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1.
Virology ; 286(2): 328-35, 2001 Aug 01.
Article in English | MEDLINE | ID: mdl-11485400

ABSTRACT

Langat (LGT) flavivirus, derived from infectious full-length cDNA clone 636, was investigated for its apoptotic activities in mouse neuroblastoma (Neuro-2a) and simian kidney (Vero and LLC-MK(2)) cells. The hallmark of apoptosis, cleavage of cellular DNA, was observed 48 h after infection of Vero, LLC-MK(2), and Neuro-2a cells by electrophoresis analysis. Apoptosis in infected cells was also confirmed by TUNEL assay. LGT-infected Neuro-2a cells showed an increase in caspase-3-like protease (DEVDase) activity. Expression of the major envelope glycoprotein (E) alone reduced cell viability in both Vero and Neuro-2a cells, and the baculovirus P35 protein, which inhibits multiple caspases, completely blocked this effect. Cleavage of cellular DNA was observed in E gene-transfected Vero cells by TUNEL assay. Expression of E protein or caspase-9 resulted in activation of caspase-3-like proteases in Neuro-2a cells. The caspase-3-like protease specific inhibitor, Ac-DEVD-CHO peptide, partially inhibited E protein- or caspase-9-induced apoptosis in Neuro-2a cells. These observations indicate that infection of cells with LGT virus or expression of LGT virus E protein induces apoptosis through a caspase-3-like protease pathway.


Subject(s)
Apoptosis , Encephalitis Viruses, Tick-Borne/pathogenicity , Neurons/virology , Viral Envelope Proteins/metabolism , Animals , Cell Line , Chlorocebus aethiops , DNA Fragmentation , Encephalitis Viruses, Tick-Borne/metabolism , Enzyme Activation , In Situ Nick-End Labeling , Mice , Peptide Hydrolases/metabolism , Tumor Cells, Cultured , Vero Cells , Viral Envelope Proteins/genetics , Virulence
2.
J Virol ; 75(17): 8259-67, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11483771

ABSTRACT

Langat virus (LGT), strain TP21, a naturally avirulent tick-borne flavivirus, was used to construct a chimeric candidate virus vaccine which contained LGT genes for premembrane (preM) and envelope (E) glycoprotein and all other sequences derived from dengue type 4 virus (DEN4). The live virus vaccine was developed to provide resistance to the highly virulent, closely related tick-borne flaviviruses that share protective E epitopes among themselves and with LGT. Toward that end the chimera, initially recovered in mosquito cells, was adapted to grow to high titer in qualified simian Vero cells. When inoculated intraperitoneally (i.p.), the Vero cell-adapted LGT TP21/DEN4 chimera remained completely attenuated for SCID mice. Significantly, the chimera protected immunocompetent mice against the most virulent tick-borne encephalitis virus (TBEV). Subsequently, rhesus monkeys were immunized in groups of 4 with 10(5) or 10(7) PFU of LGT strain TP21, with 10(5) PFU of DEN4, or with 10(3), 10(5), or 10(7) PFU of the chimera. Each of the monkeys inoculated with DEN4 or LGT TP21 became viremic, and the duration of viremia ranged from 1 to 5 days. In contrast, viremia was detected in only 1 of 12 monkeys inoculated with the LGT TP21/DEN4 chimera; in this instance the level of viremia was at the limit of detection. All monkeys immunized with the chimera or LGT TP21 virus developed a moderate to high level of neutralizing antibodies against LGT TP21 as well as TBEV and were completely protected against subsequent LGT TP21 challenge, whereas monkeys previously immunized with DEN4 virus became viremic when challenged with LGT TP21. These observations suggest that the chimera is attenuated, immunogenic, and able to induce a protective immune response. Furthermore, passive transfer of serum from monkeys immunized with chimera conferred significant protection to mice subsequently challenged with 100 i.p. 50% lethal doses of the highly virulent TBEV. The issue of transmissibility of the chimera by mosquitoes was addressed by inoculating a nonhematophagous mosquito, Toxorhynchites splendens, intrathoracically with the chimera or its DEN4 or LGT parent. Neither the LGT TP21/DEN4 vaccine candidate nor the wild-type LGT TP21 virus was able to infect this mosquito species, which is highly permissive for dengue viruses. Certain properties of the chimera, notably its attenuation for monkeys, its immunogenicity, and its failure to infect a highly permissive mosquito host, make it a promising vaccine candidate for use in immunization against severe disease caused by many tick-borne flaviviruses.


