Derivation,safety and efficacy of a Marek's disease vaccine developed from an Australian isolate of very virulent Marek's disease virus

OBJECTIVE: To develop a serotype 1 Marek's disease (MD) vaccine from a very virulent MDV (vvMDV) pathotype and demonstrate safety and efficacy against early challenge with very virulent field strains in the presence of maternal antibody. STUDY DESIGN: Strain BH 16 was isolated and attenuated by serial cell culture passage. One of two cloned passages was selected for vaccine development following early laboratory-scale protection trials in commercial birds. Comparative protection trials were carded out on the BH 16 vaccine and on a CVI 988 Rispens vaccine using commercial and SPF chickens. Challenge viruses used were either a low passage strain BH 16 virus, the Woodlands No. 1 strain or MPF 57 strain of MDV. The BH 16 vaccine was back-passaged in SPF chickens six times and virus recovered from the final passage and the original vaccine virus were tested for safety. The immunosuppressive potential of the BH 16 and Rispens vaccines was also assessed in parallel. RESULTS: The BH 16 and Rispens vaccines induced comparable levels of protection when used as monovalent or multivalent vaccines, although protection achieved with the monovalent vaccines was lower. No gross tumour formation was evident in any birds receiving the BH 16 vaccine or bird-passaged virus, although microscopic lesions were present in 2/12 birds that received the bird-passaged virus. In tests for immunosuppression, there was no histological evidence of damage to either the bursa of Fabricius or the thymus. CONCLUSION: The BH 16 vaccine was shown to be safe and at least as protective as the Rispens vaccine against three highly virulent MD challenge viruses.     

鸡传染性法氏囊病病毒适应于Vero细胞的初步研究

将国内６个省市（广东、巾东、河北、辽宁、、新疆和北京）的７株鸡传染性法氏囊病（ＩＢＤ）的法氏囊组织处理后，直接在Ｖｅｒｏ细胞上盲传３～４代，均能不同程度的产生特征性细胞病变效应（ＣＰＥ），并通过选用具有广谱性的抗法氏囊病病毒的单克隆抗体（ＩＢＩ）Ｖ－ＭｃＡｂ）采用间接免疫荧光（ＩＦＡ）和碱性磷酸酶—抗碱性磷酸酶桥联酶显色技术（ＡＰＡＡＰ）的方法对７株分离毒的Ｖｅｒｏ第８代细胞进行鉴定，又用逆转—聚合酶链式反应（ＲＴ－ＰＣＲ）对７株分离毒的第１４代Ｖｅｒｏ细胞毒进行进一步证实，结果表明这７株ＩＢＤ囊毒均适应于Ｖｅｒｏ细胞上，这是国内首次报道将组织毒直接适应于Ｖｅｒｏ传代细胞上。     

Derivation, safety and efficacy of a Marek's disease vaccine developed from an Australian isolate of very virulent Marek's disease virus

Objective To develop a serotype 1 Marek's disease (MD) vaccine from a very virulent MDV (vvMDV) pathotype and demonstrate safety and efficacy against early challenge with very virulent field strains in the presence of maternal antibody.
Study design Strain BH 16 was isolated and attenuated by serial cell culture passage. One of two cloned passages was selected for vaccine development following early laboratory-scale protection trials in commercial birds. Comparative protection trials were carried out on the BH 16 vaccine and on a CVI 988 Rispens vaccine using commercial and SPF chickens. Challenge viruses used were either a low passage strain BH 16 virus, the Woodlands No. 1 strain or MPF 57 strain of MDV. The BH 16 vaccine was back-passaged in SPF chickens six times and virus recovered from the final passage and the original vaccine virus were tested for safety. The immunosuppressive potential of the BH 16 and Rispens vaccines was also assessed in parallel.
Results The BH 16 and Rispens vaccines induced comparable levels of protection when used as monovalent or multi-valent vaccines, although protection achieved with the mono-valent vaccines was lower. No gross tumour formation was evident in any birds receiving the BH 16 vaccine or bird-passaged virus, although microscopic lesions were present in 2/12 birds that received the bird-passaged virus. In tests for immunosuppression, there was no histological evidence of damage to either the bursa of Fabricius or the thymus.
Conclusion The BH 16 vaccine was shown to be safe and at least as protective as the Rispens vaccine against three highly virulent MD challenge viruses.     

