Ethics statement

The field mission was conducted in accordance with Cambodian guidelines and received formal approval from the Ministry of Agriculture, Forestry and Fisheries (MAFF), Ministry of Health National Ethics Committee for Health Research (NECHR #008), local governments, and land/site owners. All necessary permissions were obtained prior to data collection, ensuring compliance with local regulations and ethical standards. The study prioritized biosecurity measures, minimized disruption to the environment and wildlife, and upheld principles of transparency, respect, and responsible data handling.

Collection and processing of bat bugs

The cave of Ta Rumm is located in Sampeu hill, Banan district, Battambang province, north-western Cambodia (13.022 N, 103.095 E). This cave has two vertical entrances, accessible by descending 7–8 meters using a custom-made ladder installed by local guano miners (Fig 1A and 1B). The cave hosts a roost of nearly two million wrinkle-lipped free-tailed bats (Mops plicatus) (Fig 1C), the sole species documented to inhabit this location [59]. Bat guano is harvested in this cave on average twice a month using shovels and a custom pulley system.

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Fig 1. Ta Rumm cave in Phnom Sampov and bat bug collection.

(A) IPC field team staff in personal protective equipment (B) Lower entrance of the Ta Rumm cave. IPC field team collecting bat guano and urine inside the cave. (C) Wrinkle-lipped free-tailed bat (Chaerephon plicatus). (D-E) blood engorged bat bugs (Stricticimex phnomsampovensis Suor & Maquart n.sp). (Photos: Jurre Y. Siegers/Institut Pasteur du Cambodge).

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In June 2022, the Institut Pasteur du Cambodge (IPC) field team entered the cave through the lower entrance wearing full biosafety level 3-adequate personal protective equipment to collect fresh environmental samples (guano and urine) from bats (Fig 1A- 1B). During this mission, 13 bat bugs (Fig 1D) were opportunistically collected, seven were preserved in viral transport medium (VTM) consisting of 2.95% tryptose phosphate broth, 145 mM of NaCl, 5% gelatin, 54 mM Amphotericin B, 106/L U penicillin-streptomycin, 80 mg/L gentamicin (Sigma-Aldrich, Steinheim, Germany), while six bat bugs were stored in 70% ethanol. The bat bugs in VTM were transferred into liquid nitrogen within six hours for transport back to IPC and subsequently stored at -80°C until further analysis. The ethanol preserved bat bugs were kept on ice and handed over to the Medical and Veterinary Entomology Unit at IPC for morphological identification.

The initial identification prompted the need for more specimens. Subsequently, we provided additional tubes to local bat guano collectors for further collection of bat bugs during a harvesting session in April 2023. In total, 80 specimens were stored in VTM, six in 70% ethanol and one was left in no medium for later mounting on a cover slide. These were transported on ice back to IPC within 24 hours and subsequently stored as previously described.

Morphological bat bug identification

The bat bugs were determined using the determination key provided by Usinger (1966) [60] and the descriptions provided in Ueshima (1968) [61], Klein (1969a, 1969b, 1970) [10,11,15].

Speciation (COI, 16s, and 18S) PCR and Sanger sequencing

Parasite species barcoding was performed using three PCR systems targeting invertebrate Cytochrome c oxidase subunit I (COI) [62], 16S and 18S rRNA genes [63–66]. Total nucleic acid of individual sample was extracted using Zymo Research Direct-zol RNA MiniPrep kit (Zymo Research, Cat # R2050, CA, USA) according to the manufacturer’s instructions after a homogenization step (MagNA Lyser, Roche). Following reverse transcription using SuperScript III First-Strand Synthesis Super-Mix (Invitrogen, San Diego, CA) according to the manufacturer’s instructions, cDNA was used for the different barcoding PCR protocols (Table 2).

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Table 2. Primers used for species identification.

