Development of a quantitative real-time RT-PCR assay with internal control for the laboratory detection of tick borne encephalitis virus (TBEV) RNA

Schwaiger, M.; Cassinotti, Pascal

doi:10.1016/s1386-6532(02)00168-3

Cited by 278 publications

(235 citation statements)

References 17 publications

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“…A real-time RT-PCR for the detection of TBEV by amplifying a fragment of the TBEV 39-UTR was performed for all RNA samples using primers and probes described previously (Schwaiger & Cassinotti, 2003). TBEV RNA was amplified in a 25 ml final volume containing Superscript III RT Platinum Taq (Invitrogen Life Technologies), 100 mM primer F-TBE1, 50 mM primer R-TBE1, 20 mM TBE probe/WT probe (synthesized by Applied Biosystems), 506 ROX Reference Dye (Invitrogen) and 5 ml template.…”

Section: Methodsmentioning

confidence: 99%

Prevalence and genetic variability of tick-borne encephalitis virus in host-seeking Ixodes ricinus in northern Italy

Carpi

Bertolotti

Rosati

et al. 2009

Journal of General Virology

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Tick-borne encephalitis (TBE) is a severe disease that has been endemic in north-east Italy since 1992. Over the past two decades, there has been an increase in the number of human cases reported in many European countries, including Italy. To assess the current TBE infection risk, questing ticks were collected from known TBE foci, as well as from a site in northern Italy where no human infections have been reported previously. A total of 1739 Ixodes ricinus (1485 nymphs and 254 adults) was collected and analysed for TBEV prevalence by a real-time RT-PCR targeting the 39 untranslated region. Phylogenetic analyses of the partial envelope gene were conducted on two newly sequenced TBE virus (TBEV) strains and 28 previously published sequences to investigate the genealogical relationships of the circulating TBEV strains. These phylogenetic analyses confirmed a previous report that the European TBEV subtype is the only subtype circulating within the TBE foci in north-east Italy. Interestingly, nucleotide sequence analysis revealed a high degree of divergence (mean 2.54 %) between the TBEV strains recovered in the Italian province of Trento, despite the circulation of a single TBEV subtype. This elevated genetic variability within a single TBE focus may reflect local differences in the longstanding evolutionary dynamics of TBEV at this site relative to previously characterized sites, or more recent and continuous reintroduction of various TBEV strains. INTRODUCTIONTick-borne encephalitis virus (TBEV) is a zoonotic arbovirus of significant medical importance in both Europe and Asia, causing 4500 and 11 000 cases, respectively, of human encephalitis annually (Randolph, 2006). The ecology of TBEV involves a rodent-tick transmission cycle where uninfected ticks acquire TBEV from infected ticks while co-feeding on the same competent reservoir host, mostly small mammals of the genus Apodemus (Labuda et al., 1993(Labuda et al., , 1997. The non-viraemic transmission among co-feeding ticks represents the major amplification route for TBEV, which contributes to the local specificity of infection risk and the heterogeneity of the spatial distribution of endemic sites (Randolph et al., 1999). Additionally, systemic viraemia in rodents is not sufficient to ensure TBEV transmission to ticks, as the virus itself causes a high mortality rate in the host before ticks are presented the opportunity to complete a blood meal (Randolph et al., 1996). Furthermore, the distribution of endemic tick-borne encephalitis (TBE) sites can also be influenced by multiple abiotic and biotic factors, which affect tick abundance, tick phenology, tick infestation prevalence and tick human contact rate, as well as differences in surveillance efforts (Randolph et al., 2008).TBEV belongs to the genus Flavivirus and its genome consists of a single-stranded, positive-sense RNA of approximately 11 kb, which encodes a polyprotein that is cleaved by cellular and viral proteases into three structural -the capsid, membrane and envelope proteins -and seven non-st...

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Section: Methodsmentioning

confidence: 99%

Prevalence and genetic variability of tick-borne encephalitis virus in host-seeking Ixodes ricinus in northern Italy

Carpi

Bertolotti

Rosati

et al. 2009

Journal of General Virology

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“…The protocol for the detection of TBEV has been described previously (35) and it is based on the amplification of 3' non-coding region of the TBEV genome with primers F-TBE1: GGG CGG TTC TTG TTC TCC and R-TBE1: ACA CAT CAC CTC CTT GTC AGA CT and probe TBE-P-WT: 6FAM-TGA GCC ACC ATC ACC CAG ACA CA-DB. Enteroviral RNA was detected using primers and dual labelled hydrolysis probe for amplification of 200 base pair in the 5' non-coding region.…”

Section: Patientsmentioning

confidence: 99%

Prevalence of Neurotropic Viruses in Malignant Glioma and Their Onco-Modulatory Potential

Strojnik

Duh

Lah

2017

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“…Detection of tick-borne encephalitis virus RNA TBEV real-time reverse transcriptase PCR detection in ticks was carried out with F-TBE1 and R-TBE1 primers and TBEprobe-WT probe as described (Schwaiger and Cassinotti 2003) with some modifications. Forward primer concentration of 3 lM, reverse primer concentration of 0.3 lM, and probe concentration of 0.2 lM were used, and the reverse transcription step was performed at 42°C.…”

Section: Nucleic Acid Isolationmentioning

confidence: 99%

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

Geller

Nazarova

Katargina

et al. 2013

Vector-Borne and Zoonotic Diseases

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During southward migration in the years [2006][2007][2008][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. Birdassociated B. garinii and B. valaisiana were detected in I. ricinus nymphs removed from T. merula. Rodentassociated 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.

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Development of a quantitative real-time RT-PCR assay with internal control for the laboratory detection of tick borne encephalitis virus (TBEV) RNA

Cited by 278 publications

References 17 publications

Prevalence and genetic variability of tick-borne encephalitis virus in host-seeking Ixodes ricinus in northern Italy

Prevalence and genetic variability of tick-borne encephalitis virus in host-seeking Ixodes ricinus in northern Italy

Prevalence of Neurotropic Viruses in Malignant Glioma and Their Onco-Modulatory Potential

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

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