A comprehensive bioinformatic analysis of hepatitis D virus full-length genomes.

In association with hepatitis B virus (HBV), hepatitis delta virus (HDV) is a subviral agent that may promote severe acute and chronic forms of liver disease. Based on the percentage of nucleotide identity of the genome, HDV was initially classified into three genotypes. However, since 2006, the original classification has been further expanded into eight clades/genotypes. The intergenotype divergence may be as high as 35%-40% over the entire RNA genome, whereas sequence heterogeneity among the isolates of a given genotype is <20%; furthermore, HDV recombinants have been clearly demonstrated. The genetic diversity of HDV is related to the geographic origin of the isolates. This study shows the first comprehensive bioinformatic analysis of the complete available set of HDV sequences, using both nucleotide and protein phylogenies (based on an evolutionary model selection, gamma distribution estimation, tree inference and phylogenetic distance estimation), protein composition analysis and comparison (based on the presence of invariant residues, molecular signatures, amino acid frequencies and mono- and di-amino acid compositional distances), as well as amino acid changes in sequence evolution. Taking into account the congruent and consistent results of both nucleotide and amino acid analyses of GenBank available sequences (recorded as of January, 2017), we propose that the eight hepatitis D virus genotypes may be grouped into three large genogroups fully supported by their shared characteristics.

First hepatitis B virus full-length genomic study among blood donors from Argentina: unexpected mutations in the circulating subgenotypes' proteins.

Hepatitis B virus (HBV) is a worldwide public health concern. The circulation of strains carrying mutations in the viral proteins implies both clinical and therapeutics challenges. HBV complete genomes (HBV-CGs) were reported from injecting drug users and HBV chronically infected patients from Argentina-including Amerindians-although no studies were conducted in blood donors. Here, we described HBV-CG sequences from the latter population. Some of the HBV sequences classified as B2 and C2 subgenotypes clustering together with Asian isolates, while others, such as D3, F1b, and F4, were homologous to European and Latin America sequences. New substitutions for all analyzed open reading frames and changes in the HBsAg hydrophobicity profiles were detected. Several HBV-CG subgenotypes are described for the first time in this population. Mutations observed in X, PreS, and P proteins have been associated with advanced liver disease, hepatocellular carcinoma, and/or natural resistance to nucleos(t)ide antiviral treatment. It deserves to be highlighted that these substitutions were detected in a population without epidemiological risk factors for viral infection, and most importantly, without any previous antiviral treatment (natural resistance). Regarding the remaining mutations, further research is warranted in order to determine their clinical and therapeutics relevance.

HBV-HDV en donantes de sangre: prevalencia y análisis molecular / Hepatitis B virus and Hepatitis D virus in blood donors: prevalence and molecular analysis in an Argentinean population

The aim of this report was to determine retrospectively the prevalence of hepatitis viruses infection by both HBV and HDV, and to identify the genotype in a population of blood donors. From 42,055 sample of donors, the authors study the hepatitis B virus and hepatitis D virus prevalence and molecular analysis in an Argentinean population. The results obtained are detailed in the article.

Prevalencia de infección por HTLV-I/II en donantes de sangre de la Provincia de Santa Fe, Argentina / Prevalence of HTLV-I/II infection among blood donors in Santa Fe Province, Argentina

Subsequent to the National Epidemiologic Surveillance Program developed in 1997 by the National AIDS Program, anti-HTLV-I/II antibodies among blood donors in Santa Fe Province started to be detected. On the basis of this initial finding, it was regarded of interest to evaluate the true HTLV-I/II seroprevalence in this population during a four-year survey. Thus, from 1997 up to 2002, 9425 samples were studied from 17 out of the 19 provincial departments. Out of the total sampling, 38 proved reactive by agglutination techniques, 18 of which were confirmed by western blot (WB). Out of the latter, 10 were HTLV-I/II seropositive with a final prevalence of 0.1% (10/9425), whereas 7 were indeterminate and 1 negative. Among these 10 confirmed sera, 2 (0.02%) were HTLV, 3 (0.03%) HTLV-I and 5 (0.05%) HTLV-II. It should be highlighted that the presence of HTLV-I/II infection in blood donors in Santa Fe Province was demonstrated for the first time, with a prevalence greater than that reported for blood donors in non-endemic Argentine areas. Such findings confirm the need of corresponding systematic screening through regulatory blood bank norms in Santa Fe Province.

