Pyramiding dsRNAs increases phytonematode tolerance in cotton plants.

MAIN CONCLUSION: Host-derived suppression of nematode essential genes decreases reproduction of Meloidogyne incognita in cotton. Root-knot nematodes (RKN) represent one of the most damaging plant-parasitic nematode genera worldwide. RNAi-mediated suppression of essential nematode genes provides a novel biotechnological strategy for the development of sustainable pest-control methods. Here, we used a Host Induced Gene Silencing (HIGS) approach by stacking dsRNA sequences into a T-DNA construct to target three essential RKN genes: cysteine protease (Mi-cpl), isocitrate lyase (Mi-icl), and splicing factor (Mi-sf), called dsMinc1, driven by the pUceS8.3 constitutive soybean promoter. Transgenic dsMinc1-T4 plants infected with Meloidogyne incognita showed a significant reduction in gall formation (57-64%) and egg masses production (58-67%), as well as in the estimated reproduction factor (60-78%), compared with the susceptible non-transgenic cultivar. Galls of the RNAi lines are smaller than the wild-type (WT) plants, whose root systems exhibited multiple well-developed root swellings. Transcript levels of the three RKN-targeted genes decreased 13- to 40-fold in nematodes from transgenic cotton galls, compared with those from control WT galls. Finally, the development of non-feeding males in transgenic plants was 2-6 times higher than in WT plants, indicating a stressful environment for nematode development after RKN gene silencing. Data strongly support that HIGS of essential RKN genes is an effective strategy to improve cotton plant tolerance. This study presents the first application of dsRNA sequences to target multiple genes to promote M. incognita tolerance in cotton without phenotypic penalty in transgenic plants.

Hepatitis C virus genotypes in hemodialysis patients in the Federal District, Brazil.

Hepatitis C virus (HCV) genotypes and subtypes were determined in hemodialysis patients in the Federal District, Brazil, by sequencing of the 5' noncoding (NC) and nonstructural 5B (NS5B) regions. From 761 patients, 66 anti-HCV-positive samples were tested for HCV RNA. All 51 HCV RNA-positive samples by PCR of the 5' NC region were genotyped as genotypes 1 (90.2%) and 3 (9.8%). Subtype 1a (82.3%) was the most prevalent, followed by subtypes 3a (9.8%), 1b (5.9%) and 1a/1b (2.0%). Forty-two samples could be amplified and genotyped in the NS5B region: 38 (90.5%) as genotype 1, subtypes 1a, and 8 (9.5%) as genotype 3, subtype 3a. For the 42 samples sequenced in both regions, the genotypes and subtypes determined were concordant in 100% and 95.2% of cases, respectively. Two samples presented discrepant results, with the 5' NC region not distinguishing correctly the subtypes 1a and 1b. These findings indicate that the HCV genotype 1, subtype 1a, is the most prevalent among hemodialysis patients in the Federal District, Brazil.

Hepatitis C virus genotypes in hemodialysis patients in the Federal District, Brazil / Genótipos do vírus da hepatite C em pacientes em hemodiálise no Distrito Federal, Brasil

Hepatitis C virus (HCV) genotypes and subtypes were determined in hemodialysis patients in the Federal District, Brazil, by sequencing of the 5' noncoding (NC) and nonstructural 5B (NS5B) regions. From 761 patients, 66 anti-HCV-positive samples were tested for HCV RNA. All 51 HCV RNA-positive samples by PCR of the 5' NC region were genotyped as genotypes 1 (90.2 percent) and 3 (9.8 percent). Subtype 1a (82.3 percent) was the most prevalent, followed by subtypes 3a (9.8 percent), 1b (5.9 percent) and 1a/1b (2.0 percent). Forty-two samples could be amplified and genotyped in the NS5B region: 38 (90.5 percent) as genotype 1, subtypes 1a, and 8 (9.5 percent) as genotype 3, subtype 3a. For the 42 samples sequenced in both regions, the genotypes and subtypes determined were concordant in 100 percent and 95.2 percent of cases, respectively. Two samples presented discrepant results, with the 5' NC region not distinguishing correctly the subtypes 1a and 1b. These findings indicate that the HCV genotype 1, subtype 1a, is the most prevalent among hemodialysis patients in the Federal District, Brazil.

