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Feline leukemia virus (FeLV) is a retrovirus distributed worldwide in domestic cats and with different outcomes (progressive, regressive, abortive, focal). The present study reports an epidemiological survey of FeLV frequency and the evaluation of some risk factors and the two main disease outcomes (progressive and regressive) in an urban cat population from Brazil. A total of 366 cats with sociodemographic information and p27 FeLV antigen test performed were included in the study. FeLV DNA (provirus) in the blood samples of all cats was detected via real-time polymerase chain reaction (qPCR). Plasma samples from 109 FeLV-positive and FeLV-negative cats were also submitted to reverse transcription (RT-qPCR) to determine the FeLV viral load. The results demonstrated that 112 (30.6%) cats were positive through the p27 antigen and/or qPCR. A risk factor analysis demonstrated that cats without vaccination against FeLV (OR 9.9, p < 0.001), clinically ill (OR 2.9, p < 0.001), with outdoors access (OR 2.7, p < 0.001), and exhibiting apathetic behavior (OR 3.1, p < 0.001) were more likely to be infected with FeLV. FeLV-infected cats were also more likely to present with anemia (OR 13, p < 0.001) and lymphoma (OR 13.7, p = 0.001). A comparative analysis of the different detection methods in a subset of 109 animals confirmed FeLV infection in 58 cats, including 38 (65.5%) with progressive, 16 (27.6%) with regressive, and 4 (6.9%) with probably focal outcome diseases. In conclusion, this study demonstrates a high prevalence of FeLV in this urban cat population from Brazil and highlights the need to establish more effective prevention strategies (such as viral testing, vaccination programs, specific care for FeLV-positive cats) to reduce diseases associated with this virus in Brazil.
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Bovines infected by bovine leukemia virus (BLV) are characterized by presenting low proviral load (LPL) or high proviral load (HPL). It is reported that animals with HPL in peripheral blood mononuclear cells (PBMCs) present a decrease in apoptosis, an increase in viability and the proliferation rate, while animals that maintain an LPL have an intrinsic ability to control the infection, presenting an increased apoptosis rate of their PBMCs. However, there is little information on the effect of BLV on these mechanisms when the virus infects somatic milk cells (SC). This study investigates the mechanisms underlying apoptosis in milk and blood from BLV-infected animals with HPL and LPL. Relative levels of mRNA of tumor necrosis factor-α (TNF-α), TNF receptor 1 (TNF-RI), TNF receptor 2 (TNF-RII), anti-apoptotic B-cell lymphoma 2 protein (Bcl-2), and pro-apoptotic Bcl-2-like protein 4 (Bax) were measured in SC and PBMCs using quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay. A significant decrease in the expression of TNF-α in SC from HPL animals vs non-infected bovines was observed, but the infection in SC with BLV did not show a modulation on the expression of TNF receptors. A significant increase in TNF-RI expression in PBMCs from HPL bovines compared to LPL bovines was observed. No significant differences in PBMCs between HPL and LPL compared to non-infected animals concerning TNF-α, TNF-RI, and TNF-RII expression were found. There was a significant increase of both Bcl-2 and Bax in SC from LPL compared to non-infected bovines, but the Bcl-2/Bax ratio showed an anti-apoptotic profile in LPL and HPL bovines compared to non-infected ones. Reduced mRNA expression levels of Bax were determined in the PBMCs from HPL compared to LPL subjects. In contrast, BLV-infected bovines did not differ significantly in the mRNA expression of Bax compared to non-infected bovines. Our data suggest that the increased mRNA expression of Bax corresponds to the late lactation state of bovine evaluated and the exacerbated increase of mRNA expression of Bcl-2 may be one of the mechanisms for the negative apoptosis regulation in the mammary gland induced by BLV infection. These results provide new insights into the mechanism of mammary cell death in HPL and LPL BLV-infected bovine mammary gland cells during lactation.
