Hantavirus infection--haemorrhagic fever in the Balkans--potential nephrological hazards in the Kosovo war.
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Disease Outbreaks↗
Biomedical subjects
G Darai
Publications and source records attributed to G Darai.
Chemokine homolog of molluscum contagiosum virus: sequence conservation and expression.
An analysis of the complete Molluscum contagiosum virus (MCV-1) genome sequence revealed a 104-amino-acid open reading frame (MC148R) that is structurally related to the beta (CC) family of chemokines. The predicted MCV chemokine homolog (MCCH) has a deletion in the NH2-terminal activation domain, suggesting the absence of chemoattractant activity. The principal objectives of the present study were to determine whether: (i) MCCH is conserved in independent isolates of MCV-1 and MCV-2; (ii) MCCH mRNA is expressed in vivo; and (iii) the MCCH protein is secreted from mammalian cells. The nucleotide sequence of the MCCH gene locus was determined for 27 isolates of MCV-1 and 2 of MCV-2 obtained from 29 MCV-infected individuals. In each case, the characteristic CC sequence, the NH2-terminal deletion, and the length of the open reading frame were conserved, although there were some, mostly conservative, amino acid substitutions. Since MCV cannot be propagated in cell culture, mRNA was synthesized in vitro by the early transcription apparatus in purified MCV virions. MCCH RNA was amplified by RT-PCR; the sequence included the complete open reading frame and extended 40 to 50 nucleotides past the first poxviral termination signal (TTTTTNT). Similar RT-PCR results were obtained using total cellular RNA derived from MCV-infected tissue specimens. Finally, the MCCH open reading frame was expressed in a vaccinia virus vector and the predicted size polypeptide was secreted into the medium, as determined by Western blotting. Taken together, our data support the prediction that MCV expresses a secreted chemokine homolog that could antagonize the inflammatory response in vivo.
Amino Acid Sequence↗
Is the major capsid protein of iridoviruses a suitable target for the study of viral evolution?
Iridoviruses are large cytoplasmic DNA viruses that are specific for different insect or vertebrate hosts. The major structural component of the non-enveloped icosahedral virus particles is the major capsid protein (MCP) which appears to be highly conserved among members of the family Iridoviridae, Phycodnaviridae, and African swine fever virus. The amino acid sequences of the known MCPs were used in comparative analyses to elucidate the phylogenic relationships between different cytoplasmic DNA viruses including three insect iridoviruses (Tipula iridescent virus, Simulium iridescent virus, Chilo iridescent virus), seven vertebrate iridoviruses isolated either from fish (lymphocystis disease virus, rainbow trout virus, European catfish virus, doctor fish virus), amphibians (frog virus 3), or reptiles (turtle virus 3, turtle virus 5), one member of the family Phycodnaviridae (Paramecium bursaria Chlorella virus type 1), and African swine fever virus. These analyses revealed that the amino acid sequence of the MCP is a suitable target for the study of viral evolution since it contains highly conserved domains, but is sufficiently diverse to distinguish closely related iridovirus isolates. Furthermore the results suggest that a substantial revision of the taxonomy of iridoviruses based on molecular phylogeny is required.
Amino Acid Sequence↗
Identification of a thymidylate synthase gene within the genome of Chilo iridescent virus.
