Serological evidence of Hantaan virus in Slovakia during 1989-1991.

Nephropathia epidemica was supposed to be an European form of haemorrhagic fever with renal syndrome (HFRS). However, in 1989-1991 we found serological evidence of infection with Hantaan virus in patients' sera from Slovakia. Higher titers of antibodies against Hantaan virus were detected in sera of 6 out of 75 patients with febrile disease, renal failure and clinical diagnosis of HFRS. The higher titers of antibodies against Puumala virus were detected in sera of 6 out of 75 patients. Serologic differentiation between Hantaan and Puumala infection in patients was based on the detection of higher antibody titers (4-fold or greater differences).

[Vaccination against tick-borne encephalitis in the Central Slovakia Region using a Soviet vaccine]. / Ockovanie proti kliestovej encefalitide sovietskou vakcínou v Stredoslovenskom kraji.

During the period between 1986 and 1989 in the region 1311 people working in forestry were vaccinated against tick-borne encephalitis by the inactivated Soviet vaccine. Only some of the people had prevaccination examinations only in some reactogenity of the vaccine was investigated as well as the persistence of humoral antibodies one and two years after the basic vaccination. These examinations in some of the vaccinated subjects were made in autumn 1986 and spring 1987. After two doses of vaccine in the haemagglutination-inhibition test (HIT) the seroconversion was 77% (of 182 evaluated) with a geometric mean of 37.9. One year after the primary vaccination in 53% of the examined subjects the titre of humoral antibodies dropped to negative values. Two years after the primary vaccination in the HIT antibodies were detected in 44% of 57 examined subjects. None of the immunized subjects contracted tick-borne encephalitis.

The complex of flavivirus envelope polypeptide with meningococcal proteosomes elicits formation of virus-neutralizing antibodies.

Polypeptide E of tick-borne encephalitis virus was isolated in sucrose density gradient and mixed with equal weight portion of meningococcal proteosomes in the presence of N-dodecyl-N,N-dimethylglycine. Mutual complexing of viral and bacterial molecules occurred after removal of detergent by dialysis. Complexed particles appeared in the electron microscope as 40-50 microns thick short-rod structures covered on their surface with both, delicate poppy-like grains, or envelope subunit-like clustered molecules. Even when applied without adjuvant, the complex of tick-borne encephalitis virus polypeptide E with meningococcal proteosomes elicited in mice a marked antiviral as well as antibacterial humoral response.

Detection of antibodies to the western and the eastern type of haemorrhagic fever with renal syndrome in patient's sera from Slovakia.

We present serological evidence of infection with western and eastern type of haemorrhagic fever with renal syndrome (HFRS) virus in patient's sera from Slovakia by indirect immunofluorescence. Treatment of sera with 2-mercaptoethanol decreased levels of haemagglutination-inhibition (HI) antibodies to the eastern type of HFRS suggesting primary infection of the patients by this virus.

Comparison of skalica, Hypr, and Langat viruses by kinetic haemagglutination-inhibition test.

The Skalica virus has been compared with Hypr and Langat viruses by kinetic haemagglutination-inhibition (HI) test. Using Hypr antigen, differences were observed between Skalica, Hypr, and Langat viruses. By the use of Skalica antigen, a close relationship between Hypr and Skalica viruses was detected, however, it was possible to differentiate the Langat virus. When Langat antigen was tested, a close relationship among all the three viruses was found.

To the antigenic classification of some viruses from the tick-borne encephalitis complex by monoclonal antibodies.

Two monoclonal antibodies (MoAb) to the Skalica virus from the tick-borne encephalitis (TBE) complex were used to compare Karshi and Royal Farm viruses with the Russian spring-summer encephalitis, Central European encephalitis (Hypr and Kumlinge strains) Skalica, Langat and Powassan viruses. The first MoAb was prepared by fusion of P3NS1 cells with BALB/c mouse spleen cells, immunized with the Skalica virus; it was of IgM class and reacted in haemagglutination-inhibition (HI) test (MoAb type 1). The second MoAb was of IgG class and reacted in complement-fixation (CF) test (MoAb type 2). MoAb type 1 reacted in the HI test with Russian spring-summer encephalitis (RSSE), Central European encephalitis (CEE) virus strains, Skalica and Langat viruses. No reaction was observed with Powassan, Karshi, and Royal Farm viruses. MoAb type 2 reacted in the CF test with all members of tick-borne encephalitis complex except the Powassan, Karshi, and Royal Farm viruses.

[Isolation of tick-borne encephalitis viruses from the cervical spinal cord in meningoencephalitis]. / Izolácia vírusu kliest'ovej encefalitídy z krcnej miechy pri meningoencefalitíde.

In 1988 the organs of a male were examined post mortem for the presence of viruses in isolation experiments on white suckling mice. The virus isolated from the cervical spinal cord was identified as the virus of tick-borne encephalitis. Although death from tick-borne encephalitis is rather rare in Slovakia, the disease requires great attention with special focus on preventive measures.

