REVIEW

Eckstrand et al., Journal of General Virology 2017;98:1985–1996

DOI 10.1099/jgv.0.000866

Central and peripheral reservoirs of feline immunodeficiency

virus in cats: a review
Chrissy D. Eckstrand,1,* Ellen E. Sparger2 and Brian G. Murphy3

Abstract
Infection with feline immunodeficiency virus (FIV), a lentivirus similar to human immunodeficiency virus (HIV), results in
lifelong viral persistence and progressive immunopathology in the cat. FIV has the ability to infect and produce infectious
virus in a number of different cell types. FIV provirus can also be maintained in a replication-competent but transcriptionally
quiescent state, facilitating viral persistence over time. Immediately after the initial infection, FIV infection quickly
disseminates to many anatomical compartments within the host including lymphoid organs, gastrointestinal tract and brain.
Collectively, the anatomic and cellular compartments that harbour FIV provirus constitute the viral reservoir and contain foci
of both ongoing viral replication and transcriptionally restricted virus that may persist over time. The relative importance of
the different phenotypes observed for infected cells, anatomic compartment, replication status and size of the reservoir
represent crucial areas of investigation for developing effective viral suppression and eradication therapies. In this review,
we discuss what is currently known about FIV reservoirs, and emphasize the utility of the FIV-infected cat as a model for the
HIV-infected human.

INTRODUCTION cohabitation with other cats and concurrent disease status

Feline immunodeficiency virus (FIV) is a lentivirus that
shares similarities to human immunodeficiency virus (HIV)
and is present in both domestic and feral cat (Felis catus)
populations worldwide. FIV prevalence varies by geographic
region with the incidence in North America reported at
2.5 % (2006), 5.4 % in Bangkok, Thailand (2014), 9.5 % in
Ankara, Turkey (2013), 23.2 % in Japan (2008) and 31.1 %
in peninsular Malaysia (2012), equating to millions of FIV-
infected animals worldwide [1–5].
Infection with FIV results in lifelong viral persistence and pro-
gressive immune dysfunction; however the clinical outcomes
of FIV-infected cats are variable, with many FIV-infected cats
living approximately normal lifespans [6]. Although FIV has
been associated with unfavourable outcomes such as opportu-
nistic infections, neoplasia, wasting and death, the precise
determinants of morbidity remain unclear. Contributory fac-
tors including viral/host genetics, age of host, viral load
during acute virus infection, housing environment, stress,
have been proposed [7–12]. Efforts to control lentiviral infec-
tions primarily focus on two broad strategies: preventing new
infections and suppressing viral replication in already infected
individuals, both of which are particularly relevant for HIV-
infected humans. The complete elimination of virus-infected
cells from the host is a laudable goal. However, practical and
effective viral eradication strategies do not yet exist for animals
or humans infected with lentiviruses in spite of highly effective
viral suppression therapies and several decades of research
focused on the pathogenesis of lentiviral agents [13]. The
identification of permissive cell types and anatomical com-
partments harbouring virus have been important goals in
research related to immunodeficiency-inducing lentiviruses.
Such information is critical to the development of effective
eradication therapies which depend upon the tissue penetra-
tion of various pharmaceutical agents. The relative importance
of the specific cell types and anatomical compartments that
constitute the lentiviral reservoir in an infected host remain
controversial [13, 14].

Received 28 March 2017; Accepted 15 June 2017

Author affiliations: 1Veterinary Microbiology and Pathology, College of Veterinary Medicine, 4003 Animal Disease Biotechnology Facility, Washington
State University, Pullman, WA 99163, USA; 2Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, 3115
Tupper Hall, Davis, CA 95616, USA; 3Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, 4206 Vet Med 3A,
University of California, Davis, CA 95616, USA.
*Correspondence: Chrissy D. Eckstrand, ceckstrand@vetmed.wsu.edu
Keywords: FIV; latency; lentivirus; reservoir; tissue; viral persistence.
Abbreviations: FIV, feline immunodeficiency virus; HIV, human immunodeficiency virus; SIV, simian immunodeficiency virus; FAIDS, feline acquired
immunodeficiency syndrome; Tfh, T follicular helper; IF, immunofluorescence; IHC, immunohistochemistry; RT, reverse-transcriptase; CNS, central
nervous system; ART, antiretroviral therapy; GALT, gut-associated lymphoid tissue; PBMCs, peripheral blood mononuclear cells; SPF, specific patho-
gen free; qPCR, quantitative polymerase chain reaction; VOA, viral outgrowth assay; SAHA, suberoylanilide hydroxamic acid; VPA, valproic acid; TSA,
trichostatin; NaB, sodium butyrate; DZNep, 3-deazanoplanocin A; PKC, protein kinase-C; MLN, mesenteric lymph node.

