Background

Tick-borne infections constitute a significant portion of vector-borne diseases, can generate a considerable disease burden, and present an imminent threat to public health. Ticks act as biological vectors for a wide range of microorganisms, including RNA viruses that can be transmitted to humans and animals [1].

Some examples of pathogenic viruses transmitted by ticks to humans or animals are Crimean−Congo hemorrhagic fever virus (CCHF), Nairobi sheep disease virus, tick-borne encephalitis virus, Omsk hemorrhagic fever, Kyasanur forest disease virus, Powassan virus, Alkhurma hemorrhagic fever virus, severe fever with thrombocytopenia virus, Heartland virus, Colorado fever virus, Bourbon virus, and African swine fever virus (ASF) [2, 3]. In addition, of these pathogens, **gmen tick virus (JMTV) associated with disease in humans has recently been described [4].

JMTV was first detected in 2010 from China in Rhipicephalus microplus [5]. The virus belongs to the **gmen virus group (JMV), and although it remains unclassified by the International Committee on Taxonomy of Viruses (ICTV), it is related to the Flavivirus genus of the Flaviviridae family [5]. The JMV group are single-stranded RNAs in the positive sense (ssRNA+), and unlike typical flaviviruses, it has a segmented genome [6].

The JMTV genome comprises four segments: S1, S2, S3, and S4. The S1 and S3 encode nonstructural proteins (NSPs), while the S2 and S4 encode structural proteins (VPs) [7]. S1 encodes NSP1, an RNA-dependent RNA polymerase homologous to the NS5 protein of flaviviruses; S2 presents an open reading frame (ORF) that encodes a glycoprotein (VP1), S3 encodes NSP2, homologous to the flavivirus NS3-NSP2 complex; S4 presents two ORFs to encodes two proteins, the capsid protein (VP2) and the membrane protein (VP3) [5]. Although segments S1 and S3 are closely related to the NSPs genes (NS3 and NS5) of flaviviruses, the remaining two segments (S2 and S4) are unique to these viruses and lack homologs [5].

JMTV has been identified in several regions of China in ticks and humans [4, 5, 8]. Additionally, its presence has been reported in ticks from various countries including Poland, Georgia, France, French Antilles, Laos People’s Democratic Republic, Brazil, Kenya, Turkey, Japan, Romania, Trinidad and Tobago, and Colombia [9,10,11,12,13,14,15,16,17], as well as mosquitoes (Aedes albopictus) in Italy [18]. JMTV has also been detected in vertebrates, such as the red colobus monkey (Procolobus rufomitratus) in Uganda [19], bats in Cambodia [9], cattle in Brazil [10], and rodents in China [25]. De novo assembly was performed to obtain viral contigs or scaffolds using MEGAHIT v.1.2.9 [26]. Finally, the taxonomic assignment was performed with BLASTx 2.14.1 [27]. All these tools were accessed through the online platform Galaxy [28].

Phylogenetic analysis

The obtained contigs were compared with the GenBank database using BLASTp [27]. Alignments were generated with MAFFT [29] with sequences downloaded from GenBank [30]. Phylogenetic reconstructions were performed in IQ-TREE v2.2.2.6 [31] with the maximum likelihood method [32]; the best-fit amino acid substitution models were obtained using ModelFinder, and the substitution models were chosen according to the Bayesian information criterion [33] and SH-like approximate likelihood ratio test for statistical support [34]. Trees were reconstructed using 1000 bootstraps visualized in iTOL v5 [35] and edited in Inkscape v.1.1 [36].

Results

A total of 766 ticks were collected, of which 87.3% (669/766) were R. microplus, 5.4% (42/766) Dermacentot nitens, 4.2% (32/766) Rhipicephalus linnaei, and 3.0% (23/766) Amblyomma dissimile. In total, 120 pools were generated.

In the metatranscriptome of the species R. microplus and D. nitens collected in the Cesar Department, the complete genome of JMTV was detected. Additionally, complete and partial fragments of segments 1, 2, 3, and 4 that encode the proteins NSP1, VP1, NSP2, VP2, and VP3 of JMTV were detected in R. microplus, D. nitens, and A. dissimile collected in Córdoba and Cesar (Table 1).

Table 1 Grou**s of JMTV-positive ticks by NGS
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The JMTVs detected in this study are phylogenetically related to JMTVs detected in A. albopictus from France, JMTVs detected in R. microplus from Trinidad and Tobago, JMTVs detected in R. microplus and A. variegatum from the French Antilles, and JMTVs detected in Colombia. This relationship was consistent in all constructed trees (Fig. 2). Additionally, when performing BLASTp, the amino acid sequences presented a percentage of identity between 96.23% and 99.38% with the JMTV mentioned above [7, 11, 16].

