FIV Diversity: Fivple Subtype Composition May Inﬂuence Disease

Veterinary Immunology and Immunopathology 143 (2011) 338–346

Contents lists available at ScienceDirect

Veterinary Immunology and Immunopathology

j ournal homepage: www.elsevier.com/locate/vetimm

Research paper

FIV diversity: FIVPle subtype composition may inﬂuence disease

outcome in African lions

a,∗ a b b,1

Jennifer L. Troyer , Melody E. Roelke , Jillian M. Jespersen , Natalie Baggett ,

b c,2 c,3 c

Valerie Buckley-Beason , Dan MacNulty , Meggan Craft , Craig Packer ,

b b

Jill Pecon-Slattery , Stephen J. O’Brien

Laboratory of Genomic Diversity, SAIC-Frederick, National Cancer Institute, Frederick, MD, United States

Laboratory of Genomic Diversity, National Cancer Institute, Frederick, MD, United States

Department of Ecology, Evolution, and Behavior, University of Minnesota, St. Paul, MN, United States

a r t i c l e i n f o a b s t r a c t

Keywords:

Feline immunodeﬁciency virus (FIV) infects domestic cats and at least 20 additional species

FIVPle

of non-domestic felids throughout the world. Strains speciﬁc to domestic cat (FIVFca) pro-

Lions

duce AIDS-like disease progression, sequelae and pathology providing an informative model

CDV

for HIV infection in humans. Less is known about the immunological and pathological inﬂu-

Babesia

ence of FIV in other felid species although multiple distinct strains of FIV circulate in natural

populations. As in HIV-1 and HIV-2, multiple diverse cross-species infections may have

occurred. In the Serengeti National Park, Tanzania, three divergent subtypes of lion FIV

(FIVPle) are endemic, whereby 100% of adult lions are infected with one or more of these

strains. Herein, the relative distribution of these subtypes in the population are surveyed

and, combined with observed differences in lion mortality due to secondary infections

based on FIVPle subtypes, the data suggest that FIVPle subtypes may have different patterns

of pathogenicity and transmissibility among wild lion populations.

1. Introduction et al., 1998, 2010; Henriksen et al., 1995; Stump and

VandeWoude, 2007). As with HIV and SIV models, there

Feline immunodeﬁciency virus (FIV) is a lentivirus is considerable variation in transmission, course of infec-

closely related to HIV and SIV. In domestic cats (Felis catus), tion, and outcome of FIV infections in domestic cats. Some

FIV infection results in immune pathology, secondary infec- variation likely results from host genetic restriction factors

tions, and death. The parallels between human and feline that inﬂuence the viral life cycle, similar to those described

AIDS (FAIDS) have been explored for further understand- in humans (Lochelt et al., 2005; Munk et al., 2008, 2007;

ing of HIV/AIDS transmission, infection, and pathology Troyer et al., 2008; VandeWoude et al., 2010). However,

(Bendinelli et al., 1995; Burkhard and Dean, 2003; Elder differences in pathogenicity have also been demonstrated

among genetically distinct subtypes of FIV that circulate

in domestic cats (de Monte et al., 2002; Elder et al., 2010;

∗

Corresponding author. Tel.: +1 301 486 7478; fax: +1 301 846 6100. Pedersen et al., 2001; Weaver, 2010).

E-mail address: [email protected] (J.L. Troyer). Most experimental viruses representing FIVFca subtypes

Current address: Department of Biology, Rider University, are cell-line adapted but nonetheless retain recognized

Lawrenceville, NJ, United States.

differences in pathogenicity. For example, FIV-CPG derived

Current address: Utah State University, Department of Wildland

strains generally result in high initial viral loads and a faster

Resources, Logan, UT, United States.

