Sidi et al.

BMC Veterinary Research (2022) 18:69

https://doi.org/10.1186/s12917-022-03166-y

RESEARCH Open Access

Molecular characterization of African swine

fever viruses from Burkina Faso, 2018
Moctar Sidi1^, Habibata Lamouni Zerbo1*, Bruno Lalidia Ouoba1, Tirumala Bharani K. Settypalli2,
Gregorie Bazimo1, Hamidou Sandaogo Ouandaogo1, Boubacar N’paton Sie1, Ilboudo Sidwatta Guy3,
Drabo Dji‑tombo Adama1, Joseph Savadogo3, Anne Kabore‑Ouedraogo1, Marietou Guitti Kindo1,
Jenna E. Achenbach4, Giovanni Cattoli2 and Charles E. Lamien2

Abstract
Background: African swine fever (ASF) is a viral hemorrhagic disease of domestic and wild swine. ASF has been
endemic in Burkina Faso since 2003. In October 2018, substantial pig deaths occurred in Ouagadougou and two
neighboring municipalities in central Burkina Faso. Following these mortalities, the veterinary extension services car‑
ried out investigations to begin control measures and collect samples.
Methods: We performed real-time PCR for diagnostic confirmation and molecular characterization of the virus based
on the partial P72, the complete p54, the partial CD2v, and partial B602L genes.
Results: The field study revealed that mortalities started two weeks before our investigations. The real-time PCR
results confirmed ASFV DNA in twenty samples out of sixty-two blood samples collected in four different locations.
The sequencing and phylogenetic analysis showed that ASFVs causing these outbreaks belong to genotype I and
serogroup 4. The study of the CVR showed 4 TRS variants, and that of the CD2v amino acid sequence revealed five
variants based on the number of deleted KCPPPK motifs in the C-terminal proline-reach region of the protein.
Conclusions: The existence of multiple variants in these outbreaks shows the importance of molecular characteriza‑
tion to understand the evolution of ASFV isolates and the link between epidemics.
Keywords: African swine fever, Central variable region, B646L, B602L, E183L, Burkina Faso

Background Globally, ASF continues to spread and has expanded

African swine fever (ASF) is a viral disease caused by the its geographical distribution to four continents: Africa,
ASF virus (ASFV), an Asfivirus of the family Asfarviridae Europe, Asia, and America [1, 2].
and can affect both wild and domestic pigs. In West Africa, ASF was first reported in 1978 in Sen-
ASF causes a drop in animal production and affects egal and spread to Ivory Coast in 1996, followed by Cape
livestock productivity due to high mortality that can Verde, Togo, and Nigeria in 1997, Benin and Ghana in
reach 100% in outbreaks involving highly virulent strains. 1999, and Burkina Faso in 2003 [3–7]. In 2018 alone,
Due to this high mortality, the disease has a significant Benin, Ivory Coast, Niger, Nigeria, Togo, Ghana, Senegal,
impact on low-income countries. Bissau Guinea reported 79 outbreaks [8].
Burkina Faso has an estimated pig population of
2,345,803 [9]. ASF is now endemic in the country, affect-
ing the livelihood of smallholder farmers and food
*Correspondence: [email protected]
1
security at a national level. For instance, Burkina Faso
Laboratoire National d’Elevage, Ouagadougou, Burkina Faso
Full list of author information is available at the end of the article

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Sidi et al. BMC Veterinary Research (2022) 18:69 Page 2 of 11

reported 32 ASF outbreaks to OIE between 2014 and The present study details our findings during the field
2018, affecting 14 provinces. investigation and the molecular characterization of iso-
Since there is no vaccine for ASF, disease control lates collected during these outbreaks.
mainly relies on early detection and implementation of
control measures such as animal movement restriction,
Materials and Methods
restricted access to infected areas, stamping out, and
Study area, outbreak investigation, and sample collection
proper carcass disposal [10]. Besides, is equally impor-
The study covered four farms in the peri-urban region of
tant to consistently characterize outbreak strains to
Ouagadougou (Fig. 1). All suspicions of ASF cases were
assess how the disease is spreading to better implement
from semi-intensive farms with a herd size of 30 to 100
control measures. There are molecular tools capable of
pigs. The field investigations collected information on
detecting all ASFV genotypes, serogroups, and additional
farm management, history of diseases, examination of
gene variant analysis to characterize strains within vari-
dead and sick animals, and sample collection. Blood sam-
ous genotypes [11–15].
ples were collected on EDTA-treated tubes from Sixty-
Currently, there are twenty-four known ASFV geno-
two sick animals at different locations in Ouagadougou
types [16, 17], all of which have been detected in Africa.
(Table 1) and taken to the Laboratoire National d’Elevage,
Europe and Asia [18] has experienced both genotypes I
Ouagadougou, for diagnostic confirmation.
and II.
In October 2018, there was a report of high mortality