000866

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 Eckstrand et al., Journal of General Virology 2017;98:1985–1996
Multiple factors play roles in defining a cell’s permissiveness
to lentiviral infection and productive replication and include
the appropriate membrane receptors for virus attachment,
fusion and entry, a temporally appropriate milieu of nuclear
transcription factors, activation state, target cell subset, tissue
micro-environment, and presence of specific host cellular
restriction factors like tetherin, APOBEC3 family and others
[11, 13, 15, 16]. These same factors also determine the identity
of cells that serve as reservoirs for persistent latent virus [13].
This review discusses what is currently known about the cellu-
lar and anatomical reservoirs in the FIV-infected cat and point
out similarities shared by the FIV- and HIV-infected host. For
the purpose of this review, reservoirs relevant to viral patho-
genesis will be defined as cells and tissues that persistently har-
bour replication-competent provirus. The FIV-infected cat
offers the opportunity to investigate the virologic and cellular
features of central viral reservoirs that are typically inaccessible
in living HIV-infected humans, and may also serve as a model
for assessing the effectiveness of viral suppression and eradica-
tion therapies.
FIV BACKGROUND
Feline immunodeficiency virus was initially isolated in the
mid-1980s from a cohort of group-housed domestic cats in
northern California exhibiting a variety of non-specific ill-
nesses such as gingivitis, enteritis, dermatitis, weight loss and
death [17]. Virus isolation, electron microscopy, reverse tran-
scriptase enzyme assays and Western blot analyses identified a
T-lymphotropic retrovirus in the cats with clinical disease [17,
18]. Subsequent to this discovery, investigations into naturally
and experimentally infected domestic cats have revealed a
plethora of information about the virus and disease pathogen-
esis; as a result, the FIV-infected cat has become a well-
recognized translational model for investigation of HIV path-
ogenesis, as well as development of antiviral therapeutics and
vaccines. The FIV-infected cat is the only naturally occurring
HIV animal model for a clinical immunodeficiency that is
characterized by a progressive CD4+ T cell depletion, oppor-
tunistic infections and various entities including wasting and
neurological disorders [19, 20]. As such, FIV is distinguished
from other experimental animal models such as simian immu-
nodeficiency virus (SIV)-infection of non-natural hosts
including the Asian macaque species and the HIV-infected
humanized mouse [21]. Similar to HIV-infected humans, FIV
infection follows a relatively predictable course of disease
through three phases: the acute, chronic asymptomatic and
terminal feline acquired immunodeficiency syndrome stage
(FAIDS) [22–24]. Numerous reports have described
the virologic and immunopathologic aspects of acute and early
asymptomatic phases of FIV infection through experimental
inoculation studies. However, investigation of the late asymp-
tomatic phase and transition into FAIDS has been primarily
restricted to clinical cohorts of cats naturally infected with FIV
[7, 23, 25]. This is perhaps due to the cost of maintaining and
housing experimentally infected cats in specific pathogen free
(SPF) facilities for prolonged time frames.
1986
FIV RECEPTOR USAGE
Early reports revealed the presence of FIV proviral DNA in
specific cell subsets including CD4+ and CD8+ T cells, mac-
rophages and dendritic cells by sorting of these cell popula-
tions from experimentally infected cats or analysis of tissues
by immunohistochemistry (IHC) [26–31]. Subsequent stud-
ies revealed that FIV utilizes two cell surface receptors iden-
tified as the costimulatory molecule CD134 (OX40) and
the chemokine receptor CXCR4 for cellular attachment and
entry into these specific cellular targets [32–34]. Primary
binding of the cell by the virion is mediated between the
FIV Env-SU protein and the cell surface receptor CD134, a
costimulatory marker expressed by activated CD4 T cells.
Binding instigates a conformational change in Env, which
facilitates subsequent binding between the cell surface
expressed chemokine receptor CXCR4 and the V3 region of
Env-SU [35, 36]. Binding of a primary cell surface receptor
to facilitate Env interactions with a surface chemokine
receptor is a mode of cell entry that is also well character-
ized for other immunodeficiency-inducing lentiviruses such
as HIV-1 and SIV [37, 38].
CD134 is a transmembrane glycoprotein within the tumour
necrosis factor superfamily, and serves as the initial binding
receptor equivalent to the CD4 cell surface receptor in HIV
binding [35]. Human and mouse CD134 are predominantly
expressed on activated CD4+ T cells including regulatory T
cells and T follicular helper (Tfh) cells, and more transiently
expressed on activated CD8+ T cells. CD134 expression by
natural killer (NK) cells, NKT cells and neutrophils has also
been reported [39–41]. Currently, expression of feline
CD134 has been demonstrated on the surface of CD4+ T
and CD8+ T cells, and B220 (CD45R)+ B cells, but not on
peripheral blood-derived monocytes [42, 43]. However,
feline monocyte-derived macrophages, tissue-derived CD14
+ macrophages and activated CD8+ T cells were shown to
express CD134 surface expression that was further enhanced
with lipopolysaccharide activation [33, 42]. In a separate
study, cellular CD134 mRNA was detected in feline T and B
lymphocytes, dendritic cells and macrophages; the highest
expression, which increased with activation, was detected in
activated T lymphocytes [43]. Overall, CD134 expression
correlated with immune cell populations shown to be sus-
ceptible to FIV infection in vitro or to be positive for viral
DNA in the infected cat.
Early reports showed that different CD134 binding-domain
patterns may be viral strain-dependent [35, 44, 45]. Further-
more, CD134 domains required for FIV Env binding and
infection with more pathogenic FIV strains were mapped to
two cysteine-rich domains (CRD1 and CRD2), whereas less
pathogenic strains only required binding to CRD1 based on
in vitro analyses. These findings agreed with evidence for
FIV Env-CD134 binding patterns changing over time in
individually infected animals, which may ultimately influ-
ence viral cell surface affinity due to nucleotide base changes
in env and reflect isolates sensitive to circulating anti-
CD134 autoantibodies [44–46]. Recent studies further
 Eckstrand et al., Journal of General Virology 2017;98:1985–1996
