Karel A. Schat

Characterisation of two highly oncogenic strains of Marek's disease virus 1 2

The RB-1B and ALA-8 strains of Mareks disease (MD) virus, which were isolated from chickens with MD and which had been vaccinated with the herpesvirus of turkeys (HVT), were evaluated for their oncogenic potential in genetically susceptible (P-line) and resistant (N-line, PDRC) chickens. RB-1B and ALA-8 were both highly oncogenic, causing a high incidence of MD in both susceptible and resistant birds. Vaccination of P-line birds with SB-1 or HVT did not protect satisfactorily against RB-1B. However, a bivalent vaccine consisting of SB-1 and HVT enhanced protection significantly. HVT alone, and the bivalent vaccine, protected PDRC and N-line chickens well against RB-1B, but SB-1 was less protective in PDRC birds. HVT protected equally well against challenge with ALA-8 and the standard JM-10 strain. Differences in the pathogenesis of viral infection could not be detected among ALA-8, RB-1B and JM-10 between 4-7 days post-infection (d.p.i.). However, after d.p.i. 12 RB-1B caused significantly higher levels of viral internal antigen and virus isolation rates than did JM-10 in the same genetic strain. Prior vaccination prevented the expression of ALA-8 at 5 and 20 d.p.i., but not that of RB-1B. Pathogenetic events such as expression of VIA or level of virus infection appeared to be directly related to the level of protection observed in challenged birds.

Expression of cytokine genes in Marek's disease virus-infected chickens and chicken embryo fibroblast cultures

The role of cytokines in the pathogenesis and immunity of Mareks disease (MD), a herpesvirus‐induced T‐cell lymphoma in chickens, is poorly understood. Two different experiments were used to examine the potential role of particular cytokines in the pathogenesis and immune responses of MD. First, chicken embryo fibroblasts (CEF) were stimulated with lipopolysaccharide (LPS) and/or recombinant chicken interferon‐γ (rChIFN‐γ) and used to develop techniques for examining transcription of IFN‐α, IFN‐γ, inducible nitric oxide synthase (iNOS), interleukin (IL)‐1β, IL‐2, IL‐6 and IL‐8 by reverse transcription–polymerase chain reaction (RT–PCR). Addition of LPS and/or rChIFN‐γ resulted in the up‐regulation of mRNA for iNOS, IL‐1β and IL‐6, while IFN‐γ was up‐regulated by LPS alone. IL‐2 was down‐regulated by the treatments. Second, to determine the effects of Mareks disease herpesvirus (MDV) infection on cytokine transcription in vivo, chickens were infected with MDV at 21 days of age and examined at 7 days post‐infection (p.i.) (exp. 1) or were infected with MDV at 1 day of age and examined from 3 to 15 days p.i. (exp. 2). In MDV‐infected chickens, IFN‐γ transcription was up‐regulated as early as 3 days p.i. until the termination of the experiment at 15 days p.i., while iNOS and IL‐1β were up‐regulated between 6 and 15 days p.i. Infection of 1‐day‐old chicks increased levels of mRNA for IFN‐γ and iNOS between 16‐ and 64‐fold at 9 days p.i. These results suggest that IFN‐γ and iNOS may play an important role in the pathogenesis of MD.

Inhibitory Effects of Nitric Oxide and Gamma Interferon on In Vitro and In Vivo Replication of Marek's Disease Virus

