First detection and molecular characterization of Jingmen Tick Virus with a high occurrence in Rhipicephalus (Boophilus) microplus collected from livestock in Cameroon (2024)

doi:10.21203/rs.3.rs-5294136/v1

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First detection and molecular characterization of Jingmen Tick Virus with a high occurrence in Rhipicephalus (Boophilus) microplus collected from livestock in Cameroon (2024)

https://doi.org/10.21203/rs.3.rs-5294136/v1

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Background.

Jingmen tick virus (JMTV) is a novel tick-borne virus detected for the first time in Riphicephalus (Boophilus) microplus in China. To date, there is no information regarding the circulation of JMTV in ticks collected from livestock in Cameroon. This study aims to assess the presence of JMTV in ticks collected from livestock (cattle and sheep) in an area of the Akonolinga health district, Center region, Cameroon.

Methods.

A cross sectional study was carried out during the dry season between the 5th to 14th march 2024. Ticks were collected from cattle and sheep in six sampling sites in an area approximately 30 km long and 18 km wide along the Nyong river, in central Cameroon. Ticks were identified morphologically and molecularly. Total RNA/DNA was extracted from tick pools and screened for JMTV RNA using a segment 2 RT-qPCR system. Positive JMTV pools were sequenced for partial JMTV-Segment 1 and full genome analyses.

Results.

A total of 622 ticks, organized in 251 pools were collected from 155 cattle and 9 sheep. They consisted of five species covering three genera: R. (B.) microplus (472; 75.88%), Amblyomma variegatum (118; 18.97%), Hyalomma truncatum (13; 2.09%), Hyalomma rufipes (2; 0.32%), and other Rhipicephalus spp. (17; 2.73%). The qRT-PCR screening of 251 tick pools yielded 61 JMTV-positive pools of which 58 corresponded to R. (B.) microplus. Multiple sequence analysis revealed that JMTV from the Akonolinga area shared > 95% identity with strains from Guinea, and that these strains clustered phylogenetically together.

Conclusions.

We provided molecular evidence of the presence of JMTV in R. (B.) microplus and A. variegatum collected from cattle and sheep from an area not yet recognized as endemic for this virus confirming the wide geographical distribution of JMTV.

Jingmen tick virus

ticks

cattle

sheep

Cameroon

Jingmen tick virus (JMTV) is a novel tick-borne virus characterized by a segmented RNA genome [1]. Jingmen tick virus was first described in Rhipicephalus (Boophilus) microplus ticks in the Jingmen region of the Hubei province (China) in 2010. Following this discovery, JMTV has been detected in multiple arthropod and vertebrate species, alongside other segmented flavivirus-genetically-related viruses classified as jingmenviruses, such as Alongshan virus [2]. The partial genetic relatedness concerns non-structural proteins from segments 1 and 3 similar to flavivirus NS5, NS2B and NS3. Segments 2 and have a different evolutionary origin and are to date specific to jingmenviruses [3, 4].

Jingmenviruses group phylogenetically into two main clades: one clade comprises tick- and vertebrate-associated jingmenviruses while the other includes insect- and other host-associated jingmenviruses [5].

Although the pathogenicity of JMTV remains poorly understood, the virus has been detected in multiple common species of ticks and in patients presenting with mild to severe febrile disease [6]. JMTV has been identified in serums of fatal cases of Crimean-Congo hemorrhagic fever virus (CCHFV) in Kosovo and detected in patients bitten by ticks [6, 7]. Jingmen tick virus was shown to be present and replicate in human skin tissue by in situ hybridization [6]. These results indicate that a potential pathogenicity for humans merits further investigation.

The primary mode of transmission of JMTV is believed to occur through tick bites during blood-sucking. Despite regional variations in the species of ticks reported to harbour JMTV (i.e: Haemaphysalis longicornis, R. sanguineus, Ixodes sinensis, and Amblyomma javanense), the most common species in which JMTV RNA has been detected is R. (B.) microplus [5]. Jingmen tick virus RNA has been also identified in other arthropods and mosquito. Jingmen tick virus RNA and specific antibodies were been detected in mammals such as cattle, bats, goats, and various rodent species [4], as well as in samples derived from primates and reptiles, specifically tortoises [8, 9].

