Phylogenetic analysis and pathogenicity of tick-borne encephalitis virus from Japan and far-east Russia

Phylogenetic analysis of tick-borne encephalitis (TBE) virus revealed that Hokkaido strain of TBE virus evolved several hundreds years ago in far-east Russia. TBE virus strains in Irkutsk area were identified as Siberian subtype of TBE virus. BHK-cell adapted mutant of TBE virus showed lower neuro-invasive virulence in mice than parent virus. The mutant carried one amino acid substitution in envelope protein which resulted in increase of positive charge of the protein. The mutant-infected mice showed lower virus titers in bloods and spleens than the parent-infected mice. Infectious c-DNA clone of TBE virus Hokkaido strain was successfully generated and was applied to examine the neurovirulence in mice. One amino acid change in envelope protein and 2 amino acid changes in Ns5 protein showed a synergistic effect on reduced neurovirulence in mice.     

Tick-borne encephalitis virus in northern Italy: molecular analysis, relationships with density and seasonal dynamics of Ixodes ricinus

Ixodes ricinus ticks were collected from dragging vegetation and from shot roe deer in the province of Trento and Belluno in northern Italy. Ticks were pooled for analyses and from 1060 pools of ticks collected in the province of Belluno and 12390 tick samples collected in Trentino, four proved positive by immunofluorescence microscopy using a tick-borne encephalitis (TBE)-specific antiserum. The identity of the virus isolates was determined by RT-PCR cycle sequencing and they were all found to be closely similar (> 98% nucleotide identity) to typical western European TBE complex viruses as found in Austria. The isolates from Trentino differed from the Neudorfl strain of western European TBE virus at eight nucleotide positions but as these nucleotide substitutions were all synonymous, there were no amino acid changes. These results imply that the virus isolates in Trentino have changed slightly from the typical European strains isolated in nearby Austria. The abundance of questing ticks and ticks feeding on roe deer was greater in TBE positive hunting districts than in hunting districts where TBE complex viruses were only probable or believed to be absent. In TBE positive and probable districts synchrony in the seasonal dynamics of larvae and nymphs of L. ricinus was observed. This study provides evidence to suggest that roe deer may have an important role to play in the maintenance of tick density and in the persistence of TBE virus.     

Localization of the antigenic segment of the tick-borne encephalitis virus envelope protein using monoclonal antibodies]

The largest cyanogen bromide fragment (GP-14,5; coordinates 78-176) of E protein belonging to the envelope of the tick-borne encephalitis (TBE) virus (Far Eastern subtype, strain Sofjin) interacted with five out of twelve E-specific monoclonal antibodies (MAbs). Having compared; efficiencies of some MAbs binding to the antigens of TBE viruses of Far Eastern and West European subtypes and primary structures of analogous peptides of these viruses, we suggested the epitopes of these MAbs to be located in the vicinity of 89 and/or 116-th amino acid residues of E protein. Effect of denaturing agents and reduction followed by carboxymethylation on the protein E antigenic properties was studied.     

Attenuation of Tick-Borne Encephalitis Virus Using Large-Scale Random Codon Re-encoding

Large-scale codon re-encoding (i.e. introduction of a large number of synonymous mutations) is a novel method of generating attenuated viruses. Here, it was applied to the pathogenic flavivirus, tick-borne encephalitis virus (TBEV) which causes febrile illness and encephalitis in humans in forested regions of Europe and Asia. Using an infectious clone of the Oshima 5–10 strain ("wild-type virus"), a cassette of 1.4kb located in the NS5 coding region, was modified by randomly introducing 273 synonymous mutations ("re-encoded virus"). Whilst the in cellulo replicative fitness of the re-encoded virus was only slightly reduced, the re-encoded virus displayed an attenuated phenotype in a laboratory mouse model of non-lethal encephalitis. Following intra-peritoneal inoculation of either 2.10⁵ or 2.10⁶ TCID50 of virus, the frequency of viraemia, neurovirulence (measured using weight loss and appearance of symptoms) and neuroinvasiveness (detection of virus in the brain) were significantly decreased when compared with the wild-type virus. Mice infected by wild-type or re-encoded viruses produced comparable amounts of neutralising antibodies and results of challenge experiments demonstrated that mice previously infected with the re-encoded virus were protected against subsequent infection by the wild-type virus. This constitutes evidence that a mammalian species can be protected against infection by a virulent wild-type positive-stranded RNA virus following immunisation with a derived randomly re-encoded strain. Our results demonstrate that random codon re-encoding is potentially a simple and effective method of generating live-attenuated vaccine candidates against pathogenic flaviviruses.     

