RESUMEN
Transmission of pathogens from domestic animals to wildlife populations (spill-over) has precipitated local wildlife extinctions in multiple geographic locations. Identifying such events before they cause population declines requires differentiating spillover from endemic disease, a challenge complicated by a lack of baseline data from wildlife populations that are isolated from domestic animals. We tested sera collected from 12 ocelots (Leopardus pardalis) native to Barro Colorado Island, Panama, which is free of domestic animals, for antibodies to feline herpes virus, feline calicivirus, feline corona virus, feline panleukopenia virus, canine distemper virus, and feline immunodeficiency virus (FIV), typically a species-specific infection. Samples also were tested for feline leukemia virus antigens. Positive tests results were only observed for FIV; 50% of the ocelots were positive. We hypothesize that isolation of this population has prevented introduction of pathogens typically attributed to contact with domestic animals. The high density of ocelots on Barro Colorado Island may contribute to a high prevalence of FIV infection, as would be expected with increased contact rates among conspecifics in a geographically restricted population.
Asunto(s)
Anticuerpos Antivirales/sangre , Felidae/virología , Virus de la Inmunodeficiencia Felina/inmunología , Infecciones por Lentivirus/veterinaria , Animales , Animales Domésticos/virología , Animales Salvajes/virología , Conservación de los Recursos Naturales , Femenino , Infecciones por Lentivirus/epidemiología , Infecciones por Lentivirus/transmisión , Masculino , Panamá/epidemiología , Estudios Seroepidemiológicos , Especificidad de la EspecieRESUMEN
Although lentiviruses similar to feline immunodeficiency virus (FIV) are known to infect numerous felid species, the relative utility of assays used for detecting lentiviral infection has not been compared for many of these hosts. We tested bobcats (Lynx rufus), pumas (Felis concolor), and ocelots (Leopardus pardalis) for exposure to lentivirus using five different assays: puma lentivirus (PLV), African lion lentivirus (LLV), and domestic cat FIV-based immunoblots, a commercially available enzyme-linked immunosorbent assay (ELISA) kit, and nested polymerase chain reaction (PCR). Puma lentivirus immunoblots identified more seropositive individuals than the other antibody-detection assays. The commercial ELISA provided a fair ability to recognize seropositive samples when compared with PLV immunoblot for screening bobcats and ocelots, but not pumas. Polymerase chain reaction identified fewer positive samples than PLV immunoblot for all three species. Immunoblot results were equivalent whether the sample tested was serum, plasma, or whole blood. The results from this study and previous investigations suggest that the PLV immunoblot has the greatest ability to detect reactive samples when screening wild felids of North America and is unlikely to produce false positive results. However, the commercial ELISA kit may provide an adequate alternative for screening of some species and is more easily adapted to field conditions.
Asunto(s)
Felidae/virología , Infecciones por Lentivirus/veterinaria , Lentivirus/aislamiento & purificación , Lynx/virología , Puma/virología , Animales , Animales Salvajes/virología , Diagnóstico Diferencial , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Femenino , Immunoblotting/métodos , Immunoblotting/veterinaria , Lentivirus/inmunología , Infecciones por Lentivirus/diagnóstico , Masculino , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/veterinaria , Especificidad de la EspecieRESUMEN
We recently described the development and validation of a highly sensitive and specific microsphere immunoassay capable of simultaneously quantifying three domestic cat cytokines in tissue culture supernatant. Here we describe the modification of this assay to measure interferon gamma (IFNγ), interleukin (IL)-10 and IL-12/IL-23 p40 (IL-12/23) in domestic cat plasma, report values obtained from plasma collected after feline immunodeficiency virus (FIV) exposure, and compare plasma concentrations to blood cell mRNA expression. The validated quantitation limits of this assay are 31-1000 pg/ml for IFNγ, 63-2000 pg/ml for IL-10, and 20-625 pg/ml for IL-12/23. Plasma cytokine levels from domestic cats infected with pathogenic and/or apathogenic FIV were determined at 3-4 and 7-8 weeks post-infection. IL-12/23 was elevated (p<0.05) during acute infection with both FIV strains in two similar studies, conducted five years apart in different feline cohorts (n=44 total animals). IL-12/23 concentrations ranged from 377 to 1904 pg/ml in naïve cats and 552 to 3460 pg/ml in infected cats. In contrast, the majority of plasma samples had IFNγ and IL-10 concentrations below the lowest standard tested. The inability to consistently detect levels of IFNγ and IL-10 in plasma, despite the fact that mRNA changes were detected, suggests that these cytokines may be secreted and/or cleared in a more highly regulated manner than IL-12/23, or perhaps exert local effects under tighter peripheral constraints and/or at a lower effective concentration.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida del Felino/inmunología , Inmunoensayo/métodos , Interleucina-12/sangre , Interleucina-23/sangre , Enfermedad Aguda , Animales , Gatos , Interferón gamma/sangre , Interferón gamma/genética , Interleucina-10/sangre , Interleucina-10/genética , Microesferas , ARN Mensajero/análisisRESUMEN