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Doenças dos Bovinos , Leucose Enzoótica Bovina , Vírus da Leucemia Bovina , Animais , Bovinos , Feminino , Apoptose , Proteína X Associada a bcl-2/metabolismo , Proliferação de Células , Leucócitos Mononucleares/metabolismo , Leite , Provírus/genética , Provírus/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: The prevalence of human T-lymphotropic virus type-1 (HTLV-1) infection is higher in women, and sexual intercourse has been described as an important route of male-to-female transmission. The present study aimed to quantify HTLV-1 proviral load (PVL) in vaginal fluid, and to investigate correlations with PVL in peripheral blood mononuclear cells (PBMCs). In addition, cytopathological alterations and vaginal microbiota were evaluated. METHODS: HTLV-1-infected women were consecutively recruited at a multidisciplinary center for HTLV patients in Salvador, Brazil. All women underwent gynecological examinations to obtain cervicovaginal fluid and venipuncture for blood collection. PVL, as measured by real-time quantitative polymerase chain reaction (RT-qPCR), was expressed as the number of copies of HTLV-1/106 cells in blood and vaginal fluid samples. Light microscopy was used to assess cervicovaginal cytopathology and vaginal microbiota. RESULTS: In the 56 included women (43 asymptomatic carriers and 13 diagnosed with HTLV-1-associated myelopathy/tropical spastic paraparesis-HAM/TSP), mean age was 35.9 (SD ± 7.2) years. PVL was higher in PBMCs (median: 23,264 copies/106 cells; IQR: 6776-60,036) than in vaginal fluid (451.9 copies/106 cells; IQR: 0-2490) (p < 0.0001). PVL in PBMCs was observed to correlate directly with PVL in vaginal fluid (R = 0.37, p = 0.006). PVL was detected in the vaginal fluid of 24 of 43 (55.8%) asymptomatic women compared to 12 of 13 (92.3%) HAM/TSP patients, p = 0.02. Cytopathologic analyses revealed no differences between women with detectable or undetectable PVL. CONCLUSION: HTLV-1 proviral load is detectable in vaginal fluid and correlates directly with proviral load in peripheral blood. This finding suggests that sexual transmission of HTLV-1 from females to males may occur, as well as vertical transmission, particularly in the context of vaginal delivery.
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Genetic variations in components of the immune response seem to be an important factor that contributes to the manifestation of symptoms of some diseases related to HTLV-1 infection. Nerve growth factor (NGF) and the p75 neurotrophin receptor (p75NTR) are related to the maintenance of neurons and the activation of the immune response. In this study, we evaluated the association of the NGF -198C/T, NGF Ala35Val, and p75NTR Ser205Leu polymorphisms with HTLV-1 infection and plasma cytokine levels in 166 samples from individuals infected with HTLV-1 (59 symptomatic and 107 asymptomatic). The genotyping and quantification of the proviral load were performed by real-time PCR, and cytokine levels were measured by ELISA. The NGF -198C/T and NGF Ala35Val polymorphisms were not associated with HTLV-1 infection. The frequency of the Ser/Leu genotype of p75NTR Ser205Leu was more frequent in the control group (p = 0.0385), and the Ser/Leu genotype and allele Leu were more frequent among the asymptomatic (p < 0.05), especially with respect to the HTLV-1-associated myelopathy (HAM) group (p < 0.05). The symptomatic showed a higher proviral load and higher TNF-α and IL-10 levels (p < 0.05). Asymptomatic carriers of the Ser/Leu genotype (p = 0.0797) had lower levels of proviral load and higher levels of TNF-α (p = 0.0507). Based on the results obtained, we conclude that the p75NTR Ser205Leu polymorphism may be associated with reduced susceptibility to HTLV-1 infection, a lower risk of developing symptoms, including HAM, and better infection control.
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Infecções por HTLV-I , Vírus Linfotrópico T Tipo 1 Humano , Paraparesia Espástica Tropical , Citocinas , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Fator de Crescimento Neural , Provírus/genética , Receptor de Fator de Crescimento Neural , Fator de Necrose Tumoral alfa , Carga ViralRESUMO
Droplet digital PCR (ddPCR) is a highly sensitive tool developed for the detection and quantification of short-sequence variants-a tool that offers unparalleled precision enabling measurement of smaller-fold changes. We describe here the use of ddPCR for the detection of Bovine leukemia virus (BLV) DNA provirus. Serum samples and whole blood from experimentally infected sheep and naturally infected cattle were analyzed through ddPCR to detect the BLV gp51 gene, and then compared with serologic and molecular tests. The ddPCR assay was significantly more accurate and sensitive than AGID, ELISA, nested PCR, and quantitative PCR. The limit of detection of ddPCR was 3.3 copies/µL, detecting positive experimentally infected sheep beginning at 6 d post-infection. The ddPCR methodology offers a promising tool for evaluating the BLV proviral load, particularly for the detection of low viral loads.