In vitro study of macrophage barrier function in mice vaccinated against tick-borne encephalitis and infection with flaviviruses of varying virulence

Tick-borne encephalitis (TBE) and Langat viruses were shown to be equally capable of multiplication in mouse peritoneal macrophages (PM) in vitro. The reproduction dynamics of TBE virus proved to be the same in PM of mice both highly sensitive and relatively less sensitive to TBE virus. The preliminary immunization of PM donors with commercial inactivated or experimental concentrated TBE virus vaccine produced no effect on the capacity of the virus for multiplication in PM. These facts indicate the absence of correlation between the capacity of the viruses under study for multiplication in PM in vitro and their virulence in vivo, as well as the insignificant role of circulating macrophages in the realization of the barrier function in an immune or nonimmune body.     

森林脑炎自然疫源地样本的监测及病毒的分离研究

为了解森林脑炎疫源地的分布变化趋势及样本分离病毒的特性,采集了森林脑炎高发区周边的森林全沟硬蜱、血蜱样本及森林脑炎患者的脑组织样本,用小白鼠脑内接种法检测、分离病毒分离的病毒经鉴别试验证明为森林脑炎病毒：蜱、脑两种标本检测的阳性率分别为50％和100％、结果表明森林脑炎的疫区有从林区向农业区扩散的趋势,且全沟硬蜱的带毒率较高;森脑患者的脑组织样本与蜱标本病毒的性状育差异     

Production and characterisation of monoclonal antibodies to an Indian isolate of peste des petits ruminants virus

Nine monoclonal antibodies (Mabs) were produced against an Indian isolate of peste des petits ruminants (PPR) virus. These Mabs were directed against the nucleo (N) protein and were of IgG1 isotype. The Mabs produced intranuclear or coarse granular cytoplasmic fluorescence in PPR virus infected Vero cells and did not exhibit any neutralising activity. The Mabs cross-reacted with five other local isolates of PPR virus in slot blot hybridisation, radio immunoprecipitation assay (RIPA) and fixed-cell enzyme linked immunosorbent assay (ELISA). Two of the nine Mabs cross-reacted mildly with the vaccine strain of rinderpest (RP) virus in slot blot hybridisation and fixed-cell ELISA but did not precipitate the N protein of RP virus in RIPA. The N protein specific Mabs will be highly useful in differential diagnosis of PPR from RP.     

PB2 Protein of a Highly Pathogenic Avian Influenza Virus Strain A/chicken/Yamaguchi/7/2004 (H5N1) Determines Its Replication Potential in Pigs

It has been shown that not all but most of the avian influenza viruses replicate in the upper respiratory tract of pigs (H. Kida et al., J. Gen. Virol. 75:2183-2188, 1994). It was shown that A/chicken/Yamaguchi/7/2004 (H5N1) [Ck/Yamaguchi/04 (H5N1)] did not replicate in pigs (N. Isoda et al., Arch. Virol. 151:1267-1279, 2006). In the present study, the genetic basis for this host range restriction was determined using reassortant viruses generated between Ck/Yamaguchi/04 (H5N1) and A/swine/Hokkaido/2/1981 (H1N1) [Sw/Hokkaido/81 (H1N1)]. Two in vivo-generated single-gene reassortant virus clones of the H5N1 subtype (virus clones 1 and 2), whose PB2 gene was of Sw/Hokkaido/81 (H1N1) origin and whose remaining seven genes were of Ck/Yamaguchi/04 (H5N1) origin, were recovered from the experimentally infected pigs. The replicative potential of virus clones 1 and 2 was further confirmed by using reassortant virus (rg-Ck-Sw/PB2) generated by reverse genetics. Interestingly, the PB2 gene of Ck/Yamaguchi/04 (H5N1) did not restrict the replication of Sw/Hokkaido/81 (H1N1), as determined by using reassortant virus rg-Sw-Ck/PB2. The rg-Sw-Ck/PB2 virus replicated to moderate levels and for a shorter duration than parental Sw/Hokkaido/81 (H1N1). Sequencing of two isolates recovered from the pigs inoculated with rg-Sw-Ck/PB2 revealed either the D256G or the E627K amino acid substitution in the PB2 proteins of the isolates. The D256G and E627K mutations enhanced viral polymerase activity in the mammalian cells, correlating with replication of virus in pigs. These results indicate that the PB2 protein restricts the growth of Ck/Yamaguchi/04 (H5N1) in pigs.     