3 progression to disease, especially in young cats (de Rozieres

Current address: College of Medical, Veterinary and Life Sciences,

University of Glasgow, Glasgow G12 8QQ, UK. et al., 2004a,b, 2008). In contrast, cats infected with FIV-A

J.L. Troyer et al. / Veterinary Immunology and Immunopathology 143 (2011) 338–346 339

strains often remain asymptomatic for longer periods of outcomes, in part because of the same geographic and envi-

time, with lower initial viral loads, though viral growth ronmental co-factors mentioned above.

kinetics are similar in adult cats once the acute stage of Several lines of evidence suggest that FIVPle subtypes

infection has passed (de Rozieres et al., 2008; Pedersen may be substantially different from each other. Two FIVPle

et al., 2001; Sparger et al., 1994). FIV subtype A strains are strains, FIVPle subtype E and FIVPle subtype A, circulate in

often neurotrophic and neurotoxic, producing CNS symp- Botswana while three, FIVPle subtypes A, B, and C, occur in

toms similar to those seen in HIV-1 infection (Gruol et al., the Serengeti National Park (Antunes et al., 2007; Brown

1998; Henriksen et al., 1995; Johnston et al., 2000; Meeker, et al., 1994; O’Brien et al., 2006; Troyer et al., 2004, 2005).

2007; Phillips et al., 1994, 1996; Power et al., 1998). Strains representing the predominant subtype in each of

Species-speciﬁc FIV viruses infect other felids and are these populations, FIVPle-B from the Serengeti and FIVPle-

distributed throughout the world, yet little is known about E from Botswana, have been fully sequenced revealing

their immunological and pathological effects in wild popu- remarkable differences between these subtypes (Pecon-

lations (Brown et al., 2010; Carpenter et al., 1996; Franklin Slattery et al., 2008a). While these two strains form a

et al., 2008, 2007; Olmsted et al., 1992; Troyer et al., 2005). lion-speciﬁc clade when full length viruses are aligned,

Long term surveillance of non-domestic felids infected the envelope (env) sequence by itself displays a different

with FIV, as well as evidence from free-ranging popula- phylogenetic relationship that suggests an historic recom-

tions of pumas (Puma concolor) and lions (Panthera leo), bination event between distantly related viruses, making

suggest that these viruses are ancient, host-adapted, and FIVPle-E env seemingly more similar to domestic cat FIV

have little to no negative impact on life-history param- than to FIVPle-B. In contrast, FIVPle-B env groups with

eters such as longevity (Biek et al., 2006; Carpenter and other non-domestic cat env gene sequences (Carpenter and

O’Brien, 1995; Packer et al., 1999). However, a few clin- O’Brien, 1995; Pecon-Slattery et al., 2008a; Smirnova et al.,

ical studies have revealed that individuals of these same 2005). The env gene is responsible for several aspects of

species may demonstrate FIV-associated immune deple- lentiviral pathogenicity; changes in these sequences can

tion and, in some cases, AIDS-like complications and death affect receptor binding, antibody afﬁnity, and target cell

(Brennan et al., 2006; Brown et al., 2010; Bull et al., 2002, speciﬁcity. Therefore, these differences have been hypoth-

2003; Roelke et al., 2006, 2009). Data on life history and esised to inﬂuence disease outcomes (Barlough et al., 1993;

clinical parameters are rare, and seldom collected in the Burkhard and Dean, 2003; Elder et al., 2010; Patrick et al.,

same population. 2002; VandeWoude and Apetrei, 2006).

At least six genetically distinct strains of lion FIV (FIVPle) The FIVPle subtypes circulating in Serengeti lions are

circulate in wild populations of African lions (Panthera more divergent then the FIVPle found in other African lion

leo) (Fig. 1; Antunes et al., 2008; O’Brien et al., 2006; populations. Speciﬁcally, FIVPle-C pol is as different from

Pecon-Slattery et al., 2008b; Troyer et al., 2005). FIVPle sub- the other two Serengeti subtypes as from FIV strains that

types demonstrate distinct phylogeographic distributions, infect other felid species (Troyer et al., 2005). Further,

suggesting prolonged host association, perhaps predating within-subtype diversity is much higher for FIVPle-B than

the Late-Pleistocene expansions of lions (Antunes et al., for the other two subtypes. Phylogenetic reconstruction of

