RESUMO
This study aimed to evaluate by means of Nested Polymerase Chain Reaction (nPCR), co-cultivation and sequencing, with genetic comparison between strains (mother/newborn), the occurrence of vertical transmission of Small Ruminant Lentiviruses (SRLV) from naturally occurring nannies infected for their offspring. For the detection of SRLV seropositive progenitors, blood was collected from 42 nannies in the final third of gestation in tubes with and without anticoagulant. The diagnostic tests used were Western Blot (WB) and nPCR. During the period of birth, the same blood collection procedure was performed on 73 newborns at zero hours of birth, with the same diagnostic tests. Seventeen blood samples from seven-day-old kids, proven positive for SRLV by nPCR, chosen at random, were subjected to coculture in goat synovial membrane (GSM) cells for 105 days. The pro-viral DNA extracted from the cell supernatant from the coculture was subjected to nPCR. For DNA sequencing from the nPCR products, nine positive samples were chosen at random, four nannies with their respective offspring, also positive. Each sample was performed in triplicate, thus generating 27 nPCR products of which only 19 were suitable for analysis. Among the 42 pregnant goats, in 50% (21/42) pro-viral DNA was detected by nPCR, while in the WB, only 7.14% (3/42) presented antibodies against SRLV. Regarding neonates, of the 73 kids, 34 (46.57%) were positive for the virus, using the nPCR technique, while in the serological test (WB), three positive animals (4.10%) were observed. The coculture of the 17 samples with a positive result in the nPCR was confirmed in viral isolation by amplification of the SRLV pro-viral DNA. When aligned, the pro-viral DNA sequences (nannies and their respective offspring) presented homology in relation to the standard strain CAEV Co. It was concluded that the transmission of SRLV through intrauterine route was potentially the source of infection in the newborn goats.
Assuntos
Doenças das Cabras/transmissão , Transmissão Vertical de Doenças Infecciosas/veterinária , Infecções por Lentivirus/transmissão , Provírus/isolamento & purificação , Doenças dos Ovinos/transmissão , Animais , Animais Recém-Nascidos/virologia , Linhagem Celular , DNA Viral/sangue , Feminino , Doenças das Cabras/virologia , Cabras/virologia , Lentivirus/isolamento & purificação , Infecções por Lentivirus/veterinária , Reação em Cadeia da Polimerase , Gravidez , Provírus/genética , Análise de Sequência de DNA , Ovinos/virologia , Doenças dos Ovinos/virologiaRESUMO
BACKGROUND: Infective dermatitis associated with human T-cell lymphotropic virus type-1 (HTLV-1), (IDH), is a chronic eczema occurring in HTLV-1 infected children. Rare cases of adulthood IDH have been reported and no study until now aimed to compare juvenile and adulthood IDH. METHODOLOGY/PRINCIPAL FINDINGS: Twelve cases of adulthood IDH followed for a mean time of 7.5 years were analyzed according to clinicopathological and molecular aspects, comparing them to juvenile IDH cases. Diagnosis was based on the modified major criteria used for juvenile IDH. Proviral load (PVL) assessment was performed by real-time PCR technique. Adulthood IDH presented similar clinicopathological and molecular aspects compared to juvenile IDH. The morphology of lesions and areas of involvement were similar, except for the involvement of the ankles and inframammary folds in the adulthood form. HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) occurred in six adulthood IDH patients, with almost equal frequency. However, at least in two patients, HAM/TSP appeared prior to IDH, differently from what was observed in juvenile IDH. CONCLUSIONS/SIGNIFICANCE: Adulthood IDH is similar to juvenile IDH according to clinicopathological aspects and PVL levels. Therefore, the same modified major diagnostic criteria for juvenile IDH can be applied to both forms.
Assuntos
Eczema/patologia , Eczema/virologia , Infecções por HTLV-I/complicações , Vírus Linfotrópico T Tipo 1 Humano/isolamento & purificação , Provírus/isolamento & purificação , Carga Viral , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
AIMS: To evaluate the potential use of synthetic oligonucleotides as a standard curve for proviral load (PVL) of human T-cell leukaemia virus type 1 (HTLV-1) quantification in peripheral blood mononuclear cells (PBMC) of HTLV-1-infected individuals by quantitative real-time polymerase chain reaction (qPCR) analysis. METHODS AND RESULTS: Synthetic oligonucleotides based on HTLV-1 genome were customized to use as a standard curve. Twelve anti-HTLV-1-positive samples with known HTLV-1 PVL, previously quantified by qPCR assay using TARL-2 cells as a conventional standard curve, were submitted to the new protocol. The proviral quantification levels had a high concordance with qPCR results using a conventional standard curve. The results demonstrate that the conventional standard curve can be replaced by a synthetic standard curve due to its ability to quantification based on the linearity and qPCR efficiency and similar results with a validated qPCR assay using a conventional standard curve. CONCLUSIONS: Synthetic oligonucleotides standard curves could be a very useful tool on HTLV-1 diagnosis and absolute HTLV-1 PVL quantification. SIGNIFICANCE AND IMPACT OF THE STUDY: HTLV-1 PVL determination using synthetic oligonucleotides standard curve by qPCR could be a helpful alternative for the laboratories that monitor infected patients as an important prognostic factor in HTLV-1-associated diseases progression. Also, it can decrease costs and overcome the biological limitations of the plasmid curve.