DNA was extracted from blood using DNeasy® Blood &

of pigs in Ouagadougou and surrounding areas in the Nucleic acids extraction and amplification
Kadiogo province (Fig. 1), prompting the deployment of
field investigation teams from the veterinary extension Tissue kit (Qiagen, Hilden, Germany), according to the
services to implement control measures. manufacturers’ instructions. ASFV DNA detection was

Fig.1 Map showing the ASF outbreak suspicion areas in Ouagadougou and surroundings (Kadiogo province) in central Burkina Faso. The red zones
circle indicates the confirmed outbreaks, and the green the non-confirmed ones
Sidi et al. BMC Veterinary Research (2022) 18:69 Page 3 of 11

Table 1 Outbreak informations and samples of this study

Sampling location Morbidity Mortality Sample collect date Number
of samples
collected

Ouagadougou/ Kossodo 100% 91% 10 October 2018 10

Ouagadougou/ Nioko II 100% 100% 10 October 2018 15
Saaba 30% 100% 02 October 2018 16
Ouagadougou / Tampouy 100% 57% 18 October 2018 12
Koubri 9
Total 62

performed by real time PCR, using an adaptation of a GenBank under accession numbers MT851949 to
previously described procedure [19]. MT851967 (p54 gene), MT851968 to MT851986 (p72
gene), MT851987 to MT852005 (B602L gene), and
MT852006 to MT852019 (CD2v gene). For compara-
Molecular characterization of African swine fever virus tive analysis, additional ASFV sequences were retrieved
Three different genomic targets of the ASFV genome from GenBank. Multiple sequence alignments were
were amplified and sequenced using previously described performed with MUSCLE as implemented in MEGA
methods with primers: P72-U- 5`-GGC​ACA​AGT​TCG​ software version 7 [23].
GAC​ATG​T-3` and P72-D—5`-GTA​CTG​TAA​CGC​AGC​ For phylogenetic analysis of the p72 gene fragments,
ACA​G-3` [20] for the C-terminal region of the B646L a data set of 93 nucleotide sequences (398 characters)
gene encoding the p72 protein, P54F-5’-GCC​TGC​GGA​ was prepared, including 19 sequences from this study
TTC​TGA​AGA​TA-3’and P54R- 5`-AGG​ACG​CAA​TTG​ and additional sequences from GenBank with at least
CTT​AAA​CG -3` [12] for the complete E183L gene one representative of each of the 24 known ASFV
encoding the p54 protein, ORF9RLW_F-5`-AAT​ GCG​ genotypes and the sequences of some historical sam-
CTC​AGG​ATC​TGT​TAA​ATC​GG-3` and ORF9RLW_R— ples from Burkina Faso and neighboring countries. A
5`-TCT​TCA​TGC​TCA​AAG​TGC​GTA​TAC​CT -3` [21] for Neighbor-Joining (NJ) tree was produced for the p72
the central variable region (CVR) of the B602L gene. gene in Mega 7 using the Maximum Composite Like-
To determine the serogroups of the 2018 ASFV iso- lihood method, with the data being re-sampled 1,000
lates from Burkina Faso, the partial CD2v gene [22] times using the bootstrap method.
was amplified and sequenced using two sets of primers For the p54 tree, the dataset consisted of 63 taxa (453
ga3611for- 5`-TAT​AAT​ATA​ACA​AAT​AAT​TGTAG- characters), including 19 sequences from this study and
3`, ga4220rev-5`-AGG​GAC​GCA​TGT​AGT​AAA​TAG- additional sequences from GenBank with at least one
3`, ga4124f-5`-CTG​AAT​CTA​ATG​AAG​AAG​A-3` and representative of each of the 18 ASFV p72 genotypes for
ga4698r-5`-AAG​TCT​T TG​TAG​GTT​T TT​C GT ​TCA​-3` which a p54 sequence is available. Historical sequences
to generate two overlapping fragments. Briefly, a mixture from Burkina Faso and neighboring countries were also
consisting of 12. 5 µL of the 2X Q5 High-Fidelity Master included in the dataset. A Minimum Evolution tree was
Mix (Neb Inc), 500 nM of each primer, and 2 µL of the constructed using the p-distance substitution model
template DNA was set up in a total reaction volume of and the Close-Neighbor-Interchange (CNI) algorithm.
25 µL. The following thermal profile was used for ampli- The initial tree was generated using the Neighbor-join-
fication: initial denaturation at 95 °C for 5 min, then 40 ing algorithm. All positions with less than 95% site cov-
cycles of denaturation at 95 °C for 45 s, annealing at 52 °C erage were removed. The data were re-sampled 1,000
for 45 s and elongation at 72 °C for 90 s, and final elonga- times using the bootstrap method.
tion at 72 °C for 5 min. A maximum‐likelihood (ML) tree of the partial CD2v
The amplified PCR products were purified using Wiz- amino acid sequences was constructed applying the
ard SV Gel and PCR Clean-Up kit, according to the predetermined CpREV + G model. The dataset included
manufacturer’s protocol (Promega Corporation, Madi- representatives of the eight known ASFV serogroups
son, WI, USA) and sequenced by LGC Genomics (Ber- and ASFVs clustering outside the eight established
lin, Germany). The raw sequences were assembled and serogroups. For each phylogenetic reconstruction, the
edited using Vector NTI 11.5 Software (Life Technolo- robustness of the tree topology was assessed using 1000
gies, Carlsbad, CA, USA). The nucleotide sequences bootstrap replicates.
of ASFV isolates of Burkina Faso were deposited in
Sidi et al. BMC Veterinary Research (2022) 18:69 Page 4 of 11

