Do bed bugs transmit human viruses, or do humans transmit bed bug viruses? A worldwide survey of the bed bug RNA virosphere

Abstract

Bed bugs (Hemiptera: Cimicidae ) are a globally distributed hematophagous pest that routinely feed on humans. Unlike many blood-sucking arthropods, they have never been linked to disease transmission in a natural setting, and despite interest in their role as disease vectors, little is known about the viruses that bed bugs naturally harbor. Here, we present a global-scale survey of the bed bug RNA virosphere. We sequenced the metatranscriptomes of 22 individual bed bugs ( Cimex lectularius and Cimex hemipterus ) from 8 locations around the world. We detected sequences from two known bed bug viruses (Shuangao bedbug virus 1 and Shuangao bedbug virus 2) which extends their geographical range and the host range of Shuangao bedbug virus 1 to Cimex lectularius . We identified three novel bed bug virus sequences from a tenui-like virus ( Bunyavirales ), a toti-like virus ( Ghabrivirales ), and a luteo-like virus ( Tolivirales ).
Interestingly, some of the bed bug viruses branch near to insect-transmitted plant-infecting viruses, opening questions regarding the evolution of plant virus infection. When we analyzed the putative viral sequences by their host’s collection location, we found unexpected patterns of geographical diversity that may reflect humans’ role in bed bug dispersal. Additionally, we investigated the effect that Wolbachia, the primary bed bug endosymbiont, may have on viral abundance and found that Wolbachia infection neither promotes nor inhibits viral infection. Finally, our results provide no evidence that bed bugs transmit any known human pathogenic viruses.

Full text

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RESEARCH ARTICLE
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Do bed bugs transmit human viruses, or do humans transmit bed bug viruses? A worldwide
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survey of the bed bug RNA virosphere
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Hunter K. Walt1, Jonas G. King1, Johnathan M. Sheele4, Florencia Meyer1, Jose E. Pietri2*, and
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Federico G. Hoffmann1,3,*
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1Department of Biochemistry, Molecular Biology, Entomology, and Plant Pathology, Mississippi
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State University, Starkville, MS, USA
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2Sanford School of Medicine, Division of Basic Biomedical Sciences, University of South Dakota,
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Vermillion, SD, USA
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3Institute for Genomics, Biocomputing and Biotechnology, Mississippi State University,
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Starkville, MS, USA
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4University Hospitals Cleveland Medical Center & Case Western Reserve University, Department
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of Emergency Medicine, Cleveland, OH, USA
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*Correspondence: fgh19@msstate.edu, Jose.Pietri@usd.edu
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ABSTRACT:
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Bed bugs (Hemiptera: Cimicidae) are a globally distributed hematophagous pest that
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routinely feed on humans. Unlike many blood-sucking arthropods, they have never been linked
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to disease transmission in a natural setting, and despite interest in their role as disease vectors,
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little is known about the viruses that bed bugs naturally harbor. Here, we present a global-scale
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survey of the bed bug RNA virosphere. We sequenced the metatranscriptomes of 22 individual
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bed bugs (Cimex lectularius and Cimex hemipterus) from 8 locations around the world. We
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detected sequences from two known bed bug viruses (Shuangao bedbug virus 1 and Shuangao
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bedbug virus 2) which extends their geographical range and the host range of Shuangao bedbug
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virus 1 to Cimex lectularius. We identified three novel bed bug virus sequences from a tenui-like
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virus (Bunyavirales), a toti-like virus (Ghabrivirales), and a luteo-like virus (Tolivirales).
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Interestingly, some of the bed bug viruses branch near to insect-transmitted plant-infecting
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viruses, opening questions regarding the evolution of plant virus infection. When we analyzed
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the putative viral sequences by their host's collection location, we found unexpected patterns
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of geographical diversity that may reflect humans’ role in bed bug dispersal. Additionally, we
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investigated the effect that Wolbachia, the primary bed bug endosymbiont, may have on viral
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abundance and found that Wolbachia infection neither promotes nor inhibits viral infection.
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Finally, our results provide no evidence that bed bugs transmit any known human pathogenic
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viruses.
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1. INTRODUCTION:
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Bed bugs (Hemiptera: Cimicidae) are globally distributed obligately hematophagous
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ectoparasites (Doggett et al., 2012). Some species routinely feed on humans, but unlike many
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other blood sucking insects, there is no evidence that bed bugs are human disease vectors
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(Davies et al., 2012). Several studies have been conducted to assess if bed bugs could transmit
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human pathogens, but transmission in the wild has not been documented (Blakely et al., 2018;
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El Hamzaoui et al., 2019; Goddard & deShazo, 2009; Leulmi et al., 2015; Pietri, 2020).
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Considering the consistent resurgence of bed bug populations leading to outbreaks in the last
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20-30 years, it is important to understand what microbes they harbor along with their potential
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as vectors (Davies et al., 2012; Doggett & Lee, 2023; Lewis et al., 2023).
