A five-year perspective on the situation of haemorrhagic fever with renal syndrome and status of the hantavirus reservoirs in Europe, 2005-2010.

Hantavirus infections are reported from many countries in Europe and with highly variable annual case numbers. In 2010, more than 2,000 human cases were reported in Germany, and numbers above the baseline have also been registered in other European countries. Depending on the virus type human infections are characterised by mild to severe forms of haemorrhagic fever with renal syndrome. The member laboratories of the European Network for diagnostics of Imported Viral Diseases present here an overview of the progression of human cases in the period from 2005 to 2010. Further we provide an update on the available diagnostic methods and endemic regions in their countries, with an emphasis on occurring virus types and reservoirs.

Natural hybridization between Ixodes ricinus and Ixodes persulcatus ticks evidenced by molecular genetics methods.

The recently shown phenomenon of natural hybridization between Ixodes persulcatus and Ixodes pavlovskyi ticks (Kovalev et al., 2015) stimulated similar studies in the sympatric zones of other tick species. In the present paper, 265 Ixodes ricinus and I. persulcatus ticks from Estonia were subjected to a search for interspecific hybrids based on nuclear (ITS2) and mitochondrial (cox1) markers as well as morphological features. Surprisingly, only 72.1% of ticks morphologically identified as I. ricinus actually were I. ricinus both at nuclear and mitochondrial markers, while the accuracy of morphological species identification for I. persulcatus was 99.3%. Among ticks morphologically identified as I. ricinus, 24.6% turned out to be interspecific hybrids and 3.3% were I. persulcatus. Generally, about 11% of the individuals studied were shown to be interspecific hybrids with different levels of nuclear DNA introgression. The analysis of hybrid populations proved the mating pair female I. ricinus×male I. persulcatus to form hybrids more efficiently, then female I. persulcatus×male I. ricinus. The same trend can be observed for backcrosses preferentially mating with I. ricinus. Hybridization between I. ricinus and I. persulcatus proved the existing view about their reproductive isolation to be untenable. Interspecific hybridization occurring between both closely (I. persulcatus and I. pavlovskyi) and more distantly (I. ricinus and I. persulcatus) related Ixodes species could introduce novel alleles that modify vector competence, host use or the ability to exploit diverse microhabitats.

Genetic variability of Rickettsia spp. in Ixodes persulcatus ticks from continental and island areas of the Russian Far East.

Rickettsia spp. are intracellular Gram-negative bacteria transmitted by arthropods. Two potentially pathogenic rickettsiae, Candidatus Rickettsia tarasevichiae and Rickettsia helvetica, have been found in unfed adult Ixodes persulcatus ticks. The aim of this study was to assess the prevalence and genetic variability of Rickettsia spp. in I. persulcatus ticks collected from different locations in the Russian Far East. In total, 604 adult I. persulcatus ticks collected from four sites in the Khabarovsk Territory (continental area) and one site in Sakhalin Island were examined for the presence of Rickettsia spp. by real-time PCR. Nested PCR with species-specific primers and sequencing were used for genotyping of revealed rickettsiae. The overall prevalence of Rickettsia spp. in ticks collected in different sites varied from 67.9 to 90.7%. However, the proportion of different Rickettsia species observed in ticks from Sakhalin Island significantly differed from that in ticks from the Khabarovsk Territory. In Sakhalin Island, R. helvetica prevailed in examined ticks, while Candidatus R. tarasevichiae was predominant in the Khabarovsk Territory. For gltA and ompB gene fragments, the sequences obtained for Candidatus R. tarasevichiae from all studied sites were identical to each other and to the known sequences of this species. According to sequence analysis of gltA, оmpB and sca4 genes, R. helvetica isolates from Sakhalin Island and the Khabarovsk Territory were identical to each other, but they differed from R. helvetica from other regions and from those found in other tick species. For the first time, DNA of pathogenic Rickettsia heilongjiangensis was detected in I. persulcatus ticks in two sites from the Khabarovsk Territory. The gltA, ompA and оmpB gene sequences of R. heilongjiangensis were identical to or had solitary mismatches with the corresponding sequences of R. heilongjiangensis found in other tick species.

Identification of Anaplasma phagocytophilum in tick populations in Estonia, the European part of Russia and Belarus.

Anaplasma phagocytophilum is associated with diseases of goats, sheep, cattle, dogs and horses. In the beginning of the 1990s it was identified as a human pathogen, causing human granulocytic anaplasmosis (HGA) in the USA, Europe and the far east of Russia. A. phagocytophilum is maintained in nature in an enzootic cycle including ticks as the main vector and a wide range of mammalian species as reservoirs. Ixodes ricinus and I. persulcatus ticks were collected in Estonia, Belarus and the European part of Russia and screened for the presence of A. phagocytophilum by real-time PCR. Positive samples were found only among I. ricinus, in 13.4% in the European part of Russia, 4.2% in Belarus, 1.7% in mainland Estonia and 2.6% on Saaremaa Island. Positive samples were sequenced for partial 16S rRNA, groESL and ankA genes and phylogenetic analyses were performed. The results showed that A. phagocytophilum circulating in Eastern Europe belongs to different groESL lineages and 16S rRNA gene variants and also consists of variable numbers of repetitive elements within the ankA gene.

