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Peripheral and central immune cell reservoirs in tissues from asymptomatic cats chronically infected with feline immunodeficiency virus

View Article: PubMed Central - PubMed

ABSTRACT

Feline immunodeficiency virus (FIV) infection in cats results in life-long viral persistence and progressive immunopathology. We have previously described a cohort of experimentally infected cats demonstrating a progressive decline of peripheral blood CD4+ T-cell over six years in the face of apparent peripheral viral latency. More recently we reported findings from this same cohort that revealed popliteal lymph node tissue as sites for ongoing viral replication suggesting that tissue reservoirs are important in FIV immunopathogenesis during the late asymptomatic phase of infection. Results reported herein characterize important tissue reservoirs of active viral replication during the late asymptomatic phase by examining biopsied specimens of spleen, mesenteric lymph node (MLN), and intestine from FIV-infected and uninfected control cats. Peripheral blood collected coincident with harvest of tissues demonstrated severe CD4+ T-cell depletion, undetectable plasma viral gag RNA and rarely detectable peripheral blood mononuclear cell (PBMC)-associated viral RNA (vRNA) by real-time PCR. However, vRNA was detectable in all three tissue sites from three of four FIV-infected cats despite the absence of detectable vRNA in plasma. A novel in situ hybridization assay identified B cell lymphoid follicular domains as microanatomical foci of ongoing FIV replication. Additionally, we demonstrated that CD4+ leukocyte depletion in tissues, and CD4+ and CD21+ leukocytes as important cellular reservoirs of ongoing replication. These findings revealed that tissue reservoirs support foci of ongoing viral replication, in spite of highly restricted viral replication in blood. Lentiviral eradication strategies will need address tissue viral reservoirs.

No MeSH data available.


Related in: MedlinePlus

Detection of virus infection in tissues by ex vivo viral reactivation.The protocol used for detection of leukocytes harboring replication competent proviruses is shown in this diagram. Replication competent and infectious virus was successfully propagated by ex vivo viral reactivation and detected by real-time PCR from all MLN and spleen tissue samples collected from FIV-infected cats as shown in the associated table. A positive detection is indicated by the + symbol, followed by the number of days in culture required for detection of infectious virus. A MLN was not collected from cat 165, and therefore was not tested for this cat (represented by the abbreviation N.A.). The LTNP cat 187 is designated in red and took a longer time to reactivate replication competent provirus from MLN and spleen-derived leukocytes.
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pone.0175327.g004: Detection of virus infection in tissues by ex vivo viral reactivation.The protocol used for detection of leukocytes harboring replication competent proviruses is shown in this diagram. Replication competent and infectious virus was successfully propagated by ex vivo viral reactivation and detected by real-time PCR from all MLN and spleen tissue samples collected from FIV-infected cats as shown in the associated table. A positive detection is indicated by the + symbol, followed by the number of days in culture required for detection of infectious virus. A MLN was not collected from cat 165, and therefore was not tested for this cat (represented by the abbreviation N.A.). The LTNP cat 187 is designated in red and took a longer time to reactivate replication competent provirus from MLN and spleen-derived leukocytes.

Mentions: Mesenteric lymph node and spleen-derived leukocytes cultured ex vivo from all FIV-infected cats demonstrated the capacity to produce infectious, replication competent virus as determined by an ex vivo reactivation assay (Fig 4). Clarified cell-free culture supernatants collected from unfractionated tissue-derived leukocytes cultures were passaged onto SPF FIV-negative feline PMBCs on days 7, 14, and 21. After 7 days of secondary culture, real-time PCR was performed to identify the presence or absence of vDNA in SPF PBMC genomic DNA (Fig 4). A positive PCR result indicated that the clarified supernatant contained infectious virus. Infectious virus was detected in culture supernatant at day 7 from splenic leukocytes in all cats, except for the FIV-infected LTNP animal, for which virus reactivation required 14 days in culture. Time in culture required for detectable virus production was generally longer for the MLN samples, ranging from seven days for cat 186, and 21 days for cats 184 and the LTNP cat 187. A MLN was not procured from cat 165. FIV provirus was not detected at any time point for MLN or spleen-derived leukocyte cultures from a FIV-uninfected cat (183, negative control). Statistically, a significant correlation did not exist between the unfractionated leukocyte proviral load of each tissue type from each cat and the days to reactivation (MLN r = 0.49, p = 0.67; Spleen r = -035, p = 0.65).