Subsecuentemente a que en 1997 el Programa Nacional de SIDA implementó un Programa de Vigilancia Epidemiológica a escala nacional, se comenzaron a detectar anticuerpos anti-HTLV-I/II en donantes de sangre de la Provincia de Santa Fe. En base a ese hallazgo inicial, se consideró pertinenteestimar la seroprevalencia de HTLV-I/II en donantes santafecinos en el curso de los 4 años siguientes. Así,desde 1997 hasta 2002, se estudiaron 9425 muestras provenientes de 17 de los 19 departamentos de laProvincia. Del total de muestras, 38 resultaron reactivas por técnicas de tamizaje, y de ellas 18 fueron confirmadas por western blot (WB). De esas muestras, 10 fueron HTLV-I/II seropositivas con una prevalencia finalde 0.1% (10/9425), en tanto que 7 resultaron indeterminadas y 1 negativa. De las seropositivas, 2 (0.02 %)eran HTLV, 3 (0.03 %) HTLV-I, y 5 (0.05 %) HTLV-II. Cabe destacar que por primera vez se constató lapresencia de infección por HTLV-I/II en donantes de sangre de Santa Fe, y con una prevalencia mayor a lasreferidas para donantes de sangre de áreas no endémicas de Argentina. Estos datos fundamentan la necesidadde un screening sistemático para la infección por HTLV-I/II mediante normas regulatorias en bancos desangre de esta provincia..

Specific IgA detection can be used for perinatal diagnosis of HIV in children under protocol ACTG 076.

Detection of anti-HIV-1 IgA antibodies using a modified ELISA test for the early diagnosis of perinatally acquired HIV-1 infection in children treated with protocol ACTG 076 was evaluated. A total of 177 sera were obtained from 141 infants between 1 and 12 months of age (46 were treated and 95 were non-treated with protocol ACTG 076) and tested for HIV IgA antibodies by an ELISA test after removal of IgG with recombinant protein G. Infants were classified according to CDC's classification system after a follow-up until 20 months of age. Of the 46 treated children 22 turned out to be infected and in the group of 95 untreated children, 52 were infected. All 81 samples from uninfected children treated or untreated with protocol ACTG 076 were persistently IgA-negative. HIV IgA antibodies were detected in 14 of 25 plasma samples from infected children with treatment, and in 58 of 71 samples in infected children without treatment. Considering that the sensitivity of this test is lower in children younger than 6 months the population of children studied was divided into two groups; those under and those over 6 months of age. No significant differences were observed in the detection of IgA in treated or untreated children in both age groups. The overall specificity of the test was 100 per cent; sensitivity in children older than 6 months was 76.92 per cent in treated children and 93.10 per cent in untreated children. In spite of the small number of samples studied it could be demonstrated that treatment with zidovudine does not affect the detection of IgA antibodies. This is a simple and inexpensive method that could be used for diagnosis of treated and untreated children in developing countries.

Human immunodeficiency virus bDNA assay for pediatric cases.

Techniques to quantify plasma HIV-1 RNA viral load (VL) are commercially available, and they are adequate for monitoring adults infected by HIV and treated with antiretroviral drugs. Little experience on HIV VL has been reported in pediatric cases. In Argentina, the evaluation of several assays for VL in pediatrics are now being considered. To evaluate the pediatric protocol for bDNA assay in HIV-infected children, 25 samples from HIV-infected children (according to CDC criteria for pediatric AIDS) were analyzed by using Quantiplex HIV RNA 2.0 Assay (Chiron Corporation) following the manufacturer's recommendations in a protocol that uses 50 microliters of patient's plasma (sensitivity: 10,000 copies/ml). When HIV-RNA was not detected, samples were run with the 1 ml standard bDNA protocol (sensitivity: 500 HIV-RNA c/ml). Nine samples belonged to infants under 12 months of age (group A) and 16 were over 12 months (group B). All infants under one year of age had high HIV-RNA copies in plasma. VL ranged from 30,800 to 2,560,000 RNA copies/ml (median = 362,000 c/ml) for group A and < 10,000 to 554,600 c/ml (median = < 10,000) for group B. Only 25% of children in group B had detectable HIV-RNA. By using the standard test of quantification, none of the patients had non detectable HIV-RNA, ranging between 950 and 226,200 c/ml for group B (median = 23,300 RNA c/ml). The suggested pediatric protocol could be useful in children under 12 months of age, but 1 ml standard protocol must be used for older children. Samples with undetectable results from children under one year of age should be repeated using the standard protocol.

