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.     

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.     

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.     

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.     

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.     

Peroral immunization of adult white mice with the Skalica strain from the tick-borne encephalitis virus complex.

Adult white mice immunized perorally with the infectious Skalica strain from the tick-borne encephalitis (TBE) virus complex did not show any clinical symptoms of illness. 56% of experimental animals immunized with two doses of the Skalica virus (the titer of virus was 6 x 10(10) LD50) were protected against the challenge with the Hypr strain of TBE virus. All mice immunized with the Skalica virus and having haemagglutination-inhibiting antibodies higher than 1:80 survived the challenge with the given dose of virulent TBE virus. No differences in the immunogenicity and protectivity were observed in experimental animals infected with infectious Skalica virus by oesophageal probe, or by drinking virus-containing medium. A higher protective activity against the virulent Hypr virus was observed in adult white mice immunized subcutaneously with the Skalica 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.     

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).     

Development of an enzyme-linked immunosorbent assay for serological diagnosis of tick-borne encephalitis using subviral particles

The similarity of symptoms produced by tick-borne encephalitis (TBE) and Japanese encephalitis (JE) and the high degree of cross-reactivity between TBE and JE viruses by serological tests make the development of a differential diagnostic test a priority. In this study, recombinant prM/E proteins of TBE virus strain Oshima 5-10 expressed in mammalian cells resulted in the release of subviral particles (SPs) into the culture medium. Using the SPs as antigens, enzyme-linked immunosorbent assay (ELISA) systems were developed to detect TBE virus-specific IgM and IgG antibodies, designated SP-IgG and SP-IgM ELISAs, respectively. Of 83 serum samples from encephalitis patients in Khabarovsk, Russia, which were positive with the neutralization test (NT), 82 were positive by the SP-IgG ELISA, for a sensitivity of 98.8%, which was higher than that of a commercial ELISA kit. All 12 NT-negative samples were also negative by the SP-IgG ELISA (specificity, 100%). Of 17 patient samples that were NT-positive, 16 (94.1%) were positive by the SP-IgM ELISA. Of 15 paired serum samples that yielded equivocal results by NT, 11 had positive results with the SP-IgM ELISA, indicating a diagnosis of TBE infection. The SP-IgG and SP-IgM ELISAs showed no cross-reactivity with antibodies to the JE virus. The results indicate that these ELISAs will be useful for the detection of TBE-specific antibodies.     

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.     

Analysis of the complete genome of the tick-borne flavivirus Omsk hemorrhagic fever virus

Omsk hemorrhagic fever virus (OHF) is a tick-borne flavivirus endemic to Western Siberia. This virus is the only known tick-borne flavivirus to cause hemorrhagic disease in humans in the absence of encephalitis. OHF virus circulates within a small, defined niche in which other tick-borne complex flaviviruses are also present. The objectives of this study were to genetically classify OHF virus based on its complete genome and to identify genetic determinants that might be involved in tissue tropism and viral replication leading to the disease state caused by this virus. The OHF virus genome was sequenced and phylogenetic analysis demonstrated that OHF virus falls within the tick-borne encephalitis serocomplex of flaviviruses, yet is distinct from other members of the complex, including those closely associated geographically. OHF is also distinct from Alkhurma (ALK) and Kyasanur forest disease (KFD) viruses, both of which cause disease that includes hemorrhagic and encephalitic manifestations. Several amino acid residues were found to be distinct among OHF, KFD, and ALK viruses; these residues include E-76, which is closely associated with the viral envelope protein fusion peptide. In addition, variation between the viral 5'-untranslated region of OHF and other tick-borne flaviviruses suggests potential variability in viral replication. These data demonstrate that OHF is a unique virus among the tick-borne flaviviruses and also provide insight to viral biodiversity and tropism.     

Studies on the effect of 9-(R, S)-(2,3-dihydroxypropyl)adenine on tick-borne encephalitis virus

Peroral treatment with 9-(R, S)-(2,3-dihydroxypropyl) adenine did not protect mice infected with tick-borne encephalitis (TBE) viruses (strains Hypr and Ir13) when the drug was administered at the time or infection and/or up to day 2 after infection. The analogue inhibited the haemagglutination activity of the Skalica strain in vitro.     

A case of tick-borne encephalitis in Japan and isolation of the the virus.

A case of tick-borne encephalitis (TBE) has not been reported for many years in Japan, although a serological survey of sera from domestic animals suggested the presence of TBE foci in Hokkaido, the northern island of Japan. Studies were conducted to prove the presence of an endemic focus of TBE virus in Japan by means of serology and virus isolation. In October 1993 in Hokkaido, a severe case of encephalitis in a dairy farmer's wife was diagnosed as TBE. Serological examination of paired serum specimens showed a rise in the neutralization antibody titer to Russian spring summer encephalitis virus. A seroepizootiological survey of dogs showed that the TBE-related virus was prevalent in the area. Three virus isolates were obtained from the blood of sentinel dogs, and antigenic analysis grouped the isolates into TBE-related viruses. Sequence analysis of the envelope protein gene identified one of the isolates as being of the same subtype as the Russian spring summer encephalitis (Far Eastern TBE) virus. The results provide evidence that TBE is endemic in a certain area of Japan.     

