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Alexander Plyusnin

Publications and source records attributed to Alexander Plyusnin.

At least 19 recordsLinked to original sources
PubMed2006

Puumala hantavirus in Slovenia: analyses of S and M segment sequences recovered from patients and rodents.

In Slovenia, the co-existence of Dobrava and Puumala (PUUV) hantaviruses in a single endemic region has been demonstrated. This study presents selected Slovenian HFRS cases caused by PUUV combined with genetic analysis of viral genome sequences recovered from clinical specimens and tissue samples of Clethrionomys glareolus (bank voles). Serum samples from nine HFRS patients were included in the study. Rodents study sites were selected with regard to the HFRS cases. Partial S segment sequences were recovered from all nine patients and partial M segment sequences could be recovered from seven. Partial S and M segments sequences were also recovered from five C. glareouls captured at three different study sites. The sequences from Slovenian clinical specimens and rodent tissue samples belonged to the PUUV genotype and formed a distinct genetic lineage of PUUV. Human and rodent PUUV sequences located in the closest proximity to each other on the phylogenetic trees suggest genetic links between the human cases and the hantaviral strains circulating in natural foci of this zoonotic infection. Analysis of the complete S segment sequences recovered for two wild-type PUUV strains confirmed the existence of a distinct genetic lineage and also indicated a possible quasispecies type of Slovenian PUUV.

Animals↗
PubMed2006

Genetic analysis of Thailand hantavirus in Bandicota indica trapped in Thailand.

Sixty one tissue samples from several rodent species trapped in five provinces of Thailand were examined for the presence of hantaviral markers by enzyme-immunoassay and immunoblotting. Four samples, all from the great bandicoot rat Bandicota indica, were confirmed positive for the hantaviral N-antigen. Two of them were trapped in Nakhon Pathom province, the other two in Nakhon Ratchasima province, approximately 250 km from the other trapping site. When analysed by RT-nested PCR, all four rodents were found positive for the hantaviral S- and M-segment nucleotide sequences. Genetic analysis revealed that the four newly described wild-type strains belong to Thailand hantavirus. On the phylogenetic trees they formed a well-supported cluster within the group of Murinae-associated hantaviruses and shared a recent common ancestor with Seoul virus.

Animals↗
PubMed2006

Genetic analysis of Puumala hantavirus strains from Austria.

Sequences of the Small (S) and the Medium (M) genome segments of Puumala hantavirus (PUUV) were recovered from bank voles Clethrionomys glareolus trapped at 2 locations, Klippitztörl (Carinthia) and Ernstbrunn (Lower Austria). Lung tissue samples from 12 rodents earlier found hantavirus antibody-positive were further screened for the presence of hantaviral N-antigen using immunoblotting. RNA purified from 7 N-Ag-positive samples was subjected to the reverse transcription-polymerase chain reaction with primers designed to recover the complete S segment sequence of PUUV. The amplicons of expected size (approximately 1800 bp) have been recovered from 2 samples, 1 from Klippitztörl and another from Ernstbrunn. From the same 2 samples, PCR amplicons corresponding to the partial M segment sequence (nt 2140 to 2594), have been prepared. The S and M amplicons were sequenced and subjected to genetic analyses. Recovered hantavirus genome sequences undoubtedly belonged to PUUV genotype and the corresponding wild-type hantavirus strains were designated as PUU/Klippitztörl/Cg9/1995 and PUU/Ernstbrunn/Cg641/1995. Phylogenetic analysis placed 2 Austrian PUUV strains within a distinct genetic lineage that also included strains from the neighbouring Slovenia and Croatia. This Alpe-Adrian lineage, previously designated as 'Balkan lineage', was well supported on both S and M phylogenetic trees.

Animals↗
PubMed2006

Oligomerization of hantavirus nucleocapsid protein: analysis of the N-terminal coiled-coil domain.

Hantaviruses constitute a genus in the family Bunyaviridae. They are enveloped negative-strand RNA viruses with a tripartite genome encoding the nucleocapsid (N) protein, the two surface glycoproteins Gn and Gc, and an RNA-dependent RNA polymerase. The N protein is the most abundant component of the virion; it encapsidates genomic RNA segments forming ribonucleoproteins and participates in genome transcription and replication as well as virus assembly. In the course of RNA encapsidation, N protein forms intermediate trimers via head-to-head and tail-to-tail interactions. We analyzed the amino-terminal trimerization domain (amino acid residues 1 to 77) of Tula hantavirus using computer modeling, mammalian two-hybrid assay, and immunofluorescence assay. The results obtained were consistent with the existence of an antiparallel coiled-coil stabilized by interactions between hydrophobic residues. Residues L44, V51, and L58 were important for the N-N interaction; other residues, e.g., L25 and V32, also made a contribution, albeit a modest one. Our alignments of the N-terminal domain of the hantaviral N proteins suggest the coiled-coil structure, and hence the mode of N-protein oligomerization, is conserved among hantaviruses.

