Optimization of the Light-On system in a lentiviral platform to a light-controlled expression of genes in neurons

BACKGROUND: Molecular brain therapies require the development of molecular switches to control gene expression in a limited and regulated manner in time and space. Light-switchable gene systems allow precise control of gene expression with an enhanced spatio-temporal resolution compared to chemical inducers. In this work, we adapted the existing light-switchable Light-On system into a lentiviral platform, which consists of two modules: (i) one for the expression of the blue light-switchable transactivator GAVPO and (ii) a second module containing an inducible-UAS promoter (UAS) modulated by a light-activated GAVPO. RESULTS: In the HEK293-T cell line transfected with this lentiviral plasmids system, the expression of the reporter mCherry increased between 4 to 5 fold after light induction. A time expression analysis after light induction during 24 h revealed that mRNA levels continuously increased up to 9 h, while protein levels increased throughout the experiment. Finally, transduction of cultured rat hippocampal neurons with this dual Light-On lentiviral system showed that CDNF, a potential therapeutic trophic factor, was induced only in cells exposed to blue light. CONCLUSIONS: In conclusion, the optimized lentiviral platform of the Light-On system provides an efficient way to control gene expression in neurons, suggesting that this platform could potentially be used in biomedical and neuroscience research, and eventually in brain therapies for neurodegenerative diseases.

Caprine lentivirus in sheep milk and semen / Lentivírus caprino em leite e sêmen de ovinos

With the objective of detecting the presence of caprine lentivirus (CLV) in ewe milk and in ram semen, ten matrixes and four reproducers experimentally infected with CLV were used. Samples of ewe milk were collected during the four months of lactation, five collections per animal, totaling 50 samples. Regarding the rams, eight semen collections were made per animal, during one year of experimentation, totaling 32 samples. The milk and semen samples were submitted to DNA extraction and the nested polymerase chain reaction test (nPCR) to detect CLV proviral DNA. Eight (16%) of the milk samples were positive in nPCR originating from two ewes. Only one (3.12%) semen sample was positive. The amplification products were sequenced, and were confirmed to be a CLV genomic sequence. Thus, the presence of CLV proviral DNA in sheep milk and semen was demonstrated, confirming the feasibility of infection between species, and alerting to the risk of spreading infections.(AU)

Com o objetivo de detectar a presença do lentivírus caprino (LVC) no leite de ovelhas e no sêmen de carneiros, utilizaram-se 10 matrizes e quatro reprodutores infectados experimentalmente com o LVC. Foram coletadas amostras de leite das ovelhas durante os quatro meses de lactação, ocorrendo cinco coletas por animal, totalizando 50 amostras. Quanto aos carneiros, realizaram-se oito coletas de sêmen por animal, durante um ano de experimentação, totalizando 32 amostras. As amostras de leite e de sêmen foram submetidas à extração de DNA e à prova de reação em cadeia da polimerase do tipo nested (nPCR) visando à detecção de DNA proviral do LVC. Oito (16%) amostras de leite foram positivas na nPCR oriundas de duas ovelhas. Apenas uma (3,12%) amostra de sêmen apresentou positividade. Produtos da amplificação foram sequenciados, confirmando-se tratar de sequência genômica do LVC. Dessa forma, demonstrou-se a presença do DNA proviral do LVC em leite e sêmen de ovinos, confirmando a viabilidade da infecção entre espécies e, assim, alertando sobre o risco de que a infecção seja disseminada.(AU)

Goat umbilical cord cells are permissive to small ruminant lentivirus infection in vitro

Abstract Small ruminant lentiviruses isolated from peripheral blood leukocytes and target organs can be propagated in vitro in fibroblasts derived from goat synovial membrane cells. These cells are obtained from tissues collected from embryos or fetuses and are necessary for the establishment of the fibroblast primary culture. A new alternative type of host cells, derived from goat umbilical cord, was isolated and characterized phenotypically with its main purpose being to obtain cell monolayers that could be used for the diagnosis and isolation of small ruminant lentiviruses in cell culture. To accomplish this goal, cells were isolated from umbilical cords; characterized phenotypically by flow cytometry analysis; differentiate into osteogenic, chondrogenic and adipogenic lineage; and submitted to viral challenge. The proliferation of goat umbilical cord cells was fast and cell monolayers formed after 15 days. These cells exhibited morphology, immunophenotype, growth characteristics, and lineage differentiation potential similar to mesenchymal stem cells of other origins. The goat umbilical cord derived cells stained positive for vimentin and CD90, but negative for cytokeratin, CD34 and CD105 markers. Syncytia and cell lysis were observed in cell monolayers infected by CAEV-Cork and MVV-K1514, showing that the cells are permissive to small ruminant lentivirus infection in vitro. These data demonstrate the proliferative competence of cells derived from goat umbilical cords and provide a sound basis for future research to standardize this cell lineage.

