[Creation of an avirulent and immunogenic mutant from the rhinopneumonitis virus]. / Suzdavane na avirulenten i imunogenen mutant ot virusa na rinopnevmonita.

An avirulent immunogenic virus strain mutant of the causative agent of rhinopneumonia was found to cause abortions and respiratory diseases in horses. The mutant was obtained with the use of a virulent strain that induced strongly manifested clinical symptoms of the disease, and was cultured in cell media containing 5-iodine-2-desoxiuridine as an antimetabolite, following a definite pattern. It was found that the mutant completely lost its virulence, however, it retained its immunogenicity. It likewise retained these newly acquired biologic properties with regard to its being stable and irreversible. Animals vaccinated with this mutant did not act as carriers, neither did they shed any virus. The mutant is now used in the production of a vaccine for the practice.

[Thymidine kinase activity of virulent and vaccinal strains of Aujeszky's disease virus]. / Timidin-kinazma aktivnost na virulentmi i vaksinalni shtamove na virusa na Aueski.

Studied was the timidine-kinase activity of the A2 virulent strain and the MK-25 and MK-35 vaccinal strains of the Aujeszky's disease virus via the infection of primary cell cultures of chick embryo fibroblasts and of pig kidney. It was found that the A2 strain substantially enhanced the induction of the timidine-kinase enzyme in the infected cell cultures, while the IUDR- and the BUDR-resistant vaccinal strains MK-25 and MK-35 showed 10 to 12 times lower activity. Strain MK-25 did not restore the high timidine-kinase activity of the parent A2 strain after 10 successive passages in primary chick embryo fibroblast cultures. The strongly reduced timidine-kinase activity of the two vaccinal strains (MK-25 and MK-35) could be used as a marker to control their properties. The virulent A2 strain of the Aujeszky's disease virus had a well manifested mutability when passing in cultures in the presence of IUDR.

[Creation of an avirulent immunogenic mutant of the transmissible gastroenteritis virus and its use as a vaccine]. / Suzdane na avirulenten imunogenen mutant ot virusa na transmisivniia gastroenterit i izpolzuvaneto mu kato vaksina.

The serial cultivation of a virulent strain of the virus of transmissive gastroenteritis in the presence of the antimetabolite 5-bromurazil has led after a definite number of passages to the production of a virus mutant of new biologic properties. It was established that the mutant is resistant to the antimetabolite, and it retains its infectious titer. It is avirulent to newborn and growing pigs, and confers solid immunity in the vaccinated animals which do not become virus carriers. It is stable with regard to its newly-obtained biologic properties.

[Morphology and morphogenesis of Aujeszky's disease virus (the viral mutant MK-35 and virulent strain 2)]. / Morfologiia i morfogeneza na virusa na bolestta na Aueski (virusen mutant MK-35 i virulenten shtam 2).

Ultrastructural studies were carried out on the morphology and morphogenesis of the Aujeszky's disease avirulent immunogenic mutant strain MK 35 and the virulent parental strain 2. It was found that MK 35 had a delayed cycle of replication, lasting about 10 hours as against the virulent strain 2. It also induced specific cytomorphology of the infected cells as well as the synthesis of a predominantly virus antigen in the form of empty viral capsids of tubular shape. THe culturing of the virulent strain in cell cultures in the presence of the antimetabolite 5-brom-desoxiuridine led to the synthesis of a limited amount of mature viral particles. Characteristic proved, the production, although in small amounts, of empty viral capsids. This process was observed after the 24th hour of infection of the cell cultures.

[Developing an avirulent mutant of Aujeszky's disease virus using the effect of 5 bromodesoxyuridine]. / Het ontwikkelen van een a-virulente mutant van het virus van de ziekte van Aujeszky onder invloed van de inwerking van 5 bromo-desoxy-uridine.

Chemicals are being used increasingly in the preparation of vaccines. Thus, an effective vaccine (MK25) against Aujeszky's Disease showing stable genetic characteristics was produced by having 5 iododesoxyuridine exert its action on a particular strain of virus. 5 Bromo-desoxyuridine also has an inhibitory effect on the synthesis of DNA viruses. The development of a new mutant of Aujeszky's Disease virus using this agent is reported. The resulting mutant, MK35, was found to be biologically stable, non-pathogenic for white mice, rabbits, sheep and piglets and, in addition, produced stable immunity in piglets, sheep and rabbits within four days after injection of this mutant.

[Cytological and cytochemical studies of cell cultures infected with the avirulent viral mutant MK 35 of the pseudorabies virus]. / Tsitologichni i tsitokhimichni prouchvaniia na kletuchni kulturi, zarazeni s avirulentniia virusen mutant MK 35 na Pseudorabies virus.

