Genotyping and characterization of the geographical distribution of tick-borne encephalitis virus variants with a set of molecular probes.

A panel of deoxyoligonucleotide probes for studying the genetic variability and genotyping of Tick-borne encephalitis virus (TBEV) strains by molecular hybridization of nucleic acids (MHNA) was created. This panel allows to estimate the genetic structure of individual TBEV strains, as the targets for probes are both variable and genotype (subtype)-specific sequences of all TBEV genes. With the help of this panel using the method of molecular hybridization of nucleic acids 268 archived TBEV isolates were investigated and the distribution of its genotypes and subgenotypes of genotype 3 was made more precise in the territory of Eurasia. The conclusion made earlier has confirmed that five genotypes of TBEV co-circulate in Eastern Siberia. It is generally recognized that the Far Eastern (TBE-FE), European (TBE-Eu), and Siberian (TBE-Sib) genotypes are widespread and epidemiologically important. The fourth genotype is presented by only one isolate, TBE178-79, originated from Irkutsk region, Russia. The fifth genotype includes 10 isolates, 1 of them, TBE886-84, was found earlier and recognized as unique [Zlobin et al. (2001b): Vopr Virusol 1:12-16 (Russian)].

Serological survey for viral pathogens in Turkish rodents.

Wild rodents (n = 330) were trapped around the villages of Altindere and Cosandere (Maçka, Trabzon Province), Ayder, Ortan, and Yolkiyi (Camlihemsin, Rize Province), and Bozdag (Odemis, Izmir Province) in northeastern and western Turkey during April 2004. Samples were tested for arenavirus, hantavirus, and cowpox virus (family Poxviridae, genus Orthopoxvirus, CPXV) antibodies by using immunofluorescence assays (IFAs). Antibodies against arenaviruses were found in eight of 330 (2.4%) rodents. Arenavirus sero-positive animals were found from all study sites. Antibodies to Puumala virus (family Bunyaviridae, genus Hantavirus, PUUV) were detected in four of 65 Microtus voles tested. Of the PUUV-IFA-positive voles, one Microtus guentheri lydius was caught from Izmir, and one Microtus roberti and two Microtus rossiaemeridionalis were captured near Trabzon. All 264 Apodemus spp. mice tested negative for antibodies to Saaremaa virus (family Bunyaviridae, genus Hantavirus, SAAV); the single Dryomys nitedula tested negative for both PUUV and SAAV antibodies. Only one (0.3%) of the rodents, an Apodemus sylvaticus from Trabzon area, tested seropositive to CPXV. This is the first serologic survey for rodent-borne viruses in their natural hosts in Turkey. Although these preliminary results support presence of several virus groups with zoonotic potential, additional studies are needed to identify the specific viruses that are present in these populations.

Sequence analysis of the Puumala hantavirus Sotkamo strain L segment.

Sequence of the Puumala virus (PUU) Sotkamo strain L segment is provided, completing the total genome of this prototype PUU virus strain. The L segment is 6530 nucleotides long and it can encode 2156 amino-acids-long L protein, RNA-dependent RNA polymerase. The strain Sotkamo, originally isolated in Finland, showed for the L genome segment nucleotide (84.6%) and amino acid (97.3%) homology to a previously sequenced PUU Russian isolate, strain Bashkiria/CG1820 (B1820) and the L genome segment appeared to be at least as conserved as the S segment. Phylogenetic analysis based on the S, M and L segment sequences proposes that the three viral genes have a similar evolutionary history with no evidence for genome segment reassortment. Precise sequencing of the L segment termini demonstrated that the Puumala strains differ from the conserved sequences of the other hantaviruses at two positions.

Nucleotide and deduced amino acid sequences of the M and S genome segments of a Swedish Puumala virus isolate.

The Swedish Puumala (PUU) virus strain Vindeln 83-L20, isolated from a bank vole trapped in 1983 near Vindeln, Västerbotten county, Sweden, was characterized by nucleotide sequence analysis. The coding region of the M segment was determined by PCR followed by direct sequencing and the entire S segment was characterized by cloning and nucleotide sequence analysis. The genomic organization was found to be very similar to that of other PUU virus strains regarding open reading frames, polypeptide sizes and potential glycosylation sites. According to phylogenetic analysis 83-L20 was found to represent a new lineage within the Puumala virus serotype in the Hantavirus genus. The M segment sequence of 83-L20 was found to be more closely related to the Finnish PUU virus strains than to strains from Central Europe or from Russia. The evolutionary origin of the S segment was not as clearly resolved since the branching points of all PUU virus strains in the phylogenetic tree were nearly the same.

Characterization of Tula virus antigenic determinants defined by monoclonal antibodies raised against baculovirus-expressed nucleocapsid protein.