Small ruminant lentiviruses: economic and productive losses, consequences of the disease / Lentiviroses de pequenos ruminantes: perdas produtivas e econômicas, consequências da doença

Small ruminant lentiviruses, caprine arthritis encephalitis virus, and Maedi-Visna virus cause diseases that result in significant productive losses, mostly in dairy animals. These viruses belong to the Retroviridae family, Lentivirus genus, and constitute a heterogeneous group, which may generate implications for the diagnosis and control of small ruminant lentiviruses. Losses caused by them are associated with reproductive failure, short productive life, and decreased milk production by the infected animals. In addition, these viruses may reduce milk quality, affecting the production of dairy products such as cheese. Small ruminant lentiviruses lead to indirect losses, decreasing herd value and forcing the development of epidemiological trade barriers for animal germplasm. Control of small ruminant lentiviruses is important to promote optimal milk production and to reduce costs with medicine and technical assistance. This control may vary in caprine and ovine populations of each country, according to seroprevalence, variety of breeds, and peculiarities of the practiced management.(AU)

Os lentivírus de pequenos ruminantes, o vírus da artrite encefalite caprina e o vírus Maedi-Visna causam enfermidades que ocasionam perdas produtivas significativas, principalmente em animais com aptidão leiteira. Esses vírus pertencem à família Retroviridae e ao gênero Lentivirus e formam um grupo genético heterogêneo, o que pode ocasionar implicações para o diagnóstico e o controle dos lentivírus de pequenos ruminantes. As perdas causadas pelos lentivírus de pequenos ruminantes estão relacionadas com falhas reprodutivas, vida produtiva curta e diminuição da produção leiteira dos animais infectados. Além disso, esses vírus podem promover a redução da qualidade do leite, afetando a produção de laticínios, tal como o queijo. Os lentivírus de pequenos ruminantes levam a perdas indiretas, reduzindo o valor dos rebanhos e forçando o desenvolvimento de barreiras comerciais epidemiológicas para germoplasma animal. O controle dos lentivírus de pequenos ruminantes é importante para promover uma maior produção de leite e reduzir os custos com medicamentos e assistência técnica. Esse controle pode variar de acordo com a população caprina e ovina de cada país em termos de soroprevalência, variedade de raças e particularidades do manejo adotado.(AU)

Knockdown of ubiquitin-specific peptidase 39 inhibited the growth of osteosarcoma cells and induced apoptosis in vitro

BACKGROUND: Ubiquitin specific peptidase 39 (USP39), an essential factor in the assembly of the mature spliceosome complex, has an aberrant expression in several cancer. However, its function and the corresponding mechanism on human osteosarcoma has not been fully explored yet. METHODS: The mRNA and DNA copies of USP39 were increased in osteosarcoma cancer tissues compared with the one in human normal tissues according to datasets from the publicly available Oncomine database. A further western blot analysis also demonstrated an aberrant endogenous expression of USP39 in three different osteosarcoma cells. Then lentivirus-mediated short hairpin RNA (shRNA) was designed to silence USP39 in human osteosarcoma cell line U2OS, which is used to test the impact of USP39-silencing on cellular proliferation, colony formation, cell cycle distribution and apoptosis. RESULTS: Knockdown of USP39 expression in U2OS cell significantly decreased cell proliferation, impaired colony formation ability. A further analysis indicated suppression of USP39 arrested cell cycle progression at G2/M phase via p21 dependent way. In addition, the results of Annexin V/7-AAD staining suggested the knockdown of USP39 could promote U2OS cell apoptosis through PARP cleavage. CONCLUSIONS: These results uncover the critical role of USP39 in regulating cancer cell mitosis and indicate USP39 is critical for osteosarcoma tumorigenesis.

