Monitoring of humans and animals for the presence of various Rickettsiae and Coxiella burnetii by serological methods.

Serological examination of humans in Slovakia suspected of having rickettsial infections revealed the presence of antibodies to spotted fever group rickettsiae (R. conorii, R. slovaca, and R. typhi). Of interest is the finding of serological positivity to the newly recognized "IRS" agent. Antibodies to these rickettsiae and to C. burnetii were demonstrated also in domestic and hunting dogs and pet animals. These results confirm the occurrence and possible circulation of these rickettsiae and C. burnetii in the Slovak Republic.

Processed enzymatically active protease (p15gag) of avian retrovirus obtained in an E. coli system expressing a recombinant precursor (Pr25lac-delta gag).

Processing proteases of avian and mammalian retroviruses cut the polyprotein precursors encoded by the retroviral genes into mature functional proteins. Retroviral processing proteases are still a rather poorly characterized group as to their relation to other proteases, specificity, and mechanism of enzymatic action. In avian retroviruses the generation of the processing protease itself comprises a processing cleavage event - the protease p15gag is cut off the carboxy-terminus of a gag polyprotein precursor, Pr76gag. We report here that direct and efficient production of the avian retrovirus processing protease p15gag (required for structure-function studies and rational design of inhibitors) was obtained in an E. coli system, where massive expression of a size-reduced, recombinant precursor (Pr25lac-delta gag) was accompanied by its structurally accurate processing.

Inhibition of avian myeloblastosis virus reverse transcriptase by diphosphates of acyclic phosphonylmethyl nucleotide analogues.

Diphosphates of N-(2-phosphonylmethoxyethyl) derivatives of heterocyclic bases were studied in the endogenous oligo(dT)12-18 primed reaction of reverse transcriptase from detergent-disrupted AMV(MAV) retrovirions. These diphosphates (analogues of nucleotide 5'-triphosphates) exhibited an inhibitory activity towards reverse transcriptase. This inhibitory activity was dependent on the character of the heterocyclic base and decreased in the order: 2-aminoadenine greater than adenine greater than guanine much greater than cytosine much greater than thymine greater than uracil. The 2-aminoadenine derivative was more potent than either AZT-TP or ddTTP, while PMEApp had approximately the same potency as the two reference compounds (IC50 approximately 1 microM at 20 microM competing substrate). This finding is consistent with the antiviral activity of the parent nucleotide analogues against retroviruses (including HIV).

Protein A/G dependent ELISA a promising diagnostic tool in Lyme disease seroprevalence in game animals and hunting dogs.

One of the major problems in serodiagnosis in wild animals is unavailability of specific antiglobulin conjugate. Our study focuses on validation of Protein A/G dependent ELISA in game animals like deer and mouflons as well as in hunting dogs. Binding ability of Protein A/G-conjugate to antibodies was the highest in dogs followed by fallow deer and mouflons. Three different whole cell Borrelia antigens were used to evaluate antigen dependent variation. In new Protein A/G-ELISA the highest sensitivities (90.50%, deer; 85.37%, mouflon & 94.29%, dog) were obtained by B. garinii antigen, with no statistically significant variation (chi(2), P>0.05) among all other antigens used. Average seroprevalences observed in deer, mouflons and dogs were 44.90%, 29.41% and 30.43%, respectively. Marked influence of age on seroprevalence was noticed. Protein A/G-ELISA proved to be sensitive and promising diagnostic tool in serodiagnosis of Lyme disease in game ungulates and it can be used effectively for serosurvey in different wild mammals.

Stepwise transition of aggregate structure of high-molecular-weight avian myeloblastosis virus RNA. Mode of releasing of associated 4S RNA.

Mode of releasing of associated 4S RNA species was studied during a controlled transition of aggregate structure of high-molecular-weight AMV-RNA. It has been found that associated 4S RNA constitutes 2.5% of 60S AMV-RNA complex. Approximately 60% of associated 4S RNA is successively released during treatment of viral RNA with increasing formamide concentration, concomitantly with the transition of 60S RNA aggregate through 50--55S RNA intermediate into the final 30--40S RNA subunits. 40% of 4S RNA remains associated with 30--40S RNA subunits prepared by formamide treatment and can be released from them by heating. A procedure is thus provided both for the isolation of oncornaviral RNA subunit structures deprived of various partions of associated 4S RNA and for the fractionation of 4S RNA species according to their binding affinity to the genome oncornaviral RNA.

Mathematical analysis of the oncornavirus maturation process (virion RNA conversion and morphological condensation).

