Mother-to-child transmission (MTCT) of HIV accounts for more than 90% of pediatric infections worldwide, yet the mechanism of vertical transfer remains unknown. The feline immunodeficiency virus (FIV)-infected cat is a cost-effective, small-animal model of HIV pathogenesis and MTCT, which produces a high rate of reproductive failure and fetal infection in litters delivered at early- and late-term gestation. Our previous data suggest that FIV infection may dysregulate placental cytokines and compromise pregnancy. We hypothesized that FIV-infection may cause dysregulation of placental cytokine expression, and aberrant expression of these cytokines may potentiate inflammation and transplacental infections. The purpose of this project was to evaluate feline placental immunopathology at the whole and cellular levels during early- and late-term gestation to understand how lentiviruses may perturb placental immune parameters. To determine whether placentas were vulnerable to FIV infection, we quantified the expression of the FIV receptors, CD134 and CXCR4, in RNA extracted from late-term placental tissue. We found higher expression of CD134 and CXCR4 in placentas from successful pregnancies. To evaluate relative cytokine expression in randomly-sampled, whole placental specimens, we quantified representative pro- and anti-inflammatory cytokines and a chemokine. IL-6 and IL-12p35 were increased in early-gestation, FIV-infected queens; IL-6 was increased in late-gestation, FIV-infected queens. To evaluate placental immunopathology at the cellular level, we developed a novel immunohistochemistry method to identify trophoblastic cells selectively. Trophoblasts were collected using laser capture microdissection, and RNA was extracted from captured cells. We detected expression of several anti- and pro-inflammatory cytokines and the chemokine receptor CXCR4 (the FIV co-receptor) in trophoblasts at both stages of gestation. However, we failed to detect expression of other cytokines and CD134, the FIV primary receptor. FIV infection slightly lowered expression of all cytokines at both early and late pregnancy, although only the decrease in IL-5, from early pregnancy, and IL-4 and IL-12p35, from late pregnancy, reached significant levels. Fetal non-viability was associated with decreased trophoblast expression of IL-4, IL-6, IL-12p35, and CXCR4 at early gestation and decreased expression of IL-4, IL-12p35, IL-12p40 at late gestation. Collectively, these data indicate that FIV infection negatively impacts pregnancy outcome and alters placental immunomodulation.

We are using the feline immunodeficiency virus (FIV)-infected cat to model HIV mother-to-child-transmission (MTCT). Vertical transmission of either virus may result not only in infected offspring, but also failed pregnancy.In HIV infections, maternal hematological and virological parameters predict MTCT.We hypothesized that such parameters would likewise be predictors of FIV vertical transfer. We inoculated ten cats with FIV-B-2542; 10 cats were uninoculated. Cats were allowed to breed naturally. Fetuses were delivered at approximately week 3 (early) gestation by cesarean section. Fetal and placental tissues were collected.Blood samples were collected from the day of inoculation through delivery. We quantified CD4:CD8 T cell ratios, proviral load, and plasma viremia, and monitored seroreactivity to FIV proteins in longitudinal sera from both groups of cats. We documented clinical and reproductive outcome. The infected group produced reduced litter size and more failed pregnancies; CD4:CD8 ratios were depressed by 3.5 months p.i.Proviral DNA was detected in 14 of 14 (100%) placentas tested and 12 of 14 (86%) fetuses. However, the parameters assessed were not predictive of reproductive outcome and suggested a role for placental immunopathology in compromised pregnancy.Regulatory T cells (Treg) are anti-inflammatory and essential in maintaining pregnancy.Th17 cells are pro-inflammatory and associated with pregnancy failure. The activation of these cell populations is regulated by the cytokines TGF-? and IL-6. We hypothesized that placental immunology may result from altered dynamics of these cell populations.Using immunofluorescence confocal microscopy to measure Treg and Th17 markers FoxP3 and ROR ? , respectively, we quantified these cells in placental specimens from FIV-infected and control cats at early and late (week 8) gestation.Significantly higher levels of ROR ? were measured in FIV-infected placentas at early pregnancy; these cells co-localized at the maternaletal interface. We quantified the expression of Treg immunomodulators by quantitative PCR, noting higher expression of TGF-? in infected queens.A positive correlation of ROR ? with IL-6 occurred in control placentas, as predicted, but not in infected placentas.Collectively, the data suggest that an inflammatory placental microenvironment at early pregnancy in infected queens may result, in part, from dysregulation of the Treg/Th17 balance.

Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains xiv, 144 p.; also includes graphics (some col.). Includes abstract and vita. Co-advisors: Lawrence E. Mathes and Maria H. Neff, Dept. of Veterinary Biosciences. Includes bibliographical references (p. 122-144).

In the present studies, the essential role of inducer cells for the induction of soluble anti-viral activity against feline immunodeficiency virus (FIV) was investigated. Induction of suppression of FIV replication was found to not strictly require autologous cells and was probably not FIV specific. Suppression was maximum when the inducer cells and the effector CD8+ T cells were in contact with each other, suggesting a potential role for membrane antigen interactions and/or cytokines in the induction process. Additionally, flow cytometry analysis demonstrated a significant increase in the percentage of CD8+ B7-1+ T cells in the peripheral blood of chronically FIV infected cats as compared with uninfected cats. Examination of the FIV V3-V4 envelope sequences from PBMC, lymph nodes and spleen from six cats chronically infected from three to six years with the molecular clone of FIV-PPR did not demonstrate viral variants specific for the tissues examined, emphasizing the critical role of the initial diversity and virulence of the infecting virus inoculum. Additionally, in vitro CD8+ T cell antiviral activity demonstrated by four of the six cats could have led to the control of virus replication in vivo, resulting in the uniform viral variants observed. Infection of specific pathogen free cats with FIV-TX53, an FIV isolate that belongs to an emerging subtype more closely related to FIV clade B, demonstrated an acute stage infection characterized by lymphoadenopathy and a viral dose dependent decline of CD4+/CD8+ T cell ratios below 1 by 11 weeks post infection. Interestingly, an expansion of CD8 low population of CD8+ T cells was observed in the infected cats. The soluble antiviral activity generated from inducer T cell stimulated CD8+ T cells from FIV-A-PPR infected cats also suppressed in vitro replication of the emerging FIV-TX53 and FIV-TX078 isolates. This is the first report demonstrating that the CD8+ T cell antiviral activity is inter-clade effective among FIV strains. As the success of a FIV vaccine could be hampered by occurrence of highly divergent viral variants in the fields, the exploitation of this innate, soluble anti-FIV activity could contribute to the design of novel, safe and complementary anti-FIV therapeutic strategies.

In the present studies, the essential role of inducer cells for the induction of soluble anti-viral activity against feline immunodeficiency virus (FIV) was investigated. Induction of suppression of FIV replication was found to not strictly require autologous cells and was probably not FIV specific. Suppression was maximum when the inducer cells and the effector CD8+ T cells were in contact with each other, suggesting a potential role for membrane antigen interactions and/or cytokines in the induction process. Additionally, flow cytometry analysis demonstrated a significant increase in the percentage of CD8+ B7-1+ T cells in the peripheral blood of chronically FIV infected cats as compared with uninfected cats. Examination of the FIV V3-V4 envelope sequences from PBMC, lymph nodes and spleen from six cats chronically infected from three to six years with the molecular clone of FIV-PPR did not demonstrate viral variants specific for the tissues examined, emphasizing the critical role of the initial diversity and virulence of the infecting virus inoculum. Additionally, in vitro CD8+ T cell antiviral activity demonstrated by four of the six cats could have led to the control of virus replication in vivo, resulting in the uniform viral variants observed. Infection of specific pathogen free cats with FIV-TX53, an FIV isolate that belongs to an emerging subtype more closely related to FIV clade B, demonstrated an acute stage infection characterized by lymphoadenopathy and a viral dose dependent decline of CD4+/CD8+ T cell ratios below 1 by 11 weeks post infection. Interestingly, an expansion of CD8 low population of CD8+ T cells was observed in the infected cats. The soluble antiviral activity generated from inducer T cell stimulated CD8+ T cells from FIV-A-PPR infected cats also suppressed in vitro replication of the emerging FIV-TX53 and FIV-TX078 isolates. This is the first report demonstrating that the CD8+ T cell antiviral activity is inter-clade effective among FIV strains. As the success of a FIV vaccine could be hampered by occurrence of highly divergent viral variants in the fields, the exploitation of this innate, soluble anti-FIV activity could contribute to the design of novel, safe and complementary anti-FIV therapeutic strategies.