Jingmen tick virus has a wide geographic distribution in ticks: the Americas (Brazil, Colombia, Trinidad and Tobago), Asia (China, Japan, Laos and Türkiye), Europe (France, Romania,Finland and Serbia), and Africa (Kenya, Guinea ) [5, 10]. In Africa, JMTV was also detected in R. geigyi and in R. (B.) microplus collected from domestic cattle in Guinea [11], R. (B.) microplus collected from cattle and in several tick species collected from ruminants and tortoises in Kenya [9]. In Uganda, a mosquito-associated Jingmenvirus has been detected in non-human primates [12]. However, the prevalence and distribution of JMTV in other African countries is still poorly characterised. To date, there is no information regarding the circulation of JMTV in ticks collected from livestock in Cameroon. This study aims to assess the presence of JMTV in ticks collected from livestock (cattle and sheep) in an area of the Akonolinga health district, Centre region, Cameroon.

Description of the study sites

The Akonolinga health district is located in the department of Nyong-Mfoumou, 100 km east from Yaoundé in the Central region of Cameroon, with a population of 105,789 inhabitants in 2015 (Fig. 1). The Akonolinga area lies within the forest-savannah transitional ecological zone (humid forest-savannah mosaic), thus exposing human and animal populations to both ecosystems. The environmental conditions are influenced by seasonality, as this zone has a sub-tropical climate with two rainy seasons per year (August to November; April to May) and two dry seasons (December-March and July-August) [13]. The Nyong river basin in this area, which has several tributaries throughout the villages, is characteristically known for its swampy banks, which is the contact point between human population, domestic and wild animals also providing suitable habitats for many arthropod vectors. The populations are typically rural and their main activities are farming, fishing and subsistence livestock rearing (mainly poultry, goats, sheep and pigs) within close range of human living quarters. In this area cattle and sheep are raised by the Fulani ethnic group, a pastoral community spanning Central and West Africa [14]. The Fulani graze mainly Zebu White fulani cattle on extensive communal pastures. Many herders still practice transhumance (seasonal migration) in the dry season along river valleys in search of pasture. The cattle population in the central region of Cameroon is estimated at 276,855 head. In the Akonolinga area included in this study, the cattle population is estimated at 6675 and sheep population at 1250 which reared under an open grazing system along the Nyong river mainly (unpublished data communicated by MINEPIA).

Sampling sites and data collection

Between the 5th and the 14th of March 2024 (dry season), ticks were collected from cattle and sheep in six sampling sites (Efoulan, Akam-Engali, Mvé, Akena, Essang-Ndibi and Ngoulemakong) in an area approximately of 30 km long and 18 km wide along the Nyong river, nearby the town of Akonolinga, as illustrated in Fig. 1.

Tick collection, morphological and molecular identification

At each site, all cattle and sheep available at the time of the inspection were examined for the presence of ticks (adults and immature stages). Cattle and sheep were restrained and kept standing and all the body parts of the cattle were examined. Ticks were collected from cattle and sheep using blunt steel forceps and placed inside a collection tube containing 70% ethanol. The samples were sent after obtaining import authorisation (number 13205107 from the French Ministry of Agriculture, Food and Forestry) to the virology laboratory of the Unité des Virus Emergents of the University of Corsica. The specimens were identified taxonomically [15], with the help of a veterinary entomologist of the University of Buea (Cameroon), and grouped by genus, sex, and stage, with a maximum of nine individuals by pool and stored at -80°C for further analysis. To complement morphological identification of ticks, molecular analysis of the partial sequence of the mitochondrial 12S rRNA gene sequences was performed as previously described [16]. The PCR products were examined on an ethidium bromide-stained 1.5% agarose gel and amplicons of the correct size purified for Sanger sequencing on ABI 3730xl analyzer. All tick pools detected positive for JMTV RNA have been selected for 12S rRNA gene sequencing to confirm morphological tick identifications.

RNA/DNA extraction

Pools of ticks were mechanically homogenized in 1ml Minimum Essential Medium using the TissueLyser II (QIAGEN) at 5,500 rpm for 3 minutes. Each pool was spiked with a predetermined quantity of MS2 bacteriophage prior to extraction to monitor nucleic acid extraction, reverse transcription and PCR amplification, as well as to detect any inhibitors and enzymatic reactions [17]. According to the manufacturer’s instructions, total RNA/DNA was extracted from 200µL homogenates using KingFisher and the MagMAX™ Viral/Pathogen Ultra Nucleic Acid Isolation Kit, eluted in 100 µL of buffer and stored at − 80°C.