characterized FIV Env changes associated with CD134
binding of distinctive FIV isolates that emerge in naturally
infected cats during disease progression [47]. Variants char-
acterized as Env recombinant viruses have also been
described [48]. Notably, specific changes in the env gene
sequence mapped over longitudinal time points of infection
resulted in FIV variants that lost dependence for binding to
the CRD2 domain of CD134 for virus entry. These CRD2-
independent isolates, which are characteristic of the later
stages of FIV infection, were also shown to be less efficient
for transmission and replication during acute infection
when compared to CRD2-dependent isolates in experimen-
tal infection studies. These findings generated a hypothesis
that CRD2-dependent FIV isolates, which are efficiently
transmitted in vivo and able to achieve higher virus
loads during early infection, may be analogous to CCR5-
dependent HIV-1 isolates that also transmit much more
efficiently during primary HIV-1 infection of humans when
compared to later-emerging CXCR4-tropic variants in HIV-
1 infection [49, 50]. Specific CD4+ T cell subsets that may
be specific targets for these CRD2-dependent FIV isolates
and their role in viral pathogenesis remain to be
determined.
Flow cytometric analysis revealed that chemokine receptor
CXCR4 is a co-receptor shared by both FIV and HIV-1 and
is expressed on feline peripheral blood-derived monocytes,
B-cells and activated T cells [51, 52]. These findings for
feline CXCR4+ cell subsets overall coincide with cells that
express FIV primary receptor feline CD134, and also
account for most cellular subsets shown to infected with
FIV in vivo, as described below. FIV Env domains responsi-
ble for binding of CXCR4 and CD134 have also been
mapped and found to be independent of each other, as
expected [36, 53]. Additionally, CXCR4 expression has been
detected in lymph node, thymic and bone marrow-derived
CD4+, CD8+ T lymphocytes, CD21+ B lymphocytes and
unfractionated CD4-/CD8-/CD21- cells [54]. Results from
this report also revealed FIV infection in both CXCR4+ and
CXCR4- cell populations, as determined by quantitative
polymerase chain reaction (qPCR) assay of isolated cell pop-
ulations for FIV proviral DNA. These results suggested the
possibility that FIV may be capable of infecting cells in a
CXCR4-independent manner, although this finding has not
been tested further or confirmed by subsequent studies.
Interestingly, laboratory-adapted FIV isolates have been
shown to be capable of infecting cells independent of
CD134 by use of heparin sulphate proteoglycans (HSPGs)
and DC-SIGN as binding receptors to promote infection
and facilitate binding of CXCR4 in the absence of CD134
[53]. FIV Env binding of HSPG and DC-SIGN are proper-
ties shared with HIV-1 [55, 56] wherein HIV-1 Env binding
of these receptors on dendritic cells may facilitate transmis-
sion of HIV-1 virions to T cell targets.
However, usage of these alternative receptors appears to be
restricted to laboratory-adapted FIV isolates and the role of
these receptors in FIV pathogenesis is currently unknown.
1987
Accordingly, HIV-1 and FIV utilize similar strategies for
entry of target cells by Env binding of a primary cell surface
receptor, CD4 and CD134, respectively, that facilitates Env
interactions with either the CCR5 (HIV-1 only) or CXCR4
(HIV-1 and FIV) chemokine coreceptors [37, 49]. Further-
more, targeting CD4+ T cells through either CD4 or CD134
surface proteins results in a similar immunopathogenesis,
characterized by a progressive CD4+ T cell depletion, lym-
phoid depletion and immune exhaustion during the chronic
phases of HIV-1 and FIV infections, respectively.
CELL RESERVOIRS OF FIV
Specific cell reservoirs of FIV are discussed with the under-
standing that a cellular reservoir contains integrated replica-
tion-competent provirus and persists over time. The biology
of FIV cellular reservoirs is complex and dynamic. Critical
factors include different cell types that are permissive to FIV
infection, infecting strain of FIV, changes in absolute CD4+
and CD8+ T cell numbers, cell surface receptor expression
modulation over time, stage of infection, evolution of the
viral genotype over time in the infected host, host-adaptive
immune responses and mechanisms of viral latency. Similar
to HIV-1, FIV infects and rapidly expands during the acute
stage of the disease, and then persists in peripheral blood
and tissue CD4+ T cells [26, 27, 52, 57]. However, CD4+ T
cells are not the only relevant reservoirs as the virus also
infects and disseminates to a wide variety of other leuko-
cytes during the acute stage of infection, including CD8+ T
cells, B-cells, monocytes/macrophages and other long-lived
cells such as dendritic cells and megakaryocytes [26, 27, 29,
31, 58, 59]. Evidence supporting that these leukocytes are
susceptible and permissive to FIV include PCR for proviral
DNA and viral RNA, in situ hybridization for intracellular
FIV genomic RNA and/or DNA, immunofluorescence (IF)
or IHC assessment of tissues for detection of viral antigen,
and either reverse-transcriptase (RT) activity assays
or enzyme-linked immunosorbant assay (ELISA) for detec-
tion of extracellular viral protein. Table 1 summarizes key
studies that have identified FIV cell reservoirs and the
modalities used to detect infection or susceptibility to infec-
tion. It is important to note that the leukocyte subsets that
have proven most consistently infected in vivo and suscepti-
ble to infection in vitro are CD4+ T cells and monocyte/
macrophages, which are also target cell reservoirs for HIV
[13, 49, 54, 60–62]. Evidence thus far is conflicting regard-
ing productive infection of dendritic cells in vivo [29, 30, 54,
63], although multiple studies support productive FIV infec-
tion of cultivated dendritic cells [43, 63, 64]. Likewise,
reports may demonstrate productive HIV-1 infection of
dendritic cells in vitro [65], but in vivo analyses suggest that
extracellular virions bound to the cell surface of dendritic
cells are the primary mode by which these cells transmit
virus infection to CD4 T cells [13, 66]. Evidence for produc-
tive replication of other subsets such as CD8+ T cells and B
cells in vivo or in vitro has not been reported. These findings
point once more to the similarities in cell tropism shared by
FIV and HIV-1.
 Table 1. Summary of scientific reports that identify cellular and tissue reservoirs of feline immunodeficiency virus
Author and Year Infection period FIV-infected cell type(s) identified FIV-infected tissue(s) identified Method(s) of evaluation FIV strain
(derived compartment, infection type)

