The study of the influence of the Epstein–Barr virus (EBV) on the development of colorectal cancer is of current interest, particularly in light of the active discussion of the participation of this virus in the carcinogenesis of stomach cancer. In this review, aimed at a fundamental understanding of the processes associated with the impact of EBV on the human body, attention is paid to the issues of the life cycle of the virus, its phases (latent and lytic), as well as proteins that may be detected in each of the phases. The papers reporting on the role of EBV in the development of colorectal cancer have been analyzed. A summary table indicating the population under study, the number of samples, the method, and the result obtained is provided. Given that the primary cells affected by EBV are lymphocytes, it is logical to assume the involvement of this virus in the development of inflammatory bowel diseases. The review cites studies which confirm the presence of virus DNA in tissues in the inflammatory diseases of the colon, including microscopic and ulcerative colitis. To confirm the direct impact of EBV on the development of colorectal cancer, large studies with applying various methods for detecting the virus and the mandatory description of its localization are required. Besides, it is necessary to correlate these data with the clinical and morphological characteristics of EBV. The Epstein–Barr virus (EBV) is the first detected and one of the most common oncoviruses which accounts for approximately 10–15% of all malignant neoplasms. It belongs to the group of gamma viruses and is ubiquitous in the adult population. The virus infects more than 90% of the world’s population by the age of 35, most being asymptomatic. The main pathway for its transmission is through the saliva .–81.78–81in situ hybridization, EBER cells were detected in 28 out of 58 biopsy specimens . The relationship between the presence of EBV and atypical lymphoplasmacytic infiltrate was noted in the biopsy specimens (p<0.001). However, it remains unclear what criteria were used to consider the infiltrate as atypical (the authors describe it as a disorganized infiltrate with large B lymphocytes). There is no information about where exactly the virus was found — in the infiltrate or in the cells of the intestinal epithelium, either.Thus, in a study by Nissen et al.  performeEarlier studies , 81 notein situ hybridization, EBER positive cells were identified (mainly B lymphocytes and histiocytes) in 18/30 (60.0%) cases of microscopic colitis, in 3/30 (10.0%) cases of ulcerative colitis. At the same time, the virus DNA was detected in none of the cases of irritable bowel syndrome (n=30), which is not a true inflammatory process. The authors found that EBV infection is almost always present in patients with microscopic colitis, the pathogenesis of which has not yet been established.The study by Rizzo et al.  presentsThe research data –81 confiTo date, there is no definite answer to the question about the direct effect of the EBV on the carcinogenesis of colorectal cancer. Until now, there is no generally accepted method for detecting the virus in tumor tissues of the colon, which leads to the incomparability of the results and the impossibility of forming a single basis. Most of the studies lack information about the location of the detected virus , which is fundamentally important for assessing the stage of development of the EBV, on the one hand, and establishing its prognostic role, on the other hand. In addition, it is necessary to perform correlation analysis with clinical and morphological characteristics. Special attention should be given to the study of the role of the EBV in precancerous processes of the colon and inflammatory diseases since the virus has been identified in microscopic and ulcerative colitis.


Obtaining an understanding of the mechanism underlying the interrelations between the structure and function of HIV-1 is of pivotal importance. In previous decades, this mechanism was addressed extensively in a variety of studies using conventional approaches. More recently, atomic force microscopy, which is a relatively new technique with unique capabilities, has been utilized to study HIV-1 biology. Atomic force microscopy can generate high-resolution images at the nanometer-scale and analyze the mechanical properties of individual HIV-1 virions, virus components , and infected live cells under near-physiological environments. This review describes the working principles and various imaging and analysis modes of atomic force microscopy, and elaborates on its distinctive contributions to HIV-1 research in areas such as mechanobiology and the physics of infection. Structural virology has become a field of increasing interest in recent years with the development of tools and techniques that enable researchers to study viruses as solid-state objects from a physical perspective. Investigating viruses and their infection mechanisms from this viewpoint opens an important avenue of research to study the mechanical properties of viruses, such as stiffness, elasticity, plasticity, and material fatigue. The use of traditional techniques, such as conventional electron microscopy, cryo-electron microscopy, X-ray crystallography, nuclear magnetic resonance spectroscopy, fluorescence, and circular dichroism have enabled researchers to determine the structure of viruses and other nano-molecules over the years. However, recent advances in physical and mechanical mapping techniques enable new light to be shed on virus structure and replication mechanisms. Extensive studies focused on the mechanical properties of enveloped and non-enveloped viruses have been summarized in several reviews. In the current review, we provide an overview of the physics and mechanics underlying HIV-1 infection.Advances in scanning probe microscopy were initially obtained from the development of scanning tunnelling microscopy by Binnig and Rohrer in the early 1980s . This teAn atomic force microscope (AFM) can be used to construct topographic images  or to analyze surface forces per se (using its force mode), with both the quantitative and force modes relying on the production of force-versus-distance curves. In the contact imaging mode, the AFM probe scans the surface in a zig-zag pattern, thereby generating relatively high lateral (shear) forces on the sample. These forces may eventually damage either the sample or the AFM tip , thus, tOne of the main advantages of atomic force microscopy as a high-resolution imaging technique is its ability to analyze non-fixed samples in fluid environments; thus, it is capable of measuring biological samples under near-physiological conditions. However, samples must be adhered or immobilized to a supporting surface. Thus, much care must be taken when immobilizing a soft and delicate biological sample so that it is attached sufficiently strongly to the substrate to allow successful scanning without impairing its native morphology and structure. Depending on the sample, a wide range of substrates ) can be used to adhere the biological samples to the substrate.Atomic force microscopy is finding increasing applications in the study of the mechanical and physical properties of biological samples (reviewed in ). The abIn virology, atomic force microscopy has been widely used to image single virus structure as well as individual virus components. In parallel, it has been used to study the mechanical properties of virus particles as well as interaction forces between viruses and their physical environment. In bacteriophages, atomic force microscopy studies have revealed the unique properties of the viral shell of bacteriophages , in which nucleic acid is tightly packed prior to being injected under extremely high-pressure into a bacterial cell during infection. Bacteriophage capsids are relatively stiff to protect the viral genome but also brittle, which makes them susceptible to failure. Interestingly, bacteriophages overcome the brittleness problem during capsid maturation by initially forming thin-walled capsids while maintaining intermolecular integrity . SimilarThe fine balance of mechanical properties exhibited by viral particles during the course of infection makes the field of physical virology an interesting area to explore. Since viruses and virus capsids are biologically soft in nature, it becomes imperative to understand the mechanics governing the physical properties of these small entities, especially during infection, which involves overcoming several harsh physical and chemical barriers. Extensive studies focused on the mechanical properties of enveloped and non-enveloped viruses have been summarized in several reviews ,10,17,18HIV-1 is an enveloped lentivirus studded with viral gp120/gp41 trimers. The lipid bilayer acquired during budding has a relatively low copy number of envelope protein moieties ,20. UndeHIV-1 infection begins with binding of the virus to target cells via interaction between the viral gp120 envelope protein and the cellular CD4 receptor and co-receptor CCR5 or CXCR4. This binding results in fusion between the viral and cell membranes, which permits entry of the viral core into the target cell. The viral core must travel through the viscous and densely packed cytoplasm of the target cell to the cell nucleus and dock at the narrow nuclear pore ,26,27,28The various stages of the HIV-1 life cycle have been extensively studied in detail using multiple biochemical and fluorescence techniques. From a biophysics perspective, the virus particles must overcome mechanical forces during both entry and exit from target cells. There are multiple points in the complete process at which they are vulnerable to loss of structural integrity and failure. In the proceeding sections, we will describe studies that utilized atomic force microscopy to provided new insights into the fundamental biophysical mechanism at the following mechanical checkpoints in the HIV-1 life cycle: viral entry, maturation, uncoating, and budding.Unlike other viral genomes ,30,31,32The mature and immature states of the virus have different morphologies, with the thickness of the protein inner shell significantly greater in the immature state . In an iOver the years, three models have been proposed for virus uncoating: (1) immediately upon entry into the cytoplasm ,43,44; , which has been shown to stabilize virus cores by interacting with capsid hexamers and pentamers . ApproacMechanical studies on IP6-treated virus cores showed that their initial stiffness is elevated to a value similar to the E45A hyperstable mutant. However, cores undergo complete disassembly after a relatively short time (~7 h) of reverse transcription without displaying the characteristic stiffness peak . Time-laThe above in vitro experiments provide insight into the material properties of virus cores, but it is important to understand how these attributes are relevant and what role they play during the actual infection process in vivo. The increase in stiffness can be attributed to the presence of rigid nucleic acid within the capsid, but could also be due to the generation of free pyrophosphates (PPi) that phosphorylate the inner wall of multiple virus capsids, causing minor structural changes ,71,72,73In HIV-1, budding occurs near the plasma membrane where the virus assembles all the necessary viral components and buds out while acquiring a part of the plasma membrane. The primary driving force for budding is the Gag protein, which regulates all the activities for assembly and egress of the virus. Although electron microscopy can generate images of viral budding at excellent resolutions, it is not possible to obtain real-time kinetics data using this methodology. Since HIV-1 budding occurs near the cell membrane ,75, it cIn an initial study, individual MLV budding events were monitored using an AFM  Figure . The accSeveral studies on equine infectious anemia virus (EIAV), vaccinia virus, MLV, and HIV, show the participation of actin in remodeling the cytoskeleton during viral budding ,82,83,84Analysis of the relatively fast kinetics of HIV-1 budding using standard atomic force microscopy revealed that its poor temporal resolution constitutes a weakness of this method. With the recent development of fast scanning AFMs capable of imaging soft biological samples with a temporal resolution of a few seconds, detailed characterization of HIV-1 budding has become possible.After more than three decades of development in the area of near-field microscopy, atomic force microscopy has emerged as a powerful and significant tool to image and study the mechanical properties of a wide range of biological systems. By combining force microscopy and spectroscopy, atomic force microscopy opens a doorway to characterizing important biological processes from the novel perspective of biophysics and mechanobiology. Atomic force microscopy grants key insights into the mechanical properties governing fundamental biological processes and how their physical characteristics bestow different functional advantages on small molecular entities, such as protein clusters or viruses, at different time points in their development.As elucidated by this review, atomic force microscopy provides uniquely relevant images and physical information concerning the mechanobiological properties of biological systems. Complementing traditional techniques, such as electron microscopy, X-ray crystallography, and mass-spectroscopy, atomic force microscopy greatly furthers our understanding of critical biological processes by examining their underlying physics.