Os genótipos e subtipos do vírus da hepatite C (HCV) foram determinados em pacientes em hemodiálise no Distrito Federal, Brasil, pelo sequenciamento das regiões 5' não codificante (NC) e não estrutural 5B (NS5B). De 761 pacientes, 66 amostras anti-HCV positivas foram testadas para RNA-HCV. Todas as 51 amostras RNA-HCV positivas por PCR para a região 5' NC foram genotipadas como dos genótipos 1 (90,2 por cento) e 3 (9,8 por cento). O subtipo 1a (82,3 por cento) foi o mais prevalente, seguido pelos subtipos 3a (9,8 por cento), 1b (5,9 por cento) e 1a/1b (2,0 por cento). Quarenta e duas amostras puderam ser amplificadas e genotipadas na região NS5B: 38 (90,5 por cento) como genótipo 1, subtipo 1a, e 8 (9,5 por cento) como genótipo 3, subtipo 3a. Para as 42 amostras sequenciadas nas duas regiões, os genótipos e subtipos determinados foram concordantes em 100 por cento e 95,2 por cento dos casos, respectivamente. Duas amostras apresentaram resultados discrepantes, sendo que a região 5' NC não diferenciou corretamente os subtipos 1a e 1b. Estes achados indicam que o genótipo 1, subtipo 1a, do HCV é o mais prevalente em pacientes em hemodiálise no Distrito Federal, Brasil.

Distribution of hepatitis C virus genotypes among blood donors from mid-west region of Brazil.

In order to investigate the hepatitis C virus (HCV) genotypes in mid-west region of Brazil, 250 anti-HCV positive blood donors were studied. Among them, the anti-HCV serological status was confirmed in 205 (82%). HCV RNA was detected in 165 samples, which were genotyped. HCV types 1, 2 and 3 were found in 67.9%, 3% and 29.1% of the donors, respectively. In Goiás state, subtype 1a (50%) was the most prevalent, followed by subtypes 3a (30.9%) and 1b (16.7%). In Mato Grosso state, subtype 1a was also predominant (41%), followed by subtypes 1b (29.5%) and 3a (25%). In Mato Grosso do Sul state, subtypes 1a and 1b were detected equally (36.8%), followed by 3a (21.1%). Subtype 2b was rare (2.4%, 4.5% and 5.3%, respectively). In Distrito Federal, subtype 3a (39%) was more frequent than 1a (31.7%) and the remaining (29.3%) belonged to subtype 1b.

Distribution of hepatitis C virus genotypes among blood donors from mid-west region of Brazil

Com objetivo de determinar os genótipos do vírus da hepatite C (HCV) circulantes na Região Centro-Oeste do Brasil, 250 doadores de sangue anti-HCV positivos foram estudados. Dentre eles, a positividade para anti-HCV foi confirmada em 205 (82%). O RNA-HCV foi detectado em 165 amostras, as quais foram genotipadas. Os tipos 1, 2 e 3 do HCV foram encontrados em 67,9%, 3% e 29,1% dos doadores, respectivamente. No Estado de Goiás, o subtipo 1a (50%) foi o mais prevalente, seguido pelos subtipos 3a (30,9%) e 1b (16,7%). No Estado de Mato Grosso, o subtipo 1a (41%) foi também predominante, seguido pelos subtipos 1b (29,5%) e 3a (25%). No Estado de Mato Grosso do Sul, os subtipos 1a e 1b foram igualmente detectados (36,8%), seguidos por 3a (21,1%). O subtipo 2b foi raro (2,4%, 4,5% e 5,3%, respectivamente). No Distrito Federal, o subtipo 3a (39%) foi mais freqüente que 1a (31,7%), sendo o restante (29,3%) identificado como subtipo 1b.

Antiretroviral resistance and genetic diversity of human immunodeficiency virus type 1 isolates from the Federal District, Central Brazil

