Inactivation of a European strain of West Nile virus in single- donor platelet concentrate using the INTERCEPT blood system.

BACKGROUND AND OBJECTIVE: In order to prevent West Nile virus (WNV) contaminations by transfusion, the French National Blood Service decided to evaluate the INTERCEPT Blood System's efficiency on a European strain. MATERIALS AND METHODS: Culture supernatant of WNV was used to infect six platelets concentrates. Viral titre was determined by plaque reduction neutralization test before and after viral inactivation using the INTERCEPT Blood System. RESULTS: In all assays, the absence of plaque forming unit was observed after viral inactivation. The log reduction observed ranged between > 5.1 logs to > 5.2 logs. CONCLUSION: INTERCEPT Blood System is a commercially viral inactivation method potentially useful in order to prevent WNV transmission by blood products in France during re-emerging outbreaks.

Genus Coltivirus (family Reoviridae): genomic and morphologic characterization of Old World and New World viruses.

We report a genomic and morphologic study of the European Eyach (EYA) virus (genus Coltivirus, family Reoviridae) and a comparative analysis with the American Colorado tick fever (CTF) virus (the type species of the genus). The previously established, but distant, antigenic relationship between these viruses was strengthened by genetic findings (presence of cognate genes, amino acid identity between 55 and 88%, similar conserved terminal motifs, suspected read-through phenomenon in segment 9 of both viruses) and by indistinguishable ultramicroscopic morphologies. Moreover, putative constitutive modifying enzyme activities were suspected to be carried out by homologous viral proteins (RNA-dependent RNA polymerase, methyl/guanylyl transferase, NTPase). These findings, together with the comparative analysis to genomes of southeast Asian isolates, support the recent classification of arboviruses with 12 segments of dsRNA within two distinct genera (genus Coltivirus and genus Seadornavirus) and raise interesting questions about the evolutionary origins of coltiviruses. The previously proposed hypothesis that EYA virus was derived from an ancestral virus introduced in Europe with the migration of lagomorphs from North-America, would imply a divergence date between American and European isolates of over 50 million years ago (MYA). This analysis allows for the first time to propose an evolutionary rate for virus dsRNA genomes which was found to be in the order of 10(-8) to 10(-9) mutations/nt/year, a rate similar to that of dsDNA genomes.

Sequence characterization of Ndelle virus genome segments 1, 5, 7, 8, and 10: evidence for reassignment to the genus Orthoreovirus, family Reoviridae.

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 gamma3(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.

Comparison of systems performance for TT virus detection using PCR primer sets located in non-coding and coding regions of the viral genome.

BACKGROUND: the heterogeneity of the TT virus (TTV) DNA prevalence values reported from comparable human cohorts suggests that diagnostic PCR protocols still require to be optimized. OBJECTIVES: to design TTV PCR primer sets with low genotype restriction and to compare their performances with commonly used amplification systems. STUDY DESIGN: we compared full length TTV genomic sequences and identified conserved nucleotide patterns in the 5' and 3' non-coding regions of the viral genome. This permitted to design two new primer sets usable for the PCR amplification of the most divergent human isolates of TTV described to date. The performances of these amplification systems were compared with those of three other PCR systems earlier used for prevalence studies. RESULTS: the primer systems P5Bx and P3Bx exhibited higher PCR scores than the other systems tested; 14 to 34% improvement values were obtained, and divergent positive results of earlier described PCR systems were confirmed systematically by our new detection assays. CONCLUSIONS: an optimized detection of TT virus DNA is a pre-requisite for the accurate epidemiological survey of viral infection and for the realization of phylogenetic studies. Such PCR systems with low genotype restriction will be helpful in the future for a better knowledge of natural history of TT virus infection.

Strategies for the sequence determination of viral dsRNA genomes.

The genetic study of viruses having dsRNA genomes is hampered by the technical difficulty of complete sequence determination of dsRNA. Optimised methods are described here for sequencing dsRNAs, which meet three different situations: (1) genomes that can be obtained in fairly high amounts (>20 ng per separated segment); (2) genomes with limited amounts of RNA that can be detected by electrophoretic gel separation and staining; (3) genomes that cannot be detected by electrophoretic gel separation and staining. These methods include improved Single Primer Amplification Technique protocols, an adaptation of the SMART methodology, and a new method permitting the selective enzymatic removal of dsRNA segments. Strategies permitting adaptation of these protocols to the full-length determination of dsRNA viral genomes are described. Each of the protocols is described for sequence determination of a chosen dsRNA virus.