N-glycans, in particular complex glycans, high-mannose glycans, and hybrid glycans, together with some O-glycans, participate in the glycan shield of the Ebola virus, influenza virus, human cytomegalovirus, herpes virus, human immunodeficiency virus, Lassa virus, and MERS-CoV, SARS-CoV, and SARS-CoV-2, which are responsible for respiratory syndromes. The glycans are linked to glycoproteins that occur as metastable prefusion glycoproteins on the surface of infectious virions such as gp120 of HIV, hemagglutinin of influenza, or spike proteins of beta-coronaviruses. Plant lectins with different carbohydrate-binding specificities and, especially, mannose-specific lectins from the Vicieae tribe, such as pea lectin and lentil lectin, can be used as glycan probes for targeting the glycan shield because of their specific interaction with the α1,6-fucosylated core Man3GlcNAc2, which predominantly occurs in complex and hybrid glycans. Other plant lectins with Neu5Ac specificity or GalNAc/T/Tn specificity can also serve as potential glycan probes for the often sialylated complex glycans and truncated O-glycans, respectively, which are abundantly distributed in the glycan shield of enveloped viruses. The biomedical and therapeutical potential of plant lectins as antiviral drugs is discussed.Pathogenic enveloped viruses are covered with a glycan shield that provides a dual function: the glycan structures contribute to virus protection as well as host cell recognition. The three classical types of  Many pathogenic viruses for humans are so-called enveloped viruses with a lipid bilayer that allows the infectious virions to fuse with the cell membrane, followed by the entry and replication of the viral genetic material into the host cells. Ebola virus (EBOV), influenza virus (IV), herpes simplex virus (HSV), human immunodeficiency virus (HIV), human cytomegalovirus (HCMV), Lassa virus (LASV), and the beta-coronaviruses responsible for the Middle East respiratory syndrome (MERS-CoV) and the severe acute respiratory syndrome  belong to this group of pathogenic enveloped viruses . Among tN- and O-glycans decorate the fusion glycoproteins, the three classical types of N-glycans, including complex-type glycans, high-mannose-type glycans, and hybrid-type glycans, are predominantly distributed along the fusion proteins of pathogenic enveloped viruses. In addition, a high proportion of complex glycans are α1,6-fucosylated on the first GlcNAc linked to the Asn residue and often sialylated on their terminal Gal residues ..14].N-glycan shield of enveloped viruses. Two-chain legume lectins form a particular group of Man-specific lectins that display an enhanced affinity for complex N-glycans possessing an α1,6-fucosylated trimannoside core Man3GlcNAc2 [Pisum sativum) (PsA), lentil (Lens culinaris) (LcA), Cyprus-vetch (Lathyrus ochrus) (LoL-I/II), and faba bean (Vicia faba) (VfA) belong to this group of two-chain lectins [Canavalia ensiformis) [Lathyrus ochrus isolectin-II (LoL-II) in complex with an octasaccharide derived from the human lactotransferrin (PDB code 1LGC) [Lectins from higher plants offer extreme diversity in terms of structural organization and recognition of simple and complex glycans . Owing t3GlcNAc2 ,33. Seed lectins ,35,36,37iformis) . A detaide 1LGC)  revealedLathyrus ochrus lectin with non-fucosylated trisaccharides, indicated that Vicieae lectins also interact with one of the terminal Man of the trimannosyl core from the non-fucosylated N-glycans [L. ochrus seeds interacted with the trisaccharide Manα1,3Manβ1,4GlcNAc in such a way that the terminal Man residue occupies the monosaccharide-binding site of the lectin ) for PsA, LcA, and VfA, all members of the two-chain lectins from the Vicieae tribe, yielded the best results with glycans possessing the a1,6-fucosylated Man3GlcNAc2 core. As an example, most of the top five glycans displaying the best affinity for PsA, LcA, and VfA occur in the glycan shield covering the pathogenic enveloped viruses   bark lectin I (SNA-I) specifically interacts with these sialylated termini. In this respect, the top five glycans interacting with SNA-I in glycan array experiments contained sialylated Gal residues that occur in the glycan shield of enveloped viruses , and Morniga-G from Morus nigra, should especially interact with the few O-glycans exposed at the surface of beta-coronaviruses [O-glycans interacting with Morniga-G in glycan array experiments are present in the glycan shield of the SARS-CoV-2 particles , which essentially recognize high-mannose glycans, have been identified as potential CBAs for pathogenic enveloped viruses . In thishsia sp. , was invhsia sp.  because hsia sp.  , Epipactis helleborine (EHA), Hippeastrum hybrid (HHA), Galanthus nivalis (GNA), Listera ovata (LOA), and Narcissus pseudonarcissus (NPA), have been widely investigated in this respect by Balzarini and co-workers [Musa acuminata [Canavalia ensiformis [n-specific lectins Nictaba from Nicotiana tabacum [Urtica dioica [Triticum aestivum [+ T lymphocytes [Experimental studies performed in vitro, especially on HIV-infected cultured cells, have focused on higher plant lectins and cyanobacterial lectins . Mannose-workers ,54. Othecuminata  and Con siformis  or diffe tabacum , UDA froa dioica , and WGAaestivum , were alphocytes ,100.+ T lymphocytes [N- and O-glycans by lectins.A rather different situation can occur under in vivo conditions due to the multiplicity of cells susceptible to interacting with the virus. In addition, to block the entry of HIV particles into the cells and syncytium formation, plant lectins interfere with other mechanisms of virus infection and transmission, for example, by preventing the recognition of high-mannose glycans of gp120 by the DC-SIGN receptor of dendritic cells ,102,103 phocytes , interacThe effects of plant lectins on other pathogenic enveloped viruses have been more scarcely investigated. Plant lectins were identified as blocking agents for the entry of IV ,52,57, HGriffithsia sp., also inhibited the entry of SARS-CoV and MERS-CoV, respectively, in virus-infected cultured cells [Maackia amurensis lectin, MAL, inhibited the interaction of the SARS-CoV-2 S protein with the ACE2 receptor in cell cultures and decreased the expression of inflammatory mediators associated with COVID-19 disease progression [The effects resulting from the binding of plant lectins on the different enveloped viruses also depend on the mechanisms of infection and transmission of these viruses, which may differ from those developed by HIV. In addition to preventing the entry and replication of Ebola viruses into the host cells in cell cultures, the engineered BanLec lectin, pre-administered to virus-infected mice, were highly protective against a lethal EBOV infection in vivo (~80% of mice protected) . The enged cells ,74. The gression .n-specific lectin from the stinging nettle (Urtica dioica) (UDA), was shown to prevent virus entry and replication in a dose-dependent manner and reduced the virus infectivity significantly in a lethal SARS-CoV BALB/c mouse model [Lallab purpureus), FRIL, neutralized H1N1 influenza by aggregating and trapping virions in the late endosomes of the host cells, thus preventing their nuclear internalization [The effects of plant lectins on coronaviruses were also investigated in virus-infected mice models. The (GlcNAc)se model . Under ilization . The lecAlgal and cyanobacterial Man-specific lectins, such as griffithsin and cyanovirin-N, also inhibited the entry of HIV and other enveloped viruses in the host cells in vitro and exerted in vivo cytotoxic effects very similar to those of plant lectins ,106,107.Depending on their affinity towards surface-exposed glycans of enveloped viruses, plant lectins are considered as potential CBAs useful for combating viral infections, even though little evidence exists to date for their efficacy as relevant therapeutic tools ,111,112.Griffithsia sp., for the purpose of obtaining a large-scale production of the lectin able to supply the quantities of lectins needed for therapeutic applications [Nicotiana benthamiana) leaves. In addition, griffithsin is easily purified and recovered from ensiled dried tobacco leaves, which allows for a low-cost production of lectin quickly adaptable to demand.In most higher plants, lectins of different specificities that could be used as virus blockers occur as storage proteins in seeds and other vegetative organs such as tubers and rhizomes . Man-speications ,120,121.Lathyrus ochrus lectins, were easily prepared [Most plant lectins consist of oligomeric structures built up from the non-covalent association of 15–20 kDa monomers in dimers and tetramers, more rarely in hexamers or octamers . Dependiprepared ,125. Eveprepared ,127. Thrprepared ,129. In prepared .Initially recognized as potent mitogenic proteins ,132,133,ex-vivo plasmapheresis on a Man-specific lectin-immobilized column of blood spoiled by the MERS-CoV and the Marburg viruses has proven its efficacity to purge the blood samples from virus particles [In spite of these limitations hampering the use of plant lectins as CBAs for combating pathogenic enveloped viruses, some ex vivo applications of plant lectins have been successfully developed. An important decrease in the plasma load with Ebola virus was achieved by extracorporeal affinity plasmapheresis of the contaminated blood through a GNA-immobilized matrix . Recentlarticles . Althougarticles ,150. AnoAtomic coordinates of fusion proteins, B glycoproteins, and E glycoproteins were taken from the Protein Data Bank (PDB): 7JPH (EBOV) , 6Y5G  (accessed on 22 December 2021) [The molecular surface of the lectins and envelope glycoproteins from pathogenic enveloped viruses were calculated and displayed with Chimera  and Chimer 2021)  and reprN-glycans, complex N-glycans, hybrid N-glycans, and O-glycans were built and represented with the DrawGlycan SNFG package for Mac [The illustrations of the high-mannose  for Mac . ColoredN-glycan types covering enveloped viruses, especially beta-coronaviruses [3GlcNAc2 core of N-glycans of the complex- and hybrid-type, are particularly relevant as glycan probes for the beta-coronaviruses MERS-CoV, SARS-CoV, and SARS-CoV-2 [The glycan shield covering pathogenic enveloped viruses plays a role not only in the protection of viruses but also in various important mechanisms insuring the entry and replication of viruses in the host cells . Thus, taviruses , plant lRS-CoV-2 ,106. HowDespite the accumulating evidences that plant lectins and, especially, Man-specific plant lectins, could be used as tools for preventing infection by pathogenic enveloped virus, in particular SARS-CoV-2 responsible for the COVID-19 pandemic, some unwanted characteristics of plant lectins make these molecules difficult to use for a therapeutic purpose. Due to the fact of their high molecular size, which favors the synthesis of anti-lectin antibodies, and their mitogenic/cytotoxic properties, which interfere with the cytokine response of infected individuals, their use is limited to external treatments. However, the promising results obtained with a single-mutated engineered banana lectin, BanLec, which retains its carbohydrate-binding ability but loses its mitogenic property ,47, coulN-glycan types that are linked to the receptor DPP4 for MERS-CoV and SARS-CoV viruses [In addition to lectins, other small molecules could be used as blockers for the SARS-CoV-2/ACE2 interaction. Recently, some small molecular drugs, including dyes, glycosides, tannins, and immunosuppressors, were characterized as either spike or ACE2 binders susceptible to blocking the attachment of the SARS-CoV-2 spikes to ACE2 by interfering with the ligand and/or the receptor surface . Dependi viruses  and ACE2 viruses , plant l