The rate of the maturation process of avian myeloblastosis virus experimentally estimated on the basis of genomic viral RNA conversion and morphological transition of virions was mathematically analysed. Three mathematical models were suggested and fitted to experimental data. It was found that: (a) model of simple kinetics (Model 1) does not agree with experimental data. Therefore, two hypotheses were considered in further mathematical modelling: (b) virions are identical in time of budding: maturation is dependent on the presence of a virion component which is degraded with time (Model 2). This model agrees with experimental data in all stages of the maturation process. (c) Virions are released from cells at different stages of assembly (Model 3). This model differs from experimental data especially in early stages of maturation. The hypothesis used for the construction of Model 2 seems to be the most plausible to explain the maturation process and is in agreement with data of murine leukemia virus maturation which was found to be accomplished by cleavage of p70 precursor protein.

Avian nephroblastomas induced by a retrovirus (MAV-2) lacking oncogene. I. Construction of MAV-1 and MAV-2 proviral restriction maps and preparation of specific proviral molecular subclones.

A 9.8 kb DNA fragment containing the complete MAV-1 provirus was recloned from the recombinant bacteriophage lambda 311411 (Perbal et al., 1985) into the plasmid pAT153. A detailed and precise restriction map of the obtained clone (pAT-MAV-1) was constructed. From compilation of this map and the known sequence of a variable portion of the MAV-2 env gene was a restriction map of MAV-2 deduced. Knowledge of the detailed pAT-MAV-1 map facilitated the preparation of five specific proviral subclones: pAT-U3 and pUC-U3 (both contain the U3 domain of the proviral LTR, which is MAV-specific and displays no homology with other hitherto known retroviruses including avian endogenous proviruses), pUC-RU5 (containing the R and U5 domains of the proviral LTR), pUC-UT5 (containing untranslated sequences flanking the 5' LTR), and pUC-UT3 (containing untranslated sequences flanking the 3' LTR). Thus tools for analysis of integrated MAV-2 proviruses in nephroblastomas induced by this virus were formed.

Binding properties of avian retroviral proteins. II. Binding of protein ASLV NC(p12) to viral RNA and proviral DNA.

Binding of the major avian retroviral nucleocapsid protein ASLV NC(p12) to the MAV-1 (myeloblastosis-associated virus) proviral dsDNA and viral ssRNA was analysed using electron microscopy. Specificity of the binding was estimated by special computer programs. The NC(p12) protein bound to MAV-1 proviral dsDNA (clone pAT153--MAV-1), but specificity of this binding was not found by computer evaluation. NC(p12) also bound to nondenatured 70S viral RNA at a rate of 25 +/- 3 molecules per RNA molecule. When this RNA was denatured either before or after the complexing, it showed no binding affinity for the protein. This result implies that preserved secondary structure of the viral RNA was required for the binding.

Binding properties of avian retroviral proteins. I. Preparation and basic characterization of ASLV NC(p12) and MA(p19).

Using SP-Sephadex column chromatography we isolated from an avian retrovirus, AMV(MAV), nucleic acid-binding proteins ASLV NC(p12) and MA(p19). As shown by several criteria, namely SDS-PAGE, PR(p15) protease activity, and nucleic acid binding assay with the use of both ss and ds DNAs, our NC(p12) and MA(p19) isolates are virtually pure proteins mutually not cross-contaminated. Rabbit anti-NC(p12) and anti-MA(p19) sera which we prepared did not cross-react mutually. We conclude that both NC(p12) and MA(p19) and antibodies against them are adequately pure preparations for investigating their nucleic acid binding specificities towards AMV(MAV) genomic RNA and MAV-1 proviral DNA using electron microscopy supported by computer analysis of electron micrographs.

Binding properties of avian retroviral proteins. III. Binding of protein ASLV MA(p19) to viral RNA and proviral DNA.

The binding of the avian retroviral matrix protein ASLV MA(p19) to homologous viral ssRNA and proviral dsDNA was analysed using electron microscopic methods combined with a special computer evaluation. No binding affinity of MA(p19) to homologous nondenatured or denatured viral RNA was found. In contrast, ASLV MA(p19) was shown to have one specific binding site on MAV-1 proviral dsDNA at position 6795 +/- 345 bp from the 5' end of the molecule. A second specific binding site was found in a cellular sequence.

Avian nephroblastomas induced by a retrovirus (MAV-2) lacking oncogene. III. Presence of defective MAV-2 proviruses in tumour DNA.

