Ticks and Tick-borne Diseases 8 (2017) 640–645
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Ticks and Tick-borne Diseases
journal homepage: www.elsevier.com/locate/ttbdis
Original article
Detection of a novel putative phlebovirus and first isolation of Dugbe virus MARK
from ticks in Accra, Ghana
Daisuke Kobayashia,b,1, Mitsuko Ohashia, Joseph H.N. Oseic, Esinam Agbosud, Millicent Opokuc,
Alfred Agbekudzic, Joannitta Joannidesc, Ryosuke Fujitab,e, Toshinori Sasakib, J.H. Ko dfi Bonney ,
Samuel Dadziec, Haruhiko Isawab,⁎, Kyoko Sawabeb, Nobuo Ohtaa
a Department of Environmental Parasitology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-0034, Japan
b Department of Medical Entomology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
c Department of Parasitology, Noguchi Memorial Institute for Medical Research, University of Ghana, P.O. Box LG 581, Legon, Accra, Ghana
d Department of Virology, Noguchi Memorial Institute for Medical Research, University of Ghana, P.O. Box LG 581, Legon, Accra, Ghana
e Japan Agency for Medical Research and Development, 1-7-1 Ote-machi, Chiyoda-ku, Tokyo 100-0004, Japan
A R T I C L E I N F O A B S T R A C T
Keywords: Ticks are ectoparasites that transmit various types of human and animal pathogens. In particular, emerging and
Africa re-emerging diseases caused by tick-borne viruses are public health concerns around the world. However, in
Ghana many countries of the sub-Saharan African region, epidemiological information on tick-borne viral infections is
Dugbe virus limited, and their prevalence and distribution remain largely unknown. In this study, we conducted surveillance
Nairovirus on ticks to detect medically important tick-borne bunyaviruses in three study sites in and near to Accra, the
Phlebovirus
Surveillance capital city of Ghana, in 2015. Domestic dogs and cattle were surveyed and were found to be infested with
Tick various tick species belonging to the genera Rhipicephalus, Amblyomma and Haemaphysalis. Importantly, we
detected a novel putative phlebovirus in Rhipicephalus ticks, and successfully isolated a new strain of Dugbe virus
from Am. variegatum ticks. To our knowledge, this is the first report of tick-associated viruses in Ghana other than
Crimean-Congo hemorrhagic fever virus.
1. Introduction maining largely unknown.
In the Republic of Ghana, located in West Africa, tick-borne viral
Ticks are known as important vectors of human and animal diseases in human have not yet been reported, although many potential
pathogens including protozoa, bacteria and viruses. Recently, emerging viral vectors are distributed nationwide (e.g. Hyalomma marginatum
infectious diseases caused by novel tick-borne viruses such as severe rufipes as a vector of CCFHV, Rhipicephalus decoloratus as a vector of
fever with thrombocytopenia syndrome virus, Heartland virus, and Bhanja virus, and Amblyomma variegatum as a vector of Dugbe virus)
Bourbon virus have been discovered in some parts of the world (Yu (Ntiamoa-Baidu et al., 2004) and CCHFV was recently detected in
et al., 2011; Savage et al., 2013; Kosoy et al., 2015). On the other hand, ixodid ticks (Akuffo et al., 2016). In fact, human or other animal
reported cases of infection with Crimean-Congo hemorrhagic fever infections with several tick-borne viruses, such as Bhanja virus and
virus (CCHFV) and tick-borne encephalitis virus, both of which had Dugbe virus, are present in surrounding countries (Moore et al., 1975;
been found more than half a century ago, are increasing in epidemic Matsuno et al., 2013). The present tick survey was conducted in three
proportions, and expansion of their geographical distributions have study sites in and near to Accra, the capital city of Ghana, to determine
become global public health concerns as re-emerging viral diseases the geographical distribution and infection risk of tick-borne viruses in
(Estrada-Peña and de la Fuente, 2014). In the sub-Saharan African the Greater Accra Region in Ghana.
region, tick-borne bacterial infections including tick-borne relapsing
fever and African tick bite fever are common and well documented
(Hotez and Kamath, 2009). However, information on tick-borne viral
diseases remains limited, with their prevalence and distribution re-
⁎ Corresponding author.
E-mail address: hisawa@nih.go.jp (H. Isawa).
