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Clinical Infectious Diseases
BRIEF REPORT
Zoonotic Transmission of Two New
Strains of Human T-lymphotropic
Virus Type 4 in Hunters Bitten by a
Gorilla in Central Africa
Léa Richard,1,2,3 Augustin Mouinga-Ondémé,4 Edouard Betsem,1,2,5,a
Claudia Filippone,1,2,b Eric Nerrienet,6 Mirdad Kazanji,4,c and Antoine Gessain1,2
1
Unité d’Epidémiologie et Physiopathologie des Virus Oncogènes, Institut Pasteur, 2Centre
National de la Recherche Scientifique, UMR 3569, and 3Université Paris Diderot, Cellule Pasteur,
France; 4Unité de rétrovirologie, Centre International de Recherches Médicales de Franceville,
Gabon; 5Faculty of Medicine and Biomedical Sciences, University of Yaounde I, and 6Centre
Pasteur du Cameroun, Yaoundé, Cameroon
Molecular screening of 300 at-risk people from Central Africa
identified 2 human T-lymphotropic virus (HTLV)-4–infected
individuals. A zoonotic origin of infection was suggested, as
both individuals reported being severely bitten by a gorilla during hunting activities. One strain was highly divergent and was
designated as the HTLV-4 subtype-b prototype.
Keywords. HTLV-4; gorilla; zoonosis; emergence; Central
Africa.
Primate T-lymphotropic viruses (PTLV), including human
T-lymphotropic virus (HTLV)-1/simian T-lymphotropic virus
(STLV)-1, HTLV-2/STLV-2, HTLV-3/STLV-3, and HTLV-4/
STLV-4, constitute a group of related human and simian retroviruses that share common epidemiological, biological, and molecular features. HTLV-1 and HTLV-2 are relatively widespread,
infecting millions of people worldwide. In contrast, HTLV-3
has only been observed in a few African individuals living in
close contact with infected non-human primates (NHPs), and
HTLV-4 has been identified in only 1 person living in South
Cameroon [1]. Whereas the cosmopolitan HTLV-1 subtype is
transmitted by sexual contact, blood transfusion, and breast
feeding, HTLV-1 African subtypes (b, d, e, f, g), particularly
HTLV-3 strains, are also probably acquired through interspecies
transmission from NHPs infected with STLV-1 and/or STLV-3
Received 29 March 2016; accepted 2 June 2016; published online 19 June 2016.
Presented in part: 17th International Conference on Human Retroviruses: HTLV and Related
Viruses, Trois Ilets, Martinique, 18–21 June 2015. Abstract P83.
a
Present address: Edouard Betsem, Laboratoire Mixte International de Vaccinologie, Agence
de Médecine Préventive, Bobo-Dioulasso, Burkina Faso.
b
Present address: Claudia Filippone, Unit of Virology, Institut Pasteur de Madagascar, Antananarivo, Madagascar.
c
Present address: Mirdad Kazanji, Institut Pasteur de la Guyane, Cayenne, French Guiana.
Correspondence: A. Gessain, Unité d’Epidemiologie et Physiopathologie des Virus Oncogènes, Institut Pasteur, Paris, France ([email protected]).
Clinical Infectious Diseases® 2016;63(6):800–3
© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society
of America. All rights reserved. For permissions, e-mail [email protected].
DOI: 10.1093/cid/ciw389
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viruses. We showed that a severe bite by a NHP is a major risk
factor for HTLV-1 infection in Cameroon, Central Africa [2].
The only known HTLV-4–infected individual reported contacts
with various NHPs, including gorillas, suggesting viral acquisition through NHP body fluid exposure in Cameroon [1]. The
origin of HTLV-4 remained unknown until a recent report of
the presence of STLV-4 in 6 gorillas among 58 studied in Cameroon (either wild-caught or living in captivity) [3]. The gorilla
viral strains were closely related to the prototypic human strain,
with more than 99.4% nucleotide identity. HTLV-4 may have
thus emerged from a gorilla reservoir.
To obtain a better understanding of the possible zoonotic origin of HTLV-4 and its distribution and genetic diversity, we
screened 2 available series of gorillas and highly exposed individuals from Cameroon and Gabon by molecular methods for
PTLV-4 infection.
METHODS
We screened a series of 32 wild-born and wild-caught gorillas
(17 originating from Cameroon, 15 from Gabon) for PTLV-4
infection in the context of ongoing studies on retroviral emergence in Central Africa [4, 5]. Blood samples were collected in
accordance with the rules of animal care committees. We also
included 300 humans in the study who had been previously
identified to have been bitten by NHPs, including 102 by a gorilla, 34 by a chimpanzee, and 164 by a small monkey. Human
samples had been previously tested for simian foamy virus
(SFV) and HTLV-1 infection but never specifically for HTLV-4
infection [2, 5–7]. Among them, 81 were Pygmies of the Baka or
Bakola tribes and 219 were Bantus of different groups living
in the southern forest of Cameroon (234) or in rural areas
of Gabon (66). There were 278 males and 22 women with a
mean age of 38 years. The National Ethics Committee in Cameroon, the Ministry of Health and the Ethics Committee of the
Centre International de Recherches Médicales de Franceville in
Gabon, and the Comité de Protection des Personnes and the
Commission Nationale de l′Informatique et des Libertés in
France approved the study. All individuals provided written informed consent.