Real time-qPCR of tick pools and JMTV sequencing

All ticks collected were screened for Jingmenvirus RNA using a RT-qPCR systems (gTJ-seg2) targeting the segment 2 [10], designed to detect multiple Jingmenvirus sequences, including JMTV, Yanggou tick virus (YGTV), Alongshan virus (ALSV), and Pteropus lylei jingmenvirus (PLJV). These viruses all belong to the tick-associated Jingmenvirus group. Each amplification reaction was prepared in a 25µL final volume per tube and comprised: 3µL nucleic acids, 1.25µL sense and antisense primers, 12.5µL 2x buffer, 0.5µL reverse transcriptase/Taq polymerase and 6.5µL RNAse free water. The thermal cycle used was as follows: (i) 55°C -10 min, (ii) 98°C -2 min, (iii) 40 cycles consisting of 98°C -10 sec, 55°C -10 sec, 68°C -1min 15 sec and a final step of 68°C -5min. All tick pools that tested positive in the initial screening described above were subsequently tested using three primer pairs targeting JMTV segment 1 (nt 627 to 2545) (Supplementary Material 1) [3]. The amplification reactions were carried out on a conventional PCR system (2720 thermal cycler) using the SuperScript™ IV One-Step RT-PCR System kit (ThermoFisher ref: 12594100). The amplified products were purified (NEBLabs ref: T3010) and pooled in equimolar proportion. The pooled fragments were then sequenced using the MinIon platform following the manufacturer’s instructions (Oxford Nanopore ref: SQK-LSK 109 and NBD 96). A random two-step RT-PCR was performed on the RNA extract (Merck ref : WTA2) from one JMTV-positive tick pool with a low Ct, following the manufacturer’s instructions [10]. Subsequent sequencing of the amplified RNA was carried out using the Ion Torrent S5 plateform (ThermoFisher scientifc), according to the manufacturer’s instructions.

Sequence and phylogenetic analyses

12S rRNA gene sequences and phylogenetic analyses

To confirm the identity of each 12S rRNA gene sequences (tick species), the sequences were compared with those available in the GenBank database using the BLASTn program [18]. Sequences obtained were aligned with published sequences. Tree were constructed using the maximum-likelihood with Tamura-Nei model (best DNA/Protein model (ML) ) in MEGA11 software, with 500 bootstrap [19]. The tick pools selected to identify 12S rRNA gene sequences were those showing JMTV positive results.

JMTV gene sequences and phylogenetic analyses

Reads were trimmed and assemblies were performed using Geneious Prime 2024.0.7 using the closest reference sequence to our data, determined using NCBI BLASTn [18]. For JMTV, a full genome sequence from Guinea was used as references (Genbank accession numbers MK673133 to MK673136). The most closely related sequences identified with BLAST, as well as reference JMTV sequences were used in a multiple nucleotide sequence alignment performed with MUSCLE on Geneious Prime 2024.0.7. The alignments obtained were used to infer maximum likelihood phylogenies in MEGA11, with Tamura-Nei model for the 12S ticks sequences and the General Time Reversible (GTR) substitution model for JMTV sequences, with equilibrium frequencies ML optimized and a fixed (0.0) proportion of invariable sites as determined by Smart Model Selection in PhyML (20), and with 500 bootstraps.

Statistical analysis

A 95% confidence interval (CIs) was employed to estimate the individual-level prevalence of pathogens, based on the assumption that each PCR-positive pool contained at least one positive tick. The estimation was conducted using the pooled prevalence model available on the EpiTools epidemiological calculator platform [21]. Categorical variables were described by numbers and percentages, with comparison using Chi-square test or Fisher’s exact test when appropriate. Missing values were excluded from analysis. Statistically significant was considered if p value ≤ 0.05. All the analyses were computed with R software, version 4.0.3 (4.0.3, R Core Team, 2021, R Foundation for Statistical Computing, Vienna, Austria; https://www.r-project.org/). For the spatial analysis, the exact site of each tick found attached to cattle and sheep was recorded. Each location was transferred into ArcMap v.10.8.2 software (ArcGis Desktop) and plotted on maps.

Tick infestation rates

A total of 383 cattle and 225 sheep were examined for ticks (Table 1 and Fig. 2). Among the 383 cattle, 67.37% (n = 258) were sampled in Akam-Engali, 9.40% (n = 36) in Ngoulemekong, 6.26% (n = 24) in Akena and 16.97% (n = 65) in Essang-Ndibi. Among the 225 sheep, 60.00% (n = 135) were sampled in Akam-Engali, 28.00% (n = 63) in Efoulan and 12.00% (n = 27) in Mvé (Fig. 2). The infestation rate was higher in cattle (40.47%; 155/383) than in sheep (4.00%; 9/225) (p < 0.001). The overall infestation rate in cattle and sheep was 26.97% (164/608) with the most infested cattle in Akam-Engali (55.04%; 142/258) and the most infested sheep in Efoulan (9.52%; 6/63) (Fig. 2).

