New serotype 2 and attenuated serotype 1 Marek's disease vaccine viruses: comparative efficacy

Attempts were made, through selection of optimum viral strains, to develop improved vaccines against Marek's disease (MD). Seven attenuated serotype 1 strains and 22 avirulent serotype 2 strains, both alone and in combination with the FC126 strain of serotype 3, were screened for protective efficacy against challenge with virulent and very virulent MD viral strains. The three viruses selected as most promising were evaluated alone and in various combinations and compared with commercially available vaccines, including FC126, bivalent (FC126 + SB-1), and CV1988/C, in 12 separate assays. Two of these new viruses--301B/1 (serotype 2) and Md11/75C/R2 (serotype 1)--were exceptionally protective compared with prototype vaccine strains. Four new monovalent and polyvalent vaccines based on these two isolates protected chickens better than FC126 alone or CV1988/C alone. Three of these new vaccines provided better protection than the bivalent (FC126 + SB-1) vaccine. Protective synergism was noted commonly between viruses of serotypes 2 and 3 but only sporadically between serotypes 1 and 2 or between serotypes 1 and 3. Strain CVI988/C was protective but was no better than FC126 alone, and it was less effective than bivalent (FC126 + SB-1) vaccine, even when used as a bivalent vaccine with FC126 or SB-1.     

Induction of strong protection by vaccination with partially attenuated serotype 1 Marek's disease viruses

Studies were conducted to better understand the relationship among Marcek's disease (MD) vaccine strains between induction of protective immunity and the degree of attenuation (or virulence). To obtain viruses at different stages of attenuation, very virulent plus MD strains 584A and 648A and selected clones of these strains were serially passaged in chicken and duck cells. These viruses were considered fully attenuated after passage for 70-100 times in chicken embryo cell cultures until they no longer induced gross lesions in susceptible, maternal antibody-negative (ab-) chickens. Lower passages of the same strains were considered partially attenuated, provided their virulence was less than that of the parent strain. Four of five partially attenuated preparations derived from MD virus strains 584A and 648A or the previously attenuated Md11 strain induced 28%-62% higher levels of protection in maternal antibody-positive (ab+) chickens against virulent MD challenge than the fully attenuated counterpart viruses. The partially attenuated 584A/d2/3 strain replicated in chickens but was totally nonprotective. Data from two subsequent trials in ab+ chickens confirmed that protection induced by the partly attenuated (passage 80) preparations was 79% and 118% higher, respectively, than that induced by the fully attenuated (passage 100) preparations of strain 648A. However, in one trial with ab- chickens, no difference in protection between partially and fully attenuated virus was observed. Strong protection (up to 85%) against highly virulent challenge also was provided by preparations of 648A at passages 40-60, which were moderately oncogenic when used alone. Partially attenuated strains tended to replicate to higher titers in both ab+ and ab- chickens compared with fully attenuated vaccines. Also, ab+ and ab- chickens vaccinated with partially attenuated strains developed three- to nine fold more extensive microscopic lesions in peripheral nerves at 14 and 22 days after virulent challenge than chickens vaccinated with fully attenuated strains. When measured in ab+ chickens, loss of lesion induction by 648A was achieved 30 passages earlier (at passage 70) than when measured in ab- chickens. Thus, maternal antibodies appeared to abrogate the pathogenicity of some partially attenuated strains. These studies establish for MD the principle that at least some partially attenuated MD viruses may replicate better and induce stronger immunity against virulent challenge than fully attenuated preparations of the same strain, at least when tested in ab+ chickens. Moreover, depending on passage level, partially attenuated vaccine strains may be relatively innocuous for ab+ chickens, causing few or no lesions.     

Biological diversity among serotype 2 Marek's disease viruses

Selected biological characteristics were determined for 14 low-passage serotype 2 Marek's disease virus (MDV) isolates. Four of these isolates were also tested after extensive serial passage in chicken embryo fibroblast cultures. Observations were made on replication in vitro and in vivo, pathogenicity by in ovo inoculation, antigenicity, and protection against virulent MDV challenge. Among the low-passage isolates, there were some differences in pathogenicity after in ovo inoculation but relatively little difference in other characteristics, with the exception of the HN-1 strain, which replicated more rapidly in cell culture but produced generally lower in vivo responses than other isolates. After extended in vitro passage, isolates replicated much more readily in cell culture and produced lower pathologic responses in vivo than low-passage isolates, as has been reported for serotype 1 isolates. No antigenic differences among isolates were detected, but high-passage isolates induced lower levels of precipitating antibodies than low-passage isolates, indicating a possible reduction in A antigen production. The observed diversity associated with strain and passage level may be of value in the selection of optimum vaccine strains.     