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Doenças dos Bovinos , Leucose Enzoótica Bovina , Vírus da Leucemia Bovina , Doenças dos Ovinos , Animais , Bovinos , Leucose Enzoótica Bovina/diagnóstico , Ensaio de Imunoadsorção Enzimática/veterinária , Vírus da Leucemia Bovina/genética , Provírus/genética , Reação em Cadeia da Polimerase em Tempo Real/veterinária , OvinosRESUMO
Proviral load (PVL) is one of the determining factors for the pathogenesis and clinical progression of the human T-lymphotropic virus type I (HTLV-1) infection. In the present study, we optimized a sensitive multiplex real-time PCR for the simultaneous detection and quantification of HTLV-1 proviral load and beta-globin gene as endogenous control. The values obtained for HTLV-1 PVL were used to monitor the clinical evolution in HTLV-1-infected individuals. A vector containing cloned DNA targets of the real-time PCR for the beta-globin gene and the HTLV-1pol region was constructed. For the reaction validation, we compared the amplification efficiency of the constructed vector and MT-2 cell line containing HTLV-1. The analytical sensitivity of the reaction was evaluated by the application of a standard curve with a high order of magnitude. PVL assay was evaluated on DNA samples of HTLV-1 seropositive individuals. The construct showed adequate amplification for the beta-globin and HTLV-1 pol genes when evaluated as multiplex real-time PCR (slope = 3.23/3.26, Y-intercept = 40.18/40.73, correlation coefficient r2 = 0.99/0.99, and efficiency = 103.98/102.78, respectively). The quantification of PVL using the MT-2 cell line was equivalent to the data obtained using the plasmidial curve (2.5 copies per cell). In HTLV-1-associatedmyelopathy/tropical spastic paraparesis patients, PVL was significantly higher (21315 ± 2154 copies/105 PBMC) compared to asymptomatic individuals (1253 ± 691 copies/105 PBMC). The obtained results indicate that the optimized HTLV-1 PVL assay using plasmidial curve can be applied for monitoring and follow-up of the progression of HTLV-1 disease. The use of a unique reference plasmid for both HTLV-1 and endogenous gene allows a robust and effective quantification of HTLV-1 PVL. In addition, the developed multiplex real-time PCR assay was efficient to be used as a tool to monitor HTLV-1-infected individuals.
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Infecções por HTLV-I , Vírus Linfotrópico T Tipo 1 Humano , Paraparesia Espástica Tropical , DNA Viral/análise , DNA Viral/genética , Infecções por HTLV-I/diagnóstico , Infecções por HTLV-I/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Leucócitos Mononucleares , Paraparesia Espástica Tropical/diagnóstico , Paraparesia Espástica Tropical/genética , Provírus/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Carga Viral/métodos , Globinas beta/análise , Globinas beta/genéticaRESUMO
Human T-lymphotropic virus type 1 (HTLV-1) provirus expression is mainly directed by Tax-responsive elements (TRE) within the long terminal repeats (LTR). Mutations in TRE can reduce provirus expression and since a high proviral load (PVL) is a risk factor for the development of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), we evaluated polymorphisms in the 5' LTR and the association with PVL and disease progression. HTLV-1 LTR and tax sequences derived from asymptomatic carriers (AC) and HAM/TSP patients followed in a longitudinal study were analysed according to PVL and clinical severity. Individuals infected with HTLV-1 presenting the canonical TRE, considering strain ATK-1 as the consensus, displayed sustained higher PVL. By contrast, an LTR A125G mutation in TRE was associated with slightly reduced PVL only in HAM/TSP patients, although it did not influence the speed of disease progression. Moreover, this polymorphism was frequent in Latin American strains of the HTLV-1 Cosmopolitan Transcontinental subtype. Therefore, polymorphisms in the 5' TRE of HTLV-1 may represent one of the factors influencing PVL in HAM/TSP patients, especially in the Latin American population. Indeed, higher PVL in the peripheral blood has been associated with an increased inflammatory activity in the spinal cord and to a poorer prognosis in HAM/TSP. However, this event was not associated with TRE polymorphisms.
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Produtos do Gene tax , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Paraparesia Espástica Tropical/virologia , Polimorfismo Genético , Sequências Repetidas Terminais , Carga Viral , Idoso , Doenças Assintomáticas , Portador Sadio/virologia , Progressão da Doença , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Mutação , Filogenia , Provírus/genética , Provírus/fisiologiaRESUMO
Bovine leukemia virus (BLV) main host cells are B lymphocytes. Infected animals can be classified into high or low proviral load (HPL or LPL respectively), regarding the number of proviral copies infected lymphocytes they carry. After infection, there is an overexpression of several cytokines, particularly TNF-α, which has a delicate regulation mediated by receptors TNFRI and TNFRII; the first one involved with apoptosis, while the other stimulates cell proliferation. The study aimed to quantify TNF-α and its receptors mRNA expression, and in which extent in vitro proliferation was affected, in peripheral blood mononuclear cells (PBMC) from BLV-infected animals with different proviral loads, after the addition or not of synthetic TNF-α (rTNF-α) for 48 h. PBMC from BLV-infected animals showed spontaneous proliferation after 48 h in culture but did not show changes in proliferation rates after 48 h incubation in the presence of the rTNF-α. TNF-α mRNA expression after 48 h culture without exogenous stimulation was significantly lower, regardless of the proviral load of the donor, compared to non-infected animals. In the LPL animals, the expression of TNF-α mRNA was significantly lower with respect to the control group while the expression of TNFRI mRNA was significantly increased. The HPL animals showed a significant decrease in the expression of TNF-α and TNFRII mRNA respect to the control group. After 48 h incubation with rTNF-α, PBMC from infected animals had different responses: TNF-α and TNFRI mRNA expression was reduced in PBMC from the LPL group compared to the BLV negative group, but no differences were observed in PBMC from the HPL group. TNFRII mRNA expression showed no differences between HPL, LPL, and BLV negative groups, though HPL animals expressed 10.35 times more TNFRI mRNA than LPL. These results support the hypothesis that LPL animals, when faced with viral reactivation, present a pro-apoptotic and anti-proliferative state. However, complementary studies are needed to explain the influence of TNFRII on the development of the HLP profile. On the other hand, exogenous stimulation studies reinforce the hypothesis that BLV infection compromises the immune response of the animals.