For each isolate, the CVR nucleotide sequence was inspection of the multiple sequences alignments of the
translated into amino acid, and the deduced amino acid nucleotide and amino acid sequences of the P54 gene
tetramers were matched with previously reported codes showed that, although they all belong to genotype Ia, the
[11, 13, 24–26]. The CVR sequences were analyzed 2018 isolates of Burkina Faso comprised two sub-groups
together with those of historical isolates from Burkina based on the complete length of the p54 gene (552 bp,
Faso and other Western African countries. and 564 bp). The difference was due to a deletion of 12
nucleotides. The amino acid sequences showed that the
Results deletion occurred in a region flanked by "ATGG" and
Outbreak investigations "SAHP" and containing a series of 2 or 3 repeats of the
The affected farms consisted of semi-intensive produc- four amino acids "PAAA" (Table 2) and Fig. 4.
tion systems with open-air housing and free-ranging The phylogenetic tree of the CD2v partial amino acid
animals kept under reduced biosecurity levels. The inves- sequences showed that all sequenced isolates belong
tigations revealed that the disease had started several to serogroup 4 (Fig. 5). The multiple sequence align-
days to two weeks prior to the visit by our field team to ment of the amino acid sequences of the CD2v protein
the Saaba farm. The affected farms had introduced new showed that the proline-rich region near the C-terminal
animals without observing an initial quarantine period. comprised variable number repeated units of KPCPPP
In addition, several butchers visited the farms to acquire (Fig. 6). Therefore, based on the number of deleted
animals for slaughtering. Typically, the butchers visit sev- PCPPPK units, the isolates could be segregated into
eral farms to obtain pigs, then transport the purchased groups 0, 2, 3, or 4 missing units of the KPCPPP repeats
animals to meat processing sites increasing the risk for (Table 2). Three variants with 0, 2, and 4 missing KPCPPP
further disseminating the disease. In three farms located units were present on the farm at Kossodo. The ASFVs
in the peri-urban area of Ouagadougou, 100% morbid- in Saaba had three missing KPCPPP units, those in Tam-
ity was observed, with a fatality rate varying between pouy had four missing KPCPPP units, and those in Nioko
57 and 100% (Table 1). The farm in Saaba had low mor- II had 0 units missing.
bidity, however, it presented 100% mortality (Table 1). The analysis of 20 CVRs revealed four variants of the
The first outbreak occurred in Saaba, followed by Nioko tetrameric repeat sequence (TRS) with 32, 24, 23, and 12
II, Kossodo, and Tampouy. These four localities have in TRS (Table 2).
common a network of roads connecting them, thus facili- Three out of the four CVR variants of this study
tating the trade of animals between the sites and con- (Table 2) shared motifs beginning with "ABNAAA" and
necting them to pig slaughtering sites. The diseased pigs ending with "CBNAFA" flanking additional TRS. The
showed depression, inappetence, and skin redness, espe- CVR with 12 TRS was present in Saaba, the index out-
cially on the ears with petechiae. They also had hyper- break (September 28, 2018), and in two subsequent
thermia, reduced mobility, and lethargy. Unfortunately, outbreaks in Nioko II and Kossodo (Table 2). In Nioko
fresh carcasses were not available for necropsy. II, there was an additional profile with 24 TRS, unique
to this farm, for which, unfortunately, we could not
Laboratory diagnosis sequence the p72. In Kossodo, there were two other pro-
The real-time PCR results confirmed ASFV DNA in files with 23 and 32 TRS, the former being identical to
twenty, out of sixty-two blood samples, from the four dif- the pattern found in the fourth outbreak that occurred on
ferent locations (Fig. 1) and distributed as follows: seven October 11, 2018, in Tampouy.
positives in Kossodo, seven in Nioko II, three in Saaba,
and three in Tampouy. Nine samples from Koubri tested Discussion
negatives. Since its first discovery in 2003 [5], ASF has been
endemic in Burkina Faso.
Molecular characterization An epizootic of African swine fever in 2008, and the
We successfully sequenced nineteen partial p72 and p54 one in 2018, led to significant losses in the pig production
genes, fourteen partial CD2v genes, and twenty partial sector of the country and neighboring countries of Benin,
B602L. Ivory Coast, Niger, Togo, and Ghana.
In the p72 phylogenetic analysis, all 2018 ASFV from The trade of live animal and animal products and inten-
Burkina Faso clustered within ASFV genotype I (Fig. 2), sive movement of people between the porous borders of
suggesting that only genotype I ASFVs caused these these countries can explain the transboundary persis-
outbreaks. The analysis of the p54 sequences showed tence of ASF.
that all Burkina Faso ASFV isolates of this study belong Our finding that ASF genotype I caused these out-
to the genotype Ia subgroup (Fig. 3). However, a close breaks is consistent with previous reports showing that
Sidi et al. BMC Veterinary Research (2022) 18:69 Page 5 of 11

