The macaque gut microbiome in health, lentiviral infection, and chronic enterocolitis.

The vertebrate gut harbors a vast community of bacterial mutualists, the composition of which is modulated by the host immune system. Many gastrointestinal (GI) diseases are expected to be associated with disruptions of host-bacterial interactions, but relatively few comprehensive studies have been reported. We have used the rhesus macaque model to investigate forces shaping GI bacterial communities. We used DNA bar coding and pyrosequencing to characterize 141,000 sequences of 16S rRNA genes obtained from 100 uncultured GI bacterial samples, allowing quantitative analysis of community composition in health and disease. Microbial communities of macaques were distinct from those of mice and humans in both abundance and types of taxa present. The macaque communities differed among samples from intestinal mucosa, colonic contents, and stool, paralleling studies of humans. Communities also differed among animals, over time within individual animals, and between males and females. To investigate changes associated with disease, samples of colonic contents taken at necropsy were compared between healthy animals and animals with colitis and undergoing antibiotic therapy. Communities from diseased and healthy animals also differed significantly in composition. This work provides comprehensive data and improved methods for studying the role of commensal microbiota in macaque models of GI diseases and provides a model for the large-scale screening of the human gut microbiome.

Cyclophilin A interacts with diverse lentiviral capsids.

BACKGROUND: The capsid (CA) protein of HIV-1 binds with high affinity to the host protein cyclophilin A (CypA). This binding positively affects some early stage of the viral life-cycle because prevention of binding either by drugs that occupy that active site of cyclophilin A, by mutation in HIV-1 CA, or RNAi that knocks down intracellular CypA level diminishes viral infectivity. The closely related lentivirus, SIVcpz also binds CypA, but it was thought that this interaction was limited to the HIV-1/SIVcpz lineage because other retroviruses failed to interact with CypA in a yeast two-hybrid assay. RESULTS: We find that diverse lentiviruses, FIV and SIVagmTAN also bind to CypA. Mutagenesis of FIV CA showed that an amino acid that is in a homologous position to the proline at amino acid 90 of HIV-1 CA is essential for FIV interactions with CypA. CONCLUSION: These results demonstrate that CypA binding to lentiviruses is more widespread than previously thought and suggest that this interaction is evolutionarily important for lentiviral infection.

Lentivirus-specific cytotoxic T-lymphocyte responses are rapidly lost in thymectomized cats infected with feline immunodeficiency virus.

To what extent the thymus is needed to preserve the virus-specific cytotoxic T-lymphocyte (CTL) response of lentivirus-infected adults is unclear. Presented here is the first definitive study using thymectomized (ThX) animals to directly evaluate the contribution of thymic function to lentivirus-specific CTL response and the control of lentivirus infections. ThX and mock-ThX cats were inoculated with feline immunodeficiency virus (FIV) and monitored for their FIV-specific CTL responses. Early in infection, both FIV-ThX and FIV-mock-ThX cats produced similar CTL responses, but surprisingly, after 20 weeks, the FIV-ThX cats showed a statistically significant loss of FIV-specific CTL activity, while FIV-infected cats with intact thymuses continued to maintain FIV-specific CTL. The loss of CTL did not affect plasma virus load, which remained elevated for both groups. These results emphasize the importance of thymic integrity in maintaining immunity to lentiviruses, but also bring into question the notion that virus load is regulated predominantly by the virus-specific CTL response.

Effect of preexisting FeLV infection or FeLV and feline immunodeficiency virus coinfection on pathogenicity of the small variant of Haemobartonella felis in cats.

OBJECTIVE: To investigate the effects of preexisting FeLV infection or FeLV and feline immunodeficiency (FIV) coinfection on the pathogenicity of the small variant of Haemobartonella felis (Hfsm, California variant) in cats. ANIMALS: 20 FeLV infected, 5 FeLV-FIV coinfected, and 19 retrovirus-free cats. PROCEDURES: A client-owned cat, coinfected with FeLV and Hfsm, was the source for Hfsm. Inoculum 1 (FeLV free) was obtained by passage of source Hfsm through 4 FeLV-resistant cats. Inoculum 2 was obtained by further passage of Hfsm (inoculum 1) through 2 specific pathogen-free cats. RESULTS: A mild-to-moderate anemia started 21 days after inoculation, with its nadir occurring at 35 to 42 days after inoculation. Infection with Hfsm induced greater decrease in hemoglobin concentration in FeLV infected cats, compared with retrovirus free cats. Reticulocytosis, macrocytosis, and polychromasia of erythrocytes developed in anemic cats regardless of retrovirus infection status. Mean neutrophil counts decreased during the hemolytic episode. For most cats, the anemia was transient. Four FeLV infected cats, 1 of which was also FIV infected, developed fatal FeLV-associated myeloproliferative diseases. Of the surviving cats, 8 died over the next 24 months from other FeLV-related diseases. Hemolysis did not recur after the initial episode. Inoculum 1 induced more severe anemia than inoculum 2. CONCLUSIONS AND CLINICAL RELEVANCE: Our results support the clinical observation that cats coinfected with FeLV and H felis develop more severe anemia than cats infected with H felis alone. Infection with Hfsm may induce myeloproliferative disease in FeLV infected cats. The small variant of H felis may lose pathogenicity by passage through FeLV-free cats.