Arbovirological survey in Silica plateau area, Roznava District, Czechoslovakia

The serosurveys conducted in the Silica plateau area of the Slovak karst region revealed the presence of specific neutralizing antibody against tick-borne encephalitis (TBE) virus in 18% of local inhabitants (33 examined, mostly goats and sheep farmers), 54% of goats (26 examined), 18% of sheep (120 examined) and 13% of cattle (60 examined), against Lipovník (LIP) virus in 30% of inhabitants, 88% of goats, 55% of sheep and 45% of cattle, and against Bhanja (BHA) virus in 27% of inhabitants, 46% of goats, 29% of sheep and 23% of cattle. The results of hemagglutination-inhibition tests with TBE and BHA antigens were analogous. A detailed analysis of these serologic data points to a recent enhancement of the circulation of LIP and BHA viruses and to a very low TBE virus activity in this natural focus of arboviral infections. The immunological surveys of the 32 former "Roznava disease" patients, conducted 25 years after an extensive epidemic of a TBE virus infection that originated in Roznava in 1951, revealed the presence of neutralizing (and also hemagglutination-inhibiting) antibodies against TBE virus in as many as 78% of cases. Antibodies against LIP and BHA viruses were also detectable in the sera of 16% and 9%, respectively, of these individuals. Populations of the ectoparasites examined for the presence of arbovirus comprised 231 Ixodes ricinus, 806 Dermacentor marginatus and 204 Haemaphysalis punctata ticks and 117 specimens of the louse-flies Melophagus ovinus. Two strains of arbivirus that were antigenically related to Lipovník and Tribec viruses belonging to a group of Kemerovo viruses were isolated from male and female I. ricinus ticks collected from cattle.     

First detection of TBE virus sequences in Ixodes ricinus from Friuli Venezia Giulia (Italy)

We report, for the first time, the presence of tick-borne encephalitis (TBE) virus in the tick Ixodes ricinus collected in the Friuli Venezia Giulia region of north-eastern Italy. Using molecular methods, we demonstrate that the TBE virus carried by ticks from FVG is a western European strain. Sequence analysis of the 5' NCR showed 98.4% identity to the Neudoerfl strain.     

Monoclonal antibodies against different epitopes of a 40 Kd capsid protein of infectious bursal disease viruses.

Nine monoclonal antibodies (Mab) against a 40 Kd capsid protein of infectious bursal disease virus (IBDV) strain P3009 were isolated. They were characterized by enzyme-linked immunosorbent assay, indirect fluorescent antibody staining and virus neutralization. They were divided into two groups concerning virus neutralization. Group I Mabs were able to neutralize virus infectivity; however, group II Mabs were not. Competitive binding assays using these Mabs demonstrated the existence of two distinct antigenic regions (A and B) on the 40 Kd protein. Region A was recognized by group I Mabs and region B was by group II Mabs. The binding reaction with group I Mabs was affected by denaturing of the viral proteins, indicating that the antigenic region involving neutralization was conformation-dependent. The results of enzyme-linked immunosorbent assays and virus neutralization tests suggested that group I Mabs might react with one epitope within region A and group II Mabs with 2 or 3 epitopes within region B.     

