Essential role of Drosophila Hdac1 in homeotic gene silencing.

Deacetylation of the N-terminal tails of core histones plays a crucial role in gene silencing. Rpd3 and Hda1 represent two major types of genes encoding trichostatin A-sensitive histone deacetylases. Although they have been widely found, their cellular and developmental roles remain to be elucidated in metazoa. We show that Drosophila Hdac1, an Rpd3-type gene, interacts cooperatively with Polycomb group repressors in silencing the homeotic genes that are essential for axial patterning of body segments. The biochemical copurification and cytological colocalization of HDAC1 and Polycomb group repressors strongly suggest that HDAC1 is a component of the silencing complex for chromatin modification on specific regulatory regions of homeotic genes.

The skin, tongue, and brain as favorable organs for hog cholera diagnosis by immunofluorescence.

Hog cholera virus antigens were found densely distributed in skin and tongue of pigs experimentally infected with hog cholera virus. The finding described here warrants the usage of ear biopsies for hog cholera diagnosis on a herd basis.

African swine fever virus: generation of subpopulations with altered immunogenicity and virulence following passage in cell cultures.

Virus subpopulations with variable virulence, immunogenicity, and infectivity to pigs were readily generated by passaging Tengani isolate of African swine fever virus, either biologically cloned or uncloned, in Vero cell cultures. Avirulent virus populations which account for more than 99% of virus in an uncloned preparation of the 27th passage are laboratory artefacts, perhaps do not exist in nature. Furthermore, attenuation of virulence did not occur uniformly in all subpopulations newly generated, and a continuous modulation of virus populations differing in immunogenicity and virulence took place in the same individuals inoculated with the 27th passage virus. The same virus preparation, appearing to be slightly virulent in pigs, contained at least a virulent subpopulation that was manifested only by further inoculating susceptible pigs with viremic blood collected at various times during the clinical course. A cloned virus after 23 passages in cell cultures generated a subpopulation (99.9%) which induced subclinical infection in pigs; however, the infection did not confer a solid immunity to homologous challenge with Tengani isolate in these pigs. The Tengani isolate contained subpopulations of virus with immunogenicities shared by the Lisbon '60 isolate and also contained at least one subpopulation specific for the Tengani only.

African swine fever virus: purification of capsomeres released by lipase.

The virus capsomeres of the outer and inner layers of capsids were effectively released simultaneously from purified virions by lipase digestion and were purified by a linear gradient ultracentrifugation. The capsid consisted of an array of double layers of uniformly arranged individual capsomeres where a lipid(s) served as a matrix in between the capsomeres.

Epitopic diversity of African swine fever virus.

African swine fever (ASF) is caused by an icosahedral cytoplasmic, double stranded DNA virus. In the acute form of the disease, pigs die from disseminated intravascular coagulation (DIC) with extensive damage of the free and fixed macrophage systems and the reticular epithelial cells of the thymus; mortality is virtually 100%. In recent years, subacute and chronic forms of ASF have become more prevalent in the field, especially in outbreaks occurring outside the continent of Africa, and virus isolated from these outbreaks have often been of lesser virulence. In pigs experimentally infected with such isolates, a number of immunopathological manifestations have been encountered, e.g. hypergammaglobulinemia associated with necrotizing pneumonia, persistent infection in the presence of ASF-specific antibodies, and lack of demonstrable virus neutralizing antibodies. Nevertheless, the immune systems of pigs that have clinically recovered have not been impaired by the infection. We suggest that the heterogeneous composition of the virus population in a given isolate may be one of the causes of the anomalous immune responses. When a number of biological markers, i.e., hemadsorption characteristics, plaque size, infectivity, virulence, antigenic determinants, and genomic structure, were used to characterize the virus clones derived from various ASF virus (ASFV) isolates, considerable heterogeneity was apparent. In the present investigation, 20 monoclonal antibodies (MAb), which specifically identified the 14 kDa viral protein within the cytoplasmic membrane of the infected cells, were used to determine epitopic differences among a number of virus clones derived from various isolates. All of the non-African isolates examined contained two epitopically different groups of virus clones, and the reaction profiles obtained were distinctly different from those obtained with the clones of an African isolate (Tengani). It was concluded that an ASFV isolate is composed of a biologically diverse virus population with distinctly different members which are only identified after cloning.