In the context of universal access to antiretroviral therapy, the surveillance of human immunodeficiency virus type 1 (HIV-1) genetic diversity and resistance becomes pivotal. In this work our purpose was to describe the genetic variability; prevalence of drug-resistance mutations; and genotypic resistance profiles in HIV-1 infected individuals under antiretroviral treatment, from the Federal District, Brasília, Central Brazil. The entire viral protease and codons 19 to 234 of the reverse transcriptase gene from 45 HIV-1 isolates were amplified and sequenced for subtyping and genotyping. By phylogenetic analysis, 96 percent of the samples clustered with subtype B and the remaining 4 percent with HIV-1 subtype F sequences. One major protease inhibitor resistance-associated mutation, I50V, was detected in 38 percent of the samples. Minor mutations were also found at the protease gene: L10I/V (7 percent), K20M (2 percent), M36I (11 percent), L63P (20 percent), A71T (2 percent), and V77I (7 percent). Many mutations associated with reduced susceptibility to nucleoside or non-nucleoside reverse transcriptase inhibitors were detected: M41L (11 percent), E44D (4 percent), D67N (11 percent), T69D (2 percent), K70R (11 percent), L74V (2 percent), L100I (4 percent), K103N (18 percent), V118I (9 percent), Y181C (11 percent), M184V (18 percent), G190A (4 percent), T215Y (4 percent), and K219E (4 percent). This study has shown that 84 percent of the studied population from the Federal District, showing evidences of therapy failure, presented viral genomic mutations associated with drug resistance. The main antiretrovirals to which this population showed resistance were the PI amprenavir (38 percent), the NNRTIs delavirdine, nevirapine (31 percent), and efavirenz (24 percent), and the NRTIs lamivudine (18 percent), abacavir, and zidovudine (13 percent).

Hepatitis C virus genotypes in blood donors from the Federal District, Central Brazil

The objective of this study was to characterize hepatitis C virus (HCV) genotypes in blood donors from the Federal District, Central Brazil, and to compare HCV screening by serological assays and reverse transcriptase polymerase chain reaction (RT-PCR). Plasma samples from 57 individuals with reactive or indeterminate results in serological anti-HCV screening assays (ELISA or EIA) were tested for HCV RNA by RT-PCR. The results from a confirmatory LIA serological assay were also evaluated. The 5' non-coding region of the HCV genome was amplified from 41 PCR positive samples (71.9 percent), which were further characterized by nucleotide sequencing analysis. Of these, 60.9 percent were of HCV genotype 1 and 39.1 percent of genotype 3.

Antiretroviral resistance and genetic diversity of human immunodeficiency virus type 1 isolates from the Federal District, Central Brazil.

In the context of universal access to antiretroviral therapy, the surveillance of human immunodeficiency virus type 1 (HIV-1) genetic diversity and resistance becomes pivotal. In this work our purpose was to describe the genetic variability; prevalence of drug-resistance mutations; and genotypic resistance profiles in HIV-1 infected individuals under antiretroviral treatment, from the Federal District, Brasilia, Central Brazil. The entire viral protease and codons 19 to 234 of the reverse transcriptase gene from 45 HIV-1 isolates were amplified and sequenced for subtyping and genotyping. By phylogenetic analysis, 96% of the samples clustered with subtype B and the remaining 4% with HIV-1 subtype F sequences. One major protease inhibitor resistance-associated mutation, I50V, was detected in 38% of the samples. Minor mutations were also found at the protease gene: L10I/V (7%), K20M (2%), M36I (11%), L63P (20%), A71T (2%), and V77I (7%). Many mutations associated with reduced susceptibility to nucleoside or non-nucleoside reverse transcriptase inhibitors were detected: M41L (11%), E44D (4%), D67N (11%), T69D (2%), K70R (11%), L74V (2%), L100I (4%), K103N (18%), V118I (9%), Y181C (11%), M184V (18%), G190A (4%), T215Y (4%), and K219E (4%). This study has shown that 84% of the studied population from the Federal District, showing evidences of therapy failure, presented viral genomic mutations associated with drug resistance. The main antiretrovirals to which this population showed resistance were the PI amprenavir (38%), the NNRTIs delavirdine, nevirapine (31%), and efavirenz (24%), and the NRTIs lamivudine (18%), abacavir, and zidovudine (13%).

Hepatitis C virus genotypes in blood donors from the Federal District, Central Brazil.

The objective of this study was to characterize hepatitis C virus (HCV) genotypes in blood donors from the Federal District, Central Brazil, and to compare HCV screening by serological assays and reverse transcriptase polymerase chain reaction (RT-PCR). Plasma samples from 57 individuals with reactive or indeterminate results in serological anti-HCV screening assays (ELISA or EIA) were tested for HCV RNA by RT-PCR. The results from a confirmatory LIA serological assay were also evaluated. The 5' non-coding region of the HCV genome was amplified from 41 PCR positive samples (71.9%), which were further characterized by nucleotide sequencing analysis. Of these, 60.9% were of HCV genotype 1 and 39.1% of genotype 3.