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.     

Antigenic analysis of tick-borne encephalitis complex viruses by time-resolved fluoroimmunoassay with monoclonal antibodies.

The antigenic structure of 5 strains of tick-borne encephalitis (TBE) virus and 7 other viruses of the TBE complex was examined by the highly sensitive and specific technique of time-resolved fluoroimmunoassay (TR-FIA). A collection of 8 monoclonal antibodies to the Austrian strain. Neud?rfl, was used in this study. The findings demonstrate the uniformity of the antigenic structure of TBE viruses from different geographic regions of the USSR. In addition, an epitope was detected which is characteristic of western variants of TBE virus, and another epitope was detected which permits the differentiation of the east-Siberian strain, Aina, from other TBE virus strains. The unique nature of Skalica virus was confirmed, and its similarity, but not identity, to Langat TP-21 virus was shown. Substantial variability in the antigenic structure of some TBE complex viruses was also demonstrated.     

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.     

Identification of individual antigenic epitopes of the envelope protein in the tick-borne encephalitis virus using monoclonal antibodies]

A variant analysis of virus antigens of tick-borne encephalitis complex was carried out by enzyme immunoassays and topographic mapping of this protein using a panel of monoclonal antibodies to the structural glycoprotein of tick-borne encephalitis (TBE) virus of the Far East subtype. The results of the study confirmed the existence on the structural protein of both identical determinants typical of all the antigens of the complex and of subgroup-specific determinants. The topological analysis of the epitopes binding monoclonal antibodies revealed 3 separate domains possessing different functional properties. The results of topological mapping and immune typing of TBE virus antigen were compared.     

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.     

Localization of tick-borne encephalitis virus protein E antigenic determinant recognized by antihemagglutinating monoclonal antibodies using a phage-display peptide library

Bacteriophages bearing peptides reacting with antihemagglutinating monoclonal antibodies (MAb) 10H10 to tick-borne encephalitis (TBE) virus protein E were selected from a phage-display peptide library by affinity selection and enzyme immunoassay. The library contained randomized peptides that are 6 amino acids long, fused with protein pIII and exposed on the surface of the bacteriophage. No significant homology between the sequences of selected peptides and TBE virus protein E was detected. Computer software was created to locate the conformation epitopes on the surface of protein E. Amino acids R73, C74, T76, M77, N103, C105, L107, and S112 were found to form a discontinuous epitope recognized by MAb 10H10. These amino acids are remote in the protein sequence but close in the tertiary structure and form a whole epitope located in the structural domain II of protein E. Presumably the localized amino acids bind to the cellular receptor for TBE virus.     

Serological characteristics of KAMA-51 monoclonal antibodies to the tick-borne encephalitis virus

KAMA-51 monoclonal antibodies to tick-borne encephalitis virus are produced by hybridoma obtained by fusion of splenocytes of mice immunized with this virus with myeloma X-653 cells. The antibody belongs to the IgG class, is active in the immunofluorescence test (IFT), does not react in CFT, and has no antihemagglutinating or neutralizing properties. In the IFT, it reacts with all viruses of the tick-borne encephalitis complex indicating their directivity to the groupspecific determinant of E protein. In the indirect IFT, antibody titres in the culture fluid are within the range of 1: 16-1: 62, in the ascitic fluids 1: 320-1: 640. Because of the wide range of interspecies reactions, KAMA-51 monoclonal antibody may be used for group detection of the tick-borne encephalitis complex viruses.     

The differentiation of viruses of the tick-borne encephalitis complex by means of RNA-DNA hybridization]

Nucleic acid spot hybridization with cloned cDNA of tick-borne encephalitis (TBE) virus, strain Sofjin, was used to differentiate strains of TBE and other flaviviruses. The cDNA probe reacted with strains of TBE and flaviviruses of TBE subgroup with the exception of Powassan virus. The probe did not react with viruses of Japanese encephalitis and Gendue subgroups. The viruses of TBE subgroup and some strains of TBE virus were differentiated from TBE strain Sofjin by thermal stability of RNA-DNA hybrids. Negishi and Louping ill viruses were found to be most closely related to TBE strain Sofjin among viruses of the TBE subgroup.     

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).     