Amino Acid Sequence↗
PubMed2006

Tickborne encephalitis virus, Norway and Denmark.

Serum from 2 Norwegians with tickborne encephalitis (TBE) (1 of whom was infected in Denmark) and 810 Norwegian ticks were tested for TBE virus (TBEV) RNA by reverse transcription-polymerase chain reaction. Sequencing and phylogenetic analysis were performed. This is the first genome detection of TBEV in serum from Norwegian patients.

Animals↗
PubMed2005

Phylogenetic evidence for the distinction of Saaremaa and Dobrava hantaviruses.

Dobrava virus (DOBV) and Saaremaa virus (SAAV) are two closely related hantaviruses carried by different rodent species. The distinction of these two viruses has been a matter of debate. While the phylogenies based on the viral M segment sequences were repeatedly showing monophyly of SAAV strains, some trees based on the S segment sequences were not, thus causing questions on the demarcation between these two viruses. In order to clarify this issue, the current collection of the virus S segment sequences was subjected to extensive phylogenetic analysis using maximum likelihood, maximum parsimony and distant matrix methods. In all inferred phylogenies, the SAAV sequences were monophyletic and separated from DOBV sequences, thus supporting the view that SAAV and DOBV are distinct hantavirus species. Since collection of the S segment sequences used in this study "obeyed" the molecular clock, calculations of the split of DOBV and SAAV were now repeated resulting in an estimation of 3.0-3.7 MYA that is very close to the values obtained earlier.

Genes, Viral↗
PubMed2005

Recombinant Tula hantavirus shows reduced fitness but is able to survive in the presence of a parental virus: analysis of consecutive passages in a cell culture.

Tula hantavirus carrying recombinant S RNA segment (recTULV) grew in a cell culture to the same titers as the original cell adapted variant but presented no real match to the parental virus. Our data showed that the lower competitiveness of recTULV could not be increased by pre-passaging in the cell culture. Nevertheless, the recombinant virus was able to survive in the presence of the parental virus during five consecutive passages. The observed survival time seems to be sufficient for transmission of newly formed recombinant hantaviruses in nature.

Animals↗
PubMed2005

Puumala virus in Croatia in the 2002 HFRS outbreak.

HFRS is an endemic disease throughout Croatia. The incidence of HFRS varies in a cyclic fashion, with peaks occurring every couple of years, coinciding with peaks in vole populations. PUUV was shown to be dominant pathogen during the last HFRS outbreak in Croatia in 2002. We focused our research on two newly discovered localities (Okucani and Nova Gradiska) with a high number of reported HFRS cases and a significant increase in rodent population. PUUV infection was verified in 84.2% of patients at this region during the 2002 outbreak. Genetic analysis of wild-type (wt) PUUV strains was performed. Fifty seven bank voles Clethrionomys glareolus originating from PUUV-associated HFRS areas were screened for the presence of PUUV N antigen and 15 (26%) were found positive. Total RNA isolated from rodent lung tissues was reverse transcribed followed by PCR amplification with primers specific for PUUV medium (M) or small (S) genome segments. Partial PUUV M segment sequences (approximately 450 bp long) were recovered from five bank voles and partial S segment sequences (app. 250 nt long)-from two bank voles. Genetic analysis of Croatian wt-PUUV strains revealed their close relatedness suggesting that the two localities belong to the same natural focus of infection. On phylogenetic trees Croatian PUUV strains clustered together with the strains from Slovenia and Austria forming distinct Alpe-Adrian genetic lineage.

Animals↗
PubMed2004

Genetic analysis of Crimean-congo hemorrhagic fever virus in Iran.