Praomys sp. rodent in the Central African Republic in 1981 and assigned to Ippy virus based on antigenic similarity. The strain was simultaneously sequenced on Illumina NovaSeq 6000 and MinION Mk1B devices and analysed with various bioinformatics tools. We show that the best genome coverage and depth were obtained with the Kaiju and Minimap2 classification and identification tools, on either the MinION or the Illumina reads. The genetic analysis of AnRB3214 fragments showed 68% to 79% similarity with the Mobala and Gairo mammarenaviruses at the nucleic acid level. Strain AnRB3214 had a truncated nucleoprotein smaller than that of other Old World arenaviruses. Molecular clock analysis suggests that this strain diverged from Mobala virus at least 400 years ago. Finally, this study illustrates the importance of genomics in the identification of archived viruses and expands on the diversity of African arenaviruses, because strain AnRB3214 is either a variant or a close relative of Mobala virus, and not Ippy virus.Arenaviruses represent a family of viruses that are naturally present in rodents belonging to subfamily Murinae, Neotominae or Sigmodontinae. Except for Lassa virus, little information is available on other Old-World arenaviruses. Here, we describe strain AnRB3214, a virus isolated from a presumed  Arenaviridae, order Bunyavirales. With approximately 43 species, arenaviruses are subdivided into four genera  that infect fishes, snakes or mammals1. Arenaviruses typically have ambisense, bisegmented RNA genomes. Each RNA segment encodes two non-overlapping open reading frames (ORFs) of opposite polarity. The small (S) 3500 nt genomic segment encodes the nucleocapsid protein (NP) and a glycoprotein precursor (GPC), which later matures into two membrane glycoproteins, GP1 and GP2. The large (L) ~ 7000 nt genomic segment codes for the RNA-dependent RNA polymerase (RdRp) and exceptionally a zinc-binding protein (Z) in the Mammarenavirus and Reptarenavirus genera3. Interestingly, Antennavirus has a trisegmented RNA genome, with NP and GPC being on two distinct segments4.Arenaviruses are enveloped, single-stranded RNA viruses belonging to family 5. The natural reservoirs of mammarenaviruses are generally rodent subfamilies Murinae, Neotominae and Sigmodontinae, in which they establish persistent infections with no overt pathology for the animal host7. These viruses seem to be associated with specific rodent host species, although many host-switching events have been recorded, which implies that virus-host co-evolution may not be as tightly linked as previously thought9. Most mammalian arenaviruses are non-pathogenic to humans, but seven are recognized as haemorrhagic fever viruses including the Lassa and Lujo viruses in Africa and the Sabia, Machupo, Chapare, Junin and Guaranito viruses in Latin America11. LCMV, which has a worldwide distribution, can cause neurological disease in immune-compromised individuals12. Except for Lassa virus, knowledge of OW arenavirus diversity, evolution and involvement in human diseases is sparse. Several arenaviruses of unknown pathogenic potential have been described in African rodents such as Gbagroube, Kodoko and Menekre viruses in West Africa and Mopeia, Morogoro, Luna, Merino Walk, Mariental and Okahandja viruses in southern Africa2. In Central Africa, wild and domestic rodents act as reservoirs of numerous parechoviruses, rhabdoviruses, phleboviruses, flaviviruses, picornaviruses, astroviruses, paramyxoviruses, orthopoxviruses and arenaviruses, some of which are directly responsible for human diseases, including monkeypox, Mokola virus infection or lymphocytic choriomenigitis15.Mammarenaviruses are geographically segregated into two monophyletic groups based on genomic features and antigenic properties. The New World (NW) arenaviruses, represented by the Tacaribe virus complex, are prevalent in North and Latin America; the Old World (OW) arenaviruses, represented by the Lassa-Lymphocytic choriomeningitis virus (LCMV) complex, are found in AfricaMus, Praomys, Mastomys and Arvicanthis rodents in Gabon, Cameroon and the Central African Republic (CAR)18. Seroconversion has been highlighted in the CAR for Mobala virus infection in humans in locations where the virus has been isolated from Praomys rodents19. Interestingly, seroprevalence in humans seems to reflect virus isolation in its reservoir host. Indeed, for a prevalence of 7.7% and 3.2% in Praomys sp. rodents in the Bouboui and Gomoka villages, respectively, using the indirect fluorescent antibody test (IFAT), the prevalence in humans ranges from 3.1 to 3.8%; however, in Botambi, with a prevalence of 2.2% in the reservoir, no antibodies have been detected in the human population19. Seroconversion in humans for Souris and Ippy viruses remains to be determined.Few arenaviruses are known in Central African rodents; however, LCMV, Souris, Mobala and Ippy viruses have been described in 22. The leading NGS technologies, among which Illumina, are based on the incorporation of terminating nucleotides in a DNA polymerization process. These technologies, which produce very accurate and high-quality results, are widely used. However, they are usually cumbersome and only available in well-equipped, specialized laboratory facilities that can provide all the necessary utilities, such as a stable electric supply and internet connection. On the other hand, MinION uses a different approach in which a specific nanopore protein is used to recognize a k-mer in the sequence of interest via a disturbance in the basal electrical current. This innovation led to the miniaturization of the sequencing device and the ability to perform real-time sequence analysis24. This portable tool is gaining interest because it brings more flexibility to sequencing, albeit with a high error rate, and can, therefore, be used in remote regions as a diagnosis tool or in response to outbreaks as seen in the recent Lassa, Ebola and Zika epidemics29. Despite its shortcomings, MinION sequencers can be implemented in resource-limited settings such as the CAR as a first-line tool to respond instantly to emergencies or to analyse infectious samples subject to transportation restrictions.Next-generation sequencing (NGS) platforms have been a breakthrough in many scientific fields. In virology, NGS has facilitated a better understanding of the diversity and evolutionary history of viruses, the discovery of new viruses and the rapid response to outbreaks through diagnosis of the causative agentsPraomys rodents in the CAR in 1981. The strain was sequenced using the MinION Mk1B  and Illumina NovaSeq 6000  devices and analysed with various bioinformatics pipelines.Here, we describe a divergent arenavirus, the AnRB3214 strain isolated from wild Praomys sp. rodent caught near Botambi, Ombella-M’poko prefecture, CAR in October 1981 . Only mice that died after a 7-day follow-up were harvested and their brains were collected, crushed and suspended in a 1X PBS solution, and then filtered. This filtered brain suspension was used to infect fresh confluent Vero E6 cells culture in Dulbecco’s Modified Eagle’s Medium  supplemented with 2% Foetal Calf Serum  and a 1% v/v antimicrobial solution . The cell cultures were monitored for 7 days for cytopathic effects using an inverted optical microscope. The brain suspensions and cell culture supernatant were then stored at -80 °C until further use.Total RNA was extracted from the culture supernatant using the QIAamp Viral RNA Mini kit  according to the manufacturer's protocol. These RNAs were then quantified using the Qubit RNA HS Assay kit  and stored at -80 °C.The first cDNA strand was prepared using SuperScript III reverse transcriptase and random hexamers  on total RNA. The second cDNA strand was then prepared using the NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module kit  according to the manufacturer's instructions. All final repair and adapter ligation steps were based on the use of the NEBNext Ultra II RNA Library Prep Kit for Illumina . All DNA purification steps were performed using Agencourt AMPure XP beads . The library quality was checked on the 2100 BioAnalyzer instrument  and then sequenced on Illumina NovaSeq 6000 sequencer  to obtain 2 × 150 bp paired-end reads. In parallel, the double-stranded cDNA obtained after the end repair step was used to prepare the MinION library with the PCR Barcoding kit (SQK-PBK004)  according to the manufacturer's instructions. The library was multiplexed with five other samples and loaded on a R9.4 flow cell (FLO-MIN106) and sequenced on the MinION Mk1B device in 37 h. The raw sequencing data were collected using ONT MinKNOW software (version 19.05.0).30. All the reads that have been mapped were grouped in a dataset referred to as "AnRB-3214-MinION". From this dataset, three sub-datasets were constituted in the same way as previously. Viral diversity was determined from the two above-described datasets (AnRB-3214-MinION and AnRB-3214-Illumina) and their three sub-datasets using three different taxonomic classification tools: Kaiju31, Centrifuge32, and Kraken233. These tools use reference databases in which the sequence of our AnRB-3214 strain was absent. In parallel with the use of these three taxonomic classification tools, two other traditional approaches were used for the identification of viral reads. The first approach consisted in a homology search from reference sequences using the BLASTN tool whereas the second approach was to map the reads on other reference sequences using Minimap2. To use these approaches, the genomes of viruses closest to our AnRB3214 strain were screened for in GenBank and identified as Mobala virus  and Gairo virus . Then, we screened the AnRB-3214-MinION and AnRB-3214-Illumina datasets against these Mobala and Gairo virus sequences using the BLASTN (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and