Assuntos
Infecções por HTLV-I/diagnóstico , Vírus Linfotrópico T Tipo 1 Humano/isolamento & purificação , Provírus/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Carga Viral/métodos , Adulto , DNA Viral/genética , Progressão da Doença , Genoma Viral/genética , Infecções por HTLV-I/sangue , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Leucócitos Mononucleares/virologia , Pessoa de Meia-Idade , Oligonucleotídeos/síntese química , Oligonucleotídeos/genética , Prognóstico , Provírus/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Carga Viral/normasRESUMO
INTRODUCTION: Infective dermatitis associated with HTLV-1 (IDH) is a recurrent eczema which affects children vertically infected with HTLV-1. In Bahia, Brazil, we recently reported that 47% of IDH patients also develop juvenile HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a progressive disabling disorder which is typically reported in adult HTLV-1 carriers. IDH may also predispose to adult T-cell leukemia/lymphoma, a neoplasm associated with HTLV-1. The factors relating to the development of HTLV-1-associated juvenile diseases have not yet been defined. HTLV-1 proviral load (PVL) is one of the main parameters related to the development of HTLV-1 associated diseases in adults. In the current study, we investigated the role of PVL in IDH and juvenile HAM/TSP. METHODOLOGY/PRINCIPAL FINDINGS: This is a cohort study that included fifty-nine HTLV-1 infected children and adolescents, comprising 16 asymptomatic carriers, 18 IDH patients, 20 patients with IDH and HAM/TSP (IDH/HAM/TSP) and five with HAM/TSP. These patients were followed-up for up to 14 years (median of 8 years). We found that PVL in IDH and IDH/HAM/TSP patients were similarly higher than PVL in juvenile asymptomatic carriers (p<0.0001). In those IDH patients who developed HAM/TSP during follow-up, PVL levels did not vary significantly. HAM/TSP development did not occur in those IDH patients who presented high levels of PVL. IDH remission was associated with an increase of PVL. Inter-individual differences in PVL were observed within all groups. However, intra-individual PVL did not fluctuate significantly during follow-up. CONCLUSIONS/SIGNIFICANCE: High PVL in IDH patients was not necessary indicative of progression to HAM/TSP. PVL did not decrease after IDH remission. The maintenance of high PVL after remission could favor early development of ATL. Therefore, IDH patients would have to be followed-up even after remission of IDH and for a long period of time.
Assuntos
Infecções por HTLV-I/patologia , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/isolamento & purificação , Provírus/isolamento & purificação , Carga Viral , Adolescente , Brasil , Criança , Pré-Escolar , Estudos de Coortes , Progressão da Doença , Feminino , Humanos , MasculinoRESUMO
Bovine leukemia virus (BLV) is the agent responsible for enzootic bovine leukosis, the most common neoplastic disease in cattle. The horn fly, a major hematophagous pest of cattle, is able to transmit different diseases in cattle. However, its implication in BLV transmission under a natural environment is still discussed. The objectives of this work were to determine the presence of BLV in horn flies (by sequencing) and to evaluate the ability of horn flies to transmit BLV to cattle (through an experimental assay under a natural environment). To demonstrate the presence of BLV in the flies, 40 horn flies were collected from a BLV-positive cow with a sweep net and 10 pools with four horn-fly mouthparts each were prepared. The presence of BLV was determined by nested polymerase chain reaction and sequencing. To demonstrate BLV transmission, other 40 flies were collected from the same BLV-positive cow with a sweep net. Eight homogenates containing five horn-fly mouthparts each were prepared and injected to eight cows of different breeds, and blood samples were collected every 21 days. Then, to evaluate the ability of horn flies to transmit BLV to grazing cattle under natural conditions, both infected and uninfected cattle from the experimental transmission assay were kept together in the same paddock with more than 200 horn flies per animal for 120 days. Blood samples were collected every 20 days and the number of flies was determined. The sequencing results confirmed the presence of the provirus in horn flies. The results also confirmed that BLV transmission is a possible event, at least experimentally. However, the role of horn flies as vectors of BLV under a natural grazing system is still discussed.