ABSTRACT The replication of Mareks disease herpesvirus (MDV) and herpesvirus of turkeys (HVT) in chicken embryo fibroblast (CEF) cultures was inhibited by the addition ofS-nitroso-N-acetylpenicillamine, a nitric oxide (NO)-generating compound, in a dose-dependent manner. Treatment of CEF culture, prepared from 11-day-old embryos, with recombinant chicken gamma interferon (rChIFN-γ) and lipopolysaccharide (LPS) resulted in production of NO which was suppressed by the addition ofNG-monomethyl l-arginine (NMMA), an inhibitor of inducible NO synthase (iNOS). Incubation of CEF cultures for 72 h prior to treatment with rChIFN-γ plus LPS was required for optimal NO production. Significant differences in NO production were observed in CEF derived from MDV-resistant N2a (major histocompatibility complex [MHC],B 21 B 21) and MDV-susceptible S13 (MHC,B 13 B 13) and P2a (MHC,B 19 B 19) chickens. N2a-derived CEF produced NO earlier and at higher levels than CEF from the other two lines. The lowest production of NO was detected in P2a-derived CEF. NO production in chicken splenocyte cultures followed a similar pattern, with the highest levels of NO produced in cultures from N2a chickens and the lowest levels produced in cultures from P2a chickens. Replication of MDV and HVT was significantly inhibited in CEF cultures treated with rChIFN-γ plus LPS and producing NO. The addition of NMMA to CEF treated with rChIFN-γ plus LPS reduced the inhibition. MDV infection of chickens treated withS-methylisothiourea, an inhibitor of iNOS, resulted in increased virus load compared to nontreated chickens. These results suggest that NO may play an important role in control of MDV replication in vivo.

Reproductive effort reduces long-term immune function in breeding tree swallows (Tachycineta bicolor)

We examined whether strategies of reproductive allocation may reduce long-term immunocompetence through the effects of manipulated effort on secondary or acquired immunity. We tested whether increased reproductive effort leads to reduced immune function and survival by manipulating brood size in tree swallows (Tachycineta bicolor) and exposing breeding females to a primary and secondary exposure of sheep red blood cells to elicit a humoral immune response. Females raising enlarged broods produced fewer secondary antibodies than did females raising control or reduced broods. Most importantly, individuals with high secondary responses were more likely to survive to breed 3 years after brood manipulations, suggesting that differences in disease susceptibility may be caused by trade–offs in reproductive allocation. We also found that individual quality, measured by clutch initiation date, mediated the effects of brood manipulations, with higher–quality birds showing a greater ability to deal with increases in effort.

RELATIONSHIP BETWEEN THE IMMUNOSUPPRESSIVE POTENTIAL AND THE PATHOTYPE OF MAREK'S DISEASE VIRUS ISOLATES

Isolates of Mareks disease virus (MDV) representing three pathotypes of differing virulence were compared for relative immunosuppressive properties in genetically susceptible P2a-strain and genetically resistant N2a-strain chickens. Criteria of immunosuppression were 1) persistence of early cytolytic infection (i.e., a delay or failure to enter latency) in lymphoid organs, 2) atrophy of the bursa of Fabricius and thymus as measured by organ weight proportional to body weight at 8 and 14 days postinfection (DPI), and 3) histopathologic evidence of necrosis and atrophy in lymphoid organs. No significant differences in infection level were observed among the pathotypes during the early (4-5 DPI) period of infection. However, the extent of persistent cytolytic infection at 7-8 DPI, based on numbers of tissues positive and mean scores in immunofluorescence tests, was greater (P < 0.05) for three isolates (RK1, 584A, 648A) in the highest virulence pathotype (very virulent-plus MDV [vv + MDV]) than for two isolates (JM16, GA5) in a lower virulence (virulent MDV [vMDV]) pathotype. Results from two isolates (RB1B, Md5) classified in the intermediate very virulent pathotype (very virulent MDV [vvMDV]) fell between those from the other two pathotypes. Similarly, there was a stepwise effect of viral pathotype in which the vv + MDV isolates caused the most severe damage to lymphoid organs in terms of atrophy (relative organ weights) and histopathologic changes. Organs from chickens infected with vv + MDVs showed little recovery between 8 and 14 DPI. The vMDV isolates caused the least severe damage, and lymphoid organs showed a significant return toward normal by 14 DPI; vvMDV isolates induced intermediate degrees of atrophy and recovery. The same pattern of relationship between virulence pathotype and degree of bursal and thymic atrophy was also observed in genetically resistant N2a chickens. These results suggest that the degree of immunosuppression is linked to virulence and that a simple measure of atrophic changes (relative organ weights) in the bursa of Fabricius and thymus might be useful in determining the pathotype classification of new MDV isolates. The basis for differences in immunosuppressive potential of MDV isolates needs further clarification.