Pedersen et al. [17]
Brown et al. [59]
Dow et al. [67]
Beebe et al. [103]
English et al. [26]
Toyosaki et al. [99]
Dua et al. [81]
Beebe et al. [31]
Park et al. [97]
Dean et al. [27]
Burkhard et al. [95]
Woo et al. [57]
Rogers and Hoover [94] 5–9 weeks (foetus)
Dow et al. [58]
Unknown T-lymphocyte (blood, in vivo infection) NA RT activity, Western Blot, Electron Petaluma
microscopy
NA CD4, CD8 T lymphocytes NA RT activity (supe) Petaluma, Peeper
(blood, in vitro infection) IFA (FIV polyclonal serum)
NA Astrocytes, microglia NA IFA (FIV polyclonal serum) p26 ELISA (supe) Petaluma
(primary cell culture, in vitro infection)
119–335 days Megakaryocytes, mononuclear cells Bone marrow ISH (proviral and viral RNA gag, pol, env) Petaluma
(tissue, in vivo infection)
2–4 weeks and CD4, CD8, Ig+ (blood, in vivo infection) NA PCR (proviral gag) FIV-NCSU1, Petaluma, Mt. Airy
12–18 months RT activity (supe)
5 months CD4, FDC, CD8 (tissue, in vivo infection) Lymph node IHC (gag polyclonal ab) TM-1, KYO-1, Petaluma
1–12 weeks NA CNS, tonsil, thymus, bone marrow, lymph PCR (proviral gag) Petaluma
nodes, liver, spleen, kidney, intestine, lung,
salivary gland
3 months CD3, macrophages (tissue, in vivo infection) Bone marrow, thymus, lymph nodes, tonsil, ISH (proviral and viral RNA gag, pol, env), Petaluma
spleen, intestine, kidney, lung, liver IHC (CD3, Mac387)
2–6 weeks Epithelial cells of interlobular ducts (tissue, in Salivary gland IHC (p26), PCR (proviral env) West Aust T91
vivo infection)
2–306 weeks CD4, CD8, CD21 (blood and tissue, in vivo Lymph node PCR (proviral, viral RNA gag), p24 ELISA Petaluma
infection) (supe)
8 weeks PBMCs, LNC, IEL (tissue, in vivo infection) Thymus, lymph node, spleen, intestine, Virus isolation, PCR (proviral, viral RNA gag), FIV-B-2542
vaginal mucosa, rectal mucosa ISH (whole genome)
2–20 weeks CD1, CD21/CD22, CD4/CD8 (thymus, in NA PCR (proviral and viral RNA gag) Petaluma
vivo infection)
NA PBMCs, brain, thymus, bone marrow, MLN, PCR (proviral gag) FIV-B-2542
spleen, liver
12–16 weeks Monocytes (blood, in vivo infection) NA ICC (FIV polyclonal serum), IF (FIV p26) FIV-B (2542, 2560, 2561)
PCR (FIV proviral env), ELISA (supe and FIV-AB (2531,2546)

