Terminal Noncoding Region Research Articles

Occurrence and Molecular Variability of the Main Kiwifruit Viruses in the Sichuan Province of China.

Viruses cause important yield losses in kiwifruit. Here, we studied the occurrence and population structure of the major kiwifruit viruses in the Sichuan province of China. RT-PCR results showed the presence of Actinidia virus A (AcVA), Actinidia virus B (AcVB), Actinidia chlorotic ringspot-associated virus (AcCRaV), and the cucumber mosaic virus (CMV). AcCRaV was widely distributed, followed by CMV. These two viruses were often detected in co-infection with AcVA and AcVB. The virus detection rate was positively correlated with vine age. Four phylogenetic groups of AcVA and AcVB were identified, with AcVA isolates clustering mainly in subgroup I, and AcVB isolates clustering mainly in subgroups II, III, and IV. All CMV isolates clustered in subgroup II, and AcCRaV isolates clustered in subgroup IA. The genome of AcVA and AcCRaV was under negative selection pressure, while the genome of AcVB and CMV was under positive selection pressure. All the viruses, except AcVB, were in a state of expansion. The full-length genome of the most widely distributed AcCRaV isolate in kiwifruits in the Sichuan province was characterized by sequencing. Unique eight-nucleotide (TTTTTGAT) repeats were found in the 5'-terminal non-coding region of the AcCRaV RNA3 in a possible association with reduced disease symptoms. This is the first study of kiwifruit viruses in Sichuan.

Synergistic Effect between 3'-Terminal Noncoding and Adjacent Coding Regions of the Influenza A Virus Hemagglutinin Segment on Template Preference.

The influenza A virus genome is comprised of eight single-stranded negative-sense viral RNA (vRNA) segments. Each of the eight vRNA segments contains segment-specific nonconserved noncoding regions (NCRs) of similar sequence and length in different influenza A virus strains. However, in the subtype-determinant segments, encoding hemagglutinin (HA) and neuraminidase (NA), the segment-specific noncoding regions are subtype specific, varying significantly in sequence and length at both the 3' and 5' termini among different subtypes. The significance of these subtype-specific noncoding regions (ssNCR) in the influenza virus replication cycle is not fully understood. In this study, we show that truncations of the 3'-end H1-subtype-specific noncoding region (H1-ssNCR) resulted in recombinant viruses with decreased HA vRNA replication and attenuated growth phenotype, although the vRNA replication was not affected in single-template RNP reconstitution assays. The attenuated viruses were unstable, and point mutations at nucleotide position 76 or 56 in the adjacent coding region of HA vRNA were found after serial passage. The mutations restored the HA vRNA replication and reversed the attenuated virus growth phenotype. We propose that the terminal noncoding and adjacent coding regions act synergistically to ensure optimal levels of HA vRNA replication in a multisegment environment. These results provide novel insights into the role of the 3'-end nonconserved noncoding regions and adjacent coding regions on template preference in multiple-segmented negative-strand RNA viruses. IMPORTANCE While most influenza A virus vRNA segments contain segment-specific nonconserved noncoding regions of similar length and sequence, these regions vary considerably both in length and sequence in the segments encoding HA and NA, the two major antigenic determinants of influenza A viruses. In this study, we investigated the function of the 3'-end H1-ssNCR and observed a synergistic effect between the 3'-end H1-ssNCR nucleotides and adjacent coding nucleotide(s) of the HA segment on template preference in a multisegment environment. The results unravel an additional level of complexity in the regulation of RNA replication in multiple-segmented negative-strand RNA viruses.

Small RNA-Mediated De Novo Silencing of Ac/Ds Transposons Is Initiated by Alternative Transposition in Maize.

