Demonstration of Marek's disease tumor-associated surface antigen in chickens infected with nononcogenic Marek's disease virus and herpesvirus of turkeys.

The presence of Marek's disease tumor-associated surface antigen (MATSA) was demonstrated on spleen cells from P-line chickens inoculated 5--6 days earlier with herpesvirus of turkeys and SB-1 (a nononcogenic Marek's disease virus). Antisera against MATSA expressed on five Marek's disease lymphoblastoid cell lines were able to recognize the MATSA present on SB-1-infected spleen cells. No viral membrane antigens and only a low incidence of viral internal antigens could be demonstrated.

Characterization of an apparently nononcogenic Marek's disease virus.

A new isolate of Marek's disease virus (MDV) was described. This virus, SB, and a clone, SB-1, differed from pathogenic isolates in in vitro growth characteristics as described for other apathogenic isolates. Serologically, as with other apathogenic isolates, SB could be distinguished from pathogenic MDV and the avirulent turkey herpesvirus. SB failed to induce lesions characteristic of Marek's disease (MD) during a 6- to 11-week experimental period. Also, SB was nononcogenic in immunosuppressed chickens or in chickens inoculated with this virus in ovo. However, under those conditions, SB caused a cytolytic infection. The term "nononcogenic" rather than "apopathogenic" was therefore proposed to classify this and similar isolates. SB-1 protected chickens against challenge with either virulent MDV or the non-virus-producing MD tumor transplant, JMV. Possible mechanisms of protection are discussed.

Pathogenesis of Marek's disease: early appearance of Marek's disease tumor-associated surface antigen in infected chickens.

Marek's disease tumor-associated surface antigen (MATSA) appeared in different lymphoid tissues of P-line chickens soon after they were infected with BC-1 strain of Marek's disease virus. MATSA-bearing cells first appeared in spleens by 5 days post infection (PI) and were observed through a 21-day experimental period at mean levels varying from 3.8 to 21.9% of the total cells examined. Lower percentages of MATSA-bearing cells were observed in the thymus, in the bursa of Fabricius, among peripheral blood lymphocytes, and among bone marrow cells beginning 7 days PI. The antigen was not detected on normal lymphocytes from chickens or chicken embryos, nor was it detected on cultured chicken embryo fibroblasts infected or transformed by avian RNA tumor viruses.

Latent infections with Marek's disease virus and turkey herpesvirus.

The number of plaque-forming units (PFU) from lymphocytes latently infected with oncogenic (GA-5) or nononcogenic (SB-1) Marek's disease virus (MDV) was decreased after complement lysis with antisera against thymus-derived lymphocytes or bursa-derived lymphocytes; an additive effect with dual treatment was observed. Complement lysis with antisera against Marek's disease tumor-associated surface antigen (putative tumor antigen) had little effect, which suggests an absence of the antigen on latently infected cells. None of the serum complement treatments affected the number of PFU from lymphocytes infected with turkey herpesvirus (HVT). No viral antigens were detected in spleen lymphocytes when removed from infected birds. After 42-72 hours of incubation, many cultured spleen cells from MDV-infected birds (JM-10, GA-5, and SB-1 isolates) contained viral antigen, whereas very few if any cultured cells from HVT-infected birds were positive. These data suggest that the nature of the latent infections with MDV and HVT may differ significantly.

Characterization of Marek's disease virus-infected lymphocytes: discrimination between cytolytically and latently infected cells.

Leukocyte suspensions derived from genetically Marek's disease (MD)-resistant N-line and MD-susceptible P-line chickens were fractionated at various times after exposure to the JM-10 clone of MD virus. At 3 and 5 days post exposure (DPE), during the productive-restrictive (cytolytic) phase, most infected spleen and thymus leukocytes were found to be low-density, nylon wool-adherent cells that possessed Fc receptors and surface Ia and IgM and were depleted by carbonyl iron treatment. This was true for leukocytes derived from N-line as well as those from P-line chickens. In contrast, most infected spleen cells derived from P-line chickens during the latent phase (i.e., after 7 DPE) were not found to have the above characteristics, with one exception: Ia antigen was demonstrated on the surface of latently infected cells. From these experiments it was concluded that the principal targets of the cytolytic JM-10 infection are B-cells, whereas the subsequent latent infection was found mostly in non-B-lymphocytes.