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The COI gene was targeted using primers LC01490-F and HC02198-R. During amplification, the following steps were used: 1 cycle of 94°C for 2 minutes, 40 cycles of 94°C for 30 seconds, 50°C for 30 seconds, and 72°C for 1 minute, and a final extension of 1 cycle of 72°C for 10 minutes. The 16S rRNA gene was targeted with the primers 16S LR-J and 16S LR-N. Amplification was performed with the following conditions: 1 cycle of 94°C for 2 minutes, 35 cycles of 94°C for 30 seconds, 48°C for 40 seconds, and 72°C for 60 seconds, and a final extension of 1 cycle of 72°C for 7 minutes. Finally, the 18S rRNA gene was targeted using pairs of primers 18S-1, 18S-3 and 18S-2, 18S-4. Following conditions were used for both PCR and nested PCR steps: 1 cycle of 94°C for 2 minutes, 40 cycle of 94°C for 30 seconds, 48°C for 60 seconds, and 72°C for 60 seconds, and final extension of 1 cycle of 72°C for 10 minutes.

All amplifications were performed using Invitrogen Platinum Taq polymerase and T100 Thermal Cycler, Bio-Rad. All PCR products were subsequently sequenced by Sanger sequencing (Macrogen, Inc., Seoul, Republic of Korea) in both forward and reverse directions using relevant primers from PCR systems (from the nested PCR for 18S amplification).

Phylogenetic analysis Cambodian bat bug

The obtained COI, 16S, and 18S sequences were concatenated and aligned with Geneious Prime 2023.2 (Geneious, https://www.geneious.com). Additional sequences of Cimicidae species and outgroups were obtained from the National Center for Biotechnology Information GenBank database (S1 Table). A Bayesian consensus tree was created using the MrBayes [67] Geneious plugin, with the General Time Reversible model with gamma distribution and invariant sites [68]. Chain length was set to 5,000,000, sampling frequency to 500, and burn-in length to 100,000. The random seed was set to 21,775. Phylogenetic trees were edited using TreeViewer.

Twist comprehensive viral research panel

The sequencing libraries were generated from homogenized, pooled (n = 4) bat bugs using the combination of Twist Library Preparation Enzymatic Fragmentation (EF) kit 2.0 (#104211) and hybridization with Twist Comprehensive Viral Research Panel (#1035550), according to the manufacturer’s protocol (Twist Total Nucleic Acids Library Preparation EF Kit 2.0 for Viral Pathogen Detection and Characterization Protocol and Twist Target Enrichment Standard Hybridization v1 protocol). Briefly, RNA was extracted using Direct-zol RNA Miniprep Kits (R2053) and the extracted nucleic acid was converted to cDNA using ProtoScript II First Strand cDNA Synthesis kit (E6560S) and Random Primer 6 (S1230S) from New England Biolab (NEB). The NEB Next Ultra II Non-Directional RNA second Strand Synthesis kit (E6111S) was subsequently used to convert the single-stranded cDNA to double stranded DNA (dsDNA). The Illumina TruSeq-compatible libraries were generated using the Twist Library Preparation EF kit and the generated libraries were pooled with 6 libraries per pool. Each library was measured for concentration and normalized to the same mass before pooling, with the total combined mass not exceeding 4 µg. The pooled libraries then underwent hybridization capture using the Twist Comprehensive Viral Research Panel. Following the enrichment, the enriched libraries were pooled again in equimolar ratios and sequencing with loading concentration of 6pM and spiked with 1% Phix control V3 using Illumina Miseq. The pooled library was diluted and denatured according to the standard Miseq System Denature and Dilute Libraries Guide (Document # 15039740v10), and sequenced to generate paired-end 75 bp reads using a 150 cycle Miseq V3 reagent kit (illumina, MS-102–3001). After sequencing, demultiplexed FASTQ files were generated and analyzed using Genome Detective [69] which performs quality control, de novo assembly of reads into contigs, and taxonomic assignment through similarity searches against custom made and curated viral databases. The pipeline employs a novel alignment approach that reconstructs viral genomes by reference-guided linking of de novo contigs, integrating both amino acid–level and nucleotide-level alignment scores. This combined scoring strategy improves detection and assembly of divergent viruses.

