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ABSTRACTAlthough tick-borne infectious diseases threaten human and animal health worldwide, with constantly increasing incidence, little knowledge is available regarding vector–pathogen interactions and pathogen transmission. In vivolaboratory study of these subjects using live, intact ticks is expensive, labor-intensive, and challenging from the points of view of biosafety and ethics. Several in vitromodels have been developed, including over 70 continuous cell lines derived from multiple tick species and a variety of tick organ culture systems, facilitating many research activities. However, some limitations have to be considered in the translation of the results from the in vitroenvironment to the in vivosituation of live, intact ticks, and vertebrate hosts. In this review, we describe the available in vitromodels and selected results from their application to the study of tick-borne viruses, bacteria, and protozoa, where possible comparing these results to studies in live, intact ticks. Finally, we highlight the strengths and weaknesses of in vitrotick culture models and their essential role in tick-borne pathogen research.

The American grass carp reovirus (AGCRV) Aquareovirus Gis not strongly associated with disease in fish, but it is often detected by cell culture during routine inspections of healthy fish. The cytopathic effect of AGCRV does not involve the typical syncytia associated with most aquareoviruses. Instead, the AGCRV produces a pattern of cell rounding that is very similar to that produced by rhabdoviruses, including those that are highly regulated. We have developed a quantitative polymerase chain reaction assay that can be used to identify AGCRV in cell cultures or directly on fish tissues. The assay detects as few as two copies of the plasmid template, has a coefficient of variation of 15% among assays performed on different days, and does not cross-react with any other aquareoviruses tested. Assays performed on tissues of cultured golden shiners Notemigonus crysoleucasand fathead minnow Pimephales promelasrevealed a high prevalence of infection among healthy fish but no association with disease.

AbstractThe American grass carp reovirus (AGCRV) Aquareovirus Gis not strongly associated with disease in fish, but it is often detected by cell culture during routine inspections of healthy fish. The cytopathic effect of AGCRV does not involve the typical syncytia associated with most aquareoviruses. Instead, the AGCRV produces a pattern of cell rounding that is very similar to that produced by rhabdoviruses, including those that are highly regulated. We have developed a quantitative polymerase chain reaction assay that can be used to identify AGCRV in cell cultures or directly on fish tissues. The assay detects as few as two copies of the plasmid template, has a coefficient of variation of 15% among assays performed on different days, and does not cross-react with any other aquareoviruses tested. Assays performed on tissues of cultured golden shiners Notemigonus crysoleucasand fathead minnow Pimephales promelasrevealed a high prevalence of infection among healthy fish but no association with disease.

TTV and TTMV (recently assigned to the floating genus Anellovirus) infect human populations (including healthy individuals) at high prevalence (>80%). They display notably high levels of genetic diversity, but very little is known regarding the distribution of Anellovirusgenetic groups in human populations. We analyzed the distribution of the major genetic groups of TTV and TTMV in healthy voluntary blood donors using group‐independent and group‐specific PCR amplifications systems, combined with sequence determination and phylogenetic analysis. Analysis of Anellovirusgroups revealed a non‐random pattern of group distribution with a predominant prevalence of TTV phylogenetic groups 1, 3, and 5, and of TTMV group 1. Multiple co‐infections were observed. In addition, TTMV sequences exhibiting a high genetic divergence with reference sequences were identified. This study provided the first picture of the genetic distribution of the major phylogenetic groups of members of the genus Anellovirusin a cohort of French voluntary blood donors. Obtaining such data from a reference population comprising healthy individuals was an essential step that will allow the subsequent comparative analysis of cohorts including patients with well‐characterized diseases, in order to identify any possible relationship between Anellovirusinfection and human diseases. J. Med. Virol.78:298–304, 2006. © 2005 Wiley‐Liss, Inc.