Attenuated revertant serotype 1 Marek's disease viruses: safety and protective efficacy.

In earlier studies, a revertant serotype 1 Marek's disease virus (MDV), clone Md11/75C/R2, was found to be a highly protective vaccine virus but was mildly pathogenic for susceptible chickens. The term "revertant" indicates that the virus, after attenuation, gained virulence following backpassage in chickens. The present study is an attempt to develop a more attenuated but still protective vaccine virus from Md11/75C/R2. Forty-two derivative viruses or clones from Md11/75C/R2 were evaluated. Two of these, designated clones R2/23 and R2/29, induced viremia but little or no pathology in preliminary trials and were selected for further study. In a series of nine trials, both clones provided protection against challenge with very virulent MDV strains that was superior to that induced by turkey herpesvirus (HVT) and was not significantly different (P greater than 0.05) from that induced by a bivalent (HVT + SB-1) vaccine. Both clones appeared fully attenuated based on pathogenicity tests in susceptible antibody-negative chickens. Both clones gained virulence on backpassage in chickens, but this seemed of little concern because neither virus spread by contact to other chickens. Although the two clones were very similar, clone R2/23 appeared to have a slightly lower pathogenic potential following backpassage and thus best meets the combined criteria of safety and efficacy.     

Difference in the meq gene between oncogenic and attenuated strains of Marek's disease virus serotype 1

Serotype 1 strains of Marek's disease virus (MDV1), except attenuated vaccine strains, are known to cause lymphomas in visceral organs of infected chickens. To know additional genetic differences between oncogenic and nononcogenic MDV1, polymerase chain reaction (PCR) was performed to amplify the meq gene of the viral genome. In addition to the 1,062-bp band including the native meq open reading frame (ORF), a 1.2-kb band was amplified from the DNA sample prepared from chick embryo fibroblast infected with an attenuated strain, CVI988, but not with oncogenic strains. Sequence analysis of the 1.2-kb band showed that a 178-bp sequence was inserted to the meq ORF of CVI988. This ORF could encode for the Meq protein with a different transactivator domain. Southern blot analysis also confirmed the insertion of the 178-bp sequence in the meq ORF of CVI988. This insertion of 178-bp sequence may explain the reason why CVI988 is not oncogenic.     

Early posthatch protection against Marek's disease in chickens vaccinated in ovo with a CVI988 serotype 1 vaccine

CVI988, a serotype 1 Marek's disease virus (MDV), was used as an in ovo vaccine in specific-pathogen-free chickens to determine if this virus induces early posthatch protection against Marek's disease as has been shown previously for turkey herpesvirus. MDV CVI988 was injected at embryonation day (ED) 17 (group 1) or at hatch (group 2). A third group (group 3) was left unvaccinated. At 1, 2, 3, 4, 5, and 7 days of age, chickens from each group were sampled and examined as follows: a) single-cell suspensions of spleen were inoculated onto chicken embryo fibroblast monolayers to isolate the virus; b) sections of bursal tissues were stained by indirect immunofluorescence assays with anti-pp38 monoclonal antibody to identify viral antigen expression; and c) chickens were exposed intra-abdominally to MDV RB1B, a virulent serotype 1 MDV. Results revealed that in chickens given MDV CVI988 at ED 17, virus and virus-encoded protein were not detected until chickens were 3 and 2 days old after hatching, respectively. Results also indicated that during the first 4 days after hatch, the chickens given MDV CVI988 at ED 17 were better protected against virulent MDV than those given MDV CVI988 at hatch (P < or = 0.001). These results suggested that MDV CVI988 proteins were adequately expressed in the embryo to initiate prehatch immunologic response. Additional efforts with more sensitive techniques than used in this study are needed to identify the nature of viral expression in embryos.     