Subject(s)
Dengue Virus/immunology , Dengue/prevention & control , Encephalitis Viruses, Tick-Borne/immunology , Encephalitis, Tick-Borne/prevention & control , Viral Vaccines , Animals , Antibodies, Viral/blood , Cells, Cultured , Chlorocebus aethiops , Culicidae/virology , Dengue/immunology , Dengue/virology , Dengue Virus/genetics , Dengue Virus/physiology , Encephalitis Viruses, Tick-Borne/genetics , Encephalitis Viruses, Tick-Borne/physiology , Encephalitis, Tick-Borne/immunology , Encephalitis, Tick-Borne/virology , Immunization, Passive , Macaca mulatta , Mice , Mice, Inbred BALB C , Mice, SCID , Recombinant Fusion Proteins , Vaccination , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunology , Vero Cells , Viral Vaccines/administration & dosage , Viral Vaccines/immunology , Virus Replication
3.
Virology ; 282(2): 288-300, 2001 Apr 10.
Article in English | MEDLINE | ID: mdl-11289811

ABSTRACT

Forty-five years ago a naturally attenuated tick-borne flavivirus, Langat (LGT) strain TP21, was recovered from ticks in Malaysia. Subsequently, it was tested as a live attenuated vaccine for virulent tick-borne encephalitis viruses. In a large clinical trial its attenuation was confirmed but there was evidence of a low level of residual virulence. Thirty-five years ago further attenuation of LGT TP21 was achieved by multiple passages in eggs to yield mutant E5. To study the genetic determinants of the further attenuation exhibited by E5 and to allow us to manipulate the genome of this virus for the purpose of developing a satisfactory live attenuated tick-borne flavivirus vaccine, we recovered infectious E5 virus from a full-length cDNA clone. The recombinant E5 virus (clone 651) recovered from a full-length infectious cDNA clone was more attenuated in immunodeficient mice than that of its biologically derived E5 parent. Increase in attenuation was associated with three amino acid substitutions, two located in the structural protein E and one in nonstructural protein NS4B. Subsequently an even greater degree of attenuation was achieved by creating a viable 320 nucleotide deletion in the 3'-noncoding region of infectious full-length E5 cDNA. This deletion mutant was not cytopathic in simian Vero cells and it replicated to lower titer than its E5-651 parent. In addition, the E5 3' deletion mutant was less neuroinvasive in SCID mice than its E5-651 parent. Significantly, the deletion mutant proved to be 119,750 times less neuroinvasive in SCID mice than its progenitor, LGT strain TP21. Despite its high level of attenuation, the E5 3' deletion mutant remained highly immunogenic and intraperitoneal (ip) inoculation of 10 PFU induced complete protection in Swiss mice against subsequent challenge with 2000 ip LD50 of the wild-type LGT TP21.


Subject(s)
Brain/virology , DNA, Recombinant/genetics , DNA, Viral/genetics , Encephalitis Viruses, Tick-Borne/genetics , Encephalitis Viruses, Tick-Borne/pathogenicity , Sequence Deletion/genetics , Animals , Antibodies, Viral/immunology , Base Sequence , Brain/pathology , Cell Line , Chick Embryo , Chlorocebus aethiops , Cloning, Molecular , Consensus Sequence/genetics , DNA, Complementary/genetics , Encephalitis Viruses, Tick-Borne/immunology , Encephalitis Viruses, Tick-Borne/isolation & purification , Encephalitis, Tick-Borne/immunology , Encephalitis, Tick-Borne/pathology , Encephalitis, Tick-Borne/prevention & control , Encephalitis, Tick-Borne/virology , Mice , Mice, SCID , Neutralization Tests , Ovum , Serial Passage , Survival Rate , Vaccination , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunology , Viral Plaque Assay , Viral Vaccines/genetics , Viral Vaccines/immunology , Virulence/genetics
4.
Virology ; 274(1): 26-31, 2000 Aug 15.
Article in English | MEDLINE | ID: mdl-10936085

ABSTRACT

Langat virus (LGT), a tick-borne flavivirus, is naturally attenuated for humans but it is very virulent in SCID mice. In contrast, viable recombinant chimeras of LGT (preM and E genes) and dengue type 4 virus (all other sequences) recovered in mosquito cell culture were completely attenuated in SCID mice but still capable of providing protection against LGT. To develop the chimeras into vaccine candidates, we adapted them to replicate efficiently in simian Vero cells, a satisfactory substrate for human vaccines. The adapted chimeras remained completely attenuated for SCID mice and, significantly, provided protection in immunocompetent mice against tick-borne encephalitis virus, the most virulent of the tick-borne flaviviruses.