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Large scale metatranscriptomic studies have enhanced our knowledge of viral diversity
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in invertebrates and have made detecting arthropod-associated viruses increasingly more
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feasible (Käfer et al., 2019; Li et al., 2015; Shi et al., 2016). Bed bugs have been included in some
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arthropod viromics-based studies, and four putative bed bug virus sequences have been
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detected: Serbia reo-like virus 2 (NCBI:txid2771464), Serbia picorna-like virus 2
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(NCBI:txid2771462), Shuangao bedbug virus 1 (NCBI:txid1608071) and Shuangao bedbug virus
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2 (NCBI:txid1608072) (Li et al., 2015; Zhang et al., 2020). No further studies have been
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conducted to examine the pathogenic properties of these putative viruses, but they do provide
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evidence that bed bugs encounter viral infection. Furthermore, Ling et al. (2020) took a
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metatranscriptomic approach to survey for viruses in bed bugs and found that some reads in a
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sample of recently blood fed individuals aligned to hepatitis C virus. Although there was no
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evidence that the virus was replicating (i.e., low number of reads mapped and an incomplete
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genome), their study highlights the role that bioinformatic surveillance could play in detecting
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human pathogen transmission in bed bugs. However, bed bug specific viruses were either not
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detected or not reported in their study (Ling et al., 2020).
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In this study, we conducted a worldwide survey of the bed bug RNA virosphere. Our
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aims were to search for known human viral pathogens and novel bed bug viruses that could be
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of interest to human health or biocontrol. We collected bed bugs (Cimex lectularius and Cimex
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hemipterus) from 8 distinct locations around the world and sequenced RNA libraries from 22
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individuals. We assembled bed bug metatranscriptomes, and conducted phylogenetic analyses
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on the virus sequences that we detected. We also assessed viral diversity between bed bug
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species and geographic location. Additionally, we investigated whether there is a correlation
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between Wolbachia (a bed bug endosymbiont known to have an antiviral effect when infecting
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other insect taxa) reads and bed bug virus reads in each sample (Cogni et al., 2021; Hussain et
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al., 2023; Lindsey et al., 2018; Teixeira et al., 2008; Terradas & McGraw, 2017).
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2. MATERIALS AND METHODS:
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2.1 Collection and extraction:
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The samples used in this study were obtained as part of a large international collection
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of bed bugs provided by numerous pest control companies and researches. We included Cimex
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lectularius samples from Czechia (n=3), France (n=1), the UK (n=3), Rome-Italy (n=3), Assisi-Italy
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(n=3), Ohio-USA (n=3), the Harlan lab strain of Cimex lectularius (initially collected in Fort Dix,
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New Jersey, USA and maintained at the King lab at Mississippi State University) (n=3), and
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Cimex hemipterus samples from Madagascar (n=3). Each bug was washed in 95% ethanol and
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total RNA was isolated using a standard TRIzol protocol (Invitrogen Waltham, MA) and further
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purified using NEB’s Monarch RNA cleanup kit was used (New England Biolabs Ipswich, MA).
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2.2 Library Prep and Sequencing:
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We checked total RNA quality using Nanodrop quantitation and agarose gel
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electrophoresis, and we assessed RNA integrity with an Agilent 2100 bioanalyzer. Libraries were
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prepared using a strand specific library prep with ribosomal RNA depletion. Sequencing was
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conducted on the Illumina NovaSeq 6000 instrument for 150 base pair paired-end reads,
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resulting in approximately 100-135 million reads per sample. The quality of the reads was
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inspected using FastQC v0.11.5 (Andrews, 2010) and reads were quality trimmed using
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trimmomatic v. 0.39 (Bolger et al., 2014).
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2.3 Virus Discovery:
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To enrich the data for viral reads, we filtered out Cimex, Wolbachia, and human reads
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by mapping to the Cimex lectularius (GCF_000648675.2), Cimex hemipterus (GCA_001663875.1,
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partial), Wolbachia endosymbiont of Cimex lectularius (GCF_000829315.1), and Homo sapiens
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(GCF_000001405.40) genomes using the bbsplit.sh tool of the bbmap suite (version 38.46)
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(sourceforge.net/projects/bbmap/) and retained all unmapped reads. We assembled the
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unmapped reads using Trinity v.2.11.0 (Grabherr et al., 2011) both individually and by collection
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location. We clustered the individual sample assemblies with cd-hit-est (W. Li & Godzik, 2006)
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with a sequence similarity threshold of 90%, a minimum sequence length of 500 nt, and a word
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size of 8. The assemblies were annotated using diamond blastx in the –very-sensitive mode
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with an e-value cutoff of 1e-5 (Buchfink et al., 2015, 2021) and the annotated transcripts were
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filtered for viral hits which were further inspected for false positives using NCBI’s BLASTx
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(https://blast.ncbi.nlm.nih.gov/Blast.cgi) against the nr protein database. To detect known viral
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conserved domains, we also inspected each viral hit in NCBI’s conserved domain database
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(CDD) (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) and InterProScan
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(https://www.ebi.ac.uk/interpro/search/sequence/) (Quevillon et al., 2005).
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When putative multipartite virus sequences were present, we used a viral co-occurrence
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detection method we previously described to identify the other genomic segments (Walt et al.,
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2023). Briefly, this method calculates two metrics based off sample co-occurrence. The first
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metric, Vco, determines the frequency at which a transcript occurs in a sample with a given viral
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conserved sequence (e.g., transcripts with confirmed RdRp domains detected in BLAST
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analysis). The second metric, Tco, determines if the transcripts found together with a viral
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conserved sequence occur in other samples without the viral conserved sequence. We used the
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thresholds Vco=0.75 and Tco=0.5 to determine candidate viral genomic segments. After running
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the co-occurrence analysis, we only kept candidate sequences with an ORF size > 500 nt. We
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further inspect these transcripts for conserved protein families and domains using NCBI’s CDD
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and InterProScan.