Surface antigens on Marek's disease lymphoblastoid tumor cell lines.

A total of 32 Marek's disease virus (MDV)-induced cell lines, 1 avian leukosis virus-induced cell line, and 1 reticuloendotheliosis virus (REV)-induced lymphoblastoid cell line were investigated for the presence of Fc receptors, surface IgM, and Ia-like antigen. Surface IgM and Fc receptors could not be demonstrated on any of the cell lines. Ia-like antigen was detected on all MDV-induced cell lines and on the 1 REV-induced cell line. Ia-like antigen was unrelated to other antigens reported to be present on Marek's disease tumor cells. It was also unimportant as a target antigen for MDV-related, allogeneic cell-mediated cytotoxicity. The densities of the cells of 5 MDV-induced cell lines were established on continuous Percoll gradients. Most cells had densities between 1.05 and 1.06 g/ml.

In vitro infection of lymphocytes with Marek's disease virus.

Suspension cultures of splenic lymphocytes, incubated at 41 degrees C, became infected with Marek's disease virus (MDV) following exposure to a) other infected lymphocytes, b) infected chicken kidney monolayer cultures, or c) cell-free MDV. Both viral antigen expression and virus isolation could be demonstrated after more than 40 passages made by the addition of fresh spleen cells at 2- to 3-day intervals. Susceptibility of spleen cells from bursectomized chickens was markedly lower than that of cells from intact birds. Furthermore, when spleen cell suspensions were depleted of cells having characteristics of bursa-derived cells, e.g., those with surface IgM, Fc receptors, or ability to adhere to nylon wool, the susceptibility of the cell suspension was diminished. Enrichment of the suspension with cells having those features enhanced overall susceptibility. The target cells for virus infection in vitro also were shown to be nonphagocytic, to be of low or medium density, and to bear Ia-like antigen. In vitro susceptibility to infection of spleen cells did not correlate with the genetic susceptibility of the donor to Marek's disease.

Expression of a putative tumor-associated surface antigen on normal versus Marek's disease virus-transformed lymphocytes.

Various avian tumor cell lines and normal spleen cells from 3 genetic strains of specific-pathogen-free (SPF) chickens were examined for expression of Marek's disease (MD) tumor-associated surface antigen (MATSA). Two anti-MATSA monoclonal antibodies (RPH 6 and EB 29) and a rabbit anti-MATSA antiserum were used in indirect fluorescent antibody tests, and cells were examined by fluorescence microscopy and with a fluorescence-activated cell sorter (FACS). Less than 5% MATSA-positive cells were observed in 2 non-MD tumor cell lines (LSCC-RP 9 and RECC-CU 60) with RPH 6, but 7-82% positive cells were observed with EB 29 or the rabbit antiserum. Five MD tumor cell lines (MDCC-CU 2, -CU 14, -CU 25, -CU 32, and -CU 41) had 12-72% positive cells detected with one or both monoclonals and 31-99% positive cells detected with the rabbit antiserum. Over 90% of cells in all MD lines were la and T3 positive, while values for the same parameters in LSCC-RP 9 were 100 and 3% and for RECC-CU 60, 48 and 51%, respectively. Evidence for cell-cycle-dependent expression of MATSA on MDCC-CU 2 was obtained from cell sorting experiments with the FACS and from examination of the MATSA-staining characteristics of 3 clones derived from the parent culture. Less than 5% MATSA-positive cells were observed in uncultured spleen cells from SPF chickens or in spleen cells stimulated for 48 hours with concanavalin A or phytohemagglutinin-M. However, with one exception, 10-53% of normal spleen cells were MATSA positive with RPH 6, after stimulation by mitogen for 24 or 48 hours followed by maintenance in conditioned medium (CM) for various times or after culture directly in CM for 3 days. More limited experiments with rabbit anti-MATSA antiserum yielded 55-85% MATSA-positive cells. From 60 to 97% of these MD virus-free, MATSA-positive cells were la-positive; and, in 2 cases, 89 and 90% were T3 positive.