2008). Probably as a result of the highly social nature of the three Serengeti FIVPle subtypes suggest different ances-

lions, FIVPle-infected populations have high prevalence of tral evolutionary trajectories and/or selection pressures

seropositive individuals, often approaching 100% in adults, (Troyer et al., 2004). For example, FIVPle-B is representa-

while other populations remain completely uninfected tive of a widely distributed East African clade found across

(Antunes et al., 2008; Brown et al., 1994; Troyer et al., 2005). Tanzania, Uganda, and Kenya. FIVPle-A appears to have

This “all or nothing” distribution of FIV in lion population spread from Southern Africa as the most closely related lion

makes appropriate comparisons of infected vs. uninfected viruses are found in South Africa and Botswana. Unique to

lions a challenge. Differences in FIVPle status may be con- the Serengeti, FIVPle-C is distantly related to other FIVPle

founded by important environmental parameters affecting viral subtypes (Antunes et al., 2008; O’Brien et al., 2006)

lion health including other infectious agents, prey abun- and exhibits relatively low within strain diversity consis-

dance, and water availability. tent with either recent introduction or stronger selective

Epidemiological and life history vs. clinical and pressure from the host immune system. This pattern likely

immunological studies on FIVPle-infected lions have been arose from three separate introductions of FIVPle to this

collected in different populations with only limited over- population in the recent past, a hypothesis supported by

lap. No evidence has been found of decreased lifespan in population genetic analyses of lion microsatellite loci, auto-

FIVPle-endemic populations in the Serengeti National Park somal sequences, and mitochondrial sequences (Antunes

and the Ngorongoro crater, where most lions are infected et al., 2008).

at an early age; no direct comparison with uninfected ani- Because these three divergent strains exist in a sin-

mals was possible (Packer et al., 1999). By contrast, infected gle population, it is possible to compare both population

individuals from Botswana and Tanzania demonstrated dynamics and epidemiology of these strains while reduc-

multiple clinical features of chronic immune depletion sim- ing the inﬂuence of confounding environmental factors.

ilar to human, simian, and domestic cat AIDS (Roelke et al., Here we present an analysis of FIVPle subtype composi-

2006, 2009). The phylogeographic distribution of FIVPle tion throughout the lifespan of Serengeti lions and during

raises the question as to whether FIVPle subtypes con- a deadly disease outbreak. In late 1993, an unprecedented

fer differential pathogenicity. However, to date, no study number of Serengeti lions died or disappeared; sev-

has correlated FIVPle subtypes with clinical or life history eral were observed with neurological disorders including

340 J.L. Troyer et al. / Veterinary Immunology and Immunopathology 143 (2011) 338–346

Fig. 1. Phylogenetic relationship of geographically distributed FIV subtypes from lions, domestic cats, and puma adapted from O’Brien et al. (2006).

Bootstraps and posterior probibilities of phylogentic reconstructions are shown (maximum likelihood/minimum evolution/maximum parsimony/Bayesian).

myoclonus ataxia and grand-mal seizures. From November proliferated during severe droughts. The associated tick

1993 to August 1994, the lion population in the long- infestations of the primary prey species of lions is believed

term study area in the southeastern part of the Serengeti to have led to the high levels of Babesia observed during

National Park plummeted by 39% with 78% of deaths occur- the CDV outbreak and to be an important co-factor in these

ring between the beginning of January and the end of March mortality events (Munson et al., 2008).

1994 (Fig. 2). The abruptness and extent of mortality indi-

cated the emergence of a deadly pathogenic agent, initially

suspected to be a neurotropic FIV strain. However, com-

prehensive clinical sampling and an assessment of over 100

lions during the outbreak revealed no relationship between

disease and FIV infection, excluding FIVPle as the primary

pathogen. The dead and sick lions showed reactivity to

canine distemper virus (CDV) monoclonal antibodies, and

post mortem immunohistopathology of brain tissue con-

ﬁrmed CDV as the causative agent. PCR detection and CDV

viral sequences indicated that the strain had spread from

domestic dogs living in villages adjacent to the National

Park (Carpenter et al., 1998; Munson et al., 2008; Packer

et al., 1999, 2005; Roelke-Parker et al., 1996).