Subject(s)
Dengue Virus/immunology , Encephalitis Viruses, Tick-Borne/immunology , Encephalitis, Tick-Borne/prevention & control , Vaccines, Synthetic/immunology , Viral Envelope Proteins/immunology , Viral Vaccines/immunology , Adaptation, Physiological , Animals , Chlorocebus aethiops , Dengue Virus/genetics , Encephalitis Viruses, Tick-Borne/genetics , Encephalitis Viruses, Tick-Borne/pathogenicity , Encephalitis, Tick-Borne/immunology , Female , Genetic Vectors/genetics , Genetic Vectors/immunology , Humans , Mice , Mice, Inbred BALB C , Mice, SCID , Vaccines, Synthetic/genetics , Vero Cells , Viral Envelope Proteins/genetics , Viral Vaccines/genetics , Virulence
5.
Virology ; 269(1): 225-37, 2000 Mar 30.
Article in English | MEDLINE | ID: mdl-10725214

ABSTRACT

Tick-borne flavivirus strain Langat TP21 (LGT TP21) recovered from ticks, is naturally attenuated for humans but retains demonstrable neurovirulence and peripheral virulence ("neuroinvasiveness") for mice. Previously a mutant, strain E5, less virulent for mice was derived from LGT TP21. Multiple attempts to prepare a full-length infectious TP21 cDNA from cDNA fragments cloned in E. coli were uniformly unsuccessful. A more informative sequence than that obtained from these cloned cDNA fragments and similar E5 cDNA fragments was derived from RT-PCR fragments that had not been cloned in E. coli. Comparison of the RT-PCR consensus sequence of TP21 and E5 identified only seven amino acid differences that might be responsible for the observed difference in virulence of these strains for mice. Eleven independent infectious cDNA clones of TP21 were recovered using two overlapping long RT-PCR fragments. Importantly, low-titered virus used to prepare cDNA as template for PCR was harvested early in the growth cycle to minimize the frequency of deletion mutants that accumulated late in infection. The four analyzed rescued clones exhibited clone-specific minimal divergence from the consensus sequence but this limited variation was associated with diminished peripheral virulence for immunocompetent mice. Manipulation of these clones should facilitate elucidation of LGT virulence.


Subject(s)
Cloning, Molecular , Encephalitis Viruses, Tick-Borne/genetics , Encephalitis Viruses, Tick-Borne/pathogenicity , Encephalitis, Tick-Borne/virology , Neurons/virology , Amino Acid Substitution/genetics , Animals , Cell Line , Consensus Sequence/genetics , DNA Mutational Analysis , DNA, Complementary/genetics , Encephalitis Viruses, Tick-Borne/isolation & purification , Encephalitis Viruses, Tick-Borne/physiology , Encephalitis, Tick-Borne/mortality , Encephalitis, Tick-Borne/pathology , Genetic Variation/genetics , Genome, Viral , Lethal Dose 50 , Mice , Mice, SCID , Mutation/genetics , Neurons/pathology , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Virulence/genetics , Virus Replication/physiology
6.
Proc Natl Acad Sci U S A ; 95(4): 1746-51, 1998 Feb 17.
Article in English | MEDLINE | ID: mdl-9465088

ABSTRACT

Langat virus (LGT) strain TP21 is the most attenuated of the tick-borne flaviviruses for humans. Even though LGT has low-level neurovirulence for humans, it, and its more attenuated egg-passage derivative, strain E5, exhibit significant neurovirulence and neuroinvasiveness in normal mice, albeit less than that associated with tick-borne encephalitis virus (TBEV), the most virulent of the tick-borne flaviviruses. We sought to reduce or ablate these viral phenotypes of TP21 and E5 by using a strategy that had been used successfully in the past to reduce neurovirulence and abolish neuroinvasiveness of TBEV, namely substitution of structural protein genes of the tick-borne flavivirus for the corresponding genes of dengue type 4 virus (DEN4). In pursuit of these objectives different combinations of LGT genes were substituted into the DEN4 genome but only chimeras containing LGT structural proteins premembrane (preM) and envelope glycoprotein (E) were viable. The infectious LGT(preM-E)/DEN4 chimeras were restricted in replication in simian cell cultures but grew to moderately high titer in mosquito cell culture. Also, the chimeras were at least 5,000 times less neurovirulent than their parental LGT virus in suckling mice. Significantly, the chimeras lacked detectable evidence of neuroinvasiveness after i.p. inoculation of Swiss mice or the more permissive SCID mice with 10(5) or 10(7) plaque-forming units (PFU), respectively. Nonetheless, i.p. inoculation of Swiss mice with 10 or 10(3) PFU of either chimeric virus induced LGT neutralizing antibodies and resistance to fatal encephalitis caused by i.p. challenge with LGT TP21. The implications of these observations for development of a live attenuated TBEV vaccine are discussed.