Sequence of genome segments 1, 2, and 3 of the grass carp reovirus (Genus Aquareovirus, family Reoviridae).

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.

HCV RNA in blood donors with isolated reactivities by third-generation RIBA.

BACKGROUND: The objective of this collaborative study was to learn the proportion of HCV RNA-positive samples obtained from a population of donors with isolated anti-HCV reactivities by third-generation RIBA (RIBA-3) (indeterminate results). STUDY DESIGN AND METHODS: During a 2-year period, 11 blood transfusion centers kept all samples with indeterminate RIBA-3 results to test them by PCR, using both local and commercial techniques. RESULTS: Of the 758 RIBA-3 indeterminate samples, 10 (1.3%) were positive for HCV RNA: 3. 3 percent (6/180) and 1.3 percent (4/317) of samples with anti-core or anti-NS3 reactivity, respectively, and none of the 52 and 209 samples with anti-NS4 or anti-NS5 reactivity, respectively. HCV RNA-positive donors with anti-core reactivity were infected with different subtypes (1 with HCV subtype 1b, 1 with 2, 1 with 2a/2c, 2 with 3a, and 1 with 5a), and a follow-up indicated a chronic-carrier state in two of the six donors. Acute hepatitis was diagnosed in three of the four donors with anti-NS3 reactivity alone. Two of these three were IV drug users and were infected with subtype 1a. CONCLUSION: HCV RNA-positive donors with indeterminate results in RIBA-3 are extremely rare, but they do exist. They were observed only when either anti-core or anti-NS3 was present. With such a RIBA-3 profile, PCR testing remains necessary to reveal an eventual acute or chronic HCV infection.

Sequence determination and analysis of the full-length genome of colorado tick fever virus, the type species of genus Coltivirus (Family Reoviridae).

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.

High prevalence of TT virus infection in French blood donors revealed by the use of three PCR systems.

BACKGROUND: The purpose of this study was to determine the prevalence of TT virus (TTV) infection in voluntary blood donors in Southeastern France. STUDY DESIGN AND METHODS: The sera of 289 blood donors were tested for the presence of TTV DNA by two PCR systems detecting genes located in the 5' UTR (primer set A [Set A]) and the open reading frame (ORF2) (primer set B [Set B]) of the viral genome. A randomized sample of 40 blood donors was also tested by a nested-PCR system in the ORF1 by use of primer set C (Set C). Donors were questioned for possible risk factors for virus transmission. RESULTS: In the entire population studied, 30.8 percent of blood donors tested positive with both Sets A and B, and 70.6 percent with at least one set. In the sample tested with three sets of primers, 27.5 percent of blood donors were positive in testing with all PCR systems and 80 percent with at least one system. The specificity of TTV DNA amplification was confirmed by sequencing 10 PCR products obtained with each set of primers. Statistical analysis revealed that the prevalence of TTV reactivity increased with age. CONCLUSION: The high prevalence of TTV reactivity and the absence of a pathologic condition or risk factors obviously associated with the infection in blood donors suggest that there is no need for systematic detection of TTV infection before blood donation. Further studies are required to determine if TTV isolates can be responsible for a pathologic condition in humans after blood transfusion.

TT virus: a study of molecular epidemiology and transmission of genotypes 1, 2 and 3.

BACKGROUND: TT virus (TTV) is a recently discovered virus, which is not related to any other known virus infecting humans. OBJECTIVES: To investigate: (i) the world-wide distribution of the three major TTV genotypes; and (ii) the possible routes of viral transmission. STUDY DESIGN: (i) The phylogenetic distribution of 494 TTV isolates originating from 31 countries was analysed, using partial ORF1 sequences. (ii) Faeces samples (n=22) and saliva samples (n=72) from French individuals were tested for the presence of TTV DNA. (iii) Viral titres in paired serum and saliva samples were compared. RESULTS: (i) Genotypes 1, 2 and 3 were distributed world-wide, with a high proportion of type 1 in Asia (71%) and no type 3 identified in Africa to date. In the USA, 77% of isolates were grouped in four clusters only (genetic distances <10%). This was also the case of 76% of French isolates, 76% of Japanese isolates, and 89% of Hong Kong isolates. (ii) TTV DNA was detected in 18% of faeces samples and 68% of saliva samples tested. (iii) Viral titre in saliva samples was 100-1000 times higher than that of the corresponding serum. CONCLUSIONS: (i) The observed epidemiological distribution of TTV isolates is compatible with an ancient dissemination of viral ancestors belonging to the different genotypes and a slow genetic evolution in sedentary populations. (ii) Besides the possible transmission of TTV by the parental and oral-faecal routes, the high titre of TTV DNA observed in saliva raises the hypothesis of the viral transmission by saliva droplets. This route of transmission could explain the high degree of exposure to viral infection observed in the general population.