Thesis--Auburn University, 2008. / Abstract. Vita. Includes bibliographical references (p. 60-90).

Orientador: João Pessoa Araújo Júnior / Banca: Lenice do Rosário de Souza / Banca: Alexandre Secorum Borges / Resumo: O Vírus da Imunodeficiência Felina (FIV) é um lentivírus que causa uma síndrome de imunodeficiência em gatos domésticos. O FIV tem sido particularmente utilizado em estudos de resistência viral aos análogos de nucleosídeos devido a Transcriptase Reversa (TR) apresentar propriedades físicas, catalíticas e sensibilidade às drogas semelhantes à TR do HIV. Os objetivos desse trabalho foram tratar com AZT gatos naturalmente infectados com o FIV, fazer o monitoramento da carga viral e DNA proviral por PCR em tempo real e monitoramento genético por seqüenciamento. Dos 12 animais infectados, 6 receberam o AZT na dose de 10mg/kg/dia e 6 receberam placebo. Durante 96 dias de tratamento, o plasma e sangue destes animais foram analisado com relação à carga viral e concentração relativa de DNA proviral utilizando-se a técnica de quantificação relativa por PCR em tempo real com SYBR Green, desenvolvida por nossa equipe. Além disso, foi realizado o sequenciamento genético da região que codifica a TR de 3 dos animais. Foi realizada com sucesso a padronização da PCR em tempo real para quantificação relativa do FIV. Não houve diferença estatisticamente significativa da carga viral ou do DNA proviral entre os grupos tratado e controle. O seqüenciamento genético revelou a presença de lisina na posição 41 do sítio ativo da TR. A presença deste aminoácido confere até 4 vezes menor sensibilidade ao AZT em mutantes do HIV. Por possuir alta estabilidade genética, supomos que os vírus dos demais animais não sequenciados possuem também a 41-lisina A presença da 41-lisina pode ser uma das possíveis explicações para a falha do tratamento com AZT. Outra hipótese é a de que a dose fornecida não foi adequada. / Abstract: Feline Immunodeficiency Virus (FIV) is a lentivirus which causes a progressive disruption of the host's immune functions. FIV has been particularly used as a model for studies in retroviral resistance to nucleoside analogs because its similarities in physical properties, catalytic and sensitivity in comparison with HIV/RT. The aims of this work were to treat cats naturally infected with FIV, quantify viral load and proviral DNA by real time quantitative PCR with SYBR Green and analyze the viral nucleotide sequence. From 12 animals naturally infected, 6 received AZT at a dose of 10mg/kg/day and 6 received placebo. During 96 days of treatment, viral load and concentration of proviral DNA were measured by relative quantitative real time PCR developed by our staff. The nucleotide sequence of the RT encoding region was also achieved for 3 animals. The real time PCR relative quantification was successfully standardized for FIV. There was no significant statistical difference between treated and control groups. The nucleotide sequence revealed a lysine at position 41 on the enzyme active site. This lysine confers 4-fold decreased sensitivity to AZT in HIV RT-mutants. FIV subtype B has high genetic stability and we purposed that the other virus not sequenced have the same amino acid and hypothesized that this mutations can be one of the reasons determining the failure of the treatment. The other hypothesis is that the dose was not adequate. / Mestre

The role that amniotic fluid (AF) may play in HIV vertical infection is unresolved. We used the FIV-infected cat model to study this question. We hypothesized that AF may be a source of fetal infection if the virus is present in the fluids. However, virus neutralizing (VN) antibodies in AF may limit vertical transfer. Fetuses were delivered from FIV-infected queens by cesarean section at early and late gestation. AFs were aspirated from intact fetal membranes and tested for viral antigen and RNA and for FIV-specific antibody. Randomlyselected samples were tested for VN activity using a syncytium reduction assay. Neither FIV antigen nor RNA was detected in any AFs. AFs and parallel serum samples from early and late pregnancy were positive for FIV-specific antibody. VN activity was detected in three early-term AFs and a parallel serum, but not late-term AFs. AF appears to play no appreciable role in FIV vertical transmission.

