The use of serotype 1- and serotype 3-specific polymerase chain reaction for the detection of Marek's disease virus in chickens

A serotype 1- and serotype 3-specific detection of Marek's disease virus (MDV) by polymerase chain reaction (PCR) was developed. The sensitivity of the method when applied to cell culture grown virus was comparable with that of cultivation. The method was applied to various tissue samples from chickens experimentally inoculated with serotype 1 or serotype 3 MDV.The serotype 1 strains CVI988 and RB-1B could be detected in feather follicle epithelium up to 56 and 84 days post-inoculation (p.i.), respectively, while the MDV-3 serotype was detected until 42 days p.i. The purpose of this study was to develop and evaluate a reliable and easy-to-handle method for surveillance of the occurrence of MDV in chicken flocks. We emphasize the development of a method, which can be applied to types of samples conveniently collected in the field, e.g. feather tips and blood samples. In addition, the PCR was applied to samples collected from four commercial table egg layer flocks of young stock or pullets vaccinated with either serotype 1 (CVI988) or serotype 3 (HVT) vaccine. These flocks had various clinical signs of Marek's disease. MDV-1 was detected in buffy-coat cells, spleen, liver, skin, feather tips and ovaries. The detection of MDV in feather tips appeared to be as sensitive as co-cultivation of buffy-coat cells, although an inhibiting factor was observed in extracts from feather tips of non-white chickens. This inhibition could be overcome in most extracts by applying a bovine serum albumen pretreatment. The PCR proved to be a convenient tool for the monitoring of MDV in the poultry population, and feather tips were the most convenient and sensitive samples.     

Sequential skin lesions in chickens experimentally infected with Marek's disease virus

Skin biopsies taken at weekly intervals from the same specific-pathogen free (SPF) chickens inoculated with Md/5 Marek's disease virus revealed two distinctive patterns of perifollicular cutaneous lesions, tumour-associated and non-tumour-associated. The tumour-associated pattern was subdivided into two types. The progressive type was manifested by a continuous increase of lymphoid cell aggregates (LCA) in the skin, resulting in the development of gross skin tumours with or without visceral tumours, and the regressive type showed initially increased and finally regressed LCA in the skin, associated with the development of visceral tumours. The non-tumour-associated pattern was characterized by initial transient small LCA in the skin without evidence of tumour formation. Birds with the tumour-associated pattern, regardless of type, had persistent nuclear inclusions (NI) and positive reactions against MDV1-specific phosphorylated polypeptides in the feather follicle epithelium (FFE) and initial R(1)-type (consisting mainly of small lymphocytes with a few lymphoblasts) to advanced T-type (consisting predominantly of lymphoblasts) feather pulp lesions (FPL). On the other hand, birds with the non-tumour-associated pattern formed transient NI and positive reactions against MDV1-specific phosphorylated polypeptides in the FFE and Ri-type to R(2)-type (consisting mainly of plasma cells with oedema) FPL. Antigen-positive lymphoid cells against MDV1-specific phosphorylated polypeptides were detected in both inflammatory and tumourous lesions, especially in the necrotic tumour lesions in the skin of birds showing the progressive type.     

Structural proteins of Marek's disease virus

Marek's disease virus (MDV) was propagated in roller-bottle cultures of duck embryo fibroblasts and partially purified by sucrose gradient centrifugation. Analysis of viral protein by polyacrylamide gel electrophoresis revealed that at least eight proteins (designated VPI-VPVIII) were present in MDV. The VPI is the major viral protein. At least two viral proteins, VPII and VPIV, with glucosamine label could be detected. These two peaks may represent viral glycoprotein and may be associated with the viral envelope. No host cell proteins coelectrophoresed with any viral proteins. Similar electropherograms were obtained by coelectrophoresis of MDV and herpes simplex virus proteins and of MDV and pseudorabies virus proteins.     