We developed a feline model of lentiviral cross-species transmission using a puma lentivirus (PLV or FIV(Pco)) which infects domestic cats but does not cause disease. Infection with PLV protects cats from CD4+ T-cell decline caused by subsequent infection with virulent feline immunodeficiency virus (FIV). Previous studies implicate innate immune and/or cellular restriction mechanisms for FIV disease attenuation in PLV-infected cats. In this study, we evaluated viral infection and cytokine mRNA transcription in 12 different tissue reservoirs approximately five months post infection. We quantitated tissue proviral load, viral mRNA load and relative transcription of IL-10, IL-12p40 and IFNγ from tissues of cats exposed to FIV, PLV or both viruses and analyzed these parameters using a multivariate statistical approach. The distribution and intensity of FIV infection and IFNγ transcription differed between single and co-infected cats, characterized by higher FIV proviral loads and IFNγ expression in co-infected cat tissues. Variability in FIV mRNA load and IFNγ was significantly more constrained in co-infected versus singly infected cat tissues. Single-infected:co-infected ratios of FIV mRNA load compared to FIV proviral load indicated that active viral transcription was apparently inhibited during co-infection. These results indicate that previous PLV infection increases activation of tissue innate immunity and constrains the ability of FIV to productively infect tissue reservoirs of infection for months, independent of FIV proviral load, supporting a model in which innate immunity and/or modulation of target cell susceptibility play a key role in PLV-induced protection from FIV disease.
Asunto(s)
Anticuerpos Antivirales/sangre , Enfermedades de los Gatos/inmunología , Protección Cruzada , Virus de la Inmunodeficiencia Felina/inmunología , Infecciones por Lentivirus/veterinaria , Puma/virología , Animales , Animales Domésticos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Enfermedades de los Gatos/transmisión , Enfermedades de los Gatos/virología , Gatos , Coinfección , Inmunidad Innata , Virus de la Inmunodeficiencia Felina/genética , Virus de la Inmunodeficiencia Felina/patogenicidad , Interferón gamma/genética , Interleucina-10/genética , Subunidad p40 de la Interleucina-12/genética , Infecciones por Lentivirus/inmunología , Infecciones por Lentivirus/transmisión , Infecciones por Lentivirus/virología , Análisis Multivariante , ARN Mensajero/genética , ARN Viral/genética , Carga Viral , Replicación Viral/fisiologíaRESUMEN
The use of cells as a cryopreserved, readily available reagent has facilitated high-throughput screening of new drug candidates by bioassay. This practice is considerably less labor intensive and allows more flexibility in laboratory testing than traditional continuous cell culture. We have shown that this practice can be adapted to cell proliferation and reporter gene assay formats used in routine sample testing for determination of relative potency of commercial product in a Quality Control Laboratory. The ability to use the same, optimized population of cells provides consistency in an assay over time. Measures of assay performance to indicate maintenance of the validated state of a method and to determine benefit on variation in potency results were compared between cultured and cryopreserved (frozen ready-to-plate) cells. Control of the cellular component, which is the most variable aspect of most cell based potency assays, allowed detection of more minor contributors to variability. In a cell proliferation assay format, the final result was a highly precise bioassay (repeatability CV of 2%). An improvement in process capability (Cp) was noted when ready-to-plate cells were used in the studies completed over long periods of time.
Asunto(s)
Proliferación Celular , Técnicas Genéticas , Animales , Línea Celular , Criopreservación , Ratones , Reproducibilidad de los ResultadosRESUMEN
Immune dysregulation initiated by a profound loss of CD4+ T-cells is fundamental to HIV-induced pathogenesis. Infection of domestic cats with a non-pathogenic lentivirus prevalent in the puma (puma lentivirus, PLV or FIV(pco)) prevented peripheral blood CD4+ T-cell depletion caused by subsequent virulent FIV infection. Maintenance of this critical population was not associated with a significant decrease in FIV viremia, lending support to the hypothesis that direct viral cytopathic effect is not the primary cause of immunodeficiency. Although this approach was analogous to immunization with a modified live vaccine, correlates of immunity such as a serum-neutralizing antibody or virus-specific T-cell proliferative response were not found in protected animals. Differences in cytokine transcription profile, most notably in interferon gamma, were observed between the protected and unprotected groups. These data provide support for the importance of non-adaptive enhancement of the immune response in the prevention of CD4+ T-cell loss.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Virus de la Inmunodeficiencia Felina/patogenicidad , Infecciones por Lentivirus/inmunología , Infecciones por Lentivirus/virología , Animales , Recuento de Linfocito CD4 , Gatos , Efecto Citopatogénico Viral , Femenino , Expresión Génica , Interferón gamma/genética , Infecciones por Lentivirus/sangre , Infecciones por Lentivirus/genética , Linfopenia/etiología , Linfopenia/prevención & control , Masculino , Provirus/aislamiento & purificación , ARN Mensajero/sangre , ARN Mensajero/genética , VirulenciaRESUMEN
Domestic cats develop an asymptomatic, productive infection with a feline immunodeficiency virus (PLV) derived from a naturally infected cougar (P. concolor). We previously demonstrated that there are extensive G to A substitutions, characteristic of host cytidine deaminase editing, and positive selection on reverse transcriptase in the PLV genome during this cross-species infection. In this study, we evaluated full-length viral genomes from each of four cats infected with PLV to determine if viral recombination occurred during this single source infection. Recombination rates were measurable in three of the four infected cats. In two of these animals, a single site in reverse transcriptase was under positive selection and there was significant topological incongruence among individual genes in the 3' half of the genomes. The break point was proximate to a splice site used for accessory gene expression. Our data indicate that recombination can facilitate lentivirus persistence in unfavorable environments such as a new host species.