dUTPase-minus caprine arthritis-encephalitis virus is attenuated for pathogenesis and accumulates G-to-A substitutions.

The importance of the virally encoded dUTPase for CAEV replication, invasiveness, pathogenesis, and genetic stability was investigated in goats infected by viruses with single point (DU-G) and deletion (DU-1) mutations of the dUTPase gene (DU gene). The DU gene was found to be dispensable for CAEV replication in vivo as judged by times taken to seroconvert, frequencies of viral isolation, and tissue distribution of viral RNAs. DU- reversion at week 34 in one of three goats infected with the single point mutant DU-G, however, suggested that the viral dUTPase confers some advantages for replication in vivo. Moreover, we show that dUTPase is necessary for the timely development of bilateral arthritic lesions of the carpus. Finally, dUTPase was shown to efficiently prevent accumulation of G-to-A transitions in the viral genome.

Ovine lentivirus infection: an animal model for pediatric HIV infection?

While the incidence of the human immunodeficiency virus (HIV) infection has leveled off somewhat in homosexual men, infection in women, children and adolescents is rising. Unless effective preventive measures are introduced, the number of pediatric patients with HIV and related illnesses will continue to increase. Animal models play a key role in the understanding of the pathogenesis and in the establishment of therapeutic approaches of infectious diseases. Ovine lentivirus (OvLV) comprises a subgenus of the lentivirus genus in the family Retroviridae, that shares genotypic, phenotypic and pathogenic features with HIV. Infection of sheep with OvLV results in a progressive chronic disease characterized by cachexia and chronic active inflammation in the lungs, lymph nodes, joints, mammary gland and the central nervous system. Pulmonary lesions in OvLV-affected sheep consist of lymphoid interstitial pneumonia (LIP) and lyphocytic alveolitis. Similarly, these pulmonary lesions also occur in up to 40% of HIV-infected children and in some adults with AIDS. Neonatal lambs experimentally inoculated intratracheally with OvLV develop LIP in 5 to 6 months, thus shortening by several years the natural incubation period and resembling the shorter incubation period observed in children with HIV-associated LIP. However, unlike HIV, OvLV does not infect CD4+T lymphocytes; OvLV only infects and replicates in macrophages. Recent studies indicate that macrophage tropic HIV plays an important role in disease progression. Similarities between HIV and OvLV argue for the use of ovine lentivirus infection as a model to advance in the understanding of some of the aspects of HIV infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Oligopeptide-based enzyme immunoassay for ovine lentivirus antibody detection.

Ovine progressive pneumonia virus (OPPV) is a lentivirus which causes a progressive disease in sheep. Immunodominant epitopes have been identified in the envelope gp40 glycoprotein. Synthetic peptides representing these regions are able to detect the presence of OPPV antibodies in 96% of infected sheep.

Neuropathologic aspects of lentiviral infections.

Studies of lentiviral infections of various animals and man have shown that all may invade the CNS and induce pathological lesions. This is well established in infections with VV, CAEV, SIV, HIV-1, and FIV. Although VV and CAEV do not cause an overt immunodeficiency, they share several features pertinent for the establishment of neuropathologic lesions with those that induce immunodeficiency. This holds especially true for the initial steps and early CNS lesions. 1) Infection of the CNS is from the blood stream. Although a definite proof of how the different viruses cross the blood-brain barrier remains to be brought forward there are indications that it may occur through migration of infected monocytes and/or lymphocytes into the brain. Furthermore free virus may enter the CNS, either directly or through infection of endothelial cells. 2) The lesion pattern at least in initial stages is similar; that is, it consists of meningitis, perivascular infiltrations especially of the deep white matter, and inflammation of the choroid plexus. In visna a local amplification of the inflammatory response is frequently observed in choroid plexus often with formation of active lymphoid follicles. Multinucleated giant cells are prominent in HIV-1 and SIV infections, but rare in VV, and practically nonexistent in infections with FIV and CAEV, possibly a reflection of differences in virus replication. Myelin breakdown is a feature of various lentiviral infections but its mechanisms and morphological expression may vary. Sharply demarcated plaques of primary demyelination seem to be unique for VV infection and vacuolar myelopathy for infection with HIV-1. 3) The main target cells in the brain are cells of the monocyte/macrophage/microglial lineage. In visna infected monocytes are found but evidence for infection of the enigmatic resident microglial cells is still lacking. Infection, especially productive, of neuroectodermal cells is rare, but may, however be important for viral persistence. Infection of endothelial cells occurs in the various lentiviral infections and may play a part in viral entry into the CNS and contribute to tissue damage. 4) The discrepancy between the frequency of productively infected cells and cell types infected and extent and character of pathological lesions, indicates that a mechanism other than the direct effect of the virus contributes to the evolution of CNS lesions. In HIV-1 infection evidence, mainly obtained by in vitro studies, indicates that lesions are mediated by cytokines and other toxic factors secreted by inflammatory or glial cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Use of TrpE fusion protein to identify antigenic domains within the BIV envelope protein.