Fig. 2 Neighbor-joining tree of the partial p72 gene, depicting genetic relationships of the 2018 ASFV isolates from Burkina Faso (highlighted with
red diamonds) with representatives of the 24 known ASFV genotypes. The evolutionary distances were computed using the Maximum Composite
Likelihood method. Bootstrap values > 70% are shown
Sidi et al. BMC Veterinary Research (2022) 18:69 Page 6 of 11

Fig. 3 Minimum Evolution tree, based on the full-length p54 gene sequences, depicting the genetic relationships between the 2018 ASFV isolates
from Burkina Faso (are highlighted with red diamonds) with representatives of the 18 out of 24 known ASFV genotypes. The evolutionary distances
were computed using the p‐distance method. Bootstrap values > 70% are shown

this genotype was the only one circulating in West Africa Africa, only genotype I ASFV is present in Cameroon
[16, 27–29]. [31]. Other Central African Countries also have genotype
Nonetheless, it is worth noting that ASFV genotype I, co-circulating with genotype IX in Chad and the Cen-
II has been reported recently in Nigeria [30]. In central tral African Republic, and genotype IX and XIV in DRC
Sidi et al. BMC Veterinary Research (2022) 18:69 Page 7 of 11

Table 2 Comparison of the CVR, p54 and CD2v (C-terminal) profiles of the 2018 ASF outbreak isolates from Burkina
Isolate name CVR Number of p54 (PAAA CD2v (number of Location Outbreak date
repeats (CVR) repeats) deleted PCPPPK)

