Relationship between Marek's disease virus load in peripheral blood lymphocytes at various stages of infection and clinical Marek's disease in broiler chickens.

Vaccination with herpesvirus of turkey (HVT) vaccine provides protection against clinical Marek's disease (MD) but does not preclude infection with wild-type MD virus (MDV). The quantity of MDV detected in circulating lymphocytes during the early period after infection may be a useful predictor of subsequent clinical MD later in the life. A study was designed to quantify MDV and HVT copy number in peripheral blood lymphocytes (PBL) using real-time polymerase chain reaction between days 5 and 35 post-challenge and to relate this to subsequent development of gross MD lesions. Female commercial broiler chickens were vaccinated with HVT or were sham-vaccinated at hatch, then challenged with MDV strain MPF-57 at day 2 post-vaccination and reared in positive-pressure isolators up to 56 days post-challenge, when all survivors were euthanized. All dead and euthanized chickens were examined post mortem for gross MD lesions. Birds were scored for MD lesions and mortality. MDV and HVT genome copy numbers were determined for each PBL sample. There was an increase in HVT load in PBL between days 7 and 37 post-vaccination, with marked increases between days 7 and 16 and again between days 30 and 37. There was a steady increase in MDV load to 35 days post-challenge. The mean MDV copy number (log(10)) was greater in chickens subsequently exhibiting gross MD lesions (5.05 +/- 0.21) than in those that did not (2.88 +/- 0.223), with the largest difference at 14 and 21 days post-challenge (P < 0.001). Quantification of MDV during early infection is therefore a potential tool for monitoring MD in broiler flocks.     

Cross-protection between JMV Marek's disease-derived tumour transplant, Marek's disease and turkey herpesviruses

Cross-protection tests were conducted using attenuated JMV (JMV-A) Marek's disease-derived lymphoblasts, glutaraldehyde-treated JMV tumour cells, attenuated Marek's disease virus (MDV, strain HPRS-16/att) and turkey herpesvirus (HVT, strain FC126) as vaccines, and virulent JMV and MDV (HPRS-16) for challenge. The JMV and JMV-A preparations were free of MDV, leukosis and reticuloendotheliosis viruses. Vaccination of chickens with attenuated MDV or with HVT provided good protection against both JMV lymphoblastosis and Marek's disease (MD). In one experiment HVT (cell-free) caused a better resistance to JMV than to MD. Inoculation of JMV-A always resulted in a 100% resistance to virulent JMV. However, JMV-A did not induce any appreciable resistance to MD, even when the birds were challenged with MDV by contact exposure. Control experiments revealed that high doses of normal lymphocytes from uninfected chickens also had a protective effect against JMV. The 50% protective dose varied from 10(7) to 10(8) lymphocytes. JMV tumour cells inactivated by glutaraldehyde were used in different experiments but rarely caused a clear-cut protection against virulent JMV. The results of this study suggested that a one-way relationship exists in vivo between HVT or MDV and JMV lymphoblastic leukaemia. However, resistance induced against JMV tumour cells appeared to be related to histocompatibility antigens at least as much as to tumour-specific cell surface antigens. The results obtained failed to provide clear evidence for or against vaccinal resistance to MDV being dependent on the action of a common Marek's disease tumour-associated surface antigen (MATSA) additional to the immune response to viral antigens.     

Antigen a gene of Marek's disease virus (MDV) detects two mRNA species (3.7 and 2.1 kb) in MDV- and HVT-infected cells

The gene of glycoprotein A of Marek's disease virus (MDV) detected to mRNA species (3.7 and 2.1 kb) by Northern blot hybridization. These two mRNA species were also detected in RNA extracted from herpesvirus of turkeys (HVT)-infected chick embryo fibroblasts (CEF) and from the MSB-1 cell line.     

Effect of native chicken interferon on MDV replication

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus. Its specific phosphorylated protein, pp38 has been implicated in MDV oncogenesis. In order to check whether the known anti-viral or anti-proliferative actions of interferon (IFN) are of importance in Marek's disease (MD), chicken embryo fibroblasts (CEFs) were infected with attenuated serotype-1 MDV strain CVI988, or with herpesvirus of turkeys (HVT). Different concentrations of native chicken IFN were added to the cell cultures, prior to their infection. After incubation, MDV plaques were counted. Analysis by flow cytometry for pp38 expression was performed by using three monoclonal antibodies (MAbs) and for HVT by using an anti-glycoprotein B (gB) MAb. Increasing IFN quantities caused a reduction in a stepwise manner of plaque numbers as well as a suppression of pp38 and gB expression in the CVI988- and HVT-infected cells, respectively.     