Complete sequences of two highly divergent european isolates of TT virus.

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.

Lessons from a multicentre study of the detectability of viral genomes based on a two-round quality control of GB virus C (GBV-C)/hepatitis G virus (HGV) polymerase chain reaction assay.

The aim of this study was to determine whether multicentre quality controls for the detectability of viral genomes could contribute to the improvement of diagnostic performance in the participating laboratories. The study was carried out during two successive rounds, during which 18 laboratories specialized in nucleic acid testing analyzed, through a polymerase chain reaction (PCR) assay, a common panel of GB virus C (GBV-C)/hepatitis G virus (HGV) RNA-positive and -negative samples. During the first round, the laboratories used either an 'in-house' PCR procedure or a partly standardized commercial test. After decoding the results of the first round, the procedures of the participating laboratories were compared in order to establish a consensus procedure deduced from those of the laboratories which provided the best results. During the second round, each participating laboratory could use the resulting consensus procedure, or its own procedure, or both. The results of this quality control study indicated that, whatever method used, even specialized and trained laboratories may give false-negative or false-positive results. The commercial assay did not guarantee a systematic high quality level of results. The striking heterogeneity of results observed among laboratories using the same commercial assay confirm that molecular biology methods need skilled technicians. The results of this quality control study suggest that full standardization of viral genome detection, including all steps of the procedure, is necessary and that the laboratories performing PCR should participate in repeated quality control studies, whatever technique is being used.

GBV-C/hepatitis G virus (HGV) RNA load in immunodeficient individuals and in immunocompetent individuals.

The aim of this study was to establish the mean plasma GBV-C/hepatitis G virus (HGV) RNA load in groups of GBV-C/HGV-infected individuals with varied immune status and to determine the most frequent patterns of evolution of the plasma GBV-C/HGV RNA load over time during the natural history of infection. The mean plasma GBV-C/HGV RNA load observed was, from the lowest to the highest: 5.21 log in immunodepressed multiply-transfused patients, 6.45 log in HIV-positive individuals, 6.66 log in immunocompetent multiply-transfused patients, and 6.71 log in blood donors. The difference was significant between the four groups (P < 0.0001). The most frequent pattern of evolution of the plasma GBV-C/HGV RNA load was as follows: after the primary GBV-C/HGV infection, the viral load was elevated from the onset; then, a high, persistent and relatively steady viral RNA level was the rule; and when it occurred, the loss of viremia was not preceded by a decrease before recovery from GBV-C/HGV infection.

TT virus infection in French hemodialysis patients: study of prevalence and risk factors.

The TT virus (TTV) is a recently discovered DNA virus which was first identified in patients with non-A to -G hepatitis following blood transfusion. In this study, we tested 150 attendees of two hemodialysis (HD) units of the public hospitals of Marseilles, France, for the presence of TTV genome by using a PCR-based methodology. The overall prevalence of TTV viremia was 28% (compared to 5.3% in blood donors from the same region). We demonstrated the existence of chronic infections and superinfections by strains belonging to different genotypes. The prevalence of infection was higher in patients originating from Africa, in patients with previous blood transfusion or organ transplantation, in patients with antibody to hepatitis B core antigen, and in those with diabetes mellitus. A high prevalence of TTV infection (50%) was also observed in a population of patients with diabetes mellitus but without renal disease. No significant relationship was found between TTV viremia and hepatitis C virus or GB virus C, transaminases, age, sex, and duration of HD treatment. The PCR amplification products (located in open reading frame 1 of the TTV genome) were sequenced. These genomic sequences were submitted to phylogenetic analysis by using the Jukes-Cantor algorithm for distance determination and the neighbor-joining method for tree building. In several instances, sequences from viruses isolated in a HD unit were grouped in the same phylogenetic cluster. These results together with the different distribution of cases in the two HD units suggest there is viral transmission within each.