The thymidylate synthase (TS, EC 2.1.1.45) is essential for the de novo synthesis of dTMP in pro- and eucaryotic organisms. Consequently it plays a major role in the replication of the DNA genome of a cell or a DNA virus. The gene encoding the TS of Chilo iridescent virus (CIV) was identified by nucleotide sequence analysis of the viral genome and was mapped within the EcoRI CIV DNA fragments G and R. Computer assisted analysis of the DNA nucleotide sequence between the genome coordinates 0.482 and 0.489 revealed an open reading frame (ORF) of 885 nucleotides. This ORF was found to encode a polypeptide of 295 amino acid residues (33.9 kDa) that showed significant homologies to known TS of different species including mammals, plants, fungi, protozoa, bacteria, and DNA viruses. The highest amino acid homologies were found between the CIV-TS and the TS of herpesvirus ateles (54.0%), Saccharomyces cerevisiae (51.8%), herpesvirus saimiri (51.0%), rhesus monkey rhadinovirus (50.7%), mouse (50.5%), rat (50.2%), varicella-zoster virus (50.2%), equine herpesvirus 2 (50.0%), and the human TS (48.4%). The CIV-TS contains six amino acid domains that are highly conserved in the TS of other species. Within these domains the major amino acid residues are present for which a functional role has been reported. The CIV-TS was found to be more closely related to the TS of eucaryotes than to the TS of procaryotes indicating the phylogenetic origin of the CIV-TS gene. The identification of a TS gene in the genome of CIV is the first report of a viral TS that is not encoded by a herpesvirus or a bacteriophage.
Amino Acid Sequence↗
Identification of the UL56 protein of herpes simplex virus type 1 within the virion by immuno electron microscopy.
Recently the UL56 protein of herpes simplex virus type 1 (HSV-1) was shown to be associated with the virion of HSV-1 as determined by Western blot analysis. The detection of the UL56 protein in infected cells and its association with virions of HSV-1 is of particular importance, pointing to a possible involvement of UL56 protein in virus-host interactions. In order to investigate the properties of the UL56 protein further immuno-localization was performed using rabbit hyperimmune serum against fusion recombinant UL56 protein and purified virions of HSV-1 strain F. The UL56 protein was detected in the HSV-1 virions by immuno gold negative staining.
Herpesvirus 1, Human↗
Stable expression of nucleocapsid proteins of Puumala and Hantaan virus in mammalian cells.
The development of an in vitro-system for the stable expression and the analysis of native hantavirus proteins using hantaviral cDNA is of particular interest. As a first step the expression of the hantavirus nucleocapsid (N) proteins in mammalian cells was studied in more detail. The cDNA of the S-RNA segment of Puumala virus strain CG-1820 and Hantaan virus strain 76-118 was used for the construction of eucaryotic expression vectors that allow the generation and selection of mammalian cells harboring and expressing the N protein genes of hantaviruses. A variety of conventional and novel expression vectors as well as different mammalian cell lines were screened. The expression of the N protein of Puumala virus using the pGRE5-1 vector in which the transcription is under control of inducible glucocorticoid responsive elements (GRE) revealed that the Puumala virus N protein can be expressed in Vero E6 cells efficiently without any detectable cell toxicity. From the variety of expression vectors tested, it was found that pCR3.1 is the vector of choice for stable expression of hantavirus N proteins. The successful establishment of different mammalian cell lines expressing considerable amounts of Puumala and Hantaan virus N protein indicates that the stable and efficient expression of this particular viral protein in the cell lines of three evolutionary distinct species (human, monkey, and mouse) is possible. The system described here represents the experimental basis for further studies of hantavirus infection, replication, and pathogenesis using a reverse genetics approach.
3T3 Cells↗
Hantavirus infection--new threats by an old virus.
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Orthohantavirus↗
Identification and characterization of the Tupaia herpesvirus DNA polymerase gene.
Tupaia herpesviruses (THVs) have been isolated from malignant lymphomas and from degenerating lung or spleen cell cultures of tree shrews (Tupoia spp.), but because of a lack of genetic information the final classification of THVs is still open. In the present work the viral DNA polymerase (DPOL) gene was mapped within the genome of the different THV strains using PCR and degenerate oligonucleotide primers. Nucleotide sequences of the DPOL genes of THV strains 1 to 5 were determined and used for comparative analyses. The transcriptional activity of the THV-2 DPOL gene was confirmed by RT-PCR. It was found that the different THV strains are very closely related to each other. When compared to other herpesviruses the highest amino acid sequence identities detected were with DPOLs of the murine and human cytomegaloviruses. These results justify the conclusion that THVs are members of the subfamily Betaherpes-virinae.