Minimap2 tools. Hits produced by both methods were reformatted in fastq files and remapped to obtain bam files for both BLASTN and Minimap2. Bam files were converted into consensus sequences by applying BCFtools on the results of SAMtools mpileup.The quality of the raw reads from the Illumina sequencing was first assessed, filtered, and then trimmed using CLC Genomics workbench v10.0.1 . All trimmed reads were then de novo assembled using SPAdes v3.10 to obtain two contigs corresponding to the S and L segments of an arenavirus genome. All reads were mapped back on the two contigs previously obtained using Geneious Prime . All the reads from the Illumina sequencing that have been mapped were grouped together in a constituted dataset referred to as "AnRB-3214-Illumina". Three subsets, derived from the AnRB-3214-Illumina dataset, were constituted by collecting random reads from the raw fastq files in such a way that the first dataset represented 10% of the initial reads, the second 1%, and the last 0.1%. In parallel, the raw data obtained from MinION sequencing were acquired in real-time in files containing 4000 sequences in FAST5 format. These files were base-called and demultiplexed using Guppy (version 3.4.1). As carried out for the Illumina raw reads, the MinION raw reads were mapped on the obtained sequences of the two segments using Minimap234 and manually edited. Then, phylogenetic trees were inferred for each segment using the maximum-likelihood (ML) method implemented in MEGA X (version 10.1.1) under the GTR + I + Γ4 nucleotide substitution model as determined by the best model finder in MEGA X35. The node supports were estimated using 1000 bootstrap replicates.S and L segment nucleic-acid sequences as well as the coding sequences of the NP, GPC, RdRp and Z genes were aligned using ClustalO in Unipro Ugene version 34.036 and manually edited with AliView37. Starting from this alignment, ML and Bayesian analyses were conducted. A global ML evolutionary tree was reconstructed using IQ-TREE38 according to a GTR + F + I + Γ4 substitution model, recommended by the “ModelFinder” model fitter included IQ-TREE. Branch robustness was assessed using the ultrafast bootstrap approximation with 1000 bootstrap replicates39. The resulting ML tree was visualized and edited in FigTree (https://github.com/rambaut/figtree/releases/tag/v1.4.4). TempEst v1.5 was used for investigating the temporal signal of our molecular phylogeny40. Analysis of the extreme values of residuals  permitted us to spot problematic sequences. Those genomes were discarded from the following steps. BEAST v1.10.4 was used for Bayesian Monte Carlo Markov chain (MCMC) analysis to estimate the divergence time of nodes41. We retained the simplest model: strict clock and constant population size with the HKY substitution model and four gamma categories. BEAST runs were completed checking chain convergence and sufficient sampling of the posterior space (ESS > 200). The final chronogram was generated using TreeAnnotator v1.10.441.Concurrently, a molecular clock was tested on concatenated S and L segments using Bayesian inference. S + L concatenates of genomic sequences were aligned using MAFFT v7The study was approved by the institutional committee of the Institute Pasteur of Bangui. For the experiments realised out of CAR, material transfer agreements were established with regards to national and international policies. All the experiments carried on mice were done in accordance with relevant guidelines and regulations on the use of laboratory animals including the ARRIVE guidelines.Arenaviridae family ranged from 0.02 to 0.4% whereas for reads belonging to the AnRB-3214-MinION dataset, taxonomic assignment percentages were 0.01% and 0.88% for Centrifuge32 and Kaiju31, respectively  except for the 0.1% subset for which no reads were assigned. Excluding the 0.1% subset in which no MinION reads could be classified, the percentages in the other datasets ranged from 0.03 to 1.04% for computer-diluted datasets from MinION sequencing (Table 30) and a homology search tool (BLASTN) using the sequence of the closest variant available in the GenBank . From the AnRB-3214-Illumina dataset, a total of 26,726 reads (0.29%) were identified with Minimap2, but only 2364 (0.025%) were identified with BLASTN. Finally, although only 0.45% of the reads were classified with Kaiju, almost 100% of both the S and L segments were covered. The second best coverage was obtained with Minimap2 with 95% (573 positions uncovered), but using either BLASTN or the other two classification tools (Kraken2 and Centrifuge), the coverage percentages were lower, varying between 17% (BLASTN) and 45% (Kraken2) and were concentrated on only certain regions of the different fragments  in an ambisense organization typical of arenaviruses. The S segment encodes GPC [position 146 to 1618] and NP [position 3190 to 1646] and the L segment encodes the Z protein [position 153 to 458] and the RdRp protein [position 7256 to 570].43, with respectively 64% and 56% nucleic acid similarity, evidence that they belong to two distinct virus species. Although the two strains were identified in two locations in the CAR from Arvicanthis sp. (DakAnB188d) and Praomys sp. (AnRB3214) rodents almost 10 years apart, the AnRB3214 strain was classified as an Ippy virus due to cross-reactivity with DakAnB188d. Serum-based assays were widely used for the classification of viruses in the 1970-1980s and viruses were historically assigned to antigenic groups  given their antigenic relationships.Nucleotide sequences and the deduced amino acid sequence of AnRB3214 strain were compared with those of other OW arenaviruses species. The S and L segments of the AnRB3214 strain are phylogenetically very distant from the other described Ippy virus strain DakAnB188dMastomys natalensis in Tanzania and Zambia and the Mobala virus has been identified in Bouboui village in Praomys sp. from the CAR one year prior to the isolation of strain AnRB3214 in Botambi, another village of the same prefecture. When considering each protein in their respective segments, we observed an increase in the level of conservation. At the amino acid level, there was a high level of conservation for GPC with 94%, 83% and 82% of similarity with Mobala, Gairo, Luna, and Morogoro viruses )  with Mobala virus. Molecular clock analysis shows that the two viruses diverged from one another around the year 1600 AD with a substitution rate of 4.28 × 1046. When using reference viral genome to recover arenaviruses reads in our AnRB3213 dataset, the best read identifications were obtained with Kaiju and Minimap2. Few, if any, studies have compared tools for the classification of reads, either from long reads (MinION) or short reads (Illumina) for the analysis of divergent viruses. Minimap was initially developed for long reads, but has been updated to work with smaller paired-end reads, through a genuine management of gaps30. Furthermore, Minimap2 has been shown to be a good tool for the alignment of nanopore reads, with a higher speed than GraphMap although with comparable accuracy47. Contrary to Kraken2 and Centrifuge which primarily classify nucleotides, Kaiju uses amino acid sequences instead of genomic sequences for metagenomic classification. This feature allows Kaiju to overcome the limitations introduced by the redundancy of the genetic code and therefore make it suitable for investigating divergent organisms31. For more insight on our dataset and sequencing approaches, randomly generated subsets corresponding to 10%, 1%, and 0.1% of the overall initial outputs were generated for subsequent analysis to simulate a situation similar to that of a primary biological sample, i.e. from infected rodent reservoirs with low viral load. We showed that, except for the 0.1% dilution subset using Kaiju, arenavirus reads were detected by all the classification tools, although at varying rates. However, this test also demonstrated the limitations that can arise in the detection of such viruses in their reservoirs because the corresponding reads will be present in limited amounts.In this study, the AnRB3214 strain was sequenced on two sequencing platforms, Illumina and MinION, and we used a diverse set of taxonomic classification and mapping tools to retrieve arenavirus-specific reads from the obtained datasets. We obtained 77,133 and 9,275,654 reads specific to arenaviruses from the MinION and Illumina datasets, respectively. It is no surprise that we obtained less reads from MinION sequencing as this technology produces longer reads compared to Illumina. Additionally, the high amount of reads obtained during our experiment is representative of the type of sample that was used  compared with direct sequencing of biological samples in which the viruses are present at low abundance with high levels of the host nucleic acid backgroundMurinae, which includes approximately 129 genera and 584 species. Members of about eight rodent genera are recognized as harbouring arenavirus species across the African continent7. For example, Praomys rodent species have a very extended geographic range and can be found across Sub-Saharan Africa. Ippy virus, Souris virus and Mobala virus have been isolated from Praomys rodents and Praomys clade rodents host almost all the OW arenaviruses48. Here, we described the AnRB3214 strain which was initially identified as an Ippy virus in 1981 in Botambi, CAR based on serological cross-reactivities. We demonstrated that strain AnRB3214 is distinct from the only other Ippy strain described to date, strain DakAnB188d, which also originates from the CAR. This classification was certainly due to cross-reactivity between the two viruses, because immunological assays were often used for virus identification and taxonomic assignment in the 1980s. Furthermore, “Ippy virus” has already been referred to as a complex comprising at least two viral species. Immunological microcompliment fixation data rapidly corroborated the presence of at least two distinct strains of Ippy virus, with low cross-reaction level, one of which was found in an Arvicanthis sp. rodent and the other in a Praomys sp. rodent7. With the description of the AnRB3214 strain, we confirm the huge arenavirus diversity in CAR rodents and the importance of the molecular characterization of viruses stored in biobanks.OW mammarenaviruses are zoonotic viruses hosted by rodents of subfamily 49. For OW arenaviruses, the interspecies amino acid cut-off divergences vary according to the protein: 8.6–20.4% (GPC), 10.2–21.5% (NP), 24.2–30.2% (Z) and 20.8–36.6% (L)49. Even so, some authors consider 12% divergence at the amino acid level in the NP enough to distinguish between two OW arenaviruses51. However, the latter criterion raises significant limitations, especially when considering