Although transposable elements (TEs) comprise a major fraction of many higher eukaryotic genomes, most TEs are silenced by host defense mechanisms. The means by which otherwise active TEs are recognized and silenced remains poorly understood. Here we analyzed two independent cases of spontaneous silencing of the active maize Ac/Ds transposon system. This silencing is initiated by alternative transposition, a type of aberrant transposition event that engages the termini of two nearby separate TEs. Alternative transposition during DNA replication can generate Composite Insertions that contain inverted duplications of the transposon sequences. We show that the inverted duplications of two Composite Insertions are transcribed to produce double-stranded RNAs that trigger the production of two distinct classes of small interfering RNAs: a 24-nt class complementary to the TE terminal inverted repeats and noncoding subterminal regions, and a 21- to 22-nt class corresponding to the TE transcribed regions. Plants containing these small interfering RNA-generating Composite Insertions exhibit decreased levels of Ac transcript and heritable repression of Ac/Ds transposition. Further, we demonstrate that Composite Insertions can heritably silence otherwise active elements in trans This study documents the first case of transposon silencing induced by alternative transposition and may represent a general initiating mechanism for silencing of DNA transposons.

Conserved microsatellites may contribute to stem-loop structures in 5′, 3′ terminals of Ebolavirus genomes

Microsatellites (SSRs) are ubiquitous in coding and non-coding regions of the Ebolavirus genomes. We synthetically analyzed the microsatellites in whole-genome and terminal regions of 219 Ebolavirus genomes from five species. The Ebolavirus sequences were observed with small intraspecies variations and large interspecific variations, especially in the terminal non-coding regions. Only five conserved microsatellites were detected in the complete genomes, and four of them which well base-paired to help forming conserved stem-loop structures mainly appeared in the terminal non-coding regions. These results suggest that the conserved microsatellites may be evolutionary selected to form conserved secondary structures in 5′, 3′ terminals of Ebolavirus genomes. It may help to understand the biological significance of microsatellites in Ebolavirus and also other virus genomes.

A study of the human bocavirus replicative genome structures

The complete genomes of two human bocavirus 4 (HBoV4) isolates recovered in 2011 in Novosibirsk, Russia have been determined. A set of primers was designed based on the determined and previously published HBoV sequences; this primer pair was able to detect all possible HBoV replicative intermediates. This primer set was used to assay all HBoV genotypes and detected only those structures that correspond to an episomal form of this viral genome. Also, for the first time, head-to-tail nucleotide sequences have been determined for HBoV4. Secondary structures of the terminal noncoding regions (NCRs) of episomal forms have been computed for all HBoV genotypes, as well as for the canine bocavirus. Conserved secondary structures in episomal NCRs, which are likely to play an important part in the replication of bocaviruses, were found. NCR heterogeneity in the genomes of individual HBoV isolates has been shown for the first time.

Characterization of the Noncoding Regions of the 1918 Influenza A H1N1 Virus

The terminal noncoding region (NCR) sequences of the eight gene segments of the influenza A/Brevig Mission/1/1918 (H1N1) virus were determined by rapid amplification of cDNA ends (RACE). Chimeric viruses encoding the open reading frames of the 1918 virus but flanked by either the wild-type 1918 NCR sequences or the NCR sequences of two other H1N1 virus strains, A/WSN/1933 and A/New York/312/2001, were produced. No growth differences between the NCR variant 1918 influenza viruses were noted.

Detection of a Bocavirus Circular Genome in Fecal Specimens from Children with Acute Diarrhea in Beijing, China

To determine if human bocavirus 2 (HBoV2) has a circular genome similar to the head-to-tail sequence of HBoV1 and the episomal form of HBoV3, 15 HBoV2 positive samples identified from 553 stool specimens from children with acute diarrhea were tested for a head-to-tail sequence using TaqMan-based real-time PCR. A circular genome with a head-to-tail sequence was identified in one (BJQ435) out of 15 samples tested by nested PCR. The complete circular genome of HBoV2-C1 (BJQ435) was 5307 nt in length and was flanked with a 520 nt-long terminal non-coding region (NCR). The secondary structure of HBoV2 -C1 had some differences compared to HBoV3-E1 (JN086998). Our study indicates that the HBoV genome exists in the form of a head-to-tail monomer and provides more information for understanding the HBoV replication mechanism.