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Leucose Enzoótica Bovina/imunologia , Vírus da Leucemia Bovina/fisiologia , Receptores Tipo II do Fator de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Fator de Necrose Tumoral alfa/genética , Carga Viral , Animais , Bovinos , Proliferação de Células , Citocinas/imunologia , Leucose Enzoótica Bovina/virologia , Expressão Gênica , Sistema Imunitário , Leucócitos Mononucleares/virologia , RNA Mensageiro/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
In dairy cattle infected with bovine leukemia virus (BLV), the proviral load (PVL) level is directly related to the viral transmission from infected animals to their healthy herdmates. Two contrasting phenotypic groups can be identified when assessing PVL in peripheral blood of infected cows. A large number of reports point to bovine genetic variants (single nucleotide polymorphisms) as one of the key determinants underlying PVL level. However, biological mechanisms driving BLV PVL profiles and infection progression in cattle have not yet been elucidated. In this study, we evaluated whether a set of candidate genes affecting BLV PVL level according to whole genome association studies are differentially expressed in peripheral blood mononuclear cells derived from phenotypically contrasting groups of BLV-infected cows. During a 10-mo-long sampling scheme, 129 Holstein cows were phenotyped measuring anti-BLV antibody levels, PVL quantification, and white blood cell subpopulation counts. Finally, the expression of 8 genes (BOLA-DRB3, PRRC2A, ABT1, TNF, BAG6, BOLA-A, LY6G5B, and IER3) located within the bovine major histocompatibility complex region harboring whole genome association SNP hits was evaluated in 2 phenotypic groups: high PVL (n = 7) and low PVL (n = 8). The log2 initial fluorescence value (N0) transformed mean expression values for the ABT1 transcription factor were statistically different in high- and low-PVL groups, showing a higher expression of the ABT1 gene in low-PVL cows. The PRRC2A and IER3 genes had a significant positive (correlation coefficient = 0.61) and negative (correlation coefficient = -0.45) correlation with the lymphocyte counts, respectively. Additionally, the relationships between gene expression values and lymphocyte counts were modeled using linear regressions. Lymphocyte levels in infected cows were better explained (coefficient of determination = 0.56) when fitted a multiple linear regression model using both PRRC2A and IER3 expression values as independent variables. The present study showed evidence of differential gene expression between contrasting BLV infection phenotypes. These genes have not been previously related to BLV pathobiology. This valuable information represents a step forward in understanding the BLV biology and the immune response of naturally infected cows under a commercial milk production system. Efforts to elucidate biological mechanisms leading to BLV infection progression in cows are valuable for BLV control programs. Further studies integrating genotypic data, global transcriptome analysis, and BLV progression phenotypes are needed to better understand the BLV-host interaction.
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Leucose Enzoótica Bovina/genética , Vírus da Leucemia Bovina/fisiologia , Polimorfismo de Nucleotídeo Único/genética , Animais , Bovinos , Leucose Enzoótica Bovina/virologia , Feminino , Estudo de Associação Genômica Ampla/veterinária , Contagem de Leucócitos/veterinária , Leucócitos/virologia , Leucócitos Mononucleares/virologia , Contagem de Linfócitos/veterinária , Fenótipo , Provírus/fisiologia , Carga Viral/veterináriaRESUMO
A high proviral load (PVL) is recognized as a risk factor for human T cell leukemia virus-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), but there is a lack of prospective studies evaluating whether or not HTLV-1 carriers with high PVL are at risk of developing HAM/TSP or other HTLV-1-related diseases. Here, we compare the incidence of clinical manifestations and the cytokine levels in 30 HTLV-1 carriers with high (> 50,000 copies/106 PBMC) and an equal number of subjects with low proviral load. Participants were followed for 3 to 16 years (median of 11 years). The PVL, IFN-γ, TNF, and IL-10 levels were quantified at entry and at the end of the follow-up. Among the self-reported symptoms in the initial evaluation, only the presence of paresthesia on the hands was more frequent in the group with high PVL (p < 0.04). The production of IFN-γ was higher in the group with high PVL group (median of 1308 versus 686 pg/ml, p < 0.011) when compared with the control group in the first assessment. There was no difference in the occurrence of urinary symptoms or erectile dysfunction, periodontal disease, Sicca syndrome, and neurologic signs between the two groups during the follow-up. The observation that none of the HTLV-1 carriers with high PVL and with exaggerated inflammatory response progressed to HAM/TSP indicates that other factors in addition to the PVL and an exaggerated immune response are involved in the pathogenesis of HAM/TSP.