Assuntos
Leucose Enzoótica Bovina/transmissão , Insetos Vetores/virologia , Vírus da Leucemia Bovina/isolamento & purificação , Muscidae/virologia , Animais , Argentina , Bovinos , Feminino , Insetos Vetores/fisiologia , Muscidae/fisiologia , Reação em Cadeia da Polimerase/veterinária , Provírus/isolamento & purificaçãoRESUMO
BACKGROUND: The absence of virus expression during the chronic stage of bovine leukemia virus (BLV) infection and its reactivation upon ex vivo culture has become a long-lived Dogma. During the chronic stage of BLV infection the immune response limits viral replication and the mitotic division of latently infected cells, carrying BLV provirus, allows viral expansion and disease progression towards a lymphoproliferative disorder. Several stressor factors have been associated with animal production and handling. As natural mediator of stress, glucocorticoids are strong immunosuppressive agents; moreover, they can bind long-terminal repeat region of retroviruses and induce viral expression. In the present study, we present a case report describing the spontaneous reactivation of BLV infection in naturally infected cattle. CASE PRESENTATION: In order to investigate if virus reactivation occurred in vivo during the course of BLV infection, we followed up for 328 days one Holstein cow (> 3 years) chronically infected with BLV which presented high-proviral loads. This animal was neither lactating nor pregnant. Furthermore, we investigated if a stressor stimulus, in this case the administration of a synthetic glucocorticoid (dexamethasone), could impact the course of BLV infection in three additional cattle. For the first time, we observed a high level of BLV transcripts in a total of four cattle chronically infected with BLV. The detection of viral transcripts corresponding to pol gene strongly suggests virus reactivation in these animals. Interestingly, this simultaneous virus reactivation was unrelated to dexamethasone treatment. CONCLUSIONS: We reported for the first time spontaneous and high level of BLV transcriptional activation in cattle chronically infected with BLV. Although virus reactivation was unrelated to dexamethasone treatment, other stressor stimuli might have influenced this outcome. Future studies will be necessary to understand these observations, since the spontaneous virus reactivation presented here might have implications on BLV pathogenesis and transmission.
Assuntos
Leucose Enzoótica Bovina/virologia , Vírus da Leucemia Bovina/fisiologia , Ativação Viral/fisiologia , Animais , Bovinos , Dexametasona/farmacologia , Feminino , Provírus/isolamento & purificação , Estresse Fisiológico , Ativação Viral/efeitos dos fármacosRESUMO
General knowledge on the diversity and biology of microbial viruses infecting bacterial hosts from extreme acidic environments lags behind most other econiches. In this study, we analyse the AcaML1 virus occurrence in the taxon, its genetic composition and infective behaviour under standard acidic and SOS-inducing conditions to assess its integrity and functionality. Occurrence analysis in sequenced acidithiobacilli showed that AcaML1-like proviruses are confined to the mesothermophiles Acidithiobacillus caldus and Thermithiobacillus tepidarius. Among A. caldus strains and isolates this provirus had a modest prevalence (30%). Comparative genomic analysis revealed a significant conservation with the T. tepidarius AcaML1-like provirus, excepting the tail genes, and a high conservation of the virus across strains of the A. caldus species. Such conservation extends from the modules architecture to the gene level, suggesting that organization and composition of these viruses are preserved for functional reasons. Accordingly, the AcaML1 proviruses were demonstrated to excise from their host genomes under DNA-damaging conditions triggering the SOS-response and to produce DNA-containing VLPs. Despite this fact, under the conditions evaluated (acidic) the VLPs obtained from A. caldus ATCC 51756 could not produce productive infections of a candidate sensitive strain (#6) nor trigger it lysis.
Assuntos
Acidithiobacillus/virologia , Bacteriófagos/fisiologia , Provírus/fisiologia , Acidithiobacillus/genética , Acidithiobacillus/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Provírus/genética , Provírus/isolamento & purificação , Proteínas Virais/genética , Proteínas Virais/metabolismo , Integração ViralRESUMO
The bone marrow (BM) biology during HTLV-1 infection is obscure. In this study, we investigated BM mononuclear cells and mesenchymal stromal cells (MSC) from HTLV-1 asymptomatic and symptomatic individuals. An infiltration of CD4+ T-cell lymphocytes in the BM of HTLV-1-infected individuals was observed when compared to healthy controls. The provirus detection in the BM CD4+ T cells confirmed the presence of integrated HTLV DNA. In regard to MSC, we observed that the number of fibroblast progenitor cells was lower in HTLV-1 infected individuals than in healthy controls. Isolated HTLV-1 infected BM-MSC demonstrated surface expression markers and in vitro differentiation potential similar to uninfected individuals. The presence of HTLV-1 proviral DNA in the BM-MSC of HTLV-1-infected patients was demonstrated but no p19 antigen was detected in supernatant from cultured MSC. We suppose that HTLV-1 infects human MSC probably by cell-to-cell contact from the infected CD4+ T-lymphocytes infiltrated into the bone marrow.