Amino Acid Sequence↗
The genome of molluscum contagiosum virus: analysis and comparison with other poxviruses.
Analysis of the molluscum contagiosum virus (MCV) genome revealed that it encodes approximately 182 proteins, 105 of which have direct counterparts in orthopoxviruses (OPV). The corresponding OPV proteins comprise those known to be essential for replication as well as many that are still uncharacterized, including 2 of less than 60 amino acids that had not been previously noted. The OPV proteins most highly conserved in MCV are involved in transcription; the least conserved include membrane glycoproteins. Twenty of the MCV proteins with OPV counterparts also have cellular homologs and additional MCV proteins have conserved functional motifs. Of the 77 predicted MCV proteins without OPV counterparts, 10 have similarity to other MCV proteins and/or distant similarity to proteins of other poxviruses and 16 have cellular homologs including some predicted to antagonize host defenses. Clustering poxvirus proteins by sequence similarity revealed 3 unique MCV gene families and 8 families that are conserved in MCV and OPV. Two unique families contain putative membrane receptors; the third includes 2 proteins, each containing 2 DED apoptosis signal transduction domains. Additional families with conserved patterns of cysteines and putative redox active centers were identified. Promoters, transcription termination signals, and DNA concatemer resolution sequences are highly conserved in MCV and OPV. Phylogenetic analysis suggested that MCV, OPV, and leporipoxviruses radiated from a common poxvirus ancestor after the divergence of avipoxviruses. Despite the acquisition of unique genes for host interactions and changes in GC content, the physical order and regulation of essential ancestral poxvirus genes have been largely conserved in MCV and OPV.
Amino Acid Sequence↗
The complete DNA sequence of lymphocystis disease virus.
Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease, which has been reported to occur in over 100 different fish species worldwide. LCDV is a member of the family Iridoviridae and the type species of the genus Lymphocystivirus. The virions contain a single linear double-stranded DNA molecule, which is circularly permuted, terminally redundant, and heavily methylated at cytosines in CpG sequences. The complete nucleotide sequence of LCDV-1 (flounder isolate) was determined by automated cycle sequencing and primer walking. The genome of LCDV-1 is 102.653 bp in length and contains 195 open reading frames with coding capacities ranging from 40 to 1199 amino acids. Computer-assisted analyses of the deduced amino acid sequences led to the identification of several putative gene products with significant homologies to entries in protein data banks, such as the two major subunits of the viral DNA-dependent RNA polymerase, DNA polymerase, several protein kinases, two subunits of the ribonucleoside diphosphate reductase, DNA methyltransferase, the viral major capsid protein, insulin-like growth factor, and tumor necrosis factor receptor homolog.
Base Sequence↗
Recent advances in molluscum contagiosum virus research.
Molluscum contagiosum virus (MCV) and variola virus (VAR) are the only two poxviruses that are specific for man. MCV causes skin tumors in humans and primarily in children and immunocompromised individuals. MCV is unable to replicate in tissue culture cells or animals. Recently, the DNA sequence of the 190 kbp MCV genome was reported by Senkevich et al. MCV was predicted to encode 163 proteins of which 103 were clearly related to those of smallpox virus. In contrast, it was found that MCV lacks 83 genes of VAR, including those involved in the suppression of the host response to infection, nucleotide biosynthesis, and cell proliferation. However, MCV possesses 59 genes predicted to code for novel proteins including MHC-class I, chemokine and glutathione peroxidase homologs not found in other poxviruses. The MCV genomic data allow the investigation of novel host defense mechanisms and provide new possibilities for the development of therapeutics for treatment and prevention of the MCV infection.
Animals↗
Molecular anatomy of lymphocystis disease virus.