the intra-species diversity of Lassa virus and NW arenaviruses52. Compared with Mobala virus at the amino acid level, strain AnRB3214 showed 6%, 13%, 17% and 17% genetic distance for the GPC, RdRp, Z and NP genes, respectively. Mobala virus and strain AnRB3214 were both isolated from unspecified Praomys rodents in the CAR in 1980 and 1981, respectively. Given the wide diversity of rodent species found in the forest areas of Central Africa, their morphological identification is often difficult and may sometimes require the use of molecular tools. Unfortunately, the absence of reference sequences for few rodent species mean that they cannot be identified53. Furthermore, Praomys rodents, which are subdivided into at least 20 putative species, are morphologically similar to Mastomys and Hylomyscus rodents55. Given that all these rodent species are abundant in CAR and that it is sometimes impossible to distinguish them on morphological criteria alone, it is difficult to confirm that strain AnRB3214 was isolated with certainty from a rodent of the Praomys genus. Mobala virus has also been isolated in Botambi, the same village from where strain AnRB3214 originates. Moreover, field and experimental evidence suggest that co-infections of a rodent species by two different arenaviruses are possible, although it has not yet been reported in the literature54. It is therefore difficult to conclude whether strain AnRB3214 is distinct from Mobala virus or not, especially because no further information is available for either virus. Molecular clock analysis under the strict clock model shows that the MRCA of strain AnRB3214 and Mobala virus can be dated back to year 1600 AD, almost 400 years ago. OW arenaviruses are thought to have originated nearly 3000–7000 years ago, as illustrated by LCMV speciation events which are estimated to have occurred around 1000 to 5000 years ago and the spread of Lassa virus from Nigeria to Mali and Ivory Coast some 1000–2000 years ago57. However, molecular clock analysis within Lassa virus lineages shows more contemporary virus speciation events, of less than 100 years up to 700 years59, similar to what was observed for strain AnRB3214. Additionally, the substitution rate deduced for strain AnRB3214 matches that of other arenaviruses, ranging from 3.3 × 10–4 to 6.3 × 10–4 subt/site/year, rates similar to those for Gbagroube virus, LCMV or Lassa virus60. Hence the circumstances that have led to the diversification of Mobala virus and strain AnRB3214 are probably similar to those observed within the Lassa virus lineages.Species delineation among arenaviruses is based on many criteria, including antigenic cross-reactivity, geographic distribution, host species and divergence at the amino acid level61. The N- and C-terminal domains have been shown to be the main NP regions directly involved in NP-NP oligomerisation, ribonucleoprotein complex formation and RNA synthesis. In comparison with the results obtained through an amino acid mutagenesis experiments in the N-terminus using the Tacaribe virus, a NW arenavirus62, we can conclude that the deletion observed in the AnRB3214 NP protein may not impact its biological function. However, further investigations are needed to understand the origin and consequences of this truncated nucleoprotein on viral particles.The AnRB3214 NP protein seems to be shorter than that of Mobala virus or the other OW arenaviruses, as it shows a ~ 53 aa deletion at the beginning of the N-terminal domain. Arenavirus NP proteins are essential for many aspects of the viral life cycle, such as the formation of the ribonucleoprotein complex when associated with RdRp, and the interference with the host immune system19, cross reactivity with arenavirus strain AnRB3214 cannot be rule out.More studies are needed to understand the implications of strain AnRB3214 for public health, especially in the CAR where civil unrest and political instability force the population to resort to feeding on forest-dwelling rodents. Although the prevalence of antibodies against Mobala virus in human population was associated with its prevalence in the rodent reservoirsLemniscomys striatus, Praomys spp., Arvicanthis spp. and Mastomys spp. rodents, of which 16 were isolated from Praomys spp.63. As seen in our study, these strains can represent distinct arenaviruses, especially because a specific arenavirus is usually associated with a single host species. Therefore, this study opens new ways to characterize the remaining CAR arenavirus strains, and especially those classified as Ippy virus and improve knowledge on the ecology of OW arenaviruses.OW arenavirus diversity remains to be explored. Here, we revealed that strain AnRB3214, classified as an Ippy virus, is clearly a species close or identical to Mobala virus. This article shows once again the usefulness of molecular tools for taxonomic assignment, especially considering archived viral strains obtained using classical virology tools and classified based on antigenic similarities. In the CAR, 22 viral isolates identified as Ippy virus have been reported from


Encephalitozoon intestinalis and Enterocytozoon bieneusi can cause diarrhea in humans, especially severe diarrhea in immunocompromised patients. However, there have been few studies on Enc. intestinalis and Ent. bieneusi in patients with acute diarrhea in the Republic of Korea (ROK). In this study, fecal samples were collected from 1241 patients with acute diarrhea in 2020. Among these, 24 cases of Enc. intestinalis and one case of Ent. bieneusi were detected via PCR amplification of small subunit ribosomal RNA. Genotyping of the internal transcribed spacer region sequence revealed that the detected Ent. bieneusi genotype was in Group 1. This study provides the first evidence that Ent. bieneusi exists in humans in addition to animals in the ROK. To identify the causative agent, continuous monitoring of Enc. intestinalis and Ent. bieneusi is necessary for patients with acute diarrhea in the ROK. Enterocytozoon, Pleistophora, Encephalitozoon, Vittaforma, Trachipleistophora, Brachiola, Nosema, and Microsporidium have been reported to infect humans. Encephalitozoon intestinalis and Enterocytozoon bieneusi are known to be the most common cause of intestinal diseases [Enc. cuniculi-infected pups [Microsporidia, which include more than 170 genera and 1300 species, are opportunistic protozoan pathogens that infect a variety of vertebrate and invertebrate hosts [diseases ,3. Microdiseases . Microspdiseases , and spediseases . In partted pups ,8.Ent. bieneusi and Enc. intestinalis, respectively [Ent. bieneusi and Enc. intestinalis, respectively [Ent. bieneusi [Enc. intestinalis [Ent. bieneusi in China [Enc. intestinalis [Enc. intestinalis and two with Ent. bieneusi [Enc. intestinalis and four with Ent. bieneusi of 70 healthy individuals in Slovakia [Microsporidia infections occur mainly in immunocompromised patients, such as patients with HIV, organ transplant recipients, and patients with cancer. Of 68 patients with HIV suffering from diarrhea in the United States, 25 and 5 were infected with ectively . Of 97 pectively . In Malibieneusi . In addibieneusi ,13. In Mstinalis , while 3in China . Moreovestinalis . Of 100 bieneusi . SimilarSlovakia .Enc. intestinalis were detected among 139 patients with diarrhea [Ent. bieneusi infections have been reported in the ROK, despite infections in wild animals  and livestock  [Enc. intestinalis and Ent. bieneusi and identify their genotypes in the ROK.In the Republic of Korea (ROK), little research on microsporidia has been reported. In 2011, seven cases of diarrhea . However calves) ,22,23. TEnc. intestinalis and one for Ent. bieneusi  and 12 female  patients. In contrast, Ent. bieneusi was detected in only a single male patient . Enc. intestinalis infection was not associated with sex . The prevalence of Enc. intestinalis was highest in the 30–39 age group (6.5%), followed by the 20–29 (4.7%) and 10–19 (3.5%) age groups. Ent. bieneusi was detected in the 10–19 age group, with a prevalence of 1.2%  showed 100% similarity with the small subunit ribosomal RNA (SSU rRNA) gene and were clustered with previously reported isolates , based on their molecular phylogenies. The SSU rRNA sequence of the single Ent. bieneusi case (KDCA 25) was also clustered with previously reported isolates  (J019869) .Ent. bieneusi genotype, Korea-WL2. The KDCA25 Ent. bieneusi isolate obtained in the present study was identical to LC436502 from a raccoon dog and LC436503 from a Korean water deer in the ROK [Ent. bieneusi genotypes. A neighbor-joining tree was constructed using the Ent. bieneusi ITS nucleotide sequences from humans and domestic animals. KDCA25 was clustered into Group 1, the human pathogenic group  gene, we detected one  the ROK . KDCA25 ic group .Ent. bieneusi and Enc. intestinalis are both common microsporidia species responsible for gastrointestinal diseases in humans [Ent. bieneusi is the most common microsporidial cause of intestinal diseases [Enc. intestinalis showed a higher infection rate than Ent. bieneusi in the ROK. This could have two possible explanations. First, the detection rate depends on patient condition. Ent. bieneusi infection accounts for 30–51% of all cases of diarrhea in immunocompromised patients [Enc. intestinalis and Ent. bieneusi focused on immunocompromised patients. Although this study focused on immunocompetent patients with diarrhea, it is necessary to include immunocompromised patients as a control group in future studies. Second, developed countries have reported gradual decreases in the prevalence and occurrence of Ent. bieneusi in immunocompromised patients due to the use of antiretroviral therapies and improved hygiene [n humans . Ent. bidiseases . Howeverpatients . Previou hygiene .Enc. intestinalis in patients with diarhhea in the ROK; however, we found a prevalence of 2% (24/1241). In this study, the infection rate was high in the 20–29  and 30–39  age groups. However, in previous studies, the infection rates were high in the 11–20  and 31–40  age groups. In addition, no infection rate was reported for the 21–30 age group by Kim et al. [In 2015, Kim et al.  reportedm et al.  as it coEnt. bieneusi. At least 474 ITS genotypes have been discovered, which can be systematically classified into 11 groups (Groups 1–11), with Group 1 and 2 being the major groups [The ITS domain of rRNA genes is the only known polymorphic marker of r groups ,29. At lr groups ,30,31,32r groups . Accordir groups , and thor groups . Group 5r groups . Group 6r groups , while Gr groups . Groups r groups .Ent. bieneusi isolate (KDCA 25) belonged to Group 1. The sequence matched with those previously reported from domestic wildlife [Ent. bieneusi in the ROK, except for domestic and wild animals. Previous studies on Ent. bieneusi infection have shown