the least. The two viral strains were mutually protective in that the calves were generally found to be refractory to challenge inoculation with either the homologous or the heterologous virus. Finally, both viral strains failed to evoke the production of neutralizing antibody in the experimental calves.     

Immunogenicity in Aujeszky's disease virus structural glycoprotein gVI (gp50) in swine.

Aujeszky's disease virus (ADV) envelope glycoprotein gVI (gp50) was purified from virus-infected Vero cells by ion-exchange and immunoaffinity chromatography and its usefulness as a subunit vaccine was evaluated in active and passive immunization studies. Four-week-old piglets were immunized intramuscularly (IM) with purified gVI twice two weeks apart and challenged intranasally (IN) 10 days after the second immunization with 30 LD50 (10(8)PFU) of a virulent strain of ADV. Pigs, vaccinated with 100 micrograms of purified gVI, produced virus neutralizing antibodies and did not develop clinical signs after challenge exposure. The challenge virus was not isolated from nasal swabs and tonsils of gVI-vaccinated pigs, whereas non-vaccinated control pigs developed illness after challenge exposure with the same virulent ADV strain which was later recovered from their nasal swabs and tonsils. Pregnant sows vaccinated twice with purified gVI (IM) at a three week interval produced virus neutralizing antibodies in colostrum. Four-day-old sucking piglets born of vaccinated sows were passively protected by colostral antibodies against intranasal challenge with a lethal dose of virulent ADV. Sera from gVI-vaccinated pigs were distinguished from experimentally infected swine sera by their differential reactivity in enzyme-linked immunosorbent assay (ELISA) using four major viral glycoproteins (excluding gVI) as antigen purified by the use of lentil-lectin.     

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)     

Vaccines against Aujeszky's disease: Evaluation of their efficacy under standardized laboratory conditions

Summary

A standardized test was developed to compare the efficacy of Aujeszky's disease virus (ADV) vaccines under laboratory conditions. Per test 3 groups of 6 to 8 sero‐negative pigs were used. The first vaccination was done at 10 weeks of age. One group was vaccinated once, another was vaccinated twice and the 3rd served as control. Pigs were challenge exposed to the virulent NIA‐3 strain of ADV 12 weeks after the first vaccination. Apart from mortality, average periods of growth arrest, fever and virus shedding after challenge were used as parameters to evaluate vaccine efficacy.

Two inactivated and 4 attenuated vaccines were tested. Two attenuated vaccine viruses were excreted after vaccination. Despite maximal standardization, a considerable variation still existed between the experiments in mortality and growth arrest periods of control pigs after challenge. However, the controls were always more severely affected than the vaccinated pigs. All vaccines except one were effective in preventing death after challenge, but none conferred complete protection. Most vaccinated pigs still lost weight, developed fever and shed virus after challenge. Revaccination after 3 or 4 weeks had little effect, particularly with the attenuated vaccines. The results of the present study indicate that 2 of the attenuated vaccines conferred the best protection, I attenuated vaccine appeared to be as effective as the 2 inactivated ones, and the 4th attenuated vaccine was least effective.     

Differentiation of infectious laryngotracheitis virus isolates by restriction fragment length polymorphic analysis of polymerase chain reaction products amplified from multiple genes

Infectious laryngotracheitis (ILT) has been identified in most countries around the world and remains a threat to the intensive poultry industry. Outbreaks of mild to moderate forms of ILT are common in commercial layer flocks, while sporadic outbreaks of ILT in broiler flocks have also been recognized as an emerging problem in several countries. Examination of viral isolates using restriction fragment length polymorphism of polymerase chain reaction (PCR-RFLP) from individual ILTV genes has suggested that some of these outbreaks were caused by vaccine strains. In this study, PCR-RFLP of a number of ILTV genes/genomic regions including gE, gG, TK, ICP4, ICP18.5, and open reading frame (ORF) B-TK was used to examine a number of historical and contemporary Australian ILTV isolates and vaccine strains. PCR-RFLP of gE using restriction endonuclease EaeI failed to distinguish between any of the isolates including the vaccine strains. PCR-RFLP of gG, TK, and ORFB-TK using restriction endonucleases MspI and FokI, respectively, divided all the isolates into two groups. PCR-RFLP of ICP18.5 and ICP4 using restriction endonuclease HaeIII separated the isolates into three different groups with some field isolates only able to be distinguished from vaccine strains by PCR-RFLP of ICP18.5. A combination of groupings including gG, TK, ICP4, ICP18.5, and ORFB-TK PCR-RFLP classified the ILTV isolates under investigation into five different groups with most isolates distinguishable from vaccine strains. Results from this study reveal that to achieve reliable identification of strains of ILTV, the examination of multiple gene regions will be required, and that most of the recent ILT outbreaks in Australia are not being caused by vaccine strains.     