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2.4 Phylogenetic Analysis of Viruses
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We only used transcripts with confirmed RdRp domains for phylogenetic analyses. First,
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we extracted and translated ORFs encoding for RdRp proteins using NCBI’s ORFfinder
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(https://www.ncbi.nlm.nih.gov/orffinder/) and we downloaded diverse representative
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sequences from families within relevant viral orders from NCBI. We aligned RdRp amino acid
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sequences using MAFFT v7.490 (using the E-INS-I, G-INS-i, and L-INS-i, algorithms) and MUSCLE
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v3.8.1551 (Edgar, 2004; Katoh & Standley, 2013). We compared alignment qualities using
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MUMSA (Lassmann & Sonnhammer, 2006) scores, and the highest scoring alignment was used
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for tree inference. Phylogenetic analyses were conducted with IQ-TREE2 v.2.0.7 using
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ModelFinder (Kalyaanamoorthy et al., 2017) to find the best fitting substitution model. We
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assessed branch support using ultrafast bootstrap with 1000 replicates, Shimodaira-Hasegawa-
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like approximate likelihood ratio test (SH-aLRT) with 1000 replicates, and the aBayes test
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(Anisimova et al., 2011; Minh et al., 2013; Nguyen et al., 2015). All phylogenetic trees were
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midpoint rooted (unless otherwise noted) and visualized using the Interactive Tree of Life (iTOL)
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webserver (Letunic & Bork, 2021).
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2.5 Phylogeographic Analysis
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We retrieved all transcripts of putative viral origin from each individual bed bug
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assembly using Diamond BLASTx and CD-hit output. We predicted coding sequences using
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EMBOSS’s getorf tool using the -find 2 option. We only used coding sequences with complete
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RdRp domains for phylogenetic analysis and duplicate sequences within samples were
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discarded. We conducted phylogenetic analysis in the same way as section 2.4, except that
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nucleotide sequences were used instead of amino acid. We selected closely related taxa from
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the analysis in 2.4 as outgroups for phylogeographic analysis. We calculated evolutionary
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distance as p-distance using MEGA v.11.0.13 (Tamura et al., 2021). We did not conduct
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phylogeographic analysis on Shaungao Bedbug virus 2 because it was only detected in one
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location in our study.
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2.5 Correlation of Wolbachia and Viral Read Abundance
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We mapped the trimmed read datasets to all the viral genomes detected in this study
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and the Wolbachia endosymbiont of Cimex lectularius genome (GCF_000829315.1) using
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HISAT2 v.2.2.1 (Kim et al., 2019). We used the summary file output to obtain the percent of
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reads that mapped to the virus genomes or the Wolbachia genome. To correlate the Wolbachia
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and virus read abundances, we conducted a simple linear regression analysis in R v.4.2.2. (R
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Core Team, 2020), using the percentage of reads that mapped to the viral genomes versus the
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percentage of reads mapped to the Wolbachia genome.
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3. RESULTS
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3.1 Read Mapping, Assembly, and Transcript Annotation
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To survey the bed bug virome, we sequenced the total RNA (ribosomal and small RNA
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depleted) from 22 individual bed bugs collected from the United Kingdom, France, Chechia,
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Italy, Madagascar, and the USA. To enrich for virus sequences in our bed bug samples, we
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filtered out reads that mapped to genomes of organisms that could be represented in our
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dataset. We classified an average of 85% of reads from each sample as bed bug (65.2% C.
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lectularius, 19.8% C. hemipterus), Wolbachia (2.08%), or human reads (0.07%). Those reads
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were not used for metatranscriptome assembly (Figure 2). We assembled the remaining reads
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and aligned the assemblies to NCBI’s nr protein database using diamond BLASTx, which
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returned multiple significant hits to putative bed bug virus sequences. We detected sequences
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from two known negative-sense single stranded RNA (-ssRNA) viruses associated with C.
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hemipterus: Shuangao bed bug virus 1 (Sbbv1) and Shuangao bed bug virus 2 (Sbbv2), but we
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did not detect the other two known bed bug viruses in our assemblies (C. X. Li et al., 2015;
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Zhang et al., 2020). In addition, we detected 3 novel viral sequences. These sequences all
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belong to the realm Riboviria and encode for 1.) a -ssRNA genome, 2.) a positive-sense single
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stranded RNA (+ssRNA) genome, and 3.) a double stranded RNA (dsRNA) genome. Strikingly, all
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putative viral sequences detected in this study are present intercontinentally, but none were
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detected across all samples.
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3.2 Shuangao Bedbug Virus 1
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Sbbv1 is described as an unclassified rhabdovirus (-ssRNA) (NCBI:txid1608071) in the
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NCBI database. Our phylogenetic analysis groups Sbbv1 within the Bunyavirales order
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branching sister to the Hantaviridae, which is consistent with previous studies (C. X. Li et al.,
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2015) (Figure 3). We detected Sbbv1 sequences in C. hemipterus samples from Madagascar,
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and C. lectularius samples from France and Czechia. Our results extend Sbbv1’s geographical
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range from China (where it was first detected) to Madagascar, Czechia, and France, and its host
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range to C. lectularius. We detected both known segments of Sbbv1: a ~7000 nt segment (L)
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containing the RdRp domain, and a ~3000 nt (M) segment encoding for the glycoprotein
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precursor. The complete M segment was detected in Czechia sample 3, France, and all three
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Madagascar samples, but it was only partially complete in Czechia Sample 2.