The specific reactivity of cDNA and deoxyoligonucleotide probes, complementary to the tick-borne encephalitis virus genome, with the RNA of strains of different geographical origins]

Hybridization experiments with RNA of 143 tick-borne encephalitis (TBE) virus strains isolated in different parts of the distribution area were used to study the reactivity of kDNA- and a set of 10 synthetic deoxyoligonucleotide probes. The kDNA probe under certain conditions was shown to hybridize with RNA of all the strains under study, and under other (strict) hybridization conditions did so selectively with a small number of strains. The capacity of oligonucleotide probes for hybridization with RNA of TBE virus strains varied from 12% to 100%. The differences in the hybridization activity of kDNA- and oligonucleotide probes complementary to the genomes of the Sophyin strain (Far-Eastern subtype) and Neudorffle strain (Western subtype) with TBE virus strains were used for differentiation of the strains into six genetic variants. Comparison of the reactivity of molecular probes in experiments with RNA of TBE virus strains and viruses of the TBE complex showed that the differences of the strains belonging to different genetic variants from the prototype Sophyin strain were comparable to those of some members of the TBE complex, with the exception of Powassan virus. These data attest to the necessity of further studies dealing with specification of the taxonomy of TBE complex viruses.     

Differentiation in the complement fixation test of the viruses of tick-borne encephalitis complex by means of a type-specific soluble antigen

Highly type-specific soluble antigens (SA) of viruses of the tick-borne encephalitis (TBE) complex were obtained by treatment of the sucrose-acetone antigens with 8 mol/l urea. Each SA reacted with the homologous serum only. The serum to SA of strain Sofyin and of Omsk haemorrhagic fever virus distinguished the TBE complex representatives; the difference between homologous serum titres was in the range of 4-5 two-fold dilution steps.     

Serological monitoring of arbovirus infections in the estuary of the Kuban River (the 2006-2007 data)

Solid-phase enzyme immunoassay, neutralization test, and the hemagglutination-inhibition test were used to study the sera from human beings (152 samples), agricultural animals (n = 77), hares (n = 3), and wild birds (n = 69), collected in 2006-2007 in the Kuban River estuary (Temryuk District, Krasnodar Territory). There were specific antibodies against viruses of West Nile (WH), tick-borne encephalitis (TBE) (Flaviviridae, Flavivirus), Sindbis (Togaviridae, Alphavirus), the antigenic complex of California, Batai (Bunyaviridae, Orthobunyavirus), Dhori (Orthomyxoviridae, Thogotovirus). The findings suggest the presence of arboviruses from 6 transmitting mosquitoes and ticks in the study area and human infection by the viruses of the antigenic complex of California (20-47%), Batai (3-15%), West Nile (3-12%), Dhori (2%). The index agricultural animals (horses, cattle) were observed to have specific antibodies to the viruses of WN (8-15%), TBE (0-2%), Sindbis (2-9%), the antigenic complex of California (27-54%). Out of the representatives of the wild fauna, virus-neutralizing antibodies to Sindbis virus were found in European hares (Lepus europaeus), California complex virus in gulls (Larus argentatus) and terns (Sterna hirundo), WN and Sindbis viruses in herons (Ardea purpurea), and WN and California complex viruses in bald-coots (Fulica atra).     

Pathogenicity of the skalica strain (from the tick-borne encephalitis complex) for white mice

Outbred white mice of different age were inoculated by subcutaneous (s. c.) route with the Skalica strain of tick-borne encephalitis virus (TBE). In the CNS of 3-days-old mice diffuse necrotizing encephalitis, abundant cytoplasmic fluorescence of viral antigen in nearly each neuron and high levels of virus (8.8 log LD50/mg tissue) were found. In 10-days-old mice, the extent of encephalitis and that of immunofluorescence in neurons were less widespread; the peak titre of the virus did not exceed 5.5 log LD50/mg brain tissue. In the CNS of 21-days-old mice the infectivity titre was either very low (1.5 log50/mg on day 3 post infection) or the virus was not detected at all. A few neurons revealed positive fluorescence of viral antigen in the basal ganglia in 1 out of 2 mice examined by day 3 post infection (p. i.). No virus was isolated from the CNS of 2-months-old mice observed for 53 days. In the CNS of 3 out of 10 juvenile mice examined histologically within 8 days post infection, minimal inflammatory changes were seen; foci of neurons showing positive immunofluorescence were not found. The failure to recover infectious virus from cultured brain tissue fragments coming from these mice confirmed the negative outcome of direct virus isolations. It is concluded that the Skalica strain was not pathogenic for juvenile mice when administered by s. c. route.     

Vaccines, immunoglobulins, and test systems for the prevention and diagnosis of tick-borne encephalitis

The quality of drugs used in the Russian Federation to prevent and diagnose tick-borne encephalitis (TBE) was analyzed. The TBE vaccines made in Russian were shown to be as effective as those manufactured in foreign countries and to have the similar production techniques and quality indices therefore they may be interchangeable in the population's vaccination. The specific activity of has recently increased in terms of the content of TBE virus antibodies. In Russia, a wide range of enzyme immunoassay systems have been designed and manufactured for the detection of human serum (plasma) IgM and IgG antibodies to TVE virus and for that of TBE viral antigen in the biological objects, primarily in the ticks.     

Antigenic classification and taxonomy of flaviviruses (family Flaviviridae) emphasizing a universal system for the taxonomy of viruses causing tick-borne encephalitis

For many years the expression "tick-borne encephalitis (TBE) virus" has been used to denote viruses causing the clinical entity TBE. No virus with the name "TBE virus" has been registered in the International Catalogue of Arboviruses, the generally accepted reference for naming arthropod-borne viruses, and no formal agreement has been reached within the scientific community to use the expression "TBE virus"; this term is inaccurate for indicating the etiologic agent of the disease TBE and confuses students and others regarding virus ecology, transmission, and disease severity. This paper suggests alternative names for viruses of the antigenic complex of flaviviruses (family Flaviviridae) to which viruses causing TBE belong. A classification scheme and system of taxonomy for flaviviruses is also proposed. It is suggested that universal acceptance of the term Central European encephalitis virus would improve the dialogue between laboratory virologists and clinicians and improve the understanding of the epidemiology of the tick-borne flaviviruses.