Peripheral blood buffy-coat DNA samples were subjected to
β-globin polymerase chain reaction (PCR) to ensure DNA quality and screened for the presence of PTLV-4 using 2 specific
nested PCRs targeting a 482-bp long terminal repeat (LTR)
and a 195-bp pol region, respectively [3] (Supplementary
Table 1). Synthetic DNA corresponding to the HTLV4_Cam1863LE sequence was used as a positive control and permitted the estimation of the PCR sensitivity to 1 copy in 250 ng
of DNA. A PTLV-4–specific nested tax PCR (275-bp) was
performed on gorilla samples and those from humans who had
been bitten by a gorilla [3] (Supplementary Table 1). An additional generic PTLV 319-bp env nested PCR was performed on
all gorilla and positive human samples [3] (Supplementary
Table 1).
Human plasma samples were tested with a Western blot
(WB) assay (HTLV blot2.4; MP Diagnostics) and a line immunoassay (INNO-LIA HTLVI/II Score; Innogenetics).
RESULTS
Of the 32 gorilla DNA samples screened with PTLV-4 specific
pol, LTR, tax, and generic env nested PCR, 2 were found to be
positive: 1 for LTR, pol, and tax, and the other for all PCRs.
Comparison of the NHP information suggested that the samples corresponded to the same Benito and Nyum gorillas identified in a previous study [3] but sampled 6 years earlier. The 2
captive animals were already infected in 2003. The sequences
showed 100% nt identity with those previously published [3].
Another gorilla, Mvie, was positive for env PCR and was
found to be infected with STLV-1 subtype b (HTLV-1 885-bp
env PCR; GenBank KU863538).
The screening of the 300 human DNA samples identified
2 positive samples that originated from hunters (GabL14
and GabL36) bitten by a gorilla during hunting activities in
Gabon. GabL14 was positive for pol and tax PCR; GabL36
was also positive for LTR PCR. Both samples were also positive
for the generic env PCR.
GabL36 is a 65-year-old man living in the Ogooué-Ivindo
region. He reported a severe bite on the arm by a gorilla in
1991 and was sampled in 2008. Previous studies had shown that
he was also infected with gorilla SFV (GenBank HQ450594) [5].
Generic env PCR amplified an HTLV-1 sequence. Sequencing of
a larger HTLV-1 env region demonstrated that GabL36 was also
infected by an HTLV-1 subtype b strain (GenBank KU863539).
GabL36 exhibited classic HTLV-1 serology with faint reactivity
to HTLV-2 gp46 (Figure 1A and 1B). Sequence analysis of
GabL36 HTLV-4 PCR products showed more than 99.5% nt identity with known PTLV-4 sequences (GenBank LTR KU863537,
tax KU863536). Phylogenetic analysis of the 275-bp tax region
confirmed the proximity of HTLV-4.GabL14 with the only previously known HTLV-4 (Cam1863LE) and STLV-4 strains
(Figure 1C).
GabL14 is a 58-year-old hunter living in the Ngounié region,
also sampled in 2008. When he was younger, he frequently hunted
gorillas that devastated plantations and was severely bitten on the
left thigh by an adult male silverback gorilla in 1999. Furthermore,
he does not remember serious injury when butchering gorillas,
chimpanzees, or monkeys. He is currently suffering from insulin-dependent diabetes and pulmonary tuberculosis. GabL14 plasma had a very faint HTLV-2 pattern by WB assay (Figure 1A),
whereas he exhibited an indeterminate pattern with the line immunoassay (faint seroreactivity to Envgp21, Figure 1B).
Sequence comparison of partial pol, tax, and env sequences
showed that HTLV-4.GabL14 shared only 93% to 96.3%
nt identity with the prototype HTLV-4_Cam1863LE. The complete genome of HTLV-4.GabL14, obtained with 8 successive
nested PCR amplifications (Supplementary Table 1 and Supplementary Figure 1B), is 8799-bp long (GenBank KU863535). Its
genetic organization is similar to that of other PTLV-4 strains
consisting of the gag, pro, pol, env, tax, and rex open reading
frames (ORFs), as well as an antisense ORF in the pX region
showing similarity to HBZ [8] (Supplementary Figure 1A).
While HTLV-4_Cam1863LE has 5 supplementary ORFs in
the pX region, only ORFs I–IV were observed in HTLV-4.
GabL14. Similar to other PTLV-4 sequences, the LTR has
only two 21-bp repeat sequences, and the putative tax protein
does not have a PDZ domain. However, putative HTLV-4.