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3.3 Shuangao Bedbug Virus 2
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Sbbv2 is described as an unclassified -ssRNA virus (NCBI:txid1608072) with a 10,925 bp
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monopartite genome. Our analyses show that it groups with the Rhabdoviridae family (Figure
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4), consistent with the findings of Li et al. (2015). We detected Sbbv2 sequences in C.
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hemipterus samples from Madagascar samples 1 and 3. The genomes we assembled of this
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virus were around 3 kb longer than those from Li et al. (2015), ranging from 12,802-13,477 bp.
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Our results complete the genome of Sbbv2, as the extra 3 kb contains ORFs at the beginning of
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the genome: one encoding for a rhabdovirus nucleoprotein (CDD E-value = 1.77e-06), and the
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other encoding for what we suspect is the rhabdovirus phosphoprotein (Supplementary Figure
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1). Although the latter ORF did not have any significant BLAST hits or conserved domains, we
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assume that it is the phosphoprotein based off synteny to other rhabdovirus genomes which
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generally encode for five proteins in the following order: nucleoprotein, phosphoprotein,
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matrix protein, glycoprotein, and large protein (Walker et al., 2022). Our study also extends the
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geographic range of this virus, as it was previously only detected in bed bugs from China (Li et
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al. 2015).
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3.4 Tenuiviridae
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We detected sequences that had significant BLAST hits to viruses in the family
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Phenuiviridae (-ssRNA) in C. lectularius samples from Czechia, France, Rome-Italy, and Assisi-
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Italy, and C. hemipterus samples in Madagascar. These viruses are typically multisegmented,
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and from our BLASTx-based analysis, we detected a ~9000 bp putative L segment. We found
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complete L segments in Czechia sample 3, Madagascar sample 2, Rome-Italy sample 1, and
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Assisi-Italy samples 1, 2, and 3. We found partial L segments in Czechia sample 2, France, and
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Rome-Italy sample 3. Our phylogenetic analysis groups these sequences in the family
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Phenuiviridae, sister to a clade including the tenuiviruses, which are a genus of hemipteran-
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transmitted plant infecting viruses (Figure 3). We refer to this putative virus as Cimex tenui-like
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virus 1.
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Because bunyaviruses are typically multisegmented, we used an approach previously
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described in Walt et al. (2023) to identify candidate genomic segments based off sample co-
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occurrence. We found a single transcript that met our requirements with a Vco= 0.86 and a Tco =
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0.75. The transcript is 2,851 nt long and encodes for a 902 amino acid protein. Based on
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transcript length in comparison to Cimex tenui-like virus 1’s closest BLAST hit (Solenopsis invicta
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virus 14: M segment length=2,705 bp), we propose this may be the M segment, which encodes
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a glycoprotein. InterProScan predicted two transmembrane domains in the amino acid
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sequence of this transcript, which is similar to the glycoprotein of Solenopsis invicta virus 14 as
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it also has two transmembrane domains. These results need further confirmation, as this
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transcript had no significant BLAST alignments to other known sequences, no predicted
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conserved domains in the translated protein, and no similar transcripts were found in the Italy
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samples, which also had Cimex tenui-like virus 1 sequences in them.
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3.5 Luteoviridae
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We detected a ~2800 bp luteo-like virus 1 sequence (+ssRNA) in the Harlan lab strain,
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Czechia, and France samples, all of which were C. lectularius. We found complete transcripts in
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Czechia samples 1,2, and 3, France, and Harlan Strain-USA samples 1 and 2, and found a partial
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transcript in Harlan Strain-USA sample 3. Our phylogenetic analysis groups these viruses within
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a clade of unclassified Luteoviridae, sharing a common ancestor with 4 viruses detected in
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mosquitoes, and 1 virus detected in an anal swab from a bird (Figure 5). This clade branches
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sister to a group of three viruses, Miscanthus yellow fleck virus, Rabbit luteovirus, and
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Arracacha latent virus E. Rabbit luteovirus was discovered in a rabbit but was assumed to have
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come from its diet (Tsoleridis et al., 2019), and the other two viruses are plant viruses that are
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likely transmitted by aphids, another hemipteran insect (Bolus et al., 2020). We refer to this
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putative virus as Cimex luteo-like virus 1.
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3.6 Totiviridae
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We detected a toti-like virus (dsRNA) sequence in C. lectularius samples from five
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locations: the Harlan lab strain-USA, Czechia, France, Assisi-Italy, and the UK. The complete
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genome is around 7.8 kb and encodes for two large ORFs. One ORF (~3880 nt) has significant
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BLASTx hits to other toti-like virus RdRps, and the other (~1860 nt) has significant BLAST hits to
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other toti-like virus proline-alanine rich proteins (Spear et al., 2010). We recovered whole or
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nearly complete genomes for the UK (all samples), Czechia (complete for samples 1 and 3,
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partial for sample 2), France, and all three Assisi-Italy samples. Phylogenetically, this virus
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shares a most recent common ancestor with an unclassified totivirus isolated from a flea. The
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virus forms a clade with other Hemiptera and Thysanoptera associated toti-like viruses along
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with two viruses isolated from plants (Figure 6). We hereon refer to this putative virus as Cimex
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toti-like virus 1.