1988
lysate FIV p26) FIV-A-Petaluma
Tanabe and Yamamoto [102] 24 weeks–1 year Macrophages and fibroblasts (bone marrow, NA RT (supe) FIV-UK8, Bang, Shi, Petaluma
in vivo infection)
Rogers et al. [29] 3 weeks CD3 T cells, macrophages, dendritic cells Lymph node, thymus, spleen, intestine, bone IHC, IFA (FIV polyclonal plasma) FIV-B-2542
(tissue) marrow, liver ISH (gag, envA, envB) FIV Petaluma
Joshi et al. [52] 5 years CD4+CD25+, CD4+CD25- (blood and lymph NA PCR (LTR, LTR-gag, env-gag CJs) p24 Ag NCSU1
node, in vivo and in vitro infections) capture
Hein et al. [107] 14–183 days Microglia (brain, in vivo infection) NA PCR (viral RNA gag) Wo, Womic
Troth et al. [54] 3–4 weeks CD4/CD8, B lymphocyte (CA1.2D6), CXCR NA PCR (proviral gag) FIV-C-Pgmr
+/- (blood, bone marrow, lymph node,
thymus, in vivo infection)
Reggeti et al. [43] NA CD4, CD8, CD21 (blood, in vitro infection) NA PCR (proviral LTR-gag) p24 ELISA (supe) USgaB01
Eckstrand et al., Journal of General Virology 2017;98:1985–1996