Specific and nonspecific immune responses to Marek's disease virus

Mareks disease (MD) virus (MDV) has provided an important model to study immune responses against a lymphoma-inducing herpesvirus in its natural host. Infection in chickens starts with a lytic infection in B cells, followed by a latent infection in T cells and, in susceptible birds, T cell lymphomas develop. Non-specific and specific immune responses are important for the control of virus infection and subsequent tumor development. Interferon-gamma and nitric oxide are important for the control of virus replication during the lytic phase of infection and are also important to prevent reactivation of MDV replication in latently infected and transformed cells. Cytotoxic T cells (CTLs) are the most important of the specific immune responses in MDV. In addition to antigen-specific CTL against MDV proteins pp38, glycoprotein B (gB), Meq, and ICP4, ICP27-specific CTL can also be detected as early as 6 to 7 days post infection. The epitope for gB recognized by CTLs from P2a (MHC: B(19)B(19)) chickens has been localized to the Eco47III-BamHI (nucleotides 1515-1800) fragment. A proposed model for the interactions of cytokines and immune responses as part of the pathogenesis of MD is discussed.

Attenuation of Marek's Disease Virus by Deletion of Open Reading Frame RLORF4 but Not RLORF5a

ABSTRACT Mareks disease (MD) in chickens is caused by the alphaherpesvirus MD virus (MDV) and is characterized by the development of lymphoblastoid tumors in multiple organs. The recent identification and cloning of RLORF4 and the finding that four of six attenuated strains of MDV contained deletions within RLORF4 suggested that it is involved in the attenuation process of MDV. To assess the role of RLORF4 in MD pathogenesis, its coding sequence was deleted in the pRB-1B bacterial artificial chromosome clone. Additionally, RLORF5a was deleted separately to examine its importance for oncogenesis. The sizes of plaques produced by MDV reconstituted from pRB-1BΔRLORF5a (rRB-1BΔRLORF5a) were similar to those produced by the parental pRB-1B virus (rRB-1B). In contrast, virus reconstituted from pRB-1BΔRLORF4 (rRB-1BΔRLORF4) produced significantly larger plaques. Replication of the latter virus in cultured cells was higher than that of rRB-1B or rRB-1BΔRLORF5a using quantitative PCR (qPCR) assays. In vivo, both deletion mutants and rRB-1B replicated at comparable levels at 4, 7, and 10 days postinoculation (p.i.), as determined by virus isolation and qPCR assays. At 14 days p.i., the number of PFU of virus isolated from chickens infected with rRB-1BΔRLORF4 was comparable to that from chickens infected with highly attenuated RB-1B and significantly lower than that from rRB-1B-infected birds. The number of tumors and kinetics of tumor production in chickens infected with rRB-1BΔRLORF5a were similar to those of P2a chickens infected with rRB-1B. In stark contrast, none of the chickens inoculated with rRB-1BΔRLORF4 died up to 13 weeks p.i.; however, two chickens had tumors at the termination of the experiment. The data indicate that RLORF4 is involved in attenuation of MDV, although the function of RLORF4 is still unknown.

Infection with chicken anaemia virus impairs the generation of pathogen-specific cytotoxic T lymphocytes