a total infection rate of 45.2% (71/157) in domestic animals [Ent. bieneusi infections in domestic wildlife and livestock. Microsporidian spores remain viable in water after desiccation following incubation at various temperatures, suggesting that indirect zoonotic transmission of microsporidia between animals and humans could occur through exposure to contaminated water, food, or aerosols [In this study, the wildlife , suggestwildlife . There h animals  and 18%  animals . Other s animals  and 14.9 animals , 1.9%  of the Korea Disease Control and Prevention Agency (KDCA) in 2020. The samples were examined for bacteria, viruses, and parasitic protozoa to determine the cause of diarrhea.Total DNA was extracted from 400 mg stool samples using the Fast DNA SPIN kit for Soil  according to the manufacturer’s protocol. The extracted DNA was stored at −20 °C until PCR analysis.Enc. intestinalis and Ent. bieneusi were detected using nested PCR, targeting the SSU rRNA [SSU rRNA . The priEnt. bieneusi, a 390 bp fragment of the ITS gene was amplified using nested PCR [Ent. bieneusi were as follows: the primary cycle consisted of 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 57 °C for 40 s, and 72 °C for 40 s; followed by 72 °C for 10 min; and termination at 4 °C. The second cycle consisted of 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 40 s; followed by 72 °C for 10 min; and termination at 4 °C.For genotyping sted PCR . The priEnc. intestinalis and Ent. bieneusi DNA fragments were sequenced using both forward and reverse primers (https://blast.ncbi.nlm.nih.gov/Blast.cgi accessed on 13 October 2021) using the data available on GenBank (http://www.ncbinlm.nih.gov/genbank/ accessed on 13 October 2021). Multiple alignments were conducted by ClustalW using BioEdit version 7.2.5 . To determine the similarity and difference rates between the sequences, the MegaAlign program  was employed. Finally, a phylogenetic analysis was performed with MEGA software (version 5.02) using the maximum parsimony algorithm with a Kimura two-parameter model assessed using bootstrap analysis with 1000 replications. Each sequence was identified by its accession number, host, origin, and genotype designation.The amplified  primers . The resEnc. intestinalis test positivity and factors such as sex and age group. Odds ratios and 95% confidence intervals were used to measure univariate associations. In this study, p-values < 0.05 were considered statistically significant. All statistical analyses were performed using SPSS Statistics 23.0 .Fisher’s exact test was used to assess the association between


Theba pisana) preferred taller supports as well. Results were similar in the field, where more snails were found on wider and taller supports 24 h after being released. The percentage of snails found in groups on a support was strongly density-dependent. The presence of other snails or their mucus did not affect the choices made. Overall, our results point towards the possibility of developing attractive supports to trap snails in the field.Terrestrial snails living in warm and dry climates had to develop strategies to survive. Several species climb on vertical supports when temperatures rise and spend the warmest months resting, typically in groups. Understanding this behavior could be useful in developing new management tools for species that are invasive. Here, we focused on four invasive snail species, and assessed their preferences for vertical supports varying in widths and heights under laboratory and field conditions. We also tested whether the presence of other snails from the same or different species affected these preferences. The snails strongly preferred wider supports in laboratory choice tests, and one species (Theba pisana) showed a consistent preference for taller supports as well. These results were confirmed in the field, where more snails were found on wider and taller supports 24 h after being placed in test quadrats. The percentage of snails found in groups on a support was strongly density-dependent. The presence of conspecifics or their mucus did not affect the choices of the snails, nor did the presence of snails of other species or their mucus. Taken together, these results could lead to the development of attractive supports that could be used to mass-capture snails in the field.Terrestrial snails that live in hot and dry climates have developed strategies to cope with high summer temperatures. Several species estivate during the warmest months of the years by resting on vertical supports, typically in groups. Understanding how snails choose their estivation sites and aggregate may lead to the development of new management tools in areas where these snails are invasive. Here, we investigated the preferences of four snail species for vertical supports varying in widths and heights under laboratory and field conditions, and tested whether the presence of conspecifics or snails of other species affected these preferences. The results show that the snails strongly preferred wider supports in laboratory dual-choice tests, and one species ( Mollusks have evolved in aquatic environments, but some lineages have successfully colonized terrestrial environments ,2. Out oEstivation is a diapause occurring during summer. It is an extended resting period that is associated with a reduction in the metabolic rate, including changes to enzymes and protein activities ,5. In seEstivation behavior seems to be triggered by a lack of moisture, but not by a lack of food . Some spTheba pisana (Müller) (Gastropoda: Helicidae) individuals resting in clumps on vertical supports had lower internal temperatures than isolated snails at the same height. Forming clumps may therefore benefit snails by providing them with better protection from high temperatures. It could also reduce predation risks, as seen in aggregating marine mollusks )nditions .Taken together, the results of the laboratory choice tests strongly suggest that snails mainly use visual cues to choose vertical supports.T. pisana ended up more on the black walls of the arena than on the supports (data not shown), a result consistent with Zanforlin’s findings (1976). It should also be noted that the snails exhibited climbing behavior in order to rest in their rearing containers prior to and after the tests. It is therefore likely that, while they may have moved following phototaxis, they may have also been looking for a support to rest. In the early stage of estivation, snails adjust their position until a suitable place is found [The general consensus regarding the vision of terrestrial gastropods is that they have poor vision, with eyes too underdeveloped to see objects in high resolution, and that snail eyes have evolved primarily to detect changes in light intensity . Terrestis found .Snail preferences in the laboratory were, overall, very consistent between the spring and summer tests, which seems to indicate that support preferences do not depend on the phenological status of the snail (active vs. ready to estivate). However, the choice tests in the laboratory were conducted at constant temperature and humidity, which is not like the environmental context leading to snail estivation in the field. Therefore, field tests were essential in order to validate the results obtained in the laboratory.T. pisana and C. virgata was clearly observable. More than 80% of the snails tested were found resting on vertical supports the day after being placed in the quadrats. Interestingly, the percentage of snails forming aggregations  increased with the number of snails found on a support. For T. pisana, almost all snails were found aggregated on the supports with the highest numbers of snails. This result is highly unlikely to have occurred because snails were running out of space on the supports. Indeed, the maximum space that the snails occupied on a support  only represented 2.2% of the total surface of the support (given a surface of 1 cm2 per snail). Therefore, clustering is more likely to be the result of the active choice of snails to rest near conspecifics.In the field, the estivation behavior of both More snails were found in the field on wider and taller supports. These results corroborate the results found in the laboratory and may indicate that snails respond to the same stimuli in the laboratory and in the field. It is also possible that wider supports were preferred simply because they were taking up more space in the quadrats and were encountered more often by snails. Indeed, the distribution of snails on the supports of different widths matched their expected distribution if they moved randomly and climbed on the first support they encountered . HoweverT. pisana, C. virgata, and C. acuta revealed that they did not show strong preferences toward such odors [To further explore the estivation preferences of snails, the effects of other types of support physical characteristics should be investigated . It also remains to be explored whether chemical cues, such as food odors, could be used to enhance the attractiveness of the support. Some snail species have been shown to respond to food odors under laboratory conditions ,40, but ch odors . It is ach odors . The hypIn conclusion, this study shows that consistent patterns of preferences for certain vertical supports exist in snails that climb to estivate and provides experimental settings to further explore this aspect of snail behavioral ecology. The fact that higher numbers of snails were consistently found on wider supports in the laboratory and in the field, and on taller supports in the field, is encouraging and supports the notion that optimal supports could be developed to trap snails at the beginning of the estivation period in the field. For example, supports could be placed at regular intervals along the borders of the fields to protect them before the beginning of the estivation period, and could be removed from the field once colonized by snails. The snails removed would be disposed of in order to reduce the field populations. More importantly, the snails on supports would not be estivating on crops and would not be harvested with the grain, which is the main economic issue. The practicality of this approach needs to be evaluated, and cost/benefit ratio analyses need be performed to assess under which conditions the benefit of reducing snail numbers outweighs the costs of integrating this management method for growers. More engineered products trapping snails permanently, or controlling them directly on the support, could also be developed. The fact that all four snail species exhibited similar patterns of preferences is also promising from the perspective of pest management, as it suggests that new management tools based on estivation preferences would equally work against all four species.