Development of a one-step real-time RT-PCR assay using a minor-groove-binding probe for the detection of duck Tembusu virus

The design and development of a 3'-conjugated minor-groove-binding (MGB) probe for a real-time RT-PCR assay allowing for the rapid, sensitive, and specific detection of duck Tembusu virus (DTMUV) RNA are described. This assay targeted the 3' terminal non-coding region (NCR) of the TMUV genome and detected 1 × 10¹ copies of RNA per reaction without cross-reaction with other duck pathogens. The linear range of detection was 2 × 10¹-2 × 10⁸ copies/μl. The assay was rapid, requiring just over 2.0 h, including the nucleic acid extraction step. Therefore, this assay is an excellent tool for research routine diagnostic applications, and study of the epidemiology of TMUV infections among duck flocks.

Genetic diversity of the VP1 gene of duck hepatitis virus type I (DHV-I) isolates from southeast China is related to isolate attenuation

The complete sequence of an isolate (ZJ-V) of Duck hepatitis virus I (DHV-I), originally taken from the field in southeast China was determined. It was 7691 nucleotides long and had 5′- and 3′-terminal non-coding regions of 626 and 315 nucleotides, respectively. The poly(A) tail contained at least 22 residues and the single open reading frame encoded a polypeptide of 2249 amino acids. The VP1 gene was also sequenced from nine southeast China field isolates and three attenuated DHV-I vaccine strains. In phylogenetic analysis of the isolates and other published sequences, attenuated and tissue-adapted isolates (including ZJ-V) clustered as genotypes significantly different from the field isolates that had not been passaged in chicken/duck embryos. There were two consistent amino acid substitutions (E 129 → V 129 and A 142 → S 142) between all the field isolates and all the tissue-adapted ones. The carboxyl terminal region was generally the most variable and here the four attenuated Chinese isolates showed six consistent differences from the field isolates (S 181 → L 181, H 183K 184 → R 183G 1841, N 193 → D 193, E 205 → K 205, R 217 → K 217, N 235 → D 235). It seems likely that at least some of these differences result from mutations leading to isolate attenuation.

Sequence determination and functional analysis of the Akabane virus (family Bunyaviridae) L RNA segment

Akabane virus (AKAV) causes epizootic congenital deformities in cattle, sheep, and goats. Due to the lack of a complete genome sequence, the molecular biological properties of this virus are not known. We have cloned and sequenced the functional large (L) RNA segment of AKAV, and shown that it has polymerase activity using a minireplicon system with RNA polymerase I. The complete L RNA segment is 6868 nucleotides long and encodes an L protein of 2251 amino acids, which functions as an RNA-dependent RNA polymerase. A minireplicon reporter plasmid was constructed by flanking either the firefly luciferase or the green fluorescent protein gene in the antisense orientation with the 5'- and 3'-terminal noncoding regions of the small RNA segment. HmLu-1 cells were transfected with the reporter plasmid, and the L protein and nucleoprotein (N protein) expression plasmids. The reporter activity was upregulated in a dose-dependent manner with increasing concentration of either the L or N protein expression plasmid. Furthermore, the reporter activity could be downregulated by the AKAV NSs protein as well as by other orthobunyaviruses. These results show that the AKAV minireplicon system is a powerful tool for studying transcription and for rescuing infectious viruses from cloned cDNAs.