Using Southern hybridization with a series of probes derived from the MAV provirus we searched for structurally changed MAV-2 proviruses in the DNA from MAV-2-induced nephroblastomas. Anomalous fragments of MAV-2 provirus were found in all samples analysed in more detail. Comparison of the sizes of anomalous fragments detected by different probes in the same sample indicates that the fragments, at least in a majority of cases, do not represent recombinant proviruses (which might contain transduced oncogene) but deleted proviruses only. Various types of proviral deletions occur in different nephroblastoma clones, and no preferred type of deletion was found. The defects in MAV-2 sequences appear in infected chickens de novo, because defective proviruses also occur in tumours of chickens that have been infected with the plaque-purified MAV-2 preparations and, in addition, most of the proviral defects found prevent the virus from replicating and from being transmitted by infection. The regular occurrence of defective proviruses in tumour DNA supports the concept that the proviral defects are involved in oncogenesis. Hypotheses on the substance of this involvement are discussed.

Two exons specific for the myb proto-oncogene found upstream from the avian myeloblastosis virus-transduced myb sequences.

A partial restriction map of cloned 5.42-kb chicken DNA (clone P542, Perbal et al. 1983), covering a portion of the c-myb locus, is presented. The 5' end of the v-myb gene (approximately 0.5 kb) is located at the 3' end of P542 DNA, the remainder are the cellular sequences not transduced by avian myeloblastosis virus. Two non-contiguous DNA segments were detected within these cellular sequences which code for the 5' end of c-myb mRNA. These two exons, designated e1 and e2, are separated by a approximately equal to 1.5-kb non-coding region. Both of them are transcribed into 0.4 kb located near the 5' end of c-myb mRNA. The second exon e2 (approximately equal to 0.2 kb) is flanked at its 3' end by a short non-coding region within which virus-cell recombination took place. The possible presence of a portion of this intron in the 2.1-kb v-myb mRNA is discussed.

In vitro cleavage of gag-myc fused protein P110 of a defective leukaemia virus MC29 by retroviral protease p15.

Gag-related proteins were precipitated from 35S-methionine-labelled cells of the PR-2 line derived from a hepatoma induced by virus MC29. Immunoprecipitates were incubated with viral protease p15 and the cleavage products of P110 were analysed in SDS-PAGE. The major cleavage fragment of P110 is a protein with mol. wt. of 56 000. Preparative isolation of the non-gag fragments from protein P110 by means of a gag immunosorbent showed that the 56K fragment did not contain any gag specific antigenic determinants. This finding was also confirmed by performing immunization with this fragment. Cleavage of protein P110 and the properties of cleavage products are discussed.

In vitro processing of avian oncovirus precursor polypeptide Pr76gag by virion protein p15.

In vitro cleavage by p15 virion protein of the primary translation product of the avian oncovirus gag gene, the precursor polypeptide Pr76gag, was studied in kinetic experiments. Nondenatured 35S-methionine-labelled Pr76gag, which was synthetized in reticulocyte lysate programmed by genomic AMV-RNA was used as a substrate, pure native p15 (AMV) as a protease. Reaction conditions were optimal for in vitro protein synthesis. Composition of the cleavage products was estimated by immune precipitation with monospecific antisera against internal structural virion (AMV) proteins p27, p19, p15, and p12, and their size by SDS-PAGE. Monospecificity of each of the antisera was assessed by adsorption with the three respective heterologous gag proteins, followed by immune precipitation of 35S-methionine-labelled proteins of the virion (AMV) lysate. The p15-mediated in vitro cleavage proceeds rapidly and specifically. In the early stages (10 min incubation with p15) the Pr76gag was absent, and an optimum amount of six cleavage intermediates with the following size, composition (shown in parentheses) and orientation (determined form the presence or absence of antigenic determinants of p19 or p15 proteins as N- or C-terminal moieties of the precursor) was found: N-terminal fragments of 66K (p19, p27, p12) and 60K (p19, p27); internal fragments of 37K (p27, p12); C-terminal fragments of 51K (p27, p12, p15), 32K (p12, p15) and 21K (p12, p15). These intermediates were converted into the following four final cleavage products, as the only polypeptides detected after prolonged (5 h) incubations: mature p27 and mature p15 proteins and 38K and 34K polypeptides containing only p19 antigenic determinants. Mature p12 an p19 proteins were not found among cleavage products. Autocatalytic cleavage of Pr76gag was not observed. The results hav allowed us to conclude that the arrangement of the gag proteins in the Pr76gag is N-p19-(p10?)-p27-p12-p15-C and that under in vitro conditions p15 recognizes three correct cleavage sites on native Pr76gag: one located at p12-p15 junction and two on both sides of the p27 moiety, as well as aberrant cleavage sites located inside p12 and possibly also p10 moieties.

Analysis of gag-myc proteins of partially transformation-defective mutants of avian myelocytomatosis virus strain MC29 by in vitro cleavage with protease p15: indication for the presence of specific cleavage site p15 in the myc domain of fusion protein.