Subject(s)
Dengue Virus/pathogenicity , Encephalitis Viruses, Tick-Borne/pathogenicity , Encephalitis, Tick-Borne/virology , Vaccines, Attenuated , Viral Vaccines/immunology , Amino Acid Sequence , Animals , Base Sequence , Cells, Cultured , Chimera , Dengue Virus/genetics , Encephalitis Viruses, Tick-Borne/genetics , Mice , Mice, Nude , Virus Replication
8.
FEBS Lett ; 382(3): 327-9, 1996 Mar 18.
Article in English | MEDLINE | ID: mdl-8605995

ABSTRACT

T7 RNA polymerase is shown to recognize the SP6 promoter including 17 base pairs before the transcription start site and produce the 5'-end TBEV RNA. The yield of TBEV RNA synthesized by heterologous T7 RNA polymerase from cDNA construction with SP6 promoter is higher than the RNA production by homologous SP6 RNA polymerase. The addition of 1 pmol template DNA with SP6 17 bp promoter in transcription mixture for SP6 or T7 RNA polymerases resulted in a 1-5 X 10(-2) pmol RNA production.


Subject(s)
DNA-Directed RNA Polymerases/metabolism , Encephalitis Viruses, Tick-Borne/genetics , Promoter Regions, Genetic/genetics , RNA, Viral/biosynthesis , Salmonella Phages/genetics , Transcription, Genetic , Bacteriophage T7/enzymology , Base Sequence , DNA, Complementary/metabolism , DNA, Viral/metabolism , Encephalitis Viruses, Tick-Borne/pathogenicity , Molecular Sequence Data , RNA, Viral/genetics , Salmonella Phages/enzymology , Salmonella typhimurium/virology , Viral Proteins
9.
Bioorg Khim ; 21(7): 528-34, 1995 Jul.
Article in Russian | MEDLINE | ID: mdl-7488268

ABSTRACT

Using reverse transcription and the polymerase chain reaction, cDNA fragments of noncoding regions of the tick-borne encephalitis virus (TBEV) genome were obtained. These fragments were cloned into a pGEM3 vector, and their nucleotide sequences were determined. The heterogeneity of the 3'-terminal untranslated region of the TBEV RNA was revealed. To create a stable full-size DNA copy of the TBEV genome, four cDNA variants differing in length and structure of the 3'-terminal fragment of the viral RNA were cloned into a pBR322-derived vector.


Subject(s)
DNA, Viral , Encephalitis Viruses, Tick-Borne/genetics , Genome, Viral , Base Sequence , DNA, Complementary , Molecular Sequence Data , Plasmids , RNA, Viral/genetics
10.
J Virol ; 67(8): 4956-63, 1993 Aug.
Article in English | MEDLINE | ID: mdl-8331735

ABSTRACT

Two new chimeric flaviviruses were constructed from full-length cDNAs that contained tick-borne encephalitis virus (TBEV) CME or ME structural protein genes and the remaining genes derived from dengue type 4 virus (DEN4). Studies involving mice inoculated intracerebrally with the ME chimeric virus indicated that it retained the neurovirulence of its TBEV parent from which its pre-M and E genes were derived. However, unlike parental TBEV, the chimeric virus did not produce encephalitis when mice were inoculated peripherally, indicating a loss of neuroinvasiveness. In the present study, the ME chimeric virus (vME) was subjected to mutational analysis in an attempt to reduce or ablate neurovirulence measured by direct inoculation of virus into the brain. We identified three distinct mutations that were each associated independently with a significant reduction of mouse neurovirulence of vME. These mutations ablated (i) the TBEV pre-M cleavage site, (ii) the TBEV E glycosylation site, or (iii) the first DEN4 NS1 glycosylation site. In contrast, ablation of the second DEN4 NS1 glycosylation site or the TBE pre-M glycosylation site or amino acid substitution at two positions in the TBEV E protein increased neurovirulence. The only conserved feature of the three attenuated mutants was restriction of virus yield in both simian and mosquito cells. Following parenteral inoculation, these attenuated mutants induced complete resistance in mice to fatal encephalitis caused by the highly neurovirulent vME.