猪流行性腹泻弱毒株的培育

用ＰＥＤＶＣＶ７７７株强毒适应Ｖｅｒｏ细胞系并传至１４７代。以ＰＥＤＶ沪株做效检用强毒。传代毒８３代之后适应了仔猪肾原代细胞。自９０代起进行了５次克隆纯化,克隆是在２次群斑（由５～７个单斑组成）的基础上,再进行３次单斑挑选（均是１ｍｍ以内小空斑）。以８３７斑５株继续传代并做系列试验。传代毒的毒价为１０７．０～１０７．５ＴＣＩＤ５０／０３ｍｌ。免疫接种途径为后海穴位。克隆后５代（总代次１０４代）～２５代的５批次主动免疫试验的总保护率为９５９２％（４７／４９）,对照组８８８％（１６／１８）发病。克隆后１７代～４０代的８批次被动免疫试验的总保护率为９６２％（７６／７９）,对照组１００％（１０／１０）发病。以克隆后２５代（总代次１２５代）进行稳定性试验,经６代次返祖传代毒力未返强,仍是稳定的,已符合弱毒株的标准。在与ＴＧＥ弱毒株组合制备的二联弱毒苗的初步田间试验中取得良好效果。     

Pathotyping of Newcastle Disease Viruses by RT-PCR and Restriction Enzyme Analysis

The technique of RT-PCR and restriction enzyme analysis was standardized to detect and differentiate Newcastle disease viruses. Digestion of RT-PCR-amplified, F gene sequences encoding for the cleavage activation sites of fusion protein with restriction enzymes AluI, BglI, HaeIII, HinfI, HhaI, RsaI, StyI and TaqI was carried out in order to characterize Newcastle disease viruses of varying pathogenicity. Restriction enzyme digestion of the amplicons by BglI and HhaI could group eight viruses, both field isolates and known vaccine strains, into lentogenic, mesogenic and velogenic pathotypes. By employing this technique directly on a clinical sample, Newcastle disease virus of the lentogenic pathotype could be detected.     

Tween 80: a marker for differentiation of hog cholera and bovine viral diarrhea viruses.

Markers for differentiating hog cholera and bovine viral diarrhea viruses were studied using Tween 80, chloroform, trichlorotrifluoroethane and tri (n-butyl) phosphate. Attenuated A and virulent Ames strains of hog cholera virus were employed. Moreover, the NADL PK-15 cell culture adopted strain and low cell culture passaged Purdue strain of bovine viral diarrhea virus were used. These viruses were reacted with 2,500 micrograms/ml of Tween 80 for one hour at 37 degrees C. When attenuated A and virulent Ames strains of hog cholera virus with titers greater than 10(6) and 10(5) plaque forming units respectively, were reacted with Tween 80 the titer of each strains was reduced by approximately 10(4) plaque forming units of virus. When either strain of bovine viral diarrhea virus was reacted with Tween 80, virus was not detected.     

Physical and biological properties of nairobi sheep disease virus

Thermal and pH stability of Nairobi sheep disease (NSD) virus were studied. The 180th mouse brain passage lost infectivity at a higher rate than “wild” virus at 4°C. At 37°C and neutral pH, “wild” virus again was more stable than cell culture and mouse brain attenuated strains with half-life periods of 104, 87 and 51 min, respectively. At 0°C the cell culture attenuated virus was most stable at pH 7.4 with an estimated half-life of 164 h. The density of the virus in sucrose gradients came to 1.195 g vm^?3.Metabolic growth inhibition studies using a halogenated nucleoside, and staining of RNase and DNase-treated infected cell cultures with acridine orange, indicated that NSD virus has a single stranded RNA genome. The growth of the cell culture adapted virus was assayed in monolayers of BHK21/13 cells at low multiplicity of infection. Cell-associated virus (CAV) was first detected at 6 h post-inoculation (PI). The titre increased rapidly until CPE appeared at 48 h and declined after 72 h PI. Cell-free virus (CFV) was first detected at 10 h PI. The titre of CFV increased up to 72 h, but on average was two log units less than the CAV titre.     

