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Molecular characterization of a reptarenavirus detected in a Colombian Red-Tailed Boa (Boa constrictor imperator)

Abstract

The global decline in biodiversity is a matter of great concern for members of the class Reptilia. Reptarenaviruses infect snakes, and have been linked to various clinical conditions, such as Boid Inclusion Body Disease (BIBD) in snakes belonging to the families Boidae and Pythonidae. However, there is a scarcity of information regarding reptarenaviruses found in snakes in both the United States and globally. This study aimed to contribute to the understanding of reptarenavirus diversity by molecularly characterizing a reptarenavirus detected in a Colombian Red-Tailed Boa (Boa constrictor imperator). Using a metagenomics approach, we successfully identified, and de novo assembled the whole genomic sequences of a reptarenavirus in a Colombian Red-Tailed Boa manifesting clinically relevant symptoms consistent with BIBD. The analysis showed that the Colombian Red-Tailed Boa in this study carried the University of Giessen virus (UGV-1) S or S6 (UGV/S6) segment and L genotype 7. The prevalence of the UGV/S6 genotype, in line with prior research findings, implies that this genotype may possess specific advantageous characteristics or adaptations that give it a competitive edge over other genotypes in the host population. This research underscores the importance of monitoring and characterizing viral pathogens in captive and wild snake populations. Knowledge of such viruses is crucial for the development of effective diagnostic methods, potential intervention strategies, and the conservation of vulnerable reptilian species. Additionally, our study provides valuable insights for future studies focusing on the evolutionary history, molecular epidemiology, and biological properties of reptarenaviruses in boas and other snake species.

Introduction

The global decline in biodiversity is a matter of great concern for members of the class Reptilia, comprising reptiles such as snakes, lizards, turtles, and crocodiles [1]. These diverse and ancient creatures play vital roles in ecosystems worldwide, contributing to the balance of nature and maintaining healthy habitats [2]. Boid Inclusion Body Disease (BIBD) is a potentially fatal viral disease affecting boas and pythons, characterized by the formation of eosinophilic or amphophilic intracytoplasmic inclusion bodies (IBs) within almost all cell types [3,4,5]. The existing body of literature indicates that several of the more than 100 known boa and python species are susceptible to BIBD [5]. BIBD can lead to progressive and degenerative clinical signs, including neurologic abnormalities such as star-gazing and head tremors, as well as respiratory signs like mouth breathing and increased respiratory effort [5, 6]. Unfortunately, there is currently no known curative therapy for BIBD, emphasizing the need for a deeper understanding of the underlying viral agents and their characteristics [7]. Previous studies have reported the association between reptarenaviruses and Boid Inclusion Body Disease in various snake species, highlighting the importance of further investigations [6, 8].

The family Arenaviridae currently comprises five genera, namely, Antennavirus, Hartmanivirus, Innmovirus, Mammarenavirus, and Reptarenavirus [9]. Viruses in the genus Reptarenavirus infect snakes, and some reptarenaviruses cause boid inclusion body disease (BIBD) [8,9,10].

The genome of reptarenaviruses produce enveloped virions containing bisegmented negative-sense RNA with an ambisense coding strategy [9]. The long (L) segment encodes the zinc finger matrix protein (ZP) and RNA-directed RNA polymerase (RdRp) [9]. The short (S) segment of the genome codes for the glycoprotein precursor (GPC) and the nucleoprotein (NP) [9]. For segmented RNA viruses like arenaviruses, the potential for generating novel genotypes is substantial due to their unique genetic makeup. These viruses can undergo mutation, recombination, and reassortment, processes that contribute to their genetic diversity and may ultimately lead to the emergence of a new lineage of arenaviruses [11]. Moreover, the occurrence of recombination and reassortment events in natural arenavirus infections has been definitively documented through genetic analyses. Such genetic exchanges can lead to the emergence of novel strains with altered pathogenic properties or expanded host ranges, underscoring the importance of monitoring and understanding these processes for effective surveillance and control of arenavirus infections [10].