Human immunodeficiency virus bDNA assay for pediatric cases

Techniques to quantify plasma HIV-1 RNA viral load (VL) are commercially available, and they are adequate for monitoring adults infected by HIV and treated with antiretroviral drugs. Little experience on HIV VL has been reported in pediatric cases. In Argentina, the evaluation of several assays for VL in pediatrics are now being considered. To evaluate the pediatric protocol for bDNA assay in HIV-infected children, 25 samples from HIV-infected children (according to CDC criteria for pediatric AIDS) were analyzed by using Quantiplex HIV RNA 2.0 Assay (Chiron Corporation) following the manufacturer's recommendations in a protocol that uses 50 microliters of patient's plasma (sensitivity: 10,000 copies/ml). When HIV-RNA was not detected, samples were run with the 1 ml standard bDNA protocol (sensitivity: 500 HIV-RNA c/ml). Nine samples belonged to infants under 12 months of age (group A) and 16 were over 12 months (group B). All infants under one year of age had high HIV-RNA copies in plasma. VL ranged from 30,800 to 2,560,000 RNA copies/ml (median = 362,000 c/ml) for group A and < 10,000 to 554,600 c/ml (median = < 10,000) for group B. Only 25 of children in group B had detectable HIV-RNA. By using the standard test of quantification, none of the patients had non detectable HIV-RNA, ranging between 950 and 226,200 c/ml for group B (median = 23,300 RNA c/ml). The suggested pediatric protocol could be useful in children under 12 months of age, but 1 ml standard protocol must be used for older children. Samples with undetectable results from children under one year of age should be repeated using the standard protocol.

Seroepidemiological and molecular studies of human T cell lymphotropic virus type II, subtype b, in isolated groups of Mataco and Toba Indians of northern Argentina.

We studied plasma samples from 2082 Mataco Indians living in 22 different communities in the western part of Formosa Province, northern Argentina. Samples were screened for HTLV-I/II antibodies by particle agglutination assay. All positive or borderline samples were then tested by an immunofluorescence assay (IFA) on C19 HTLV-II-producing cells. Western blot was used for confirmation of all IFA-positive plasma samples. The crude HTLV-II seroprevalence was 3.0% (62 of 2051), and 0.9% (5 of 588) in children less than 10 years old. The latter result suggests ongoing mother-to-child transmission, probably by breast feeding. There was a marked increase in HTLV-II seroprevalence with age (0.9%, 0-10 years; 1.6%, 11-20 years; 4.4%, 21-30 years; 3.4%, 31-40 years; 7.2%, 41-50 years; 5.7%, >50 years) in both male (p = 0.002) and female subjects (p = 0.00002). None of the 80 non-Indian inhabitants tested was HTLV-I/II seropositive. In a second study, among 105 Toba Indians from a village (Primavera) of the eastern part of this region, 23 were HTLV-II seropositive with a seroprevalence of 59% in those more than 40 years old. From seven of the Indians from Primavera, three others from neighboring regions (including two Tobas and one Pilaga), and one intravenous drug user (IVDU) from Rosario, DNA was extracted from peripheral blood mononuclear cells, and the gp21 transmembrane-encoding gene (590 bp) was amplified by PCR, cloned, and sequenced. LTR sequences were also obtained from the Pilaga, the IVDU, and one Toba. Molecular and phylogenetic analyses revealed that the Indians were all infected with closely related HTLV-II molecular strains belonging to the b subtype, while the IVDU was infected with an HTLV-II subtype a variant. Such data help to make a phylogenetic atlas of HTLV-II among Amerindian tribes and are crucial to gain new insights into the origin and modes of dissemination of this human retrovirus in the Americas.

[Molecular characterization of HTLV-II from an intravenous drug addict with AIDS in Argentina]. / Caracterización molecular de HTLV-II de un paciente drogadicto endovenoso con SIDA de la Argentina.

Ten intravenous drug user AIDS patients were studied to determine the presence of HTLV-I/II infection. Sera were screened by particle agglutination test and by an in house indirect immunofluorescence assay using MT2 (HTLV-I) and C19 (HTLV-II) producing cell lines. Out of the ten sera, one was confirmed HTLV-II positive by Western blot. Peripheral blood mononuclear cells were obtained from this patient, separated through a Ficoll-Hypaque gradient and DNA was extracted and amplified by semi-nested PCR. The amplified product obtained was cloned and sequenced. Results demonstrated the presence of HTLV-II proviral DNA and comparison of the obtained sequence with different HTLV-II prototypes led to classify the virus into subtype a. This is the first molecular characterization of HTLV-II in an intravenous drug user in Argentina.