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3.7 Phylogeography of Bed Bug Viruses
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All the virus sequences that we detected were distributed across geographically distant
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locations, so we investigated viral diversity between localities. We hypothesized that viral
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evolutionary relationships would reflect the differences in host species, and that within a host
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species, phylogenetic relationships would be reflective of geographic distance (Ballinger et al.,
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2022; Longdon et al., 2014). Specifically, we would expect C. lectularius and C. hemipterus to
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harbor closely related but distinct viral populations, and within C. lectularius, we would expect
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samples from Europe to be distinct from those rom North America. We only used sequences
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with complete RdRp domains and only included the unique sequences within individuals for our
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phylogeographic analysis.
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3.7.1 Cimex tenui-like virus 1
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We found Cimex tenui-like virus 1 sequences in both C. lectularius, and C. hemipterus. In
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the case of C. lectularius, sequences from this virus were found in one individual from Rome-
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Italy, all three individuals from Assisi-Italy, one individual from Madagascar, the individual from
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France, and one individual from Czechia. Interestingly, the Italian samples form a distinct clade
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from the rest of the world (Figure 7A). When we grouped the samples by clade: Italy, world,
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and outgroup and assessed evolutionary distance between the groups, the mean p-distance
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between the Italian clade and the rest of the world clade was 22.4%, while the “within group”
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mean p-distance was 3.0% and 1.1% for the Italian clade and the rest of the world, respectively
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(Table 1). Interestingly, the “within group” distance is three times higher in the samples that
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come from the same country (Italy) than those collected in different countries (France, Chechia,
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and Madagascar).
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Because the samples collected in Italy had a higher “within group” mean than the
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samples from the rest of the world, we computed pairwise comparisons of p-distance for the
263
samples used in the phylogeny in Figure 7A (Table 2). We found that one sequence of Cimex
264
tenui-like virus 1 (Rome-Italy 1) is the driver of the viral genetic diversity within the group of
265
samples collected in Italy, since the Assisi-Italy samples are identical, but the Rome-Italy 1
266
sample is 7.5% different from all the Assisi-Italy samples at the nucleotide level (Table 2).
267
Furthermore, the France and Czechia 3 Cimex tenui-like virus 1 sequences are nearly identical,
268
while the Madagascar 2 Cimex tenui-like virus 1 sequence is only 1.7% different from both the
269
France and the Czechia samples (Table 2), even though the Madagascar samples are C.
270
hemipterus, while the rest of the samples are C. lectularius.
271
3.7.2 Cimex toti-like virus 1
272
We only found Cimex toti-like virus 1 sequences in C. lectularius samples. We found
273
them in two of the three Czechia individuals, the France individual, all Assisi-Italy individuals,
274
and all UK individuals. Once again, the samples collected from Italy form a distinct clade from
275
the rest of the world (Figure 7B). We grouped the samples by the Italy clade, the rest of the
276
world clade and the outgroup and found that there was little difference between the Italy
277
viruses (“within group” mean p-distance = 0.3%), while rest of the world clade were more
278
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15
genetically distant (“within group” mean p-distance = 10%) (Table 1). The “between group”
279
mean distance between the virus sequences detected in Italy and the virus sequences detected
280
from the rest of the world is 16.4%. Within the rest of the world clade, the viruses from Czechia
281
form one group and the France and UK viruses form another group, following expected
282
geographic patterns.
283
3.7.3 Cimex luteo-like virus 1
284
We only found Cimex luteo-like virus 1 sequences in C. lectularius, and we detected
285
them in all three Czechia samples, two of the Harlan Strain-USA samples, and the France
286
individual. The tree has low resolution other than one branch containing the viral sequences
287
detected in the Harlan Strain-USA bugs (Figure 7C). All the Cimex luteo-like virus 1 RdRp-
288
encoding transcripts have a mean p-distance of 4%.
289
3.7.4 Shuangao Bedbug Virus 1
290
We detected Sbbv1 sequences in all three C. hemipterus individuals collected from
291
Madagascar, and one C. lectularius individual from Czechia. The Madagascar samples and the
292
previously described Sbbv1 sample (detected in China) group together with 100% support
293
(Figure 7D). This reflects host taxonomy, as the Sbbv1 sample that was first detected in China
294
was also detected in C. hemipterus. Because the bed bugs collected in Czechia were C.
295
lectularius, our study expands the host range of Sbbv1 to C. lectularius. Overall, these viruses
296
are very similar to each other with an average p-distance of 2% in the RdRp-encoding
297
transcripts.
298
3.8 Influence of Wolbachia on Viral Abundance
299
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Many hypotheses have been proposed to understand why bed bugs have never been
300
linked to pathogen transmission, including one involving their Wolbachia endosymbiont as a
301
potential factor (Pietri, 2020). Wolbachia has formed a nutritional symbiosis with bed bugs, as it
302
provides them with B-vitamins (Hosokawa et al., 2010). Other studies have shown that
303
Wolbachia colonization can confer resistance to viral infection in other insects such as
304
Drosophila, mosquitoes, and even hemipteran insects (Cogni et al., 2021; Gong et al., 2020;
305
Lindsey et al., 2018; Teixeira et al., 2008). A study investigating the influence of bed bug
306
Wolbachia on feline calicivirus titer has been conducted, but there was no evidence that the
307
virus ever replicated inside of the bed bugs (Fisher et al., 2019). To investigate the effects that
308
Wolbachia may have on viral abundance, we mapped all reads to the viral genomes detected in
309
this study and the Wolbachia endosymbiont of Cimex lectularius genome. Table 3 shows the
310
percentage of reads that mapped to the virus and Wolbachia genomes from each sample. We
311
used these values to investigate the influence of Wolbachia reads on viral reads and we found
312
no correlation (y = 0.41 - 0.042x, R = -0.14, p = 0.53) between percent Wolbachia reads and
313
percent virus reads in a sample (Figure 8).