Table 1

Rates of tick infestation by animal species and sampling sites.
Sites	Status	Cattle	Sheep	Total
Akam-Engali	Examined	258	135	393
	Infested	142 (55.04%)	3 (2.22%)	145 (36.90%)
Ngoulemakong	Examined	36	0	36
	Infested	3 (8.33%)	0	3 (8.33%)
Akena	Examined	24	0	24
	Infested	7 (29.17%)	0	7 (29.17%)
Essang-Ndibi	Examined	65	0	65
	Infested	3 (4.62%)	0	3 (4.62%)
Efoulan	Examined	0	63	63
	Infested	0	6 (9.52%)	6 (9.52%)
Mve	Examined	0	27	27
	Infested	0	0 (0.00%)	0 (0.00%)
Total	Examined	383	225	608
	Infested	155(40.47%)	9 (4.00%)	164 (26.97%)

Morphological and molecular identification of ticks

A total of 622 ticks, organized in 251 pools containing an average of 4 ticks (range 1–9 ticks), have been collected from 155 cattle (589 ticks organized in 237 pools) and from 9 sheep (33 ticks organized in 14 pools). The overall adult male to female ratio in the collected ticks was 0.47:1 (193 males, 411 females), with a ratio of 0.46:1 (179 males, 392 females) in cattle and of 0.74:1 (14 males, 19 females) in sheep. Because of engorged status and/or absence of some morphological criteria, some Hyalomma spp. and Rhipicephalus spp. were not identified to the species level. The 622 ticks collected comprised four main species in three genera: R. (B.) microplus (472; 75.88%), Amblyomma variegatum (118; 18.97%), Hyalomma truncatum (13; 2.09%), Hyalomma rufipes (2; 0.32%), and other Rhipicephalus spp. (17; 2.73%) (Table 2). In cattle, the predominant tick species was R. (B.) microplus (453 of 589; 76.91%), followed by A. variegatum (105 of 589; 17.83%). Similarly, in sheep, R. (B.) microplus was the most common tick species (19 of 33; 57.58%) followed by A. variegatum (13 of 33; 39.39%) (Table 2).

Table 2

Percentage of distribution of tick species among cattle and sheep.
Tick species (N = 622)	n	%
Rhipicephalus (B.) microplus	472	75.88
Cattle	453	76.91
Sheep	19	57.58
Amblyomma variegatum	118	18.97
Cattle	105	17.83
Sheep	13	39.39
Hyalomma truncatum	13	2.09
Cattle	13	2.21
Sheep	0	0.00
Hyalomma rufipes	2	0.32
Cattle	2	0.34
Sheep	0	0.00
Rhipicephalus spp.	17	2.73
Cattle	16	2.72
Sheep	1	3.03
Overall cattle	589	94.69
Overall sheep	33	5.31
Total	622

Phylogenetic analysis of R. (B.) microplus

Among the 163 tick pools classified as R. (B.) microplus based on morphological and taxonomic criteria, 38 were further validated through amplification and sequencing of a partial fragment of their 12S rRNA gene. (Fig. 3). The 38 12S rRNA segment sequences detected in R. (B.) microplus were all 100% identical thus two sequences were used for the construction of the tree. These sequences displayed nucleotides (nt) identities ranging from 96–100% with R. (B.) microplus detected in other regions worldwide. These sequences from a monophyletic clade with sequences reported in Uruguay (EU921763.1), Tanzania (EU921765.1), Brazil (EU921760.1; MT231546.1; OR880377; MN081899.1), South Africa (EU921764.1; KY676828.1), Argentina (EU921758.1), Uganda (KY688459.1 ; OR880375.1), Mozambique (EU921766.1), Democratic Republic of Congo (MF479199.1), Burundi (MZ361320.1) andBenin (KY676828.1), with 100% nt identity.