BKF_2018_01 ABNAAA​AAA​ACA​AAA​AACBNAFA 23 3 2 Kossodo 28-Sep-18

BKF_2018_02 ABNAAA​AAA​ACA​AAA​AACBNAFA 23 3 2 Kossodo 28-Sep-18
BKF_2018_03 ABNAAA​AAA​ACA​AAA​AACBNAFA 23 3 2 Kossodo 28-Sep-18
BKF_2018_05 ABNAAA​AAC​BNAAA​AAC​BNAAA​AAA​ACBNAFA 32 3 4 Kossodo 28-Sep-18
BKF_2018_06 ABNAAACBNAFA 12 2 0 Kossodo 28-Sep-18
BKF_2018_07 ABNAAACBNAFA 12 2 0 Kossodo 28-Sep-18
BKF_2018_08 ABNAAACBNAFA 12 2 0 Kossodo 28-Sep-18
BKF_2018_09 AAAABNABBNABBAABBNABNABA 24 2 ND Nioko II 29-Sep-18
BKF_2018_10 ABNAAACBNAFA 12 2 0 Nioko II 29-Sep-18
BKF_2018_11 ABNAAACBNAFA 12 2 0 Nioko II 29-Sep-18
BKF_2018_12 ABNAAACBNAFA 12 2 0 Nioko II 29-Sep-18
BKF_2018_13 ABNAAACBNAFA 12 2 0 Nioko II 29-Sep-18
BKF_2018_14 ABNAAACBNAFA 12 2 0 Nioko II 29-Sep-18
BKF_2018_15 ABNAAACBNAFA 12 2 0 Nioko II 29-Sep-18
BKF_2018_16 ABNAAACBNAFA 12 2 3 Saaba 23-Sep-18
BKF_2018_17 ABNAAACBNAFA 12 2 3 Saaba 23-Sep-18
BKF_2018_18 ABNAAACBNAFA 12 2 3 Saaba 23-Sep-18
BKF_2018_19 ABNAAA​AAC​BNAAA​AAC​BNAAA​AAA​ACBNAFA 32 3 4 Tampouy 11-Oct-18
BKF_2018_20 ABNAAA​AAC​BNAAA​AAC​BNAAA​AAA​ACBNAFA 32 3 4 Tampouy 11-Oct-18
BKF_2018_21 ABNAAA​AAC​BNAAA​AAC​BNAAA​AAA​ACBNAFA 32 3 4 Tampouy 11-Oct-18

Fig. 4 Comparison of the 2018 ASFV isolates from Burkina Faso using the amino acid sequences of the p54 protein. The partial representation of
the multiple sequence alignment shows the amino acid sequences variation among the isolates (inside the red box)

(See figure on next page.)

Fig. 5 Maximum Likelihood tree based on the partial amino acid sequences of the CD2v protein. The tree shows the relationship between the 2018
ASFV isolates from Burkina Faso (highlighted with red diamonds) and the representatives of the eight known ASFV serogroups and ASFVs clustering
outside the eight established serogroups. The General Reversible Chloroplast Model with Gamma distribution (cpREV + G) was used. Bootstrap
values higher than 70% are shown
Sidi et al. BMC Veterinary Research (2022) 18:69 Page 8 of 11

Fig. 5 (See legend on previous page.)

Sidi et al. BMC Veterinary Research (2022) 18:69 Page 9 of 11

Fig. 6 Comparison of the 2018 ASFV isolates from Burkina Faso using the C-terminal of the CD2v protein. The partial representation of the multiple
sequence alignment shows the amino acid sequences variation in the C-terminal of the CD2v protein (inside the red box)

