Epidemiological and Ecological Studies of Tick-borne Encephalitis Virus

Abstract: Ticks are blood-sucking parasites that are an inconvenience for both humans and animals. The tick by itself is normally harmless unless they attack in excessive numbers. The harm from ticks stems from them being excellent vectors for other parasites, in the form of bacteria and virus that via the ticks are provided a bridge to move across the blood streams of different animals, including humans.One of the most pathogenic tick-borne disease for humans is caused by a flavivirus, the tick-borne encephalitis virus (TBEV). Each year approximately 10 000 individuals on the Eurasian continent develop neurological disease, in the form of meningitis, encephalitis, myelitis and radiculitis, following a bite by a TBEV infected tick.To evaluate the risk of TBEV infection after a tick-bite, we have developed a study to investigate ticks that have bitten humans and to follow up the tick-bitten humans to investigate if they get infected, and if they develop symptoms, and further trace the virus back to the tick that is infected with TBEV. Ticks, blood samples, and questionnaires were collected in collaboration with 34 primary health care centers in Sweden and on the Åland Islands during 2008 and 2009.Several demographical and biological factors were investigated regarding the interaction between ticks and humans. The main finding was that men removed the ticks later than women, and that both older men and older women removed the ticks later than younger individuals. This could in part explain why older individuals in general, and men in particular, are at greater risk of acquiring tick-borne encephalitis (TBE).Furthermore, the prevalence of TBEV in ticks that have bitten humans were investigated, in order to correlate the copy number of TBEV in the tick and the tick feeding-time to the risk of developing symptomatic and asymptomatic infection. This entailed the development of new methodology for tick analysis and TBEV real-time PCR. The result showed a very low risk of TBEV infection in the studied areas, only 5 of 2167 investigated ticks contained TBEV. Three of the individuals bitten by TBEV infected ticks were vaccinated and did not develop symptoms of TBEV infection. One unvaccinated individual got bitten by a tick containing 1800 virus copies, with a feeding-time of 12-24h, and interestingly showed no signs of infection. Another unvaccinated individual got bitten by a tick containing 7.7 million virus copies, with a feedingtime of >60h. This individual developed symptoms consistent with a 1st phase of TBE, including fever and headache, but did not develop the 2nd neurological phase of TBEV infection. Despite only  finding 5 ticks infected with TBEV, a correlation between the virus load in the tick and the tick feeding-time was observed. In 2 other individuals, TBEV antibody seroconversion was detected during the 3 month study period, one without symptoms, while the other experienced symptoms consistent with the 1st phase of TBE. These observations support the hypothesis that a higher virus amount in the tick and a longer feeding time increases the risk of TBEV infection.To further examine TBEV in ticks that have bitten humans and find factors that may predict the risk of human infection and development of TBE, we characterized several TBEV strains genetically. Including TBEV strains isolated from ticks that have bitten human, from questing field-collected ticks, and TBEV strains isolated from patients with TBE. In one of the ticks detached from a human after >60h of feeding, there was a heterogeneous population of TBEV quasispecies with varying poly(A) length in the 3’ untranslated region of the genome was observed. These variations might have implications for differences in virulence between TBEV strains, and the heterogeneous quasispecies population observed could be the virus adapting from replication in tick cells to mammalian cells.We also investigated the response to TBEV vaccination in relation to 14 health-related factors in a population of older individuals on the Åland Islands. Blood samples, questionnaires, and vaccination records were collected from 533 individuals. Three different serological assays to characterize antibody response to TBEV vaccination were used. The main finding was that the number of vaccine doses in relation to age was the most important factor determining successful vaccination. The response to each vaccination dose declined linearly with age, and as much as 47%  of individuals 50 years or older that had taken 3 vaccine doses were seronegative, compared to 23% that had taken 4 doses and 6% with 5 doses. Comparison between the serological assays revealed that the cutoffs determining the balance between sensitivity and specificity differed, but not the overall accuracy.Taken together, these results contribute to a better understanding of the TBEV epidemiology and can provide tools in the prevention of TBE.