Cytological and cytochemical studies were carried out of cell cultures of chick embryo fibroblasts infected with the vaccinal mutant strain MK 35 (4.8 X 10(7) PFU) of the Pseudorabies virus. It was found that the first cytologic changes in the infected cultures presented definite focal character. In the first hours of infection several rounded cells were included only, while later on an increasing number of cells were involved, and the foci grew in size. The cytoplasma of the infected cells contained inclusions which were small, spherical, basophilic, and Foelgen positive; six hours later the cytoplasmatic inclusions became larger, oval, acidophilic, surrounded by a brighter area, and were Fölgen-negative. Parallel to the cytologic changes in the infected cell cultures there set in metabolic disturbances and changes in the enzymatic activity. The infected cells showed enhanced lactate dehydrogenase, diphosphopyridin-nucleotide diaphorase, thiamine pyrophosphatase, and alkaline phosphatase activity and suppressed succinate dehydrogenase and acid phosphatase activity. These studies were said to reveal new biologic properties of the vaccinal mutant virus.

[Use of the passive hemagglutination reaction to study Aujeszky's disease]. / Prilozhenie na reaktsiia pasivna khemaglutinatsiia za prouchvane bolestta na Aueski.

Experiments were carried out with the use of the passive hemagglutination test to study Aujeszky's disease, the antigen employed being the avirulent MK-35 strain of the disease virus. It was established that the passive hemmagglutination reaction could be referred to as a specific and rapid method in the study and diagnosis of the disease. Results showed that the vaccinal MK-35 strain was successfully used as an antigen in the test. In the treatment with an MK-25 vaccine there were predominantly antibodies detectable with the passive hemagglutination reaction.

[Creation of an avirulent mutant of Aujeszky's disease virus by exposure to 5-bromodeoxyuridine]. / Suzdavane na avirulenten mutant ot virusa na Aueski pod vuzdeistvieto na 5-bromdoezoksiuridin.

A mutant with new biologic properties has been obtained at the cultivation of a virulent strain of the Aujeszky's disease virus in chick embryo fibroblast cultures, following a definite number of passages. It has proved resistant when culturing at the present of 5-brom-desoxyuridine and 5-iod-2-desoxyuridine, retaining its infections titer. It produces small plaques and destroys cell cultures of a tiny granular structure, the plaque-forming titer being 5. 6X10-7. The mutant has been shown to be avirulent for sheep, suckling pigs, and rabbits. In confers solid and lasting immunity in vaccinated animals which are virus-free and show no virus excretion. The newly obtained MK-35 mutant has proved to be stable in terms of its new biologic properties.

[Biological properties of the MK 35 mutant of Aujeszky's disease virus (Pseudorabies virus)]. / Biologichni svoistva na mutant MK 35 na virusa na bolestta na Aueski (pseudorabies virus).

Clinical, virological, and morphological studies were carried out with a total of 15 twenty-day-old pigs and 55 rabbits of 2.5 kg body weight. All were experimentally infected with a new mutant, MK 35 (3.5 x 10(7) plaque forming units) of the pseudorabies virus. The changes taking place in the test animals were followed up in their dynamics from the first to the thirtieth day after injecting the virus. No clinical signs speaking of the development of Aujeszky-s disease were noted, neither was the virus isolated from the tonsils, lungs, lymph nodes, and the central nervous system of the treated animals. Histologically, there was in the central nervous system a slightly expressed nonsuppurative meningoencephalitis which was most clearly manifested on the fourth day after the animals were infected. Later on the changes appeared to be more slightly visible, and on the 8-10th day they were no longer seen. At the 48th hour following the inoculation and up to the end of the experiment eosinophil leukocytes were present in the tonsils, lymph nodes, and spleen of all infected animals as well as within the parenchyma of the adrenal glands of the pigs only. The presence of eosinophil leukocytes in the enumerated organs of the test animals is referred to as the morphologic manifestation of immunogenesis--the result of an enhanced defense status of the host induced by the MK 35 mutant of the pseudorabic virus.

[Inactivated vaccines against rhinopneumonitis in horses]. / Inaktivirani vaksini sreshtu rinopnevmonita po konete.

Attempts were made to produce inactivated vaccines against horse Herpes virus 1, using various inactivating agents and adjuvants, Best results were obtained with vaccine No 3 (glutaraldehide inactivator and "CTC" adjuvant). Used were two strains of the virus (St. Karaja and Varna). isolated in this country in cell cultures of a sucking pig kidney. Vaccine No 3 showed good immunogenic properties. Its application resulted in the full cease of abortions and respiratory diseases on the base of infection with the horse Herpes virus 1. The vaccination protects newborn colts from rhinopneumonitis if reimmunization of mares is carried out in the 6th-7th month of pregnancy.

[Complement fixation reaction studies in rhinopneumonitis of horses]. / Prouchvaniia vurkhu reaktsiiata za svurzvane na komplementa pri rinopnevmonita po konete.