Serotype 1 viruses modified by backpassage or insertional mutagenesis: approaching the threshold of vaccine efficacy in Marek's disease

Improved vaccines to control Marek's disease (MD) in chickens are desired by the poultry industry but have been difficult to develop. Studies were conducted to evaluate strategies for deriving MD vaccines of high protective efficacy, irrespective of virulence. Candidate viruses from parent strains representing v and vv+ pathotypes were modified by cell culture passage, backpassage in chickens, or insertional mutagenesis following cocultivation with retroviruses. Ten strains considered most likely to exhibit high protective efficacy were selected for further study. The ability of these modified viruses to protect commercial or maternal antibody-positive (ab+) chickens against virulent MD virus (MDV) challenge was compared with that of strain CVI988, the standard commercial MD vaccine. Modified strains were also evaluated for the ability to induce lymphomas or other pathologic changes in ab+ and antibody-negative (ab-) chickens. Two of the 10 modified viruses, strains RM1 and CVI988/BP5, provided high levels of protection against highly virulent MDV challenge. The magnitude of protection was greater than that of one laboratory and two commercial preparations of CV1988, but was approximately equal to that of two other commercial preparations of CVI988 in laboratory and field tests. Three of the strains, including RMI and CVI988/BP5, induced lymphoid organ atrophy in ab-chicks but not in ab+ commercial chicks, a property designated here as L phenotype. Seven strains, including two L+ strains, were mildly oncogenic for ab- chicks, a property designated here as O phenotype. Five of these strains caused no tumors in ab+ chickens. The two fully attenuated strains induced neither lymphomas nor lymphoid organ atrophy. The L and O phenotypes appeared not to be linked, and both (especially the L phenotype) appeared associated with high levels of protection. These studies also illustrated differences in the protective efficacy of different preparations of CVI988 vaccine, indicating the need to choose carefully the most protective strains as controls for efficacy studies. A new vv+ strain, designated as 686, is described and appears useful as a challenge virus; it is the most virulent of the 48 field isolates of MDV thus far pathotyped in this laboratory. These findings support the conclusion that new virus strains with high levels of protective immunity comparable to that of CVI988 can be developed. However, the question of whether strains can be developed that exceed the efficacy of current CVI988-based vaccines remains unanswered. After more than 30 years of unsuccessful endeavor by many laboratories toward this goal, it now may be useful to consider whether the efficacy of MD vaccines is limited by some type of biologic threshold.     

鸡马立克氏病基因工程二价苗重组病毒分子生物学特性鉴定

用磷酸钙沉淀法将MD Ⅰ型病毒gB基因插入到HVT病毒载体中制成MD重组病毒DNA，再将重组DNA转染于鸡胚成纤维细胞上，形成的病毒蚀斑与HVT病毒蚀斑形态相似，经复制2～3代后，提纯病毒DNA，PCR扩增后电泳分析，凝胶上形成一条2．9kb左右的条带。将重组病毒负染后电镜观察，病毒粒子形态与HVT病毒相似，再将重组病毒的DNA进行测序，结果表明，gB基因已经重组在二价病毒中，并能够稳定遗传。     

Diversity (polymorphism) of the meq gene in the attenuated Marek's disease virus (MDV) serotype 1 and MDV-transformed cell lines

The meq gene encoding a 339-amino-acid bZIP transactivator protein has been identified as a candidate oncogene of Marek's disease virus serotype 1 (MDV1), which induces malignant lymphomas in chickens. We have previously reported that, in addition to meq, L-meq, in which a 180-bp sequence is inserted into the region encoding the transactivation domain of meq, is also detected in chickens experimentally infected with MDV. To further analyze the diversity in meq, PCR was performed using a primer set which specifically amplify the proline-rich repeat (PRR) region in the transactivation domain of meq. In CVI988/R6, a vaccine strain of MDV1, and JM, an MDV1 strain attenuated by prolonged passage in vitro, a major band of a 0.8 kb corresponding to L-meq as well as a minor band of 0.6 kb corresponding to meq was detected by PCR. Furthermore, extra 0.5- and 0.3-kb bands, corresponding to genes termed as short meq (S-meq), and very short meq (VS-meq), respectively, were also detected. These genes were also detected in MDV-transformed cell lines, MSB1 and MTB1. In Md5, an oncogenic MDV1, attenuated by prolonged passage in vitro, the 0.6-kb meq was consistently detected, and 0.5-kb S-meq was occasionally detected. This diversity in meq was due to the difference in the copy number of the PRR region: L-meq and meq contained 9 and 6 copies of PRR while 4 and 2 copies of PRR were present in S-meq and VS-meq, respectively. Thus, the meq gene is polymorphic in the attenuated MDV1 and the MDV-transformed cell lines, and gene products from different meq genes may have different functions from each other.     