Crimean-Congo hemorrhagic fever (CCHF) is a potentially fatal disease caused by a tick-borne virus in the family Bunyavridae. The disease occurs in parts of Africa, Asia, Middle East, and Eastern Europe. During recent years, an increasing number of human CCHF cases have been diagnosed in Iran, but very little information is available on the prevalence and genetic diversity of CCHFV in Iran. In the present study, CCHF virus (CCHFV) isolates from nine Iranian patients infected during 2002 were examined genetically. Nucleotide sequencing of the S- and M-segments, encoding the nucleocapsid protein (NP) and the glycoproteins, respectively, revealed that the different isolates were related closely to each other with nucleotide sequence identities exceeding 98% for both S- and M-segments. Phylogenetic analysis of partial S-segment nucleotide sequences showed that the viruses clustered along with strains from Pakistan and Madagascar in one distinct lineage. Phylogenetic analysis also demonstrated that the Iranian isolates examined in this study and the previously published CCHFV strain ArTeh193-3 clustered into different genetic groups, indicating that at least two genetic lineages of CCHFV could be co-circulating in Iran.

Adolescent↗
PubMed2004

Characterization of tick-borne encephalitis virus from Estonia.

Tick-borne encephalitis virus (TBEV) is a severe problem in Estonia. In the present article the first genetic analysis of Estonian TBEV strains is described. In total, seven TBEV strains were isolated from ticks (Ixodes ricinus and I. persulcaus), rodents (Apodemus agrarius and Cletrionomys glareolus), and serum from a tick-borne encephalitis (TBE) patient. The nucleic acid sequences of the viral genome encoding almost the complete E protein (nt 41-1250) and the 3'-NCR-termini of the Estonian TBEV strains were determined by direct sequencing of RT-PCR products. The results showed that all three known TBEV subtypes, Western TBEV (W-TBEV), Far-Eastern TBEV (FE-TBEV), and Siberian TBEV (S-TBEV), co-circulate in Estonia. The Estonian TBEV strains of the S-TBEV and W-TBEV subtypes clustered with the previously reported strains from Latvia and Lithuania. Within the FE-TBEV subtype, however, the Estonian strain Est2546 clustered together with the strain Sofjin, originating from the Far-East of Russia, but not with the strain RK1424, isolated in the neighboring Latvia. This suggests a different evolutionary history for the Estonian and the Latvian strains in the FE-TBEV subtype. The Estonian TBEV strain (Est3535), which belonged to the S-TBEV subtype, had an organization of the 3'-NCR similar to that of strains from the Far-East of Russia (Irkutsk). The 3'-NCRs of Estonian strains of the W-TBEV subtype (Est3051, Est3053, Est3476, and Est3509) were very similar to those of the strain Ljubljana I from the Balkans. In the 3'-NCR sequence of the Estonian strain Est2546, which belonged to the FE-TBEV subtype, a deletion from position 10461 to 10810 extending approximately 10 nucleotides into the core element, was detected.

Amino Acid Sequence↗
PubMed2004

Saaremaa hantavirus in Denmark.

Most human hantavirus infections in Denmark have been registered on the Fyn island with Puumala hantavirus hosted by Clethrionomys glareolus as the causative agent. To search for other known European hantaviruses in Denmark, we analyzed 101 Apodemus spp. mice trapped on the Lolland island. Genome sequences of Saaremaa hantavirus (SAAV) were recovered from one of the A. agrarius, thus suggesting SAAV presence in Denmark. We also analyzed serum samples from a patient from Lolland with a history of hantavirus-like infection, probably caused by SAAV. Thus, in addition to Puumala virus, SAAV can impose a threat for the human health in Denmark.

Animals↗
PubMed2004

Identification of Seoul hantavirus in Rattus norvegicus in Indonesia.

The first genetic evidence for the presence of Seoul hantavirus (SEOV) in Indonesia is presented. Partial M segment sequence was recovered from the lung tissue of Rattus norvegicus trapped in central Jakarta. The sequence belongs to SEOV genotype and is most closely related to the strain B-1 from Japan.

Animals↗
PubMed2004

Tula hantavirus L protein is a 250 kDa perinuclear membrane-associated protein.

The complete open reading frame of Tula hantavirus (TULV) L RNA was cloned in three parts. The middle third (nt 2191-4344) could be expressed in E. coli and was used to immunize rabbits. The resultant antiserum was then used to immunoblot concentrated TULV and infected Vero E6 cells. The L protein of a hantavirus was detected, for the first time, in infected cells and was found to be expressed as a single protein with an apparent molecular mass of 250 kDa in both virions and infected cells. Using the antiserum, the expression level of the L protein was followed and image analysis of immunoblots indicated that there were 10(4) copies per cell at the peak level of expression. The antiserum was also used to detect the L protein in cell fractionation studies. In cells infected with TULV and cells expressing recombinant L, the protein pelleted with the microsomal membrane fraction. The membrane association was confirmed with membrane flotation assays. To visualize L protein localization in cells, a fusion protein of L and enhanced green fluorescent protein, L-EGFP, was expressed in Vero E6 cells with a plasmid-driven T7 expression system. L-EGFP localized in the perinuclear region where it had partial co-localization with the Golgi matrix protein GM130 and the TULV nucleocapsid protein.