Tula virus was recently discovered by RT-PCR in lung samples from European common voles (Microtus arvalis and M. rossiaemeridionalis). Since virus isolation attempts had been unsuccessful, no antigen was available for analysis or for use in immunoassays. To circumvent this, complete Tula virus nucleocapsid protein (bac-TUL-N) was expressed in recombinant baculovirus. Rodent antibody end-point titers to bac-TUL-N and to truncated N fragments indicated that the NH2-terminal region is the major antigenic target and revealed a high cross-reactivity to Puumala virus N. Immunizations with crude bac-TUL-N preparations evoked high antibody responses to native hantavirus N in Balb/c mice and six monoclonal antibodies (Mabs) were generated. Epitope mapping of the Mabs, based on a competitive assay, reactivities to truncated recombinant N fragments, and reactivity patterns to different hantavirus strains, identified five recognition sites on Tula virus N. One epitope, which was identified as specific for Tula virus, was located in a region of N which is highly variable among the hantaviruses (aa 226-293), and four epitopes were mapped to the NH2-terminal region of the protein (aa 1-61). One epitope was expressed only in Tula and Prospect Hill viruses, one epitope in Tula, Prospect Hill, Khabarovsk, and Sin Nombre viruses, while two epitopes were conserved in all examined hantaviruses carried by rodents within the subfamily Arvicolinae of the Muridae family.

Genetic variation of wild Puumala viruses within the serotype, local rodent populations and individual animal.

Reverse transcriptase polymerase chain reaction cloning and sequencing were used to determine the range of S gene/N protein variability in wild Puumala virus (PUU) strains and to study phylogenetic relationships between two groups of strains which originated from Finland and from European Russia. Analyses of the nucleotide and predicted amino acid sequences showed: (1) all PUU strains shared a common ancient ancestor; and (2) the more recent ancestors were different for the Finnish branch and the Russian branch of PUU strains. A cluster of amino acid substitutions in the N protein of Finnish strains was found; this cluster was located within a highly variable region of the molecule carrying B-cell epitopes (Vapalahti et al., J. Med. Virol., 1995, in press). Different levels of S gene/N protein diversity of PUU were revealed supporting the view of geographical clustering of genetic variants. Puumala virus from individual voles was found to be a complex mixture of closely related variants-quasispecies. The ratio of non-silent to silent nucleotide mutations registered in the S genes/N proteins of PUU quasispecies was 4- to 16-fold higher than that in Puumala virus strains, resulting in a more wide range of quasispecies N protein sequence diversity.

Genetic variation in Tula hantaviruses: sequence analysis of the S and M segments of strains from Central Europe.

Hantavirus carried by the European common vole Microtus arvalis from Moravia (Czech Republic) was analyzed by RT-PCR-sequencing and by reactivity with a panel of monoclonal antibodies (MAbs). Sequencing of the full-length S segment and the proximal part of the M segment showed that the virus belonged to genotype Tula (TUL) we discovered earlier in Microtus arvalis from Central Russia. This finding supported the concept of host dependence of hantaviruses. Phylogenetic analyses suggested a similar evolutionary history for S and M genes of TUL strains; thus far there is no evidence for reassortment in TUL. Geographic clustering of TUL genetic variants was observed and different levels of the genetic variability were revealed resembling those estimated for another hantavirus, Puumala (PUU). Comparison of the deduced N protein sequence from Russia and from Moravia showed that genetic drift in TUL occurred not only by accumulation of point mutations but also by the deletion of a nucleotide triplet. It encoded Ser252 which was located within a highly variable hydrophilic part of the N protein carrying B-cell epitopes and presumably forming a loop. Analysis of naturally expressed TUL N-antigen derived from lung tissue of infected voles with MAbs indicated antigenic heterogeneity among TUL strains.

Spatial and temporal dynamics of Puumala hantavirus infection in red bank vole (Clethrionomys glareolus) populations in Belgium.

Dynamics of hantavirus infection and population densities in rodents were investigated from 1996 to 1999 in southern Belgium. Evidence of Puumala infection was restricted to Clethrionomys glareolus. Although the serotype was not determined, antibodies against hantavirus were also found in eight Apodemus sylvaticus. In fall 1996, the seroprevalence in C. glareolus was high (20.1%, 37 of 184) and the infection was widely distributed in the area studied whereas a focal occurrence of positive rodents and lower seroprevalence rates were recorded in spring 1997 (14.3%, six of 42), fall 1997 (6. 6%, 11 of 166), spring 1998 (6.4%, three of 47) and fall 1998 (6.7%, 11 of 165). A pullulation of rodents was observed in spring 1999 and was associated with a markedly higher seroprevalence in C. glareolus (47.7%, 189 of 396). In all seasons, infection rates in adults were higher than in juveniles and subadults. No significant difference of prevalence was recorded between males and females. In two trapping sites, the temporary disappearance of positive animals after a crash in rodent populations suggests that a threshold in density is necessary for the maintenance of the enzootic cycle.