Cell-specific antiviral activity of 1-(2-fluoro-2-deoxy-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC) against Marek's disease herpesvirus and turkey herpesvirus.

Three new fluoroarabinosylpyrimidine nucleosides (FIAC, FIAU and FMAU) were tested for in vitro activity against oncogenic and nononcogenic strains of Marek's disease virus (MDV) and herpesvirus of turkeys (HVT). Marek's disease is a herpesvirus-induced lymphoma in chickens. Nononcogenic strains of MDV and HVT can protect against this disease. All viruses were inhibited by 1 microM of these drugs in chick kidney cell (CKC) cultures, but only FMAU and FIAU were active in chicken embryo fibroblast (CEF) and spleen cell cultures. It was determined that whereas CKC produced the enzyme 2'-deoxycytidine-deaminase which is needed to deaminate FIAC to FIAU, CEF were devoid of this enzyme activity. In addition, the deaminase inhibitor 3,4,5,6-tetrahydrouridine prevented the antiviral activity of FIAC in CKC. FMAU was not active against two Marek's disease-derived lymphoblastoid tumor cell lines.

Stages of Marek's disease virus latency defined by variable sensitivity to interferon modulation of viral antigen expression.

Cytokines in conditioned medium can suppress expression of viral internal antigens (VIA) in lymphocytes latently infected with Marek's disease virus. In the present study, conditioned media produced by spleen cells stimulated with concanavalin A or by mixed-lymphocyte reaction had significantly greater (P < 0.05) VIA-suppressive activity with lymphocytes harvested from birds at 14 days post infection than with those collected at 7 days. This finding defines two stages during the latent period in which sensitivity of lymphocytes to cytokine modulation of viral expression differs. Suppression involved proteins representing immediate-early, early and late viral antigens. Physico-chemical characterization of the suppressive factor in conditioned medium was consistent with that expected of interferon. Indeed, natural interferon prepared from avian reovirus-exposed chicken embryo cells, and recombinant chicken interferon, both mimicked the activity of conditioned medium and were more suppressive with lymphocytes from the later stage of latency.

Control of avian encephalomyelitis: a historical account.

Avian encephalomyelitis control methods were not developed until the 1950s although the disease had been discovered and described over 20 yr earlier. Inability to transmit the infection by other than intracerebral inoculation, lack of suitable immunologic methods, the unknowing use of immune chickens or embryos for experimental studies, and reliance on a highly adapted strain of virus rather than fresh field isolates were the main reasons for a general lack of progress. In the absence of supportive experimental data, at least two commercial breeding organizations turned to the use of a crude chicken brain-propagated virus for vaccination of breeder replacement flocks in the 1950s. This control procedure turned out to be practical and efficacious. Development of suitable embryo infection methods and immunologic tests and the chance finding that antibody-free flocks were essential for experimental studies led to the development of embryo-susceptibility tests to identify immune breeder flocks and formed the basis for another commercially applied control program, the testing and selection of only immune flocks for hatching purposes. The application of the new testing methods coupled with a switch from the adapted Van Roekel strain of virus to fresh field isolates for experimentation resulted in a rapid unraveling of the epizootiology and pathogenesis of the disease and also to the development of a safe and effective vaccine that was licensed for administration to breeder replacements in 1962.

Papovavirus infection in hand-fed parrots: virus isolation and pathology.