Soroprevalência da infecção por lentivírus de pequenos ruminantes em abatedouros do estado de Pernambuco, Brasil / Seroprevalence small ruminant lentiviruses in the state of Pernambuco slaughterhouses, Brazil

A soroprevalência da infecção por lentivírus de pequenos ruminantes (LVPR) foi determinada em amostras de soros sanguíneos de caprinos e ovinos de aptidão cárnea provenientes de abatedouros de dez municípios do estado de Pernambuco, Brasil. O diagnóstico sorológico ocorreu por meio da imunodifusão em gel de agarose (micro-IDGA) com antígenos dos vírus artrite encefalite caprina (CAE)/Maedi-Visna. Entre as 369 amostras de caprinos, 7(1,89%) (0,8-3,9%) eram soropositivas, e, entre as 383 de ovinos, 1 (0,26%) (0,0-1,4%) estava infectada. Os 7 caprinos soropositivos procederam dos abatedouros públicos dos municípios de Gravatá (n=2), Sertânia (n=4) e Timbaúba (n=1), e o ovino soropositivo veio do abatedouro público de Serra Talhada. A soroprevalência da infecção por LVPR em pequenos ruminantes oriundos de abatedouros do estado de Pernambuco, de 1,06% (8/752), é considerada baixa.(AU)

The prevalence of lentivirus infection of small ruminants (LVPR) was determined in samples of serum from goats and sheep in slaughterhouses from ten districts of Pernambuco State. The serological test was used in agarose gel immunodiffusion (AGID) with antigen caprine arthritis and encephalitis virus (CAE)/Maedi Visna virus. Among the 369 blood serum samples of goats examined, seven (1.89%) (0.8-3.9%) were seropositive, and among the 383 sheep samples examined, just one (0.26%) (0.0-1.4%) was infected. The seven seropositive goats came from public slaughterhouses from Gravatá (n=2), Sertânia (n=4) and Timbaúba (n=1), and the soropositive sheep was from a public slaughterhouse of Serra Talhada. The soroprevalence of LVPR infection in small ruminants from Pernambuco's slaughterhouses, of 1.06% (8/752), is considered low.(AU)

Frequência de ovinos soropositivos para lentivírus de pequenos ruminantes no município de Colinas do Tocantins, estado do Tocantins, Brasil / Frequency of antibodies against ovine Lentivirus in sheep in Colinas do Tocantins, Tocantins state, Brazil

Maedi-Visna (MV) é uma enfermidade causada por lentivírus de pequenos ruminantes com evolução crônica e em grande parte dos casos sinais clínicos inaparentes. O diagnóstico da doença é baseado em sinais clínicos e dados epidemiológicos, sendo a imunodifusão em gel de ágar (IDGA) o método padrão para a detecção sorológica de anticorpos contra o lentivírus. Sabendo que o estado do Tocantins possui potencial para o desenvolvimento da ovinocultura e que grande parte dos produtores de Colinas do Tocantins, no referido estado, possui interesse em estabelecer criação racional, esta pesquisa teve por objetivo a realização de um estudo acerca da soroprevalência da doença. Foram coletadas 369 amostras de sangue de ovinos, independentemente de raça, sexo e idade, de diferentes propriedades rurais do município para diagnóstico de MV utilizando a técnica de IDGA. Após as análises laboratoriais, para avaliação dos resultados no tocante às categorias, foi utilizado o teste exato de Fisher e também foi calculado o odds ratio , com intervalo de confiança de 95% para verificação da idade como possível fator de risco ou de proteção. Constatou-se que 6 animais (1,62%) se apresentaram positivos no IDGA. Diante desses resultados, foi possível concluir que a frequência de ovinos soropositivos no município é baixa.(AU)

Maedi-Visna is a disease caused by a small ruminant lentivirus, with a chronic evolution and, in most cases, unapparent signs. Its diagnosis is based on clinical signs and epidemiological data, with the agar gel immunodiffusion (AGID) test being the classical method for detecting antibodies against lentiviruses. Considering that the state of Tocantins has the potential to develop sheep breeding and that the majority of producers from Colinas do Tocantins city has shown an interest in establishing a rational breeding, this study aimed to determine the prevalence of such disease in that region. A total of 369 blood samples was drawn from sheep bred in several farms of that town, regardless of breed, gender, and age. Every sample underwent an AGID to test for Maedi-Visna. After the laboratory analyses were concluded, the Fisher's exact test was used to evaluate the results against the categories. Also, the odds ratio, with 95% confidence interval, was calculated to check whether age played a role as either a risk or protective factor in these results. It was found that six animals (1.62%) were positive in AGID, therefore concluding that the frequency of seropositivity in that region is low.(AU)