Antigenic analysis of tick-borne encephalitis virus group using monoclonal antibodies and immunofluorescence

The study included 18 monoclonal antibodies (MAb) to E- or NS1-antigens tested by immunofluorescence with tick-borne encephalitis (TBE) complex viruses. MAb were induced to 3 strains of TBE virus: the pathogenic 4072 strain isolated from a patient; the Skalica strain of low pathogenicity; and the Neidorf strain isolated from ticks. According to their reactivity to complex viruses, MAb comprised 3 groups: monospecific for TBE virus (T6, T15) which detected tick-borne encephalitis virus alone; widely cross-reactive with 4-6 viruses of the complex (NEK, KEN, T7, T9); and partially complex-reactive (T11, T12, T13, T33/3) and bound to 2-3 viruses of the complex. T13 and T33/3 MAb reacted with the Omsk hemorrhagic fever virus to the same degree or stronger than with TBE virus. The cross-reactivity was more marked in anti-E-than in anti-NS1 MAb. The similarity of the Langat viruses and the Skalica strain was confirmed. Using anti-NS1 MAb in tests with non-fixed cells, the release of NS1-antigen was found to begin at hour 18 (time of observation). The results of the study may be useful for improvement of laboratory diagnosis of TBE and evaluation of the capacity of a vaccine to induce cross immunity to viruses of the TBE complex.     

Passive immunization of mice with monoclonal antibodies raised against tick-borne encephalitis virus

Summary Adult Balb/c mice were passively immunized with monoclonal antibodies (100 µg/mouse) raised against tick-borne encephalitis (TBE) virus then challenged 24 hours later s. c. with 10 LD₅₀ of TBE virus (Nëudorfl isolate). None of the mice showed evidence of premature death although all except one of the monoclonal antibodies tested are capable of enhancing the infectivity of TBE virus in the Fc receptor-bearing mouse macrophage-like cell line P 388 D1. The ability of monoclonal antibodies to neutralize TBE virusin vitro, and to fix complement was examined, and of these properties only a single monoclonal antibody, which was able to neutralize virus, was also able to protect mice against virus challenge.     

Sequencing of prototype viruses in the Venezuelan equine encephalitis antigenic complex.

The 5' nontranslated region (5'NTR) and nonstructural region nucleotide sequences of nine enzootic Venezuelan equine encephalitis (VEE) virus strains were determined, thus completing the genomic RNA sequences of all prototype strains. The full-length genomes, representing VEE virus antigenic subtypes I-VI, range in size from 11.3 to 11.5 kilobases, with 48-53% overall G+C contents. Size disparities result from subtype-related differences in the number and length of direct repeats in the C-terminal nonstructural protein 3 (nsP3) domain coding sequence and the 3'NTR, while G+C content disparities are attributable to strain-specific variations in base composition at the wobble position of the polyprotein codons. Highly-conserved protein components and one nonconserved protein domain constitute the VEE virus replicase polyproteins. Approximately 80% of deduced nsP1 and nsP4 amino acid residues are invariant, compared to less than 20% of C-terminal nsP3 domain residues. In two enzootic strains, C-terminal nsP3 domain sequences degenerate into little more than repetitive serine-rich blocks. Nonstructural region sequence information drawn from a cross-section of VEE virus subtypes clarifies features of alphavirus conserved sequence elements and proteinase recognition signals. As well, whole-genome comparative analysis supports the reclassification of VEE subtype-variety IF and subtype II viruses.     

Monoclonal antibodies to tick-borne encephalitis (TBE) virus: their use for differentiation of the TBE complex viruses.

Monoclonal antibodies (MoAbs) to Central European tick-borne encephalitis virus (strain Hypr) were used for differentiation of eight viruses of the TBE complex by indirect immunofluorescence. MoAb 11/B3 (in Western blot recognizing 52 and 70 kD polypeptides) reacted with five out of the eight TBE complex viruses, MoAb 13/E5 (anti-52 kD protein) reacted with the western or eastern subtype of TBE virus only, while MoAb 12/G4 (anti-70 kD protein) distinguished the western subtype of TBE virus from the rest of the TBE complex. These three MoAbs were able to differentiate the virulent strain Hypr from attenuated strains Skalica and Hy-HK-18-"3". MoAb 2/10C (anti-56 and 70 kD proteins) which reacted with all viruses of the TBE complex, recognized both virulent and attenuated strains of TBE virus.     

Dual infection of Ixodes ricinus ticks with two viruses of the tick-borne encephalitis complex

Ixodes ricinus female ticks were inoculated with Skalica (SK) virus (non-pathogenic for adult albino mice by subcutaneous route) and 14 days later they were challenged with strain 198 of Tick-borne Encephalitis (TBE) virus (highly pathogenic for adult albino mice by subcutaneous route). After additional 14 days of incubation, 42.9 to 65.0% of the adult (10-12 g) albino mice infested with these double infected ticks developed antibodies to TBE without signs of sickness (transmission of SK virus), while paralysis or death was registered in 35.0 to 57.1% of infested mice (transmission of strain 198) depending on the concentration of strain 198 used for inoculation of ticks. However, a low degree of interference to superinfection with strain 198 was observed, when the dissected tick salivary glands were examined by subcutaneous inoculation of adult albino mice (more than 90% of examined salivary glands contained strain 198 virus).