Upsurge of tick-borne encephalitis in the Baltic States at the time of political transition, independent of changes in public health practices.

Despite evidence that socio-economic factors associated with political transition played a major causal role in the abrupt upsurge in tick-borne encephalitis (TBE) in the newly independent Baltic States, doubts are still repeatedly expressed about the importance of these factors relative to changes in public health practices that may have affected merely the registration of cases. In response to these doubts, evidence of relevant practices of surveillance, registration, diagnosis, awareness and immunization is presented as taken from archived data and interviews with experienced medical practitioners. There were changes that could have had neutral, negative or positive impacts on recorded TBE incidence, but the variable timing in these changes at both national and regional levels is not consistent with their having been responsible for the epidemiological patterns observed in the early 1990 s.

Diagnostic potential of puumala virus nucleocapsid protein expressed in Drosophila melanogaster cells.

Puumala virus (PUU) nucleocapsid protein (N) was expressed in insect cells by using the Drosophila Expression System (DES; Invitrogen BV, Groningen, The Netherlands). Stable transfectants were established by hygromycin B selection and showed continuous expression of the recombinant protein (DES-PUU-N) for at least 5 months. The antigenic property of DES-PUU-N was shown to be identical to that of native PUU N when examined with a panel of hantavirus-specific monoclonal antibodies. Enzyme-linked immunosorbent assays (ELISAs) for detection of human immunoglobulin M (IgM) and IgG antibodies were established by using DES-PUU-N as antigen and were compared to assays based on native N. The ELISAs were evaluated for patient diagnosis and seroepidemiological purposes with panels of sera collected from patients with hemorrhagic fever with renal syndrome (HFRS) and from healthy blood donors. Equally high sensitivities and specificities for detection of PUU-specific IgM in acute-phase HFRS patient sera were obtained by the ELISA based on DES-PUU-N and the assay based on the native antigen. For detection of PUU-specific IgG, the ELISA based on monoclonal antibody-captured DES-PUU-N antigen showed optimal sensitivity and specificity.

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.

Human Dobrava hantavirus infections in Estonia.

PIP: Outbreaks of severe hemorrhagic fever with renal syndrome (HFRS) caused by Dobrava hantavirus (DOB) have been reported in Albania, Greece, and Bosnia-Herzegovina. A seroepidemiologic study conducted by the authors in Estonia provided the first evidence of human DOB infection in a Baltic country. Hantavirus infection was first confirmed in Estonia in 1986 and believed related to Puumala hantavirus (PUU). To enable precise serotyping, serum samples were collected from 1728 healthy people in 11 counties in Estonia. Of 51 samples confirmed as positive, 34 showed the highest reactivities to PUU, while 17 reacted best with DOB antigen. In a further analysis of 11 selected positive samples, six showed the presence of neutralizing antibodies specific to PUU and five reacted specifically with DOB. These findings suggest that both BOB and PUU cause human infections in Estonia.^ieng

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.

Tick-borne encephalitis virus in Lithuania.

Tick-borne encephalitis (TBE) is a severe problem in Lithuania, indicated by the 171 to 645 serologically confirmed cases that occurred each year between 1993 and 1999. In the present report, the first isolation and partial genetic analysis of a Lithuanian TBE virus (TBEV) strain isolated from a patient's serum sample is described. The patient was bitten by a tick while visiting the Lazdijai district (Veisiejai forest) in the southernmost part of Lithuania, a geographical area where Ixodes ricinus but not Ixodes persulcatus ticks are known to be present. The E protein-encoding viral gene sequence (nt 74-1273) recovered from the TBEV isolate showed the closest similarity to previously characterized European strains of the Western TBEV subtype, including the prototype TBEV strain Neudoerfl and those from neighbouring Latvia. Accordingly, the Lithuanian isolate was placed within the Western genetic lineage of TBEV in phylogenetic trees.

Isolation and characterization of Dobrava hantavirus carried by the striped field mouse (Apodemus agrarius) in Estonia.

Dobrava hantavirus (DOB) was isolated from the striped field mouse (Apodemus agrarius) trapped on Saaremaa Island, Estonia, and its genetic and antigenic characteristics were subsequently analysed. Phylogenetic analysis showed that the Estonian DOB strain, together with several wild strains carried by Apodemus agrarius, forms a well-supported lineage within the DOB clade. The topography of the trees calculated for the S, M and L nucleotide sequences of the Estonian DOB suggests a similar evolutionary history for all three genes of this virus and, therefore, the absence of heterologous reassortment in its evolution. A cross-neutralization comparison of the Estonian virus with the prototype DOB, isolated from a yellow-necked mouse (A. flavicollis) in Slovenia, revealed 2- to 4-fold differences in the end-point titres of rabbit and human antisera. When studied with a panel of 25 monoclonal antibodies (MAbs), the Estonian and Slovenian DOB isolates showed similar antigenic patterns that could be distinguished by two MAbs. Genetic comparison showed sequence differences in all three genome segments of the two DOB isolates, including an additional N-glycosylation site in the deduced sequence of the G2 protein from the Estonian virus. Whether any of these mutations relates to the different rodent hosts rather than to the distant geographical origin of the two isolates remains to be resolved. Taken together, our observations suggest that A. agrarius, which is known to harbour Hantaan virus in Asia, carries another hantavirus, DOB, in north-east Europe.