Virus Pneumonia of Pigs; Attempts at Propagation of the Causative Agent in Cell Cultures and Chicken Embryos

Two strains of the agent of virus pneumonia, were tested for the ability to propagate in 12 types of cell cultures and in chicken embryos. The 5 primary cell cultures used were: swine kidney, lung, bone marrow, testicle, and chicken embryo kidney; and the 7 serial passage cell cultures were: swine kidney, kidney-tumor, testicle, bone-marrow, bovine kidney, and human cervical carcinoma (HeLa). The agent of virus pneumonia was propagated in primary swine kidney and in HeLa cell cultures as shown by the production of typical gross and microscopic lesions in pigs inoculated with cell future fluids. Third passage cell culture fluids, produced typical gross lesions in pigs, but fourth passage cell culture fluids produced only microscopic lesions, and no lesions were produced by sixth and eleventh passage fluids. Control pigs receiving fluids from uninoculated cell cultures remained free of gross or microscopic lesions, as did uninoculated controls. Cytopathic effects were not detected in any of the inoculated cell cultures and no cellular changes were detected by staining with Giemsa stain or acridine orange.

Neither lesions nor deaths occurred in chicken embryos inoculated with both strains of virus pneumonia virus. Pneumonia was not produced in pigs inoculated with suspensions from second chicken embryo passage of the 2 strains inoculated by the chorioallantioic sac, the amniotic sac, and the yolk sac routes.

Identical gross and microscopic lesions were produced in pigs inoculated with either pneumonic lung suspensions or with virulent cell culture fluids. Gross lesions consisted of areas of light to reddish-purple consolidation usually limited to the anterior, cardiac, and intermediate lobes of the lungs. Pleuritis and pericarditis were never present in experimentally produced virus pneumonia. The microscopic lesions were characterized by: 1. perivascular and peribronchiolar lymphoid infiltration and hyperplasia, 2. alveolar interstitial thickening and infiltration, and 3. alveolar exudates consisting of alveolar cells, lymphocytes, plasma cells, and neutrophiles.

     

Characterization of infectious laryngotracheitis viruses, antigenic comparison by kinetics of neutralization and immunization studies.

Five isolates of infectious laryngotracheitis virus were compared by pock formation on the chorioallantoic membrane of embryonated eggs, plaque size in chicken embryo kidney tissue culture, and antigenic relationship using reciprocal kinetics of neutralization. The A4557-5 strain of infectious laryngotracheitis virus, which causes mild respiratory disease, produced pocks with a zone of edema on the chorioallantoic membrane. A virulent virus (Virus 1), isolated from an outbreak of severe disease characterized by a diphtheritic laryngotracheitis, produced the largest plaques in chicken embryo kidney cell culture. Other virulent viruses (Viruses 2, 3 and V154) did not have unique growth characteristics when grown on the chorioallantoic membrane or in chicken embryo kidney cell culture. All viruses were closely related antigenically as shown by kinetics of neutralization but viruses 2 and 3 were not homogeneous with the other three viruses when neutralized by anti-V154 chicken serum. Following aerosol infection, chickens infected with the A4557-5 virus were immune to challenge with virulent V154 virus. However, in comparison to SA-2 virus, this virus was a less effective immunizing agent when administered by the vent or drinking water methods.     