Animals↗
PubMed2004

Tula hantavirus infection of Vero E6 cells induces apoptosis involving caspase 8 activation.

Hantaviruses are known to cause two severe human diseases: haemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. The mechanisms of pathogenesis of these two diseases are progressively becoming understood. Recently, two hantaviruses, Hantaan and Prospect Hill were reported to cause programmed cell death of Vero E6 cells. This study shows that Tula hantavirus (TULV) infection efficiently triggers an apoptotic programme in infected Vero E6 cells, and that the replication of TULV is required for the activation of caspase 3 and the cleavage of poly (ADP-ribose) polymerase, two molecular hallmarks of apoptosis. The enforced treatment of infected Vero E6 cells with tumour necrosis factor alpha (TNF-alpha), but not interferon alpha (IFN-alpha), advanced the time course of apoptosis. Furthermore, caspase 8 was activated on day 4 post-infection, the same day when caspase 3 was activated. TNF receptor 1 was induced during a late stage of TULV infection. These data suggest that, unlike during influenza A virus infection, TNF-alpha, but not type I IFN-alpha/beta, may contribute significantly to apoptosis in a synergistic manner with TULV propagation. Interestingly, pretreatment with a broad-spectrum caspase inhibitor, z-VAD-fmk, efficiently inhibited apoptosis of TULV-infected Vero E6 cells. Taken together, these results suggest that TULV replication initiates a typical apoptotic programme involving caspase 8 activation.

Animals↗
PubMed2004

Oligomerization of Hantavirus N protein: C-terminal alpha-helices interact to form a shared hydrophobic space.

The structure of the nucleocapsid protein of bunyaviruses has not been defined. Earlier we have shown that Tula hantavirus N protein oligomerization is dependent on the C-terminal domains. Of them, the helix-loop-helix motif was found to be an essential structure. Computer modeling predicted that oligomerization occurs via helix protrusions, and the shared hydrophobic space formed by amino acids residues 380-IILLF-384 in the first helix and 413-LI-414 in the second helix is responsible for stabilizing the interaction. The model was validated by two approaches. First, analysis of the oligomerization capacity of the N protein mutants performed with the mammalian two-hybrid system showed that both preservation of the helix structure and formation of the shared hydrophobic space are crucial for the interaction. Second, oligomerization was shown to be a prerequisite for the granular pattern of transiently expressed N protein in transfected cells. N protein trimerization was supported by three-dimensional reconstruction of the N protein by electron microscopy after negative staining. Finally, we discuss how N protein trimerization could occur.

Amino Acid Sequence↗
PubMed2003

Genetic characterization of new Dobrava hantavirus isolate from Greece.

The first complete genome sequence of Dobrava hantavirus isolated from yellow-necked mouse Apodemus flavicollis trapped in the northeastern Greece is described. The S, M, and L segments of the Greek isolate of Dobrava virus are 1673, 3635, and 6532 nucleotides (nt) long, respectively, and encode the nucleocapsid (N) protein of 429 amino acids (aa), glycoprotein precursor of 1135 aa, and the L protein of 2151 aa. N protein contains three cysteine residues conserved in all known hantaviruses, as well as structural domains responsible for the RNA binding and presumable interaction with the apoptosis enhancer Daxx. All cysteine residues and glycosylation sites that are conserved among G1G2 sequences of all hantaviruses species were also found in the Greek isolate. The L protein contains all the polymerase motifs and structural domains found in other hantavirus polymerases. Comparison of the Greek isolate of Dobrava virus with other hantaviruses showed the highest level of sequence homology with Dobrava virus isolate from Slovenia. Other hantaviruses carried by Murinae rodents (Saaremaa, Hantaan, Seoul, and Thailand viruses) were more divergent and hantaviruses carried by Arvicolinae or Sigmodontinae rodents showed the highest genetic diversity with the Greek isolate of Dobrava. The results of phylogenetic analyses confirmed these observations and showed a monophily of all the Dobrava virus strains that, in turn, shared more ancient ancestors first with Saaremaa virus and then with other Murinae-borne hantaviruses.

Amino Acid Sequence↗