Diversity of African swine fever virus.

An African swine fever virus is an heterogeneous population, consisting of clones having different biological characteristics in respect to hemadsorption, virulence, infectivity, plaque size, and antigenic determinants. The following observations were made: Nonhemadsorbing virus (NHV) have been segregated from field isolates from Haiti (HT-1) and a bone marrow- and buffy coat-passaged Portuguese isolate (L'60BM89BC1) and appear as a major, minor, or equal mixture with hemadsorbing viruses in the virus population. Biological characteristics of the virus inoculated into pigs often differed from viruses isolated later from the same pigs. Virulence and nonhemadsorbing characteristics of isolated clones were genetically stable. The lethal effect of 2 NHV clones of L'60BM89BC1 virus was dose-dependent; small doses of virus induced immunologic deaths or recoveries from the clinical disease in pigs, and large doses induced acute deaths. The NHV of Lisbon isolate of 1960 (L'60) and HT-1 isolate share the same antigenic determinants for inducing protection. Tengani isolate contained clones of distinctly different antigenic determinants, not shared by L'60 or HT-1 isolate that enabled it to overcome the protection induced by the other clones. Passaging of an African swine fever virus isolate in pigs or cell cultures may readily alter the proportions of the different clones in the population and thereby change its overall characteristics. A new virus population with atypical hemadsorption was found in HT-1 field isolate and L'60BM89BC1 virus.

Virulence in African swine fever: its measurement and implications.

A method of measuring and expressing the virulence of African swine fever virus in numerical terms was developed. Seventeen viruses (13 hemadsorbing and 4 nonhemadsorbing) were tested and classified into 3 groups: highly infectious and highly virulent, highly infectious and moderately virulent, and slightly infectious and slightly virulent. This classification was based on the number of 50% hemadsorption unit (HA50) or TCID50 required to produce 1 LD50 for swine, the number of HA50 or TCID50 required to produce one 50% pig infectious dose (PID50), and the number of PID50 required for each LD50. The virulent virus (group 1) required less than or equal to 10 virus units (HA50 or TCID50) for 1 PID50 and LD50 (highly infectious and highly lethal), respectively, and had a ratio of 1.0 for PID50/LD50, ie, all infected pigs died from acute African swine fever. Tengani, L'60, and DR-I isolants and nonhemadsorbing viruses of Haiti-1 isolant belong to this group. The moderately virulent virus (group 2) required less than 10 virus units for 1 PID50 and 20 to 562 virus units for 1 LD50 (highly infectious and moderately lethal), respectively, and recoveries from the clinical disease and immunologic deaths were frequent. Madrid '75, Haiti-1, DR-II, and BR-I isolants belong to this group. The slightly virulent virus (group 3) required 56 to 10,000 virus units for 1 PID50 and 56,200 to 3,120,000 virus units for 1 LD50 (slightly infectious and less lethal).(ABSTRACT TRUNCATED AT 250 WORDS)

African swine fever virus DNA: restriction endonuclease cleavage patterns of wild-type, Vero cell-adapted and plaque-purified virus.

DNA from African swine fever (ASF) virus was isolated and was characterized by two restriction enzymes, SmaI and EcoRI. Although both enzymes can distinguish Vero cell-adapted ASF isolates by characteristic restriction endonuclease cleavage patterns, all ASF isolates examined exhibited a high degree of similarity, as measured by co-migration of most of the DNA fragments. The molecular weight of ASF DNA, based on size estimates of DNA fragments from cleavage patterns, ranged from 93 x 10(6) to 100 x 10(6). Virus genome heterogeneity was observed in uncloned, cell culture-adapted ASF isolates as well as in a plaque-purified virus after serial passage in Vero cells. In contrast to the rather minor differences in restriction pattern among the Vero cell-adapted isolates, a major alteration in restriction endonuclease cleavage sites was observed during adaptation of the wild-type virus to cell culture.

New method of antibody detection by indirect immunoperoxidase plaque staining for serodiagnosis of African swine fever.

An indirect immunoperoxidase plaque-staining method was developed for detecting antibody to African swine fever virus infection. In both sensitivity and specificity, the test was comparable to indirect immunofluorescence. Because it has all of the desirable features of the indirect immunofluorescence test and may also be readily used for testing large numbers of sera, the indirect immunoperoxidase plaque-staining method can be used as a single and final serodiagnostic test in a large-scale survey of African swine fever. The indirect immunoperoxidase plaque-staining method should be applicable to other viruses that can be adapted to and grown in cell cultures.