Aspectos imunológicos e genéticos da expressão integrina A4B7 na história natural e na intervenção terapêutica da infecção por lentivírus no homem e em modelos animais / [Immunological and genetic aspects of the A4B7 integrin expression in natural history and in the therapeutic intervention of lentivirus infection in man and in animal models]

Integrinas são proteínas envolvidas na migração e adesão celular. Estudos recentes mostraram que a integrina α4β7 interage com a proteína gp120 do HIV-1, auxiliando a entrada do vírus na célula hospedeira através de sinapses virológicas. Já foi demonstrada uma maior prevalência do genótipo C-C do SNP rs1449263, localizado na região promotora do gene itga4 (que codifica a subunidade α4 da integrina), em pacientes com esclerose múltipla. Estes pacientes apresentam uma alta expressão de integrina, sugerindo o envolvimento deste SNP na superexpressão da mesma. A interação de α4β7 com seu ligante natural, MAdCAM, pode ser uma importante fonte de estímulo, capaz de gerar sinais intracelulares culminando com a ativação de linfócitos T. Diversos estudos tem avaliado o papel da integrina α4β7 na transmissão do HIV em mucosas, bem como a utilização de anticorpos bloqueadores desta proteína como novas terapias para prevenção e tratamento da infecção. Neste sentido, a utilização de primatas não-humanos como modelos de estudo da infecção por SIV/HIV é de suma importância e utilidade. Neste estudo, avaliamos a prevalência dos diferentes genótipos do SNP rs1449263 e o perfil de expressão da integrina α4 em indivíduos de três coortes diferentes que compreendem adultos HIV+ e HIV-, e em crianças HIV+ ou expostas ao vírus e não-infectadas. A partir de PBMCs foram isolados DNA genômico e RNA para a avaliação dos genótipos encontrados e expressão relativa dos genes itga4, itgb7 e itgae. Esta última pode também se ligar a subunidade β7 e competir com α4. Não foram observadas associações entre os genótipos e alelos do SNP estudado e a aquisição do HIV ou progressão para a Aids...

Integrins are transmembrane glycoproteins found in many vertebrate cell types. Recent studies show that HIV gp120 can bind the α4β7 integrin, providing a favorable environment for virus transmission between cells. α4β7 is a homing molecule to the gut-associated lymphocyte tissue (GALT). This protein has an important role during the initial course of HIV infection, since intense viral replication and lymphocyte depletion is observed in the GALT during this phase. A higher prevalence of the C-C genotype in the SNP rs1449263, located in the promoter region of the itga4 gene (that encodes α4 integrin), has been shown in multiple sclerosis patients, possibly related to a higher expression of this gene. The interaction between α4β7 and its natural ligand MAdCAM can lead to cell activation, promoting proliferation of T lymphocytes. Several studies highlight the role of α4β7 in the mucosal transmission of HIV as well as its possible use as a target for HIV prevention and antiviral therapy. Studies with non-human primates are useful in this field, as these animals have similarities with humans and can be infected with SIV. In the present study, we assessed the prevalence of different SNP rs1449263 genotypes in three different cohorts harboring HIV+ and HIV- adults, as well as HIV+ or exposed-uninfected children. SNP genotyping and gene expression analysis were carried out from DNA and RNA isolated from PBMCs. No association between SNP genotype and HIV acquisition or progression to AIDS was observed in the cohorts. With respect to itga4, itgb7 (β7) and itgae (αE) gene expression, a higher level of itga4 mRNA was observed in allele C carriers, compared to non-carriers...

Knockdown of ZFR suppresses cell proliferation and invasion of human pancreatic cancer

BACKGROUND: Zinc finger RNA binding protein (ZFR) is involved in the regulation of growth and cancer development. However, little is known about ZFR function in pancreatic cancer. METHODS: Herein, to investigate whether ZFR is involved in tumor growth, Oncomine microarray data was firstly used to evaluate ZFR gene expression in human pancreatic tumors. Then short hairpin RNA (shRNA) targeting ZFR was designed and delivered into PANC-1 pancreatic cancer cells to knock down ZFR expression. Cell viability, cell proliferation and cell cycle analysis after ZFR knockdown were determined by MTT, colony forming and FACS, respectively. In addition, cell migration and invasion were assessed using the Transwell system. RESULTS: The expression of ZFR was significantly higher in pancreatic tumors than normal pancreas tissues by Oncomine database analysis. Knockdown of ZFR by shRNA-expressing lentivirus significantly decreased the viability and invasion ability of pancreatic cancer cells. Moreover, FACS analysis showed that knockdown of ZFR in PANC-1 cells caused a significant cell cycle arrest at G0/G1 phase. Furthermore, knockdown of ZFR decreased the levels of CDK2, CDK4, CyclinA and CyclinD1 and enhanced the expression of p27, which has evidenced by qRT-PCR and Western blot analysis. CONCLUSIONS: Knockdown of ZFR might provide a novel alternative to targeted therapy of pancreatic cancer and deserves further investigation.