In ovo assay for Marek''s disease virus and turkey herpesvirus.

Marek's disease virus (MDV) and the turkey herpesvirus (HVT) may be assayed on the chorioallantoic membrane (CAM) of the chicken embryo after intravenous inoculation of chicken embryo fibroblasts (CEF) or chicken blood leukocytes infected with these viruses. Free HVT, MDV associated with Marek's tumor cells, and lymphoblastoid cell lines derived from Marek's tumors, may be assayed in the same way. The intravenous assay is quicker than the yolk sac assay and somewhat more sensitive than in vitro or conventional CAM assay after direct inoculation of the CAM. The optimal time for inoculation was day 10 of embryo incubation; therafter the log-10 CAM lesions decreased as a negative linear function of embryo age at the time of inoculation. The log-10 CAM lesions increased as a positive linear function of the time since inoculation. The optimal time for counts was day 5 after inoculation. The log-10 CAM lesions was a linear function of the log-10 cells in the inoculum; the slope was 1.0. Venous in ovo inoculation caused as increase in the weight of the spleen proportional to the number of CAM lesions. Repression of the splenomegaly, by prior X irradiation of the embryo, did not reduce the number of CAM lesions. Embryols from lines inbred for susceptibility to Marek's disease produced more CAM lesions than embryos from resistant lines. This difference did not depend on prior exposure of the mothers to MDV or HVT.     

Suppression and enhancement of mitogen response in chickens infected with Marek''s disease virus and the herpesvirus of turkeys.

The kinetics of phytohemagglutinin (PHA) response of peripheral blood lymphocytes from chickens infected with oncogenic Marek's disease (MD) virus (MDV) or nononcogenic herpesvirus of turkeys (HVT) was studied with a whole blood microassay. At about 7 days after inoculation, a depression in PHA response was observed in MDV-inoculated resistant line N or susceptible line 7(2) chickens and in HVT-inoculated line 7(2) chickens. All chickens initially regained their PHA responsiveness. Susceptible chickens that died of MD or developed MD lymphoma in later stages of virus infection showed a second severe depression in PHA response. No depression was observed in HVT-vaccinated chickens when challenged with MDV. The PHA response of MDV-inoculated chickens that survived MD, HVT-inoculated chickens, and HVT-vaccinated MDV-challenged chickens showed evidence of enhancement. The depression of PHA response was studied and was attributed to the suppressive effect of macrophages on T-cell response, a finding consistent with our previous studies on MDV suppression of PHA response.     

The expression of Marek's disease tumour-associated surface antigen in various avian species

Marek's disease tumour-associated surface antigen (MATSA) was detected on lymphoid cells from chickens infected with Marek's disease virus (MDV) or the herpesvirus of turkeys (HVT), from a chicken-quail hybrid infected with MDV, and from turkeys infected with the GA strain of MDV, but not on cells from turkeys infected with HVT or the HPRS-16 strain of MDV or from MDV-infected Japanese quails or bobwhite quails, despite the presence of lymphomas in some Japanese quails. Two complementary explanations are offered for these observations/Firstly, certain combinations of virus strain and host species may not result in malignant transformation although infection has occurred; secondly, if, as seems likely, MATSA is a modified host antigen, antigens on malignant cells arising in a foreign host may not be recognised by an antiserum prepared against MATSA of chicken origin.     

Development of a cell line system susceptible to infection with vaccine strains of MDV

Despite reliance on the need to continually prepare fresh cultures of chick embryo fibroblasts (CEFs) to make Marek's disease (MD) vaccines, MD vaccines are the most widely used vaccines in the poultry industry. Preparation of CEF's accounts for approximately 40% of the costs associated with producing MD vaccines. A significant reduction in MD vaccine production costs could be realized if a continuous cell lines were available for MD vaccine production. Recently, we reported development and characterization of a cell line system (OCL) that supports growth and replication of oncogenic serotype 1 Marek's disease virus (MDV). Here we report development of three cell line systems for production of MD vaccine. These cell lines support the growth and replication of attenuated serotype 1 MDV (CVI-OCL), serotype 2 MDV (SB1-OCL) and serotype 3 MDV (HVT-OCL). MDV is maintained in a stable state in the OCL cells and the infected cells can be continuously grown. The vaccines made from these cell lines are safe and protect White Leghorn chickens against challenge with very virulent serotype 1 MDV, similar to traditional vaccines made from CEF cells. These cell line systems can significantly reduce the costs associated with MD vaccine production. Furthermore, the increased stability of MDV and the potential for positive selection of recombinant MDV suggest that OCL will be ideal for production of more effective MDV vaccines using recombinant DNA technology.     