Peripheral and central immune cell reservoirs in tissues from asymptomatic cats chronically infected with feline immunodeficiency virus
Detection of virus infection in tissues by ex vivo viral reactivation.The protocol used for detection of leukocytes harboring replication competent proviruses is shown in this diagram. Replication competent and infectious virus was successfully propagated by ex vivo viral reactivation and detected by real-time PCR from all MLN and spleen tissue samples collected from FIV-infected cats as shown in the associated table. A positive detection is indicated by the + symbol, followed by the number of days in culture required for detection of infectious virus. A MLN was not collected from cat 165, and therefore was not tested for this cat (represented by the abbreviation N.A.). The LTNP cat 187 is designated in red and took a longer time to reactivate replication competent provirus from MLN and spleen-derived leukocytes.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5383277&req=5

pone.0175327.g004: Detection of virus infection in tissues by ex vivo viral reactivation.The protocol used for detection of leukocytes harboring replication competent proviruses is shown in this diagram. Replication competent and infectious virus was successfully propagated by ex vivo viral reactivation and detected by real-time PCR from all MLN and spleen tissue samples collected from FIV-infected cats as shown in the associated table. A positive detection is indicated by the + symbol, followed by the number of days in culture required for detection of infectious virus. A MLN was not collected from cat 165, and therefore was not tested for this cat (represented by the abbreviation N.A.). The LTNP cat 187 is designated in red and took a longer time to reactivate replication competent provirus from MLN and spleen-derived leukocytes.
Mentions: Mesenteric lymph node and spleen-derived leukocytes cultured ex vivo from all FIV-infected cats demonstrated the capacity to produce infectious, replication competent virus as determined by an ex vivo reactivation assay (Fig 4). Clarified cell-free culture supernatants collected from unfractionated tissue-derived leukocytes cultures were passaged onto SPF FIV-negative feline PMBCs on days 7, 14, and 21. After 7 days of secondary culture, real-time PCR was performed to identify the presence or absence of vDNA in SPF PBMC genomic DNA (Fig 4). A positive PCR result indicated that the clarified supernatant contained infectious virus. Infectious virus was detected in culture supernatant at day 7 from splenic leukocytes in all cats, except for the FIV-infected LTNP animal, for which virus reactivation required 14 days in culture. Time in culture required for detectable virus production was generally longer for the MLN samples, ranging from seven days for cat 186, and 21 days for cats 184 and the LTNP cat 187. A MLN was not procured from cat 165. FIV provirus was not detected at any time point for MLN or spleen-derived leukocyte cultures from a FIV-uninfected cat (183, negative control). Statistically, a significant correlation did not exist between the unfractionated leukocyte proviral load of each tissue type from each cat and the days to reactivation (MLN r = 0.49, p = 0.67; Spleen r = -035, p = 0.65).

View Article: PubMed Central - PubMed

ABSTRACT

Feline immunodeficiency virus (FIV) infection in cats results in life-long viral persistence and progressive immunopathology. We have previously described a cohort of experimentally infected cats demonstrating a progressive decline of peripheral blood CD4+ T-cell over six years in the face of apparent peripheral viral latency. More recently we reported findings from this same cohort that revealed popliteal lymph node tissue as sites for ongoing viral replication suggesting that tissue reservoirs are important in FIV immunopathogenesis during the late asymptomatic phase of infection. Results reported herein characterize important tissue reservoirs of active viral replication during the late asymptomatic phase by examining biopsied specimens of spleen, mesenteric lymph node (MLN), and intestine from FIV-infected and uninfected control cats. Peripheral blood collected coincident with harvest of tissues demonstrated severe CD4+ T-cell depletion, undetectable plasma viral gag RNA and rarely detectable peripheral blood mononuclear cell (PBMC)-associated viral RNA (vRNA) by real-time PCR. However, vRNA was detectable in all three tissue sites from three of four FIV-infected cats despite the absence of detectable vRNA in plasma. A novel in situ hybridization assay identified B cell lymphoid follicular domains as microanatomical foci of ongoing FIV replication. Additionally, we demonstrated that CD4+ leukocyte depletion in tissues, and CD4+ and CD21+ leukocytes as important cellular reservoirs of ongoing replication. These findings revealed that tissue reservoirs support foci of ongoing viral replication, in spite of highly restricted viral replication in blood. Lentiviral eradication strategies will need address tissue viral reservoirs.

No MeSH data available.


Related in: MedlinePlus