1 Research Fellow of Japan Society for the Promotion of Science.
http://dx.doi.org/10.1016/j.ttbdis.2017.04.010
Received 7 December 2016; Received in revised form 7 March 2017; Accepted 14 April 2017
Available online 23 April 2017
1877-959X/ © 2017 Elsevier GmbH. All rights reserved.
D. Kobayashi et al. Ticks and Tick-borne Diseases 8 (2017) 640–645
Fig. 1. Geographical location of the tick collection sites in Ghana. (A) The Greater Accra Region, which is the tick collection area in this study, is shown in gray. (B) Enlarged figure of the
Greater Accra Region. Each tick collection site (Mobore, Pokuasi, and Korle Gonno) is indicated by a black dot.
2. Materials and methods and Lanciotti, 2009) and the partial L protein gene in the phlebovirus L
segment (TBPVL2759F and TBPVL3267R; Matsuno et al., 2015),
2.1. Study sites, tick collections and identifications respectively. In addition, for the purpose of phylogenetic analysis of
nairoviruses, we also used another primer set (6942+ and 7385−;
Tick collections were performed in the following three study sites; Honig et al., 2004) to amplify the genomic region including the
Mobore (N5°82′, W0°02′), Pokuasi (N5°69′, W0°28′), and Korle Gonno conserved motifs in the L protein genes of nairoviruses. All primer sets
(N5°53′, W0°23′) districts within the Greater Accra Region, Ghana were used under a common RT-PCR thermal condition: RT at 50 °C for
(Fig. 1) in September 2015. Mobore is a rural site in coastal scrub 30 min; termination of RT at 94 °C for 2 min; and 35 cycles for PCR at
outside the city of Accra, where many domestic animals (cattle or goats) 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 30 s. The amplified products
are reared. There are some remaining bushes for the animals to be were confirmed by agarose gel electrophoresis and directly sequenced.
grazed, and they are often put out to graze in the bush. Korle Gonno is a Virus isolation using cell cultures was performed as previously
long-established residential district of urban Accra, and crammed with described (Ejiri et al., 2015). In brief, the filtrates were inoculated into
human residences. Thus, the district has very few domestic animals and monolayers of BHK-21 (Syrian hamster kidney) cells (Japan Health
sparse bushes. Pokuasi is one of the cities located outside the boundary Science Research Resources Bank, Osaka, Japan) and incubated at 37 °C
of urban Accra, and there are some bushes around the residences. In in 5% CO2 conditions for 7 days. The development of cytopathic effects
this study site, we came across goats and sheep occasionally, but no (CPE) was monitored by comparing with mock-inoculated controls.
pastured cattle. Therefore, Pokuasi is intermediate between the other After three additional blind passages, the supernatants were harvested
two sites being more developed than Mobore but still outside the city and cryopreserved at −80 °C until further analysis.
boundary. It was unclear whether any tick control for domestic animals
were practiced at each study site (Awumbila, 1996). 2.3. Next generation sequencing of the cell culture supernatants
In these three study sites, ticks infesting domestic animals (dogs and
cattle) were collected. The collected ticks were identified to genus and, To confirm and identify the isolated viruses, next generation
where possible, species level according to the identification keys of sequencing (NGS) was performed on the cell culture supernatants after
Walker et al. (2003), separated according to instar and sex and stored at the blind passages. However, the supernatant from the pool 15AC-25,
−80 °C until virus detection and isolation commenced. from which DUGV was isolated, was omitted from this NGS analysis.
The basic approach of NGS has been described previously (Kobayashi
2.2. Virus detection and isolation et al., 2016). In brief, the supernatants of 4 to 8 individual pools were
mixed together, and then 4 units of TURBO DNase (Life Technologies),
Collected ticks were separated into a maximum of 17 individuals 4 units of Baseline ZERO DNase (Epicentre, Madison, WI), and 0.4 μg of
per pool (Supplementary Table S1) and homogenized using the disposal RNase A (Wako Pure Chemical Industries, Osaka, Japan) were added to
tissue homogenizer (BioMasher II, Nippi Inc., Tokyo, Japan) in 500 μl of 390 μl of each mixed supernatant. After incubation at 37 °C for 1 hr,
Eagle's minimum essential medium (MEM, Sigma–Aldrich, St. Louis, total RNA was extracted and the cDNA synthesis and amplification were
MO) supplemented with 2% heat-inactivated fetal bovine serum performed using SeqPlex RNA Amplification kit (Sigma-Aldrich). The
(Sigma–Aldrich), 2% non-essential amino acids (Sigma–Aldrich), amplified cDNAs were subjected to NGS using the Ion PGM system
200 U/ml penicillin (Life Technologies, Carlsbad, CA), 200 μg/ml (Thermo Fisher Scientific, Waltham, MA). The resultant data was
streptomycin (Life Technologies), 5 μg/ml fungizone (Life analyzed by the CLC genomics workbench software (CLC bio, Aarhus,
Technologies). Subsequently, each homogenate was passed through a Denmark).