Sedimentation coefficient of African swine fever virus.

The sedimentation coefficient of the infective unit of African swine fever in tissue culture harvest fluids was measured in a preparative ultracentrifuge. The boundary locator method used also permitted making an estimate of heterogeneity. The sedimentation coefficient ranged from 3,000 to 8,000 Svedberg units, representing many classes of infective particles. Electron microscopy on culture fluids from infected cells showed many kinds of virus-containing units. Sucrose-CsCl gradient centrifugation was used to concentrate and to purify (partly) African swine fever virus for analytical ultracentrifugation. The optical patterns of the physical particles revealed a range of coefficients from 1,800 to 3,200 Svedberg units in tris-buffered saline solution at 20 C and buoyant densities from 1.19 to 1.24 g/ml in CsCl. The disparity of these values from those obtained by preparative ultracentrifugation indicates a change in the virus structure or a selection of viral populations on purification (or both).

Replication of African swine fever virus in cell cultures.

Infection-specific, nonstructural, African swine fever virus antigens, as visualized by the immunofluorescence technique, appeared as fine stipplings, evenly distributed in the cytoplasm and nucleus of Vero cells and in monocytes by postinoculation hour (PIH) 3. Cytoplasmic inclusion bodies (IB) appeared at PIH 4 and continued to increase in size up to PIH 8. The viral DNA was solely synthesized within the IB of infected monocytes, as evidenced by an autoradiographic chase experiment; maximum synthesis occurred at PIH 5. The 2,300-S and 2,500-S (sedimentation coefficient) structural viral antigens were visualized in the IB at PIH 5 and 6, respectively. The infective new progeny of African swine fever virus were formed between PIH 7 and 8, and the cell surface viral antigens were produced between PIH 8 and 9. Free virus was released between PIH 10 and 11. Infected cells detached from the glass surface from PIH 13. The 5-iodo-2'-deoxyuridine interfered with the formation of infective virus, but not interfere with the production of viral antigens.

Electron microscopic observation of African swine fever virus development in Vero cells.

African swine fever virus emerges from infected Vero cells either from areas of smooth cell surface or from microvilli. The two patterns may occur at different sites on the same cell and are unique for this virus. The scanning electron micrographs supplement regular thin section views.

African swine fever: microplaque assay by an immunoperoxidase method.

A microplaque assay for Vero cell-adapted Lisborn '60 strain of African swine fever virus (L'60-uncloned) and a large plaque-forming strain cloned from the L'60-uncloned strain was developed by an immunoperoxidase method. The immunoperoxidase method can be used to stain microplaques of 3 days after inoculation, whereas the conventional plaque assay requires 5 to 7 days to develop visible plaques. A linear relationship between viral concentration in the inoculum and plaque numbers was observed. Viral titers obtained by both microplaque assay and conventional plaque assay were comparable, and both methods were reproducible and reliable. The viral titer obtained by either one of the plaque assay methods was approximately 0.9 log10 lower than that obtained by the hemadsorption test.

Cellular immunity demonstrated in pigs infected with African swine fever virus.

Twenty-two pigs infected with African swine fever virus (ASFV) were used to demonstrate delayed hypersensitivity (DH) in vitro by the leukocyte migration-inhibition test. The results indicated that ASFV-infected pigs developed DH against ASFV antigen (ASF antigen) as early as 20 days after inoculation, and the presence of viremia did not interfere with the leukocyte migration-inhibition test. Three ASFV-infected pigs that were also sensitized to mycobacterium developed DH against both ASF antigen and mycobacterium. The conclusion was that the cellular immune system is not impaired by ASFV infection in pigs.

T and B lymphocytes in porcine blood.