Papovavirus infection was diagnosed in 44 parrots of at least 18 species exclusive of the budgerigar (Melopsittacus undulatus). The birds were 14 days to 4 months old and had been removed from parental care and hand-fed as nestlings. The birds had been unexpectedly found dead after having evidenced no premonitory signs of illness, or they died following a short (12-to-48-hour) period of lassitude and anorexia. In most cases, necropsies revealed pallor, multiple hemorrhages, splenomegaly, and hepatomegaly with multifocal necrosis. Histological lesions included multifocal to diffuse hepatic necrosis that spared the periportal hepatocytes, karyomegaly of splenic reticuloendothelial cells and cells in other tissues, membranous glomerulopathy, and necrosis of bursal medullary lymphocytes. Papovaviruses were isolated from two cases, and papovavirus infection was confirmed in 27 of the birds by the fluorescent-antibody test using a conjugate against a papovavirus isolated from a budgerigar.

Marek's disease tumor-associated surface antigen (MATSA) in resistant versus susceptible chickens.

The appearance of MATSA-bearing cells in the spleens of genetically susceptible (P-line) and resistant (N-line) chickens followed similar patterns at 4 and 6 days postinoculation (PI), with 4 to 10% of the cells positive in indirect fluorescent-antibody tests. Levels of MATSA-positive cells at 10, 14, and 21 days PI continued at 6-8% in P-line birds but dropped from about 8% to below 1 or 2% in N-line birds. This pattern was seen also in virus-isolation rates from the same spleen samples. Viral internal antigens (VIA) were seen equally and with decreasing incidence in both groups at 4-10 days PI. VIA were not detected at 14 days but reappeared at 21 days, with higher levels in P-lines than inN-lines. It was concluded that genetic resistance to Marek's disease is not related to events which result in production of the putative tumor antigen, MATSA, but rather to host-controlled factors which terminate those events.

In vitro isolation, propagation, and characterization of duck hepatitis virus type III.

The in vitro isolation, propagation, and characterization of duck hepatitis virus Type III (DHV-III), is described. This virus, which is serologically distinct from the classical (Type I) DHV, replicated in liver and kidney cell cultures of duck origin. Replication was limited in chicken and quail kidney and duck embryo fibroblast cultures. It did not replicate in a variety of other cell cultures of avian or mammalian origin. The virus was grown successfully in embryonating eggs of ducks, but not of chickens. DHV-III passed through a 50-nm membrane filter, was stable at pH 3.0 and resisted treatment with 5% chloroform. Virus growth was not inhibited by treatment with 5-iodo-2-deoxyuridine. Electron-microscope examination revealed crystalline arrays in the cytoplasm; virus particles had cubic symmetry, and were about 30 nm in diameter. By these properties, this virus can be classified as a member of the picornavirus group.

Adenoviruses of chickens: serologic groups.

By literature review and experimental studies, 57 adenovirus isolants from chickens were grouped into 10 distinct serotypes. In plaque-reduction tests, 20 antibody units were reacted with 32-320 plaque-forming units of virus. Viruses that were neutralized (80% or greater reduction in titer) by a given serum were considered to belong to that serotype. Used to distinguish among serotypes were their antisera. One-way neutralization occurred in only two instances; in both cases it was traced to contamination of the virus inocula used to induce specific antiserum. Recloning corrected the problem. Of the 10 serotypes described, all are known to exist in the United States, 7 were found in Northern Ireland, and at least 6 have been shown to occur in Japan.

Proliferation of chicken lymphoblastoid cells after in vitro infection with Marek's disease virus.