ABSTRACTThe genotype distribution of hepatitis C virus (HCV) in blood donors from southeast France was tracked for a period of 13 years (1991 to 2003). Virus genomes from 321 samples were analyzed by amplification and sequencing of the NS5b and E1 regions. The most frequent genotypes were 1b (30.2%), 1a (27.7%), and 3a (22.4%). Although it was less common, genotype 2 was characterized by the presence of strains belonging to 11 different subtypes, including 5 that had never been characterized. Genotypes 1a, 1b, 3a, and 4a presented typical “epidemic” profiles, with a large number of isolates per subtype and short mean genetic distances between isolates. Type 2 isolates displayed a typical “endemic” profile, with a large number of subtypes and very few isolates in each subtype. The epidemiology of HCV infection in southeast France changed radically during the study period in relation to modifications in the etiology of infection. We observed the emergence of new epidemic subtypes (subtypes 1a and 3) linked to intravenous drug use and a decrease in the types linked to blood transfusion and nosocomial infection (epidemic subtype 1b and endemic type 2). Comparison of strains from blood donors with strains from a cohort of inpatients in the same region during 2001 and 2002 demonstrated for the first time that the monitoring of blood donors is a generally valid indicator of HCV epidemiology in terms of genotype distribution.

ABSTRACTVP6, VP7, VP9, VP10, VP11, and VP12 of Colorado tick fever virus (CTF virus), a virus member of the genus Coltivirus, family Reoviridae, were expressed in bacteria with the pGEX-4T-2 vector. A partial sequence of VP7 (designated pVP7) was chosen to elaborate an enzyme-linked immunosorbent assay (ELISA) for detecting anti-CTF virus immunoglobulin G (IgG) antibodies in humans. This was based on two observations: (i) among all expressed proteins, pVP7 showed the highest immunoreactivity to an anti-CTF virus hyperimmune ascitic fluid; (ii) to provide the highest selectivity of antibody detection, the expressed sequence was chosen within a region which is highly divergent (49% amino acid identity) from the homologous sequence of another coltivirus, the Eyach virus. The pVP7 ELISA was evaluated with 368 serum samples from French blood donors and found to provide 98.1% specificity. Assays with the Calisher set of human serum samples, positive for anti-CTF virus antibodies (C. H. Calisher, J. D. Poland, S. B. Calisher, and L. A Warmoth, J. Clin. Microbiol. 22:84-88, 1985), showed that the pVP7 ELISA provided 100% sensitivity for the tested population. After elaboration of recombinant-protein-based ELISAs for diagnosis of infections with members of the viral genera Orbivirus, Orthoreovirus, and Rotavirus, it was shown that a recombinant protein could be used to detect antibodies to the human pathogen Colorado tick fever virus.

TT virus-like minivirus (TLMV) was recently discovered as a human circovirus. Little is known about its natural history and molecular epidemiology. A study of TLMV infection is described in a population of French hemodialysis patients. TLMV DNA was tested by seminested PCR system located in the noncoding region in 81 patients divided into seven groups according to the origin of their renal disease. Quantitation of TLMV DNA in serum was carried out. Sequences from 28 patients were compared with 40 sequences retrieved from databases and 53 TLMV sequences cloned from the serum of a single patient. The prevalence of TLMV DNA in hemodialysis patients was 95.1%. In this study, 24 samples (29.6%) presented viral loads of > 125 equivalents of plasmid (Ep)/ml, and only 6 (7.8%) had viral loads of > 125 × 10² Ep/ml. A significant correlation (P < 0.029) was found between viral loads of > 125 × 10² Ep/ml and the neoplastic origin of end-stage renal disease. Analysis of 53 sequences cloned from a single individual demonstrated high sequence variability, as shown by the genetic distance of 40.2%. This genetic distance is comparable to that between the most divergent sequences of TLMV reported to date (43.5%). These data suggest that TLMV viral load is possibly related to the level of immunocompetence of hemodialysis patients; the genetic diversity of TLMV is extremely high; and co-infection by different strains is possible. J. Med. Virol. 67:630–635, 2002. © 2002 Wiley-Liss, Inc.

The full-length nucleotide sequences of genome segments 1, 5, 7, 8 and 10 from Ndelle virus (NDEV) have been characterized. Comparison of the deduced protein amino acid sequences with those of other member viruses of the family Reoviridae demonstrates that NDEV was originally assigned incorrectly to the genus Orbivirus (aa identity values of <20%). In contrast, high levels of amino acid identity were found with members of the species Mammalian orthoreovirus (MRV); for example, amino acid identity in γ3(Pol) is between 91 and 97%. These findings, together with previous antigenic analyses, provide evidence that NDEV should be reclassified as a new serotype (designated MRV-4) within the Mammalian orthoreovirus species.