Subject(s)
Brain/microbiology , Dengue Virus/genetics , Dengue/physiopathology , Encephalitis Viruses, Tick-Borne/genetics , Encephalitis, Tick-Borne/physiopathology , Mutagenesis, Site-Directed , Point Mutation , Virulence/genetics , Amino Acid Sequence , Animals , Base Sequence , Brain/pathology , Cell Line , Cells, Cultured , Chimera , Dengue Virus/growth & development , Dengue Virus/pathogenicity , Encephalitis Viruses, Tick-Borne/growth & development , Encephalitis Viruses, Tick-Borne/pathogenicity , Glycosylation , Kinetics , Methionine/metabolism , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Oligodeoxyribonucleotides , Plasmids , Restriction Mapping , Transfection , Viral Plaque Assay , Viral Proteins/biosynthesis , Viral Proteins/genetics
11.
Proc Natl Acad Sci U S A ; 89(21): 10532-6, 1992 Nov 01.
Article in English | MEDLINE | ID: mdl-1438242

ABSTRACT

Dengue type 4 virus (DEN4) cDNA was used as a vector to express genes of the distantly related tick-borne encephalitis virus (TBEV). Full-length chimeric TBEV/DEN4 cDNAs were constructed by substituting TBEV genes coding for proteins such as capsid (C); pre-membrane, which is the precursor of membrane (M); envelope (E); or nonstructural protein NS1 for the corresponding DEN4 sequences. RNA transcripts prepared from cDNAs were used to transfect permissive simian cells. Two viable chimeric viruses that contained TBEV CME or ME genes were recovered. Compared with DEN4, chimeric TBE(ME)/DEN4 virus [designated vTBE(ME)/DEN4] produced larger plaques and grew to higher titer in simian cells. In contrast, vTBE(ME)/DEN4 produced smaller plaques on mosquito cells and grew to lower titer than DEN4. Analysis of viral RNA and proteins produced in vTBE(ME)/DEN4- and DEN4-infected mosquito or simian cells revealed that the chimera was restricted in its ability to enter and replicate in mosquito cells. In contrast, vTBE(ME)/DEN4 entered simian cells efficiently and its RNA was replicated more rapidly in these cells than was parental DEN4 RNA. Following intracerebral inoculation, vTBE(ME)/DEN4 caused fatal encephalitis in both suckling and adult mice, while nearly all mice inoculated by the same route with DEN4 did not develop disease. Unlike wild-type TBEV, vTBE(ME)/DEN4 did not cause encephalitis when adult mice were inoculated by a peripheral route. Adult mice previously inoculated with the chimera by a peripheral route were completely resistant to subsequent intraperitoneal challenge with 10(3) times the median lethal dose of TBEV, whereas mice previously inoculated with DEN4 were not protected. These findings indicate that (i) the TBEV M and E genes of the chimeric virus are major protective antigens and induce resistance to lethal TBEV challenge and (ii) other regions of the TBEV genome are essential for the ability of this virus to spread from a peripheral site to the brain. Success in constructing a viable TBEV/DEN4 chimera that retains the protective antigens of TBEV but lacks its peripheral invasiveness provides a strategy for the development of live attenuated TBEV vaccines.


Subject(s)
Chimera , Dengue Virus/genetics , Encephalitis Viruses, Tick-Borne/genetics , Genes, Viral , Amino Acid Sequence , Animals , Base Sequence , Cell Line , DNA, Viral/genetics , Dengue Virus/growth & development , Dengue Virus/pathogenicity , Encephalitis Viruses, Tick-Borne/growth & development , Encephalitis Viruses, Tick-Borne/pathogenicity , Encephalitis, Tick-Borne/microbiology , Female , Genome, Viral , Introns , Mice , Mice, Inbred BALB C , Molecular Sequence Data , RNA, Viral/biosynthesis , Transcription, Genetic , Viral Plaque Assay , Viral Proteins/biosynthesis , Virulence
12.
Vopr Virusol ; 37(5-6): 248-52, 1992.
Article in Russian | MEDLINE | ID: mdl-1290224

ABSTRACT

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.