314
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4. Discussion:
315
4.1 Detection and Phylogenetics of Bed Bug viruses
316
Bed bugs are a worldwide urban pest, that have undergone population resurgence for
317
the last 20-30 years. Although their capacity to transmit human disease remains unknown,
318
interest in their vector competence is high because of the increasing frequency in outbreaks
319
(Doggett & Lee, 2023). We did not detect any known human viruses, but our study supports
320
that metatranscriptomic surveillance is a useful technique to detect what known or emerging
321
pathogens bed bugs could potentially transmit. We detected two previously known virus
322
sequences associated with C. hemipterus, and 3 novel putative bed bug viruses.
323
The two previously detected bed bug viruses, Sbbv1 and Sbbv2 had been found in China
324
associated with the tropical bed bug, C. hemipterus (C. X. Li et al., 2015). Our phylogeny groups
325
Sbbv1 with an insect virus sister to the Hantaviridae (Figure 3). This agrees with the findings of
326
Käfer et al. (2019) and supports the hypothesis that the Hantaviridae may have originated from
327
arthropod viruses, and subsequently shifted to infecting vertebrate hosts (Marklewitz et al.,
328
2015). Although Sbbv1 shares a common ancestor with the hantaviruses, it is unknown whether
329
it is of concern to humans. We extend Sbbv1’s geographical range from China to Czechia,
330
Madagascar, and France, and extend its host range to C. lectularius, as it had previously only
331
been detected in the tropical bed bug, C. hemipterus (C. X. Li et al., 2015).
332
Sbbv2 provides an interesting insight to plant-infecting rhabdovirus evolution, as many
333
economically important plant rhabdoviruses are transmitted by hemipteran insects (Whitfield
334
et al., 2018). Our phylogeny agrees with Longdon et al. (2015), as Sbbv2 groups with insect-
335
specific clade of rhabdoviruses that shares a common ancestor with the cytorhabdoviruses and
336
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the nucleorhabdoviruses (Figure 4). This supports the hypothesis that these viruses infected
337
hemipteran insects before they infected plants (Longdon et al., 2015). It is interesting to note
338
that Sbbv2 has persisted in the bed bug lineage despite shifts in feeding strategy from plants to
339
other insects, to obligate blood feeders (Johnson et al., 2018). Our bunyavirus phylogeny
340
(Figure 3) also suggests that the tenuiviruses, which are important insect-transmitted plant
341
viruses, infected their insect hosts before evolving the ability to infect plants. This is indicated
342
by Cimex tenui-like virus 1 branching sister to the tenuiviruses, and another insect virus, Wuhan
343
horsefly virus, branching with this group (Figure 3).
344
Totiviruses are dsRNA viruses that typically infect fungi, but there is a growing number
345
of toti-like viruses detected in arthropod and vertebrate metatranscriptomic studies (Tighe et
346
al., 2022). According to our phylogenetic analysis, Cimex toti-like virus 1 falls within a clade of
347
arthropod and plant infecting viruses, along with a toti-like virus 1 detected in an anal swab
348
from a bird (GenBank: QKN88741.1) (Figure 6). Interestingly Cimex toti-like virus 1 shares a
349
most recent common ancestor with a virus detected in fleas, which are also obligate blood
350
feeders (Harvey et al., 2019). This supports the hypothesis that similarities in ecological niche
351
could be more correlative of viral similarity than taxonomic relatedness (C. X. Li et al., 2015).
352
Most other viruses in this clade are Hemiptera or Thysanoptera-associated (a sister group to
353
the Hemiptera) viruses.
354
4.2 Phylogeography of bed bug viruses
355
Although our study design limited an extensive phylogeographic analysis, we found
356
unprecedented patterns of viral diversity. First, we found that bedbug viruses detected in this
357
study are not geographically restricted and can infect more than one host species. We detected
358
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sequences from four of the five viruses in this study intercontinentally and sequences from two
359
out of five viruses were found in both C. lectularius and C. hemipterus. (Figure 7). Second, if we
360
found viruses that were present in both bed bug species, we expected these viruses to form
361
distinct clades reflecting bed bug taxonomy, as differences in host receptors between species
362
would add selective pressures to viral infection (Longdon et al., 2014). Our results did not
363
match that expectation, as the Cimex tenui-like virus 1 detected in the C. hemipterus
364
(Madagascar) samples grouped with the Cimex tenui-like virus 1 sequences from C. lectularius
365
(France and Czechia), while other Cimex tenui-like virus 1 sequences detected in C. lectularius
366
formed their own distinct clade (Rome-Italy and Assisi-Italy) (Figure 7A). Third, we expected
367
that viruses from similar geographic location would form distinct phylogenetic groups. This
368
trend was generally followed, but strikingly, in every case where viral sequences were detected
369
in samples from Italy, the sequences form their own distinct clades separated from the rest of
370
Europe (Figure 7A&B). Furthermore, even though the Cimex tenui-like virus 1 sequences from
371
Italy group together phylogenetically, there is higher mean evolutionary distance within these
372
samples than within the Cimex tenui-like virus 1 sequences from the rest of the world, which
373
include samples from France, Czechia and Madagascar, and include two different host species
374
(Tables 1 & 2). Previous studies of bed bug phylogeography have found low genetic diversity
375
within bed bug infestation sites, but high genetic diversity between infestation sites even of
376
relatively close proximity, which could be due to their dependence on humans for dispersal
377
(Fountain et al., 2014; Saenz et al., 2012). Bed bug dispersal by humans could also explain the
378
unexpected patterns of bed bug virus phylogeography, as the distinct groups of bed bug viruses
379
could be explained by Italy’s popularity as a travel destination, with Rome being a hotspot for
380
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tourism, and Assisi being a frequent site of pilgrimage. Along with this, the similarity between
381
viruses detected in Madagascar and Europe could reflect travel between these two places.