Molecular characterization of the sarcoplasmic calcium-binding protein (SCP) from crayfish Procambarus clarkii

Sarcoplasmic Calcium-binding Protein (SCP) is believed to function as the invertebrate equivalent of vertebrate parvalbumin, namely to “buffer” cytosolic Ca 2+. We have cloned and characterized a novel SCP from axial abdominal muscle of crayfish Procambarus clarkii (referred to as pcSCP1), and have examined tissue specific distribution and expression as a function of molting stage in non-epithelial and epithelial tissues. The complete sequence of pcSCP1 consists of 1052 bp with a 579 bp open reading frame, coding for 193 amino acid residues (molecular mass of 21.8 kDa). There is a 387 bp 3′ terminal non-coding region with a poly (A) tail. The deduced pcSCP1 protein sequence matched most closely with published SCP sequences from another crayfish Astacus leptodactylus (92.8%) and from shrimp (78.6–81.2%) and fruit fly (53%). Real-time PCR analysis confirmed that pcSCP1 is ubiquitously expressed in all tissues tested (gill, hepatopancreas, intestine, antennal gland, muscle); however it is most abundant in muscle particularly in the axial abdominal muscle. The real-time PCR analysis revealed that pcSCP1 expression is downregulated in pre- and postmolt stages compared with intermolt. Epithelial (hepatopancreas and antennal gland) SCP expression exhibited a more dramatic decrease than that observed in muscle. Expression trends for pcSCP1 paralleled published trends for sarco/endoplasmic reticular calcium ATPase (SERCA), suggesting that their cellular function in regulating intracellular Ca 2+ is linked.

First Report of Turnip mosaic virus in Rhubarb in Alaska.

In July 2003, noticeable red lesions were observed on rhubarb leaves (Rheum rhababarum cv. Kerwin) from a plant at the Arctic Plant Germplasm Research and Introduction Project in Palmer, AK. Extracts of leaf tissue tested positive for a potyvirus using indirect enzyme-linked immunosorbent assay (ELISA) and western blots with a monoclonal antibody specific to the potyvirus group (Agdia, Inc., Elkhart, IN). During the following growing season (June 2004), obvious chlorotic ringspots developed into red lesions on the same plant and an adjacent plant of the same cultivar. Partially purified particles that were isolated from the infected rhubarb plants were mechanically inoculated to an experimental host range (number of infected plants per total number of plants), resulting in lesions on leaves of Rheum palmatum (1 of 2) and Chenopodium amaranticolor (3 of 5) but none on C. quinoa (0 of 4). The leaves with local lesions from C. amaranticolor were ground in phosphate buffer (1 g of tissue per 10 ml of buffer), and the extract rubbed onto a set of plants resulting in lesions on R. hybridum (raponticum) (1 of 2), C. amaranticolor (1 of 4), and C. quinoa (1 of 4). The original diseased rhubarb plants and experimental symptomatic plants were confirmed to have a potyvirus using ELISA. Subsequent compound direct ELISA and western blot assays revealed that the virus reacted strongly to monoclonal or polyclonal antibodies to Turnip mosaic virus (TuMV) (Agdia, Inc.). Total RNA was extracted from leaves of the naturally infected rhubarb plants with an RNeasy Plant Mini Kit (Qiagen Sciences, Germantown, Maryland), and used in reverse-transcription-polymerase chain reaction (RT-PCR) with specific primers for TuMV (1) predicted to amplify a 1,134-bp 3'-terminal cDNA fragment encompassing the 3'-end of the nuclear inclusion protein gene (NIb), the coat protein gene, and the 3'-nontranslated region. A PCR product of approximately the expected size was obtained and then sequenced. Sequences (1,077 nt) that corresponded to the TuMV coat protein gene and 3'-terminal noncoding region were submitted to Genbank (Accession No. AY744930). Blast searches against NCBI (National Center for Biotechnology Information) contained high identities to many TuMV isolates with up to 96% (1,043 of 1,077) nucleotide identity (i.e., GenBank Accession No. AF169561). Similar high identities of up to 97% at the amino acid level occurred within the coat protein coding region (i.e., GenBank Accession No. BAC02892.1). Infected rhubarb plants were removed from the site and none of the remaining 109 plants tested positive for TuMV using ELISA. On the basis of the mechanical transmission to plant hosts, the definitive TuMV serology, and the consensus of sequenced regions with TuMV, we concluded that the causal agent of the diseased rhubarb plants was TuMV. Although TuMV has a wide plant host range occurring worldwide (2), to our knowledge, this is the first report of TuMV in rhubarb in Alaska and the first time that TuMV has been detected in Alaska.