A similar deadly outbreak of CDV occurred in the nearby

Ngorongoro Crater in 2001, while 5 other CDV outbreaks Fig. 2. Study population over time. In the time-period shown here, the

in the area over a 20 year time span were not associated population rose and fell cyclically until 1994, when it plummeted due to

the high-mortality CDV epidemic shown in grey; in 1999 the population

with any measureable mortality (Munson et al., 2008). The

returned to previous levels (Packer et al., 2005). The black bar indicates

high-mortality of the 1994 Serengeti and 2001 Ngoron-

the time period from 1984 through 1995 when samples were assessed for

goro CDV outbreaks were apparently linked to co-infection

FIVPle subtype distribution. The three dark grey bars (above the back bar)

with high levels of Babesia, a tick-borne hemoparasite that represent periods of high intensity sampling.

J.L. Troyer et al. / Veterinary Immunology and Immunopathology 143 (2011) 338–346 341

Although FIVPle was not the primary cause of the fatal 2 2 contingency tables of survival vs. death for subtype

outbreaks, the clinical evidence that FIVPle can lead to B vs. other subtypes. In all cases, two tailed p-values are

immune suppression in lions raises the question of whether reported. Survival analysis was performed using Gompertz

FIVPle could have played a supportive or interactive regression (parametric hazard model) on survival curves

role. We therefore treated these die-offs as “naturalis- produced by a LOWESS smoothing algorithm.

tic experiments”: challenges to lions’ health that allowed

an opportunity to test whether different strains of FIVPle 3. Results

might have had differential inﬂuences on CDV alone or on

CDV/Babesia combined pathogenesis. Subtype-speciﬁc primers (Troyer et al., 2004) were used

to examine FIVPle circulating in blood samples from 216

2. Materials and methods lions collected over 15 years starting in 1984 and ending

in 1999. As seen in a previous study of sixty-nine of these

2.1. FIVPle subtype designation lions (Troyer et al., 2004), there was a high incidence of

co-infections with multiple subtypes of FIV; 35% of the ani-

Blood samples from lions, had been used as a source mals were infected with more than one subtype at the time

of DNA, with subtype-speciﬁc PCR performed (reported in of sampling. Overall there was a large difference in sub-

Troyer et al., 2004). The generated FIV sequences had been type frequency, with 12% of the population infected with

assigned to subtypes A, B, and C based on phylogenetic FIVPle-A, 57% infected with FIVPle-C, and 69% infected with

analysis of the resultant 300 bp sequences from the pol gene FIVPle-B. These frequencies varied substantially with age

region produced by the strain-speciﬁc PCR primers (Troyer (Fig. 3): FIVPle-B was already common in cubs of one year

et al., 2004, 2005; Antunes et al., 2008). For the current of age (67%; N = 6) and subsequently increased between

study, novel analyses used the previously generated FIVPle the ages of 3 and 4 years old from close to 60% (N = 25)

subtypes that had been determined for each lion at one col- to over 80% (N = 13). In contrast the FIVPle-C strain was ini-

lection time point and which were assumed to remain the tially rare in yearlings (17%; N = 6) and increased by the age

same throughout the life of the lion. Whenever possible, a of 2 years (69%; N = 42). The incidence of FIVPle-A remained

collection time point nearest to the CDV/Babesia high mor- low throughout the lifespan of these lions.

tality outbreak was used. For three lions, subtype data was Long-term survival analysis was performed for lions

available at time points 5–10 years apart; in all three cases that were not involved in the high mortality CDV outbreaks

subtype composition was consistent over time. The novel (N = 97). Measuring longer-term mortality must control for

analyses were as follows. the sex of the sampled animal as males have shorter life

expectancies than females and males often disperse from

2.2. Age and longevity analysis the study area where their fates cannot be monitored.

Females infected with subtype B demonstrated signiﬁ-

Birth dates, capture dates, and death dates were used cantly longer post-sampling lifespan than those infected

to determine subtype distribution of the population as a with A or C (Gompertz parametric hazard model; z = 2.36,

function of age and longevity as a function of subtype. P = 0.018; data not shown), though this effect was not seen