NovaSeq

Sequencing libraries were generated from Coltivirus infected Vero cell supernatants using the NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs, MA, USA) following the manufacturer’s instructions. First-strand cDNA synthesis was performed using the ProtoScript II First Strand cDNA Synthesis Kit (New England Biolabs, MA, USA), followed by second-strand synthesis with the NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs, MA, USA). The resulting double-stranded DNA was fragmented to an average size of approximately 400 bp before adapter ligation. Adapter-ligated DNA fragments were then amplified by PCR using the NEBNext Multiplex Oligos for Illumina. At each step, purification was performed using AMPure XP beads (Beckman Coulter), and DNA quantification was carried out with a Qubit 4.0 fluorometer. Final libraries were sequenced on an Illumina NovaSeq X platform (Macrogen, Inc.), generating paired-end 150 bp (PE150) reads.

Phylogenetic analysis coltivirus

A total number of 96 Reovirales including species in the genus, Mycoreovirus, Cypovirus, Dinovernavirus, Oryzavirus, Fijivirus, Aquareovirus, Orthoreoviris, unclassified Reovirales, and all species of Coltiviruses were obtained from NCBI GenBank (assessed on 2025-01-26). A full list of the GenBank protein accession numbers is provided as S3 Table. Sequences were aligned with MAFFT v.7.490 [70], option L-INS-i and trimmed using trimAL [71], option -gappyout. Phylogenetic trees were constructed using IQ-TREE v.2.0.3 [72] using ModelFinder with the best-fit amino acid substitution model Q.pfam+F + I + R6 for RdRp and LG + F + R3 for VP2 chosen according to Bayesian Information Criterion (BIC), with 1,000 bootstrap replicates used to assess branch support. Trees were visualized and annotated using FigTree v.1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/) and Adobe Illustrator 2024.

Genome annotation

Coltivirus genomes were annotated with Geneious Prime v2025.1.2 (Biomatters Ltd., New Zealand). Briefly, all available Coltivirus sequences were downloaded from the NCBI database (accessed 29 October 2025). Each newly generated segment was aligned to annotated Coltivirus reference sequences using the Annotate from Database function implemented in Geneious. Start and stop codons as well as reading frames were manually inspected and adjusted where necessary. Final annotations were verified by visual inspection of translated coding sequences and exported in GenBank format for downstream analyses and submission to GenBank. The viral sequences were deposited in GenBank under accession numbers PZ358652–PZ358681.

Real-Time quantitative PCR (RT-qPCR)

A duplex real-time quantitative PCR (RT-qPCR) system was developed to target the viral RNA-dependent RNA polymerase (RdRp) gene. The sequence of this gene was aligned using CLC Genomics Workbench 5.5 with the closest available Coltivirus sequences, - Tai Forest reovirus (accession: KX989543.1) and Kundal virus (accession: NC_055248.1). Based on this alignment, two probes were designed: one targeting the newly identified Stricticimex coltivirus sequence and another targeting the Tai Forest reovirus sequence (Table 3). Prior to RNA extraction, samples underwent homogenization using a MagNA Lyser instrument (Roche) with 1.2–1.4 mm ceramic beads (Saint-Gobain, China). Viral RNA was extracted from individual samples using the Direct-zol RNA MiniPrep Kit (Zymo Research, Cat #R2050, CA, USA), following the manufacturer’s instructions. Bat bug RNA samples were screened using the developed duplex real-time PCR. A 25µL reaction mix contained 5 µL of cDNA, 12.5 µL of 2x reaction buffer provided with the Superscript III one-step RT-PCR system with Platinum Taq Polymerase (Invitrogen, Darmstadt, Germany) containing 0.5µL of a 50mM MgSO₄ solution (Invitrogen) and 0.3µL of 20-40U/µL of RNAsin ribonuclease inhibitor (Promega). Primers and probes were used at final concentrations of 200nM. PCR cycling conditions were as follows: 50°C for 15 minutes followed by 95°C for 5 minutes before 45 cycles of 95°C for 15 seconds, 56°C for 30 seconds and 72°C for 1 minute, using CFX9 Real-Time PCR detection system (Bio-Rad).

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Table 3. Primers and probe designed to detect Stricticimex coltivirus (SCCV).