sterile 0.45 μm filter (Merck Millipore, Darmstadt, Germany) and total
RNA was extracted from 200 μl of the resultant filtrates using ISOGEN II 2.4. Phylogenetic analysis
reagent (Nippon Gene, Tokyo, Japan) for reverse transcription-poly-
merase chain reaction (RT-PCR)-based virus detection. The RT-PCR for Phylogenetic analyses of phleboviruses and nairoviruses were
detection of nairoviruses (genus Nairovirus, family Bunyaviridae) and performed based on the amino acid (aa) or nucleotide (nt) sequences
phleboviruses (genus Phlebovirus, family Bunyaviridae) was carried out derived from the RT-PCR amplicons using the primers described above.
using the PrimeScript One Step RT-PCR Kit Ver. 2 (Takara, Shiga, Multiple alignments with aa or nt sequences from related viruses were
Japan) with universal primers to amplify the partial N protein gene in conducted using the Clustal W method. The phylogenetic analysis was
the nairovirus S segment (Nairo forward and Nairo reverse; Lambert carried out using the Neighbor-Joining method (Saitou and Nei, 1987).
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D. Kobayashi et al. Ticks and Tick-borne Diseases 8 (2017) 640–645
Table 1
Number of ticks collected in Accra, Ghana in September 2015.
Collection site Tick species Total no. of individuals Stages Host animals
Nymph Adult male Adult female
Mobore Am. variegatum 26 0 26 0 Dogs
25 0 25 0 Cattle
Amblyomma spp. 18 5 0 13 Dogs
4 0 0 4 Cattle
Haemaphysalis spp. 23 6 2 15 Dogs
Rhipicephalus spp. 15 4 5 6 Dogs
1 0 1 0 Cattle
Pokuasi Rhipicephalus spp. 25 7 10 8 Dogs
Korle Gonno Rhipicephalus spp. 16 0 15 1 Dogs
Total 153 22 84 47
These analyses were performed in MEGA 7 (Kumar et al., 2016). not a tick-borne virus or a tick-only virus at present.
Several new phleboviruses provisionally categorized as “the un-
3. Results and discussion classified group” within TBPV have been discovered recently (Tokarz
et al., 2014; Matsuno et al., 2015; Li et al., 2015; Papa et al., 2016);
A total of 153 ticks were collected directly from domestic animals however, the viral M segment of these viruses have not been identified
(dogs and cattle) in three collection sites in September 2015 (Table 1). and viable virus isolates have not yet been obtained so far (Tokarz et al.,
Twenty-nine tick pools derived from 153 individuals (Supplementary 2014; Li et al., 2015). On the other hand, these viral sequences were not
Table S1) were subjected to virus detection by RT-PCR. Two pools of amplified by PCR from the host tick genomes (Tokarz et al., 2014). In
Am. variegatum ticks collected from Mobore were positive for nairo- addition, ANTV, a putative phlebovirus most closely related to ODWV,
viruses (genus Nairovirus, family Bunyaviridae) whilst four pools of was not detected as its DNA forms in the host ticks (Papa et al., 2016).
Rhipicephalus ticks collected from Pokuasi and Korle Gonno were These studies indicated that these viral sequences were not derived
positive for phleboviruses (genus Phlebovirus, family Bunyaviridae) from endogenous virus-like elements in the host tick genomes. How-
(Table 2). An almost identical 487-nt sequence was obtained from 2 of 4 ever, in this study, we did not confirm the presence or absence of the
phlebovirus-positive pools from Pokuasi and Korle Gonno, and were DNA forms of ODWV in the host ticks, and failed to isolate viable
tentatively named Odaw virus (ODWV) strains 15AC-T33 (GenBank viruses from any of the four phlebovirus-positive pools (two pools
accession no. LC193451) and 15AC-T35 (GenBank accession no. identified as ODWV from Pokuasi and Korle Gonno, and the other two
LC193452), respectively. However attempts made to sequence the unindentifiable pools from Pokuasi) (Table 2). Therefore, we could not
amplicons from the other two phlebovirus-positive pools from Pokuasi exclude the possibility that the ODWV sequences are derived from
and two nairovirus-positive pools from Mobore were not successful. To endogenous viral-like elements in the host tick genome at present.
