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Autor: Karel Křivanec

Co vlastně taky ještě dělám?

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Volume 6, Number 4, 2006
© Mary Ann Liebert, Inc.

Research Paper
Tick-Borne Encephalitis Virus in Clethrionomys glareolus in the Czech Republic



A total of 474 specimens from 157 rodents caught at the military training area of Boletice in the south of the Czech Republic from May to November 1999 were screened for TBEV by nested PCR. TBEV-specific RNA was amplified from lung, kidney, and spleen derived from one Clethrionomys glareolus in the first RT-PCR round. Sequence analysis revealed a 100% identity to the TBEV strain Neudoerfl. TBEV presence in the sample was confirmed by mouse brain passage of backup samples and cell culture. The results support the observation that hantaviruses and TBEV transmission can occur sympatrically in the same rodent population. Key Words: Tick-borne encephalitis virus—Flavivirus—Virus transmission—Co-infection. Vector-Borne Zoonotic Dis. 6, 379–381.


Tick-borne encephalitis virus (TBEV) is a flavivirus occurring across the Eurasian continent from central Europe to the far-east of Russia. The clinical picture induced in man ranges from mild infection to encephalitis, the severity of which increases from western to eastern strains. TBEV has long been believed to cycle between viremic mammalian hosts and ticks which is supported by virus isolation from Clethrionomys glareolus, Apodemus flavicollis, Apodemus sylvaticus, and Sorex araneus (Kozuch et al. 1990, 1995). In recent years evidence has accumulated indicating that virus transmission from tick to tick seems to occur during co-feeding of ticks on a host animal which does not develop a significant viremia. During an infected tick’s blood meal the virus replicates locally in the skin and appears to travel to the feeding site of an uninfected tick via infected migrating cells of the host immune system. Since immune wild rodents still support virus transmission between co-feeding ticks, host viremia appears not to be a perquisite for but a secondary product of this process (Labuda et al. 1993, 1996, 1997). A hallmark of TBEV is its focal occurrence in nature and consequently the focal occurrence of disease in humans. This focal occurrence seems to rest on the coincident presence of Ixodes ricinus larvae and nymphs feeding on their rodent hosts in certain climatically favoured foci (Randolph et al. 1999).


We screened for TBEV in a rodent population caught at the military training area of Boletice close to Ceské Budejovice in the south of the Czech Republic from May to November 1999 and analysed kidney, lung and spleen of 157 rodents (Apodemus flavicollis 77, Apodemus sylvaticus 34, Mus musculus 2, Clethrionomys glareolus 41, Microtus arvalis 2, Microtus agrestis 1) and 1 shrew (Sorex araneus). 10–50 mg of each tissue sample was homogenized in a FastPrep machine, and total RNA was extracted using the FastRNA-Kit-Green (Qbiogene, Germany). We screened the extracted RNA for TBEV by nested PCR using published primers (Schrader and Suss 1999). RT-PCR was performed with up to 1 !g of extracted RNA in a 50-!L volume using the Titan-One-Tube-RT-PCR system (Roche, Germany), 400 nM first round primers, 5 mM DTT, and 200 !M dNTPs. The following optimized temperature profile was used; first round RT-PCR: RT-step 50°C/30 min, 94°C/2 min, two-step PCR (and nested PCR): 94°C/30 sec, 69°C/45 sec for 40 cycles and final hold at 68°C/7 min. 2 !L of the reaction mix were used in a 50-!L nested PCR containing 400 nM primers, 200 !M dNTPs, and 5 !L of 10! PCRbuffer (Boehringer, Germany). The RNA was prepared in Göttingen and PCR was performed in München. Out of 474 specimen analysed TBEV-specific RNA was amplified from all three organs derived from one Clethrionomys glareolus in the first RT-PCR round. Sequence analysis revealed a 100% identity to the TBEV strain Neudoerfl (accession no. U27495) (Mandl et al. 1988) isolated from a Mus musculus in Austria. Backup tissue samples (lung, kidney, spleen) of the PCR positive rodent stored in Ceske Budejovice, where homogenised and passaged twice in suckling mice brain and once in PS cells. TBEV growth was confirmed by a plaque reduction test.


The results obtained in inoculated mice and cell culture indicate that an infectious TBEV virus was present in the tissue samples from a trapped Clethrionomys glareolus. Sequence identity between the partial sequence of this isolate and the sequence of the Neudörfl strain is not surprising, since both the isolation sites are geographically just about 200 km apart to each other and the investigated 5\"-nontranslated region can not be regarded as highly informative when it comes to discrimination of closely related flaviviruses. However, this isolation confirms previous TBEV isolates from this rodent species in Slovakia (Kozuch et al. 1990, 1995). The virus isolation in suckling mice was performed with a homogenate from lung, kidney and spleen. This seems to argue for a high viral load in tissues of a TBEV-infected rodent and would at least support that TBEV viremia does occur in wild rodents as shown experimentally (Chunikhin and Kurenkov 1979). All rodent samples from Boletice had previously also been screened for hantaviral RNA. Dobrava virus sequences were obtained from one Cl. glareolus and one A. sylvaticus (Weidmann et al. 2005). Therefore, our results also support the observation that hantaviruses and TBEV transmission can occur sympatrically in the same rodent population (Kozuch et al. 1979, Sibold et al. 1999). The ecology of virus transmission in central Europe seems to be dependent on rodent population fluctuations (Olsson et al. 2002). More concerted research is needed to follow these fluctuations in rodent populations and the concomitant carrier state of these rodents for TBEV, hantaviruses and other agents.


This research was supported by the Bundesministerium für Verteidigung, Germany (grant InSanI 0598-V4301/030-V4304).


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