Tickborne Meningoencephalitis, First Case after 19 Years in Northeastern Germany

Tickborne Meningoencephalitis, First Case after 19 Years in Northeastern Germany

Complete nucleotide sequence of Pelargonium zonate spot virus and its relationship with the family Bromoviridae.

The complete sequence of the Pelargonium zonate spot virus (PZSV) genome was determined. It comprises 8477 nt, distributed in three positive-strand RNA species encoding four proteins. RNA-1 is 3383 nt long, with an ORF that encodes a polypeptide with a molecular mass of 108 419 Da (denoted protein 1a). This protein contains the conserved sequence motifs I-III of type I methyltransferases and the seven consensus motifs of the helicases of superfamily 1. RNA-2 is 2435 nt long and encodes a major polypeptide with a molecular mass of 78 944 Da (denoted protein 2a), which shows identity to the RNA-dependent RNA polymerases of positive-strand RNA viruses. RNA-3 is 2659 nt long and contains two major ORFs. The first ORF is located in the 5' portion of the genome and sequence comparison of the putative translation product revealed similarities with the 30K superfamily of virus movement proteins. The second ORF is located in the 3' half and encodes the viral coat protein, which is expressed via a subgenomic RNA, RNA-4. The transcription initiation site of RNA-4 maps to the intergenic region of RNA-3. The organization of the PZSV genome, including the primary structure of terminal non-coding regions, strongly suggests that this virus belongs to the family Bromoviridae. The overall biological and genomic characteristics of PZSV indicate affinities in diverging directions with one or other of the virus species in this family, thus enabling it to be considered as a possible representative of a new genus within the family Bromoviridae.

The 3' terminal region is strictly required for clover yellow vein virus genome replication.

To identify the cis-element in the 3' terminal region of infectious cDNA required for replication of clover yellow vein virus (ClYVV), a series of mutants with duplications or deletions of the 3' terminal non-coding region (3'-NCR) of the genome that did not affect the ORFs in the genome was constructed. These were tested for infectivity, and the 3' terminal regions of their progeny RNAs were sequenced. Deletion mutants that lacked portions of the 3'-NCR were not infectious. Various mutants with duplicated 3' terminal sequences were infective only when the authentic 3' terminal sequence was restored, probably by recombination, and none of the constructs retained the original sequence in progeny viral RNA. When a coat protein gene sequence of bean yellow mosaic virus (BYMV) followed by a termination codon was introduced between the nuclear inclusion b and coat protein genes, infective progeny were generated. Sequence analyses of the progeny viruses showed that the coat protein gene was a chimera of the BYMV N-terminal and CIYVV C-terminal portions. These results suggest that the 3'-NCR of ClYVV contains cis-acting elements and is strictly required for genome replication.

Nucleotides at the extremities of the viral RNA of influenza C virus are involved in type-specific interactions with the polymerase complex.

Influenza A and C viruses share common sequences in the terminal noncoding regions of the viral RNA segments. Differences at the 5'- and 3'-ends exist, however, that could contribute to the specificity with which the transcription/replication signals are recognized by the cognate polymerase complexes. Previously, by making use of a transient expression system for the transcription and replication of a reporter RNA template bearing either type A or type C extremities, it was shown that a type C RNA template is transcribed and replicated with equal efficiency by either the type A or the type C polymerase complex, whereas a type A RNA template is less efficiently transcribed and replicated by the type C polymerase complex than by the type A complex. To explore the contribution of the nucleotides at the extremities of the RNAs to this type-specificity, the effect of mutations introduced either alone or in combination at nucleotide 5 at the 3'-end and at nucleotides 3', 6' or 8' at the 5'-end of type A or C RNA templates were studied in the presence of either the type A or the type C polymerase complex. The results indicate that the nature of nucleotides 5 and 6' contribute to type-specificity. Moreover, these results underline the importance of the base pairing between nucleotide 3' and 8' at the 5'-end of the RNA. Thus, it could be suggested that the nature of the nucleotides as well as the stability of the secondary structure at the extremities of the viral RNA are important determinants of type-specificity.