Two subpopulations of lymphocytes in the peripheral blood of young, conventionally reared Tamworth pigs had the following cell-surface markers analogous to those of human lymphocytes; (1) T cells--receptor(s) for ovine and caprine erythrocytes, permitting spontaneous formation of rosettes and the presence of the thymus-derived antigen; and (2) B cells--membrane-associated immunoglobulin and receptor(s) for complement. The T cells could be identified by either the erythrocyte rosettes or the cytotoxicity test, and the B cells could be identified by either the erythrocyte-antibody-complement rosette test or the immunofluorescence test for surface immunoglobulin. Because of simplicity, the cytotoxicity and immunofluorescence tests are recommended. The cell losses in ficoll-diatrizoate gradient centrifugation were studied with 9 blood samples. Of the total T and B cells, 71.7% were in fraction 1, and 28.3% were in erythrocyte fraction 2. Recovery rates of total leukocytes varied widely in relation to leukocyte numbers present in the original blood; the range was between 52.6 and 93.8%. Moreover, evidence did not indicate that either T or B cells were selectively sedimented in fraction 2. Analysis of the lymphocyte-rich fractions (fraction 1) from 37 blood samples by ficoll-diatrizoate gradient centrifugation indicated the following: (1) Approximately 2.7 X 10(6) T cells and 0.9 X 10(6) B cells could be obtained in the fraction from 1 ml of peripheral blood; (2) T and B cell populations were independently distributed; hence, no definite ratio between T and B cells applied to an individual animal; (3) approximately 20% of the lymphocytes did not have markers as either T or B cells; and (4) an average of 26% cells were peroxidase-positive leukocytes, chiefly monocytes.

Immunofluorescent studies on chronic pneumonia in swine with experimentally induced African swine fever.

Chronic pneumonia experimentally produced in 14 pigs with African swine fever (ASF) virus was studied by immunofluorescene (IF) and histopathologic techniques. Frozen sections prepared from pulmonary tissues of the infected pigs were stained with fluorescein-conjugated antiserums against ASF viral antigen, porcine immunoglobulin G (IgG), procine complement (C), and porcine fibrinogen. The viral antigen(s) was mainly seen in macrophages and cell debris in alveolar walls and lumens. This finding indicates that the virus replicated in the cytoplasm of alveolar macrophages that subsequently degenerated and released the viral antigen. Diffuse immunoglobulin (Ig) deposition was found in necrotic cells and debris. Immunoglobulin also was seen bound to intracytoplasmic inclusion bodies in some degenerating alveolar macrophages. This finding indicates that antibody against ASF viral antigen(s) excluded from blood circulation or produced by local immunocytes (or both) reacted with viral antigen at intramacrophage and extramacrophage levels and resulted in the formation of insoluble antigen-antibody (Ag-Ab) complexes. The participation of C in the immune complex was evident in the early stage of the pneumonia, but was less evident in the subsequent extensive, progressive necrotic processes. Fibrin deposits were visible only in the early necrotic area of alveolar walls and lumens. Possible mechanisms inducing extensive necrosis are discussed.

Pathologic features of chronic pneumonia in pigs with experimentally induced African swine fever.

Chronic pneumonia developed in 14 pigs inoculated with an attenuated strain of African swine fever (ASF) virus. The pathogenesis of the pneumonia was as follows: (1) Interalveolar septums became thickened by accumulation of lymphocytes and monocytes; (2) lung developed focal areas of lymphocytes and macrophages; (3) necrosis began abruptly in these foci, beginning with the cells in the alveolar lumens, developing in centrifugal direction, and eventually affecting all structures in its path; (4) necrotic tissue became calcified; and (5) a mantle of mononuclear cells (including plasma cells) and fibrous tissue formed around the necrotic area. Viremia occurred in the 14 pigs at postinoculation day (PID) 14, and precipitating antibody was increased significantly at PID 58.

Immunofluorescence plaque assay for African swine fever virus.

Suitably diluted cell culture adapted African swine fever virus preparations were inoculated on VERO cell monolayers and grown on coverslips. Gum tragacanth was used as an overlay. After three days incubation at 37 degrees C the infected cultures were fixed with acetone and stained with fluorescent antibody conjugate. Fluorescing plaques consisted of 20-30 infected cells. THREE STATISTICAL CRITERIA FOR A QUANTITATIVELY RELIABLE ASSAY WERE MET: the Poisson distribution for plaque counts, linearity of the relationship between the concentration of virus and the plaque count and reproducibility of replicate titrations. The method is suitable for counts up to at least 70 plaques per 5 cm(2) coverslip and computed titers are reproducible within 0.16 log units with a total of 300 plaques enumerated.