Julie Terwee

Feline Lentivirus Evolution in Cross-Species Infection Reveals Extensive G-to-A Mutation and Selection on Key Residues in the Viral Polymerase

ABSTRACT 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.

ASSESSING FLAVIVIRUS, LENTIVIRUS, AND HERPESVIRUS EXPOSURE IN FREE-RANGING RING-TAILED LEMURS IN SOUTHWESTERN MADAGASCAR

The ring-tailed lemur (Lemur catta) is an endangered species found in southwestern Madagascar, and understanding infectious disease susceptibility is an essential step towards the preservation of wild and captive lemur populations. Lemurs are primates that are widely dispersed throughout the island of Madagascar and may serve as hosts or reservoirs for zoonotic infections. The aim of this study was to determine the prevalence of antibodies to West Nile virus (WNV), simian immunodeficiency virus (SIV), and herpes simplex virus type 1 (HSV-1) in a population of free-ranging ring-tailed lemur from the Beza Mahafaly Special Reserve, Madagascar. Samples were collected from 50 animals during field capture studies in June and July 2004 and assayed for presence of viral antibodies during the 12 mo following collection. Forty-seven of the 50 lemurs sampled had antibodies against WNV detectable by epitope-blocking enzyme-linked immuno-sorbent assay (ELISA). In addition, 50 of 50 samples had titers against WNV ranging from 80 to ≥ 1,280 using plaque reduction neutralization test (PRNT90). Ten lemurs had antibodies against lentiviral antigens as determined by Western blot analysis. None of the lemurs had antibodies against HSV-1 using ELISA.

Frequent Transmission of Immunodeficiency Viruses among Bobcats and Pumas

ABSTRACT With the exception of human immunodeficiency virus (HIV), which emerged in humans after cross-species transmissions of simian immunodeficiency viruses from nonhuman primates, immunodeficiency viruses of the family Lentiviridae represent species-specific viruses that rarely cross species barriers to infect new hosts. Among the Felidae, numerous immunodeficiency-like lentiviruses have been documented, but only a few cross-species transmissions have been recorded, and these have not been perpetuated in the recipient species. Lentivirus seroprevalence was determined for 79 bobcats (Lynx rufus) and 31 pumas (Puma concolor) from well-defined populations in Southern California. Partial genomic sequences were subsequently obtained from 18 and 12 seropositive bobcats and pumas, respectively. Genotypes were analyzed for phylogenic relatedness and genotypic composition among the study set and archived feline lentivirus sequences. This investigation of feline immunodeficiency virus infection in bobcats and pumas of Southern California provides evidence that cross-species infection has occurred frequently among these animals. The data suggest that transmission has occurred in multiple locations and are most consistent with the spread of the virus from bobcats to pumas. Although the ultimate causes remain unknown, these transmission events may occur as a result of puma predation on bobcats, a situation similar to that which fostered transmission of HIV to humans, and likely represent the emergence of a lentivirus with relaxed barriers to cross-species transmission. This unusual observation provides a valuable opportunity to evaluate the ecological, behavioral, and molecular conditions that favor repeated transmissions and persistence of lentivirus between species.

Puma Lentivirus Is Controlled in Domestic Cats after Mucosal Exposure in the Absence of Conventional Indicators of Immunity

ABSTRACT 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.

Prevention of immunodeficiency virus induced CD4+ T-cell depletion by prior infection with a non-pathogenic virus.

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.

Temporal association of large granular lymphocytosis, neutropenia, proviral load, and FasL mRNA in cats with acute feline immunodeficiency virus infection.

During acute feline immunodeficiency virus-C(PGammar) (FIV-C-PG) infection, we observed that cats develop large granular lymphocyte (LGL) lymphocytosis concurrent with a marked neutropenia that is temporally associated with the rise and fall of FIV-C-PG proviral loads. LGLs, generally considered to be analogous to natural killer (NK) cells, can also be highly cytolytic CD8/CD57 T cells. Neutropenia has been reported during acute human immunodeficiency virus (HIV-1) infection, but there is a paucity of information describing the pathogenesis of this condition. During HIV-1 infection, LGLs have been shown to be both CD16(+) NK cells and CD8(+)/CD57(+) T cells, but an association with neutropenia has not been described. However, neutropenia with concurrent LGL lymphocytosis has been demonstrated in both LGL leukemia and common variable immunodeficiency syndrome in people, and in both syndromes, an increase in soluble Fas ligand (FasL) has been associated with neutrophil apoptosis leading to neutropenia. Flow cytometric analysis demonstrated increases in CD56 and CD8 peripheral blood cell surface expression during acute FIV-C-PG infection. Expression of FasL mRNA was increased at the same time points as these peripheral hematologic abnormalities, and also decreased as FIV-C-PG proviral load reached set point. We describe an interesting temporal association between innate immune responses and viral load during acute FIV-C-PG infection, which has similarities to HIV-1 infection and other immune dyscrasias of people, and which may contribute to the neutropenia and LGL lymphocytosis during FIV-C-PG infection.

Variability in assays used for detection of lentiviral infection in bobcats (Lynx rufus), pumas (Puma concolor), and ocelots (Leopardus pardalis).

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.

Multivariate Statistical Analyses Demonstrate Unique Host Immune Responses to Single and Dual Lentiviral Infection

Background Feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV) are recently identified lentiviruses that cause progressive immune decline and ultimately death in infected cats and humans. It is of great interest to understand how to prevent immune system collapse caused by these lentiviruses. We recently described that disease caused by a virulent FIV strain in cats can be attenuated if animals are first infected with a feline immunodeficiency virus derived from a wild cougar. The detailed temporal tracking of cat immunological parameters in response to two viral infections resulted in high-dimensional datasets containing variables that exhibit strong co-variation. Initial analyses of these complex data using univariate statistical techniques did not account for interactions among immunological response variables and therefore potentially obscured significant effects between infection state and immunological parameters. Methodology and Principal Findings Here, we apply a suite of multivariate statistical tools, including Principal Component Analysis, MANOVA and Linear Discriminant Analysis, to temporal immunological data resulting from FIV superinfection in domestic cats. We investigated the co-variation among immunological responses, the differences in immune parameters among four groups of five cats each (uninfected, single and dual infected animals), and the “immune profiles” that discriminate among them over the first four weeks following superinfection. Dual infected cats mount an immune response by 24 days post superinfection that is characterized by elevated levels of CD8 and CD25 cells and increased expression of IL4 and IFNγ, and FAS. This profile discriminates dual infected cats from cats infected with FIV alone, which show high IL-10 and lower numbers of CD8 and CD25 cells. Conclusions Multivariate statistical analyses demonstrate both the dynamic nature of the immune response to FIV single and dual infection and the development of a unique immunological profile in dual infected cats, which are protected from immune decline.

Ocelots on Barro Colorado Island are infected with feline immunodeficiency virus but not other common feline and canine viruses.

Transmission of pathogens from domestic animals to wildlife populations (spillover) 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.

Microsphere immunoassay for the detection of cytokines in domestic cat (Felis catus) plasma: elevated IL-12/23 in acute feline immunodeficiency virus infections.

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.