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 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.

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.

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.

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.

Seoul hantavirus in Europe: first demonstration of the virus genome in wild Rattus norvegicus captured in France.

Although rats (Rattus rattus or Rattus norvegicus) worldwide have been found to carry Seoul hantavirus, there are at present only a very few reports of confirmed human Seoul hantavirus infections outside Asia, where the virus, in certain areas, is responsible for approximately 25% of the human hantavirus infections. In Europe, no confirmed human infections outside laboratories have been described, and although rats occasionally have been found to be antibody positive, the viral genome has not been demonstrated in these animals. The present report describes the first confirmed finding of Seoul hantavirus in R. norvegicus captured in Europe.

Hantavirus infections in Latvia.

In order to investigate the presence of hantavirus infections in Latvia, 333 randomly selected human serum samples were screened using an enzyme-linked immunoassay. Fifteen samples were positive for hantavirus-specific IgG and were subsequently serotyped using a focus reduction neutralization test. Fourteen of these samples neutralized at least one of the hantaviruses included in the test, indicating a 4.2% overall seroprevalence in Latvia. Among 14 focus reduction neutralization test-positive sera, specific reactivity (at least 4-fold higher endpoint titer) of neutralizing antibodies was as follows: six sera were specific for Saaremaa hantavirus, three showed equal titers to Saaremaa and Dobrava hantaviruses, and five showed the highest endpoint titers to Puumala hantavirus. Hantavirus infections were confirmed in individuals residing in 11 of 26 investigated counties. The sex ratio was 1:2.5 (M:F), and the antibody prevalence increased with age. This is the first report on the presence of hantavirus infections in Latvia, and the results indicate that two hantaviruses pathogenic to man, Saaremaa virus and Puumala virus, are widely distributed in this country.

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.

Tula hantavirus in Belgium.

European common voles (Microtus arvalis), captured in Belgium in 1999, were proven by molecular as well as by serological techniques to be infected with Tula hantavirus (TULV). This is the first evidence for the presence of TULV in this country. No indication of spill-over infections of Puumala virus, known to be highly endemic among bank voles (Clethrionomys glareolus) within the same geographical regions as the trapped TULV-infected common voles, was observed. Together with previous reports on the circulation of TULV in eastern/central Europe, this finding suggests a more wide-spread circulation of this hantavirus serotype throughout the continent.

Serological divergence of Dobrava and Saaremaa hantaviruses: evidence for two distinct serotypes.

In order to investigate the serological relationship of Dobrava hantavirus (DOBV, originating from Slovenia) and the Dobrava-like Saaremaa virus (SAAV, recently discovered in Estonia) we analysed 37 human serum samples, 24 from Estonia and 13 from the Balkans, by focus reduction neutralization test (FRNT). Most of the Estonian sera (19), including all sera from Saaremaa island (12), reacted with higher FRNT end-point titres to the local SAAV; the majority of them (15 and 11, respectively), with at least fourfold or higher titres to SAAV than to DOBV. In contrast, out of the 13 sera collected in Slovenia, Bosnia-Herzegovina and Greece, only one reacted more strongly with SAAV (with a twofold higher titre), while 10 of these sera reacted more strongly with the local DOBV (9/10 with fourfold or higher titres). These results indicate that DOBV and SAAV define unique hantavirus serotypes.

Wild-type Puumala hantavirus infection induces cytokines, C-reactive protein, creatinine, and nitric oxide in cynomolgus macaques.

Hantaviruses cause two severe human diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Approximately 200,000 cases are reported annually, and there is to date no specific treatment available. A major obstacle in studying the medical aspects of HFRS and HPS has been the lack of an adequate animal model. Here we show that infection of cynomolgus macaques by wild-type Puumala hantavirus resulted in typical signs of HFRS including lethargy, anorexia, proteinuria, and/or hematuria, in addition to cytokine (interleukin 6 [IL-6], IL-10, and tumor necrosis factor alpha), C-reactive protein, creatinine, and nitric oxide responses. Viral RNA was detected in plasma from days 3 to 7 postinoculation until days 24 to 28 postinoculation, infectious virus was recovered, and the virus-specific immune responses (immunoglobulin M [IgM], IgG, and neutralizing antibodies) mimicked those seen in humans. The results indicated that the monkey model will provide a valuable tool for studies of pathogenesis, candidate vaccines, and antivirals for hantavirus disease.

Characterization of tick-borne encephalitis virus from Latvia: evidence for co-circulation of three distinct subtypes.

Viruses of the tick-borne encephalitis (TBE) antigenic complex within the family Flaviviridae cause a variety of diseases, including uncomplicated febrile illness, meningoencephalitis, and hemorrhagic fever. Different domesticated animals or wildlife species often act as reservoir hosts and ixodid ticks serve as vectors. Although TBE is a serious problem in Latvia, the knowledge concerning TBE virus (TBEV) strains circulating in the country is most limited. Only two strains (Latvia-1-96 isolated from a TBE patient, and RK1424 originating from an Ixodes persulcatus tick), which belonged to the Siberian and the Far Eastern subtypes of TBEV, respectively, have previously been characterized. In the present study, we concentrated on the western and central regions of Latvia, with predominantly Ixodes ricinus ticks. Five virus strains were isolated from serum samples of patients with clinical symptoms of an acute TBE infection. Nucleotide sequences encoding the envelope (E) protein of TBEV, which were recovered from the five TBEV isolates, showed the highest level of identity to the corresponding sequences of the prototype strain Neudoerfl and other European strains of the Western TBEV subtype characterized previously. Accordingly, phylogenetic analysis placed the new Latvian isolates within the Western genetic lineage of TBEV. Taken together with earlier observations, the results proved that all three TBEV subtypes are co-circulating in Latvia and indicated that the genetic diversity of TBEV within certain geographical areas is much more complex than previously believed.

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.

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.

Isolation of Dobrava virus from Apodemus flavicollis in Greece.

Dobrava virus (DOBV) carried by Apodemus flavicollis is the causative agent of severe hemorrhagic fever with renal syndrome (HFRS). DOBV was isolated from an A. flavicollis mouse trapped in northeastern Greece. This is the third DOBV cell culture isolate in the world, clustering together with other Greek DOBV sequences from HFRS patients and rodents.

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.