assess the phylogenetic position of ODWV with known and referenced Further studies of the unclassified TBPV group viruses including ODWV
tick-borne phlebovirus (TBPV) sequences, we performed phylogenetic are needed to understand the infection dynamics and to assess the
analysis based on the partial aa sequence deduced from the nt sequence potential threat to public health.
of the viral L segment (Fig. 2). The ODWV strains were found within the During virus isolation, one of the two nairovirus-positive pools of
clade of “the unclassified group” with other referenced strains of TBPV, Am. variegatum ticks (pool No. 15AC-T25; Table 2) caused a severe CPE
and were most closely related to Antigone virus (ANTV) from Rhipice- in BHK-21 cells. To identify the causative agent, RNA extraction was
phalus sanguineus sensu lato ticks in Greece (Papa et al., 2016). The performed on the supernatant and RT-PCR was carried out using the
ODWV and ANTV strains (GenBank accession nos. KU375577, nairovirus-specific primer set. The resultant 442-nt amplicon was
KU375576, and KP164566; Papa et al., 2016) shared 77–79% nt sequenced, and subjected to phylogenetic analysis among known
sequence identities and 95–96% aa identities. Since the ODWV nairoviruses. The isolate 15AC-T25 (GenBank accession no.
sequences were detected from the tick pools including engorged ticks, LC193450) formed a robust clade with DUGV (Fig. 3). Therefore, the
it is unclear whether the putative phlebovirus strains were derived from study successfully isolated DUGV from male Am. variegatum collected
the tick samples, blood of the host animals, or both. In addition, we from Mobore and designated this isolate as DUGV strain 15AC-T25. On
cannot rule out the possibility that ODWV is a dog-infecting virus but the other hand, through the NGS analyses of the cell culture super-
Table 2
List of tick pools positive for nairovirus and phlebovirus by the RT-PCR assays.
Pool No. Collection site Tick species Number, instar, and condition of pooled ticks Host animals Results of detection Results of isolation
15AC-T25 Mobore Amblyomma variegatum 8 adult males (non-enga) Cattle Positive for nairovirus DUGV
15AC-T26 Mobore Amblyomma variegatum 7 adult males (non-eng) Cattle Positive for nairovirus N.I.c
15AC-T33 Pokuasi Rhipicephalus spp. 1 adult male (non-eng), 1 adult female (engb) Dogs Positive for phlebovirus N.I.
(ODWV)
15AC-T34 Pokuasi Rhipicephalus spp. 3 adult males (non-eng), 2 adult females (eng), 1 Dogs Positive for phlebovirus N.I.
nymph (non-eng)
15AC-T35 Korle Gonno Rhipicephalus spp. 15 adult males (non-eng), 1 adult female (eng) Dogs Positive for phlebovirus N.I.
(ODWV)
15AC-T36 Pokuasi Rhipicephalus spp. 6 adult males (non-eng), 5 adult females (eng), 6 Dogs Positive for phlebovirus N.I.
nymphs (non-eng)
a Non-engorged.
b Engorged (partially or fully engorged).
c Not isolated.
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D. Kobayashi et al. Ticks and Tick-borne Diseases 8 (2017) 640–645
Fig. 2. Phylogenetic relationships between ODWV and other phleboviruses based on the aa sequence similarities of the RdRp encoded by the L segment. Phylogenetic analysis based on
160-aa sequence deduced from partial sequence of the viral L segment was performed using the Neighbor-Joining method. The percentage of replicate trees in which the associated taxa
clustered together in the bootstrap test (1000 replicates) are shown at the nodes (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the
evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method (Zuckerkandl and Pauling, 1965). The strains
of ODWV are indicated with black arrows. Phleboviruses and their genome sequences used in this analysis are indicated by virus name with Genbank accession number in parentheses.
natants, no virus-related sequences were identified from other pools Taken together with these previous findings, our present results
including another nairovirus-positive pool (15AC-T26) and four phle- suggested that DUGV might circulate between domestic animals and
bovirus-positive pools (15AC-T33–36). DUGV is a member of the Am. variegatum ticks in the rural area of the Greater Accra region,
Nairobi sheep disease serogroup within the genus Nairovirus, family although a more detailed investigation is required to assess the natural
Bunyaviridae (Fig. 3). This virus was first isolated from Am. variegatum history of DUGV in the study site. A couple of decades ago, Am.