Differentiation of strains of equine arteritis virus of differing virulence to horses by growth in equine endothelial cells

OBJECTIVE: To compare growth characteristics of strains of equine arteritis virus (EAV) of differing virulence to horses in rabbit kidney (RK)-13 cells and equine endothelial cells (EECs) cultured from the pulmonary artery of a foal. SAMPLE POPULATION: 13 strains of EAV, including 11 field isolates of differing virulence to horses; the highly virulent, horse-adapted Bucyrus strain; and the modified-live virus (MLV) vaccine derived from it. PROCEDURE: The growth characteristics of the 13 strains were compared in EECs and RK-13 cells. Viral nucleoprotein expression, cytopathogenicity, and plaque size were compared to determine whether growth characteristics of the 13 strains were predictive of their virulence to horses. RESULTS: Cytopathogenicity, viral nucleoprotein expression, and plaque size induced by all 13 viruses were similar in RK-13 cells, whereas virulent strains of EAV caused significantly larger plaques in EECs than did the avirulent strains of EAV. Paradoxically, the highly attenuated MLV vaccine and 1 field isolate of EAV caused plaques in EECs that were larger than those caused by any of the other viruses, and sequence analysis confirmed the field isolate of EAV to be indistinguishable from the MLV vaccine. CONCLUSIONS AND CLINICAL RELEVANCE: With the notable exception of the MLV vaccine, growth of the various strains of EAV in EECs was predictive of their individual virulence to horses. Thus, EECs provide a relevant and useful model to further characterize determinants of virulence and attenuation amongst strains of EAV.     

Atypical Myxomatosis – Virus Isolation,Experimental Infection of Rabbits and Restriction Endonuclease Analysis of the Isolate

Atypical form of myxomatosis, which caused non‐lethal and clinically mild disease in domestic rabbits 1 month after immunization with a commercially available vaccine MXT, is described. The isolated myxoma virus designated as Litovel 2 (Li‐2) did not induce systemic disease following subcutaneous and intradermal applications in susceptible experimental rabbits but led to the immune response demonstrated by ELISA. No severe disease was induced in those Li‐2 inoculated rabbits by challenge with the virulent strains Lausanne (Lu) or Sanar (SA), while the control animals showed nodular form of myxomatosis with lethal course of the illness. Restriction fragment length polymorphism (RFLP) of genomic DNA with KpnI and BamHI endonucleases was used for genetic characterization of the Li‐2 isolate, the vaccine strain MXT and both virulent strains Lu and SA, respectively. In general, RFLP analysis has shown to be informative for inferring genetic relatedness between myxoma viruses. Based on restriction endonuclease DNA fragment size distribution, it was evident that the pathogenic strain SA is genetically related to the reference strain Lu and the isolate Li‐2 is more related, but not identical, to the vaccination strain MXT.     

应用PCR检测鸡传染性喉气管炎病毒

根据传染性喉气管炎病毒(ILTV)TK基因序列，设计、合成1对引物，应用PCR技术对ILTV以色列疫苗株、河南分离株（ILTV-CG和ILTV-XY）进行PCR扩增，均能扩增出预期大小的目的片段，测序分析和酶切分析证实了PCR产物的特异性，而对其它禽病原体的扩增均为阴性。PCR检测ILTV DNA的最小检测量为21 pg。应用PCR检测人工接种后不同〖JP2〗天数采集的鸡的结膜拭子，接种后第2~5 d均能检测到ILTV。该方法可用于鸡传染性喉气管炎病的诊断和临诊样品检测。     

猪多杀性巴氏杆菌对HeLa细胞附着能力的研究

本研究通过猪肺疫的活菌疫苗和死菌疫苗多杀性巴氏杆菌菌株(Pasteurella multocida,Pm)对小鼠的毒力试验测定它们的毒力性。结果表明,死菌疫苗Pm的毒力性比活菌疫苗Pm的强,即死亡率分别为10 0 %和0 %。通过两菌株对He L a细胞的附着试验测定它们的附着能力,结果证明强毒菌的附着能力明显地比弱毒菌强(P<0 .0 1) ,平均附着数分别为11.96和2 .4 4 ;从上述菌株细胞荚膜中分别提取荚膜蛋白,用SDS- PAGE分离测定两菌株荚膜蛋白质结构,结果表明39k Da荚膜蛋白是强毒菌的特异性蛋白。以上研究结果证明Pm的毒力与He L a细胞的附着能力是密切相关的,同时暗示本菌39k Da荚膜蛋白可能与它们的毒力和He L a细胞的附着能力有关     

Survival and immunization of raccoons after exposure to pseudorabies (Aujeszky's disease) virus gene-deleted vaccines