Subject(s)
DNA Probes/genetics , DNA, Viral/genetics , DNA/genetics , Encephalitis Viruses, Tick-Borne/genetics , Genome, Viral , Oligonucleotide Probes/genetics , RNA, Viral/genetics , Animals , Base Sequence , Encephalitis Viruses, Tick-Borne/isolation & purification , Genetic Variation/genetics , Molecular Sequence Data , Nucleic Acid Hybridization/methods , RNA, Viral/isolation & purification , Russia , Ticks/microbiology
13.
FEBS Lett ; 297(1-2): 67-9, 1992 Feb 03.
Article in English | MEDLINE | ID: mdl-1551439

ABSTRACT

Using monoclonal antibodies to the tick-borne encephalitis virus (TBE) nonstructural protein NS3 two forms of this protein were revealed in TBE-infected mammalian cells: a full-length form (69 kDa) and a short form (49 kDa) which has not been observed before and was called NS3'. Recombinant plasmids were constructed and various fragments of the TBE NS3 gene were expressed in rabbit reticulocyte lysate. By analyzing immune precipitates of 35S-labeled translation products, we could monitor and localize internal cleavage of NS3, due to which the NS3' protein was generated.


Subject(s)
Encephalitis Viruses, Tick-Borne/metabolism , Viral Nonstructural Proteins , Viral Proteins/genetics , Animals , Base Sequence , Blotting, Western , Cells, Cultured , DNA, Viral/genetics , Electrophoresis, Polyacrylamide Gel , Humans , Molecular Sequence Data , Plasmids , Protein Biosynthesis , RNA Helicases , RNA, Messenger/genetics , Serine Endopeptidases , Swine , Transcription, Genetic
14.
Mol Biol (Mosk) ; 26(1): 158-67, 1992.
Article in Russian | MEDLINE | ID: mdl-1508165

ABSTRACT

On the base of two overlapping cDNA-clones of tick-borne encephalitis virus (TBEV) genome and synthetic DNA fragments full DNA-copy of the TBEV NS3 protein gene was constructed and expressed in the E. coli cells. It was demonstrated that the relatively low biosynthesis level of full-length NS3 protein in the bacteria was due to the toxicity of the N-terminal region of the protein, consisting of it's first 180 amino acid residues. A form of the gene with deletion of nucleotides coding for the toxic region (called NS3*) was constructed and effective bacterial product of NS3* protein was obtained. The panel of monoclonal antibodies to TBEV NS1 and NS3 proteins was generated. According to the results of experiments of the binding of the monoclonal antibodies 18B2 to the bacterial products of NS3 and NS3* genes it was concluded, that the antigenic determinant recognized by these antibodies was located between 174 and 236 amino acids of TBEV NS3 protein.


Subject(s)
Encephalitis Viruses, Tick-Borne/genetics , Escherichia coli/genetics , Genes, Bacterial , Viral Nonstructural Proteins , Viral Proteins/genetics , Antibodies, Monoclonal , Base Sequence , Blotting, Western , Cloning, Molecular , DNA , Electrophoresis, Polyacrylamide Gel , Gene Expression , Molecular Sequence Data , Plasmids , RNA Helicases , Serine Endopeptidases
16.
Med Parazitol (Mosk) ; (2): 48-50, 1991.
Article in Russian | MEDLINE | ID: mdl-2067497

ABSTRACT

Nucleic acids of Ixodes persulcatus were studied by molecular hybridization in the natural focus of tick-borne encephalitis in Kholmsk District of Sakhalin Province. The studies have shown wide dissemination of viral RNA in the focus. The infectivity of ticks in various sites of habitation varied from 3.5 to 18.5%, their number fluctuating from 0.4 to 300 and more imago per flag-hour. The most active part of the natural focus has been determined using zoological-viral indexes. The viral strain of tick-borne encephalitis has been isolated.


Subject(s)
Disease Reservoirs , Encephalitis, Tick-Borne/microbiology , Animals , Arachnid Vectors/analysis , Arachnid Vectors/microbiology , Encephalitis Viruses, Tick-Borne/isolation & purification , Encephalitis, Tick-Borne/transmission , Female , Male , Mice , Population Density , RNA, Viral/analysis , Siberia , Ticks/analysis , Ticks/microbiology
17.
Bioorg Khim ; 17(3): 334-42, 1991 Mar.
Article in Russian | MEDLINE | ID: mdl-1712201

ABSTRACT

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.