Quantification of HTLV-II proviral copies by competitive polymerase chain reaction in peripheral blood mononuclear cells of Italian injecting drug users, central Africans, and Amerindians.

To better correlate the burden of human T cell leukemia virus type I (HTLV-I) and type II (HTLV-II) infection with diagnostic and prognostic markers, we developed a new competitive polymerase chain reaction (PCR) assay for the quantitative determination of proviral copy numbers in infected cells. A competitive plasmid was constructed that carried a 112-bp fragment from a highly conserved region of the HTLV tax gene and that was further modified by inserting a sequence of 24 bp. This competitive PCR assay system can be used for the quantification of HTLV-I and HTLV-II proviral DNA as demonstrated by using HTLV-I- and HTLV-II-infected cell lines and/or patient material. We determined the HTLV-II proviral load in peripheral blood mononuclear cells (PBMCs) of 11 Italian injecting drug users (IDUs) infected by this virus and in PBMCs of 10 seropositive Amerindian and Central African individuals from endemically infected ethnic groups. A great variation was observed in the number of HTLV-II proviral sequences in the PBMCs of Italian drug abusers, ranging from 5-10 to 16,239 copies/10(5) cells. There was no clear-cut correlation between proviral load, CD8 count, stage of HIV-1 infection, and therapy. A considerable variation in HTLV-II proviral load was also observed in PBMCs of Amerindians and Central Africans with no correlation between the amount of HTLV-II provirus and the geographic origin of the infected individuals.

Isolation and molecular characterization of a human T-cell lymphotropic virus type II (HTLV-II), subtype B, from a healthy Pygmy living in a remote area of Cameroon: an ancient origin for HTLV-II in Africa.

We report characterization of a human T-cell lymphotropic virus type II (HTLV-II) isolated from an interleukin 2-dependent CD8 T-cell line derived from peripheral blood mononuclear cells of a healthy, HTLV-II-seropositive female Bakola Pygmy, aged 59, living in a remote equatorial forest area in south Cameroon. This HTLLV-II isolate, designated PYGCAM-1, reacted in an indirect immunofluorescence assay with HTLV-II and HTLV-I polyclonal antibodies and with an HTLV-I/II gp46 monoclonal antibody but not with HTLV-I gag p19 or p24 monoclonal antibodies. The cell line produced HTLV-I/II p24 core antigen and retroviral particles. The entire env gene (1462 bp) and most of the long terminal repeat (715 bp) of the PYGCAM-1 provirus were amplified by the polymerase chain reaction using HTLV-II-specific primers. Comparison with the long terminal repeat and envelope sequences of prototype HTLV-II strains indicated that PYGCAM-1 belongs to the subtype B group, as it has only 0.5-2% nucleotide divergence from HTLV-II B strains. The finding of antibodies to HTLV-II in sera taken from the father of the woman in 1984 and from three unrelated members of the same population strongly suggests that PYGCAM-1 is a genuine HTLV-II that has been present in this isolated population for a long time. The low genetic divergence of this African isolate from American isolates raises questions about the genetic variability over time and the origin and dissemination of HTLV-II, hitherto considered to be predominantly a New World virus.

[Evaluation of a new rapid ELISA technique for detecting human cytomegalovirus antibodies (CMV). Applications in emergency blood transfusion and in organ transplantation]. / Evaluation d'une nouvelle technique rapide par Elisa de dépistage des anticorps anticytomégalovirus humain (CMV). Applications pour les transfusions sanguines en urgence et dans un contexte de transplantation d'organes.

Immunocubes can detect very low HCMV IgG levels with at least the same sensitivity as the reference technique: ELISA. Samples containing bacteria showed no particular interference on the test, and results for non-infected material were in agreement with those of ELISA. The rapid test did not give any false positive reactions even with samples from patients with very high antiherpes immunoglobulins levels. Samples from transplant patients at the beginning of seroconversion or diluted to IgG titers corresponding to ELISA cut-off values were always detected as positive by immunocubes. In view of the rapidity of the test, its high specificity, its ability to detect levels corresponding to extremely low IgG signals in ELISA, and the possibility of conserving objective proof of the test, we conclude that this rapid immunocube technique is of great interest for current serologic screening in an emergency context, especially for transfusion and organ transplantation.