Gag-related proteins were immunoprecipitated from [35S]methionine-labelled cells of the PR-2 line which contained p110 protein as well as from quail cells transformed by deletion mutants 10A, 10C, and 10H of MC29 virus. Immunoprecipitates were incubated with oncoviral protease p15 and cleavage products were analyzed in SDS-PAGE. The major 56K fragment (F56) of p110 was further analyzed by tryptic peptide mapping. The results showed that except for the myc domain of p110, a portion of p27 is also present in F56. Cleavage of 100K, 95K, and 90K proteins coded by three MC29 deletion mutants resulted in major fragments 66K, 60K, and 56K, respectively. The existence of further cleavage fragments and presence of the p15 specific cleavage site in the myc domain of MC29 specific proteins is discussed.

A search for c-myb gene regulatory sequences: cloning and restriction analysis of the 18-kb BamHI fragment of chicken chromosomal DNA, containing the 5' part of the c-myb gene.

The 18-kb BamHI fragment of the chicken chromosomal DNA derived from the 5' end of the myb proto-oncogene has been cloned. The 5' part of this clone represented by a 7.2-kb BamHI-EcoRI fragment has been analysed by means of restriction mapping, which revealed the existence of about 2.5 kb of the CpG dinucleotide-rich sequence within this fragment. A short probe prepared from the CpG-rich sequence hybridizes with the 3.6-kb c-myb mRNA. Based on our results and published c-myb cDNA sequence, we conclude that the cloned fragment contains c-myb promoter.

Monoclonal antibodies against RNA-dependent DNA polymerase from myeloblastosis-associated viruses.

Two lines of hybridomas, RT-12 and RT-14, secreting monoclonal antibodies against the reverse transcriptase from myeloblastosis-associated viruses have been prepared. The monoclonal antibodies RT-12 and RT-14 specifically react with reverse transcriptase, as has been shown by radioimmunoassay and enzyme-linked immunoassay techniques after SDS-PAGE and blotting to nitrocellulose membranes. It has been shown that antigenic determinants for RT-12 and RT-14 are stable to SDS denaturation, hence they belong to a linear type; they are located on the alpha subunit of the enzyme.

Avian nephroblastomas induced by a retrovirus (MAV-2) lacking oncogene. II. Search for common sites of proviral integration in tumour DNA.

To demonstrate the existence of a common site of integration in independent tumour clones, restriction mapping of the vicinity of integrated MAV-2 proviruses in nephroblastoma DNA was performed, using Southern hybridization with an MAV-2 specific probe U3(pAT). The results have shown that (1) nephroblastomas are of semiclonal origin. (2) Nephroblastoma cells contain an average of 5 clonally located integrated proviruses per diploid genome; they do not contain any detectable amount of non-integrated proviruses. (3) In the DNA from independent nephroblastoma clones, there appear at an increased frequency Tth111I fragments of 14.6 and 17.8 kb that hybridize with the U3(pAT) probe. Considering a random selection of integration sites, such coincidence is of little probability. Thus we suppose that these fragments represent a common site(s) of integration with an MAV-2 proviral insert. Two hypotheses concerning possible mechanisms of nephroblastoma induction are discussed: proto-oncogene insertional activation and anti-oncogene insertional inactivation.

Biological activity of proviral DNA isolated from cells infected with MAV-2(0) virus.

The ability of DNA isolated from avian myeloblastosis-associated virus MAV-2(0)-infected fibroblasts to induce the synthesis of virus upon transfection of susceptible but not resistant cells was demonstrated. The virus obtained, when inoculated into 12-day-old embryos, led to the manifestation of osteopetrosis after prolonged incubation and to the induction of nephroblastomas in some cases. Positive transfection with DNA from the non-virus producing myeloblast cell line was not detected on bone marrow cells and fibroblasts preinfected with tdB77-C virus. After transfection with DNA from virus-producing myeloblasts, the reproduction of non-transforming virus was observed. This virus did not induce myeloblastosis upon infection of chickens but induced nephroblastomas on rare occasions.

Prevalence of antibodies to Encephalitozoon cuniculi (microsporidia) in angora goats--a potential risk of infection for breeders.

The presence of antibodies against Encephalitozoon cuniculi in Angora goats was detected by the method of indirect immunofluorescence (IFAT). The animals reacting at the titre 1: 64 and more were considered positive. Of the total number of 48 sera examined, 4 were positive at the titre 1: 32 and 2 were positive at the titre 1: 64. The occurrence of antibodies against E. cuniculi indicates that one of the causes of disorders in the reproductive cycle in Angora goats may be microsporidia Encephalitozoon cuniculi, and that these animals may be potential sources of infection for people.