Mixed infection by tick-borne encephalitis virus and Borrelia in ticks

To investigate the relationships between tick-borne encephalitis (TBE) virus and the bacterial spirochaete Borrelia burgdorferi sensu lato in vectors with mixed infections, unfed adult Ixodes persulcatus ticks were collected by flagging from vegetation in southern-taiga forests of the Pre-Urals region of Russia where both infections circulate sympatrically. Prevalences of TBE and Borrelia infections in a total of 4234 ticks were compared over 5 years. No significant differences were revealed between the prevalence of Borrelia infection in ticks with and without TBE virus (29.4+/-7.8% vs 23+/-3.6%), or between the prevalence of TBE virus infection in ticks with and without Borrelia (24.0+/-6.6% vs 18.4+/-3.4%). In ticks with mixed infection (40/689 = 5.8%), concentrations of TBE virus and Borrelia were not significantly correlated with one another. Field observations showed parallel trends in the prevalence of these pathogens in tick populations from year to year (1993-1997) indicating that, in I. persulcatus with mixed infection, Borrelia and TBE virus do not seem to interfere with each other and are apparently not involved in any antagonistic relationships.     

Highly efficient induction of protective immunity by a vaccinia virus vector defective in late gene expression

Vaccinia viruses defective in the essential gene coding for the enzyme uracil DNA glycosylase (UDG) do not undergo DNA replication and do not express late genes in wild-type cells. A UDG-deficient vaccinia virus vector carrying the tick-borne encephalitis (TBE) virus prM/E gene, termed vD4-prME, was constructed, and its potential as a vaccine vector was evaluated. High-level expression of the prM/E antigens could be demonstrated in infected complementing cells, and moderate levels were found under noncomplementing conditions. The vD4-prME vector was used to vaccinate mice; animals receiving single vaccination doses as low as 10(4) PFU were fully protected against challenge with high doses of virulent TBE virus. Single vaccination doses of 10(3) PFU were sufficient to induce significant neutralizing antibody titers. With the corresponding replicating virus, doses at least 10-fold higher were needed to achieve protection. The data indicate that late gene expression of the vaccine vector is not required for successful vaccination; early vaccinia virus gene expression induces a potent protective immune response. The new vaccinia virus-based defective vectors are therefore promising live vaccines for prophylaxis and cancer immunotherapy.     

Geographical distribution of arboviruses in Yugoslavia.

Studies of arboviruses started in Yugoslavia in 1953 following the isolation of TBE virus which caused a severe epidemic that year. Until now the following viruses have been proven to circulate in the country: tick-borne encephalitis (TBE), Crimean-Congo hemorrhagic fever (CCHF), Bhanja (BHA), sandfly fever (SF), Tahyna (TAH), Calovo (CVO), West Nile (WN), dengue (DEN), Jug Bogdanovac (JB), and Hantaviruses. TBE virus is endemic in the north-west part of the country, causing also epidemics in cyclical intervals. Its typical clinical picture is aseptic meningitis, but severe cases with paralysis have also been described. The bite of ticks is confirmed in about 80% of cases. CCF caused a small epidemic with ten clinical cases in Macedonia in 1976. Bhanja virus was isolated on the Dalmatian island of Brac in 1977, the antibody rate there, determined by the HI method, being about 31%. The first human disease in the world was caused by the Yugoslav Bhanja virus strain. Sandfly fever is still active in the country. The Naples type is prevailing and has proved hazardous for newcomers. Hantaviruses have been studied since 1980. They caused severe epidemics (1967, 1980, 1989) and sporadic cases all over the country. Three different strains are in circulation. Further studies are needed for the rest of the above mentioned viruses to learn more about their significance in human pathology.     