Historically, the detection and identification of reptarenaviruses have been challenging due to the lack of specific diagnostic assays and the limited availability of complete viral genome sequences. However, recent advances in metagenomics approaches have revolutionized the field of viral discovery and characterization [12]. Metagenomics techniques allow for the identification and sequencing of viral genomes directly from clinical samples, bypassing the need for virus isolation or culture. These methods involve the extraction of total nucleic acids from the sample, followed by next-generation sequencing and bioinformatics analysis to identify and assemble viral sequences [13, 14]. Such approaches have proven to be particularly effective in uncovering novel viral agents, including reptarenaviruses, and elucidating their genetic composition [7, 15, 16].

The objective of this study was to employ a metagenomics approach to identify and molecularly characterize a reptarenavirus in a Colombian Red-Tailed Boa with BIBD symptoms. Such information is vital for development of diagnostic assays for early detection and monitoring of viral infections in snake populations. Furthermore, it provides valuable insights for the implementation of effective preventive strategies to mitigate the transmission and spread of Boid Inclusion Body Disease among snakes.

Materials and methods

Sample collection

Blood sample was collected from the tail vein of a 2-year-old female Colombian Red-Tailed Boa that was presented to the Avian and Exotics department at the University of Tennessee College of Veterinary Medicine (UTCVM). The snake was brought in due to clinical signs of whole-body twitching and excessive yawning.

Reverse transcription-polymerase chain reaction (RT-PCR) screening

The RNA was extract from the blood sample and purified using MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit (Thermo Fisher Scientific, USA). The RNA was reverse transcribed into cDNA using SuperScript™ III Reverse Transcriptase (Thermo Fisher Scientific, USA) and random hexamer oligonucleotide [10]. Diluted cDNA was used as template in RT-PCR reactions using degenerate primers targeting the glycoprotein gene, as previously reported by other researchers [10].

Library preparation and sequencing

The nucleic acid was extracted and purified using MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit (Thermo Fisher Scientific, USA). Quantity and quality of the RNA were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) and Qubit fluorometer (Fisher, Waltham, MA). Host DNA was removed by DNase treatment using heat-labile Double-Strand Specific DNase (ArcticZymes) followed by inactivation at 58 °C for 5 min.

For the first strand cDNA synthesis, the ProtoScript® II First Strand cDNA Synthesis Kit (New England Biolabs, MA, USA) was used following the manufacturer’s instructions. For the second strand cDNA synthesis, the NEBNext® Ultra™ II Directional RNA Second Strand Synthesis Module (New England Biolabs, MA, USA) was used following the manufacturer’s instructions. Sequencing libraries were prepared using the Nextera DNA Flex kit (Illumina, Inc., USA) according to the manufacturer’s instructions. Paired-end 2 × 150 bp sequencing was performed on an Illumina Miniseq in the virology and molecular diagnostic laboratory at the University of Tennessee, Knoxville TN, USA.

Sequence analysis

The sequence analysis was performed using the Sunbeam pipeline (version 3.1.1), a comprehensive and flexible bioinformatics tool for metagenomic data analysis [17]. First, the raw sequence reads obtained from the metagenomic sequencing of the reptarenavirus-infected Colombian Red-Tailed Boa were subjected to quality control using FastQC (version 0.12.1) [18] to assess read quality and detect any potential issues or biases. Adapter trimming and quality filtering were performed using Cutadapt (version 4.4) [19] and Trimmomatic (version 0.39) [20] to remove low-quality bases and adapter sequences. Sequence complexity in each read was assessed using Komplexity (version 0.3.6). Following the quality control steps, the preprocessed reads were classified taxonomically using Kraken 2 (version 2.1.3) [21]. Taxonomic classification was performed using Kraken 2 on preprocessed Illumina reads. Kraken 2 represents the most recent iteration of the Kraken taxonomic classification system. It employs precise k-mer matching to achieve rapid and accurate classification. In this process, Kraken constructs an index of all k-mers found in the reference genomes and assigns each k-mer to the least common ancestor (LCA) of all species possessing that particular k-mer. Then Kraken matches the k-mers contained in the reads to this index and eventually assigns the reads to the taxon with the most fitting k-mers by following the path from the root of the tree [21]. To construct a standard database, which includes RefSeq complete bacterial, archaeal, and viral genomes, the human genome, and a collection of known vectors (UniVec_Core), the process utilized 32 threads with default parameters. These operations were conducted on a Jetstream2 instance, specifically an m3.xl storage optimized instance with 125 gigabytes (GB) of RAM [22]. The Kraken2 result was subsequently visualized using Pavian with rank codes representing the taxonomic ranks of domain (D), kingdom (K), phylum (P), family (F), or genus (G) [23].