There is an error in the Funding statement. The correct number for “National Natural Science Foundation of China” is “81925026,” rather than “81925062.”Abstract appears below:In the original article, we missed to include the word “virion” when referring to PVVPs in the Abstract section. The corrected Sp), the sensitivity (Sn) of VirionFinder is approximately 10–34% much higher than the Sn of these tools on both complete and partial proteins. When evaluating related tools using real virome data, the recognition rate of PVVP-like sequences of VirionFinder is also much higher than that of the other tools. We expect that VirionFinder will be a powerful tool for identifying novel virion proteins from both complete prokaryote virus genomes and viral metagenomic data. VirionFinder is freely available at https://github.com/zhenchengfang/VirionFinder.”“Viruses are some of the most abundant biological entities on Earth, and prokaryote virus are the dominant members of the viral community. Because of the diversity of prokaryote virus, functional annotation cannot be performed on a large number of genes from newly discovered prokaryote virus by searching the current database; therefore, the development of an alignment-free algorithm for functional annotation of prokaryote virus proteins is important to understand the viral community. The identification of prokaryote virus virion proteins (PVVPs) is a critical step for many viral analyses, such as species classification, phylogenetic analysis and the exploration of how prokaryote virus interact with their hosts. Although a series of PVVP prediction tools have been developed, the performance of these tools is still not satisfactory. Moreover, viral metagenomic data contains fragmented sequences, leading to the existence of some incomplete genes. Therefore, a tool that can identify partial PVVPs is also needed. In this work, we present a novel algorithm, called VirionFinder, to identify the complete and partial PVVPs from non-prokaryote virus virion proteins (non-PVVPs). VirionFinder uses the sequence and biochemical properties of 20 amino acids as the mathematical model to encode the protein sequences and uses a deep learning technique to identify whether a given protein is a PVVP. Compared with the state-of-the-art tools using artificial benchmark datasets, the results show that under the same specificity (The authors apologize for these errors and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Wolbachia are associated with multiple mutualistic effects on insect biology, including nutritional and antiviral properties. Members of the genus Wolbachia naturally occur in fly species of the genus Drosophila, providing an operational model host for studying how virome composition may be affected by its presence. Drosophila simulans populations can carry a variety of strains of members of the genus Wolbachia, with the wAu strain associated with strong antiviral protection under experimental conditions. We used D. simulans sampled from the Perth Hills, Western Australia, to investigate the potential virus protective effect of the wAu strain of Wolbachia on individual wild-caught flies. Our data revealed no appreciable variation in virus composition and abundance between individuals infected or uninfected with Wolbachia associated with the presence or absence of wAu. However, it remains unclear whether wAu might affect viral infection and host survival by increasing tolerance rather than inducing complete resistance. These data also provide new insights into the natural virome diversity of D. simulans. Despite the small number of individuals sampled, we identified a repertoire of RNA viruses, including nora virus, galbut virus, thika virus and La Jolla virus, that have been identified in other species of the genus Drosophila. Chaq virus-like sequences associated with galbut virus were also detected. In addition, we identified five novel viruses from the families Reoviridae, Tombusviridae, Mitoviridae and Bunyaviridae. Overall, this study highlights the complex interaction between Wolbachia and RNA virus infections and provides a baseline description of the natural virome of D. simulans.The endosymbiont bacteria of the genus  The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.Wolbachia  are widespread endosymbionts of arthropods and nematodes  that can establish interactions with their hosts ranging from parasitic to mutualistic [Wolbachia is substantial and currently represented by 11 distinctive supergroups (denoted A–J), although the majority of strains of members of the genus Wolbachia belong to supergroups A and B [Wolbachia primarily spread by vertical inheritance through transovarian transmission. However, the presence of members of the genus Wolbachia in a diverse range of host species indicates that horizontal transmission, probably through antagonistic interactions , also contributes to the dissemination of the bacteria in nature [The members of the alpha-proteobacterium genus ualistic . The gen A and B  that are A and B . Althoug A and B , such th A and B . Membersn nature .Wolbachia in insects is often associated with their ability to manipulate host reproductive mechanisms and induce a range of alterations, including parthenogenesis, feminization, cytoplasmic incompatibility and sex-ratio distortion [Wolbachia–host interactions has revealed a variety of mutualistic effects on host biology [Asobara tabida, the presence of some strains of members of the genus Wolbachia has been positively associated with developmental processes, fertility and host viability [Wolbachia and the bedbug Cimex lectularius as well as Wolbachia-infected planthoppers, has been suggested as a means to explain B vitamin supplementation [The occurrence of bacteria of the genus stortion . Among tstortion . In addi biology . For insiability . Furtherentation .Wolbachia infection in insects is its potential for virus-blocking, which also provides a basis for intervention strategies based on the control of arbovirus transmission. This seemingly antiviral effect of members of the genus Wolbachia has been well documented in some species of insects, including flies and mosquitoes. A striking example involves the transinfection of Aedes aegypti mosquitoes with a strain of the genus Wolbachia infecting Drosophila melanogaster (wMel). A. aegypti is the primary vector of a number of important arboviruses, including members of the species Dengue virus, Zika virus and Chikungunya virus, and the establishment of the wMel strain in wild mosquito populations represents a powerful and promising approach to decrease virus transmission [Wolbachia can modify the host environment or boost basal immunity to viruses by pre-stimulating the immune response of their hosts [Wolbachia include gene expression of the Toll pathway, RNA interference, and modification of the host oxidative environment that probably trigger an antiviral immune response and hence limit infection [Arguably the most important outcome of smission . Althougir hosts . Potentinfection .A. aegypti mosquitoes, members of the genus Wolbachia naturally occur in species of the genus Drosophila, providing a valuable model system to study Wolbachia-related virus protection [Drosophila can carry a diverse array of insect-specific viruses belonging to the families Picornaviridae, Dicistroviridae, Bunyaviridae, Reoviridae and Iflaviridae amongst others [Wolbachia in D. melanogaster has been associated with increased survival and different levels of resistance to laboratory viral infections in fly stocks under experimental conditions [Wolbachia-infected flies containing the dicistrovirus Drosophila C virus (DCV) showed a delay in mortality compared with Wolbachia-free flies [Wolbachia on viral protection, as well as on virus prevalence and abundance in field-collected flies [Wolbachia on virus composition in members of the genus Drosophila in nature.Unlike otection . Naturalt others . The co-nditions . For exaee flies . In conted flies . Such coD. simulans is thought to have been in East Africa or Madagascar, this species now has a cosmopolitan distribution [D. simulans has been recorded along both the east and west coasts as well as Tasmania, with the earliest record dating to 1956 [D. simulans and Wolbachia into Australia, where wild fly populations occur near human settlements, feeding and breeding on a variety of horticultural crops [Wolbachia from supergroups A and B can naturally occur in populations of D. simulans  [wAu is associated with strong antiviral protection against Flock House virus (FHV) (Nodaviridae) and DCV (Dicistroviridae) under experimental conditions [wAu infection in Australia was one of the first infections by members of the genus Wolbachia identified as showing no cytoplasmic incompatibility, despite being widespread at a low to intermediate frequency [wAu increased in frequency along the east coast of Australia until it was replaced by wRi that exhibits cytoplasmic incompatibility. However, unlike wAu, wRi has not yet reached the Australian west coast [D. simulans flies from Western Australia, and investigated how this virome diversity might be affected by the presence of the wAu strain of Wolbachia.Although the origin of ribution . In Aust to 1956 . Human mal crops . Severaland wNo) . Of thesnditions . The wAurequency . wAu incst coast . In thisWolbachia at two other locations in the area  was also established with additional samples. Taxonomic identification to the species level was conducted on the basis of the morphology of reproductive traits of males and via DNA barcoding (cox1 gene marker). Field-collected flies were maintained at 19 °C under standard laboratory conditions until F1 offspring were raised. Parental and F1 generations were then stored at −80 °C until molecular processing.Flies used for the virus work performed here were collected at Raeburn Orchards in the Perth Hills in Western Australia , in July 2018 using banana bait. The frequency of members of the genus Wolbachia infection of field females was determined using F1 offspring from each field female. Note that wAu is transmitted at 100 % from field females to the F1 laboratory generation [et al. [Wolbachia was conducted using a real-time PCR/ high-resolution melt assay (RT/HRM) and strain-specific primers targeting a 340 bp region of the surface protein of Wolbachia (wsp) gene for wRi and wAu strains. The assay was run following the protocol of Kriesner et al. [Wolbachia wsp gene sequences for wRi (CP001391.1) and wAu (LK055284.1) with BBMap v.37.98 (minid=0.95) (available at https://sourceforge.net/projects/bbmap/).neration . DNA extneration  as adapt [et al. . Screenir et al. . In addiWolbachia on virome composition in D. simulans. Specimens were rinsed three times in RNA and DNA-free PBS solution (GIBCO). Total RNA from individual flies was extracted using the RNeasy Plus Mini Kit (Qiagen) following the manufacturer’s instructions. RNA-seq libraries were constructed using a TruSeq total RNA Library Preparation Kit (Illumina). Host ribosomal depletion was performed using a Ribo-Zero Gold rRNA Removal Kit (Human/Mouse/Rat) (Illumina) and paired-end transcriptome sequencing was performed on the HiSeq2500 platform (Illumina). Libraries from Wolbachia-negative and -positive infected flies were run in two separate lanes.We screened a total of 16 individual flies to assess the effect of infection with members of the genus de novo assembly of RNA-Seq data was performed using MEGAHIT v.1.1.3, with default parameters [−05. To identify protein-encoding sequences, open reading frames (ORFs) were predicted in positive and reverse-complement strands, with a minimum length of 600 nt between two stop codons using the GetOrf program (EMBOSS) [http://hmmer.org/) was used to perform sequence-profile searches against the Pfam HMM database. To expand the de novo assembled contigs of known viruses, the reads were mapped against reference genomic sequences. Provisional virus names were derived from geographic locations in the Perth Hills, Western Australia.The overall quality assessment of reads was conducted in FastQC