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Cell lines

All cell lines were obtained from either the American Type Culture Collection (ATCC) or the European Collection of Cell Cultures (ECACC), except for the Rhileki cell line, which was provided by G.J. Smith (Duke-NUS, Singapore), and the Aag-2 cell line, provided by J. Pompon (Duke-NUS, Singapore). The Rhileki cell line is a spontaneously immortalized, clonal cell line derived from kidney tissue of a Rhinolophus lepidus bat (NUS-IACUC B01/12), as previously described [73]. The invertebrate cell lines Aag-2 (Aedes aegypti) and C6/36 (Aedes albopictus, CRL-1660) were maintained in Leibovitz L-15 medium (Sigma-Aldrich) supplemented with 5% fetal calf serum (FCS; Gibco), 10% tryptose phosphate broth (Sigma-Aldrich), 2 mM L-glutamine (Gibco), and 100 U/mL penicillin-streptomycin (Pen/Strep; Gibco) at 28°C. Vertebrate cell lines were cultured at 37°C with 5% CO₂. Most of these, including BHK-21 (baby hamster kidney, Mesocricetus auratus, CCL-10C) and VeroE6 (simian kidney, Cercopithecus aethiops, C1008), were grown in Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich) supplemented with 10% FCS and 100 U/mL Pen/Strep. Caco-2 cells (human adenocarcinoma, HTB-37) were additionally supplemented with 1 mM HEPES (Gibco), while Rhileki cells were supplemented with 1% non-essential amino acids (Gibco) and 1 mM sodium pyruvate (Gibco). HeLa (human adenocarcinoma, CRM-CCL-2) and MDCK (Madin-Darby canine kidney, Canis familiaris, CCL-34) cells were maintained in Minimum Essential Medium (MEM; Sigma-Aldrich) supplemented with 10% FCS and 100 U/mL Pen/Strep. For viral infection experiments, the FCS content in the culture medium was reduced to 3% for invertebrate cell lines and 5% for vertebrate cell lines (Table 3).

Virus isolation and propagation

For virus isolation, homogenized bat bug samples were sterile-filtered using a 0.45 µm filter (Millipore, Burlington, MA, USA) and inoculated to cell monolayers in six-well plates at approximately 80% confluence. Inoculated cells were maintained in DMEM supplemented with 2% FCS, Pen/Strep, and 0.25 μg/mL amphotericin B (Gibco) for seven days. Cell morphology and cytopathic effects were automatically monitored every six hours using an IncuCyte S3 live-cell imaging system (Sartorius, Göttingen, Germany). At the end of the incubation period, cell supernatants were collected by centrifugation and cells were harvested by trypsinization with 0.05% trypsin-EDTA (Gibco). All samples were subsequently analyzed by RT-qPCR. Following initial isolation in VeroE6 cells, the virus was further passaged in this cell line, and virus-containing VeroE6 culture supernatants were used for inoculating growth kinetic experiments.

Description of Stricticimex phnomsampovensis

Upon examination, the Stricticimex specimens appeared to belong to a new species, closely related to Stricticimex parvus and are described below. The updated list of bat bugs in Cambodia, their references, location and known hosts are provided in Table 1. The type series is kept in the collection of the Institut Pasteur du Cambodge, Phnom Penh.

Holotype. Fig 2. Male. Head: length: 0.44, slightly longer than wide (0,41), interocular space 5,3 times as wide as eye; Head with 7 setae forming a “y” shape, joining together in the vertex; labrum with 10 pairs of bristles; 5 bristles (3 longer and 2 medium) along inner margin of each eye in addition to about 3 pairs of vertex. Antennae measuring 1.7mm long, always less than 2 mm long; Segments I-IV: 0,15; 0,42; 0,7; 0,43. Second antennal segment as long as width of head, 0,42: 0, 41. Second and fourth segments are subequal. Rostrum 0,48 size of segments I-III: 0,2:0,18:0,1. Pronotum 0,6 wide, twice as wide as long (0,6: 0,28) and straight on the inner sides, narrowed and rounded laterally and posteriorly. Longest bristles at sides as long as those on pronotum. Hemelytral pads are almost twice as large as long (0.37:0.19) and straight on inner sides, narrowed and rounded laterally and posteriorly. Longest bristles at sides about as long as those on the pronotum. Abdomen suboval; second and third segments with 3–4 ill-defined rows of bristles, remaining segments with 2 rows of bristles and with cluster of bristles on terminal segment. Ventral surface with much finer and numerous bristles on the posterior part of each segment. Base of paramere forming an acute angle, then continuing by curving sideways, paramere half as long as the genital segment at base (Fig 2). Legs long and slender; hind femora about 4,5 times as long as the greatest width, 0.79:0.18; tibiae 2 times longer than femora, 1.6:0.79.