A simple method of preparing a complement-fixing antigen from the virus of haemorrhagic fever with renal syndrome (Western type).

Different methods of the preparation of haemorrhagic fever with renal syndrome (HFRS) antigen for complement-fixation (CF) test are described. The antigens were prepared from the organs of suckling white rats inoculated with the Western type of HRFS by precipitation with polyethylene glycol, by fluorocarbon treatment and or by sucrose-acetone extraction. The highest CF titre was obtained by acetone precipitation of 20% brain suspension in isotonic sucrose.

Detection of the antigen and antibodies to the eastern subtype of haemorrhagic fever with renal syndrome virus in small rodents in Slovakia.

Direct enzyme-linked immunosorbent assay (ELISA) was used for the demonstration of haemorrhagic fever with renal syndrome (HFRS) virus antigen in lung tissue of small rodents trapped in Eastern and Western Slovakia. The eastern subtype of HFRS virus antigen was demonstrated in the lungs of Apodemus agrarius and of the western subtype in the lungs of Microtus arvalis. Antibodies to HFRS virus antigen have been detected in Apodemus species (A. agrarius and A. flavicollis) in higher titres to the Eastern subtype.

Antigenic variation of the viruses belonging to the tick-borne encephalitis complex as revealed by human convalescent serum and monoclonal antibodies.

Solid-phase enzyme-linked immunoassay (ELISA) was used for the detection of antigenic relationships and/or differences among the viruses belonging to the tick-borne encephalitis (TBE) complex. Monoclonal antibodies of IgM class with haemagglutination-inhibiting activity to the Skalica strain of TBE virus were used to compare the TBE complex viruses. Antigenic analysis of 9 viruses of the TBE complex, isolated from Eurasia and America showed close relationships among them. Nevertheless, it was possible to differentiate the Skalica strain from Langat, louping-ill and Omsk haemorrhagic fever (OHF) viruses by ELISA when monoclonal antibodies and antigens were diluted 1:10,000. Monoclonal antibodies to the Russian spring-summer encephalitis virus did not react with the Skalica strain in immunofluorescence test. By the use of convalescent serum no reaction was found with louping-ill, Russian spring-summer encephalitis, Powassan and OHF viruses in haemagglutination-inhibition (HI) test.

Studies on the ecology of tick-borne encephalitis virus in the Carpathian and Pannonian types of natural foci.

During the years 1972-1982, the role of Ixodes ricinus ticks was studied as vectors of tick-borne encephalitis (TBE) virus in the Carpathian and Pannonian types of natural foci. The proportion of TBE virus-infected Ixodes ricinus ticks in the Carpathian elementary foci varied from 0.37 to 4.1 per cent, while in the Pannonic elementary foci it ranged from 0.07 to 6 per cent, respectively. TBE virus was isolated from organs of small rodents in both the Carpathian and Pannonian types of natural foci. A correlation was established between the percentage of infected ticks and the proportion of seropositive inhabitants.

Preparation and characterization of hybridomas secreting monoclonal antibodies to tick-borne encephalitis virus.

Monoclonal antibodies (MA) were prepared to two strains of tick-borne encephalitis (TBE) virus: strain 4072 isolated from a patient in the U.S.S.R. and low-pathogenic for mice strain Skalica, isolated from a bank vole (Clethrionomys glareolus) in Slovakia. MA specific to the 4072 and Skalica strains were produced by hybridomas of the KEN (60 clones) and NEK (65 clones) series, respectively. Chromosomal analysis of MA producing 114 hybridoma clones of both series revealed a great variability in the number of chromosomes either in the range of given clones or between individual clones. The hybridoma cells under study possessed a high degree of transformation manifested by good growth in the mouse peritoneal cavity and marked accumulation in ascitic fluid (AF).

Two kinds of monoclonal antibodies to tick-borne encephalitis virus.

Two types of monoclonal antibodies (MA) of the KEN and NEK series prepared to tick-borne encephalitis (TBE) virus differed in the spectrum of their reactivity in serological tests and in their ability to react with individual representatives of the TBE virus complex. The KEN series MA were induced to the 4072 strain isolated from the blood of a patient in the U.S.S.R. The NEK series MA were prepared to the Skalica strain isolated from a bank vole in Czechoslovakia. Both groups of MA belonged to IgG class, reacted in immunofluorescence (IF) test, but possessed no haemagglutination inhibiting (HI) activity. MA of the NEK series reacted in the IF and complement fixation (CF) tests with all members of the TBE virus complex, except of the Powassan virus. MA of the KEN series had no CF activity and in the IF test, they did not react with Powassan and Langat TP-21 viruses and with the Skalica strain of TBE virus.

Studies on preparation of a tick-borne encephalitis (TBE) vaccine from the Skalica strain.