Swim training and the genetic expression of adipokines in monosodium glutamate-treated obese rats

Objective The aim of this study was to evaluate the genetic expression of adipokines in the adipocytes of monosodium glutamate (MSG)-treated obese rats submitted to physical activity.Materials and methods Obesity was induced by neonatal MSG administration. Exercised rats (MSG and control) were subjected to swim training for 30 min for 10 weeks, whereas their respective controls remained sedentary. Total RNA was obtained from sections of the mesenteric adipose tissue of the rats. mRNA levels of adiponectin (Adipoq), tumor necrosis factor alpha (Tnf), peroxisome proliferator-activated receptor alpha (Ppara), and peroxisome proliferator-activated receptor gamma (Pparg) adipokines were quantified by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).Results In the exercise-trained control group, the expression of Adipoq increased compared to the sedentary control, which was not observed in the MSG-obese rats. Increased levels of Tnf in MSG-obese rats were not reversed by the swim training. The expression of Ppara was higher in sedentary MSG-obese rats compared to the sedentary control. Swimming increased this adipokine expression in the exercise-trained control rats compared to the sedentary ones. mRNA levels of Pparg were higher in the sedentary MSG-rats compared to the sedentary control; however, the exercise did not influenced its expression in the groups analyzed.Conclusions In conclusion, regular physical activity was not capable to correct the expression of proinflammatory adipokines in MSG-obese rat adipocytes.

Lentivirus mediated silencing of Ubiquitin Specific Peptidase 39 inhibits cell proliferation of human hepatocellular carcinoma cells in vitro

BACKGROUND: Ubiquitin Specific Peptidase 39 (USP39) is a 65 kDa SR-related protein involved in RNA splicing. Previous studies showed that USP39 is related with tumorigenesis of human breast cancer cells. RESULTS: In the present study, we investigated the functions of USP39 in human hepatocellular carcinoma (HCC) cell line SMMC-7721. We knocked down the expression of USP39 through lentivirus mediated RNA interference. The results of qRT-PCR and western blotting assay showed that both the mRNA and protein levels were suppressed efficiently after USP39 specific shRNA was delivered into SMMC-7721 cells. Cell growth was significantly inhibited as determined by MTT assay. Crystal violet staining indicated that colony numbers and sizes were both reduced after knock-down of USP39. Furthermore, suppression of USP39 arrested cell cycle progression at G2/M phase in SMMC-7721cells. In addition, Annexin V showed that downregulation of USP39 significantly increased the population of apoptotic cells. CONCLUSIONS: All our results suggest that USP39 is important for HCC cell proliferation and is a potential target for molecular therapy of HCC.

Targeting PPM1D by lentivirus-mediated RNA interference inhibits the tumorigenicity of bladder cancer cells

Protein phosphatase magnesium/manganese-dependent 1D (PPM1D) is a p53-induced phosphatase that functions as a negative regulator of stress response pathways and has oncogenic properties. However, the functional role of PPM1D in bladder cancer (BC) remains largely unknown. In the present study, lentivirus vectors carrying small hairpin RNA (shRNA) targeting PPM1D were used to explore the effects of PPM1D knockdown on BC cell proliferation and tumorigenesis. shRNA-mediated knockdown of PPM1D significantly inhibited cell growth and colony forming ability in the BC cell lines 5637 and T24. Flow cytometric analysis showed that PPM1D silencing increased the proportion of cells in the G0/G1 phase. Downregulation of PPM1D also inhibited 5637 cell tumorigenicity in nude mice. The results of the present study suggest that PPM1D plays a potentially important role in BC tumorigenicity, and lentivirus-mediated delivery of shRNA against PPM1D might be a promising therapeutic strategy for the treatment of BC.

Lentiviral-mediated RNAi targeting caspase-3 inhibits apoptosis induced by serum deprivation in rat endplate chondrocytes in vitro

Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.