Marek's disease virus gene clones encoding virus-specific phosphorylated polypeptides and serological characterization of fusion proteins

Marek's disease virus (MDV) gene clones, RA2 and GA8, constructed inE. coli bacteriophage lambda-gt11 (gt11) were identified by a monoclonal antibody (MAb), H19.47, against a putative transformation-related viral antigen consisting of a complex of three phosphorylated polypeptides, pp41, pp38, and pp24. Both recombinants have a MDV-DNA insert of about 0.5 kb and are mapped to the region ofBamHI-H orEcoRI-X fragments of the MDV genome by Southern blot hybridization. Immunoblot and immunoprecipitation with H19.47 identified a recombinant beta-galactosidase-MDV 140-kD fusion protein for RA2 and a 127-kD fusion protein for GA8.Immunoprecipitation of³⁵S-methionine-labeled, MDV-infected chicken embryo fibroblasts (CEF) with antisera against RA2 and GA8 fusion proteins recognized five polypeptides, of which three (p41, p38, and p24) are specified by H19.47 and the remaining two, p135 and p20, have not been previously identified. Immunoprecipitation of³²P-phosphate-labeled or³H-glucosamine-labeled, GA-MDV-infected CEF with the antiserum against RA2 fusion protein identified a phosphorylated polypeptide of 38 kD and two glycoproteins of 60 and 49 kD, respectively. The antisera against recombinant fusion proteins thus revealed the existence of epitopes common to the phosphorylated polypeptides and other MDV-specific polypeptides.Sera from chickens or mice hyperimmunized with the purified fusion proteins reacted with serotype 1, MDV-infected CEF in the fluorescent antibody (FA) test to significant titers. These immune sera did not react with either serotype II or III, indicating the serotype specificity of the phosphorylated polypeptides.Requests for reprints should be addressed to Lucy F. Lee, USDA, Agricultural Research Service, Regional Research Laboratory, 3606 East Mount Hope Road, East Lansing, MI 48823, USA.     

Replicating Marek's disease virus (MDV) serotype 2 DNA with inserted MDV serotype 1 DNA sequences in a Marek's disease lymphoblastoid cell line MSB1-41C

Summary We characterized the properties of herpes-type viruses which grew well in a Marek's disease lymphoblastoid cell line, MSB1-41C, inducing cytopathic effect characterized by the formation of syncytial giant cells. Examination of the infectious virus by field inversion gel electrophoresis revealed the presence of DNA of about 180 kbp in both the culture fluid and cell fractions of the infected MSB-41C cells. The DNA was found to consist of Marek's disease virus (MDV) serotype 2 (MDV2) and MDV serotype 1 (MDV1) DNA by Southern blot hybridization. The MDV1 DNA consisted of sequences mainly from the long inverted repeats including multiple copies of 132 bp direct tandem repeats. Molecular cloning of BamHI digests of the MDV2 DNA revealed a fragment of MDV1 DNA and MDV2 DNA fused together, indicating that the recombinant MDV2 DNA had been generated by genetic recombination with the latent MDV1 DNA.     

Attenuation of lymphoid leukosis enhancement by serotype 2 Marek's disease virus

Serotype 2 Marek's disease virus (MDV), already known to augment the protective efficacy of turkey herpes virus (HVT) vaccine against MD, may also enhance the frequency of lymphoid leukosis (LL) in retrovirus-infected chickens of certain strains. LL enhancement refers to an increase in frequency and decrease in latent period for development of LL, and is measured by comparison of tumour responses in retro-virus-infected, MD-vaccinated chickens to those of retrovirus-infected control chickens. LL enhancement has been documented with both of the serotype 2 MDV strains tested thus far. These experiments were conducted to investigate whether LL enhancement ability varied within a larger collection of serotype 2 strains or was influenced by serial passage in cell culture. Each of seven low-passage serotype 2 MDV strains enhanced LL responses. The HN-1 strain was competent for LL enhancement at passage 19, but LL enhancement ability was absent at passages 26, 27 and 40. Enhancement of LL by strains 471B/1, 281MI/1, 287C/1 and 298B/1 was reduced by 40 to 64 serial passages in chicken embryo fibroblast cultures. Strains SB-1 and 301B/1 continued to demonstrate enhancement of LL through 66 and 40 passages, respectively. Thus, LL enhancement appeared to be a general property of serotype 2 MDV, but was susceptible in five of seven strains to reduction (attenuation) by cell culture passage. Enhancement of LL by serotype 2 MDV was attenuated more rapidly by cell culture passage than was the ability of such viruses to protect against virulent MDV challenge either alone or in combination with HVT. One such LL enhancement-attenuated strain, 471B/1 (passages 33 and 40), when combined with HVT, induced protection against MD challenge (76 and 78%) that was comparable with that induced by SB-1 + HVT (82%) or 301B/1 + HVT (89%). This or similar serotype 2 viruses could provide suitable protection against MD with a reduced risk of LL enhancement.     