Assuntos
Células da Medula Óssea/virologia , DNA Viral/isolamento & purificação , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/isolamento & purificação , Células-Tronco Mesenquimais/virologia , Provírus/isolamento & purificação , Idoso , Infecções Assintomáticas , Linfócitos T CD4-Positivos/virologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Meios de Cultura , DNA Viral/genética , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/ultraestrutura , Pessoa de Meia-Idade , Provírus/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/análiseRESUMO
BACKGROUND: Infection with Human T cell Leukemia Virus type 1 can be associated with myelopathy/tropical spastic paraparesis (HAM/TSP) and other inflammatory diseases. Lymphocytes from about half of Human T cell Leukemia Virus type 1-infected subjects spontaneously proliferate in vitro, and how this phenomenon relates to symptomatic disease and viral burden is poorly understood. OBJECTIVE: To evaluate T-cell proliferation in vitro among patients co-infected with Human T cell Leukemia Virus type 1/Hepatitis C Virus/Human Immunodeficiency Virus type 1. MATERIAL AND METHODS: From 610 Human T cell Leukemia Virus-infected patients of the Human T cell Leukemia Virus outpatient clinic from Institute of Infectious Diseases "Emilio Ribas" in São Paulo, 273 agreed to participate: 72 had HAM/TSP (excluded from this analysis) and 201 were asymptomatic, a classification performed during a regular neurological appointment. We selected the subgroup made up only by the 201 asymptomatic subjects to avoid bias by the clinical status as a confounder effect, who had laboratory results of Human T cell Leukemia Virus type 1 proviral load and T-cell proliferation assay in our database. They were further grouped according to their serological status in four categories: 121 Human T cell Leukemia Virus type 1 asymptomatic mono-infected carriers; 32 Human T cell Leukemia Virus type 1/Hepatitis C Virus, 29 Human T cell Leukemia Virus type 1/Human Immunodeficiency Virus type 1, and 19 Human T cell Leukemia Virus type 1/Human Immunodeficiency Virus type 1/Hepatitis C Virus co-infected patients. Clinical data were obtained from medical records and interviews. DNA Human T cell Leukemia Virus type 1 proviral load (PVL) and T-cell proliferation (LPA) assay were performed for all samples. RESULTS: From a total of 273 subjects with Human T cell Leukemia Virus type 1, 80 presented co-infections: 29 had Human Immunodeficiency Virus type 1, 32 had Hepatitis C Virus, and 19 had Human Immunodeficiency Virus type 1 and Hepatitis C Virus. Comparing the groups based on their serological status, independently of being asymptomatic carriers, we observed a significant increase of PVL (p<0.001) and LPA (p=0.001). However, when groups were stratified according to their clinical and serological status, there was no significant increase in Human T cell Leukemia Virus type 1 PVL and LPA. CONCLUSION: No significant increase of basal T-cell proliferation among Human T cell Leukemia Virus type 1 co-infected was observed. This interaction may be implicated in liver damage, worsening the prognosis of co-infected patients or, on the contrary, inducing a higher spontaneous clearance of Hepatitis C Virus infection in Human T cell Leukemia Virus type 1 co-infected patients.
Assuntos
Infecções Assintomáticas , Proliferação de Células/fisiologia , Coinfecção/virologia , Infecções por HIV/complicações , Infecções por HTLV-I/virologia , Hepatite C/complicações , Linfócitos T/citologia , Adolescente , Adulto , Brasil/epidemiologia , Portador Sadio , Coinfecção/epidemiologia , DNA Viral/análise , DNA Viral/genética , Feminino , Infecções por HIV/epidemiologia , Infecções por HIV/imunologia , HIV-1/isolamento & purificação , Infecções por HTLV-I/imunologia , Hepatite C/epidemiologia , Hepatite C/imunologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Masculino , Pessoa de Meia-Idade , Paraparesia Espástica Tropical , Provírus/isolamento & purificação , Fatores Sexuais , Carga Viral , Adulto JovemRESUMO
Increased access to highly active antiretroviral therapy (HAART) by human immunodeficiency virus postive (HIVâº) individuals has become a reality worldwide. In Brazil, HAART currently reaches over half of HIV-infected subjects. In the context of a remarkable HIV-1 genetic variability, highly related variants, called quasispecies, are generated. HIV quasispecies generated during infection can influence virus persistence and pathogenicity, representing a challenge to treatment. However, the clinical relevance of minority quasispecies is still uncertain. In this study, we have determined the archived proviral sequences, viral subtype and drug resistance mutations from a cohort of HIV⺠patients with undetectable viral load undergoing HAART as first-line therapy using next-generation sequencing for near full-length virus genome (NFLG) assembly. HIV-1 consensus sequences representing NFLG were obtained for eleven patients, while for another twelve varying genome coverage rates were obtained. Phylogenetic analysis showed the predominance of subtype B (83%; 19/23). Considering the minority variants, 18 patients carried archived virus harboring at least one mutation conferring antiretroviral resistance; for six patients, the mutations correlated with the current ARVs used. These data highlight the importance of monitoring HIV minority drug resistant variants and their clinical impact, to guide future regimen switches and improve HIV treatment success.