Infection with chicken anaemia virus (CAV), a circovirus, can result in immunosuppression and subsequent increased susceptibility to secondary infections. This is the first report of impairment of pathogen‐specific cytotoxic T lymphocytes (CTL) after natural and experimental infection of chickens with CAV and Mareks disease virus (MDV) or reticuloendotheliosis virus (REV). MDV‐ and REV‐specific CTL were generated at 7 days post infection by 9–30‐day‐old‐chickens that were positive for maternal antibodies to CAV at 9–17 days of age. Replication of CAV could not be demonstrated in these chickens using quantitative real‐time polymerase chain reaction (PCR) and reverse transcriptase (RT)–PCR assays. In contrast, REV‐specific CTL failed to develop when chickens negative for maternal antibodies at 9–17 days of age were infected. Infection with CAV at 45 days of age after CAV maternal antibodies had waned also caused a decreased REV‐specific CTL response. In these chickens increased levels of CAV DNA of up to 107 copy numbers per µg DNA and increased relative transcript levels of CAV by up to a factor of 106 were detected by quantitative real‐time PCR and RT–PCR. Interleukin (IL)‐1β and IL‐2 mRNA levels were not significantly affected by CAV infection at 7 or 14 days p.i. Similar assays for interferon‐γ (IFN‐γ) transcripts demonstrated a 10‐fold increase in IFN‐γ mRNA levels at 7 days post infection following REV or REV + CAV infection, while CAV alone caused a two‐ to fourfold increase. These results show a strong link between CAV antibody status, CAV replication, and the ability to generate REV‐specific CTL. It is likely that the immunosuppressive effects of subclinical infection have previously been underestimated.

Influence of Genetic Resistance of the Chicken and Virulence of Marek's Disease Virus (MDV) on Nitric Oxide Responses After MDV Infection

SUMMARY. Nitric oxide (NO), a free radical produced by the enzyme NO synthase (NOS), is a potent antiviral agent in addition to having immune regulating functions. Recently, it was reported that chickens resistant (N2a, MHC: B21B21) to the development of Mareks disease (MD) had a greater potential to produce NO than MD-susceptible chickens (P2a, MHC: B19B19). This difference was shown by measuring NO levels in chick embryo fibroblast cultures obtained from these chickens after treatment with lipopolysaccharide and recombinant chicken interferon-gamma (IFN-γ). To extend these results, the levels of NO in blood plasma from N2a and P2a chickens inoculated with the nonattenuated JM-16 strain of MD virus (MDV) were examined. In four out of five experiments, N2a chickens had increased NO levels at 7 days postinoculation (DPI). In contrast, P2a chickens challenged with JM-16 had a significant increase in NO in only one of four experiments, and in that experiment the increase was delayed (10 DPI) compared with N2a chickens. Attenuation abrogated MDV-induced NO in chickens. Inoculation with MDV strains ranging from mild to very virulent plus showed that the more virulent strains induced the highest level of NO in blood plasma, suggesting a role of NO in the pathogenesis of MD with more virulent strains. On the basis of quantitative real-time reverse transcription–polymerase chain reaction (RT-PCR) assays for analysis of mRNA expression, IFN-γ does not appear to be the primary inducer of inducible (i)NOS gene expression during MDV infection. iNOS gene expression and NO production are mediated during the cytolytic phase of MDV infection on the basis of real-time RT-PCR assays with primers specific for glycoprotein B, a late gene expressed only during the cytolytic phase of MDV infection. These findings implicate NO as a factor potentially involved in increasing virulence of MDV, possibly through immune suppression.

Identification of the chicken anemia agent, reproduction of the disease, and serological survey in the United States.

An agent with antigenic, physicochemical, and pathological characteristics of chicken anemia agent (CAA) was isolated from broiler chickens and was designated chicken infectious anemia (CIA)-1. CIA-1 was resistant to chloroform treatment and passed through 50-nm-diameter-pore membranes. When inoculated in embryonally bursectomized and/or intact chickens, CIA-1 produced signs and lesions characteristic of CIA: low hematocrit values, pale bone marrow, thymic and bursal atrophy, and enlarged liver. Microscopic lesions were a reflection of macroscopic observations. When injected into 4-week-old chickens CIA-1 induced antibodies against the Cux-1 CAA isolate. In CsCl, CIA-1 had a density of 1.36 g/ml. Antibodies against CAA were found in breeder and commercial chicken flocks from Arkansas, Connecticut, Georgia, Kansas, Maine, Michigan, New York, and Pennsylvania. The age of these flocks ranged from 10 to 78 weeks.