Fig. 2
figure 2

Phylogenetic reconstructions with amino acids of the four segments of JMTV. A Phylogenetic tree of the NSP1 protein built with 45 sequences (35 downloaded from GenBank and 10 own). B Phylogenetic tree of the VP1 protein built with 37 sequences (28 downloaded from GenBank and 9 own). C Phylogenetic tree of the NSP2 protein built with 35 sequences (28 downloaded from GenBank and 7 own). D VP2 phylogenetic tree built with 35 sequences (25 downloaded from GenBank and 10 own). E VP3 phylogenetic tree built with 39 sequences (33 downloaded from GenBank and 6 own). These five trees were rooted with JMTV (QFR36167) and JMTV (QFR36160). The sequences generated in this study are highlighted in red. Trees were constructed using the substitution models JTTDCMut + G4 for NSP1, FLU + F + G4 for VP1, FLU + G4 for VP2, JTT + G4 for NSP2, and VP3 protein

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Additionally, there is a phylogenetic relationship between the JMTVs detected in this study and those detected in humans in Kosovo, given that in trees A, D, and E the sequences of these groups are in the same cluster (Fig. 2, tree A, D, E).

Discussion

JMTV was detected in R. microplus and D. nitens ticks collected from cattle, and A. dissimile collected from iguanas in the Colombian Caribbean (Table 1). This is the third JMTV study on R. microplus and the first virus report on D. nitens and A. dissimile from Colombia.

JMTV was first identified in R. microplus ticks in the **gmen area of Hubei Province, China, in 2010 [5], then in 2012 in Uganda, JMTV was detected by NGS in the plasma of a red colobus monkey (Procolobus rufomitratus) [19]. Between 2013 and 2015, 12 human sera samples collected in Kosovo were analyzed, and patients with CCHF coinfected with JMTV were detected [21]. Between 2014 and 2016, in France, French Antilles, Laos People’s Democratic Republic, and Cambodia, JMTV was detected in Ixodes ricinus, R. microplus, Amblyomma testudinarium, and bats, respectively [9]. In 2017, JMTV was isolated in BME/CTVM23 cells. NGS sequenced the whole genome in Amblyomma javanense collected from Chinese pangolins [4]. That same year, human patients positive for JMV were reported in China. The infection was confirmed by RT-PCR assay in blood in 86 patients with fever, headache, and a history of tick bites. Serological assays showed seroconversion in 19 patients [22]. In Kenya, between 2013 and 2019, ticks of the Amblyomma and Rhipicephalus species were collected, and JMTV was detected by PCR and NGS [11].

Recently, in 2023, in China, JMTV was detected in R. microplus ticks through NGS (MGISEQ-2000), which PCR confirmed, and the complete genome was obtained with Sanger sequencing [8]. Furthermore, they detected JMTV in I. ricinus in Poland and Georgia by NGS (nanopore) and PCR [3].

In Latin America, there are a few reports of JMTV. In Brazil, between 2015 and 2016, complete and partial JMTV genomes were obtained by high-throughput sequencing in 67% (4/6) of tick pools and 14% (5/36) of bovine sera [10]. In Colombia, in the Department of Antioquía, between 2013 and 2018, several JMTV segments were detected by NGS in ticks of the genus Rhiphicephalus [16, 17]. Many of the JMTV reports with NGS carry out a confirmation with PCR; however, in this study, complete genomes were recovered. Thus, confirmation was unnecessary.

JMTV has a worldwide distribution and has been detected in six genera of ticks: Rhipicephalus, Amblyomma, Dermacentor, Haemaphysalis, Hyalomma, and Ixodes, and 26 species [7, 37]. This work provides two new species of ticks where JMTV was detected: D. nitens and A. dissimile. However, R. linnaei was negative despite previous positive reports for this tick species [5].

Interestingly, in the present work, JMTV was detected in D. nitens parasitizing cattle, which suggests transmission to cattle by these tick species, given that there are reports of this virus in cattle from Brazil [10]. This finding indicates a probable risk of transmission to different species of ticks, animals, and humans.

Phylogenetic analyses of five JMTV proteins showed that, in four of the trees obtained (Fig. 2, tree A, C–E), most of the viruses were grouped in two clusters, one of them associated with different species of bats and ticks and the other with humans and different species of ticks.

Interestingly, our sequences of the NSP1, VP2, and VP3 trees were placed in a clade closely related to the JMTVs detected in humans from Kosovo [21]. This is a finding that should alert the medical community in the tropical regions of Colombia.

Given the close linkage between the sole JMTV sequence identified in A. albopictus in Italy and the strain characterized in the current study (Fig. 2, tree A), it is necessary to carry out epidemiological surveillance of JMTV in mosquitoes from tropical areas of Colombia.

Unfortunately, for the phylogenetic analyses, only the VP1 of JMTV previously reported in Colombia could be included given the limited availability of data; however, it presents a high phylogenetic relationship with the JMTV detected in D. nitens from Cesar, reported in the present study.

It is necessary to develop serological tests with the exclusive segments of circulating JMTV to avoid cross-reactions with other endemic flaviviruses and generate tools to contribute to diagnosing febrile syndrome without an apparent focus.

Conclusions

This is the first work in the Colombian Caribbean on ticks of importance in human and animal health. JMTV is one of the emerging viruses with a potential risk of causing epidemiological outbreaks because they are endemic globally. They are closely associated with arthropods and have caused sporadic cases of febrile illness in humans with a history of tick bites. These results are essential to establish surveillance measures to prevent possible outbreaks of these pathogens.