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Portador Sadio/imunologia , Infecções por HTLV-I/imunologia , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Leucócitos Mononucleares/imunologia , Provírus/imunologia , Adulto , Idoso , Portador Sadio/diagnóstico , Portador Sadio/virologia , Disfunção Erétil/diagnóstico , Disfunção Erétil/genética , Disfunção Erétil/imunologia , Disfunção Erétil/virologia , Feminino , Expressão Gênica , Infecções por HTLV-I/diagnóstico , Infecções por HTLV-I/genética , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/crescimento & desenvolvimento , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Leucócitos Mononucleares/virologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Noctúria/diagnóstico , Noctúria/genética , Noctúria/imunologia , Noctúria/virologia , Provírus/crescimento & desenvolvimento , Síndrome de Sjogren/diagnóstico , Síndrome de Sjogren/genética , Síndrome de Sjogren/imunologia , Síndrome de Sjogren/virologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Carga Viral/imunologiaRESUMO
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.
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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
Human T-lymphotropic virus type 1 (HTLV-1) deregulates the immune system and cell cycle, resulting in loss of immune tolerance and disease, including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Three prime repair exonuclease 1 (TREX1) maintains innate immune tolerance of the host and host-cell permissiveness to retroviral infections. TREX1 polymorphisms may influence the course of infection and autoimmune manifestations. The influence of TREX1 531C/T polymorphism was investigated in HTLV-1 infection and development of symptoms among 151 persons infected with HTLV-1 (32 HAM/TSP, 19 rheumatologic manifestations, two dermatitis, five more than one diagnosis, two probable HAM/TSP, and 91 asymptomatic individuals) and 100 uninfected persons in the control group. Polymorphism genotyping and proviral load quantification were performed by real-time polymerase chain reaction (PCR) and antinuclear antibodies (ANAs) were screened by an indirect immunofluorescence assay. No statistically significant difference was found in polymorphism genotype and allele frequencies between the infected and control groups. HAM/TSP patients showed higher frequency of TT genotype than asymptomatic persons (p = 0.0339). Proviral load was significantly higher among individuals with CT/TT genotypes and CC genotype carriers had lower proviral load and higher levels of proinflammatory cytokines. ANAs were present only in the HAM/TSP group. TREX1 531C>T polymorphism seems to be associated with TREX-1 regulation and HTLV-1 infection.
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Exodesoxirribonucleases/genética , Predisposição Genética para Doença , Infecções por HTLV-I/genética , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Fosfoproteínas/genética , Polimorfismo de Nucleotídeo Único , Carga Viral , Alelos , Feminino , Frequência do Gene , Estudos de Associação Genética , Genótipo , Interações Hospedeiro-Patógeno/genética , Humanos , MasculinoRESUMO
BACKGROUND: Persistence of latent, replication-competent provirus in CD4+ T cells of human immunodeficiency virus (HIV)-infected individuals on antiretroviral treatment (ART) is the main obstacle for virus eradication. Methylation of the proviral 5' long terminal repeat (LTR) promoter region has been proposed as a possible mechanism contributing to HIV latency; however, conflicting observations exist regarding its relevance. We assessed 5'-LTR methylation profiles in total CD4+ T cells from blood of 12 participants on short-term ART (30 months) followed up for 2 years, and a cross-sectional group of participants with long-term ART (6-15 years), using next generation sequencing. We then looked for associations between specific 5'-LTR methylation patterns and baseline and follow-up clinical characteristics. RESULTS: 5'-LTR methylation was observed in all participants and behaved dynamically. The number of 5'-LTR variants found per sample ranged from 1 to 13, with median sequencing depth of 16270× (IQR 4107×-46760×). An overall significant 5'-LTR methylation increase was observed at month 42 compared to month 30 (median CpG Methylation Index: 74.7% vs. 0%, p = 0.025). This methylation increase was evident in a subset of participants (methylation increase group), while the rest maintained fairly high and constant methylation (constant methylation group). Persons in the methylation increase group were younger, had higher CD4+ T cell gain, larger CD8% decrease, and larger CD4/CD8 ratio change after 48 months on ART (all p < 0.001). Using principal component analysis, the constant methylation and methylation increase groups showed low evidence of separation along time (factor 2: p = 0.04). Variance was largely explained (21%) by age, CD4+/CD8+ T cell change, and CD4+ T cell subpopulation proportions. Persons with long-term ART showed overall high methylation (median CpG Methylation Index: 78%; IQR 71-87%). No differences were observed in residual plasma viral load or proviral load comparing individuals on short-term (both at 30 or 42 months) and long-term ART. CONCLUSIONS: Our study shows evidence that HIV 5'-LTR methylation in total CD4+ T cells is dynamic along time and that it can follow different temporal patterns that are associated with a combination of baseline and follow-up clinical characteristics. These observations may account for differences observed between previous contrasting studies.