Activated, thymus-derived (T) lymphoblasts were exposed to Marek's disease virus and cultivated in attempts to induce in vitro transformation. After 9 to 15 days, colonies or small clusters of proliferating lymphoblasts were observed in cultures from three of a total of 122 attempts. These developed into proliferating cell cultures that resembled conventional Marek's disease (MD) lymphoblastoid cell lines in terms of growth characteristics and morphology. All proliferative cultures were unusual in that 1) the expression of viral internal antigens consistently or periodically was very high (up to 30% of all cells) and 2) the cells deteriorated and/or proliferation ceased in all cases after culture periods of 45-176 days. The proliferative cultures were all characterized as CD2+ and CD3+, Ia-bearing T cells; one was CD4+/CD8- and TCR2+, the other two were CD4-/CD8- and TCR1+. The latter two are the only cultures of MD-infected cells known to be TCR1+.

In vitro inactivation of cell-free Marek's disease herpesvirus by immune peripheral blood lymphocytes.

The effect of peripheral blood lymphocytes (PBL) on cell-free Marek's disease virus (MDV) was studied in vitro in PBL from intact or embryonal bursectomized (Bx) chicks infected with the apathogenic SB-1 isolate of MDV. The PBL were harvested purified and were mixed, 200 or 2,000 to 1, with cell-free GA-5 of MDV. Virus survival was assayed after incubation for 1 hour at 36 C by the standard assay for cell-free MDV. PBL from intact but not Bx chickens were able to inactivate virus. When the effective suspension of PBL was depleted of macrophages, inactivation no longer occurred, suggesting that it resulted from cooperation between macrophages and B-cells.

Pathologic and serologic characterization of a variant of duck hepatitis type I virus.

A duck hepatitis virus (DHV), isolated from ducks on a farm in Virginia in 1963, was shown to be only partially related to DHV type I (DHV-I) in cross-neutralization and in cross-protection tests. The virus, named DHV-Ia, apparently is a serologic variant of DHV-I; both viruses are serologically distinct from DHV type III. Pathologic responses to DHV-Ia were similar to those described for DHV-I infection.

An adenovirus survey of poultry flocks during the growing and laying periods.

Chicken adenoviruses were isolated from asymptomatic chickens on each of 7 farms tested; a majority of them induced cytopathology by the second serial passage. Although adenoviruses were isolated from chickens ranging in age from 8 to 34 weeks, the highest isolation rate was from those 8 to 14 weeks. Eight serotypes (1, 2, 4, 5, 6, 7, 8, and 9) were identified in a relatively small geographic area; serotypes 1, 4, 7, and 9 were isolated most frequently. Multiple serotypes were found on 6 of 7 farms, with 1 farm having 4 serotypes identified. Repeat isolations of the same serotypes were noted on 2 farms. The pattern of virus isolations was generally related to age but varied according to farm. Although new serotypes kept appearing, even after birds came into lay, no clinical problems were associated with them. The incidence of precipitin reactors tended to be low (10-40%) in immature birds but reached 80% or more after sexual maturity. The "antiserum pool" modification of the VN test was found to be accurate and less cumbersome than the conventional procedure for typing isolants.

Characterization of paramyxo-, herpes-, and orbiviruses isolated from psittacine birds.

Isolates of paramyxo-, herpes-, and orbiviruses from psittacine birds were characterized in the course of studies in cell cultures. The LBD-1 isolate, from a lovebird, was grown in chick kidney (CK) cells. It had the properties of a paramyxovirus but was found tobe serologically distinct from known avian paramyxoviruses. This virus was pathogenic for Japanese quail but not for young chickens or budgerigars. RSL-1 and -2, isolated from diseased rosellas, were propagated in chicken embryo fibroblasts and CK cells, and were found similar to other herpesviruses in physicochemical and morphological properties. They were immunologically related to the psittacine herpesvirus which causes Pacheco's parrot disease, and when inoculated into budgerigars produced clinical disease and death with hepatic lesions. The CKT-1 and PKT-1 isolates, from a cockatiel and a budgerigar, grew in fibroblastic cell cultures from several species. Their physicochemical properties suggested that they should be classified as members of the orbivirus group. They were mildly pathogenic for budgerigars.