Screening for HBV, HCV and HIV genomes in blood donations: shortcomings of pooling revealed by a multicentre study simulating real-time testing.

This study was undertaken in order to determine whether screening of viremic blood donations by testing of pooled donor samples could constitute a technically feasible transfusional safety measure. A pilot study of real-time simulation, on a day-to-day basis, of screening of three viral genomes (hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV)) was conducted by five French Blood Centers on plasma samples collected from blood donors and studied within undiluted samples and within sample pools of various sizes. This study was carried out within time conditions compatible with the release of platelets. For the detection of HCV and HIV genomes, the five laboratories achieved a sensitivity that decreased with the size of the sample pool. Four were successful in detecting all undiluted samples. In the 1/10 diluted samples, four failed to detect one HIV or HCV sample. In the 1/100 diluted samples, all laboratories failed to detect one or more HIV or HCV samples. For HBV genome, no participating laboratories detected all of the samples of the panel, even undiluted samples, and the sample pooling considerably affected sensitivity. The improvement and standardization of assays needs to be attained, and training of laboratories appears to be a step crucial for routine screening of viral genomes in blood donations.

Determination and phylogenetic analysis of partial sequences from TT virus isolates.

Sera from French in-patients were tested for the presence of the TT virus (TTV) genome using PCR and degenerate primers located in ORF1. Thirty-six sequences were determined and compared with those deposited in databases, revealing a high degree of genetic variability between TTV isolates (up to 47% for amino acid sequences). Phylogenetic analysis demonstrated the existence of three main groups corresponding to the previously described genotypes 1 and 2 and to a new genotype 3. Isolates could be assigned to distinct genotypes if their genetic distance was > 27%. No comparable genetic criteria were found for the definition of sub-types in the region studied. A 15-31 month follow-up of three haemodialysis patients proved the existence of chronic infection by TTV. In one patient, two strains belonging to different genotypes were detected at the same time. Sequences of both ORF1 and ORF2 remained unchanged for a given strain during the follow-up.

Prevalence of GB virus type C/hepatitis G virus RNA and anti-E2 among blood donors in Southeastern France.

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. STUDY DESIGN AND METHODS: Sera were tested for the presence of GBV-C/HGV RNA by reverse transcriptase-polymerase chain reaction and that of antibodies to the GBV-C/HGV E2 (anti-E2) antigen by an enzyme-linked immunosorbent assay. A first cohort (1660 blood donors) was tested prospectively and a second cohort (238 samples with hepatitis markers) was tested retrospectively. Donors in the prospective study were questioned for possible risk factors of virus transmission. Amplification products were sequenced and subjected to phylogenetic analysis. RESULTS: Approximately 2.6 percent of individuals accepted for blood donation and 15.4 percent with positive hepatitis C virus serologic tests carried GBV-C/HGV RNA. Anti-E2 was detected in these two populations in approximately 12 percent and 48 percent of donors, respectively. Moderate relative risks were found only in tattooed or pierced individuals (1.82) and health care workers (2.45). Almost all strains were located in the same phylogenetic branch as HGV Group 2. CONCLUSION: Though a large proportion of the donors tested have been in contact with GBV-C/HGV, no elevated relative risk of infection was identified. The phylogenetic distribution of viral strains suggests that the infection is endemic in this population.

High prevalence of GB-C/hepatitis G virus in a Brazilian population with helminth infection.

A study of GB-C virus/Hepatitis G virus (GBV-C/ HGV) infection was carried out in a rural population of Northeastern Brazil, in which the prevalence of schistosomiasis is 80-90%. Despite the absence of parenteral risk exposure, the prevalence of GBV-C/HGV markers of infection was found to be unusually increased: viremia, 16.4%; specific antibody, 18.3%. It is therefore suspected that helminth infection influenced the immune response to GBV-C/HGV infection by shifting the balance of cytokine responses from Th1 to Th2, resulting in a delayed viral clearance. Phylogenetic analysis of viral isolates did not provide evidence for high rates of sexual or mother-to-infant viral transmission. The study revealed that viral strains belonged to types 1 and 2 only (predominant in Africa and Europe, respectively), suggesting that GBV-C/HGV was introduced into the New World by white conquerors and black slaves since the 16th century.