Macrophages, dendritic cells (bone marrow,

in vitro infection)
Sprague et al. [64] NA Dendritic cells (blood, in vitro infection) NA PCR (proviral gag), p26 ELISA, Electron FIV-C-Pgmr
microscopy
Miller et al. [104] 350 days NA Brain PCR (viral RNA gag) Pcenv, PPR, C36
Hood et al. [63] 1–3 years Monocytes, dendritic cells, CD8 lymphocytes NA PCR (undescribed viral RNA in supe) NCSU1
(blood, in vivo infection)
Eckstrand et al. [109] 5.1 years CD4, CD21 (blood and lymph node, in vivo Lymph node PCR (proviral and viral RNA gag), Western FIV-C-Pgmr
infection) blot, IHC (polyclonal FIV plasma), ex vivo
reactivation
Eckstrand et al. [92] 6.1 years CD4, CD8, CD21 (blood, lymph node, spleen, Lymph node, spleen, small intestine PCR (proviral and viral RNA gag), ISH (whole FIV-C-Pgmr
in vivo infection) genome), ex vivo reactivation

ab, antibody; ELISA, enzyme-linked immunosorbent assay; ICC, immunocytochemistry; IFA, immunofluorescent antibody; IHC, immunohistochemistry; ISH, in situ hybridization; NA, not
applicable; PCR, polymerase chain reaction; PBMC, peripheral blood mononuclear cell; RT, reverse transcriptase; supe, supernatant.
 Eckstrand et al., Journal of General Virology 2017;98:1985–1996
FIV is capable of infecting and replicating in cell types other
than traditional leukocytes. Feline central nervous system
(CNS)-derived glial cells, including astrocytes and micro-
glia, have been shown to support FIV replication in vivo
and in vitro [67–71]. Infection of the feline CNS by FIV is
proposed to result from trafficking of infected B and T
cells, as well as macrophages, through the blood–brain bar-
rier and into the choroid plexus based on experimental cat
inoculation studies [72] and ex vivo choroid plexus epithe-
lial cultures [73]. It is perhaps not surprising that microglia
can support FIV infection, as these cells arise from bone
marrow myeloid progenitor cells similar to other tissue-
based macrophages. However, it is puzzling that astrocytes
seem to support FIV replication, as these glial cells are
derived from neuroectoderm. Billaud et al. demonstrated
that astrocytes not only support FIV replication in vitro, but
that viral replication is dependent upon the env sequence
and that the replication rate in astrocytes differs between
FIV strains [70]. However, other studies failed to show pro-
ductive FIV infection of astrocytes [74]. Similarly, findings
for HIV-1 infection of astrocytes have been conflicting.
Astrocytes were shown to support HIV infection in both in
vivo and in vitro studies, with the human mannose receptor
reported to be essential for CD4-independent infection of
this cell type [75, 76]. However, more recent studies
reported inconsistent findings regarding HIV infection of
astrocytes and suggest that infection of astrocytes is highly
restricted, may involve CD81 vesicles and may internalize
virus particles without productive virus replication [77, 78].
Regardless, the overall findings indicate that astrocyte infec-
tion or internalization lead to cellular dysfunction and facili-
tate transfer and dissemination of HIV-1 within the CNS.
The role and nature of FIV interactions with astrocytes in
virus-induced disease of the CNS, and similarities shared
with HIV, remain to be determined.
There are relatively few reports describing longitudinal
studies designed to investigate changes within infected leu-
kocyte subsets according to the stage of FIV infection. Evi-
dence that proviral DNA loads may shift between cellular
subsets as disease progresses, especially as CD4+ T cells are
depleted, has been presented in a limited number of reports
[26, 27, 79]. The hallmark of FIV infection in the acute and
early asymptomatic phases of infection is an inverted
peripheral blood CD4:CD8 T cell ratio initially due to a
decrease in CD4+ T cells and in some cases a concurrent
expansion of CD8+ T cells [60, 80–82]. The abrupt decline
in peripheral CD4+ T cell count typically rebounds,
although a return to pre-infection values may not be
observed. This decline in peripheral CD4+ T cells counts
during acute infection has been reported to coincide with
even more severe declines in CD4+ T cell frequencies in
lymphoid tissues with the severity dependent on the FIV
isolate used for inoculation and route of infection [60].
Importantly, relatively few studies have monitored the fre-
quency of CD4+ and CD8+ T cell subsets into the late stages
of infection. However, findings from these few studies,
including experimental and natural infections, show that
1989
CD4+ T cell counts continue to progressively decline to
very low levels in blood and lymphoid tissues and may be
associated with declining peripheral CD8+ T cell frequen-
cies and clinical signs of immunodeficiencies [7, 19, 83].
Interestingly, significant declines in CD4+ T cells counts are
not always associated with clinical signs of acquired immu-
nodeficiencies and other factors such as environmental
stresses may determine disease onset [7, 24, 79].
Early studies investigating FIV-infected target cell popula-
tions during experimental infection relied on IHC analysis
with a limited array of cell surface markers, in situ hybrid-
ization for viral nucleic markers and PCR analysis fre-
quently dependent on nested-PCR protocols. Based on
these reports [26, 27, 31, 81], highest viral loads were
detected in CD4+ T cells. However, findings regarding shifts
in proviral DNA loads to other subsets varied between these
early studies to reflect greater loads in B cells, CD8+ T cells
or macrophages depending on the virus isolate tested. Later
reports and summaries [29, 54, 60, 79, 84, 85] also show
variable findings. Yet, overall, these later studies reported
that CD4+ T cells followed by CD21+ B cells and mono-
cyte/macrophages have the highest proviral DNA loads in
blood and lymphoid tissue, including the mucosa of the gas-
trointestinal tract. Dendritic cells have also been reported to
be positive for proviral DNA in the few studies that analysed
this subset, although the relevance of FIV infection of den-
dritic cells in vivo is still poorly understood [29, 30, 54]. Tar-
geting of CD4+ T cells for a progressive depletion in the
periphery and in lymphoid tissues along with targeting of
other immune cell subsets including monocyte/macro-
phages, and dendritic cells parallels findings reported for
HIV-1 infection in humans and correlates with the onset of
a clinical immunodeficiency in the FIV-infected cat.
FIV TISSUE RESERVOIRS
From a broader perspective, anatomical sites that harbour
FIV-infected cells have also been an active area of investiga-
tion in research focused on lentiviral pathogenesis. The
topic of anatomical reservoirs of lentiviral persistence
became particularly important with the advent of antiretro-
viral drug therapy (ART) for HIV-infected humans. While
ART is highly effective for suppressing HIV viral replica-
tion, there is evidence that incomplete anatomically local-
ized suppression of viral replication occurs due to
inadequate tissue drug concentrations in particular ana-
tomic sites such as the brain [86–90]. As is true for cellular
reservoirs, technical challenges impede the identification of
tissue reservoirs infected with replication-competent provi-
rus. Multiple methodologies have been utilized to identify
FIV-infected cells in tissues, including PCR to identify pro-
virus in tissue homogenates, tissue localization of virus
by IHC and in situ hybridization, electron microscopy, and
virus isolation/outgrowth assays. Optimally, biologically rel-
evant tissue reservoirs should be identified using a combina-
tion of these techniques.
 Eckstrand et al., Journal of General Virology 2017;98:1985–1996
Anatomical reservoirs frequently discussed in the HIV field
are the brain, gut-associated lymphoid tissue (GALT),
lymph nodes and the reproductive tract [13, 14, 88, 91].
Many of these tissues have been investigated as tissue reser-
voirs in the FIV-infected cat as well, particularly during the
acute and early asymptomatic phases of infection [60, 62,
79, 92]. A thorough understanding of tissue reservoir distri-
bution and biology requires an understanding of the early