Lymphocystis disease (LD) has been reported to occur in over one hundred different species of fish worldwide. The disease is caused by lymphocystis disease virus (LCDV), a member of the iridovirus family. Numerous fish species that play an important role in fishery and fish farming are highly susceptible to LCDV infection. The infected animals develop disseminated clusters of aberrant hypertrophied cells within their connective tissue, the so-called lymphocystis cells. In the cytoplasm of these cells a massive accumulation of virions can be observed. As a first step towards understanding the mechanisms of viral infection and pathogenesis the complete genomic nucleotide sequence of lymphocystis disease virus type 1 (LCDV-1; flounder isolate) was determined. LCDV-1 is the type species of the genus Lymphocystivirus within the family Iridoviridae. The virions contain a single linear double-stranded DNA molecule that is circularly permuted, terminally redundant and heavily methylated. Since there is no convenient cell system for virus replication we determined the complete nucleotide sequence of the viral genome (102,653 base pairs). Computer assisted analyses of 195 potential open reading frames resulted in the identification of a number of putative gene products with significant homology to functionally characterized proteins of other species.
Animals↗
Gene therapy: from retrovirus to triplex DNA repair.
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Animals↗
The DNA sequence of Chilo iridescent virus between the genome coordinates 0.101 and 0.391; similarities in coding strategy between insect and vertebrate iridoviruses.
Chilo iridescent virus (CIV), the type species of the genus Iridovirus within the family Iridoviridae, is highly pathogenic for larvae of important pest insects. The virions contain a single linear double-stranded DNA molecule (209 kbp) that is circularly permuted and terminally redundant. The nucleotide sequence of the viral genome between the genome coordinates 0.101 and 0.391 (60,170 bp) was determined by automated cycle sequencing. This particular region of the CIV genome contains 112 open reading frames (ORFs) with coding capacities for 50 to 1186 amino acids. The alignment of the deduced amino acid sequences with well-characterized proteins stored in protein databases led to the identification of several genes with significant homologies, such as the largest subunit of the DNA-dependent RNA polymerase, large subunit of the ribonucleoside-diphosphate reductase, endonuclease, protein-tyrosine phosphatase, helicase, global transactivator, two apoptosis inhibitor homologs, antibiotic peptide homolog, and others. The highest homologies were detected between putative viral gene products of CIV and the corresponding viral proteins of lymphocystis disease virus of fish (LCDV), which belongs to the genus Lymphocystivirus within the iridovirus family.
Amino Acid Sequence↗
A major antigenic domain of hantaviruses is located on the aminoproximal site of the viral nucleocapsid protein.
Hantavirus nucleocapsid protein has recently been shown to be an immunodominant antigen in hemorrhagic with renal syndrome (HFRS) inducing an early and long-lasting immune response. Recombinant proteins representing various regions of the nucleocapsid proteins as well as segments of the G1 and the G2 glycoproteins of hantavirus strains CG18-20 (Puumala serotype) and Hantaan 76-118 have been expressed in E. coli. The antigenicity of these proteins was tested in enzyme immunoassays and immunoblots. These studies revealed that human IgG immune response is primarily directed against epitopes located within the amino acid residues 1 to 119 of the amino terminus of viral nucleocapsid proteins. This fragment was recognized by all HFRS patient sera tested (n = 128). The corresponding enzyme immunoassays proved to be more sensitive than the indirect immunofluorescence assays. Furthermore, the majority of bank vole monoclonal antibodies raised against Puumala virus reacted specifically with this site. A recombinant G1 protein (aa 59 to 401) derived from the CG 18-20 strain was recognized by 19 out of 20 sera from HFRS patients.
Animals↗
Genome sequence of a human tumorigenic poxvirus: prediction of specific host response-evasion genes.
Molluscum contagiosum virus (MCV) commonly causes asymptomatic cutaneous neoplasms in children and sexually active adults as well as persistent opportunistic acquired immunodeficiency syndrome (AIDS)-associated disease. Sequencing the 190-kilobase pair genome of MCV has now revealed that the virus potentially encodes 163 proteins, of which 103 have homologs in the smallpox virus. MCV lacks counterparts to 83 genes of the smallpox virus, including those important in suppression of host responses to infection, nucleotide biosynthesis, and cell proliferation. MCV possesses 59 genes that are predicted to encode previously uncharacterized proteins, including major histocompatibility complex class I, chemokine, and glutathione peroxidase homologs, which suggests that there are MCV-specific strategies for coexistence with the human host.