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Metilação de DNA , Infecções por HIV/tratamento farmacológico , Repetição Terminal Longa de HIV , HIV-1/fisiologia , Adulto , Fatores Etários , Terapia Antirretroviral de Alta Atividade , Linfócitos T CD4-Positivos/virologia , Estudos Transversais , Feminino , Infecções por HIV/virologia , HIV-1/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Análise de Componente Principal , Provírus/genética , Provírus/fisiologia , Análise de Sequência de RNA , Latência ViralRESUMO
INTRODUCTION: Human T-cell lymphotropic virus type 1 (HTLV-1) is sexually transmitted and causes persistent infection. This virus induces activation of the immune system and production of inflammatory cytokines. This study aimed to assess the cytokine profile and cytopathological findings in the cervicovaginal fluid of asymptomatic HTLV-1-infected women. METHODS: HTLV-1-infected and uninfected women were selected at the Centro de Atendimento ao Portador de HTLV in Salvador-Brazil. None of the included HTLV-1-infected women reported any HTLV-1-associated diseases. All volunteers underwent gynecological examination to collect cervicovaginal fluid. Cytokine quantification was performed using the Cytometric Bead Array (CBA) Human Th1/Th2/Th17 kit. Light microscopy was used to evaluate cervicovaginal cytopathology. In addition, proviral load in cervicovaginal fluid and peripheral blood was measured by real-time quantitative polymerase chain reaction. RESULTS: 112 women (63 HTLV-1-infected and 49 uninfected) were evaluated. No differences were found with respect to cytopathological cervicovaginal findings between the groups. IL-2, TNF, IL-4, IL-10, and IL-17 levels were significantly higher in cervicovaginal fluid of the HTLV-1-infected women than in uninfected women (p<0.05). Conversely, IFN-γ was found to be lower in the HTLV-1-infected women (p<0.001) compared to uninfected individuals. Cervicovaginal proviral load was detectable in 53% of the HTLV-1-infected women and was found to be consistently lower than the proviral load in peripheral blood. CONCLUSIONS: HTLV-1 infection induces immune activation in cervicovaginal environment, characterized by elevated concentrations of Th1, Th2, and IL17 in the cervicovaginal fluid.
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Líquidos Corporais/química , Colo do Útero/patologia , Citocinas/análise , Infecções por HTLV-I/patologia , Vagina/patologia , Adulto , Líquidos Corporais/imunologia , Colo do Útero/imunologia , Colo do Útero/virologia , Estudos Transversais , Ensaio de Imunoadsorção Enzimática , Feminino , Infecções por HTLV-I/imunologia , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/isolamento & purificação , Humanos , Interleucina-17/imunologia , Leucócitos Mononucleares/virologia , Classe Social , Estatísticas não Paramétricas , Células Th1/imunologia , Células Th2/imunologia , Vagina/imunologia , Vagina/virologia , Carga ViralRESUMO
ABSTRACT Introduction: Human T-cell lymphotropic virus type 1 (HTLV-1) is sexually transmitted and causes persistent infection. This virus induces activation of the immune system and production of inflammatory cytokines. This study aimed to assess the cytokine profile and cytopathological findings in the cervicovaginal fluid of asymptomatic HTLV-1-infected women. Methods: HTLV-1-infected and uninfected women were selected at the Centro de Atendimento ao Portador de HTLV in Salvador-Brazil. None of the included HTLV-1-infected women reported any HTLV-1-associated diseases. All volunteers underwent gynecological examination to collect cervicovaginal fluid. Cytokine quantification was performed using the Cytometric Bead Array (CBA) Human Th1/Th2/Th17 kit. Light microscopy was used to evaluate cervicovaginal cytopathology. In addition, proviral load in cervicovaginal fluid and peripheral blood was measured by real-time quantitative polymerase chain reaction. Results: 112 women (63 HTLV-1-infected and 49 uninfected) were evaluated. No differences were found with respect to cytopathological cervicovaginal findings between the groups. IL-2, TNF, IL-4, IL-10, and IL-17 levels were significantly higher in cervicovaginal fluid of the HTLV-1-infected women than in uninfected women (p < 0.05). Conversely, IFN-γ was found to be lower in the HTLV-1-infected women (p < 0.001) compared to uninfected individuals. Cervicovaginal proviral load was detectable in 53% of the HTLV-1-infected women and was found to be consistently lower than the proviral load in peripheral blood. Conclusions: HTLV-1 infection induces immune activation in cervicovaginal environment, characterized by elevated concentrations of Th1, Th2, and IL17 in the cervicovaginal fluid.