382
4.3 Wolbachia influence on viral abundance
383
As an additional exploration of our dataset, we investigated if the amount of Wolbachia
384
reads was correlated with viral abundance. It has been hypothesized that Wolbachia could have
385
a protective effect against viral infection in bed bugs, similar to what has been seen in
386
mosquitoes and Drosophila (Cogni et al., 2021; Fisher et al., 2019; Hussain et al., 2023; Lindsey
387
et al., 2018; Teixeira et al., 2008; Terradas & McGraw, 2017). We used percentage of reads
388
mapped to Wolbachia and percent mapped to the viral genomes detected in this study as a
389
proxy of abundance. We found that there was no correlation between Wolbachia and virus
390
abundance when a potential outlier sample was present (Figure 8). These results indicate that
391
unlike Diptera-associated Wolbachia, bed bug Wolbachia may not confer viral resistance.
392
Although our experimental design was not ideal to test Wolbachia’s influence on bed bug virus
393
fitness, these results provide a preliminary look into how Wolbachia may affect viruses that
394
replicate inside of bed bugs.
395
5. Conclusions
396
Our study opens interesting questions about the bed bug virosphere but does not
397
provide evidence that bed bugs transmit human viruses. On the contrary, humans may drive
398
bed bug virus diversity by facilitating dispersal and local extinction of host populations
399
(Fountain et al., 2014). Future studies should assess the pathogenicity and transmission routes
400
of these viruses to have a more comprehensive understanding of their potential in biocontrol or
401
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as emerging diseases. Along with this, a more comprehensive sampling strategy and
402
phylogeographic analysis could shed light on the interesting patterns of bed bug virus dispersal.
403
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404
Figure 1: Locations of sample collection sites. Samples were collected at nine distinct sites
405
around the world: France (Cimex lectularius, n=1), the UK (Cimex lectularius, n=3), Czechia (CR)
406
(Cimex lectularius, n=3), Rome-Italy (Cimex lectularius, n=3), Assisi-Italy (Cimex lectularius, n=3),
407
Madagascar (Cimex hemipterus, n=3), Ohio-USA (Cimex lectularius, n=3), and the King lab at
408
Mississippi State University-USA (Harlan Strain-USA Cimex lectularius, n=3).
409
410
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412
Figure 2: Percentage of reads mapped to the bed bug genomes, human genome, and
413
Wolbachia genome per sample.
414
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416
Figure 3: Phylogeny of the Bunyavirales including viruses detected in this study. Viruses found
417
in this study are labeled by their location, and their branches are colored red. Dark red dots
418
indicate bootstrap support greater than 75. Silhouette images represent the general host taxa
419
of a virus or virus clade. The tree supports that we detected Shuangao bedbug virus 1 in
420
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multiple samples, and that it branches sister to the Hantaviridae (a clade of viruses that infects
421
vertebrates) along with another insect-associated virus. Cimex tenui-like virus 1 groups within
422
the family Phenuivirudae. Notably, Cimex tenui-like virus 1 branches sister to a clade including
423
the Tenuiviridae genus (highlighted in green), which is comprised of important hemipteran-
424
transmitted plant viruses.
425
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426
Figure 4: Phylogeny of the Mononegavirales with expansive Rhabdoviridae representation.
427
Red dots indicate bootstrap support greater than 75. Sbbv2 is shown by a red branch. Non-
428
rhabdovirus mononegaviruses are shown in yellow, while rhabdoviruses are shown in blue.
429
Silhouette images represent the general host taxa of a virus or virus clade. Our phylogeny
430
supports that Sbbv2 branches with other insect-associated viruses before the
431
cytorhabdoviruses, nucleorhabdoviruses, and other plant infecting Rhabdoviridae.
432
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433
Figure 5: Phylogeny of the Tolivirales, including the ssRNA(+) virus discovered in this study.
434
A.) Full phylogeny of the Tolivirales. The red branch indicates the Cimex luteo-like virus 1. Red
435
dots along the tree show bootstrap support greater than or equal to 75. B.) Zoomed in view of
436
the clade that Cimex luteo-like virus 1 groups in. Each virus is labeled by the respective location
437
in which it was found. Dark red dots indicate bootstrap support greater than 75. Silhouette
438
images represent the general host taxa of a virus or virus clade. Cimex luteo-like virus 1 groups
439
with a clade of mosquito-associated viruses, sister to a clade of plant viruses that are putatively
440
transmitted by aphids.
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442
Figure 6: Phylogeny of the Ghrabrivirales, including the dsRNA virus discovered in this study.
443
A.) Phylogeny of the Ghrabrivirales including diverse taxa from all families. Cimex toti-like virus
444
1 is shown on red branches and the clade it groups with is highlighted in red. Dots indicate a
445
bootstrap value of 75 or above. B.) Zoomed-in view of the clade that Cimex toti-like virus 1
446
groups in. Silhouette images represent the general host taxa of a virus or virus clade.