Comparative studies on Marek's disease virus and herpesvirus of turkey DNAs.

DNA of Marek's disease virus (MDV) was compared to that of herpes virus of turkey (HVT). Centrifugation of the two virus DNAs in neutral glycerol and CsCl density gradients showed that the MDV genome was slightly larger than that of HVT and that the buoyant density (1.705 g/ml) of MDV DNA in CsCl gradients was slightly lower than that (1.707 g/ml) of HVT DNA. MDV and HVT DNAs were digested with either EcoRI or HindIII restriction endonuclease and analysed by 0.5% agarose gel electrophoresis. The cleavage patterns of HindIII or EcoRI DNA digests of two strains of these two viruses showed general similarities between the strains, but not between MDV and HVT. However, a few fragments of EcoRI or HindIII digests of MDV DNA co-migrated with those of HVT DNA. DNA-DNA reassociation kinetics and DNA-RNA hybridization between the two viruses indicated that MDV and HVT DNAs share detectable homology, although it is less than 5%. The DNA of a HVT variant, which has lost the ability to protect chickens from Marek's disease, appeared similar to DNA of the vaccine strain in the size buoyant density and in its restriction endonuclease cleavage pattern.     

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.     

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.     

Effects of age at infection with serotype 2 Marek's disease virus on enhancement of avian leukosis virus-induced lymphomas

Inoculation of susceptible, 15I(5x)7(1) chickens with serotype 2 Marek's disease virus (MDV) at various ages had no influence on the development of avian leukosis virus (ALV)-induced viraemia or antibody in chickens infected with ALV and turkey herpesvirus (HVT) at hatch. However, the incidence of ALV-induced lymphoma (LL) was significantly higher in chickens infected with ALV and HVT at hatch and inoculated with serotype 2 MDV up to 6 weeks of age than in chickens receiving serotype 2 MDV at 8 to 10 weeks, uninoculated chickens, or chickens inoculated only with HVT. Metastatic LL in the viscera was more frequently observed in chickens inoculated with serotype 2 MDV at hatch and 2 weeks than in chickens inoculated at 6 weeks of age. In another experiment, chickens inoculated with serotype 2 MDV and HVT at hatch and infected with ALV at 2 or 4 weeks of age developed significantly higher incidence of LL than in uninoculated chickens or chickens inoculated only with HVT. The data suggest that enhancement of LL can occur in chickens infected with serotype 2 MDV within 6 weeks after infection with ALV at hatch. The data also suggest that infection of chickens with serotype 2 MDV at hatch may increase the rate of metastasis of LL and may interfere with age resistance to development of LL.     

Anti-viral immunity against Marek's disease virus infected chicken kidney cells

The mechanism of immunity conferred by herpesvirus of turkeys (HVT) was investigated using Marek's disease virus (MDV)-infected chicken kidney cell (CKC) cultures as target cells. Peripheral blood lymphocytes (PBL) from normal or HVT-vaccinated chickens killed MDV-infected target cells in the presence of anti-MDV or anti-HVT serum, whereas normal lymphocytes and HVT-sensitised lymphocytes themselves had no specific cytotoxic effect. Cell-mediated cytotoxicity of PBL from HVT-vaccinated chicken was exhibited specifically against HVT-infected CKC, and that of PBL from chickens inoculated with MDV was exhibited against MDV-infected CKC. From these results, it was concluded that antibody-dependent cellular cytotoxicity to MDV-infected cells may contribute to resistance to Marek's disease induced by HVT vaccination.     

Use of Marek's disease vaccines: could they be driving the virus to increasing virulence?

Marek's disease (MD) is an economically important neoplastic disease of poultry. MD almost devastated the poultry industry in the 1960s but the disease was brought under control after Marek's disease herpesvirus (MDV) was identified and vaccines were developed. This is the first effective use of an antiviral vaccination to prevent a naturally occurring cancer in any species. MDV infection has many effects. Initially causing a cytolytic infection in B-lymphocytes, MDV infects activated T-lymphocytes where it becomes latent. In susceptible chicken genotypes MDV transforms CD4+ lymphocytes, causing visceral lymphomas and/or neural lesions and paralysis. Fully productive infection and shedding of infectious virus only occurs in the feather-follicle epithelium. Vaccination of newly-hatched chicks with live vaccines has been widely used to successfully control MD since the early 1970s. However, vaccinated chickens still become infected and shed MDV. Vaccine breaks have occurred with regularity and there is evidence that the use of MD vaccines could be driving MDV to greater virulence. MD continues to be a threat and a number of strategies have been adopted such as the use of more potent vaccines and vaccination of the embryonic stage to provide earlier protection. Recombinant MD vaccines are useful vectors and are being exploited to carry both viral and host genes to enhance protective immune responses. The future aim must be to develop a sustainable vaccine strategy that does not drive MDV to increased virulence.     