Genetic characterization of Puumala hantavirus strains from Belgium: evidence for a distinct phylogenetic lineage.

Puumala hantavirus (PUUV) sequences were recovered from red bank voles (Clethrionomys glareolus) trapped between 1996 and 1998 in four localities of southern Belgium: Thuin, Montbliart, Momignies and Couvin. In addition, three PUUV isolates originating from bank voles trapped in the 1980s in southern (Montbliart) and northern (Turnhout) Belgium were genetically characterized. Analysis of the complete S and partial M segment sequences showed that the Belgian PUUV strains constitute a genetic lineage, distinct from other known PUUV lineages from Europe and Japan. This lineage also includes a wild strain (Cg-Erft) originating from a neighbouring area of Germany. Within the Belgian lineage, geographical clustering of genetic variants was observed. In the Montbliart site, the range of diversity between the most temporally distant strains (from 1986 and 1996-1998) was higher than between those from 1996 and 1998, suggesting slight genetic drift via accumulation of neutral or quasi-neutral substitutions with time.

Molecular epidemiology of viral pathogens and tracing of transmission routes: hepatitis-, calici- and hantaviruses.

BACKGROUND: The need to rapidly identify new therapeutic drugs and vaccines for clinically important viral infections has resulted in intensive study of the molecular properties of viruses. Modern molecular techniques have provided tools for tracing infections and studying the evolution of viruses. OBJECTIVE STUDY AND DESIGN: Two examples illustrating how modern molecular techniques can be used in clinical virology and molecular epidemiology (hepatitis and caliciviruses), and one example documenting their importance in basic research (hantaviruses) will be discussed. RESULTS AND CONCLUSIONS: Water- and food-borne outbreaks caused by the faeco-orally spread hepatitis A virus (HAV) are common in areas lacking proper sanitation, but they are possible also in countries with low seroprevalence. In water epidemics, the sequence comparisons between the virus from patients and from water have been used successfully. Hepatitis B virus variants are clinically important and challenge the diagnostic tests and prophylactic measures. Some hepatitis C (HCV) genotypes appear to be associated with more severe pathology and others respond better to antiviral treatment. Nosocomial and occupational infections are not rare, and the source can be identified by phylogenetic analysis of nucleotide sequences obtained from the infected individuals. The overwhelming role of Norwalk-like caliciviruses (NLV) in adult diarrhoea and especially in food- and water-borne epidemics has become apparent during the last decade. Methods are under development for detecting these viruses, not only from patient samples and water, but also from other environmental samples (e.g. foodstuff and surface swabs). The analysis of the genetic variation and evolution of the Old World hantaviruses in their carrier rodents has shown that the extent of genetic diversity correlates with geographical distance. As a rule, phylogenetic relationships of hantaviruses resemble those of their rodent hosts, suggesting virus-host co-evolution. Exceptional host-switch events allow a study on still radiating hantavirus species. There is suggestive evidence that natural reassortant hantaviruses are involved in human infection.

Tick-borne virus diseases of human interest in Europe.

Several human diseases in Europe are caused by viruses transmitted by tick bite. These viruses belong to the genus Flavivirus, and include tick-borne encephalitis virus, Omsk haemorrhagic fever virus, louping ill virus, Powassan virus, Nairovirus (Crimean-Congo haemorrhagic fever virus) and Coltivirus (Eyach virus). All of these viruses cause more or less severe neurological diseases, and some are also responsible for haemorrhagic fever. The epidemiology, clinical picture and methods for diagnosis are detailed in this review. Most of these viral pathogens are classified as Biosafety Level 3 or 4 agents, and therefore some of them have been classified in Categories A-C of potential bioterrorism agents by the Centers for Disease Control and Prevention. Their ability to cause severe disease in man means that these viruses, as well as any clinical samples suspected of containing them, must be handled with specific and stringent precautions.

Serological and genetic evidence for the presence of Seoul hantavirus in Rattus norvegicus in Flanders, Belgium.

Seoul hantavirus (SEOV), carried by Rattus rattus (black rat) and R. norvegicus (Norway, brown rat), was reported to circulate as well as cause HFRS cases in Asia. As Rattus sp. are present worldwide, SEOV has the potential to cause human disease worldwide. In Europe however, only SEOV prevalence in rats from France was reported and no confirmed cases of SEOV infection were published. We here report genetic and serological evidence for the presence of SEOV virus in brown rat populations in Belgium. We also serologically screened an at-risk group that was in contact with R. norvegicus on a daily basis and found no evidence for SEOV infection.