Then viral reads were assembled into contigs using MEGAHIT (version 1.2.9) [24]. All of the sequence analysis described in this study was conducted utilizing the Jetstream2 cloud computing resource which is supported by the National Science Foundation [22]. The assembled contigs and sequences were edited and aligned with Geneious Prime® v.2023.1.2 [25]. The open reading frame (ORF) of the viral genome was predicted using ORF finder at NCBI (https://www.ncbi.nlm.nih.gov/orffinder, accessed 15 June 2023) with default parameters. We used the Pairwise Sequence Comparison (PASC) web tool [26] (https://www.ncbi.nlm.nih.gov/sutils/pasc/viridty.cgi?textpage=overview, accessed 20 July 2023), recommended by the Arenaviridae study group of the International Committee on Taxonomy of Viruses (ICTV) for arenavirus classification [26,27,28], to analyze the identified reptarenavirus segments.

Phylogenetic analysis

L segment nucleic-acid sequences corresponding to the reptarenavirus genotypes L1 to L23 (Table 1), as well as nucleic-acid sequences corresponding to reptarenavirus genotypes S1 to S11 were retrieved from GenBank (Table 2). These sequences were then aligned with the nucleotide sequences of the reptarenavirus identified in our study using the MAFFT E-INS-i algorithm. Then, phylogenetic trees were inferred for each segment using the maximum-likelihood (ML) method implemented in MEGA11 (version 11.0.13) under the GTR + I + Γ4 nucleotide substitution model as determined by the best model finder in MEGA 11 [29]. The node supports were estimated using 1000 bootstrap replicates.

Table 1 Reptarenavirus L segment genotypes and their corresponding accessions numbers retrieved from GenBank database
Table 2 Reptarenavirus S segment genotypes and their corresponding accessions numbers retrieved from GenBank database

The amino acid sequences of the RdRp proteins associated with reptarenavirus genotypes L1 to L23, as well as the amino acid sequences of the NP and GPC proteins associated with reptarenavirus genotypes S1 to S11, were retrieved from GenBank. These sequences were then aligned with the amino acid sequences of the reptarenavirus identified in our study using the MAFFT E-INS-i algorithm. Maximum likelihood phylogenies were created using PhyML plugin for Geneious Prime® (version 2023.2.1), 100 bootstrap replicates, and otherwise default parameters.

Results

Detection of reptarenavirus RNA

In boas, antemortem BIBD diagnosis relies on the detection of inclusion bodies (IBs) in histological specimens or blood smears [5, 15, 30,31,32,33,34,35]. Nonetheless, studies have demonstrated that not all cases of reptarenavirus infection result in BIBD, with some infected snakes remaining free of intracytoplasmic inclusion bodies (IBs) and clinically healthy for an extended period, often spanning years [4, 30, 31, 33, 36,37,38]. In such cases, molecular-based diagnostic methods could offer an alternative, providing sensitive and specific tests for accurate detection [39]. As a result, we only performed reverse transcription-PCR (RT-PCR), which yielded an amplicon of the correct size, consistent with the target gene.

Taxonomic classification results using Kraken 2

Metagenomic sequencing was conducted on the clinical samples obtained from the Colombian Red-Tailed Boa. Out of the total 887 viral reads analyzed, a substantial proportion of 854 reads (96.3%) were classified as reptarenavirus Fig. 1.