In a controlled experiment, 16 wild-trapped raccoons were exposed to 1 of 2 genetically modified live pseudorabies virus (PRV) vaccines used in swine. One vaccine had genes deleted for thymidine kinase (TK(-)) and glycoprotein G (gG(-)); the other had an additional deletion for glycoprotein E (gE(-)). These vaccines were administered orally and intranasally at four dose levels: 10(3), 10(4), 10(5), and 10(6) TCID(50). The 21 days survival rate was 37.5% for the gG(-)TK(-) vaccine; all of the survivors developed antibodies to PRV. All animals receiving the gG(-)gE(-)TK(-) vaccine survived; 75% (all except the lowest dose) developed anti-PRV antibodies. Survivors were challenged intranasally with a 3.2x10(3) TCID(50) dose of the virulent wildtype PRV Shope strain. Two of the remaining three gG(-)TK(-) vaccinated raccoons survived the challenge; for the gG(-)gE(-)TK(-) vaccine, the survival rate was 50% (4/8). The raccoons with higher vaccine-induced antibody titers were more likely to survive the challenge with the virulent PRV; there was a 100% mortality rate for raccoons lacking detectable anti-PRV antibodies. This experiment indicates that exposure of raccoons to modified live gene-deleted PRV vaccines may result in an immune response, and that this immunity provides some protection against exposure to virulent virus.     

Studies on immunisation of pigs with the Bartha strain of Aujeszky's disease virus.

The K strain of Aujeszky's disease virus (ADV) grown in Vero cells was used to vaccinate pigs. Following intramuscular inoculation, the pigs remained healthy, no vaccine virus was excreted and virus could be detected only at the inoculation site. One inoculation gave good protection against challenge with a virulent strain of ADV, and the amount of virulent ADV excreted was geatly curtailed. Following vaccination only low leads of serum neutralizing antibody were detected (geometric mean titre 1/2), but three weeks after challenge very high levels were found (GMT 1/1773). Intranasal vaccination gave similar results. There was minimal excretion of vaccine virus. The clinical reaction on challenge was less severe than in the intramuscularly challenged group, although lower antibody levels were detected three wekks following challenge (GMT 1/483). A field trial, using this strain given subcutaneously, indicated that one inoculation of this vaccine is effective.     

Viral protein 1 sequence analysis of three infectious bursal disease virus strains: a very virulent virus, its attenuated form, and an attenuated vaccine

Comparisons between sequences of very virulent, virulent, and attenuated strains of the infectious bursal disease virus (IBDV) may indicate sites on the genome co-inciding with virulence. In an attempt to detect if such sites exist on the coding region of segment B, viral protein 1 (VP1) (encoded for by segment B) of a very virulent Israeli virus, IL3; its attenuated strain, IL4; and the attenuated Winterfield vaccine 2512 were cloned and sequenced. A comparison was made among them and with six other published sequences of segment B. Six nucleic acids distinguished between IL3 and IL4, three of which were predicted to be expressed as amino acids. A striking similarity between the VP1 sequences of 2512 and P2 (an attenuated German strain) was discovered. Although conclusions could not be drawn concerning attenuation sites on VP1, the analysis performed on the VP1 sequences of the two Israeli strains and the Winterfield 2512 strain sheds light on the phylogeny of IBDV and contributes to the accumulating information that may lead to the identification of virulence-related sites of this virus.     

Marker vaccines, virus protein-specific antibody assays and the control of Aujeszky's disease

Vaccination of pigs is widely practised to control Aujeszky's disease (AD). Molecular biological research revealed that several conventionally attenuated virus vaccines harbour deletions in their genomes. The deleted genes are nonessential for virus replication and can be involved in the expression of virulence. These findings have prompted several groups to construct well-characterized deletion mutants of AD virus that do not express either glycoprotein gI, gX or gIII. These mutants have also been rendered thymidine kinase negative. Although data on vaccine efficacy and safety have been published, widely varying test conditions have made it impossible to identify the most efficacious deletion mutant vaccine(s). Vaccination enhances the amount of virus required for infection and reduces, but does not prevent, the shedding of virulent virus and the establishment of latency in pigs infected with virulent AD virus. Therefore, while a vaccination programme will reduce the circulation of virus in the field, it will not eliminate AD virus from pig populations. To eradicate AD, the ability to differentiate infected from vaccinated pigs is crucial. The use of marker vaccines enables us to identify infected pigs in vaccinated populations by detecting antibodies against the protein whose gene is deleted from vaccine strains. The antibody response to gI appears to persist for more than 2 years, and all of about 300 field strains tested so far express gI. The use of vaccines lacking gI in combination with an enzyme linked immunosorbent assay to detect antibodies to gI and culling of gI-seropositive pigs, may help to eradicate AD in countries where vaccination is widely practised.