[12]. The analysis of the CVR in the 2018 samples showed Our study suggests that the proline-rich region near
four variants of the tetrameric repeats. An earlier study the C-terminal of the CD2v gene is suitable for genotype
of samples collected between 2007 and 2010 [28] showed I ASFV isolates discrimination.
ten variants of the tetrameric repeats, all different from Using genotype I and serogroup 4 isolates recovered
those found in this study, making fourteen variants iden- during 2018 outbreaks in central Burkina Faso, we have
tified in the country, between 2007 and 2010 and 2018. shown that the number of deleted KPCPPP units in the
In this study, there were three TRSs variants, two p54 proline-rich region of the CD2v protein varied substan-
profiles, three CD2v variants in Kossodo, and two differ- tially, enabling it to be an additional mean to discriminate
ent TRSs variant Nioko II. ASFVs. Hence, our study confirms an earlier report that
Saaba, Nioko II, and Kossodo shared a common ASFV analysis of the number of PCPPPK repeats provided an
variant with 12 TRS in the CVR, suggesting an epide- additional mean, similar to the analysis of the CVR pro-
miological link between those three outbreaks. The vari- file [32]. Further investigations will establish whether
ant with 12 TRS from Saaba had three KPCPPP units this approach is suitable to analyses of other ASFV sero-
deleted at the C-terminal of its CD2v, differentiating it groups and genotypes.
from the 12 TRS variants of Nioko II and Kossodo with Surprisingly, our field investigations determined that
no missing KPCPPP unit. Saaba, Nioko II, and Kossodo the pig breeders know little about common diseases of
are three nearby locations with direct connections and swine, especially African swine fever. In addition, there
frequent trade activities. The onset dates of these out- is insufficient technical support and minimal effort to
breaks, between September 23 and September 28, 2018, increase pig breeder and community awareness of ASF.
support the hypothesis that the disease spread from the The pig production system in Burkina Faso is based
first outbreak in Saaba to the neighboring sites of Nioko mainly on a traditional extensive system with household
II and Kossodo. Similarly, ASFV BKF_2018_05, a 32 TRS free-ranging pigs and small to medium semi-intensive
variant collected in Kossodo on September 28, 2018, had farming systems mainly in peri-urban areas. The housing
identical TRS, CD2v, and p54 profiles to all ASFV col- comprises shelters made with local material in extensive
lected in Tampouy during an outbreak that started on systems or semi-modern to modern housing in the semi-
October 11, 2018. The detection of identical viruses and intensive system [33]. In most farms, brewers’ grains and
the date of the outbreaks in the two locations suggest a swill are the primary feeds for the pigs.
spread of ASFV from Kossodo to Tampouy. Near and between the farms, there is a lack of proper
It is unclear why multiple ASFV variants are present in biosecurity practices, promoting several diseases, includ-
Kossodo and Nioko II but could suggest multiple intro- ing ASF [33]. Additionally, other domestic animals like
ductions of the disease within those farms. It is also pos- cattle, sheep, goats, dogs, rodents, and birds are free-
sible that the virus mutated while spreading within those ranging and share common space with pigs; therefore,
farms. Previous reports have suggested changes in the they could also spread diseases. These conditions raise
CVR sequence during adaptation of ASFV to cell cultures the need for increased awareness of transboundary
[21] and during ASF epidemics [12, 16]. animal diseases and continued rapid diagnostics and
Sidi et al. BMC Veterinary Research (2022) 18:69 Page 10 of 11

analysis to identify and understand future outbreaks. further ethical approval, as the purpose is to maintain the health and welfare
of animals. The outbreaks investigation was authorized by the General Directo‑
For instance, additional outbreaks of ASF occurred in rate of Veterinary Services (letter N°2018_11_MRAH/SG/DGSV). Informed oral
new locations in the country since those described in consent to participate was obtained from the owners.
this paper. Although there was no evidence for an epide-
Consent for publication
miological link between the recent outbreaks and those Not applicable
described in this paper, the continuous occurrence of
ASF outbreaks highlights inadequate disease manage- Competing interests
All authors declared that they have no competing interests.
ment. This instigated the veterinary authorities to start
implementing active surveillance to better monitor ASF Author details
and characterize the circulating genotypes of the ASF 1
Laboratoire National d’Elevage, Ouagadougou, Burkina Faso. 2 Animal Produc‑
tion and Health Laboratory, Joint FAO/IAEA Division of Nuclear Techniques
virus.” in Food and Agriculture, Department of Nuclear Sciences and Applications,
International Atomic Energy Agency, Vienna, Austria. 3 Direction Générale Des
Services Vétérinaire, Ouagadougou, Burkina Faso. 4 Battelle Memorial Institute,
Conclusion 1001 Research Park blvd, Charlottesville, VA 22901, USA.
We have shown that ASFVs belonging to genotype I,
Received: 8 October 2021 Accepted: 20 January 2022
serogroup 4, caused four outbreaks in October 2018 in
Burkina Faso. There were 4 TRS variants based on the
CVR analysis and five based on the number of deleted
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