A gengivite crônica e intratável observada em gatos infectados pelo vírus da imunodeficiência felina (FIV) é um problema bastante freqüente na clínica de pequenos animais. O papel do FIV na etiologia da estomatite persistente ainda está por ser determinado. As manifestações orais são freqüentemente os primeiros sintomas observados em pacientes humanos infectados pelo HIV e podem ser usadas como indicadores da progressão da doença. O objetivo do presente estudo foi quantificar os linfócitos T CD4+, T CD8+ e a razão CD4+/CD8+ em uma colônia de gatos com gengivite crônica e naturalmente infectados pelo FIV. Para tanto, foram utilizados 20 gatos, todos apresentando gengivite com graus variando de 1 a 4. Desse total, 10 gatos não eram infectados pelo FIV e os outros 10 felinos eram infectados pelo FIV. Utilizou-se como controle 20 gatos sem gengivite, sendo 10 infectados pelo FIV e outros 10 não infectados pelo Retrovírus. As contagens dos linfócitos T CD4+ e CD8+ foram realizadas utilizando-se a técnica de citometria de fluxo. Os resultados obtidos demonstraram que os gatos com gengivite e infectados pelo FIV apresentaram uma contagem significativamente menor de linfócitos T CD4+ quando comparado aos gatos com gengivite e não infectados pelo FIV. Não houve diferença significativa na contagem de linfócitos T CD8+ entre os gatos com gengivite, infectados ou não pelo FIV. A razão CD4+/CD8+ também se mostrou em declínio nos gatos com gengivite e infectados pelo FIV. Concluiu-se que nas condições do presente estudo, a infecção pelo FIV compromete a resposta imunológica de felino diante da inflamação gengival. / Chronic and intractable gingivitis in FIV-infected cats is a relatively common clinical problem in veterinary practice. The role of FIV in the etiology of persistent stomatitis is still undetermined. Oral manifestations often found in HIV-infected people are frequently the first clinical sign of the infection and can be considered as an indicator of the progression of the HIV infection. The purpose of this study was to evaluate the CD4+ and CD8+ T-lymphocytes count and CD4+:CD8+ ratio in a colony of cats with chronic gingivitis. To achieve these goals, a colony of twenty domestic shorthair cats was used. All cats had some degree of gingival inflammation with scores ranging from 1 through 4. Ten cats were FIV-positive and ten were FIV-negative. As a control, twenty cats without gingivitis were used (ten cats were FIV-positive and ten were FIV-negative). CD4+ and CD8+ T-lymphocytes counts were performed by means of flow cytometry in all forty cats and results compared. The results showed that cats with gingivitis and FIV-infected had a lower CD4+ T cells count than cats with gingivitis but not FIV-infected. There was no difference in CD8+ T lymphocytes count among the cats with gingivitis infected or not with the FIV. The CD4+:CD8+ ratio was lower in cats with gingivitis and FIV-infected. One can conclude that FIV infection induces immunological disorders in cats with gingival inflammation.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-08-24T19:19:55Z No. of bitstreams: 1 2016 - Marli Sidoni.pdf: 1735106 bytes, checksum: 03907fc3437a44bf96b94d6040184407 (MD5) / Made available in DSpace on 2018-08-24T19:19:55Z (GMT). No. of bitstreams: 1 2016 - Marli Sidoni.pdf: 1735106 bytes, checksum: 03907fc3437a44bf96b94d6040184407 (MD5) Previous issue date: 2016-12-01 / This work focused on deploying IgY technology into developing ELISA immunoenzymatic test to FIV diagnose, contributing to the establishment of a production model for a kit in the field of veterinary medicine. The first stage of this work consisted in analyzing the literature of the veterinary documents aimed to diagnose animal diseases, as stated in our current legislation. The access to both national and international optimized the enlightenment of developing parameters and method validation criteria for the development of this new product, named ELISA r-p24 IgY. The second stage consisted in establishing purification production and physicochemical characterization of the recombinant protein p24 of FIV. The biological