Assuntos
Antirretrovirais/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/classificação , Provírus/classificação , Quase-Espécies , Brasil , Genoma Viral , HIV-1/genética , HIV-1/isolamento & purificação , Humanos , Provírus/genética , Provírus/isolamento & purificação , Análise de Sequência de DNA , Carga Viral , Sequenciamento Completo do GenomaRESUMO
The lifetime risk of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) development differs among ethnic groups. To better understand these differences, this prospective cohort study was conducted to investigate the cytokine profile and the HTLV-1 proviral load (PVL) in Japanese and non-Japanese populations with HAM/TSP and asymptomatic carriers (ACs). The serum IL-2, IL-4, IL-6, IL-10, IL-17, TNF-α, and IFN-γ levels were quantified using the Cytometric Bead Array in 40 HTLV-1-infected patients (11 HAM/TSP and 29 ACs) and 18 healthy controls (HCs) in Brazil. Among ACs, 15 were Japanese descendants and 14 were non-Japanese. Of 11 patients with HAM/TSP, only one was a Japanese descendant. The HTLV-1 PVL was quantified by real-time PCR. The HTLV-1 PVL was 2.7-fold higher in HAM/TSP patients than ACs. Regardless of the clinical outcome, the PVL was significantly higher in patients younger than 60 years than older patients. The HAM/TSP and ACs had higher IL-10 serum concentrations than that of HCs. The ACs also showed higher IL-6 serum levels than those of HCs. According to age, the IL-10 and IL-6 levels were higher in ACs non-Japanese patients older than 60 years. HAM/TSP patients showed a positive correlation between IL-6 and IL-17 and a negative correlation between the PVL and IL-17 and IFN-γ. In the all ACs, a significant positive correlation was observed between IL-2 and IL-17 and a negative correlation was detected between IL-10 and TNF-α. Only 6.25% of the Japanese patients were symptomatic carriers, compared with 41.67% of the non-Japanese patients. In conclusion, this study showed that high levels of HTLV-1 PVL was intrinsicaly associated with the development of HAM/TSP. A higher HTLV-1 PVL and IL10 levels found in non-Japanese ACs over 60 years old, which compared with the Japanese group depicts that the ethnic background may interfere in the host immune status. More researches also need to be undertaken regarding the host genetic background to better understand the low frequency of HAM/TSP in Japanese HTLV-1-infected individuals.
Assuntos
Citocinas/sangue , Infecções por HTLV-I/imunologia , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano , Idoso , Povo Asiático , Brasil/epidemiologia , Portador Sadio/epidemiologia , Portador Sadio/imunologia , Portador Sadio/virologia , Estudos de Casos e Controles , Estudos de Coortes , Emigrantes e Imigrantes , Feminino , Infecções por HTLV-I/epidemiologia , Vírus Linfotrópico T Tipo 1 Humano/isolamento & purificação , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Humanos , Japão/etnologia , Masculino , Pessoa de Meia-Idade , Paraparesia Espástica Tropical/epidemiologia , Paraparesia Espástica Tropical/imunologia , Paraparesia Espástica Tropical/virologia , Estudos Prospectivos , Provírus/isolamento & purificação , Carga ViralRESUMO
A cross-sectional study was carried out on cats attending the Small Animal Hospital at the Faculty of Veterinary Sciences of the University of Buenos Aires to assess the prevalence and associated risk factors of Feline immunodeficiency virus (FIV) and Feline leukemia virus (FeLV) in the city of Buenos Aires, Argentina. Blood samples from 255 cats with symptoms compatible with FIV or FeLV infection, collected between 2009 and 2013 were analyzed by serology (immunochromatography, IA) and by hemi-nested PCR (n-PCR). The IA and n-PCR assays showed similar percentages of positivity for FIV while the n-PCR test was more sensitive for FeLV. Differences between the diagnostic tests and their choice according to the age of the animal are discussed. The clinical histories of ninety of the 255 cats showed blood profiles similar to others previously reported and revealed a higher risk of infection in male adult cats with outdoor access.
Assuntos
Cromatografia de Afinidade/métodos , Síndrome de Imunodeficiência Adquirida Felina/diagnóstico , Vírus da Imunodeficiência Felina/isolamento & purificação , Vírus da Leucemia Felina/isolamento & purificação , Leucemia Felina/diagnóstico , Reação em Cadeia da Polimerase/métodos , Viremia/diagnóstico , Animais , Argentina/epidemiologia , Gatos/virologia , Estudos Transversais , DNA Viral/análise , Síndrome de Imunodeficiência Adquirida Felina/epidemiologia , Síndrome de Imunodeficiência Adquirida Felina/virologia , Feminino , Vírus da Imunodeficiência Felina/genética , Vírus da Imunodeficiência Felina/imunologia , Vírus da Leucemia Felina/genética , Vírus da Leucemia Felina/imunologia , Leucemia Felina/epidemiologia , Leucemia Felina/virologia , Masculino , Prevalência , Provírus/isolamento & purificação , Kit de Reagentes para Diagnóstico , Fatores de Risco , Sensibilidade e Especificidade , Viremia/epidemiologia , Viremia/virologiaRESUMO
In this work, we studied seven groups of pregnant heifers from a consortium of dairy farms heavily infected with bovine leukemia virus (BLV). ELISA testing showed that the seroprevalence ranges of BLV in heifers between 36.1 and 66.5 %. No significant differences in proviral load were found when comparing heifers with adult cattle. Before their first delivery, more than 9.8 % of heifers show a high proviral load. Because BLV infection can occur during the first two years of life, the rationale of any strategy should be to take action as early as possible after birth.