CIAPIN1 gene silencing enhances chemosensitivity in a drug-resistant animal model in vivo

Overexpression of cytokine-induced apoptosis inhibitor 1 (CIAPIN1) contributes to multidrug resistance (MDR) in breast cancer. This study aimed to evaluate the potential of CIAPIN1 gene silencing by RNA interference (RNAi) as a treatment for drug-resistant breast cancer and to investigate the effect of CIAPIN1 on the drug resistance of breast cancer in vivo. We used lentivirus-vector-based RNAi to knock down CIAPIN1 in nude mice bearing MDR breast cancer tumors and found that lentivirus-vector-mediated silencing of CIAPIN1 could efficiently and significantly inhibit tumor growth when combined with chemotherapy in vivo. Furthermore, Western blot analysis showed that both CIAPIN1 and P-glycoprotein expression were efficiently downregulated, and P53 was upregulated, after RNAi. Therefore, we concluded that lentivirus-vector-mediated RNAi targeting of CIAPIN1 is a potential approach to reverse MDR of breast cancer. In addition, CIAPIN1 may participate in MDR of breast cancer by regulating P-glycoprotein and P53 expression.

Ferramentas diagnósticas de Lentivirose de Pequenos Ruminantes: padronização da técnica de ELISA indireto / Diagnostic tools of small ruminant lentiviruses: standardization of indirect ELISA

As Lentiviroses de Pequenos Ruminantes (LVPR) incluem a Maedi-Visna (MV) em ovinos e a Artrite Encefalite Caprina (CAE). Essas enfermidades estão difundidas no mundo e são responsáveis por grandes perdas na produtividade destes animais. Os LVPR são vírus RNA da subfamília Lentivirinae que causam uma infecção persistente, sendo a detecção precoce uma das formas mais eficientes para limitar sua disseminação no rebanho. Visando contribuir com essas questões, este experimento foi realizado na Universidade Federal do Piauí (UFPI) em parceria com a Embrapa Caprinos e Ovinos, com o objetivo de padronizar a técnica de ensaio imunoenzimático indireto e compará-lo com a imunodifusão em gel de agarose no diagnóstico da CAE. Foram utilizadas 696 amostras de soros de caprinos machos e fêmeas oriundas do banco de soros da Unidade de Pesquisa de LVPR do Centro de Ciências Agrárias da UFPI. As amostras foram coletadas no período de janeiro de 2007 a março de 2010. Na padronização, verificou-se que 0,25 µg de proteína/poço, diluição de 1:200 do soro e concentração de 1:3.000 do conjugado anticorpo anti-IgG cabra apresentaram os melhores resultados. O ponto de corte obtido foi de 0,36. Na comparação, o Imunodifusão em Gel de Ágar (IDGA) detectou 128 (18,4%) amostras positivas, e o ELISA indireto (ELISA-i), 259 (37,2%). A sensibilidade e a especificidade do teste ELISA-i com relação ao IDGA foi de 94,5% e 75,7%, respectivamente. Verificou-se maior índice de positividade em caprinos acima de seis meses (p < 0,05), e nos machos obteve-se prevalência de 56,7% em comparação às fêmeas, 35,4%, (p < 0,01).(AU)

The Small Ruminant Lentiviruses (SRLVs) include Maedi-Visna (MV) of sheep and Caprine Arthritis-Encephalitis (CAE). These diseases are widespread and responsible for major production losses regarding sheep and goats. The SRLV is a RNA virus of the subfamily Lentivirus genus that causes persistent infections in goats. Early detection is one of the best ways to limit its spread in the herd. To contribute to these issues, this experiment was conducted at Universidade Federal do Piauí in partnership with Embrapa Goats and Sheep, with the objective of standardizing the technique of indirect ELISA (i-ELISA) and to compare it with Immunodiffusion in Agarose Gel to diagnose Caprine Lentiviruses (LC). Six hundred ninety six serum samples were used from the University Veterinary Hospital, Universidade Federal do Piauí, from January 2007 to March 2010. Standardization showed that 0.25 µg protein/well, a 1:200 dilution of the serum and concentration of 1:3,000 of the conjugated anti-goat IgG presented the best results. It was observed that the Agar Gel Immunodiffusion (AGID) detected 128 (18.4%) positive samples, and ELISA, 259 (37.2%). The sensitivity and specificity of i-ELISA regarding AGID were 94.5% and 75.7%, respectively. A higher prevalence was observed among animals older than six months (p < 0.05). The prevalence among males was of 56.7%, and among females, 35.4% (p < 0.01).(AU)