Fig. 1
figure 1

Sankey diagrams of Kraken 2 report. The width of the flow is proportional to the number of reads. The number above each node is the number of k-mer hits. A rank code, indicating domain (D), phylum (P), family (F), or genus (G) was used. Out of the total 887 viral reads analyzed, a substantial proportion of 854 reads (96.3%) were classified as reptarenavirus

Open reading frame analysis and genome organization

The sequence reads obtained from our dataset were assembled into two distinct segments: a large (L) segment (7019 base pairs (bps)) and a small (S) segment (3352 bps). The complete genome sequence for L and S segments was deposited in NCBI (https://www.ncbi.nlm.nih.gov/) under accession numbers OR194165 and OR194166, respectively. Stenglein et al. [15] reported that the L and S segments of reptarenaviruses have the capacity to recombine and reassort within their host, leading to the emergence of different genotypes. Specifically, they identified S1-11 and L1-23 segment genotypes in the reptarenaviruses sequenced in the USA, indicating the genetic plasticity and evolution of these viruses within their host populations. Within genotypes, the L segment sequences showed a mean pairwise nucleotide identity of 96%, while between genotypes, the sequences shared 65% identity. For the S segments, the mean pairwise nucleotide identity within genotypes was 96%, and between genotypes, it was 64%.

We used the Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed 15 April 2023) and the Pairwise Sequence Comparison (PASC) web tool [26] (https://www.ncbi.nlm.nih.gov/sutils/pasc/viridty.cgi?textpage=overview, accessed 20 July 2023), recommended by the Arenaviridae study group of the International Committee on Taxonomy of Viruses (ICTV) for arenavirus classification [26,27,28], to analyze the identified reptarenavirus segments.

The analysis showed that the Colombian Red-Tailed Boa in this study carried the University of Giessen virus (UGV-1) S or S6 (UGV/S6) segment (BLAST, 97.88% identical to NC_039005.1 and 97.02% identical to KP071573.1) and L genotype 7 (PASC, 97.2% identical to KP071566.1; BLAST, 98.77% identical to KP071566.1). The PASC tool for genotyping the S segment was not operational at the time of writing this manuscript.

Phylogenetic analysis

The phylogenetic analysis revealed that RI2796 L segment clustered tightly with the L7 genotype sequences (Fig. 2A) while RI2796 S segment clustered tightly with the University of Giessen virus (UGV-1) or S6 (UGV/S6) genotype (Fig. 2B). The clustering of RI2796 S segment with the UGV/S6 genotype in our analysis is consistent with these earlier findings, further reinforcing the prevalence and persistence of the UGV/S6 genotype within reptarenavirus populations. Phylogenetic analysis of Reptarenavirus RdRps (Fig. 3A), NPs (Fig. 3B), and GPCs (Fig. 3C) confirm that the Colombian Red-Tailed Boa of this study appeared to carry only a single pair of L and S segments.

Fig. 2
figure 2

(A) Phylogenetic tree based on the reptarenavirus complete L segments, with ML and Bayesian methods, using the evolutionary model GTR + G + l. (B) Phylogenetic tree based on the reptarenavirus complete S segments, with ML and Bayesian methods, using the evolutionary model GTR + G + l. Evolutionary analysis was conducted in MEGA 11. Blue triangles indicate the sequences identified in this study. Numbers above branches indicate bootstrap values

Fig. 3
figure 3

Phylogenetic analysis of Reptarenavirus RdRps, NPs, and GPCs. (A) A phylogenetic tree based on the RdRp amino acid sequences of the viruses identified in this study and those available in GenBank. (B) Phylogenetic tree based on the NP amino acid sequences of the viruses identified in this study and those available in GenBank. (C) A phylogenetic tree based on the GPC amino acid sequences of the viruses identified in this study and those available in GenBank. RdRp, NP, and GPC amino acid sequences from this study are highlighted in blue. Numbers above branches indicate bootstrap values