ticks from Nigeria in 1964 (Causey, 1970), and is known to be variegatum was reported as a common tick species on cattle, goat, and
distributed at least in other nine African countries so far (Senegal, sheep in throughout the Ghana (Koney and Morrow, 1990; Koney et al.,
Cameroon, Central African Republic, Ethiopia, Sudan, Uganda, Chad, 1996; Morrow et al., 1996; Walker and Koney, 1999). In our present
Kenya, and Egypt) (Darwish et al., 1976; Wood et al., 1978; Hoogstraal, study, Am. variegatum was common in Mobore (a rural area), but was
1979). Our study represents the first documented isolation of DUGV in not collected in Pokuasi (a peri-urban area) and Korle Gonno (an urban
Ghana. In previous studies, DUGV has been frequently isolated from area) within the Greater Accra region. Thus, we think that the
Am. variegatum ticks and cattle in some African countries (Causey, differences in surrounding environment may reflect not only the tick
1970; Williams et al., 1972). Antibodies to the virus have been detected fauna but also the prevalence of tick-borne viruses.
in the serum of cattle in Central African Republic (Guilherme et al., In conclusion, the study reported tick surveillance carried out in
1996). In addition, the experimental infection study of DUGV showed Greater Accra region and importantly detected a novel putative
that the virus replicates efficiently in Am. variegatum ticks (Steele and phlebovirus, named ODWV, in Rhipicephalus ticks. The study success-
Nuttall, 1989). Therefore, it appears that Am. variegatum is a potential fully isolated the first Ghanaian strain of DUGV from Am. variegatum
vector of DUGV, and cattle are natural host candidates of DUGV. To ticks. Future nationwide survey is needed to assess the current status of
date, a few human cases of DUGV infection with fever and thrombo- the prevalence and risk of tick-borne virus infections in Ghana.
cytopenia have been reported (Moore et al., 1975; Burt et al., 1996).
Although human infection of DUGV is relatively rare, the virus has been
associated with febrile illness among children (Moore et al., 1975).
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D. Kobayashi et al. Ticks and Tick-borne Diseases 8 (2017) 640–645
Fig. 3. Phylogenetic relationships between DUGV strain 15AC-T25, known DUGV strains and other nairoviruses based on the nt sequence similarities of the viral L segment. Phylogenetic
analysis based on partial 442-nt sequence of the viral L segment was performed using the Neighbor-Joining method. The percentages of 1000 bootstrap replication were indicated at the
nodes. The DUGV strain 15AC-T25 is indicated with a black arrow. Nairoviruses and their genome sequences used in this analysis are indicated by virus name with GenBank accession
number in parentheses.
Acknowledgement borne viral diseases. Antiviral Res. 108, 104–128.
Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap.
Evolution 39, 783–791.
We thank Mayumi Shimura for supporting our work in Ghana. The Guilherme, J.M., Gonella-Legall, C., Legall, F., Nakoume, E., Vincent, J., 1996.
authors are also grateful to all the staff of Noguchi Memorial Institute Seroprevalence of five arboviruses in Zebu cattle in the Central African Republic.
for Medical Research for their support. In addition, we are deeply Trans. R. Soc. Trop. Med. Hyg. 90, 31–33.Honig, J.E., Osborne, J.C., Nichol, S.T., 2004. The high genetic variation of viruses of the
grateful to animal owners and cooperating people for supporting our genus Nairovirus reflects the diversity of their predominant tick hosts. Virology 318,
work in the study sites. This work was supported by grants from the J- 10–16.
GRID (Japan Initiative for Global Research Network on Infectious Hoogstraal, H., 1979. Review Article: The epidemiology of tick-borne Crimean-Congo
hemorrhagic fever in Asia, Europe, and Africa. J. Med. Entmol. 15, 307–417.
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Infectious Diseases from Japan Agency for Medical Research and their prevalence, distribution, and disease burden. PLoS Negl. Trop. Dis. 3, e412.
Development, AMED. In addition, this work was supported by JSPS Kobayashi, D., Isawa, H., Ejiri, H., Sasaki, T., Sunahara, T., Futami, K., Tsuda, Y.,
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