Enhancement of reticuloendotheliosis virus-induced bursal lymphomas by serotype 2 Marek's disease virus

The effect of serotype 2 and 3 Marek's disease virus (MDV) vaccines on the pathogenesis of reticuloendotheliosis virus (REV)-induced bursal and non-bursal lymphomas was examined in chickens of RPRL lines 15I(5) X 7(1) and 6(3) X 0, respectively. At hatch, chickens were vaccinated with strain 301B/1 of serotype 2 MDV or strain FC126 of turkey herpesvirus (HVT), a serotype 3 MDV and inoculated with spleen necrosis virus (SNV), a non-defective strain of REV. In another experiment, bursas from 14-week-old 15I(1) X 7(1) chickens coinfected with strain 301B of serotype 2 MDV and SNV strain of REV at hatch were examined microscopically for REV-induced transformed follicles by methyl green pyronin stain and for the presence of MDV by in situ hybridization. The incidence of REV-induced bursal lymphomas was significantly higher in 15I(5) X 7(1) chickens vaccinated with serotype 2 MDV than in unvaccinated chickens or chickens vaccinated with HVT. On the other hand, the incidence of REV induced nonbursal lymphoma in chickens of line 63 X 0 vaccinated with serotype 2 MDV was comparable to that in unvaccinated chickens or chickens vaccinated with HVT. The average number of hyperplastic follicles in bursas from REV-infected 15I(5) X 7(1) chickens was significantly higher in chickens vaccinated with serotype 2 MDV than that in unvaccinated chickens or chickens vaccinated with HVT, and the MDV was more frequently detected in REV-transformed than in untransformed bursal follicles. Data from this study suggest that serotype 2, but not serotype 3, of MDV may enhance the development of REV-induced bursal lymphomas, and that neither serotype 2 nor serotype 3 MDV influence the development of REV-induced nonbursal lymphomas. The data also suggest that the enhancement effects of serotype 2 MDV on REV bursal lymphomas may be at the stage of formation of hyperplastic or transformed bursal follicles.     

Multiplication of turkey herpes virus and Marek's disease virus in chick embryo skin cell cultures.

A simple method for the in vitro cultivation of chick embryo skin epithelial cells was developed and the replication of turkey herpes virus (HVT) and Marek's disease herpes virus (MDHV) in this system was studied. A high percentage of cells in monolayers inoculated with HVT (19·7 per cent) and MDHV (4·6 per cent) was infected on the fourth day post-incubation. Titres of infectious cells in the culture fluid or cell-free virus in the cell extract were low and cell-free virus was not detected in the culture medium. Enveloped particles of HVT, but not of MDHV, were detected by electron microscopy.     

Influence of serotype and virus strain on synergism between Marek's disease vaccine viruses

The enhanced protective effect (synergism) when certain Marek's disease (MD) vaccine viruses are combined has been widely used in the development of improved vaccines, but the mechanism is poorly understood. To better characterize the basis for synergism among MD vaccine viruses, three vaccine viruses from each of the three MD viral serotypes were evaluated alone and in various combinations for protection against early challenge with very virulent MD viruses in four replicate trials. Synergism seemed to be influenced by viral serotype because significant enhancement occurred frequently between viruses of serotypes 2 and 3 (five of nine bivalent vaccines positive), but rarely between viruses of serotypes 1 and 3 (one of nine bivalent vaccines positive) and 1 and 2 (one of nine bivalent vaccines positive), and was not detectable between viruses of the same serotype (none of nine bivalent vaccines positive). With some exceptions, the degree of synergism tended to vary inversely with the mean protective efficacy of the most protective component virus. Little effect of virus dose, virus dose ratio or type and route of viral challenge was noted. The combination of strains 281MI/1 (serotype 2) and WTHV-1/1 (serotype 3), both poorly protective as monovalent vaccines, consistently demonstrated high levels of synergism (over 300%) in antibody-positive chickens challenged 5 days post-vaccination with Md5 virus. This protocol may be a useful model system for further studies on mechanisms of synergism. However, mixtures that optimize synergism are not necessarily as protective as commercial vaccines.     

Central nervous system lesions induced experimentally by a very virulent strain of Marek's disease virus in Marek's disease-resistant chickens.