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Fig 2. Stricticimex phnomsampovensis Suor & Maquart n.sp description.

(A) Drawing of Stricticimex phnomsampovensis Suor & Maquart n.sp lateral view (left) and dorsal view (right) (B) Photo Male genital) (C) Male genital drawing.

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Allotype female. Like male. Paragenital sinus sinuate broadly on hind margin of third tergite sublaterally. Ectospermalege broad at opening, directed inwardly and then bent downward. Pronotum width/length: 0,68/0.3. Hemelytras width/length: 0.20/0.38. Hind tibia/femur; 1,86/0,94.

Etymology. The name refers to the location where this new species was found (Phnom Sampov).

Ecology. This species was found in a cave dominated by Mopsplicatus (Buchanan, 1800), probably feeding off the bats, but they were aggressive, readily feeding on humans entering in the cave (Fig 1E).

Differential diagnosis. The new species, close to S. parvus differs by the following characters: (i) Clypeus short and stout while it is more elongated and pointing forward in S. parvus; (ii) Hemelytrals pads twice as long as wide, where it is square-shaped in S. parvus; (iii) Hind tibiae 2 times longer than femora, while it is 1,5 times longer in S. parvus; (iv) Shape of paramere, usually straight or directed downward for S. parvus but curving sidewise on the left for S. phnomsampovensis with a more acute angle (obtuse angle for S. parvus) at the phallobase.

Authorship. The authors of the new taxon, Stricticimex phnomsampovensis, are Suor and Maquart, and are different from the authors of this article, in accordance with Article 50.1 and Recommendation 50A of the International Code of Zoological Nomenclature (ICZN, 2012, https://www.iczn.org/the-code/the-code-online/).

Updated Key to the Species of Stricticimex (revision of Usinger’s 1966 key in Ueshima, 1968)

1. 1.Fore femora slightly longer than tibiae. Size small, pronotum 0.6 mm wide. India.........pattoni (Horvath)
2. Fore femora is slightly shorter than tibiae. Size medium to large, pronotum 0.7 mm or more in width...2
3. 2.Second antennal segment shorter or as long as than width of head……...............................................3
4. Second antennal segment much longer than width of head………...……………………….….................6
5. 3.Hind femora less than 4 times as long as wide. South Africa..................... transversus Ferris & Usinger
6. Hind femora 4 times or more as long as wide…......................................................................................4
7. 4.Second antennal segment as long as the lengh of the pronotum at median line or longer……………...5
8. Second antennal segment shorter than length of pronotum. Interocular space wider than the length of second antennal segment. Size medium. Pronotum 1.0 mm wide. Egypt. …………….... namru Usinger
9. 5.Clypeus pointing forward, hemelytrals pads as long as wide, hind tibiae 1,5 times longer than femora. Obtuse angle at the base of the paramere, continuing almost in a straight line or slightly downward......................................................................................................................................... parvus Ueshima
10. Clypeus short and stout, hemelytrals pads twice as long as wide, hind tibiae 2 times longer than femora. Base of paramere forming an acute angle, curving sidewise. …...…..phnomsampovensis n.sp.
11. 6.Third antennal segment more than twice as long as fourth. Size large, pronotum 1.0 mm
12. or more in width. South Africa…...................................................................antennatus Ferris & Usinger
13. Third antennal segment less than twice as long as fourth. Size smaller, pronotum less than 1 mm wide………………..…………………………………………………………………………….7
14. 7.Last antennal segment longer than width of head. Longest bristles at sides of
15. pronotum, wing pads and abdomen about 0.37 mm. Kenya.......................intermedius Ferris & Usinger
16. Last antennal segment subequal to width of head. Longest bristles at sides of pronotum, wing pads and abdomen about 0.31 mm. Democratic Republic of Congo. ………................ brevispinosus Usinger

Phylogenetic analysis of Stricticimex phnomsampovensis

A BLAST search of the COI sequences obtained from bat bugs collected in Ta Rumm Cave revealed the closest match to Cyanolicimex patagonicus (Haematosiphoninae), with a percentage identity of 82.77% and query coverage of 98% (GenBank: MG596833). The 16S sequences showed similarity to Leptocimex inordinatus (Cacodminae), with a 100% percentage identity but low query coverage (65–66%; GenBank: KT592539), indicating potential genetic divergence.