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448
Figure 7: Phlyogeographic analysis of the putative bed bug viruses detected in this study. Only
449
the coding sequences where an RdRp domain was detected were used. All trees were rooted
450
using the closest phylogenetic neighbors from phylogenetic analyses in Figures 4-7. Dark red
451
dots indicate bootstrap support greater than 75. Samples from C. lectularius hosts are indicated
452
in blue text, and samples from C. hemipterus hosts are indicated in red text. A.)
453
Phlyogeographic analysis of Cimex tenui-like virus 1. B.) Phylogeographic analysis of Cimex toti-
454
like virus 1. C.) Phylogeographic analysis of Cimex luteo-like virus 1. D.) Phylogeographic
455
analysis of Sbbv1.
456
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458
Figure 8: Correlation of the percentage of reads mapped to Wolbachia endosymbiont of
459
Cimex lectularius and number of reads mapped to the viruses detected in this study. There is
460
no correlation between percent of Wolbachia reads present in a sample and percent of RNA
461
virus reads present in a sample.
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Table 1: Mean evolutionary distance between distinct clades of viruses from phylogeographic
463
analysis. Only Cimex tenui-like virus 1 and Cimex toti-like virus 1 are shown as they had highly
464
supported trees containing many viral genomes. Distinct phylogenetic clades were formed from
465
the Italy samples, the rest of the world, and the outgroups.
466
467
Cimex tenui-like virus 1
P-distance
Italy
Rest of the World
Outgroup
Italy
3.0%
Rest of the World
22.4%
1.1%
Outgroup
55.8%
55.9%
36.5%
Cimex toti-like virus 1
Italy
0.3%
Rest of the World
16.4%
10.0%
Outgroup
51.5%
51.8%
50.8%
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Table 2: Pairwise p-distances of Cimex tenui-like virus 1 RDRP coding sequences. The matrix includes the outgroup sequences used
469
in Figure 7A. RHBTV = Rice hoja blanca tenuivirus, RSV = Rice Stripe Virus, MSTV = Maize Stripe Tenuivirus.
470
471
Rome-
Italy 1
Assisi-
Italy 1
Assisi-
Italy 2 (1)
Assisi-
Italy 2 (2)
Assisi-
Italy 3
Czechia 3
France
Madagascar 2
RHBTV
RSV
MSTV
Rome-Italy 1
-
Assisi-Italy 1
7.5%
-
Assisi-Italy 2 (1)
7.5%
0.0%
-
Assisi-Italy 2 (2)
7.5%
0.0%
0.0%
-
Assisi-Italy 3
7.5%
0.0%
0.0%
0.0%
-
Czechia 3
23.1%
20.6%
22.6%
21.3%
22.6%
-
France
23.1%
22.6%
22.6%
20.6%
22.6%
0.0%
-
Madagascar 2
23.0%
22.6%
22.6%
21.3%
22.6%
1.7%
1.7%
-
RHBTV
56.3%
56.3%
56.3%
54.9%
56.2%
56.2%
55.3%
56.0%
-
RSV
56.7%
56.5%
56.5%
54.9%
56.5%
56.7%
56.1%
56.8%
39.5%
-
MSTV
55.1%
55.4%
55.4%
54.4%
55.4%
55.4%
54.8%
55.4%
39.3%
30.7%
-
472
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33
Table 3: Percent reads mapped to the viruses detected in this study, or Wolbachia
473
endosymbiont of Cimex lectularius.
474
Sample
% Reads
Mapped to
Viruses
% Reads mapped
to Wolbachia
Czechia 1
0.02
0.74
Czechia 2
3.88
0.35
Czechia 3
0.09
1.39
Harlan Strain-USA 1
0.00
0.40
Harlan Strain-USA 2
2.18
0.30
Harlan Strain-USA 3
0.00
0.19
France
0.34
0.43
Rome-Italy 1
0.01
0.76
Rome-Italy 2
0.00
0.41
Rome-Italy 3
0.00
0.34
Assisi-Italy 1
0.01
0.27
Assisi-Italy 2
0.09
0.75
Assisi-Italy 3
0.02
0.23
Madagascar 1
0.03
1.25
Madagascar 2
0.03
0.87
Madagascar 3
0.08
3.26
Ohio-USA 1
0.00
14.90
Ohio-USA 2
0.00
3.35
Ohio-USA 3
0.00
1.64
United Kingdom 1
0.37
3.09
United Kingdom 2
0.31
1.08
United Kingdom 3
0.13
0.99
475
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FUNDING STATEMENT:
476
This work was funded in part by a US HUD Office of Lead Hazard Control and Healthy Homes,
477
Healthy Homes Technical Studies grant (SDHHU0074-22) to JEP, and a Foundation for Food and
478
Agriculture Research New Innovator Award to JGK (534275).
479
480
ACKNOWLEDGMENTS:
481
The authors would like to thank Matthew J. Ballinger for his helpful insight and comments on
482
our manuscript.
483
484
DATA AVAILABILITY:
485
All newly reported virus genome sequences used in this study will be made available in
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GenBank, and all reads generated in this study will be deposited to NCBI’s Sequence Read
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Archive (SRA) database.
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SUPPLEMENTARY DATA:
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Supplementary data will be available online.
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CONFLICT OF INTEREST:
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The authors declare no conflict of interest
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.CC-BY 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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