The histological lesions of central nervous system (CNS) in Marek's disease-resistant chickens inoculated with a very virulent Marek's disease virus strain (vvMDV), Md/5, were examined. This vvMDV induced high incidences of CNS lesions as well as visceral and peripheral nerve lesions. The principal histopatho-logical changes of CNS consisted of non-suppurative meningoencephalomyelitis and lymphomatous lesion, which were categorized into two types, non-necrotizing and necrotizing. The main changes in the former type were perivascular cuffing of lymphoid cells of variable thickness. Although diffuse lymphomatous lesions were sometimes observed, gliosis, neuronal and axonal degeneration, and satellitosis as well as demyelination in the myelinated fibre paths, were infrequent. The most significant change of the latter type was necrotizing lymphomatous and, sometimes, non-suppurative inflammatory lesions (malacia). In the malacic foci, all the cells, including infiltrating lymphoid cells, neurons and glial cells, were necrotic or degenerated. The malacic lesions were frequently accompanied by fibrinoid necrosis of blood vessels. The present results indicate that necrotizing vasculitis associated with vvMDV may lead to ischaemic damage which in turn induces necrotizing lymphomatous and sometimes, meningoencephalomyelitis lesions. In addition, it is considered that vvMDV can induce a high incidence not only of CNS lesions, but also of visceral and nerve lesions in Marek's disease-resistant chickens.     

Identification of serotype ii infectious bursal disease virus proteins

Polyacrylamide gel electrophoresis (PAGE) of serotype II IBDV (OH and MO strains) purified from infected Vero cells resolved a previously undetected major viral polypeptide, VP2. The molecular weight (MW) of VP2 was different between the two strains of serotype II. It was 43.5 kDa in strain OH and 44 kDa in strain MO. This was higher than the MW of VP2 in SAL strain of serotype I IBDV which was 41 kDa. VPX (50 kDa), VP3 (33 kDa) and VP4 (30.5 kDa) were similar in both serotype II virus strains but were also of higher MW than VPX (48 kDa), VP3 (32 kDa) and VP4 (30 kDa) of SAL virus. VP1 (80 kDa) had the same MW in both serotypes.     

Heat inactivation of serotype 1 infectious bursal disease virus.

Heat inactivation curves for infectious bursal disease virus serotype 1 (IBDV) strain 52/70 in bursal homogenate supernatant were constructed for 70 degrees C, 75 degrees C and 80 degrees C. Biphasic, multiple kinetics curves were produced with an initial rapid drop in infectivity followed by a more gradual decline. In this second phase the approximate times taken to reduce the infectivity by 1 log(10) at each temperature were: 70 degrees C (18.8 min), 75 degrees C (11.4 min) and 80 degrees C (3.0 min), confirming the heat resistance of this virus.     

Isolation of Marek's disease virus: revisited.

Splenocytes from chickens infected with low-passage stocks of Marek's disease virus (MDV) RB-1B, a very virulent (vv) strain and vv+ RK-1 were used to compare the efficacy of chick kidney cells (CKC), chicken embryo fibroblasts (CEF) and chicken embryo kidney cells (CEKC) for virus isolation. CKC were superior to CEF and CEKC. MDV foci were present at 4 days post infection in CKC but not until 6 days post infection in CEF or CEKC. Virus yield was higher in CKC than in CEF or CEKC at 6 days post infection. Passage of RB-1B in CKC yielded a significantly higher virus increase than with CEF or CEKC. The same was true for RK-1 comparing CKC with CEKC. Interestingly, RK-1-infected CEF were negative or had very low number of foci in passage 1, but virus yield increased 500-fold to 600-fold on passage in CKC, CEF, and CEKC. Recommendations on procedures for successful virus isolation are provided.     

Protection studies against Marek's disease using baculovirus-expressed glycoproteins B and C of Marek's disease virus type 1

Glycoproteins B (MDV1-gB) and C (MDV1-gC) of Marek's disease virus type 1 (MDV 1) expressed by baculovirus recombinants [rAcNPV(s)] were investigated for their ability to confer protective immunity in chickens against virulent MDV1 challenge. Immunization of chickens with rAcNPV-expressed MDV1-gB induced antibody against MDV1 and resulted in strong protection against the lethal challenge with MDV1. In contrast, immunization of chickens with rAcNPV-expressed MDV1-gC induced antibody against MDV1, but failed to protect against challenge with the virulent MDV1. Co-immunization with both rAcNPV-expressed MDV1-gB and MDV1-gC seemed to show no synergistic effect.     