Analysis of the 18S gene fragment revealed high similarity to multiple species across different subfamilies, including Cimex latipennis (Cimicinae, 98.9% identity, 100% query coverage; GenBank: KF018720), Latrocimex spectans (Latrocimicinae, 98.7% identity, 99–100% query coverage; GenBank: MZ378786), and Cimex sp. (Cimicinae, 98.5–98.8% identity, 99% query coverage; GenBank: EU683122). Notably, trimmed sequence comparisons showed 100% similarity among our specimens.

The Bayesian consensus tree, constructed using concatenated sequences of COI, 16S, and 18S rRNA gene fragments, provides strong support for the classification of this newly discovered species within the Cacodminae subfamily (Fig 3). This phylogenetic placement, combined with the genetic divergence observed, suggests Stricticimex phnomsampovensis represents a novel species within bat-associated Cimicidae (Fig 3).

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Fig 3. Bayesian consensus tree of family Cimicidae (including all six described subfamilies) based on concatenated sequences of COI, 16S, and 18S rRNA gene fragments.

GenBank accession numbers are indicated in S1 Table. The scale bar indicates the number of substitutions per site. Main host groups of each species are indicated with circles (purple: bats; blue: birds; green: humans; gray: unknown host). Animal silhouettes used in this figure were sourced from PhyloPic (https://www.phylopic.org/).

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Identification and Phylogenetic analysis of a Novel Coltivirus in Cambodian Bat Bugs

A novel coltivirus, Stricticimex coltivirus (SCCV), was identified in Stricticimex phnomsampovensis collected from Ta Rumm Cave. The high level of amino acid identity between proteins of SCCV and Tai Forest reovirus (TFRV) and other coltiviruses species suggest that SCCV belongs to the same genus (Table 4). Phylogenetic analysis of the available viral genomes revealed that all identified proteins were most closely related to members of the genus coltivirus, which includes the tick-borne pathogenic CTFV, other tick-associated viruses, and TFRV, previously identified in African free tailed bats (Figs 4 and 5). Further in-depth analysis of the RNA-dependent RNA polymerase (RdRp) gene, based on amino acid sequences, showed the highest similarity to TFRV, with nucleotide identity ranging from 74.6% to 75.1% and amino acid similarity between 88.8% and 89.0% (Fig 4 and Table 4). Comparisons of other identified viral proteins revealed amino acid similarities exceeding 70% for VP5, > 80% for VP1, VP3, and VP10, and >90% for VP2, VP8, VP9, and VP11, all of which were most similar to TFRV. Notably, VP4 displayed a partially shared similarity with Kundal (Table 4). Due to the lack of available nucleotide or protein sequences for VP6, VP7, and VP12 from TFRV, no contigs were generated for these gene segments, leaving their genomic composition unresolved.

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Table 4. Nucleotide and amino acid similarities between Stricticimex coltivirus, Tai Forest Reovirus and Kundal virus.

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Fig 4. Maximum likelihood tree of the RdRp protein from Coltiviruses and other Reovirales.

Bootstrap values are indicated at key nodes. Stricticimex coltivirus (SCCV) in bold red. Shading corresponding to the different Reovirales genera. Tip shading and branch style reflect amino acid lengths of the sequence used to generate phylogenetic tree. Animal symbols within the Coltivirus genus represent the isolation host. Animal silhouettes used in this figure were sourced from PhyloPic (https://www.phylopic.org/).

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Fig 5. Maximum likelihood tree of the VP2 protein from Coltiviruses and other Reovirales.