Assuntos
Anticorpos Antivirais/sangue , Leucose Enzoótica Bovina/epidemiologia , Leucose Enzoótica Bovina/virologia , Vírus da Leucemia Bovina/imunologia , Vírus da Leucemia Bovina/isolamento & purificação , Animais , Bovinos , Ensaio de Imunoadsorção Enzimática , Feminino , Gravidez , Provírus/isolamento & purificação , Estudos Soroepidemiológicos , Fatores de Tempo , Carga ViralRESUMO
Introduction: To date there has been no statistical evaluation of the profiles of immunoglobulin classes and viral replication as variables in the study of HTLV-1 infection and circulation among families in virus-endemic areas of Colombia. Objective: To evaluate the correlation of several immunological and molecular characteristics with the transmission and circulation of HTLV-1 among families in the town of Tumaco. Materials and methods: Plasma levels of HTLV-1 specific immunoglobulin classes IgG, IgM and IgA1, as well as IgG and sIgA in oral fluids, were calculated for 32 members of 10 family groups from Tumaco in which the mother and at least one child were infected with the virus. Levels of the different immunoglobulin classes were correlated with viral RNA circulating in plasma or oral fluids and the proviral burden as detected by RT-PCR. Results: Significant differences were determined between mothers and carrier children for immunoglobulin levels (p=0.037) and proviral burden (p=0.002). The overall estimate of IgG in plasma and sIgA in oral fluids could be correlated with the circulation of free viral RNA in both fluids and high proviral burden, and associated with HAM/TSP mothers. The detection of anti- tax IgG in plasma revealed differences between HAM/TSP mothers and their offspring. Conclusion: The study of immunological and molecular variables permitted the analysis of HTLV-1 circulation among families of Tumaco. The strong correlation between levels of IgM specific for the virus and viral RNA circulating in fluids indirectly confirmed the transmission of HTLV-1 among families.
Introducción. Todavía no hay una evaluación estadística de los perfiles de las clases de inmuno- globulina s y la replicación viral, como variables para estudiar la infección y la circulació n del HTLV-1 en familias de zonas endémicas en Colombia. Objetivo. Evaluar la correlación de varias características inmunológicas y moleculares, con la transmisión y circulación del virus en familias del municipio de Tumaco. Materiales y métodos. Se calcularon los niveles de IgG, IgM e IgA1 en plasma, e IgG y IgA secretoria en fluido oral, de 32 miembros de 10 grupos familiares de Tumaco, en los que la madre y, al menos, un hijo estaban infectados con el virus. La concentración de las diferentes clases de inmunoglobulinas se pudo correlacionar con la circulación de ARN viral libre en plasma y fluido oral, y la carga proviral, según su detección mediante reacción en cadena de la polimerasa de transcripción inversa. Resultados. Se encontraron diferencias significativas en los niveles de inmunoglobulinas (p=0,037) y en la carga proviral (p=0,002) entre madres e hijos portadores. La estimación total de IgG en plasma e IgA secretoria en fluido oral, se pudo correlacionar con la circulación de ARN viral libre en ambos fluidos y una alta carga proviral, y se asoció con las madres paraparesia espástica tropical o mielopatía asociada con el HTLV-1. La detección en plasma de IgG anti-Tax reveló diferencias entre ellas y sus hijos. Conclusión. El estudio de las variables inmunológicas y moleculares permitió analizar la circulación del HTLV-1 en familias de Tumaco. La fuerte asociación entre los niveles de IgM específica para el virus y el ARN viral circulante en los fluidos y la carga proviral, confirmó indirectamente la transmisión intrafamiliar del virus.
Assuntos
Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , RNA Viral/análise , Vírus Linfotrópico T Tipo 1 Humano/isolamento & purificação , Anticorpos Anti-HTLV-I/análise , Infecções por HTLV-I/epidemiologia , Saúde da Família , Viremia/imunologia , Viremia/epidemiologia , Viremia/virologia , Aleitamento Materno/efeitos adversos , RNA Viral/sangue , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Anticorpos Anti-HTLV-I/sangue , Infecções por HTLV-I/imunologia , Infecções por HTLV-I/transmissão , Infecções por HTLV-I/virologia , Estudos Soroepidemiológicos , Estudos Transversais , Provírus/isolamento & purificação , Colômbia/epidemiologia , Transmissão Vertical de Doenças Infecciosas , Doenças Endêmicas , MãesRESUMO
Bovine Leukemia Virus (BLV) is endemic in Argentina, where the individual prevalence is higher than 80% in dairy farms. The aim of this work was to find preliminary evidence to know if the high level of infection of the dam would implicate a higher challenge to her own offspring. We collected 65 sets of samples consisting of dam's blood and colostrum from two heavily infected dairy farms, and investigated the correlation between the dam's blood proviral load and the presence of provirus in colostrum. We also described the dual antibody/provirus profile in the colostrum. Provirus was detected in 69.23% of the colostrum samples, mostly from dams with a high proviral load, 36/45 (80%). Colostrum proviral load was significantly higher in dams with high blood proviral load (p<0.0001). Provirus was detected in colostrum samples all along the antibody distribution, even in those with a low amount of antibodies. These results show that even when high blood proviral load dams offer higher levels of infected cells to their offspring through colostrum they also offer higher levels of protection of antibodies. On the contrary, low blood proviral load dams also offer infected cells but a poor content of antibodies, suggesting that these animals could play an important role in the epidemiological cycle of transmission.