Discussion

The first comprehensive conservation assessment of reptiles found that 21.1% of the animals were classed as Vulnerable, Endangered or Critically Endangered [40]. This is significantly more than birds, of which 13.6% are threatened [40]. Reptarenaviruses cause Boid Inclusion Body Disease (BIBD), a potentially fatal disease occurring in boas and pythons across the globe. This makes the study of reptarenaviruses all the more crucial, as BIBD poses a significant threat to these reptile species and the ecosystems they inhabit [38]. However, there is a lack of comprehensive information regarding reptarenaviruses isolated from snakes. Thus, this study aimed to contribute to the understanding of reptarenavirus diversity by applying a metagenomic approach to characterize a reptarenavirus in a Colombian Red-Tailed Boa (Boa constrictor imperator) with BIBD symptoms. Furthermore, this study underscores the importance of employing metagenomic sequencing as a diagnostic tool to detect highly divergent viruses, particularly within the Arenaviridae family.

In boas, antemortem BIBD diagnosis relies on the detection of inclusion bodies (IBs) in histological specimens or blood smears [5, 15, 30,31,32,33,34,35]. Nonetheless, studies have demonstrated that not all cases of reptarenavirus infection result in BIBD, with some infected snakes remaining free of intracytoplasmic inclusion bodies (IBs) and clinically healthy for an extended period, often spanning years [4, 30, 31, 33, 36,37,38]. To address this complexity, Thiele, Tanja et al. proposed a classification system comprising three categories: reptarenavirus infection (IB-negative), nonclinical BIBD (IB-positive without clinical disease), and clinical BIBD (IB-positive with clinical disease) [39]. Snakes with nonclinical BIBD and those serving as asymptomatic carriers of reptarenaviruses act as a hidden reservoir of infection for other animals, facilitating the introduction, spread, and persistence of these viruses within colonies. This emphasizes the critical need for the development of a precise and sensitive molecular diagnostic method to screen these clinically healthy carriers for the presence of reptarenaviruses [39].

By utilizing a metagenomics approach, we successfully identified and assembled the complete genomic sequences of a reptarenavirus isolated from a Colombian Red-Tailed Boa. Metatranscriptomic analysis revealed the presence of one reptarenavirus L segment (L7 genotype) and one reptarenavirus S segment (UGV/S6 genotype). This observation is consistent with previous studies that have consistently reported the prevalence and persistence of the UGV/S6 genotype across different reptilian populations. The dominance of the UGV/S6 genotype suggests that it possesses certain advantageous traits or adaptations, enabling it to outcompete other genotypes within the host population. Several factors may contribute to the observed dominance of the UGV/S6 genotype. Firstly, genetic factors such as mutations or genetic variations may confer a selective advantage to the UGV/S6 genotype, enabling it to replicate more efficiently or evade the host immune response effectively. Additionally, viral factors, including virulence factors or replication strategies, might play a role in the competitive advantage of the UGV/S6 genotype. Future research is needed to investigate the molecular mechanisms underlying this dominance and identify the specific genetic or viral factors involved.

The Colombian Red-Tailed Boa of this study appeared to carry only a single pair of L and S segments. The current knowledge on reptarenaviruses presents a very different scenario in which the majority of snakes with BIBD carry several L and S segments of different genotypes [7, 8, 10, 32, 33]. The identification of multiple genotypes within the same snake provides evidence for the potential coinfection and coexistence of reptarenaviruses. These coinfections may result from encounters with multiple viral strains or subsequent infections occurring in snakes already carrying one genotype or it may be due to a sampling bias [39]. The coexistence of multiple reptarenavirus subtypes in snakes highlights the complex dynamics and potential for viral interactions within reptile host populations [32, 37].

This observation also raises questions regarding the consequences of reptarenavirus coinfections in snakes. Coinfections may influence viral pathogenesis, host immune responses, and viral evolution. Further investigations are warranted to elucidate the impact of these coinfections on the health and fitness of snakes, as well as their implications for the transmission and spread of reptarenaviruses within snake populations.