GabL14 tax protein has an additional amino acid compared
with other PTLV-4 strains. The overall nt identity was only
93%–93.3%: from 91.9% for the LTR to 98% for the rex ORF
(Supplementary Table 2).
Phylogenetic analyses of the concatenated gag-pol-env-tax
genes (5820-bp) clearly demonstrated that HTLV-4.GabL14
segregates strongly with PTLV-4 sequences, although it is
quite divergent from them (Figure 1D).
DISCUSSION
Here, we demonstrated the presence of HTLV-4 infection in 2
hunters of NHPs living in Gabon, Central Africa. The fact that
these viruses were found exclusively in 2 persons severely bitten
by a gorilla (2/102) and not in persons bitten by a chimpanzee
(0/34) or a small monkey (0/164) suggests zoonotic transmission of this retrovirus to humans through a bite from these animals. The presence of an HTLV-1 infection of genotype b (the
subtype present in gorillas) in 1 of the 2 HTLV-4–infected
hunters (GabL36), as well as an infection by a gorilla SFV (a
virus mainly present in saliva and thus nearly exclusively transmitted through severe bites), strongly reinforces the very probable zoonotic origin of HTLV-4 infection in this hunter.
The bites occurred years before blood samples had been collected (17 and 9 years for the 2 hunters) in which infection
was shown. Our results thus indicate the chronic persistence of
this virus in humans, which is the case for the other human retroviruses HTLV-1 and human immunodeficiency virus type 1
and type 2. Our data also demonstrate HTLV-4 chronic persistence in gorillas with no evident genetic variability during 6 years.
GabL14 is infected with a divergent HTLV-4 strain with an
overall variability of approximately 7% at the nucleotide level
relative to the only other known HTLV-4 strain. This new strain
can thus be provisionally considered as the prototype strain of
genotype b of HTLV-4 with the original one being subtype a.
GabL14 had a very faint HTLV-2 serology, whereas the only
other known HTLV-4–infected human and the few STLV-4–
infected gorillas exhibited an HTLV-2 or indeterminate
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Figure 1. Serological patterns of human T-lymphotropic virus type 4 (HTLV-4)–infected individuals (A and B) and phylogenetic analyses of the 2 strains (C and D). A and B,
Western blot analysis (A; HTLV blot 2,4; MP diagnostics) and line immunoassay (B; INNO-LIA HTLV I/II Score; Innogenetics). Lane 1, HTLV-1 positive control; lane 2, HTLV-2
positive control; lane 3, HTLV-3 positive control (Lobak18); lane 4, negative control; lane 5, newly identified HTLV1/4.GabL36; lane 6, newly identified HTLV-4.GabL14. C and D,
Unrooted phylogenetic trees generated with the neighbor joining method (PAUP v4.0) based on a 252-bp tax region from the new strains of HTLV-4 from GabL14 and GabL36
(GenBank KU863536) (C) or 5820-bp concatenated gag-pol-env-tax genes from new GabL14 strain (GenBank KU863535) (D) and complete primate T-lymphotropic virus (PTLV)
sequences available in GenBank. The bootstrap values (1000 replicates) are indicated on the branches of the tree. The branch length is drawn to scale, and the bar indicates 0.1
nucleotide replacements per site. Human and simian strains are indicated in dark red and black, respectively. Abbreviation: STLV, simian T-lymphotropic virus.
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serology [3, 9]. This emphasizes the need to develop better serological methods to detect and discriminate PTLV infections.
Further studies on gorillas and human populations exposed
to NHPs are needed to assess the prevalence of PTLV-4 in gorilla and human populations, estimate the transmission rate,
and search for possible associated diseases. In conclusion, our
study reinforces the fact that gorillas should now be considered
as a keystone reservoir for infectious agents that can be transmitted to humans.
Supplementary Data
Supplementary materials are available at http://cid.oxfordjournals.org.
Consisting of data provided by the author to benefit the reader, the posted
materials are not copyedited and are the sole responsibility of the author, so
questions or comments should be addressed to the author.
Notes
Financial support. This work was supported by the Institut Pasteur and
the Investissement d’Avenir program, as part of a Laboratoire d’Excellence
(LabEx) research program: Integrative Biology of Emerging Infectious Diseases. L. R. was supported by the Bourse de l′Ecole Normale Supérieure, Faculté Paris Diderot. C. F. was supported by the EU PF7 EDENext (n°261504)
and EMPERIE (n°223498). E. B. was supported by the Service de Coopération et de l’Action Culturelle of the French Embassy in Yaounde, Cameroon,
the Institut National pour le Cancer, and the Virus Cancer Prevention Association in Paris, France (Prof. Guy de Thé). The part of the study that took
place in Gabon was supported by the Centre International de Recherches
Médicales de Franceville, funded by the Gabonese Government, Total
Gabon, and the French Foreign Ministry.
Potential conflicts of interest. All authors: No potential conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
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