Detection of RNA JMTV in ticks collected from animals

The qRT-PCR screening of 251 tick pools yielded 61 tick-associated jingmenviruses positive pools (Table 3). One pool (ID 642-1) consisting of a single individual male of R. (B.) microplus tick, collected from one sheep (16.67%; 1 out of 6 sheep) in the Efoulan region, tested positive for tick-associated jingmenvirus RNA. The overall estimated individual-level prevalence of in sheep was 3.03% (95% CI: 0.18–12.66). The other 60 positive tick pools belonged at 95.00% (n = 57) to R. (B.) microplus, at 33.30% (n = 2) A. variegatum and at 1.60% (n = 1) Rhipicephalus spp and were collected from cattle. Specifically, 47 pools were collected from 39 cattle from Akam-Engali, 12 pools from two cattle from Akena, and 1 pool from one cattle from Ngoulemakong, with respective prevalence of cattle with positive ticks of 27.46% (39/142), 28.57% (2/7), and 33.33% (1/3). (Table 3 and Fig. 2). The overall estimated individual-level tick-associated jingmenvirus RNA prevalence was of 12.11% (95% CI: 9.41–15.23), with a value of 16.34% (95% CI: 12.65–20.57) in R. (B.) microplus tick pools and of 1.91% (95% CI: 0.325.79) in A. variegatum tick pools. One pool consisted of nymphs (Rhipicephalus spp). The estimated individual-level prevalence was of 12.91% [95% CI: 9.60–16.2%] in male tick pools (n = 15) and of 9.17% [95% CI: 5.48–14.09%] in female tick pools (n = 44).

Table 3

Characteristics of JMTV-positive tick pools in cattle and sheep.
Animal	Sample site	Animal ID	Tick pool ID	Pool size (number of ticks/pool)	Tick species	Sex	Ct
Cattle	Akam-Engali	1113	1113_2*	1	R. (B.) microplus	Female	27
		1100	1100-1*	9	Rhipicephalus spp.	Nymphe	19
		1091	1091-1	1	R. (B.) microplus	Female	28
		1117	1117-2*	1	R. (B.) microplus	Female	19
		1053	1053-1	6	R. (B.) microplus	Male	24
			1053-3	1	Amblyomma variegatum	Male	22
			1053-4	1	R. (B.) microplus	Female	22
		1097	1097-1	1	R. (B.) microplus	Male	22
		1161	1161-1	6	R. (B.) microplus	Female	35
		1096	1096-1*	4	R. (B.) microplus	Male	24
		1189	1189-1	2	R. (B.) microplus	Female	31
		422	422-1	4	R. (B.) microplus	Female	31
		8	8_2	5	R. (B.) microplus	Female	21
		426	426-1*	6	R. (B.) microplus	Female	26
		426	426-2*	3	R. (B.) microplus	Male	21
		44	44 − 1	4	R. (B.) microplus	Female	30
		1169	1169-1	3	R. (B.) microplus	Female	20
		45	45 − 1	4	R. (B.) microplus	Female	27
		1150	1150-2*	1	R. (B.) microplus	Female	23
		1023	1023-1*	4	R. (B.) microplus	Female	28
		354	354-2	3	R. (B.) microplus	Female	34
		1168	1168-1*	5	R. (B.) microplus	Female	28
		1025	1025-1*	5	R. (B.) microplus	Female	21
		1025	1025-2	4	R. (B.) microplus	Female	38
		1015	1015-1*	5	R. (B.) microplus	Female	23
		424	424-1*	6	R. (B.) microplus	Female	25
		1195	1195-1*	4	R. (B.) microplus	Female	24
		141	141-1*	2	R. (B.) microplus	Female	20
		1109	1109-1*	6	R. (B.) microplus	Female	24
		181	181-2	4	R. (B.) microplus	Male	23
		172	172-1*	5	R. (B.) microplus	Male	20
		1137	1137-1	6	R. (B.) microplus	Female	27
			1137-2	2	R. (B.) microplus	Male	32
			1137-3*	7	R. (B.) microplus	Female	17
		428	428-1	3	R. (B.) microplus	Female	40
		176	176-1*	1	R. (B.) microplus	Female	19
		1198	1198-1*	5	R. (B.) microplus	Female	22
		161	161-1	2	R. (B.) microplus	Male	37
		161	161-2*	1	R. (B.) microplus	Female	20
		178	178-2*	1	R. (B.) microplus	Male	23
		17	17_1*	2	R. (B.) microplus	Male	17
		171	171-1*	7	R. (B.) microplus	Male	24
		171	171-2	2	Amblyomma variegatum	Male	33
		1054	1054-1*	3	R. (B.) microplus	Male	22
		167	167-2*	3	R. (B.) microplus	Female	20
		357	357-1	6	R. (B.) microplus	Female	31
		1042	1042-2*	1	R. (B.) microplus	Female	23
	Akena	92	92 − 1	1	R. (B.) microplus	Female	38
			92 − 3	1	R. (B.) microplus	Female	34
			92 − 6*	1	R. (B.) microplus	Female	20
			92 − 7	2	R. (B.) microplus	Female	23
			92 − 9*	4	R. (B.) microplus	Female	22
		82	82 − 1	6	R. (B.) microplus	Female	32
			82 − 2	6	R. (B.) microplus	Female	30
			82 − 3	6	R. (B.) microplus	Female	28
			82 − 4	6	R. (B.) microplus	Female	19
			82 − 5	6	R. (B.) microplus	Female	22
			82 − 6*	6	R. (B.) microplus	Female	19
			82 − 7*	6	R. (B.) microplus	Female	17
	Ngoulemakons	580	580-1	6	R. (B.) microplus	Male	27
Sheep	Efoulan	642	642-1	1	R. (B.) microplus	Male	33
*Confirmed to be JMTV by sequencing