Nine different recombinant clones spanning various regions of the bovine immunodeficiency-like virus (BIV) envelope gene open reading frame were generated. These clones span the entire external glycoprotein as well as the transmembrane glycoprotein region. These proteins were expressed as fusions to the TrpE protein in E. coli. The levels of recombinant protein expressed varied, some clones expressed enough protein that can be detected in a Coomassie blue-stained gel, whereas other proteins could only be detected by Western blot analyses. A recombinant env protein representing the extracellular domain of the env protein was detected by BIV-infected bovine sera. In addition, a 134 amino acid peptide which may represent a major immunoreactive epitope was identified. This peptide is located at the amino terminus of the transmembrane glycoprotein and was specifically recognized by all BIV-infected calf sera tested. The identification of this epitope and the use of recombinant envelope protein will enable us to develop a more effective screening test to study the epidemiology of BIV infection.

Clinical aspects of Chlamydia psittaci infection in cats infected with feline immunodeficiency virus.

Two groups of six cats were established, one a control group and one infected with feline immunodeficiency virus (FIV) 18 months previously. The cats in both groups were inoculated with Chlamydia psittaci and the clinical progression of the infection was monitored by means of a clinical scoring system for 10 months. Haematological, serological and viral and chlamydial isolation studies were also made. The response of the FIV infected group to treatment with oxytetracycline was monitored in the 11th and 12th months. The FIV infection prolonged the duration of the clinical signs resulting from the infection with C psittaci and led to the development of chronic conjunctivitis. The haematological and antibody responses to C psittaci were comparable in the two groups. However, it was possible to isolate C psittaci from the FIV-infected cats up to day 270, when the treatment began, but only up to day 70 in the control group. In addition, it appeared that the infection with a secondary pathogen may have accelerated the clinical progression of the FIV infection.

Identification of three feline immunodeficiency virus (FIV) env gene subtypes and comparison of the FIV and human immunodeficiency virus type 1 evolutionary patterns.

Feline immunodeficiency virus (FIV) is a lentivirus associated with AIDS-like illnesses in cats. As such, FIV appears to be a feline analog of human immunodeficiency virus (HIV). A hallmark of HIV infection is the large degree of viral genetic diversity that can develop within an infected individual and the even greater and continually increasing level of diversity among virus isolates from different individuals. Our goal in this study was to determine patterns of FIV genetic diversity by focusing on a 684-nucleotide region encompassing variable regions V3, V4, and V5 of the FIV env gene in order to establish parallels and distinctions between FIV and HIV type 1 (HIV-1). Our data demonstrate that, like HIV-1, FIV can be separated into distinct envelope sequence subtypes (three are described here). Similar to that found for HIV-1, the pairwise sequence divergence within an FIV subtype ranged from 2.5 to 15.0%, whereas that between subtypes ranged from 17.8 to 26.2%. However, the high number of synonymous nucleotide changes among FIV V3 to V5 env sequences may also include a significant number of back mutations and suggests that the evolutionary distances among FIV subtypes are underestimated. Although only a few subtype B viruses were available for examination, the pattern of diversity between the FIV A and B subtypes was found to be significantly distinct; subtype B sequences had proportionally fewer mutations that changed amino acids, compared with silent changes, suggesting a more advanced state of adaptation to the host. No similar distinction was evident for HIV-1 subtypes. The diversity of FIV genomes within individual infected cats was found to be as high as 3.7% yet twofold lower than that within HIV-1-infected people over a comparable region of the env gene. Despite these differences, significant parallels between patterns of FIV evolution and HIV-1 evolution exist, indicating that a wide array of potentially divergent virus challenges need to be considered in FIV vaccine and pathogenesis studies.