Conclusion

This study represents a significant contribution to the field by identifying and characterizing a reptarenavirus in a Colombian Red-Tailed Boa. The detection of this novel virus not only enhances our knowledge of viral diversity in reptilian species but also sheds light on the evolutionary relationships within the Reptarenavirus genus. Further investigations are warranted to determine the prevalence, pathogenicity, and zoonotic potential of these viruses. Understanding their impact on both captive and wild reptile populations is of utmost importance. Moreover, future research should focus on developing diagnostic assays for early detection, implementing preventive strategies, and elucidating the mechanisms of transmission. By unraveling the mysteries surrounding these reptarenaviruses, we can better safeguard the health and conservation of reptiles, as well as mitigate potential risks to human health.

Data Availability

The complete genome sequence for L and S segments was deposited in NCBI (https://www.ncbi.nlm.nih.gov/) under accession numbers OR194165 and OR194166, respectively (BioProject accession number PRJNA982190). The corresponding raw reads from the Illumina sequencing are available in the Sequence Read Archive (SRA) under the accession number SRR24908467.

References

  1. Gibbons JW, Scott DE, Ryan TJ, Buhlmann KA, Tuberville TD, Metts BS, Greene JL, Mills T, Leiden Y, Poppy S. The Global decline of reptiles, Déjà Vu amphibians: Reptile species are declining on a global scale. Six significant threats to reptile populations are habitat loss and degradation, introduced invasive species, environmental pollution, Disease, unsustainable use, and global climate change. Bioscience. 2000;50(8):653–66.

    Article  Google Scholar 

  2. Robinson JE, Griffiths RA, John FAVS, Roberts DL. Dynamics of the global trade in live reptiles: shifting trends in production and consequences for sustainability. Biol Conserv. 2015;184:42–50.

    Article  Google Scholar 

  3. Wozniak E, McBride J, DeNardo D, Tarara R, Wong V, Osburn B. Isolation and characterization of an antigenically distinct 68-kd protein from nonviral intracytoplasmic inclusions in Boa constrictors chronically infected with the inclusion body Disease virus (IBDV: Retroviridae). Vet Pathol. 2000;37(5):449–59.

    Article  CAS  PubMed  Google Scholar 

  4. Simard J, Marschang RE, Leineweber C, Hellebuyck T. Prevalence of inclusion body Disease and associated comorbidity in captive collections of boid and pythonid snakes in Belgium. PLoS ONE. 2020;15(3):e0229667.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Chang L-W, Jacobson ER. Inclusion body Disease, a worldwide Infectious Disease of boid snakes: a review. J Exotic Pet Med. 2010;19(3):216–25.

    Article  Google Scholar 

  6. Alfaro-Alarcón A, Hetzel U, Smura T, Baggio F, Morales JA, Kipar A, Hepojoki J. Boid inclusion body Disease is also a Disease of wild Boa constrictors. Microbiol Spectr. 2022;10(5):e01705–01722.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Argenta FF, Hepojoki J, Smura T, Szirovicza L, Hammerschmitt ME, Driemeier D, Kipar A, Hetzel U. Identification of reptarenaviruses, hartmaniviruses, and a novel chuvirus in captive native Brazilian boa constrictors with boid inclusion body Disease. J Virol. 2020;94(11):e00001–00020.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Hepojoki J, Salmenperä P, Sironen T, Hetzel U, Korzyukov Y, Kipar A, Vapalahti O. Arenavirus coinfections are common in snakes with boid inclusion body Disease. J Virol. 2015;89(16):8657–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Radoshitzky SR, Buchmeier MJ, Charrel RN, Gonzalez JJ, Günther S, Hepojoki J, Kuhn JH, Lukashevich IS, Romanowski V, Salvato MS et al. ICTV Virus Taxonomy Profile: Arenaviridae 2023. J Gen Virol 2023, 104(9).

  10. Stenglein MD, Jacobson ER, Chang L-W, Sanders C, Hawkins MG, Guzman DSM, Drazenovich T, Dunker F, Kamaka EK, Fisher D. Widespread recombination, reassortment, and transmission of unbalanced compound viral genotypes in natural arenavirus Infections. PLoS Pathog. 2015;11(5):e1004900.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Emonet SF, de la Torre JC, Domingo E, Sevilla N. Arenavirus genetic diversity and its biological implications. Infect Genet Evol. 2009;9(4):417–29.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Marschang RE, Salzmann E, Pees M. Diagnostics of Infectious respiratory pathogens in reptiles. Veterinary Clinics: Exotic Animal Practice. 2021;24(2):369–95.