(Table placed at the end of the document according to journal guidelines)

Out of the 42 cattle with at least one JMTV-positive pool, 40.47% (n = 17) had all their ticks pooled in one sample, while 59.25% (n = 25) had ticks divided into multiple pools, ranging from two to 10 tick pools. Of the cattle with multiple tick pools, 20.00% (5 of 25) had all pools positive to JMTV, while 80.00% (20/25) had a mix of positive and negative pools. The percentage of JMTV-positive pools per animal are shown in Supplementary Table 2.

Phylogenetic analyses of JMTV based on partial S1 sequences

We obtained usable partial sequences (1256 nucleotides) of segment 1 for 30 of the 61 JMTV-positive tick pools collected from 29 cattle (26 tick pools from 25 cattle of Akam-Engali and four tick pools from two cattle of Akena). We identified and removed identical sequences, selecting nine representative sequences to include in the phylogenetic analysis. Partial sequences obtained in this study showed an average nucleotide identity of 99.21%. These sequences were most closely related to and clustered phylogenetically with JMTV strains from Guinea, within clade I, which includes sequences from Africa, Asia and South America (Fig. 4). Our sequences from Cameroon showed (i) 86.73% -98.83%nt identity (average 97.56%) to JMTV detected ticks from Guinea; (ii) 89.72–90.82% (average 90.21%) nt identity to JMTV from strains identified in Kenya; and (iii) 88.97–89.63% (average 89.28%) nt identity to JMTV strain from Uganda.

Full genome sequences

The JMTV-positive tick sample (82-7BOV) from the Akena site was selected among the positive CCHFV samples due its low Ct and good sequence quality. Direct Next Generation Sequencing (NGS) provided partial nucleotide sequences of the four segments as: S1 2961 bp, S2 2812 bp, S3 2670 bp and S4 2725 bp. All the four segments of JMTV were closely related to JMTV sequences detected in ticks from Guinea with nt identity ranging from 97–99% (Fig. 5). The sequences of the four segments of JMTV of the present study showed also close nt identity with the strains of JMTV detected in Brazil with nt identity ranging from 89–98%. Compared with other JMTV genome reported in Africa, the JMTV sequences obtained in this study share nt identity of 85%-90% with JMTV strain RC27 detected in plasma of primates in Uganda and 86%-89% with JMTV strains detected in ticks collected in Kenya. (Fig. 5).

Here, we report for the first time the detection of JMTV mainly in R. (B.) microplus collected during the dry season from cattle and sheep in a forest-savannah transitional ecological zone of Akonolinga, in central Cameroon.

R. (B.) microplus, primarily parasitizing cattle, is widespread in tropical and subtropical regions, including India, Malaysia, China, Central and South America, and Australia, where it has been present for decades. It subsequently spread to Southern, Eastern, and West Africa and was first reported in Cameroon in 2019 [22, 23]. Here, R. (B.) microplus was the predominant tick species found on cattle and sheep. This is congruent with data reported in the Menoua and Noun division of the West region of Cameroon [24]. However, R. (B.) decoloratus was described as the most prevalent species in three studies conducted in Cameroon [22, 25, 26]. Nevertheless, our data suggested the replacement of the native species R. (B.) decoloratus with the exotic species R. (B.) microplus. A similar observation was reported in the Soutpansberg region, limpopo province in South Africa [21]. The high abundance of this tick in our study confirms the ongoing circulation of R. (B.) microplus in central Cameroon and indicates the progressive establishment of this species in the region.