Evaluation of ELISA for detection of antibodies to CAEV in milk.

Milk was found to be a suitable alternative specimen for the serological diagnosis of caprine arthritis-encephalitis virus (CAEV) using the ELISA. The relative sensitivity and specificity of testing individual milk samples as compared to individual sera was 96.4 and 97.3% respectively. The overall agreement between the testing of milk and sera was 96.9% and the correlation coefficient between testing sera and milk, 0.94%. The testing of bulk milk could be used to predict approximately the prevalence of CAEV infection in a dairy goat flock. It was estimated that a prevalence of about 1.6% to 7.5% could be detected in the ELISA when bulk milk samples from two infected goat flocks were tested. Either chilled milk samples or milk samples treated with merthiolate were found to be suitable for testing.

Detection of antibodies to caprine arthritis-encephalitis virus using recombinant gag proteins.

The coding sequences of the core proteins p17 and p28 of caprine arthritis-encephalitis virus (CAEV) were amplified using the polymerase chain reaction and cloned into the plasmid expression vector p-GEX-2T. Both p17 and p28 were expressed as fusion proteins with glutathione S-transferase. The recombinant proteins were affinity purified from induced bacterial lysates using glutathione-agarose beads. The purified proteins were used in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies against CAEV in goat sera and milk samples. Three different ELISA tests were developed based on p17, p28 or the combination of these two recombinant proteins (p17 + p28). A comparison was made to an ELISA based on purified whole virus particles and to agar immunodiffusion test (AGID). Sera with conflicting results in the different ELISA tests were examined by Western blotting. There was a high correlation between the ELISA tests based on p17 + p28 recombinant proteins and whole virus ELISA, with an estimated kappa value of 0.92. Only 72-75% of the sera that tested positive in these two ELISA tests were positive in AGID. Antibodies to CAEV were detected in significantly more animals when serum samples were tested compared to milk samples. Based on the time and materials required to prepare the reagents, the recombinant based ELISA test was less expensive than the whole virus ELISA.

Feline immunodeficiency virus infection of cats as a model to test the effect of certain in vitro selection pressures on the infectivity and virulence of resultant lentivirus variants.

Three groups of specific pathogen-free (SPF) domestic cats, each containing 5 animals, were infected with one of three closely related FIV variants and monitored for 36 weeks. A fourth group of 5 cats was sham-infected and served as uninfected controls. FIV variants included: (1) a fully virulent animal passaged FIV-Petaluma; (2) a Crandell feline kidney (CrFK) cell-adapted FIV-Petaluma (FIV-CrFK); and (3) a variant of FIV-CrFK (FIV-CrFKAZT) that had been selected in vitro for resistance to azidothymidine. Cats infected with fully virulent FIV-Petaluma strongly seroconverted, became persistently viremic, and exhibited lymphadenopathy, neutropenia, and inversion of the CD4+:CD8+ T cell ratio. Cats infected with FIV-CrFK seroconverted but the antibody responses were much weaker and more variable; two of the cats became transiently viremic and no hematologic abnormalities or clinical signs of illness other than a very mild lymphadenopathy were observed. None of the five cats inoculated with FIV-CrFKAZT seroconverted, became viremic, or exhibited any gross or hematologic signs of disease, even though proviral DNA was transiently detected in tissue following inoculation. This study demonstrates that the FIV infection model can be used to assess differences in the virulence of FIV variants, including variants selected for antiretroviral drug resistance.

Delayed seroconversion following naturally acquired caprine arthritis-encephalitis virus infection in goats.

One hundred eight milking goats from a dairy that had been using a modified caprine arthritis-encephalitis virus (CAEV) eradication program were tested for CAEV antibodies by serologic methods and for proviral CAEV DNA by use of polymerase chain reaction (PCR) technology. All goats were free of clinical symptoms of CAEV infection. Twenty-seven of the 108 goats were considered seropositive, on the basis of ELISA results. Proviral CAEV DNA was detected, using PCR techniques, in mononuclear leukocytes in blood samples obtained from 25 of the these 27 seropositive goats. Twenty of the 81 seronegative goats also had positive PCR test results. Ten of these goats seroconverted by 8 months later, and virus was readily isolated from mononuclear leukocytes in venous blood samples after the goats had seroconverted. Virus was also isolated from mononuclear leukocytes in blood samples collected from 4 of 11 goats that were seronegative, but had positive PCR test results. These results indicated that seroconversion can be delayed for many months following natural infection with CAEV. Delayed seroconversion appears to be a feature of CAEV infection, which may have direct implications for CAEV eradication programs and epidemiologic studies that rely on serologic methods to detect infected goats.