    Google Scholar 

  13. Mokili JL, Rohwer F, Dutilh BE. Metagenomics and future perspectives in virus discovery. Curr Opin Virol. 2012;2(1):63–77.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Barzon L, Lavezzo E, Militello V, Toppo S, Palù G. Applications of next-generation sequencing technologies to diagnostic virology. Int J Mol Sci. 2011;12(11):7861–84.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Stenglein MD, Sanders C, Kistler AL, Ruby JG, Franco JY, Reavill DR, Dunker F, DeRisi JL. Identification, characterization, and in vitro culture of highly divergent arenaviruses from boa constrictors and annulated tree boas: candidate etiological agents for snake inclusion body Disease. MBio. 2012;3(4):e00180–00112.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ihász K, Marton S, Fehér E, Bányai K, Farkas SL. Genetic characterisation of a novel reptarenavirus detected in a dead pet red-tailed boa (Boa constrictor). Acta Veterinaria Hungarica. 2022;70(1):77–82.

    Google Scholar 

  17. Clarke EL, Taylor LJ, Zhao C, Connell A, Lee JJ, Fett B, Bushman FD, Bittinger K. Sunbeam: an extensible pipeline for analyzing metagenomic sequencing experiments. Microbiome. 2019;7(1):46.

    Article  PubMed  PubMed Central  Google Scholar 

  18. BabrahamBioinformatics. FastQC. https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ 2023

  19. Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J 2011, 17.

  20. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Wood DE, Lu J, Langmead B. Improved metagenomic analysis with Kraken 2. Genome Biol. 2019;20(1):257.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Hancock DY, Fischer J, Lowe JM, Snapp-Childs W, Pierce M, Marru S, Coulter JE, Vaughn M, Beck B, Merchant N, et al. Jetstream2: accelerating cloud computing via Jetstream. Practice and experience in Advanced Research Computing. Boston, MA, USA: Association for Computing Machinery; 2021. Article 11.

    Google Scholar 

  23. Breitwieser FP, Salzberg SL. Pavian: interactive analysis of metagenomics data for microbiome studies and pathogen identification. Bioinformatics. 2020;36(4):1303–4.

    Article  CAS  PubMed  Google Scholar 

  24. Li D, Liu CM, Luo R, Sadakane K, Lam TW. MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de bruijn graph. Bioinformatics. 2015;31(10):1674–6.

    Article  CAS  PubMed  Google Scholar 

  25. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012;28(12):1647–9.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Pairwise Sequence Comparison. (PASC) web tool [https://www.ncbi.nlm.nih.gov/sutils/pasc/viridty.cgi?textpage=overview.

  27. NCBI Virus [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information. 2004 – 2023 08 08 [https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/]

  28. Radoshitzky SR, Bao Y, Buchmeier MJ, Charrel RN, Clawson AN, Clegg CS, DeRisi JL, Emonet S, Gonzalez JP, Kuhn JH, et al. Past, present, and future of arenavirus taxonomy. Arch Virol. 2015;160(7):1851–74.

    Article  CAS  PubMed  Google Scholar 

  29. Tamura K, Stecher G, Kumar S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol. 2021;38(7):3022–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Chang L, Fu D, Stenglein MD, Hernandez JA, DeRisi JL, Jacobson ER. Detection and prevalence of boid inclusion body Disease in collections of boas and pythons using immunological assays. Vet J. 2016;218:13–8.