Pesquisa de anticorpos contra maedi visna em ovinos nas microregiões de Botucatu, Campinas, Piedade e São Paulo, Estado de São Paulo / Survey for antibodies against maedi-visna virus in sheep in the regions of Botucatu, Campinas, São Paulo and Piedade, State of São Paulo, Brazil

O objetivo deste estudo foi avaliar a ocorrência da infecção pelo vírus Maedi-Visna em ovinos criados nas microrregiões de Botucatu, Campinas, Piedade e São Paulo do Estado de São Paulo. As amostras de soro sanguíneo foram colhidas de 226 ovinos e foi realizada a técnica de imunodifusão em gel de ágar para a detecção de anticorpos antivírus Maedi-Visna e verificou-se que nenhuma das amostras testadas foi sororeagente. Dessa forma, faz-se necessário um estudo mais amplo no estado, a fim de se confirmar a baixa ocorrência e importância da enfermidade no estado.

Survey for antibodies against maedi-visna virus in sheep in the regions of Botucatu, Campinas, São Paulo and Piedade, state of São Paulo, Brazil. The purpose of the study was to evaluate the occurrence of infection with maedi-visna virus in sheep raised in the regions of Botucatu, Campinas, Piedade and São Paulo, state of São Paulo, Brazil, that showed symptoms of the disease. Blood serum samples collected from 226 sheep were submitted to the agar gel immunodiffusion technique for detection of antibodies against maedi-visna virus, and none of the samples tested was serum reactive. In conclusion, the maedi-visna virus has low frequency in animals raised in the regions studied.

Alterações histopatológicas da glândula mamária e qualidade do leite de cabras naturalmente infectadas com o CAEV / Histopathological changes in the mammary gland and milk quality of goats naturally infected with CAEV

Avaliou-se a influência do vírus da CAE nas características físico-químicas de amostras de leite de 54 cabras, sem predileção racial, distribuindo-as em dois grupos: cabras positivas e negativas para o teste de imunodifusão em gel de agarose. As amostras de leite foram submetidas à análise ultrassônica para obtenção de parâmetros físico-químicos - gordura, extrato seco, proteínas, lactose e densidade; realização de microbiologia - bactérias mesófilas (UCF/mL). Foram coletadas amostras de tecido mamário para exame histopatológico e imunohistoquímica. Não houve diferença significativa das características avaliadas entre os dois grupos; no microbiológico, não houve relação direta da presença de mesófilas associada à infecção pelo CAEV. Na histopatologia, observaram-se áreas com infiltração celular de monócitos, polimorfonucleares, plasmócitos, fibrose, ausência de morfologia normal do parênquima mamário, denotando processo inflamatório crônico; e foi confirmada a presença do vírus na glândula pela imunohistoquímica. Os resultados não mostraram relação direta da incidência da CAE como fator negativo no desenvolvimento do rebanho.

Aiming to evaluate the influence of CAE viruses in the chemical and physical characteristics of milk, the samples were collected from 54 goats, without racial predilection, these were divided into two groups: goats positive and negative according results of test Agarose Gel Immunodiffusion. Milk samples were ultrasonic analyzed to obtain physicochemical parameters (fat, solids, protein, lactose and density); performance microbiology (mesophilic bacteria - CFU/mL) and mammary gland samples were collected for evaluation histopathology and immunohistochemistry. The results of physical-chemical analysis showed no significant difference between the milk samples of two groups. In the microbiological analysis showed the presence of aerobic mesophilic bacteria, but this change is not associated with the presence of CAEV infection. On histopathology, there were areas with infiltration of mononuclear-leukocyte and polymorph nuclear, plasma cells, fibrosis and absence of normal morphology of the mammary tissue, indicating a chronic inflammatory process; and confirmed the presence of virus, in the gland, by immunohistochemistry. The results showed no direct relationship between incidence of CAE in the herd as a negative factor for its development, however it is known that the disease in its chronic nature, causes reduction in the productivity of the herd.