Diethylether-treated vaccine against tick-borne encephalitis (TBE) represents a new type vaccine consisting of lipid-free and antigenically efficient components instead of whole virus particles. The TBE virus strain designated Skalica was used for vaccine preparation. This strain is thermosensitive, produces small plaques under agar overlay, is nonpathogenic for adult white mice following subcutaneous (s.c.) application and causes threshold viraemia in host animals. The vaccine was harmless and immunogenic as evidenced by experiments on white mice. Antibodies to TBE virus strain Ir 13 present in human healthy population of a natural TBE focus showed similar levels when tested with the Skalica strain. The Skalica virus strain can be recommended for preparation of the vaccine against TBE.

Serological survey with the antigen of haemorrhagic fever virus with renal syndrome in small rodents in Slovakia.

Results of serological survey in small rodents with the antigen of haemorrhagic fever with renal syndrome (HFRS) virus indicate the existence of a natural focus of this virus in Slovakia. Antibodies were found in the following free-living rodent species: Clethrionomys glareolus, Apodemus agrarius, Apodemus sylvaticus, Pitymys subterraneus, Microtus arvalis and Microtus economus. A total 10 out of 120 (8.2%) small rodents trapped in investigated localities of Western and Eastern Slovakia had antibodies to the HFRS virus antigen as detected by complement fixation (CF) and/or immunofluorescence (IF) tests.

Role of macrophages in the pathogenesis of experimental tick-borne encephalitis in mice.

In vivo phagocytosis activity of macrophages (PAM) was temporarily suppressed in mice by application of a suspension of microscopic from particles. As demonstrated, a reversible block of 70% of PAM was accompanied by a marked increase of the lethality during the acute tick-borne encephalitis (TBE) virus infection. Asymptomatic persistence of TBE virus in the brain was 4 times more frequent in mice with PAM defect than in immuno-competent mice. Suppression of PAM during the first 48 hr post infection (p.i.) did not affect interaction of B-, T-lymphocytes and macrophages. Cytotoxic activity of splenocytes against TBE virus-infected mouse embryo fibroblasts (MEF) was alike irrespective of whether cytotoxic cells were collected from mice inoculated or not inoculated with microscopic iron suspension. Similarly, frequency of seroconversion did not differ in these groups of mice. Adoptively transferred peritoneal macrophages from TBE virus-infected or intact mice did not exert any protective activity. The presence of splenic macrophages was necessary neither in adoptive immunity transfer in vivo nor in cytotoxic activity of T-lymphocytes directed to virus-infected targets in vitro. It was further found that peritoneal macrophages (PM) both from TBE virus immunized and non immunized donors in the presence of antibodies to TBE virus acquired the capability to kill TBE virus-infected target cells. Antibody-dependent cytotoxicity (ADC) of macrophages was associated with population of phagocytic cels.(ABSTRACT TRUNCATED AT 250 WORDS)

Haemorrhagic fever virus with renal syndrome in small rodents in Czechoslovakia.

The antigen of haemorrhagic fever with renal syndrome (HFRS) was detected in the lungs of the following free-living small rodents trapped in different localities in Eastern Slovakia: Clethrionomys glareolus (2 positive samples of 7), Apodemus flavicollis (1 sample of 24) and Apodemus agrarius (7 positive samples of 66). The virus was first identified by indirect fluorescent antibody (FA) staining using human convalescent serum from a case of epidemic nephropathy (NE) of Scandinavia. The lung suspensions selected according to positive immunofluorescence were inoculated by intramuscular (i.m.) route into suckling rats; the antigen prepared from the lungs of these rats by sucrose-acetone extraction reacted with the prototype human serum in complement-fixation (CF) reaction. The results of the latter assay were in good agreement with those of the indirect FA staining.

Purification of tick-borne encephalitis virus by affinity chromatography using monoclonal antibody.

The application of a simple technique for purification of tick-borne encephalitis (TBE) virus is described. TBE virus was grown in chick embryo cell (CE) cultures and the virus was concentrated by differential centrifugation. Final purification was made by the filtration through Sepharose column to which monoclonal antibodies to TBE virus had been bound. The method was effective in eliminating avian retroviruses.

Antigenic relationships among viruses of the tick-borne encephalitis complex as studied by monoclonal antibodies.

Tick-borne encephalitis (TBE) monoclonal antibodies showed haemagglutination-inhibiting (HI) activity against viruses belonging to the TBE complex except of Powassan virus. The HI titre with Kyasanur forest disease virus was lower than with tick-borne encephalitis virus, when monoclonal antibodies were incubated with the antigen at +4 degrees C for 30 min. Langat virus could be distinguished from other viruses of the TBE complex when the antigen was incubated with monoclonal antibodies at +4 degrees C for 18 hr. A close antigenic relationship was demonstrated between tick-borne encephalitis, louping-ill, Negishi and Omsk haemorrhagic fever viruses.