Viral zoonoses in Estonia : hantaviruses and tick-borne encephalitis virus : Identification, prevalence, serological and genetic relationships

Sammanfattning: Hantaviruses and tick-borne encephalitis virus (TBEV) cause serious human infections. Hantaviruses are transmitted to humans by wild rodents and cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome. Two clinical courses of HERS are recognized in Europe: a severe, with a reported mortality rate up to 14%, and a milder with a mortality rate of approx. 0,1%. The more severe form is associated with Dobrava virus (DOBV), transmitted by Apodemus flavicollis, and the milder one with Puumala virus (PUUV), transmitted by Clethrionomys glareolus, and probably also with Saaremaa (SAAV) transmitted by Apodemus agrarius. In Estonia, only a few cases of HERS are officially registered each year, in contrast to a large number of patients in the neighbouring countries, Finland, Sweden and Russia. Therefore, one of our goals was to study the presence and distribution of hantaviruses in Estonia and their significance for human morbidity. We described the first serologically confirmed cases of HFRS in Estonia, with evidence of PUUV and SAAV as the causative agents. In general, a mild clinical course was observed in the patients. The most common symptoms were fever, headache, microscopic hematuria and backache. Laboratory findings included elevated serum creatinine concentrations and proteinuria. Antibodies to PUUV as well as to SAAV, were found in the human population throughout Estonia. Our studies revealed highly variable hantavirus seroprevalence rates. The highest prevalence rates to SAAV were found on Saaremaa and Vormsi islands (23,1% and 7,3% respectively) and to PUUV in the central/south-western parts of Estonia (up to 18,4% in Raplamaa county). The relatively high prevalence suggested that a significant number of HERS cases remain undiagnosed. Our studies showed that SAAV and DOBV represent distinct serotypes within the genus Hantavirus. We revealed a clear serological distinction between SAAV and DOBV as 23 human sera reacted with at least fourfold higher neutralizing end-point titers to one of the two viruses. These findings, together with the facts that SAAV and DOBV are carried by different rodent hosts (A. agrarius and A. flavicollis) and that the two viruses possess different pathogenicity for humans, suggested that SAAV and DOBV represent distinct hantaviruses. Two rodent species, C. glareolus and A. agrarius, were found positive for hantavirus antigen at rates of 13,7% and 4,5% of the investigated rodents, respectively. Analysis of viral sequences recovered from infected C. glareolus tissue samples showed that the infecting virus belonged to the PUUV genotype, confirming circulation of this hantavirus in mainland Estonia. Together with earlier findings on SAAV in A. agrarius, the results revealed that both hantaviruses, PUUV and SAAV, are common in Estonia. Phylogenetic analysis revealed that the Estonian PUUV strains formed a distinct genetic lineage placed in the closest proximity to Russian strains. TBEV is an arthropod-borne virus, which is transmitted to vertebrates by chronically infected ticks. Tick-borne encephalitis (TBE), caused by TBEV is a well recognized serious health problem in Estonia. At the beginning of the 1990s, the TBE morbidity increased continuously, with a sharp rise in 1997, and remains to date at one of the highest levels in Europe. Before our study very little was known about the circulation of TBEV and the reasons for the high TBE morbidity in Estonia. Therefore, another important goal was to isolate and analyze TBEV strains circulating in the country. The results of our studies show that all three known TBEV subtypes: Western (W-TBEV), Siberian (S-TBEV) and FarEastern (FE-TBEV) are co-circulating in Estonia. Phylogenetic analysis showed clustering of the Estonian strains of S-T13EV and W-T13EV subtypes with the strains from Latvia and Lithuania, which is in agreement with the geographical clustering of TBEV variants. Within the S-TBEV subtype, the Baltic strains form a well supported lineage of their own. This new lineage is distinguished from the lineages that include strains from Siberia and the Far East. We also found an additional signature amino acid residue at position 175 of the E protein sequence, which is unique for all Baltic S-T13EV strains. In contrast, within the FE-T13EV subtype, the Baltic strains from Estonia and Latvia do not cluster together as expected, but instead share the most recent common respective ancestors with strains from the Far-East and Ural.