Assuntos
Humanos , Feminino , Adulto , Vagina/patologia , Líquidos Corporais/química , Infecções por HTLV-I/patologia , Colo do Útero/patologia , Citocinas/análise , Classe Social , Vagina/imunologia , Vagina/virologia , Líquidos Corporais/imunologia , Ensaio de Imunoadsorção Enzimática , Leucócitos Mononucleares/virologia , Vírus Linfotrópico T Tipo 1 Humano/isolamento & purificação , Infecções por HTLV-I/imunologia , Infecções por HTLV-I/virologia , Colo do Útero/imunologia , Colo do Útero/virologia , Estudos Transversais , Células Th2/imunologia , Células Th1/imunologia , Estatísticas não Paramétricas , Carga Viral , Interleucina-17/imunologiaRESUMO
Feline foamy virus (FFV) and feline leukemia virus (FeLV) belong to the Retroviridae family. While disease has not been reported for FFV infection, FeLV infection can cause anemia and immunosuppression (progressive infection). Co-infection with FFV/FeLV allows evaluation of the pathogenic potential and epidemiology of FFV infection in cats with FeLV pathology. Blood and buccal swab samples from 81 cats were collected in Rio de Janeiro. Plasma was serologically tested for FeLV. DNA extracted from peripheral blood mononuclear cells and buccal swabs was used to PCR detect FFV and FeLV. A qPCR was developed to detect and measure FFV proviral loads (pVLs) in cats. FeLV qPCR was performed using previous methods. The median log10 pVL of FFV mono-infected individuals was lower than found in FFV/FeLV co-infected cats in buccal swabs (p = 0.003). We found 78% of cats had detectable buccal FFV DNA in FFV mono-infected and FFV co-infected FeLV-progressive cats, while in FeLV-regressive cats (those without signs of disease) 22% of cats had detectable buccal FFV DNA (p = 0.004). Our results suggest that regressive FeLV infection may reduce FFV saliva transmission, the main mode of FV transmission. We did not find evidence of differences in pathogenicity in FFV mono- and -dually infected cats. In summary, we show that FVs may interact with FeLV within the same host. Our study supports the utility of cats naturally co-infected with retroviruses as a model to investigate the impact of FV on immunocompromised mammalian hosts.
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Doenças do Gato/virologia , Coinfecção/veterinária , Vírus da Leucemia Felina , Infecções por Retroviridae/veterinária , Spumavirus , Infecções Tumorais por Vírus/veterinária , Animais , Brasil , Gatos , Coinfecção/virologia , DNA Viral/sangue , Feminino , Masculino , Provírus , Reação em Cadeia da Polimerase em Tempo Real , Infecções por Retroviridae/sangue , Infecções Tumorais por Vírus/sangue , Carga Viral/veterinária , Replicação ViralRESUMO
Bovine leukemia virus (BLV) is one of the most important virus in dairy cattle. The infection behavior follows what we call the iceberg phenomenon: 60% of infected animals do not show clinical signs; 30% develop persistent lymphocytosis (PL); and the remaining 10%, die due to lymphosarcoma. BLV transmission depends on infected cell exchange and thus, proviral load is determinant. Understanding the mechanisms by which cattle governs the control of viral dissemination will be desirable for designing effective therapeutic or preventive strategies for BLV. The development of high proviral load (HPL) or low proviral load (LPL) might be associated to genetic factors and humoral immune responses, however cellular responses are not fully described. It is known that BLV affects cellular homeostasis: proliferation and apoptosis. It is also known that the BLV tropism is directed towards B lymphocytes, and that lymphocytotic animals have elevated amounts of these cells. Usually, when an animal is infected by BLV, the B markers that increase are CD21, CD5 and CD11b. This increase could be related to the modulation of apoptosis in these cells. This is the first work in which animals infected with BLV are classified according to their proviral load and the subpopulations of B and T lymphocytes are evaluated in terms of their percentage in peripheral blood and its stage of apoptosis and viability. PBMCs from HPL animals proliferated more than LPL and non-infected animals. CD11b+/CD5+ lymphocytes in LPL animals presented greater early and late apoptosis than HPL animals and cells of HPL animals had increased viability than LPL animals. Our results confirm that BLV alters the mechanism of apoptosis and proliferation of infected cells.