pathogenesis and dissemination of virus.
FIV inoculation in cats is believed to occur primarily
through blood transmission during fighting (biting), how-
ever many other routes of experimental inoculation have
proven to be effective for viral transmission, including oral,
rectal/vaginal mucosal transfer, intravenous, intramuscular,
intraperitoneal, subcutaneous injection and transplacental
transfer [17, 19, 93–97]. Infection can be achieved by inocu-
lation of either cell-associated or cell-free virus, with cell-
free virus being apparently more efficient (at least with
FIV-C) [30, 60, 93]. Viral inoculation is followed by robust
cell-associated viral replication in the peripheral blood,
resulting a transient plasma viraemia and systemic viral dis-
semination [60, 81, 98]. Obert and Hoover demonstrated
that within 2 days of oro-nasal or vaginal mucosal exposure,
FIV traffics from the mucosa to the regional lymph nodes
via dendritic cells or CD3+ T cells transfer with subsequent
systemic spread to secondary and tertiary lymphoid organs
[30]. Other important target tissues in early FIV dissemina-
tion include additional lymphoid organs such as the intesti-
nal tract mucosa, thymus, spleen, tonsil and bone marrow
[27, 29, 31, 81, 94, 99–103]. Fig. 1 summarizes important
tissue and cellular reservoirs known to harbour virus in
FIV-infected cats.
Additionally FIV-infected cells can be detected in the CNS,
liver, kidney, lung and salivary gland during the acute phase
[74, 81, 94, 97]. Table 1 lists the studies of tissue reservoirs
of FIV. Rogers et al. described a modified IHC technique for
the detection of FIV protein (undefined FIV antigen) in for-
malin-fixed, paraffin-embedded tissue by using feline serum
from an experimentally FIV-infected cat [29]. The study
demonstrated that, within 3 weeks of inoculation, FIV dis-
seminates to many tissue types including lymph nodes,
spleen, intestinal lamina propria, intestinal Peyer’s patches
(lymphoid follicles), thymus, bone marrow and resident tis-
sue histiocytic cells such as Kupffer cells of the liver [29].
Interestingly, brain was not identified as a tissue for FIV
infection. However, other reports have demonstrated that
different FIV strains vary in their tropism for the brain and
for different compartments of the brain [70, 104]. These
findings point to some similarities in viral pathogenesis for
the CNS shared by FIV and HIV-1 ([74, 105] – review with
references therein). Indeed, based on multiple reports, the
brain has proven to be an important target during early FIV
infection and, similar to HIV, has been associated with vari-
ous pathologic features such as encephalitis, microgial acti-
vation and, in some cases, degenerative changes like myelin
pallor ([74, 105] and references therein; [104, 106]).
1990
Multiple reports have shown virus localized to various com-
partments of the brain by qPCR or by in situ hybridization
for FIV nucleic acid. However, relatively few in vivo studies
have tracked specific cell populations within the CNS that
are infected with FIV, with a few reports confirming infec-
tion of resident microglia in experimentally infected cats
[69, 107]. FIV infection of the CNS warrants further scru-
tiny to determine the value of this animal model for testing
therapeutic approaches to purge the HIV-1 reservoirs
within the CNS.
There are fewer studies that have examined the long-term
persistence of FIV in tissue sites. However, several studies
indicate that tissues targeted by FIV in the acute phase also
harbour replication-competent provirus in the later stages
of infection. Dean et al. demonstrated the presence of repli-
cation-competent provirus in the peripheral blood and
lymph node out to 70 months (5.8 years) after infection with
FIV [27]. Pistello et al. demonstrated the presence of abun-
dant provirus in lymphoid tissues, and lesser amounts in
the brain, lung and kidney 3 years post super-inoculation
(inoculation followed by second exposure) with FIV-Petal-
uma [108]. Infection of bone marrow cells in naturally
infected asymptomatic cats has also been reported, although
the identity of infected subsets was not determined [25].
Two recent investigations into tissue reservoirs by our labo-
ratory demonstrated the presence of proviral DNA within
the popliteal lymph node, mesenteric lymph node, spleen
and mucosa of the small intestines during the late asymp-
tomatic phase of infection (5–6 years post inoculation), in
the face of a relatively quiescent or inactive viral harbour
replication-competent provirus in spite of relatively quies-
cent or inactive viral replication in the peripheral blood [79,
92, 109]. Interestingly, our studies revealed that, firstly,
lymph node germinal centres contained an increased fre-
quency of CD3+ T cells compared to uninfected cat tissues.
These observations suggested a proliferation of resident of
Tfh cells or infiltration of CD8+ T cells into the B cell fol-
licles, although CD8+ T cells are generally restricted from
entering B cell follicles. Secondly, in our most recent report,
a novel in situ hybridization technique (RNAscope) for
detection of FIV RNA identified germinal centres in the
mesenteric lymph node, spleen and intestinal Peyer’s
patches as microanatomical reservoirs of viral transcription
in the chronically FIV-infected cat [92].
FIV LATENCY
Multiple investigative modalities have been used to identify
cellular reservoirs of FIV, including the detection and quan-
tification of proviral DNA, viral RNA, viral protein, reverse
transcriptase activity and other techniques described in
detail in a recent review by Ammersbach and Bienzle [110].
While the detection and quantification of these various viral
components can provide supportive evidence of viral repli-
cation, they do not ultimately prove a cell’s ability to pro-
duce infectious virus. The ‘gold standard assay’ for the
identification of replication-competent and infectious lenti-
viral reservoir cells is the ex vivo viral outgrowth assay
 Eckstrand et al., Journal of General Virology 2017;98:1985–1996
Tissue reservoirs
Lymph nodes
Bone marrow
Reproductive tract
Gastrointestinal tract
Peyer’s patches
Spleen
Thymus
Blood
Brain
Fig. 1. Tissue and cellular reservoirs of feline immunodeficiency virus in the infected cat.
(VOA) which has been used extensively for investigation of
HIV-1 latency in humans [111] and in our laboratory for
detection of cells containing transcriptionally silent infec-
tious provirus in cats chronically infected with FIV [84, 92].
For assessment of virus infection by VOA, an enriched
(purified) cell type such as CD4+ T cells are obtained from
an infected individual and are cultured ex vivo. To reverse
potential viral latency, cultured cells are stimulated for vary-
ing amounts of time with mitogenic compounds such as
phytohemagglutinin or concanalin-A and/or allogeneic
peripheral blood mononuclear cells (PBMCs) to instigate a
mixed lymphocyte reaction [112]. In the VOA, ex vivo stim-
ulation is an attempt to ‘reverse latency’ by activating viral
transcription, viral protein production, virion assembly and
release from cells containing replication-competent provi-
rus. For detection of FIV infection, cell-free (clarified) cul-
ture supernatant from the activated cells is then passaged
onto uninfected SPF feline PBMCs or another susceptible
cell type. These cells are then cultured, and subsequently
tested for the presence of proviral DNA by standard or
quantitative PCR. Detection of proviral DNA in the second-
ary SPF cell culture confirms that supernatant from the pri-
mary cell culture contained replication-competent and
infectious virus. The VOA can also be used to quantify the
size of the reservoir by initially preparing serial dilutions of
the reservoir cell of interest [113]. Although this assay is
most sensitive for virus detection, major limitations of the
VOA are that ex vivo stimulation may fail to reverse latency
(induce viral transcription) of all the replication-competent
proviruses present within the tested reservoir population, is
labour intensive and may yield variable results [13]. As a