Identification of a bluetongue virus serotype 1-specific ovine helper T-cell determinant in outer capsid protein VP2.

Ovine T-cell lines (including one clone [101A]), which are specific for Bluetongue virus serotype 1 (BTV1), have been established and characterized. Although these T-cell lines react with different isolates of BTV1 (including those from South Africa, Australia, Nigeria, and Cameroon), they do not react with heterologous BTV serotypes. Antigen specificity of these T-cells was studied using purified virus particles, infectious subviral particles (ISVP) and cores, or using individual BTV structural proteins that were either isolated by SDS-PAGE or expressed by recombinant strains of vaccinia virus. The results showed that each of the T-cell lines reacted with outer capsid protein VP2 (the BTV protein exhibiting most serotype-specific variation and the major neutralization antigen). However, all of the uncloned T-cell lines also reacted with either the core structural proteins or the outer capsid protein VP5. In contrast, the T-cell clone 101A only reacted with outer capsid protein VP2. Cell surface marker analysis showed that 101A has a helper T-cell phenotype (CD5+, CD4+, CD8-, T-19-). The T-cell lines and clone 101A all produced large amounts of interleukin 2 (IL-2) when stimulated with purified BTV1 virus particles, or with VP2 (up to 120 IU/ml from 2 x 10(5) T-cells). BTV serotype-specific antigenic sites, for B cells and at least one site for ovine helper T-cells, are therefore located within VP2.     

A study of vaccination against Marek's disease with an attenuated Marek's disease virus

Approximately 2,000 day-old white leghorn chickens were distributed into 10 pens and half the pens vaccinated with the attenuated strain of HPRS-16. At 72 weeks of age, mortality from non-specific death and Marek's disease was 17.6% and 15.6% respectively, in vaccinated chickens compared with 14.2% and 51.7% in unvaccinated chickens. Body weights of vaccinated chickens were 5.6% higher at 8 weeks of age than unvaccinated chickens. Vaccinated chickens consumed 2.3% more feed and the hen housed egg production and hen day egg production were 58.7% and 7.0% greater than unvaccinated chickens. Vaccination resulted in a 3.8 fold increase in net margin over food and bird costs per bird housed at 16 weeks. Active antibody production to 'A' antigen occurred later and in a smaller proportion of vaccinated chickens than unvaccinated chickens. Viraemia due to vaccination was detected at 2 weeks of age and viraemia due to field virus was detected at 5 weeks of age. The incidence and titres of viraemia due to field virus were higher in unvaccinated chickens compared with vaccinated chickens. No evidence of spread of vaccine virus to unvaccinated chickens could be found. Acute, classical and apathogenic strains of field virus were isolated from vaccinated chickens and strains of field virus were found to persist throughout the life of the vaccinated chickens. Mild classical and/or apathogenic strains were first apparent at 18 weeks of age and increased in proportion thereafter, forming the majority of isolates from 52 weeks of age. Data on individual birds suggested a direct relationship between virus titre and lesion (or Marek's disease) frequency.     

Use of recombinant pp38 antigen of Marek''s disease virus to identify serotype 1-specific antibodies in chicken sera by Western blotting

A fowlpox recombinant expressing the pp38 antigen of Marek's disease virus has been constructed. Production of pp38 in chick embryo fibroblasts (CEF) infected at a m.o.i. of 1 pfu/cell occurred over a period of 5 days and reached a peak at 72 h after infection. The pp38 antigen could be released from infected cells by freezing and thawing. Western blot analysis showed that denatured pp38 antigen reacted with antisera from chickens inoculated with serotype 1 MDV but failed to react with antisera from chickens inoculated with MDV serotype 2 or HVT. The results suggest that MDV pp38 contains a serotype 1-specific epitope which becomes available upon denaturation of the antigen and that this could be exploited to identify MDV-specific antibodies in epidemiological studies. The relationship between pp38 and the related polypeptides pp24 and pp41 in MDV-infected cells was also examined. The results suggest that pp24 and pp38 are synthesised independently and that MDV coded proteins (probably a protein kinase) might be required to convert pp38 to pp41.