Selection of foot-and-mouth disease viruses by passage in bovine and swine kidney cell cultures

Two uncloned populations of foot-and-mouth disease virus, one pathogenic for adult mice and the other nonpathogenic, were passaged in cultures of primary bovine kidney (BK) cells and a line of pig kidney (MVPK) cells. Within 10 passages in MVPK cells, the nonpathogenic virus became pathogenic for adult mice, but similar passages of this virus in BK cells did not affect its pathogenicity. In contrast, passage of the pathogenic virus in MVPK cells resulted in a decrease in pathogenicity, but again passage in BK cells had no effect on this characteristics. Neither of the viruses changed in pathogenicity for infant mice during the four passages that were tested. The nonpathogenic virus passaged in BK cells was more infectious for BK than for MVPK cells, but after passage in MVPK cells, this virus was about equally infectious for the two types of cells. Infectivity of the pathogenic virus was relatively unchanged by passage in either type of cell. The parent nonpathogenic virus and the 10th BK cell passage of this virus were much more resistant to adsorption with homogenized mouse kidney than were the MVPK cell passages of nonpathogenic virus. The parent and passaged pathogenic viruses were readily adsorbed. The results demonstrated that passage of the two viruses in MVPK cells had a pronounced selective effect.     

金丝雀痘诊断与痘病毒致弱试验

本试验对送检的金丝雀经实验室诊断，确认为金丝雀痘，对分离的痘病毒进行动物致病性试验、病毒分离传代和致试验、致弱毒保护力试验和返强试验。结果表明：金丝雀痘病毒经鸡胚盲传３后能适鸡胚并致死金丝雀，８代后无致病性，１３例毒性快速通过本动物３次无返强现象，金丝雀用第１３代毒刺种３０天能抵抗强毒攻击。     

African swine fever virus: generation of subpopulations with altered immunogenicity and virulence following passage in cell cultures.

Virus subpopulations with variable virulence, immunogenicity, and infectivity to pigs were readily generated by passaging Tengani isolate of African swine fever virus, either biologically cloned or uncloned, in Vero cell cultures. Avirulent virus populations which account for more than 99% of virus in an uncloned preparation of the 27th passage are laboratory artefacts, perhaps do not exist in nature. Furthermore, attenuation of virulence did not occur uniformly in all subpopulations newly generated, and a continuous modulation of virus populations differing in immunogenicity and virulence took place in the same individuals inoculated with the 27th passage virus. The same virus preparation, appearing to be slightly virulent in pigs, contained at least a virulent subpopulation that was manifested only by further inoculating susceptible pigs with viremic blood collected at various times during the clinical course. A cloned virus after 23 passages in cell cultures generated a subpopulation (99.9%) which induced subclinical infection in pigs; however, the infection did not confer a solid immunity to homologous challenge with Tengani isolate in these pigs. The Tengani isolate contained subpopulations of virus with immunogenicities shared by the Lisbon '60 isolate and also contained at least one subpopulation specific for the Tengani only.     

Swine Interferon I. Induction in Porcine Cell Cultures with Viral and Synthetic Inducers

The production of interferon by porcine kidney (PK₁₅) cell culture in response to viral and synthetic inducers was studied. The inducers used included a synthetic double-stranded polyribonucleotide, polyriboinosinic-polyribocytidylic acid (Poly I:C), swine influenza virus and three strains of pseudorabies virus. Following exposure to these inducers cell culture fluids were examined for interferon by the plaque-reduction method.

The Poly I:C and the swine influenza virus induced production of interferon by PK₁₅ cell cultures, whereas, all three strains of pseudorabies virus at the two concentrations tested failed to induce production of interferon in vitro.

The antiviral substance produced in PK₁₅ cells was identified as an interferon because it was pH stable, non-dialyzable, sensitive to trypsin, non-sedimentable, relatively heat stable, host-species specific and it possessed broad-spectrum antiviral activity. The latter was demonstrated by inhibition of vesicular stomatitis, vaccinia and pseudorabies viruses. Differences in interferon activity against the different viruses were observed.

     

Effect of cell-culture passage on the pathogenicity and immunogenicity of two variant strains of infectious bursal disease virus.