Construction and expression of eukaryotic expressing vector pCH510 of polypeptide CH50 and its chemotaxis and antitumor function by in vivo transfection.

To construct an eukaryotic expressing vector that expresses CH50, a recombinant Cell I-Hep II bifunctional-domain polypeptide of human fibronectin, and to investigate the chemotaxis to immune cells and the inhibitory effect on the growth of tumor by the expression of the plasmid in vivo, the plasmid was constructed by DNA recombination. Gene transfection was performed in vitro and in vivo. The expressed product was identified by Western blot. The chemotaxis after gene transfection in vivo was observed by histotomy and staining of muscle tissues. The inhibition of gene transfection on solid tumor was observed in mice. The results showed that plasmid pCH510 was constructed by the recombination of the 5'-terminal noncoding region and signal peptide coding region of human fibronectin cDNA and cDNA fragment coding CH50 polypeptide with a 3'-terminal noncoding region of human FN cDNA, and the insertion of the recombinated fragment into plasmid pcDNA3. 1. After transfection with plasmid pCH510, NIH3T3 cells could produce CH50 polypeptide. The transfection of plasmid pCH510 by the injection in muscle of mouse could produce the effects of chemotaxis on immune cells and the inhibition on the growth of solid tumor. It is concluded that plasmid pCH510 can express in cells and in vivo in mouse. The expression of the plasmid in vivo has a chemotactic effect on immune cells and can inhibit the growth of solid tumor.

Attenuated lapinized chinese strain of classical swine fever virus: complete nucleotide sequence and character of 3'-noncoding region.

The complete nucleotide sequence including precise 5'- and 3'-terminal non-coding regions (NCRs) of the attenuated lapinized Chinese strain (HCLV) of Classical Swine Fever Virus (CSFV) was determined from overlapping cDNA clones constructed by separated RT-PCR and rapid amplification of cDNA ends (RACE) methods. The genomic RNA of the HCLV strain consists of 12.310 nucleotides (nts) including 374 nts and 242nts in the 5'- and 3'-NCRs, respectively. It contains one large open reading frame (ORF) encoding a polyprotein of 3,898 amino acids with a calculated molecular weight of 437.6 kDa. There is one notable insertion of 12 continuous nts, CTTTTTTCTTTT in the 3'-NCR of HCLV genomic cDNA when compared with its parental virulent Shimen strain. Sequence alignment of partial 3'-NCR reveals two groups of CSFV vaccine strains carrying similar T-rich insertions at different positions in this region. Computer-predicted secondary structures suggest that T-rich insertion greatly change the structures and thus decrease the promoter functions of 3'-NCRs during the replications of these two groups of CSFV vaccine strains.

Construction of eukaryotic expressing vector pCH503 of CH50 and its chemotaxis and anti-tumor function by expression in vivo in mice.

An eukaryotic expressing vector that expresses CH50, a recombinant polypeptide of human fibronectin, in mice was constructed, and its chemotactic and anti-tumor function by in vivo gene transfection was investigated. The plasmid was constructed by recombination techniques. The cDNA fragment coding CH50 polypeptide from a prokaryotic expressing vector of CH50 was ligated with 5'-terminal noncoding region and coding region of signal peptide of mouse IFN-gamma cDNA at 5' side and 3'-terminal noncoden region of human FN cDNA at 3' side. The recombinant cDNA was inserted into plasmid pREP8. The resulted expressing plasmid was designated as pCH503. The macrophages transfected with pCH503 in vivo and cultured in vitro could produce CH50. The expressed product was identified by heparin-affinity chromatography and SDS-PAGE. By counting and Giemsa-staining of coeliac cells and histotomy and staining of muscle tissue, the chemotaxis on immune cells was observed after transfection of pCH503 either in peritoneal cavity or in muscle. The inhibition of gene transfection of pCH503 on melanoma was observed in mice. The number of melanoma nodes in mice was reduced by 50%-60% after coeliac transfection with pCH503. The pCH503, an eukaryotic expressing vector of CH50, can express in vivo in mice. The transfection of pCH503 in vivo has the chemotaxis on immune cells and can inhibit the formation of tumor nodes, suggesting that plasmid pCH503 is potentially useful in combined treatment of tumor.