Characterization of four very virulent Argentinian strains of Marek's disease virus and the influence of one of those isolates on synergism between Marek's disease vaccine viruses.

Isolates of Marek's disease virus (MDV) from vaccinated flocks in Argentina were characterized as very virulent (vv) and very virulent plus (vv+) strains. Experimental infection with these viruses caused a high incidence of Marek's disease in both resistant N-2a line and susceptible P-2a line birds. Vaccine viruses from each of the three Marek's disease viral serotypes were evaluated alone and in various combinations for protection against challenge with a vvMDV called NULP-1. Vaccination of P-2a birds with HVT did not protect satisfactorily against any of the vv and vv+MDV strains isolated. However, CVI988/Rispens vaccine alone or combined with serotype 2 and/or serotype 3 vaccine strains enhanced protection significantly against NULP-1. Serotype 2 plus serotype 3 vaccines also provided significant protection when challenged with this strain. This is one the first reports of the occurrence of vvMDV and vv+MDV in Argentina and Latin America. It is also a preliminary evaluation of the synergistic protective effect of different vaccine viruses with local MDV strains. However, further studies are needed to evaluate the real role of these and other Marek's disease isolates in 'vaccination failures' and the influence of serotype and virus strain on synergism between Marek's disease vaccine viruses.     

A recombinant fowlpox virus vaccine expressing glycoprotein B gene from CVI988/Rispens strain of MDV: protection studies in different chickens

Recombinant fowlpox virus (rFPV) was constructed to express glycoprotein B (gB) gene from CVI988/Rispens strain of Marek's disease virus (MDV). The rFPV-gB/R alone and in combination with herpesvirus of turkey (HVT) preparations were evaluated for their protective efficacy against challenge with very virulent MDV strains Md5 and RB1B in different chickens. The rFPV-gB/R alone induced protection comparable to that by HVT vaccines in both Ab- SPF chickens and Ab+ production chickens. Significant protective synergism was observed in one of these two types of commercial production chickens when rFPV-gB/R was combined with HVT of either cell-associated or cell-free preparations. Immunogenesis studies showed that rFPV-gB/R, just like conventional vaccines, significantly reduced the level of viremia, splenocytes infection and feather follicle shedding of challenge virus in vaccinated chickens.     

Pathogenicity and Antigenicity of Clones from Strains of Marek's Disease Virus and the Herpesvirus of Turkeys

Virus was extracted by filtration from chicken embryo fibroblast cultures infected with the JM, high passage JM(JMHP), GA, and RPL39 strains of Marek's disease virus (MDV) and from the herpesvirus of turkeys (HVT) and purified by cloning. The plaques produced by clones of HVT, JMHP, and other MDV strains differed in morphology from one another. Clones of MDV varied greatly in pathogenicity for chickens, but JMHP and HVT were nonpathogenic. Two pathogenic clones of JM virus and a clone of JMHP virus lacked the A precipitin antigen present in all other clones tested. All clones had at least one B antigen in common. HVT and MDV clones with and without the A precipitin antigen could be distinguished from each other by the indirect fluorescent antibody test. Changes in virus-host cell relationships, loss of pathogenicity, and loss of the A antigens were independent events.     

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.     

Heat-shock protein 70 is associated with the entry of Marek's disease virus into fibroblast

Literature pertaining to the interactions between Marek's disease virus (MDV) entry-related glycoproteins and corresponding receptors is still limited. Results from a Western blot analysis of cellular proteins for virus receptors and co-immunoprecipitation suggest that heat shock protein 70 (HSP70) is a potential cellular receptor for MDV glycoprotein gH. Plaque inhibition assays confirm the involvement of HSP70 in the early stages of MDV entry into chicken embryo fibroblasts (CEF). The present work supports that HSP70 is implicated in the MDV entry process by binding to gH, and enhances the understanding of multifunctional HSP70 and the MDV infection process.