result, apparently ‘non-inducible’ cells harbouring replica-
tion-competent provirus will fail to be identified [114].
1991
Cellular reservoirs
Lymphocytes
CD4+T cells
CD8+T cells
CD21+B cells
Antigen processing/
presenting cells
Monocytes
Macrophages
Dendritic cells
CNS specific
Microglia
Astrocytes
Lentiviral latency refers to cells with integrated replication-
competent provirus that are transcriptionally inactive.
Latently infected cells can be identified by detecting proviral
DNA with a concurrent absence of detectable viral RNA
and viral proteins. Lentiviral latency allows the integrated
and replication-competent provirus to remain essentially
invisible to both the host immune system and antiretroviral
therapies and thus allows the virus to persist over time
within the infected cell and host. Latently infected cells are
thus of key importance when considering viral reservoirs,
particularly within a host on ART. McDonnel et al.
reviewed FIV latency and the strengths of the FIV-infected
cat as a model of the HIV-infected human [61]. Different
cohorts of naturally and experimentally infected cats have
demonstrated either undetectable or consistently detectable
plasma viraemia during the asymptomatic phase, which
may be due to differences in viral strains, inoculating dose,
age of the animal or other factors [7, 84, 98, 104, 115–118].
The cellular mechanisms involved in FIV latency have been
identified in both in vitro and in vivo infection systems [52,
84, 119–122]. McDonnel et al. reported that peripheral
blood-derived CD4+ T cells latently infected with replica-
tion-competent, infectious virus were infrequent (estimated
at approximately 1 cell in 105 peripheral CD4+ T cells). This
report also revealed that latent infection was characterized
by the association of the FIV 5¢ long terminal repeat
(LTR) promoter with host histone proteins that were hyper-
methylated and deacetylated, consistent with a condensed
chromatin state [123]. Subsequent reports demonstrated
that latently FIV-infected cells could be reactivated ex vivo
through the administration of histone deacetylase inhibitors
suberoylanilide hydroxamic acid, valproic acid,
trichostatin A, and sodium butyrate (NaB) and a histone
 Eckstrand et al., Journal of General Virology 2017;98:1985–1996
methyltransferase inhibitor 3-deazanoplanocin A [124,
125]. Similar findings were reported in two studies by Tada-
fumi and Chan who used histone deacytelase NaB and pro-
tein kinase-C inhibitors, respectively, to reverse FIV latency
[120, 126]. Lastly, only short, abortive transcripts (R region
of the LTR) along with an association of RNA polymerase II
with the LTR by chromosomal immunoprecipitation were
detected in cells latently infected with FIV [123]. These
findings provided indirect and direct evidence that RNA
polymerase II activity is paused on the FIV promoter, a
finding also reported for cells latently infected with HIV
[123, 127, 128]. These findings collectively reveal that circu-
lating CD4+ T cells serve as a reservoir for latent FIV infec-
tions with latency characterized by at least one similar
regulatory mechanism, repressive chromatin, that has also
been described for HIV-1 latency in patients on ART [129].
Assogba et al. suggested that low dose viral inocula (102–103
copies), delivered by the mucosal route, and CD8+ T cells
may also play important roles in inducing latency in CD4+
cells and continued viral suppression [121]. The same study
described an infection state proposed as latency, in which
proviral DNA was undetectable in blood- and tissue-derived
cells harvested from experimentally infected cats [121].
However, activation of cultivated tissue lysates depleted of
CD8+ T cells ex vivo resulted in virus production. The iden-
tity of cellular subsets serving as reservoirs for latent virus
within these infected tissues was not determined. A different
report showed that FIV preferentially replicated in activated
CD4+CD25+ T cells and that CD4+CD25+ T cells served as
a productive reservoir resistant to apoptotic cell death [52].
In contrast, CD4+CD25- cells were shown to be reservoirs
for latent FIV with the potential to reactivate. This observa-
tion suggests that less-activated CD4+ T cells, as defined by
the absence of CD25 expression, may preferentially harbour
latent FIV infection – a finding that agrees with the observa-
tion of resting CD4+ T cells being a major reservoir for
latent HIV-1 infection in patients on ART [13]. CD4+ T
cells are the most frequently investigated cell type with
regards to FIV latency. However, testing with additional
markers for feline T cell activation and memory will be nec-
essary to fully characterize the CD4+ T cell subsets that are
the predominant reservoirs for latent FIV infection and to
determine similarities between latency FIV and HIV-1 for
CD4 + T cell infections. Finally, a previous report revealed
that monocytes isolated 12–16 weeks after experimental FIV
inoculation contained detectable proviral DNA in the
absence of detectable viral protein. Moreover, virus produc-
tion from these monocytes could be subsequently induced
when stimulated ex vivo [58]. Monocytes and macrophages
have also been identified as reservoirs for latent HIV-1
infection in patients on ART [13]. Taken together, observa-
tions to date suggest that FIV may share similarities in cellu-
lar reservoirs and molecular mechanisms for latency with
HIV-1, although FIV latency remains less well characterized
compared to HIV-1 latency, particularly regarding mecha-
nisms of latency. These few reports described for FIV sug-
gest that epigenetic factors, early virus infection events such
1992
inoculating virus concentrations, immune restriction and
cell activation, may play a role in FIV latency, factors shared
by HIV-1 latency. One report has described FIV proviral
DNA integration sites, which were revealed to be similar to
those for HIV-1 [130]. However, any relationship between
FIV integration sites and virus latency is currently undeter-
mined. Furthermore, in contrast to HIV-1 infection, FIV
latency may be apparent in the absence of ART and a com-
mon characteristic in cats naturally infected with FIV, par-
ticularly in environments free of stress and exposure to
other pathogens, such as a single-cat household [7]. How-
ever, this dissimilarity suggests that FIV infection may pro-
vide a unique opportunity to examine mechanisms and
interventions of lentiviral latency in the absence of ART.
CONCLUSIONS
Since the initial identification of FIV, there has been an
extensive history of investigations into cellular and tissue
viral reservoirs of FIV that have greatly contributed to
understanding the pathogenesis of disease in the FIV-
infected cat. Additionally, there is no shortage of research
and review papers highlighting the strengths and advantages
of using the FIV-infected cat as an animal model for the
HIV-infected human. Many tissue reservoirs of FIV are
very similar to HIV and include (but are not limited to)
lymphoid tissues, including the gastrointestinal tract, and
the CNS. FIV infects similar leukocyte subsets as HIV and
most likely utilizes the same cells, such as dendritic cells and
astrocytes, to facilitate infection of T cell targets. Impor-
tantly, FIV infection offers the opportunity to investigate
the relevance of cellular and tissue reservoirs that may not
be readily accessible in HIV-infected people (i.e. CNS). Fur-
thermore, examination of the differences between FIV and
HIV-1 infections, including receptor usage and clinical out-
comes, may also provide unique insights to the viral mecha-
nisms of lentiviral pathogenesis. As the lentivirus field
evolves to investigate new antiretroviral and eradication
strategies such as the gene-editing tool, CRISPR, one should
consider the advantages of utilizing the affordable, outbred,
tractable, naturally occurring FIV cat disease model for test-
ing the safety and efficacy of such therapies.
Funding information
The authors received no specific grant from any funding agency.
Conflicts of interest
The authors declare that there are no conflicts of interest.
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