It was established that the complement binding reaction (CBR) is a suitable and very fast method for horse rhino-pneumonitis diagnostics. Cell cultural virus produced in cell cultures of pig kidneys was used as antigen. The antigen lots tested have no anticomplementary properties. Highest complement binding activity was evident in the non-diluted antigen, which discovered specific antibodies in immune serums. The CBR specificity was tested by the aid of homologous and heterologous serums and antigens. The titers of complement binding antibodies in the serums of 255 horses recovered from the disease varied from 1:10 to 1:320, and in some cases even to 1:640. The blood samples investigated were from studs having rhinopneumonitis proven by clinical observations, by virus neutralizing reaction and by virus isolation. In almost all animals under study neutralizing and complement binding antibodies were established simultaneously.

[Action of actidion on the replication of the transmissible gastroenteritis virus]. / Deistvie na aktidiona vurkhu razmnozhenieto na virusa na transmisivniia gastroenterit.

Explored was the possibility of inhibiting the replication of the virus of the transmissive gastroenteritis by means of actidione. It was found that in certain concentrations the antibiotic completely inhibited the replication of this virus. In individual strains the process of replication was not influenced by the various concentrations applied. It was demonstrated that when the antibiotic was added to the infected cell cultures during individual phases its varying concentrations influenced the virus replication. When actidione was added to the cells at the time of virus adsorption it did not inhibit the synthesis of the virus. Similar were the results when the cells were cultivated 24 hours with specified concentrations of actidione prior to their infection with the virus.

[Pathogenesis of Aujeszky's disease]. / Vurkhu patogenezata na bolestta na Aueski.

Results are presented of studies on the various localization of the virus of Aujeszky's disease (the avirulent mutant strain MK and the virulent strain 2) in the organism of experimentally infected pigs. At the oral infection of pigs with strain MK the virus has been isolated from the submandibular lymph nodes of the animals only, the pigs being killed on the second and fifth day following infection with a low plaque-forming titer. However, from pigs that have been infected subcutaneously no virus was isolated. In experiments with the virulent strain 2, 15 out of 20 pigs have died up to the seventh day following infection. The examination has revealed that a virus of a high plaque-forming titer was present in almost all organs. Histologic investigations in the experiments with the virulent strain 2 have revealed the presence of a strongly manifested nonsuppurative encephalitis, interstitial pneumonia, necroses, and nuclear inclusion bodies. In the experiments with strain MK these changes have been much more slightly expressed, and after the tenth day of infection they tended to disappear. In the authors' opinion the changes are due to the 'interaction' between the viral particles of the vaccinal virus and the competent cells, being the cytologic manifestation of the enhanced immunobiologic status of the host organism. This is an expression of the cell manifestation of immunogenesis and not of the toxic injury of cells which has been observed in experiments with the virulent strain.

[Test of an attenuated viral strain of TGE as a vaccine]. / Izpitvane atenuiran virusen shtam na TGE kato vaksina.

A live vaccine was produced using a local strain P of the virus of the transmissive gastroenteritis, arrenuated in cell cultures. The vaccinated pregnant sows had high-titer serum and colostrum virus-neutralizing antibodies. In the first days following farrowing there were in the colostrum ummunoglobulins of the Igg class that prevailed, however, IgA and IgM proved more effective in the virus-neutralizing test. Newborn pigs acquired passive immunity at about the 24th hour after the intake of colostrum. They withstood a challenge with a virulent virus.

[Action of actidione on the multiplication of Aujeszky's disease virus]. / Deistvieto na aktidiona vurkhu razmnozhenieto na virusa na bolestta na Aueski

Studied was the effect of actidione on the propagation of the Aujeszky's disease virus (strains 2,7,10--virulent,, and strain MK--avirulent virus mutant). It was found that at a concentration of 2 microng/cm3 actidione discontinues the reproduction of the most virulent strain 2 when the antibiotic was added immediately after the adsorption of the virus on the cells. The decrease in the virulence of the virus strain was associated with the decrease in the concentration of the antibiotic needed for the inhibition of its propagation. Thus, the reproduction of the more slightly virulent strain 10 was fully inhibited at actidione concentration of 0.75 microng/cm3, while that of the avirulent strain MK was discontinued at 0.4 microng/cm3. The preliminary cultivation of the cells with 1 microng/cm3 actidione for 24 hours and the adsorption of the virus on the cells jointly with actidione showed no effect on the vitality of the virus particles.