A monovalent attenuated serotype 2 bluetongue virus vaccine confers homologous protection in sheep

An outbreak of bluetongue caused by bluetongue virus serotype 2 virus in certain Mediterranean countries during 1999/2000, presented an opportunity to produce a monovalent type 2 vaccine. Since no data have been published previously on the protection conferred by the current live attenuated bluetongue vaccine strains used in the polyvalent vaccine, a challenge experiment was performed to determine the degree of homologous protection induced by the type 2 vaccine strain. The standard vaccine dose of 5 x 10(4) pfu of vaccine conferred 99.7% protection against clinical disease and no viraemia was detected in the vaccinates.     

马立克氏病病毒致弱毒株的致病性及致瘤相关基因序列分析

为培育及鉴定马立克氏病病毒(MDV)致弱病毒株,本研究将4株现地流行MDV强毒株(L-ZY、L-CZ、L-MS和L-SY株),在体外经CEF连续传代至110代,获得相应的4株高代次病毒株(L-ZYp 110C、L-CZp110C、L-MSp110C和L-SYp110C株).其中的L-MSp110C和L-SYp110C株对SPF鸡的致病性试验结果显示:以2×103 pfu和2×104 pfu的剂量接种1日龄SPF鸡,在试验期(12周)内试验鸡均没有引起马立克氏病病变;高代次病毒株在体外增殖能力增强,而在体内增殖能力显著低于亲本病毒株.与亲本病毒株基因序列比对发现:4株高代次病毒株病毒基因组中Meq和RLORF 12基因均有不同程度的缺失和插入;132 bpr序列拷贝数均显著增加;其中3株病毒株的RLORF4基因存在大片段缺失.以上结果表明,MDV强毒株体外传代至110代后,病毒增殖能力和主要致瘤相关基因的遗传特性均发生了改变,致弱毒株的毒力已完全弱化.该研究为进一步筛选MD弱毒疫苗提供了实验依据.     

Trials with Marek's disease vaccines prepared from a turkey herpes virus and an attenuated Marek's disease virus.

In field trials involving over 224,000 fowls in 11 different commercial flocks, three vaccines were used, namely a freeze-dried vaccine prepared from a turkey herpes virus, a cell-associated virus vaccine prepared from the same isolate and a cell-associated vaccine prepared from a strain of Marek's disease virus isolated from a fowl. The mortality from Marek's disease was reduced by 80 per cent to 95 per cent in birds vaccinated with the freeze-dried vaccine. Cell associated vaccines gave slightly less protection.     

Development of a quantitative-competitive polymerase chain reaction assay for serotype 1 Marek's disease virus

We have developed a quantitative-competitive (QC) polymerase chain reaction (PCR) for the detection of Marek's disease virus (MDV) DNA. The assay utilizes a competitor DNA that differs from the viral DNA of interest by having a small insertion. The competitor DNA acts as an internal standard for the estimation of viral DNA in an unknown sample. The amount of viral DNA in a sample is quantitated by coamplification in the presence of a known amount of competitor DNA. The same PCR primers that amplify the viral DNA also amplify the competitor DNA. When the amount of competitor is equal to the amount of viral DNA in a sample, there is equal amplification of the competitor and the virus. Thus, we are able to quantitate the viral DNA in an unknown sample. To establish the utility of this assay, in vivo correlations between virulence and virus replication were studied. Our data demonstrated that a more virulent strain of MDV (648A) replicated better in thymus during cytolytic infection than did a less virulent strain (GA). However, no differences in virus titer were observed when these two viruses were propagated in tissue culture. Our data are consistent with the generally held idea that "hot" strains of MDV replicate earlier and better in birds. Thus, QC-PCR is extremely specific and sensitive to measure MDV DNA over a wide range and can be applied to in vivo studies of viral pathogenesis.     

The early pathogenesis in chicken inoculated with non-pathogenic serotype 2 Marek's disease virus.

A newly cloned serotype 2 Marek's disease virus (MDV), strain ML-6, was inoculated via the nasal cavity in specific-pathogen-free chicks to examine early virus replication and the expression of Marek's disease (MD)-related antigens. Following inoculation, viral intracellular antigens (VIAs) were detected in lymphoid organs (bursas and spleens) between 5 and 14 days post inoculation (PI), in feather follicles between 14 and 30 days PI, and in lungs at 3 days PI by the immunohistopathological staining of avidin-biotin-peroxidase complex method. But, very few VIAs were expressed in the thymuses between 5 and 14 days PI. However, MD tumor-associated surface antigens were not detected in any organs. Viruses were isolated from separated spleen cells at 14 and 30 days PI. Fluorescent antibodies of convalescent sera were also detected after 10 days PI. As most of the VIAs were detectable in B-cells in bursas and spleens. B-cells were considered to be the main first target cells for the serotype 2 MDV infection.