Amino Acid Sequence↗
Conservation of DNA sequence in the predicted major late promoter regions of selected mastadenoviruses.
The major late promoter (MLP) of the subgroup C human adenoviruses is a preeminent model for the study of the mechanisms of basal and activated transcription, both in vivo and in vitro. However, while the structure and function of the human virus MLP has been the subject of extensive investigation, the conservation of the various promoter elements among the adenoviruses from different species has not been examined. Conservation of specific elements would strongly suggest the importance and universality of their function. To address this issue, sequences were obtained from cloned DNAs of several representative Mastadenoviridae, mouse adenovirus type 1 (MAV-1), Tupaia adenovirus type 1 (TAV-1), and two bovine adenoviruses of two distinct subgroups, BAV-3 and BAV-7. The results of the sequencing studies showed that the TATA box and an upstream inverted CAAT box are conserved in all species and that the binding site for transcription factor USF is present in all except MAV-1, in which a sequence similar to an Sp1-binding site is present at a similar position. The initiator element (INR) sequence is not well conserved, and only one or other of the two downstream activating elements, DE1 and DE2, is predicted to be present in the nonprimate virus MLP regions. Ribonuclease protection assays on RNA isolated from MAV-1-infected cells late in infection indicated that the predicted MLP is functional, and transcription initiation and splice donor sites were identified. The human virus MLP is embedded in the essential DNA polymerase sequence on the opposite DNA strand. The primary amino acid sequences of the C-terminal regions of the predicted DNA polymerases show strong conservation of sequence motifs observed in replicative polymerases ranging from prokaryotes to mammals, and additional regions of strong conservation among the adenovirus polymerases. Pairwise comparisons between the newly sequenced regions of the polymerases and previously published sequences show that BAV-7 is most dissimilar to all others, while TAV-1 has a greater similarity to the primate sequences than to the others. The sequence data from both strands were also used to construct phylogenetic trees, based on BAV-7 as the outgroup. The trees constructed from the two sets of sequences are broadly similar, showing close relationships between primate viruses, but differing in the order of divergence of TAV-1 and MAV-1 branches.
Animals↗
Identification of the gene encoding the DNA (cytosine-5) methyltransferase of lymphocystis disease virus.
The gene encoding the DNA (cytosine-5) methyltransferase (m5C-MTase) of lymphocystis disease virus (flounder isolate, LCDV-1) has been identified by polymerase chain reaction (PCR) using oligonucleotide primers synthesized corresponding to different regions of the m5C-MTase gene of frog virus 3 (FV3). A DNA fragment of 487 bp was amplified using oligonucleotide primers L3 and R4 which correspond to the nucleotide positions 87 to 109 and 530 to 550 of the m5C-MTase gene of FV3, respectively. The DNA nucleotide sequence of the PCR product was determined by direct cycle sequencing. The alignment of the deduced amino acid sequence derived from the PCR product and the m5C-MTase protein of FV3 revealed a homology of 55.4% identity and 29.1% similarity. The amino acid sequence which was found to be significantly homologous to the amino acid sequence deduced from the nucleotide sequence of the PCR product was located at the amino acid position 37 to 175 of the m5C-MTase of FV3 indicating the specificity of the amplified PCR product. The DNA nucleotide sequence of the LCDV-1 genome corresponding to the 5' and 3' termini of the m5C-MTase gene was determined by primer walking. The locus of the m5C-MTase gene of LCDV-1 was identified within the EcoRI DNA fragment G of LCDV-1 (7.9 kbp; 0.947 to 0.034 map units). The m5C-MTase gene of LCDV-1 comprises 684 nucleotides coding for a putative protein of 228 amino acid residues. A high degree of amino acid sequence homology (53.3% identity and 25.8% similarity) was detected between the m5C-MTase of LCDV-1 and FV3.
Amino Acid Sequence↗