Immunoprotective human monoclonal antibodies against five major serotypes of Pseudomonas aeruginosa

Human monoclonal antibodies (Mabs) against the O antigens of Pseudomonas aeruginosa lipopolysaccharides (LPS) were produced by cell fusion between human tonsillar lymphocytes and P3-X63-Ag8-U1 (P3U1) mouse myeloma cells. To obtain human Mabs efficiently, 6 d culture supernatants of pokeweed-mitogen-stimulated lymphocytes (21 cultures from peripheral blood and 76 from tonsils) were assayed by ELISA. Five tonsillar lymphocytes which produced IgG antibody specific for P. aeruginosa LPS were preselected for fusion. The human Mabs, named P1-1 (IgG2, kappa), P5-1 (IgG2, lambda), P7-1 (IgG2, lambda), P8-1 (IgG2, lambda) and P10-1 (IgG2, kappa), bound with high specificity to Homma standard serotype strains A, E, B, G and I, respectively, and recognized O antigens. Each Mab showed opsonophagocytic killing activity of the corresponding serotype strain. Four of the Mabs caused agglutination at a very low concentration; a rather higher concentration of P7-1 was required for this effect. Although all the Mabs conferred type-specific protection against peritoneal infection, the strongly agglutinating Mabs provided better protection than the moderately agglutinating P7-1. The protective activity of P8-1 was estimated in compromised mice. A low dose (PD50 0.5-0.6 microgram per mouse) of P8-1 prevented subcutaneous infection in burned mice and peritoneal infection in leucopenic mice. All the hybridomas described here could be cultured in serum-free medium, and they have continued to secrete human Mabs for more than 14 months at rates of 10-20 micrograms per 10(6) cells in 24 h. These results suggested that these five human Mabs specific for O antigens might be useful in the prophylaxis and treatment of P. aeruginosa infections.     

Neutralizing single-chain antibodies against the tick-borne encephalitis virus

A recombinant pSC13D6 plasmid DNA was constructed based on cDNA fragments of genes encoding variable domains of heavy and light chains of the MKA13D6 monoclonal antibody against glycoprotein of the tick-borne encephalitis (TBE) virus. This plasmid provided expression in Escherichia coli cells of the scl3D6 single-chain antibody against the TBE virus. The produced antibodies could bind to the TBE virus, strain 205, and the TBE virus recombinant E protein. The affinity constant of purified scl3D6 was (3.0 ± 0.2) × 10⁷ M^?1 for the equilibrium state and (2.8 ± 0.3) × 10⁷ M^?1 in the case of antigen-antibody formation on the surface. The obtained single-chain antibody could inhibit the infection potency of the TBE virus on a monolayer of eukaryotic cells. The calculated IC₅₀ value for scl3D6 was 16.7 μg/ml.     

The relationship of the antigenic characteristics of the tick-borne encephalitis virus to the level of the protective activity of inactivated cultured vaccines]

As shown in this study, the immunization of animals with killed vaccines against tick-borne encephalitis (TBE) leads to the formation of specific immunity, depending on the antigenic structure of the vaccine strain and the test strains used for challenge. Vaccines obtained on the basis of the TBE virus strain of the Eastern antigenic variant induced the development of a wider spectrum of specific protective activity than vaccines obtained on the basis of the TBE virus strain of the Western antigenic variant.     

Presence of poly(A) in a flavivirus: significant differences between the 3' noncoding regions of the genomic RNAs of tick-borne encephalitis virus strains.

A poly(A) tail was identified on the 3' end of the prototype tick-borne encephalitis (TBE) virus strain Neudoerfl. This is in contrast to the general lack of poly(A) in the genomic RNAs of mosquito-borne flaviviruses analyzed so far. Analysis of several closely related strains of TBE virus, however, revealed the existence of two different types of 3' noncoding (NC) regions. One type (represented by strain Neudoerfl) is only 114 nucleotides long and carries a 3'-terminal poly(A) structure. This was also found in several TBE virus strains isolated from different geographic regions over a period of almost 30 years. The other type (represented by strain Hypr) is 461 nucleotides long and not polyadenylated. The sequence homology between the two types of TBE virus 3' NC regions terminates at a specific position 81 nucleotides after the stop codon. The second type of 3' NC region more closely resembles the common flavivirus pattern, including the potential for the formation of a 3'-terminal hairpin structure. However, it lacks primary sequence elements that are conserved among other flavivirus genomes.