To date, tick-borne flaviviruses responsible for hemorrhagic fever in humans have been isolated in Siberia (Omsk hemorrhagic fever virus), India (Kyasanur Forest disease virus, KFDV), and in Saudi Arabia (Alkhurma virus, ALKV). Prior to this study, only partial coding sequences of these severe pathogens had been determined. We report here the complete coding sequence of ALK virus, which was determined to be 10,248 nucleotides (nt) long, and to encode a single 3,416 amino acid polyprotein. Independent analyses of the complete polyprotein and the envelope protein provided genetic and phylogenetic evidence that ALKV belongs to the tick-borne flavivirus group, within which it is most closely related to KFDV. Analysis of structural genes, genetic distances, and evolutionary relationship indicate that ALKV and KFDV derived from a common phylogenetic ancestor and constitute two genetic subtypes of the same virus species according to current genetic criteria of classification.

The genome segments 1, 2, and 3 of the grass carp reovirus (GCRV), a tentative species assigned to genus Aquareovirus, family Reoviridae, were sequenced. The respective segments 1, 2, and 3 were 3949, 3877, and 3702 nucleotides long. Conserved motifs 5′ (GUUAUUU) and 3′ (UUCAUC) were found at the ends of each segment. Each segment contains a single ORF and the negative strand does not permit identification of consistent ORFs. Sequence analysis revealed that VP2 is the viral polymerase, while VP1 might represent the viral guanylyl/methyl transferase (involved in the capping process of RNA transcripts) and VP3 the NTPase/helicase (involved in the transcription and capping of viral RNAs). The highest amino acid identities (26–41%) were found with orthoreovirus proteins. Further genomic characterization should provide insight about the genetic relationships between GCRV, aquareoviruses, and orthoreoviruses. It should also permit to precise the taxonomic status of these different viruses.

The Colorado tick fever virus (CTFV) is the type species of genus Coltivirus, family Reoviridae. Its genome consisting of 12 segments of dsRNA was completely sequenced. It was found to be 29,174 nucleotides long (the longest of all Reoviridae genomes characterized to date). Conserved sequences at the 5′ end (SACUUUUGY) and at the 3′ end (WUGCAGUS) of the 12 segments were identified. The analysis of the putative proteins deduced from the nucleotide sequences permitted to identify functional motifs. In particular, the VP1 was identified unambiguously as the viral RNA dependent RNA pylmerase (RDRP) (VP1pol), with a GDD located at a similar position to Reoviridae RDRPs. In other genes, RGD cell-binding, NTPAse, single strand binding protein and kinase motifs were identified. Comparison with Reoviridae proteins showed significant similarities to RDRPs (CTFV-VP1) and sigma C protein of orthoreovirus (CTFV-VP6). Similarities to nonviral enzymatic proteins, such as methyltransferases, NTPAses, RNA replication factors, were also identified.

TT virus is a virus distantly related to the Circoviridae family. We report here the complete genome characterization of two European human isolates (T3PB and TUPB) using a new and simple protocol for sequencing GC-rich genomic regions. Sequence analysis confirmed the existence of two major ORFs, of a CAV-like VP2 motif in ORF2 and of potential stem-loop structures in non-coding regions. Phylogenetic analyses based on complete genomic sequences of human isolates suggested that three different lineages exist at least. The first lineage includes genotypes 1, 2, and 3, and two other lineages include viruses related to the Japanese SANBAN and to the North American TUS01 isolates respectively. Sequence comparison made it possible to assign strain T3PB to genotype 3, and strain TUPB to the TUS01 group. Consequently, this study reports the first full-length sequence of a genotype 3 isolate and demonstrates that viruses belonging to the TUS01 lineage are present in the Old Word.

We determined the full-length coding sequence of a GB-C/hepatitis G (GBV-C/HGV) virus isolate (AUQ) obtained from a French blood donor. The genome of AUQ strain contains a long open reading frame encoding 2842 amino acid residues. Comparison with the 33 complete genome sequences so far available from the databases indicates that the full-length sequence of GBV-C/HGV AUQ showed 9.0–14.1% nucleotide sequence divergence but only 1.6–5.3% at the amino acid level. Analysis of the potential cleavage sites of the polyprotein found that they were remarkably conserved among all sequences. Although phylogenetic studies based on partial genomic sequences suggested a clusterization according to the geographical origin, analysis based on full-length polyprotein did not provide similar conclusions.

BACKGROUND:The purpose of the study was to analyze serologic and molecular markers of the GB virus type C/hepatitis G virus (GBV‐C/HGV) infection in voluntary blood donors from Southeastern France.