Because of the inherent difﬁculties of making meaning- in males (data not shown).

ful comparisons of all possible subtype combinations given Short-term survival of lions during the 1994 Serengeti

the large number of classes and relatively few individuals and 2001 Ngorongoro Crater CDV outbreaks was com-

in each class, analysis was performed for simpliﬁed cate- pared with infection status of FIVPle-A, FIVPle-B and FIVPle-C

gories: presence of FIVPle subtypes A, B, or C compared to (Fig. 4). Two different sets of animals were examined;

each other or presence of FIVPle-B compared to presence of all lions with FIVPle subtype data that were alive in the

non-FIVPle-B subtypes. Serengeti (N-115) and Ngorongoro (N = 4) study areas at

the beginning of their respective outbreaks and a subset of

2.3. Survival of CDV/Babesia outbreaks these lions that were shown to be in areas with elevated

levels of Babesia and had known CDV exposure (N = 73).

Lions that were alive at the beginning of the outbreak Overall, 66% of lions survived the outbreak. However, only

were classiﬁed as surviving the outbreak if they lived 57% of the higher-risk subset survived. In all cases, lions

through October of the following year. Any lion that died infected with FIVPle-B singly or in co-infections with other

between January of the outbreak year and October of the subtypes exhibited mortality rates similar to the popula-

following year were considered to have died during the tion at large. In contrast, lions infected with C singly had

outbreak. The majority of deaths were conﬁned to a much higher than expected mortality (Chi squared = 5.591, two

narrower time window (Fig. 2). tailed p = 0.0181; Fig. 4). The survival disadvantage exhib-

ited by lions infected with subtype C was most pronounced

2.4. Statistical analysis when all lions in the population were considered; in the

high-risk subset, the trend remained, but was not signiﬁ-

Expected survival was determined empirically from the cant (Chi squared = 3.1, two tailed p = 0.076; Fig. 4b).

general and high-risk populations. Chi-squared analysis The primary disparity in survival observed was between

with 1 degree of freedom was performed for each possi- subtypes B and C; subtype A was rare and usually in

ble FIVPle subtype category to determine deviations from combination with other subtypes, complicating analysis.

expected values. Fisher’s exact test was used to compare However, since lions with subtype A were also more likely

342 J.L. Troyer et al. / Veterinary Immunology and Immunopathology 143 (2011) 338–346

Fig. 3. FIVPle subtype distribution by age of lion at time of sampling. An average of 19 lions were included per age class with a range of 6 (for yearlings) to

42 (for 2-year-olds). Because of co-infections, some lions were included in multiple subtype categories.

Fig. 4. Number of lions by subtype composition that survived (light grey bars) or died/disappeared (dark grey bars) during the high mortality CDV/Babesia

outbreaks. Lions were infected with one, two, or three subtypes. (A) Total number of lions in each possible subtype category at the onset of the outbreak,

divided by survival class. (B) Number of high-risk lions in each possible subtype category at the onset of the outbreak, divided by survival class. The scale

is set the same as part A for comparison. Two-tailed Chi-squared p-values are shown in (A) and (B); expected values were based on the survival rates of

the population in question. (C) Survival rates comparing lions infected with B alone or in any combination to those infected with A and/or C but not B.

Two-tailed Fisher’s exact test p-values are shown.