    Article  CAS  PubMed  Google Scholar 

  31. Hetzel U, Sironen T, Laurinmäki P, Liljeroos L, Patjas A, Henttonen H, Vaheri A, Artelt A, Kipar A, Butcher SJ, et al. Isolation, identification, and characterization of novel arenaviruses, the etiological agents of boid inclusion body Disease. J Virol. 2013;87(20):10918–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Keller S, Hetzel U, Sironen T, Korzyukov Y, Vapalahti O, Kipar A, Hepojoki J. Co-infecting reptarenaviruses can be vertically transmitted in boa constrictor. PLoS Pathog. 2017;13(1):e1006179.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Windbichler K, Michalopoulou E, Palamides P, Pesch T, Jelinek C, Vapalahti O, Kipar A, Hetzel U, Hepojoki J. Antibody response in snakes with boid inclusion body Disease. PLoS ONE. 2019;14(9):e0221863.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Chang LW, Fu A, Wozniak E, Chow M, Duke DG, Green L, Kelley K, Hernandez JA, Jacobson ER. Immunohistochemical detection of a unique protein within cells of snakes having inclusion body Disease, a world-wide Disease seen in members of the families Boidae and Pythonidae. PLoS ONE. 2013;8(12):e82916.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Keilwerth M, Bühler I, Hoffmann R, Soliman H, El-Matbouli M. [Inclusion body Disease (IBD of Boids)--a haematological, histological and electron microscopical study]. Berl Munch Tierarztl Wochenschr. 2012;125(9–10):411–7.

    PubMed  Google Scholar 

  36. Aqrawi T, Stöhr AC, Knauf-Witzens T, Krengel A, Heckers KO, Marschang RE. Identification of snake arenaviruses in live boas and pythons in a zoo in Germany. Tierarztl Prax Ausg K Kleintiere Heimtiere. 2015;43(4):239–47.

    Article  CAS  PubMed  Google Scholar 

  37. Stenglein MD, Sanchez-Migallon Guzman D, Garcia VE, Layton ML, Hoon-Hanks LL, Boback SM, Keel MK, Drazenovich T, Hawkins MG, DeRisi JL. Differential Disease susceptibilities in experimentally reptarenavirus-infected boa constrictors and ball pythons. J Virol. 2017;91(15):e00451–00417.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Hetzel U, Korzyukov Y, Keller S, Szirovicza L, Pesch T, Vapalahti O, Kipar A, Hepojoki J. Experimental reptarenavirus Infection of Boa constrictor and Python regius. J Virol. 2021;95(7):e01968–01920.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Thiele T, Baggio F, Prähauser B, Subira AR, Michalopoulou E, Kipar A, Hetzel U, Hepojoki J. Reptarenavirus S segment RNA levels correlate with the Presence of inclusion bodies and the number of L segments in snakes with Reptarenavirus Infection—lessons learned from a large breeding colony. Microbiol Spectr. 2023;11(3):e05065–05022.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Cox N, Young BE, Bowles P, Fernandez M, Marin J, Rapacciuolo G, Böhm M, Brooks TM, Hedges SB, Hilton-Taylor C, et al. A global reptile assessment highlights shared conservation needs of tetrapods. Nature. 2022;605(7909):285–90.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Funding

This work used Bridges-2 at Pittsburgh Supercomputing Center and Jetstream2 cloud computing resource through allocation AGR220002 from the Advanced Cyberinfrastructure Coordination Ecosystem: Services & Support (ACCESS) program, which is supported by National Science Foundation grants #2138259, #2138286, #2138307, #2137603, and #2138296.

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Conceived, designed, and performed experiments: MAA, AR. Analyzed the data: MAA, OKE. Participated in writing of the manuscript: MAA, OKE. All authors read and approved the final manuscript.

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Correspondence to Mohamed A. Abouelkhair.

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This study did not include experiments involving live animals. The study was exempt from review by the Institutional Animal Care and Use Committee (IACUC) as it solely utilized surplus material from clinical sample that was originally submitted for diagnostic purposes with the client consent. This surplus material was utilized for metagenomic sequencing in order to obtain the necessary data for this study.

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The authors declare no competing interests.

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Abouelkhair, M.A., Roozitalab, A. & Elsakhawy, O.K. Molecular characterization of a reptarenavirus detected in a Colombian Red-Tailed Boa (Boa constrictor imperator). Virol J 20, 265 (2023). https://doi.org/10.1186/s12985-023-02237-2

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