[Study of Aujeszky's disease virus replication by using an immunofluorescent method]. / Prouchvane replikatsiiata na virusa na bolestta na Aueski s pomoshta na imunofluorestsentniia metod

Studied was the interrelationship between the Aujeszky's disease virus (the avirulent mutant strain MK and the virulent strain 2) and chick embryo cells during cultivation with and without 5-iod-2-desoxiuridine. In experiments with the virulent strain the presence of a granular antigen could be observed at the 4th hour following the infection of the nucleus. After the 10th hour the antigen showed migration from the nucleus toward the cytoplasm. After the 24th hour a complete destruction of the cellular monolayer was observed, along with the production of polynuclear cells. In the experiments with strain MK the antigen seemed to appear first in the cytoplasm after the 10th hour of infecting the cells, and after the 12th hour it was found in the nucleoplasm too. In this case full destruction of the cells set in after the 48th hour of infection. In the cultivation of strain MK in cell cultures containing 5-iod-2-desoxiuridine no disturbances in the normal cycle of reproduction was observed. The virus replicated equally well both with and without the antimetabolite. In the cultivation of the virulent strain 2 in cell cultures that contained 5-iod-2-desoxiuridine a coarse, granular antigen was noticed in the cytoplasm of single cells after the eighth hour of infection. After the 12th hour the nuclei of these cells manifested slight flourescence (hardly visible single granules). After the 48th hour the nuclei of the involved cells were swollen, and the cytoplasm presented slight, diffuse fluorescence, some of the cells being destroyed.

[Action of rifampicin on the reproduction of Aujeszky's disease virus]. / Deistvieto na rifampitsina vurkhu razmnozhenieto na virusa na bolestta na Aueski

The study on the effect of rifampicin on the reproduction of Aujeszky's disease virus (strains 2, 5, 7, 10, 12, and 17, which are virulent, and strain MK, which is an avirulent virus mutant) in cell cultures revealed that the formation of plaques is inhibited, though to a slighter extent, at a concentration of the inhibiting agent of 100 mug/ml, the value of their titer lowering by one logarithm. All virus strains have proved equally susceptible to the effect of rifampicin. During the virus multiplication in the presence of rifampicin the diameter of the plaques formed decreases from 2.8 to 0.5 mm, and with the avirulent strain MK it decreases up to 0.2 mm. They have rose tint in contrast to the plaques formed by strain 2, which are colourless. Rifampicin has no effect on virus reproduction when the cells are cultivated 24 hours prior to adding the antibiotic or when the virus adsorption on the cells takes place simultaneously with rifampicin. At lower rates the latter does not alter the reproduction process of the virus, however, at doses higher than 10 mug/ml it becomes toxic for the cells. Rifampicin inhibits the multiplication of the virus if it is added to the infected cell cultures up to the 12th hour following the adsorption of the virus. When 25-diacetyl-rifampicin is added to the nutrient medium the number of the plaques formed decreases but no change in their diameter occurs. However, with the avirulent MK strain both the number of plaques and their size decreases. Rifampicin B and rifampicin SV have no effect on the reproduction of Aujeszky's disease virus.

[Use of the direct immunofluorescence method for determination of the TGE viral antigen in swine]. / Izpolzvane na prekiia imunofluorestsenten metod za ustanoviavane virusa na TGE po svinete

Followed up were the appearance and accumulation of the transmissive gastroenteritis (TGE) virus antigen in infected cells of pig kidney and thyroid. Used were specific conjugates, such introduced by the authors and some imported from Czechoslovakia. The cell cultures were infected with strains of the TGE virus, isolated in Bulgaria or supplied from Czechoslovakia. It was found that the Bulgarian strains were the same as those from Czechoslovakia and the United States. A viral antigen was found as early as the sixth hour following the infection of the cell cultures and it appeared under the form of cytoplasmatic yellow-green fluorescence. By the 12th, and up to the 18th hour the number of the specifically fluorescing cells as well as the intensity of fluorescence itself increased. Highest values in this respect were noticed in the interval between the 36th and 48th hour after the cultures had been infected.

[Enzyme activity in cell cultures infected with herpesvirus suis]. / Enzymaktivität bei mit Herpesvirus suis infizierten Zellkulturen

The activity of succinate dehydrogenase (SDH) and lactage dehydrogenase (LDH) was studied in chick-embryo fibroblast cultures after inoculation of the virulent strain "A2" and the avirulent strain "MK" of herpesvirus suum. Strain "A2" reduced SDH activity, and so did strain MK, but here the decrease of enzyme activity was slower, and it did not become evident until the 24th hour. LDH activity fluctuated after "A2" infection but was generally increased, while there was no change in LDH activity, compared with uninfected control cells, after "MK" infection. When interaction of cell and virus took place in the presence of 5-iodo-2-desoxyuridine (IUDR), strain "A2" produced little change in the enzymes, but "MK" infection was accompanied by a definite fall in SDH and a slight increase in LDH. The presence of IUDR inhibited the proliferation of the virulent strain but had no apparent effect on proliferation of the attenuated strain "MK". Investigation of the enzyme activity of cells infected with Aujeszky's disease virus has revealed new biological properties of the virus, which might serve to distinguish between different strains of the virus.