Irina Golovljova

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.

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.

Human Dobrava hantavirus infections in Estonia

n 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.n

Detection and Characterization of Babesia Species in Ixodes Ticks in Estonia

The presence of Babesia spp. was studied in 2603 Ixodes ricinus and Ixodes persulcatus ticks collected at seven sites in Estonia. By reverse line blot screening, Babesia spp. was detected in 36 (1.4%) ticks, among them 18 (0.7%) were further recognized by a Babesia microti probe, 3 (0.1%) by a Babesia divergens probe, and the other 15 (0.6%) were recognized only by the universal Babesia spp. catch all probe. Sequence analyses of 6 of these 15 samples revealed that all of them belonged to Babesia sp. EU1. B. microti was detected in both tick species I. ricinus and I. persulcatus at the seven sites, whereas B. divergens-like and Babesia sp. EU1 were found only in I. persulcatus and I. ricinus, respectively. Genetic characterization based on partial 18S rRNA showed that the Estonian sequences of B. microti, B. divergens-like, and Babesia sp. EU1 share a high rate of similarity and are closely related to sequences from other European countries, Siberia, and United States. The present study demonstrated for the first time the existence and distribution of Babesia spp. in I. persulcatus and I. ricinus ticks in Estonia.

Puumala and Dobrava hantaviruses causing hemorrhagic fever with renal syndrome in Estonia.

Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome in the Americas [1]. Puumala hantavirus (PUUV) causes a milder form of HFRS and is reported most frequently in northern Europe and in Russia, while Dobrava hantavirus (DOBV) has been connected to a more severe form of disease in the Balkans [1–3]. Recently, DOBV was isolated from the striped field mouse (Apodemus agrarius) in Estonia [4, 5], and human infections have been demonstrated throughout the country by serological screening of healthy blood donors [6]. In this report, we present the first evidence that both PUUV and DOBV cause HFRS in Estonia.

Characterization of hemorrhagic fever with renal syndrome caused by hantaviruses, Estonia.

Thirty cases of hemorrhagic fever with renal syndrome (HFRS) due to Puumala virus (PUUV), Saaremaa virus (SAAV), and Dobrava virus infection were confirmed in Estonia. Except for the levels of serum creatinine, no remarkable differences were found in the clinical course of HFRS caused by PUUV and SAAV.

Tick-Borne Pathogens in Ticks Feeding on Migratory Passerines in Western Part of Estonia

During southward migration in the years 2006-2009, 178 migratory passerines of 24 bird species infested with ticks were captured at bird stations in Western Estonia. In total, 249 nymphal ticks were removed and analyzed individually for the presence of Borrelia burgdorferi sensu lato (s.l.), tick-borne encephalitis virus (TBEV), and Anaplasma phagocytophilum. The majority of ticks were collected from Acrocephalus (58%), Turdus (13%), Sylvia (8%), and Parus (6%) bird species. Tick-borne pathogens were detected in nymphs removed from Acrocephalus, Turdus, and Parus bird species. TBEV of the European subtype was detected in 1 I. ricinus nymph removed from A. palustris. B. burgdorferi s.l. DNA was found in 11 ticks (4.4%) collected from Turdus and Parus species. Bird-associated B. garinii and B. valaisiana were detected in I. ricinus nymphs removed from T. merula. Rodent-associated B. afzelii was detected in 3 I. ricinus nymphs from 2 P. major birds. One of the B. afzelii-positive nymphs was infected with a mix of 2 B. afzelii strains, whereas 1 of these strains was also detected in another nymph feeding on the same great tit. The sharing of the same B. afzelii strain by 2 nymphs indicates a possible transmission of B. afzelii by co-feeding on a bird. A. phagocytophilum DNA was detected in 1 I. ricinus nymph feeding on a T. iliacus. The results of the study confirm the possible role of migratory birds in the dispersal of ticks infected with tick-borne pathogens along the southward migration route via Estonia.