Obtenção de um pseudotipo repórter do vírus do oeste do Nilo baseado em vetor lentiviral / Obtaining a pseudotype reporter for West Nile virus-based lentiviral vector

O Vírus do Oeste do Nilo (WNV) é um flavivírus que se mantém na natureza em ciclos alternados de infecção entre aves silvestres e mosquitos hematófagos, principalmente do gênero Culex. Ocasionalmente, o vírus pode ser transmitido aos mamíferos por mosquitos que se alimentaram previamente de aves viremicas. Humanos e equinos são altamente suscetíveis a infecção pelo WNV, e frequentemente desenvolvem febre, que pode ser seguida por infecção do sistema nervoso central e meningoencefalite, podendo ser fatal. Já houve registros do vírus na África, Oriente Médio e Ásia desde 1937. Foi detectado em Nova York em 1999 e se disseminou rapidamente pelos Estados Unidos e mais tarde para o Canadá. A rápida disseminação da infecção em direção ao sul sugere que novas evidências sorológicas e virológicas serão relatadas nos próximos anos na América Central e do Sul. No ano passado, evidências sorológicas do virus foram registradas em equinos na região do Pantanal do Brasil. Este trabalho teve como objetivo desenvolver um pseudotipo repórter para o WNV com base em um vetor retroviral a ser utilizado em testes de neutralização. Para tanto, foi desenvolvido um plasmídeo recombinante capaz de expressar as glicoproteínas do envelope do WNV. Este plasmídeo foi co-transfectado em células eucarióticas junto a outros dois plasmídeos (pHP e pTY-GFP), o que permitiu a formação das partículas. Estas possuem o capsídeo e o genoma defectivo do vírus da imunodeficiência humana do tipo 1 (HIV-1), um gene repórter gfp (green fluorescente protein) e as glicoproteínas do envelope do WNV. Nossos resultados mostraram que as glicoproteínas do envelope do WNV foram expressas, e que as partículas pseudotipo foram formadas. Essas partículas poderão ser uma ferramenta molecular muito eficiente para estudos morfofuncionais das glicoproteínas do envelope, tais como, tropismo, mecanismos de entrada (inibidores e facilitadores). Uma melhor compreensão destes mecanismos é essencial para o desenvolvimento de técnicas de diagnóstico, profilaxia e tratamento.

Prevalência da infecção por lentivírus de pequenos ruminantes em caprinos em Teresina, Piauí / Prevalence of small ruminants lentiviruses infection in goats from Teresina, Piauí, Brazil

The prevalence of anti-lentiviruses antibodies of small ruminants was investigated in goat herds in the city of Teresina, PI, Brazil. A seroepidemiological survey was conducted involving 480 animals, apparently healthy, belonging to six rural properties. The diagnostic test was the agar gel immunodiffusion (AGID), using antigens produced from cellular cultures infected with caprine arthritis encephalitis virus (CAEV Cork). Prevalences by gender and age were estimated considering sampling fractions for each farm. A general prevalence of 4.2 percent, was observerved, being 4.2 percent for females and 3.6 percent for males. Prevalences were higher among older goats. Regarding the breed standard, 23.5 percent were of the Anglo Nubian, 5.9 percent of the Boer, 35.3 percent Anglo Nubian x Boer crossbred, and 35.3 percent of undefined breed. It is concluded that small ruminant lentiviruses are endemic among goat herds of Teresina.

Evaluation of a recombinant p24 antigen for the detection of feline immunodeficiency virus-specific antibodies / Avaliação do antígeno recombinante p24 para a detecção de anticorpos específicos do vírus da imunodeficiência felina

Feline Immunodeficiency Virus is a worldwide infection and is considered a significant pathogen. The diagnosis of FIV infections is mainly based on commercially available rapid tests that are highly expensive in Brazil, hence it is rarely performed in the country. Furthermore, lentiviruses grow slowly and poorly in tissue cultures, making the production of viral antigen by classic means and thus the establishment of FIV immunodiagnosis impracticable. In order to deal with this, recombinant DNA techniques were adopted to produce the protein p24, a viral capsid antigen. The protein's reactivity evaluation analyzed by Western blot indicated that this recombinant antigen can be a useful tool for the immunodiagnostic of FIV infections.

O vírus da imunodeficiência felina tem distribuição mundial e é considerado um patógeno significativo. No Brasil, a prática diagnóstica é baseada principalmente em teste rápidos, importados e de custo elevado, disponíveis comercialmente. Devido ao seu custo proibitivo em nosso país, o diagnóstico da infecção pelo FIV é raramente realizado. Ademais, os lentivírus se multiplicam lenta e pobremente em cultura de células, o que torna a produção de antígeno por meios clássicos e o estabelecimento do imunodiagnóstico impraticável. Com o objetivo de lidar com esta questão, técnicas de DNA recombinante foram utilizadas para produção de um antígeno do capsídeo viral, a proteína p24. A avaliação da reatividade realizada por Western blot indicou que este antígeno recombinante pode ser útil para o imunodiagnóstico de infecções pelo FIV.