R. (B.) microplus is widely recognized as the most significant ectoparasite and vector of livestock diseases [26]. Additionally, its resistance to safe and affordable pyrethroid insecticides represent a serious problem for endemic countries [27]. It is known to spread various animal pathogens, including Babesia bigemina, B. bovis, and Anaplasma marginale [23], and has been associated with the transmission of JMTV, first identified in this tick species in China in 2010 [1]. Since then, JMTV has been detected in R. (B.) microplus across several countries, such as French Antilles, Brazil, Colombia, Trinidad and Tobago [4]. In this study, JMTV has primarily been detected in R. (B.) microplus ticks. Phylogenetic analysis of these ticks showed that 12S rRNA gene is highly conserved, with tick sequences from this study identical to one another and to those previously reported from Brazil, Uruguay, Tanzania, South Africa, Argentina, and Uganda, Mozambique, Democratic Republic of Congo, Burundi and Benin. The similarity of our sequences with those from Brazil could be explained by the movement of R. (B.) microplus from Brazil to West Africa through the cattle trade, and subsequently from West Africa to Central Africa, particularly to Cameroon, which is located along a major transboundary cattle trade route between these two regions. The phylogenetic results also suggest that the tick might have been introduced to Cameroon from Southern and Eastern and western Africa, where the invasion of this species has been extensively documented [23].

In comparison to previous similar studies investigating JMTV in ticks collected from ruminants, the JMTV prevalence observed in the present study, estimated as individual-level prevalence of JMTV, was in agreement with prevalence values reported in ticks ranging from 53 to 63% in China, 25–67% in Brazil, 6–46% in Trinidad & Tobago and 24–77% in the French-Antilles [5]. A recent study conducted in Corsica (France) reported a minimum infection rate (MIR) of 0.54% in ticks (mainly R. bursa) collected from cattle [10]. In addition, in Kenya, detection rates of 0.0%, 0.3%, and 1.8% were observed in cattle, goats, and sheep, respectively [9]. The positive samples for which no sequences were obtained cannot be definitively classified as JMTV, as the detection method used also amplifies ALSV, PLJV, and YGTV. However, it is highly likely that these sequences correspond to JMTV, given that the coexistence of different Jingmenvirus species within the same ecosystem has never been observed [5].

JMTV has been detected in various tick species, primarily within the Rhipicephalus spp. and including other genera such as Amblyomma, Dermacentor, Haemaphysalis, Hyalomma, and Ixodes [5]. In our study, we found that for most animals with positive tick pools, not all tick pools from the same animal tested positive for JMTV. While we cannot draw definitive conclusions about the roles of the ticks and the animals in the transmission of JMTV, these observations allow us to suggest that the ticks themselves may carry the virus independently of the animal’s viremia, raising the possibility that positive ticks could act as vectors for JMTV transmission. However, the exact role of these tick species in JMTV transmission remains unclear and requires further investigation.

The genomic sequences of a new jingmenvirus species were identified in a human sample from Cameroon in a study conducted between 2015 and 2019. Phylogenetic analysis revealed that this virus clusters with insect-associated jingmenviruses and is phylogenetically distinct from JMTV, which belongs to the tick-associated jingmenviruses [12]. In our study, the JMTV sequences belong to the African-Asian-South American clade I that is distinct from the Caribbean-European clade II. This finding is congruent with the geographic allocation reported so far with JMTV. JMTV Cameroon sequences are most closely related to those from Guinea [11] ; interestingly, Guinea and Cameroon are 2500 km distant. An important feature of JMTV epidemiology is the apparent existence of long-distance dispersals. As it is unlikely for ticks themselves to move over long distances, the most plausible route is human-mediated transportation of infected cattle and sheep [1]. The JMTV sequences identified in Kenya and Uganda, although still classified within the same clade, demonstrate a higher degree of genetic variation when compared to those from Guinea and Cameroon. Before drawing definitive conclusions, we need to consider the absence of epidemiological studies on JMTV and jingmenviruses in general, in particular in Africa. A better idea of the dispersion dynamics of JMTV could be obtained by performing similar studies in countries located between Guinea and Cameroon for instance. In Cameroon, where cattle farming is a vital resource for about 30% of the rural population, the well-structured livestock trade network facilitates cross-border animal movement, increasing the likelihood of JMTV dispersals, especially to neighboring regions such as Gabon, Congo and Equatorial Guinea [28].