Multilocus sequence analysis of Borrelia burgdorferi sensu lato isolates from Western Siberia, Russia and Northern Mongolia

Lyme borreliosis (LB) is the most frequently recorded tick-transmitted disease in Eurasia. Tomsk Province, Western Siberia in Russia and Selenge Aimag in Northern Mongolia are leading regions in the LB incidence rate in these countries. Spirochaetes of the Borrelia burgdorferi sensu lato (s.l.) complex isolated from Ixodes ticks from Tomsk Province (nu202f=u202f56) and Ixodes persulcatus ticks from Selenge Aimag (nu202f=u202f5) were genetically characterized using Multi Locus Sequence Typing (MLST), analysis of the 5S23S rRNA intergenic spacer (IGS) amplicons, and p83/100 gene sequencing. According to MLST, B. afzelii (nu202f=u202f26), B. bavariensis (nu202f=u202f23), B. garinii (nu202f=u202f11), and B. valaisiana (nu202f=u202f1) isolates were detected in Tomsk Province, while B. afzelii and B. bavariensis isolates were identified in Selenge Aimag. Of the 32 revealed sequence types (ST), 21 STs were new and 14 of the new STs belonged to B. afzelii. Several STs of B. afzelii, B. garinii and B. valaisiana identified in this study clustered with European STs found in I. ricinus ticks. Analysis of the 5S23S IGS demonstrated that the studied Borrelia strains showed RFLP pattern characteristic for the following 5S23S IGS types: VS461 (B. afselii), NT29 (B. bavariensis), 20047 (B. bavariensis and B. garinii), VS116 (B. valaisiana), and three new groups (B. afzelii and B. bavariensis). Notably, this is the first report of Asian B. bavariensis possessing a 5S23S IGS RFLP pattern identical to 20047, and analysis of the 5S23S IGS did not provide correct determination of Borrelia species occurring in Asia. Genotyping of Borrelia strains using the clpA, pepX, and p83/100 genes demonstrated the same result as genotyping based on MLST; and further investigations are required to confirm that these three genetic loci could be used for determination of bacterial species from the B. burgdorferi s.l. complex because data based on single loci may be misleading.

Identification of I. ricinus, I. persulcatus and I. trianguliceps species by multiplex PCR

Correct identification of tick species is an essential requirement for any scientific study engaged in tick-associated research. However, morphological identification can lead to misinterpretations, especially when dealing with vector-host research and sub-adult, engorged or damaged specimens. To overcome this limitation, we developed a novel assay to discriminate between Ixodes ricinus, I. persulcatus and I. trianguliceps species collected from rodents or vegetation, using the second internal transcribed spacer (ITS2) as a genetic marker. This single tube multiplex PCR allows specific amplification of targeted species and produces rapid and accurate results. The specificity was confirmed by sequencing the ITS2 and partial 16S rRNA genes from ticks collected from Estonia, Latvia, Sweden and Russia. We tested the assay in a large-scale experiment, and a total of 1284 ticks removed from rodents and shrews were successfully identified at species level.

Hepatitis E virus infection in different groups of Estonian patients and people who inject drugs

BACKGROUNDnPreviously we demonstrated a high prevalence of hepatitis E virus (HEV) in domestic pigs and wild boars, the main reservoir and possible source of HEV infections in humans. But so far there are no reports about spread of HEV in Estonian human population.nnnOBJECTIVESnThe present study aimed to determine the prevalence and genotyping of HEV in different groups of the Estonian adult population.nnnSTUDY DESIGNnTotally 1426 human serum samples were tested (763 patients with clinically diagnosed nonA/B/C hepatitis, 176 hemodialysis patients, 282 patients with suspected HEV infection and 205 people who injected drugs (PWID)). Presence of anti-HEVantibodies was assessed by ELISA and confirmed by immunoblotting. All anti-HEV positive sera were analyzed for RNA by qPCR. Amplified ORF2 region was sequenced and used for phylogenetic analysis.nnnRESULTSnAntibody assay revealed 49 samples from 1426 (3.4%) with acute (17) or past (32) HEV infection. HEV RNA was detected in 10 anti-HEV IgM positive samples, including 9 samples from patients with suspected HEV infection and 1 hemodialysis patient. Anti-HEV IgG were found in 7.8% patients with suspected HEV infection, in 4% hemodialysis patients, in 2.4% PWID and in 1.96% patients with nonA/B/C hepatitis. All groups demonstrated a trend to share of anti-HEV seroprevalence increasing with age. Phylogenetic analysis of 9 HEV RNA sequences revealed that 3 sequences belonged to HEV genotype 1; 6 ones to genotype 3 (1 sequence belonged to sub-genotype 3a, two ones - sub-genotype 3e, and three ones - to sub-genotype 3f).nnnCONCLUSIONSnDespite the high seroprevalence among domestic pigs, no evidence of HEV transmission from Estonian pigs to humans was found. The results of our study suggest that HEV infections in Estonia are most likely associated with travel or with consumption of imported food products.