Hantavirus nucleocapsid protein: a multifunctional molecule with both housekeeping and ambassadorial duties.

In recent years important progress has been made studying the nucleocapsid (N) protein of hantaviruses. The N protein presents a good example of a multifunctional viral macromolecule. It is a major structural component of a virion that encapsidates viral RNA (vRNA). It also interacts with the virus polymerase (L protein) and one of the glycoproteins. On top of these "house keeping" duties, the N protein performs interactive "ambassadorial" functions interfering with important regulatory pathways in the infected cells.

L protein, the RNA-dependent RNA polymerase of hantaviruses.

L protein of hantaviruses is the RNA transcriptase and replicase that transcribes mRNAs and replicates the genomic RNA using antigenomic RNA as an intermediate. It also appears to have endonuclease activity. In this review, the current knowledge on the hantavirus L protein is presented including sequence motifs conserved in RNA polymerases, mechanisms of RNA synthesis and also the most recent findings on homologous RNA recombination and membrane association.

Genetics of hantaviruses: implications to taxonomy.

Hantaviruses (genus Hantavirus, family Bunyaviridae) represent a prime example of emerging viruses. Since isolation of the prototype Hantaan virus in the late 70s more than 20 new species have been described and the number is increasing fast thus demanding for a more refined classification. Taking into account that hantaviruses are difficult to isolate in cell culture, one should not be surprised that most of the "newcomers" were first described as distinct hantavirus genotypes. Moreover, the only "solid" characteristics of many hantavirus species still exist in the form of nucleotide sequences of their genome. The relatively short history of hantavirology can thus be taken to illustrate how genetics can contribute to (and even, perhaps, dominate) discovery, characterization and classification of viruses. In this review the following aspects of hantavirus genetics are discussed: (i) genome structure; (ii) genetic diversity and evolution; and (iii) use of genetic criteria in current taxonomy of hantaviruses. In addition, several examples of classification of hantavirus species (New York virus, Saaremaa virus and Hokkaido virus) are given, and future prospects are analyzed.

Molecular evolution of Puumala hantavirus.

Puumala virus (PUUV) is a negative-stranded RNA virus in the genus Hantavirus, family Bunyaviridae. In this study, detailed phylogenetic analysis was performed on 42 complete S segment sequences of PUUV originated from several European countries, Russia, and Japan, the largest set available thus far for hantaviruses. The results show that PUUV sequences form seven distinct and well-supported genetic lineages; within these lineages, geographical clustering of genetic variants is observed. The overall phylogeny of PUUV is star-like, suggesting an early split of genetic lineages. The individual PUUV lineages appear to be independent, with the only exception to this being the Finnish and the Russian lineages that are closely connected to each other. Two strains of PUUV-like virus from Japan form the most ancestral lineage diverging from PUUV. Recombination points within the S segment were searched for and evidence for intralineage recombination events was seen in the Finnish, Russian, Danish, and Belgian lineages of PUUV. Molecular clock analysis showed that PUUV is a stable virus, evolving slowly at a rate of 0.7 x 10(-7) to 2.2 x 10(-6) nt substitutions per site per year.

Completion of the Tula hantavirus genome sequence: properties of the L segment and heterogeneity found in the 3' termini of S and L genome RNAs.

In this study the L segment and the 5' and 3' termini of the S, M and L segments of the prototype Tula hantavirus (TUL) were sequenced, thus completing the first determination of the genome sequence of a hantavirus that has not been linked to any human disease. The TUL L segment comprises 6541 nt with one ORF of 6459 nt in the antigenome sense. This ORF potentially encodes a 2153 aa protein with a predicted molecular mass of 247 kDa. The amino acid sequence includes all the motifs conserved in RNA-dependent RNA polymerases. The 5' termini of all three genome RNAs (vRNAs) had the expected sequences conserved in hantaviruses. The 3' termini of M vRNAs were also conserved. However, the 3' termini of S and L vRNAs were heterogeneous as most of the sequenced 3' termini had either deletions of 1 to 22 nt or an extra 1 to 3 nt. No increase in the level of heterogeneity was seen in vRNAs of virions collected 3, 6, 9 and 12 days post-infection, suggesting that the heterogeneity already exists at the early stages of infection. The S and L vRNAs from infected cells had more truncated 3' termini than vRNAs from pelleted virus. Heterogeneity of the 3' termini of genome RNAs could decrease the efficiency of antigenome and mRNA syntheses and contribute to the slow growth observed for TUL and other hantaviruses in cell culture.