Asunto(s)
Genoma Viral , Virus de la Inmunodeficiencia Felina/crecimiento & desarrollo , Virus de la Inmunodeficiencia Felina/genética , Infecciones por Lentivirus/virología , Recombinación Genética , Adaptación Biológica , Animales , Secuencia de Bases , Gatos , Modelos Animales de Enfermedad , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , ADN Polimerasa Dirigida por ARN/genética , Selección Genética , Homología de Secuencia de Ácido NucleicoRESUMEN
Factors that restrict a virus from establishing productive infection in a new host species are important to understand because cross-species transmission events are often associated with emergent viral diseases. To determine the evolutionary pressures on viruses in new host species, we evaluated the molecular evolution of a feline immunodeficiency virus derived from a wild cougar, Puma concolor, during infection of domestic cats. Analyses were based on the coding portion of genome sequences recovered at intervals over 37 weeks of infection of six cats inoculated by either intravenous or oral-nasal routes. All cats inoculated intravenously, but only one inoculated orally-nasally, became persistently viremic. There were notable accumulations of lethal errors and predominance of G-to-A alterations throughout the genome, which were marked in the viral polymerase gene, pol. Viral structural (env and gag) and accessory (vif and orfA) genes evolved neutrally or were under purifying selection. However, sites under positive selection were identified in reverse transcriptase that involved residues in the nucleotide binding pocket or those contacting the RNA-DNA duplex. The findings of extensive G-to-A alterations in this cross-species infection are consistent with the recently described editing of host cytidine deaminase on lentivirus genomes. Additionally, we demonstrate that the primary site of hypermutation is the viral pol gene and the dominant selective force acting on this feline immunodeficiency virus as it replicates in a new host species is on key residues of the virus polymerase.
Asunto(s)
Evolución Molecular , Síndrome de Inmunodeficiencia Adquirida del Felino/virología , Genes pol , Virus de la Inmunodeficiencia Felina/patogenicidad , Mutación , Selección Genética , Animales , Animales Domésticos , Enfermedades de los Gatos/inmunología , Enfermedades de los Gatos/fisiopatología , Enfermedades de los Gatos/virología , Gatos , Síndrome de Inmunodeficiencia Adquirida del Felino/fisiopatología , Virus de la Inmunodeficiencia Felina/genética , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Puma , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
A high percentage of free-ranging pumas (Felis concolor) are infected with feline lentiviruses (puma lentivirus, feline immunodeficiency virus Pco [FIV-Pco], referred to here as PLV) without evidence of disease. PLV establishes productive infection in domestic cats following parenteral exposure but, in contrast to domestic cat FIV, it does not cause T-cell dysregulation. Here we report that cats exposed to PLV oro-nasally became infected yet rapidly cleared peripheral blood mononuclear cell (PBMC) proviral load in the absence of a correlative specific immune response. Two groups of four specific-pathogen-free cats were exposed to PLV via the mucosal (oro-nasal) or parenteral (i.v.) route. All animals were PBMC culture positive and PCR positive within 3 weeks postinfection and seroconverted without exhibiting clinical disease; however, three or four oro-nasally infected animals cleared circulating proviral DNA within 3 months. Antibody titers reached higher levels in animals that remained persistently infected. PLV antigen-induced proliferation was slightly greater in mucosally inoculated animals, but no differences were noted in cytotoxic T-lymphocyte responses or cytokine profiles between groups. The distribution of virus was predominantly gastrointestinal as opposed to lymphoid in all animals in which virus was detected at necropsy. Possible mechanisms for viral clearance include differences in viral fitness required for crossing mucosal surfaces, a threshold dose requirement for persistence, or an undetected sterilizing host immune response. This is the first report of control of a productive feline or primate lentivirus infection in postnatally exposed, seropositive animals. Mechanisms underlying this observation will provide clues to containment of immunodeficiency disease and could prompt reexamination of vaccine-induced immunity against human immunodeficiency virus and other lentiviruses.