Assuntos
Anticorpos Antivirais/análise , Colostro/virologia , Leucose Enzoótica Bovina/epidemiologia , Vírus da Leucemia Bovina/isolamento & purificação , Provírus/isolamento & purificação , Animais , Anticorpos Antivirais/sangue , Argentina/epidemiologia , Bovinos , Colostro/imunologia , Leucose Enzoótica Bovina/imunologia , Leucose Enzoótica Bovina/transmissão , Feminino , Vírus da Leucemia Bovina/imunologia , Gravidez , Prevalência , Provírus/imunologia , Carga ViralRESUMO
INTRODUCTION: To date there has been no statistical evaluation of the profiles of immunoglobulin classes and viral replication as variables in the study of HTLV-1 infection and circulation among families in virus-endemic areas of Colombia. OBJECTIVE: To evaluate the correlation of several immunological and molecular characteristics with the transmission and circulation of HTLV-1 among families in the town of Tumaco. MATERIALS AND METHODS: Plasma levels of HTLV-1 specific immunoglobulin classes IgG, IgM and IgA1, as well as IgG and sIgA in oral fluids, were calculated for 32 members of 10 family groups from Tumaco in which the mother and at least one child were infected with the virus. Levels of the different immunoglobulin classes were correlated with viral RNA circulating in plasma or oral fluids and the proviral burden as detected by RT-PCR. RESULTS: Significant differences were determined between mothers and carrier children for immunoglobulin levels (p=0.037) and proviral burden (p=0.002). The overall estimate of IgG in plasma and sIgA in oral fluids could be correlated with the circulation of free viral RNA in both fluids and high proviral burden, and associated with HAM/TSP mothers. The detection of anti- tax IgG in plasma revealed differences between HAM/TSP mothers and their offspring. CONCLUSION: The study of immunological and molecular variables permitted the analysis of HTLV-1 circulation among families of Tumaco. The strong correlation between levels of IgM specific for the virus and viral RNA circulating in fluids indirectly confirmed the transmission of HTLV-1 among families.
Assuntos
Saúde da Família , Anticorpos Anti-HTLV-I/análise , Infecções por HTLV-I/epidemiologia , Vírus Linfotrópico T Tipo 1 Humano/isolamento & purificação , RNA Viral/análise , Adolescente , Adulto , Aleitamento Materno/efeitos adversos , Criança , Pré-Escolar , Colômbia/epidemiologia , Estudos Transversais , Doenças Endêmicas , Feminino , Anticorpos Anti-HTLV-I/sangue , Infecções por HTLV-I/imunologia , Infecções por HTLV-I/transmissão , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Humanos , Transmissão Vertical de Doenças Infecciosas , Masculino , Pessoa de Meia-Idade , Mães , Provírus/isolamento & purificação , RNA Viral/sangue , Estudos Soroepidemiológicos , Viremia/epidemiologia , Viremia/imunologia , Viremia/virologia , Adulto JovemRESUMO
A quantitative real-time PCR (qPCR) assay using SYBR Green dye was established in order to detect and quantify the proviral DNA of HTLV-1 in peripheral blood mononuclear cells (PBMCs). Primers were designed, and the assay was standardized to amplify a novel, conserved HTLV-1 tax region. Proviral load was normalized to the amount of cellular DNA by quantitation of the human albumin gene. Firstly, the qPCR was assessed determining the specificity, sensitivity, dynamic range and intra- and inter-assay reproducibility of the technique. The limit of detection as determined by PROBIT analysis using dilutions of the standard was 2.97 copies. The assay had an excellent dynamic range from 105 to 10¹ copies per reaction and good intra- and inter-assay reproducibility, CVs less than 2%. Secondly, the performance of the qPCR was tested on 40 HTLV-1 seropositive individuals. Proviral load for HTLV-1 carriers ranged from 2.2×10² to more than 8.3×104 copies/106 PBMCs. The high sensitivity and wide dynamic range allowed the determination of a broad range of HTLV-1 proviral loads in infected individuals. This assay is a valuable alternative diagnostic tool when current available serological assays are insufficient. In addition, it will facilitate the study of the relationship between proviral load and pathogenesis.