Three points were highlighted in this study: first, R. (B.) microplus, was the main tick collected from cattle and sheep suggesting the replacement of the native species R. (B) decoloratus with the exotic species R. (B.) microplus; second, JMTV RNA was detected in almost 30% of R. (B.) microplus tick pools collected from cattle suggesting that this species plays a major role in the transmission of JMTV; third, JMTV strains from Cameroon clusterized within a clade closely related to JMTV and KINV viruses detected from R. (B.) microplus and from R. geigyi respectively collected from cattle in Guinea (West Africa).

To the best of our knowledge, we provided here the first work in Cameroon and Central Africa with a detection of JMTV in ticks collected from cattle and sheep. JMTV is an emerging virus with a potential risk of causing epidemiological outbreaks as endemic globally. Additional epidemiological studies, including antibody testing in livestock and humans, are required to evaluate the possible health risks associated with JMTV in the study region. The establishment of the exotic species R. (B.) microplus in Cameroon, raises concerns about potential outbreaks of various animal pathogens transmitted by this tick. Increased vigilance and enhanced surveillance are crucial to prevent and manage potential animal disease outbreaks associated with this tick in the region.

JMTV

Jingmen tick virus

NGS

Next-generation sequencing and qRT-PCR:Quantitative reverse transcription-PCR

Acknowledgments. We would like to thank all the cattle and sheep keepers whom collaborate in good with their time and herd and make data collection possible and MINEPIA for helping us to contact herders.

Funding Statement. This work was supported by PFI 7736A1 of the Ministere de l’Europe et des Affairs Etrangers, programme 209 and by the European Commission (European Virus Archive Global project (EVA GLOBAL, grant agreement No 871029) of the Horizon 2020 research and innovation programme). The material was provided by the European virus archive-Marseille (EVAM) under the label technological platforms of Aix-Marseille University.

Ethical consideration and authorization. The study protocol was implemented with approval from the Regional Delegation of the Ministry of Livestock, Fisheries, and Animal Industries (MINEPIA), authorizations N°1325/L/MINEPIA/SG/DREPIA-CE/SRDPIA. Oral consent for blood and ticks sampling was obtained from the animal’s owners. Pastoralist herdsmen were visited either at their homestead or at a convenient location in the vicinity where the cattle could be examined. The translator/research assistant explained the project in either Fulfulde, Pidgin, English or French and the herdsman or farmer was asked to give verbal consent to participate in the study.

Availability of data and materials. Data will be available on request by email to the corresponding author. All the nucleotide sequences were deposited in GenBank under accession numbers PQ478049-PQ478060 for JMTV genome sequences and PQ480081- PQ480082 for R. (B.) microplus sequences (12s rRNA).

Authors’ contributions. PK performed the laboratory work and contributed to the writing of the manuscript. EL conducted the S1 sequencing, prepared samples for full genome sequencing, and reviewed the final version of the article. MG created the maps and also reviewed the final version of the article. GP carried out the full genome sequencing. AC reviewed the article and helped in the phylogenetic tree construction. AP collected and identified the ticks and reviewed the article. SM contributed to field organization, and LT participated in the fieldwork. XL and RC reviewed the final version of the article and provided critical revisions. AF contributed to the collection of ticks, performed laboratory work, and co-wrote the manuscript.

Consent for publication. Not applicable

Competing interests. The author(s) declare(s) that they have no competing interests

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No competing interests reported.

Additionalfile1.xlsx
Additional file 1: Conventional PCR primers for amplifying the S1 segment sequences of JMTV.
Additionalfile2.xlsx
Additional file 2: Percentage of JMTV pool positivity per cattle.

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First detection and molecular characterization of Jingmen Tick Virus with a high occurrence in Rhipicephalus (Boophilus) microplus collected from livestock in Cameroon (2024)

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Abstract

Background.

Methods.

Results.

Conclusions.

Figures

Introduction

Materials and Methods

Description of the study sites

Sampling sites and data collection

Tick collection, morphological and molecular identification

RNA/DNA extraction

Real time-qPCR of tick pools and JMTV sequencing

Sequence and phylogenetic analyses

12S rRNA gene sequences and phylogenetic analyses

JMTV gene sequences and phylogenetic analyses

Statistical analysis

Results

Tick infestation rates

Morphological and molecular identification of ticks

Detection of RNA JMTV in ticks collected from animals

Phylogenetic analyses of JMTV based on partial S1 sequences

Full genome sequences

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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