activity maintenance was proved by way of Western Blot test with the banda presence of approximately 25 kDa, referring to the p24 protein. The third stage was to obtain IgY cat anti-IgG, derived from hens inoculation. The kinetics were monitored by ELISA and the outcome demonstrated that as of the second week, there was a gradual increase in antibody in the yolk, and remained high throughout the period of five months. Reference to the chicken 1, the average concentration was 40,1 mg/mL e for the chicken 2 was 32,2 mg/mL, throughout the period of 5 months. The fourth stage was the use of IgY technology to develop, standardize e validate the r-p24 IgY ELISA related to its use to diagnose the infection caused by FIV. The results were: 99% accuracy, 97.7% sensitivity, 99.5% specificity and 99.1% kappa index. In the fifth stage was carried out a comparative study between ELISA r-p24 IgY and ELISA r-p24 IgG, and it was demonstrated superior performance of the ELISA r-p24 IgY. The ELISA r-p24 IgY to have favorable characteristics from a commercial perspective, such as high precision and maintenance of reactivity for a minimum period of 12 months. Therefore, the above described procedure was efficient and enabled the development of a FIV test. The predominance of IgY technology may contribute to research and development of new tests, following both international regulation related to animal welfare and validation, thus boosting national development of diagnostic kits, for the benefit of human health or animal. / O objetivo deste trabalho foi aplicar a tecnologia IgY no desenvolvimento de um teste imunoenzim?tico, ELISA, para o diagn?stico do FIV, contribuindo no estabelecimento de um modelo de produ??o para um kit nacional na ?rea de medicina veterin?ria. A primeira etapa do desenvolvimento deste trabalho consistiu na revis?o da literatura dos documentos pr?prios para produtos de uso veterin?rio, destinados a diagnosticar doen?as dos animais, disponibilizados na legisla??o vigente. A consulta aos documentos nacionais e internacionais potencializou o esclarecimento de par?metros de desempenho ou crit?rios de valida??o de m?todos, para o desenvolvimento deste novo produto, denominado ELISA r-p24 IgY. A segunda etapa consistiu no estabelecimento da produ??o, purifica??o e caracteriza??o f?sico-qu?mica da prote?na recombinante p24 do FIV. A preserva??o da atividade biol?gica foi demonstrada por Western Blot com a presen?a de uma banda pept?dica de aproximadamente 25 kDa, referente ? prote?na r-p24. A terceira etapa deste trabalho, consistiu na obten??o de anticorpos IgY anti-IgG de gato, a partir da inocula??o em galinhas poedeiras. A cin?tica foi acompanhada por ELISA demonstrando um aumento gradativo do t?tulo de anticorpos na gema a partir da segunda semana, com um aumento significativo no 2? m?s, e mantendo-se elevado durante todo o per?odo de cinco meses. As concentra??es m?dias de prote?nas na galinha 1 foi de 40,1 mg/mL a partir de uma gema e na galinha 2 foi de 32,2 mg/mL por gema, no per?odo de 5 meses. A quarta etapa deste trabalho consistiu no emprego da tecnologia IgY para o desenvolvimento, padroniza??o e a valida??o do teste de Elisa r-p24 IgY para o diagn?stico da infec??o causada pelo FIV. Os resultados obtidos foram: a acur?cia de 99%, a sensibilidade de 97,7%, a especificidade de 99,5%, e o ?ndice kappa de 99,1%. Na quinta etapa deste trabalho realizou-se o estudo comparativo do ELISA r-p24 IgY frente ao ELISA r-p24 IgG, e foi demonstrado o desempenho superior no ELISA r-p24 IgY. A valida??o do ELISA r-p24 IgY mostrou caracter?sticas desej?veis para o uso comercial, tais como alta precis?o e manuten??o da reatividade por um per?odo m?nimo de 12 meses. Conclui-se que o procedimento elaborado foi eficiente e possibilitou o desenvolvimento de um teste para o diagn?stico do FIV. O dom?nio da Tecnologia IgY poder? contribuir com a pesquisa e o desenvolvimento de novos ensaios atendendo ?s normas e diretrizes nacionais e internacionais, tanto de bem-estar animal como de valida??o, impulsionando o desenvolvimento nacional de kits diagn?sticos de interesse em sa?de humana ou animal.