Assuntos
Genes pX , Infecções por HTLV-I/diagnóstico , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/isolamento & purificação , Provírus/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Carga Viral/métodos , Primers do DNA/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Provírus/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Carga Viral/normasRESUMO
BACKGROUND: The Human T-cell Leukemia Virus type 1 (HTLV-1) is the causative agent of several inflammatory diseases, including HTLV-1-associated inflammatory myopathies (HAIM). Little is known about the virological and immunological characteristics of this viral disease. OBJECTIVES: To characterize the histological and virological features of HAIM patients, in order to better understand the pathogenetic mechanisms and unravel new biological markers of this disease. STUDY DESIGN: We conducted a retrospective study on 13 patients with HAIM, based on blood and muscle samples. We included blood samples from HTLV-1-infected individuals without myopathy as controls. Muscle biopsies were used for a broad immunohistological evaluation of tissue damage and inflammation, as well as identification of infected cells through in situ hybridization. DNA extracted from patients' PBMC was used to identify the virus genotype by sequencing and to assess the proviral load by quantitative PCR. Anti-viral antibodies in plasma samples were titrated by indirect immunofluorescence. RESULTS: Patients originate from HTLV-1 endemic areas, the West Indies and West Africa. Histological alterations and inflammation in patients muscles were mostly moderate, with classical features of idiopathic myositis and rare HTLV-1-infected infiltrating cells. In all patients, HTLV-1 belonged to the A subtype, transcontinental subgroup. Anti-HTLV-1 antibodies titers were high, but the proviral load was not elevated compared to asymptomatic HTLV-1 carriers. CONCLUSION: We show here that muscle inflammation is moderate in HAIM, and accompanied by a low HTLV-1 proviral load, suggesting that the pathogenetic events do not exactly mirror those of other HTLV-1-associated inflammatory diseases.
Assuntos
Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/isolamento & purificação , Inflamação/virologia , Miosite/virologia , Adulto , África Ocidental , Idoso , Idoso de 80 Anos ou mais , Feminino , Vírus Linfotrópico T Tipo 1 Humano/classificação , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia , Provírus/isolamento & purificação , RNA Mensageiro/análise , RNA Viral/análise , Estudos Retrospectivos , Estatísticas não Paramétricas , Carga Viral , Índias OcidentaisRESUMO
This study compared the proviral load and the plasma cytokine profiles (interleukin-IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ) in 87 HTLV-1-infected individuals, including 28 with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), 32 with possible pHAM/TSP and 27 asymptomatic carriers (AC). The control group was composed by 21 HTLV-1-seronegative individuals. Our finding demonstrated that HAM/TSP group presented higher proviral load as compared to all other HTLV-1 groups (p<0.0001). The HAM/TSP group showed higher serum concentration of IL-6 (p=0.0009) as compared to all other groups. Moreover, higher serum concentration of IFN-γ (p=0.0118) and IL-4 (p=0.0166) were observed in HAM/TSP group as compared to the healthy controls. Additionally, the HAM/TSP group also showed higher serum concentration of TNF-α (p=0.0239) and IFN-γ (p=0.0118) as compared to AC. No differences in the serum concentration of IL-2 and IL-10 were observed among the groups. The analysis of cytokine balance demonstrated that HAM/TSP presented higher pro-inflammatory profile with enhanced IFN-γ/IL-10 and IFN-γ/IL-4 ratio as compared to AC and pHAM/TSP. Further analysis pointed out to a positive correlation between the IFN-γ response and the proviral load in AC. Conversely, a negative association between TNF-α and IL-2 with the proviral load was the hallmark of HAM/TSP group. These findings suggested that the proviral load and the pro-inflammatory cytokine profile may be independent events in the peripheral blood of HAM/TSP individuals. The knowledge about the existence of individual virological/immunological behavior upon HTLV-1 infection, may guide to the establishment of more effective therapeutic interventions.
Assuntos
Citocinas/sangue , Infecções por HTLV-I/imunologia , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/isolamento & purificação , Provírus/isolamento & purificação , Carga Viral , Adulto , Idoso , Infecções Assintomáticas , Feminino , Infecções por HTLV-I/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Soro/química , Soro/virologia , Adulto JovemRESUMO
Bovine leukemia virus (BLV)-infected cattle were classified by their proviral load into low and high proviral load profiles (LPL and HPL, respectively). Blood from these animals was used to infect sheep to obtain multiple identical copies of integrated provirus. An env fragment of BLV was amplified from all infected sheep and sequenced. The sequences that were obtained were compared to already published BLV genome sequence, resulting in three clusters. Mutations could not be attributed to the passage of provirus from cattle to sheep and subsequent amplification and sequencing. The description of two different proviral load profiles, the association of the BoLA-DRB3.2 0902 allele with the LPL profile, the availability of complete BLV sequences, and the comparison of a variable region of the env gene from carefully characterized cattle are still not enough to explain the presence of animals in every herd that are resistant to BLV dissemination.