Two variant strains of infectious bursal disease virus, IN and E, were adapted and passaged in an established cell line (BGM-70) 30 times and 40 times, respectively. Passage in cell culture resulted in loss of pathogenicity. However, both viruses maintained their antigenicity and immunogenicity, as demonstrated by the immunofluorescence and virus-neutralization tests and by the satisfactory protection induced by vaccinating specific-pathogen-free (SPF) chickens with inactivated preparations of both passaged viruses. No protection was induced when the passaged viruses were given to SPF chickens as live vaccines. It is speculated that the passaged viruses might have lost some ability to replicate in their natural host, resulting in lack of protection.     

Preparation and evaluation of chicken embryo-adapted fowl adenovirus serotype 4 vaccine in broiler chickens

The current study was planned to develop an efficient vaccine against hydropericardium syndrome virus (HSV). Currently, formalin-inactivated liver organ vaccines failed to protect the Pakistan broiler industry from this destructive disease of economic importance. A field isolate of the pathogenic hydropericardium syndrome virus was adapted to chicken embryos after four blind passages. The chicken embryo-adapted virus was further serially passaged (12 times) to get complete attenuation. Groups of broiler chickens free from maternal antibodies against HSV at the age of 14 days were immunized either with 16th passage attenuated HSV vaccine or commercially formalized liver organ vaccine. The antibody response, measured by enzyme-linked immunosorbent assay was significantly higher (P < 0.05) in the group immunized with the 16th passage attenuated HSV vaccine compared to the group immunized with liver organ vaccine at 7, 14, and 21 days post-immunization. At 24 days of age, the broiler chickens in each group were challenged with 10^3.83 embryo infectious dose₅₀ of pathogenic HSV and were observed for 7 days post-challenge. Vaccination with the 16th passage attenuated HSV gave 94.73% protection as validated on the basis of clinical signs (5.26%), gross lesions in the liver and heart (5.26%), histopathological lesions in the liver (1.5 ± 0.20), and mortality (5.26%). The birds inoculated with liver organ vaccine showed significantly low (p < 0.05; 55%) protection estimated on the basis of clinical signs (40%), gross lesions in the liver and heart (45%), histopathological lesions in the liver (2.7 ± 0.72), and mortality (35%). Birds in the unvaccinated control group showed high morbidity (84%), mortality (70%), gross (85%), and histopathological lesions (3.79 ± 0.14) with only 10% protection. In conclusion, this newly developed HSV vaccine proved to be immunogenic and has potential for controlling HSV infections in chickens.     

表达传染性喉气管炎病毒gB基因重组鸡痘病毒疫苗的遗传稳定性评价

为了检测表达鸡传染性喉气管炎病毒糖蛋白gB基因重组鸡痘病毒（rFPV-ILTVgB)的稳定性，我们对重组病毒进行6代和16代克隆纯化，对纯化后的病毒通过转瓶连续培养传20代，结果发现第6代纯化病毒（rFPV-ILTVgB-C6)在传代过程中有白斑出现，说明病毒纯度不够；经过16代纯化的病毒（rFPV-ILTVgB-C16)对细胞的嗜性加强，病毒的效价进一步升高，20代传代后蓝斑率仍然为100％，PCR的检测进一步证明插入的外源基因能在病毒的长期传代过程中稳定地遗传。抽取病毒细胞传代过程中的第5，10，15，20代次的病毒翅膀内侧无血管处皮下接种2周龄SPF鸡，20天后分为两组，分别用传染性喉气管炎病毒WG株和鸡痘病毒102株攻击，结果不同代次的重组病毒均可以使免疫鸡抵抗传染性喉气管炎病毒和鸡痘病毒强毒的攻击，鸡体内病毒传代试验表明，重组病毒在接种部位仅存在6天，之后就检测不到病毒，重组病毒通过SPF鸡连续传代，不存在病毒返祖的可能，由此可以得出一个结论：rFPV-ILTVgB在结构上和免疫原性上是稳定的，无论是在体外传代，还是在体内均可以稳定遗传，不存在因为接种重组疫苗而给免疫鸡群带来安全威胁的可能，完全可以作为疫苗毒株进行商品化开发。