Bootstrap values are indicated at key nodes. Stricticimex coltivirus (SCCV) in bold red. Shading corresponding to the different Reovirales genera. Tip shading and branch style reflect amino acid lengths of the sequence used to generate phylogenetic tree. Animal symbols within the Coltivirus genus represent the isolation host. Animal silhouettes used in this figure were sourced from PhyloPic (https://www.phylopic.org/).

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Phylogenetic analyses of the conserved RdRp and VP2 protein sequences revealed that the SCCV clusters within a distinct clade alongside known coltivirus and coltivirus-like viruses detected in bats (Figs 4 and 5). Notably, SCCV is most closely related to Tai Forest reovirus (TFRV), originally isolated in 2006 from free-tailed bats (Mops aloysiisabaudiae) in Côte d’Ivoire. More distantly, and with greater uncertainty due to partial RdRp and VP2 sequences, SCCV also shares similarities with coltivirus-like viruses detected in Taphozous melanopogon bats detected in Guangxi, China in 2017.

The strong bootstrap support for the clustering of SCCV sequences with TFRV indicates a relatively close evolutionary relationship (Figs 4 and 5). Phylogenetic analysis of the RdRp and VP2 proteins further reveals that bat-associated coltiviruses form a monophyletic clade within the coltivirus genus. However, the presence of partial protein sequences introduces some uncertainty at certain branching points and may not fully capture the true evolutionary relationships.

Taken together, the sequence data, gene segment identities, and phylogenetic placement strongly support SCCV as a novel coltivirus species, expanding our understanding of bat-associated reoviruses. Further studies are needed to fill phylogenetic gaps and refine its evolutionary history and clarify its relationship to other Coltivirus members and whether bats and bat-associated ectoparasites occupy a phylogenetic niche within the Coltivirus genus.

Coltivirus prevalence in Cambodian bat bugs

A total of 87 individual bat bugs collected from Ta Rumm Cave in April 2023 were screened for coltivirus using a newly developed real-time PCR assay targeting the RdRp gene. Sixteen (18.4%) tested positive, with Ct values ranging from 23.02 to 40.58 (mean Ct: 30.18). All positive samples were identified as the SCCV based on a specifically designed probe, while no samples tested positive for Tai Forest reovirus (TFRV).

Virus Isolation

Virus isolation was attempted using homogenized bat bugs to inoculate C6/36 (Aedes albopictus), BHK-21 (Mesocricetus auratus), Rhileki (Rhinolophus lepidus), and VeroE6 (Chlorocebus sabaeus) cell lines. Initial inoculations included samples with Ct values ranging from 23.02 to 40.58. Successful virus isolation was achieved in VeroE6 cells from samples with Ct values of 23.02 and 25.29. The presence of the virus was confirmed in the culture supernatant across three subsequent passages using the SCCV specific RT-PCR assay.

Virus growth kinetics

The replication dynamics of one SCCV isolate was assessed in invertebrate (Aag-2, C6/36) and vertebrate (BHK-21, Caco-2, MDCK, Rhileki, VeroE6) cell lines over 12 days. Culture supernatants and cells were analyzed by RT-PCR to detect the presence of SCCV (Fig 6). Viral entry was observed in all tested cell lines at some point during the experiment (Fig 6B). However, productive viral replication, indicated by the release of virus into the culture supernatant, was only detected in Caco-2, HeLa, BHK-21, and VeroE6 cells. Among these, BHK-21 and VeroE6 cells exhibited the highest viral titers, with replication increasing over time (Fig 6A).

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Fig 6. In vivo growth kinetics of Stricticimex coltivirus (SCCV).

Various invertebrate (AAg2 in red; C6/36 in blue and shades of green) and vertebrate cell lines (Vero E6 in brown; BHK-21 in green; Caco-2 in purple; HeLa in orange; MDCK in yellow and shades of blue), including a bat-derived cell line (in pink/violet), were inoculated with the SCCV isolate (passage 3 in Vero E6) for 1 hour at 37 °C. Supernatants (A) and cells (B) were collected every 24 hours and analyzed by RT-PCR. Experiments were conducted in biological duplicates. The Ct values are plotted inversely to intuitively demonstrate viral growth.

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