Subject(s)
Antibodies, Monoclonal , Antigens, Viral/immunology , Encephalitis Viruses, Tick-Borne/immunology , Viral Envelope Proteins/immunology , Blotting, Western , Cyanogen Bromide , Electrophoresis, Polyacrylamide Gel , Epitopes/immunology , Restriction Mapping , Viral Envelope Proteins/genetics
18.
Biomed Sci ; 2(2): 183-6, 1991.
Article in English | MEDLINE | ID: mdl-1772972

ABSTRACT

Porcine embryo kidney cells infected by tick-borne encephalitis virus (TBEV) were fractionated into nuclear, membrane, and cytoplasmic fractions. To identify proteins involved in the initiation of RNA replication at different stages of infection a highly specific affinity labelling technique was used. In samples of the nuclear fraction taken from cells 45 h after infection (late stage), affinity labelling with aldehyde-containing derivatives of ATP and elongation of this label with [alpha-32P]GTP identified a polypeptide with a molecular mass of about 69 kDa. By means of affinity labelling with aldehyde-containing analogues of GMP, GDP, and GTP as initiation substrates and [alpha-32P]ATP as the elongation substrate, a polypeptide of 100 kDa was selectively modified in the nuclear fraction of cells at the early stages of infection (8 h). These proteins were immunostained with TBEV-specific antibodies, and were identified as the nonstructural TBEV proteins NS3 and NS5, respectively. It was concluded that NS3 and NS5 take part in the initiation of TBEV genome replication at the late and early stages of infection, respectively.


Subject(s)
Encephalitis Viruses, Tick-Borne/enzymology , RNA, Viral/biosynthesis , RNA-Dependent RNA Polymerase/metabolism , Animals , Cell Nucleus/physiology , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Embryo, Mammalian , Encephalitis Viruses, Tick-Borne/genetics , Kidney , Macromolecular Substances , Molecular Weight , RNA-Dependent RNA Polymerase/analysis , Swine , Viral Proteins/biosynthesis , Viral Proteins/isolation & purification
19.
Vopr Virusol ; 36(1): 27-31, 1991.
Article in Russian | MEDLINE | ID: mdl-1713371

ABSTRACT

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.


Subject(s)
DNA, Viral/genetics , Encephalitis Viruses, Tick-Borne/classification , Nucleic Acid Hybridization/genetics , RNA, Viral/genetics , Animals , Brain , DNA/genetics , DNA Probes/isolation & purification , Encephalitis Viruses, Tick-Borne/genetics , Genes, Viral/genetics , Genetic Techniques , Mice , RNA/isolation & purification
20.
Acta Virol ; 35(1): 71-80, 1991 Jan.
Article in English | MEDLINE | ID: mdl-1683119

ABSTRACT

Recombinant plasmid DNA was used as a probe to detect tick-borne encephalitis (TBE) virus RNA during incubation period, acute disease and persistent infection of syrian hamsters. Within the first three weeks post-infection the results of direct virus isolation and RNA detection in the brain agreed by a rate of 100%, the virus titre ranging between 10(1.9) to 10(10.5) LD50/ml and viral RNA concentration at 1-1000 pg. At the same time TBE virus RNA was detected in the spleen when the virus titre was greater than or equal to 10(6.5) LD50/ml. By 8 months post infection (p.i.) viral RNA was found in the brain, liver, and spleen in the absence of infectious TBE virus. No viral RNA was present in the thymus. In addition, electron microscopic findings in hamster brain confirmed the hypothesis that TBE virus persistence was accompanied by formation of virus-specific structures but impaired virion maturation.


Subject(s)
DNA Probes , DNA, Viral , Encephalitis Viruses, Tick-Borne/isolation & purification , Encephalitis, Tick-Borne/microbiology , RNA, Viral/analysis , Acute Disease , Animals , Brain/microbiology , Brain/pathology , Chronic Disease , Cricetinae , DNA/genetics , DNA, Recombinant/genetics , Encephalitis Viruses, Tick-Borne/genetics , Encephalitis, Tick-Borne/pathology , Liver/microbiology , Mesocricetus/microbiology , Nucleic Acid Hybridization , Spleen/microbiology , Thymus Gland/microbiology
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