Annual and seasonal variation of tick-borne encephalitis virus (TBEV) prevalence in ticks in selected hot spot areas in Germany using a nRT-PCR: Results from 1997 and 1998

The prevalence of tick-borne encephalitis virus (TBEV) in Ixodes ricinus tick populations in endemic areas of Germany with the highest TBE risk is unknown. Annual and seasonal differences in TBEV prevalence have also not been studied. Against this background, in May 1997 we started a systematic virus surveillance programme in ticks collected in locations known to have a high incidence of autochthonous TBE cases. These were 5 locations in Baden-Württemberg (Black Forest) and 8 locations in Bavaria (surrounding Passau). Field-collected ticks were randomly assigned to pools of 10 adults or 20 nymphs, respectively. The tick pools were tested for the presence of TBEV-RNA using a newly developed, sensitive nested reverse transcriptase polymerase chain reaction assay (nRT-PCR). The primer pairs were selected from the 5'-terminal noncoding region, a highly conserved part of the virus. The specificity was tested by computer homology searches of sequences, as well as by sequencing of the first and the second amplificates, by Southern blot hybridisation with a DIG-labelled oligonucleotide probe, and by restriction enzyme analysis. The method has proved to be very sensitive, with a detection limit of 20 fg of TBEV RNA per PCR run, or a single positive tick. Based on biostatistical considerations a sample size of at least 1000 ticks per estimation point was chosen. The estimated TBEV prevalence and confidence intervals (CI) were calculated from the nRT-PCR results of pooled samples (10 adults or 20 nymphs) using appropriate formulae for pooled testing. In order to identify the estimated TBEV prevalence as well as to assess the influence of annual and seasonal factors on TBEV prevalence, ticks were sampled twice a year (May and September) in 1997 and 1998 at exactly identical sites. These sites were selected because they were known to have had the highest incidence of autochthonous TBE cases during the previous 10 years. On sampling days, relevant local meteorological data were also noted. In total, 8500 I. ricinus ticks were investigated in this study, 4270 (3540 nymphs, 730 adults) from the Black Forest habitats, and 4230 (3680 nymphs, 550 adults) from the Bavarian locations. In the foci near Freiburg (Black Forest), the estimated virus prevalence was relatively high in the whole tick population, during 1997 with only slight seasonal differences [3.4% (confidence interval, CI, 2.3-4.8%) in May and 2.9% (CI 1.7-4.5%) in September]. In contrast, in 1998, in the same foci the estimated TBEV prevalence was considerably lower [1.1% (CI 0.5-2.0%) in May and 0.6% (CI 0.2-1.4%) in September]. Thus, while the seasonal differences again remained low, the annual variation was marked. In the Bavarian foci in 1997, the estimated virus prevalence of the whole tick population studied was lower than in the Black Forest foci and the seasonal fluctuations were low: in May 1997 0.9% (CI 0.4-1.8%) of the ticks were positive, in September 1.1% (CI 0.5-1.9%). In 1998, in May 2.0% (CI 1.1-3.3%) of the ticks were positive, and in September 1.1% (CI 0.5-2.1%). For the whole study period, every 50th to 100th I. ricinus nymph or adult in the Passau region was calculated to give a positive signal in the nRT-PCR. The TBEV prevalence data indicate that residents and visitors of areas in Germany known for high endemic activity take a significant risk of contracting TBEV infection, if bitten by ticks. In addition, the data suggest that annual fluctuations may exist in the whole tick population studied. Seasonal fluctuations of the virus prevalence in ticks were small.