J.L. Troyer et al. / Veterinary Immunology and Immunopathology 143 (2011) 338–346 343

Fig. 5. Lowess curves of (A) short-term age-speciﬁc survival of lions that lived (1) or died (0) between the onset of CDV and the following October and (B)

post-CDV onset lifespan (in years) of lions sampled during the CDV/Babesia outbreaks for FIV+ animals that were not infected with the B-subtype (left) and

those that were infected with B (right).

to die than those with B, subsequent analysis compared that were not involved in the outbreak) there was a trend

lions infected with subtype B alone or in combination to for the subsequent lifespan of FIVPle-B infected lions that

those infected with subtype A and/or C, but not with B. survived the outbreak to be greater than that of lions with

Overall survival of the CDV/Babesia outbreak was signiﬁ- the other strains (Fig. 5b).

cantly greater for lions infected with subtype B compared

to those infected with either or both of the other sub- 4. Discussion

types (Fig. 4c; N = 119; Fisher’s exact test, two tailed

p = 0.0152). The trend remained the same for the high- Lentiviruses evolve rapidly and demonstrate strong

risk subset (Fig. 4c; N = 73; Fisher’s exact test, two tailed species ﬁdelity. However, divergent strains infect individ-

p = 0.0731). ual species, sometimes as a result of multiple introductions

The major difference between the whole data set and and sometimes through viral evolution post-introduction.

the high-risk lions consisted of lions from 4 prides that These strains may differ in virulence, host-cell tropism,

had negative CDV titers or low Babesia levels (N = 46); a pathogenicity and clinical outcome. In free-ranging African

majority of the lions from these 4 prides (70%) survived lions, FIVPle-A, FIVPle-B, and FIVPle-C demonstrated differ-

and were infected with either subtype B or subtypes B ential pathology and transmission. Older lions (>5 years

and C together. The overall effect of subtype on surviving old) infected with FIVPle-C or A alone were more likely

these outbreaks was apparently due to the lower survival to die than those infected with FIVPle-B. The differences

of adults between 5 and 10 yrs of age that lacked the B in outcome between subtypes were less apparent in lions

subtype. FIVPle subtype may also have had an effect on the with high Babesia levels and CDV then in the population

survival of younger animals: survival of non-B individuals at large. This suggests the possibility of increased immune

peaked at 3 yrs of age (each arm of the non-B spline was suppression with these subtype compared with FIVPle-B.

signiﬁcant at P < 0.05; Fig. 5a). In addition (similar to lions It also indicates that chronic effects of FIVPle are impor-

344 J.L. Troyer et al. / Veterinary Immunology and Immunopathology 143 (2011) 338–346

tant to long-term survival and differ according to subtype. Conﬂict of interest statement

Phylogenetic evidence supports the notion that subtype B

has been in residence longer and therefore it is possible All authors declare no conﬂict of interest.

that it is more host-adapted than the other two subtypes.

While speculative, this could explain the lack of FIV-related Acknowledgements

pathology in the lions of the Serengeti, where FIVPle-B is the

predominant circulating strain. We thank Randy Johnson and Rachel Simmons for

The change in subtype frequency relative to age statistical assistance and consultation. This research was

observed here raises the intriguing possibility that FIVPle supported in part by the Intramural Research Program

subtypes may also have different modes of transmission. of the NIH, National Cancer Institute, Center for Cancer

67% of lions were infected with FIVPle-B by their ﬁrst year, Research and has also been funded in part with federal

suggesting possible maternal transmission of this subtype. funds from the National Cancer Institute and National

In contrast, the prevalence of FIVPle-C rose substantially Institutes of Health, under contract HHSN26120080001E.

between the ages of one and two years, when non-sexual Samples were collected in full compliance with speciﬁc fed-

social contact and aggression begins, suggesting biting as eral permits (CITES; Endangered and Threatened Species)

the primary mode of transmission. Finally FIVPle-B preva- issued to the National Cancer Institute, principal investi-

lence rose between the ages of two and three years. Since gator S.J. O’Brien, by the U.S. Fish and Wildlife Service of

this corresponds to the age of sexual maturity, it sug- the Department of the Interior. The content of this publica-

gests sexual transmission of FIVPle-B. The dearth of FIVPle tion does not necessarily reﬂect the views or policies of the

subtypes A and C in older lions combined with the dispro- Department of Health and Human Services, nor does men-

portionate mortality of very young and very old lions may tion of trade names, commercial products, or organizations

account for the subtype differences observed for disease imply endorsement by the U.S. Government.

outcome.

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