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Apoptose , Leucose Enzoótica Bovina/imunologia , Vírus da Leucemia Bovina/imunologia , Subpopulações de Linfócitos/imunologia , Carga Viral/veterinária , Animais , Bovinos , Proliferação de Células , Células Cultivadas , FemininoRESUMO
BACKGROUND: HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a progressive neurological and inflammatory disease, associated with HTLV-1 infection. HAM/TSP neurological disease is a consequence of an inflammatory reaction, and adaptive immune responses, through the secretion of anti-inflammatory and pro-inflammatory cytokines, play an important role in the outcome of infection and disease progression. Studies addressing the association between cytokines functional single nucleotide polymorphisms and HAM/TSP development are scarce. METHODS: The genetic polymorphisms of cytokine genes were evaluated in HAM/TSP patients (n = 68) and in asymptomatic HTLV-1 positive carriers (n = 83) from Rio de Janeiro, Brazil, in a case-control study. HTLV-1 infected patients were genotyped for SNPs in five cytokine genes: TNFA-308G/A, IL6-174G/C, IFNG + 874 T/A, TGFB at the codons + 10 T/C and + 25G/C, IL10-592C/A and -819C/T, and -1082A/G and proviral load (PVL) was quantified. Associations between genotypes, haplotypes, clinical outcome and pro viral load were evaluated. RESULTS: Lack of association between the cytokine polymorphisms and disease outcome was observed. The genotypes TNFA-308GG, IL6-174GG/GC, IL10-592AA and -819CC and TGFb1 high producers phenotypes were correlated with higher PVL in HAM/TSP patients versus asymptomatic carriers. CONCLUSIONS: We did not observe association between cytokine polymorphisms and risk for HAM/TSP development in Brazilian HTLV-1 infected individuals, regardless of differences in PVL between HAM/TSP versus asymptomatic carriers in specific cytokine polymorphisms.
Assuntos
Citocinas/genética , Vírus Linfotrópico T Tipo 1 Humano , Inflamação/genética , Paraparesia Espástica Tropical/genética , Paraparesia Espástica Tropical/virologia , Polimorfismo de Nucleotídeo Único , Adulto , Idoso , Brasil/epidemiologia , Estudos de Casos e Controles , Progressão da Doença , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Infecções por HTLV-I/epidemiologia , Infecções por HTLV-I/genética , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Humanos , Inflamação/complicações , Masculino , Pessoa de Meia-Idade , Paraparesia Espástica Tropical/epidemiologia , Carga ViralRESUMO
Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL). Although efficient eradication programs have been successfully implemented in most European countries and Oceania, BLV infection rates are still high worldwide. BLV naturally infects cattle, inducing a persistent infection with diverse clinical outcomes. The virus infects lymphocytes and integrates a DNA intermediate as a provirus into the genome of the cells. Therefore, exposure to biological fluids contaminated with infected lymphocytes potentially spreads the virus. Vertical transmission may occur in utero or during delivery, and about 10% of calves born to BLV-infected dams are already infected at birth. Most frequently, transmission from dams to their offspring occurs through the ingestion of infected colostrum or milk. Therefore, although EBL is not a disease specific to the neonatal period, during this period the calves are at special risk of becoming infected, especially in dairy farms, where they ingest colostrum and/or raw milk either naturally or artificially. Calves infected during the first week of life could play an active role in early propagation of BLV to susceptible animals. This review discusses the main factors that contribute to neonatal BLV infection in dairy herds, as well as different approaches and management practices that could be implemented to reduce the risk of BLV transmission during this period, aiming to decrease BLV infection in dairy herds.
RESUMO
An elevated human T cell leukemia virus type 1 (HTLV-1) proviral load (PVL) is an important risk factor for HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), although there is a considerable frequency of asymptomatic carriers (AC) with high PVL in blood. Our objective was to evaluate whether PVL quantified in cerebrospinal fluid (CSF) is helpful to distinguish AC from HAM when AC have high PVL in blood (ACH). ACH (n = 7) were characterized to have high PVL in blood by quantification of samples collected over time (mean 7 years). HAM patients (n = 14) also had analyzed blood samples collected at different times (mean 9 years). Comparing paired CSF and blood samples of each individual, CSF PVL mean was 4.7-fold higher than blood PVL in the ACH group and 10.8-fold in the HAM group. CSF PVL was significantly greater than blood PVL in the HAM group (p = 0.004), but not in the ACH group. Important to highlight, CSF PVL was not significantly different between the ACH and the HAM groups. These results suggested that significantly higher PVL in CSF than in blood is a hallmark of HAM/TSP patients, but this is also true for asymptomatic carriers with high PVL in blood, thus reducing its usefulness as a marker for HAM/TSP. A greater number of ACH should be analyzed, but whether they will eventually develop HAM/TSP or why they have not developed the disease are still questions to be clarified. Longitudinal studies are necessary to answer these questions.