and Trimmomatic . A de norameters . Assemblrameters , with a (EMBOSS) . Functio(EMBOSS) , and theWolbachia (16S and cox1) and D. simulans (rpl32 and cox1), as well as against all the RNA viruses identified in the annotation analyses. Mapping was performed using BBMap v.37.98 (minid=0.95) (available at https://sourceforge.net/projects/bbmap/).Viral abundance was assessed using the number of reads per million (RPM). This metric quantifies the number of reads per million mapped to a given contig assembly over the total number of reads. RPM values lower than 0.1 % of the highest count for each virus across samples were presumed to be index-hopping artefacts and excluded from the remaining analyses . To compD. simulans were compared with homologous reference sequences retrieved from the NCBI GenBank database and aligned with MAFF v7.450  [Drosophila, taxonomic profiling and read mapping to components of the fly microbiome and diet were conducted using the CCMetagen software (default settings) [RNA viral sequences identified in gorithm) . Phylogegorithm)  approachgorithm) . Nodal settings)  and BBMaWolbachia. Comparisons were made using raw and log-transformed data corresponding to RPM values  for each library. All analyses were performed using R software package rstatix (available at https://rpkgs.datanovia.com/rstatix/).The assumption of data normality was assessed by visual inspection and using Kolmogorov–Smirnov (K-S) and Shapiro–Wilk’s tests. As the data was not normally distributed, a Mann–Whitney–Wilcoxon test was used to compare the RNA virome composition with respect to the presence/absence of members of the genus Wolbachia infection through their F1s. The overall prevalence of members of the genus Wolbachia was 63.6 % (173 out of 272), with frequencies at the three sampled locations varying from 54.8 %  to 63.8 %  and 72.5 % . We randomly selected 16 flies from the Raeburn Orchard field females for individual sequencing and RNA virus screening, representing eight Wolbachia-positive and eight Wolbachia-negative specimens.A total of 272 female flies were wild-caught in the Perth Hills, Western Australia and tested for Wolbachia strain in D. simulans using sequence-specific primers targeting the wsp gene. We further confirmed the occurrence of Wolbachia by mapping the reads back to the wRi and wAu wsp genes. Most of the Wolbachia-infected flies showed a median coverage >100 reads, number of mapping reads >40, and coverage percentage >90 % to the reference wAu strain, confirming that infected flies harbour wAu rather than wRi. No reads mapped to the wsp gene for library RAPP88  despite the positive infection status determined using a Wolbachia-specific qPCR assay.We identified the 16S and cox1 in Wolbachia and rpl32 and cox1 in D. simulans. This provided an internal control to identify any effect on viral abundance due to potential biases introduced during RNA extraction or library preparation. Although, as expected, there was moderate variation in the abundance values, expression levels of reference maker genes were relatively stable across libraries in both Wolbachia and D. simulans  to the RdRp of known RNA viruses, whereas the newly discovered viruses shared only between 32.6 and 62.6% amino acid identity to the best viral hit  , La Jolla virus (Iflaviridae), thika virus (Picornaviridae) and nora virus (Picornaviridae) (Bunyaviridae), Araluen mito-like virus (Mitoviridae), Carmel mito-like virus (Mitoviridae), Lesley reo-like virus (Reoviridae), and Cannin tombus-like virus (Tombusviridae) (wAu-negative library (RAPN56)  , and S4.viridae) . In addiviridae) . SimilarD. simulans, and hence might be associated with the fly diet or microbiome. Specifically, these viruses were closely related to Phytomonas sp. TCC231 leishbunyavirus 1 (in the case of Raeburn bunya-like virus), Leptomonas pyrrhocoris RNA virus (Cannin tombus-like virus) and two mito-like viruses  , respectively. Hence, multiple microorganisms were present within individual fly libraries which may explain the occurrence of viruses not directly associated with Drosophila (files available at https://doi.org/10.6084/m9.figshare.c.5466690). In contrast, Lesley reo-like virus is likely to be a bona fide arthropod virus since it grouped with viruses previously detected in odonates and mosquitoes. In addition, it exhibited only approximately 24 % nucleotide similarity with those reoviruses previously reported to be contaminants in Drosophila cell culture [D. simulans virome. The five newly identified viruses in this study corresponded to full or nearly complete genomes (see below). However, for the majority of the known viruses of Drosophila we only were able to identify ORFs encoding the RdRp: the exceptions were La Jolla virus and thika virus, for which we also predicted structural components corresponding to coat and capsid proteins.Some of the newly discovered RNA viruses identified here were probably infecting hosts other than  culture . This inD. simulans in relation to the wAu infection status (n=9) of viruses in Wolbachia-negative flies compared with Wolbachia-positive flies (n=6). Among these, galbut virus, nora virus, thika virus, as well as three novel viruses identified in this study – Raeburn bunya-like virus, Araluen mito-like virus and Cannin tombus-like virus – were present in D. simulans regardless of Wolbachia infection. Likewise, ‘chaq virus-like’ sequences were observed co-occurring with galbut virus in the two groups of D. simulans. In contrast, La Jolla virus, as well as the novel Carmel mito-like virus and Lesley reo-like virus, were only found in wAu-negative flies. Overall, assembled viral contigs displayed high sequence similarity at the nucleotide and amino acid level within and between libraries and regardless of the presence or absence of members of the genus Wolbachia (Table S3).We next characterized the virome profile present in n status  and S4. Wolbachia on the abundance of RNA viruses present in wAu-infected and wAu-uninfected flies. Overall, the number of non-rRNA reads represented approximately 50 % of the total number of reads (n=743 389 696 pair-end reads) . Furthermore, the RPM values among viruses infecting Wolbachia-negative and -positive infected flies was highly heterogeneous, ranging from 47 to 232 346 and 5 to 37 688 virus RPM, respectively. With the exception of thika virus, viruses present in both wAu-positive and wAu-negative flies were 1.87–40.17-fold more abundant in the wAu-negative individuals than wAu-positive D. simulans. In contrast, the abundance of thika virus was 0.39-fold higher in the Wolbachia-positive flies , whilst the newly discovered Lesley reo-like virus was detected in four out of eight libraries (RPM=3360–8749) (Table S2). Although an interesting result, the limited sample size (n=16) means that these observations should be taken with caution and that larger sample sizes are needed for corroboration.We also assessed the potential effect of infection with members of the genus Wolbachia infection has been widely documented in natural populations of Drosophila [D. simulans is commonly used as an experimental model to investigate the interactions within the tripartite Drosophila–Wolbachia–virus system. In Australia, D. simulans can be naturally infected with two strains of Wolbachia from supergroup A wAu and wRi. While wRi has been gradually displacing wAu in eastern Australia, reflected in the changing infection frequencies in surveyed populations since 2004, D. simulans from the west coast of Australia only harbour wAu [D. simulans populations inhabiting the east and west coasts of Australia and the challenging environmental conditions posed by the intervening desert [The occurrence and spread of osophila . Indeed,bour wAu . A simplg desert .Wolbachia infection across samples by identifying the wsp, 16S and cox1 marker genes. The lack of reads mapping to the library RAPP88 might reflect either low levels of wsp RNA molecules present in the input for library preparation or high variability compared with the reference sequence. Although Wolbachia density was not experimentally assessed, the similar levels of 16S and cox-1 abundance across libraries indicate no appreciable biases in the library preparation and RNA sequencing steps.We corroborated the presence of wAu strain in Western Australia exceeded 50 % in D. simulans [Wolbachia might be present in a significant proportion of the natural fly population, at least around Perth. Although wAu does not cause cytoplasmic incompatibility, its spread is suggested to confer fitness advantages  to the host, including antiviral protection [D. simulans populations [Wolbachia-infected and -uninfected D. simulans in western Australia revealed no clear effect of Wolbachia infection on virome composition and viral abundance between Wolbachia infected and uninfected animals. Although our analysis is based on a small sample of individual flies, the apparent absence of a Wolbachia-mediated virus protection effect in natural D. simulans is compatible with previous findings on D. melanogaster naturally infected with wMel in eastern Australia [Wolbachia infection status and Wolbachia density. Even so, the absence of a significant association between wAu infection and virus diversity does not necessarily translate into a homogeneous effect of wAu on the different viruses identified here. For example, it is plausible that the restricted presence of La Jolla virus and the newly identified Lesley reo-like virus in Wolbachia-free flies could reflect some effect of antiviral protection in D. simulans [D. melanogaster, where La Jolla virus was widely distributed across different libraries [wAu-virus interactions, studies based on larger sample sizes are clearly needed to determine whether the apparent association between La Jolla virus and Wolbachia-uninfected flies observed here is an artefact due to small sample sizes. Indeed, it is notable that La Jolla virus was so rarely detected in the D. simulans flies studied here.Estimates from previous surveys indicated that the frequency of the simulans . This isotection , that miulations . Howeverustralia , in whicsimulans . Indeed,ibraries . AlthougwAu strain of Wolbachia has a protective role against virus infection in D. simulans when flies are challenged with FHV and DCV in a laboratory setting [wAu strain is protective against the dengue (DENV) and zika (ZIKV) viruses in Aedes aegypti mosquitoes [Wolbachia-mediated antiviral protection contrasts with those obtained previously, it is likely that differences may depend on Wolbachia–host species combinations and natural or artificial viral infections, which may also explain the contrasting results for La Jolla virus. Indeed, most of the available studies have documented the antiviral effect in transinfected insect hosts with non-natural strains of Wolbachia and viruses under laboratory conditions, as opposed to the study of the natural virome undertaken here.It has previously been shown that the  setting . Moreovesquitoes . AlthougWolbachia–virus–host interactions. Here, we collected flies during the Western Australian winter (mean temperature of 21 °C daylight time) and the specimens were maintained at 19 °C under laboratory conditions. Lower temperatures have been associated with an increase in viral resistance against DCV in D. melanogaster infected with wMel and wMelCS [Wolbachia-mediated virus blocking in natural populations [Wolbachia-conferred antiviral protection. On the other hand, the presence of Wolbachia has been suggested to influence host temperature preferences. For instance, wRi and wHo strains seem to manipulate D. simulans flies to seek cooler temperatures [wAu and D. simulans need to be tested, this indicates that the results observed here as well as a protective scenario might be temperature-dependent. This highlights the importance of careful future studies of the interactions within the host–virus–Wolbachia system along with environmental factors in natural populations [It is noteworthy that ecological variables such as temperature might affect d wMelCS . Similarulations . In thiseratures . Althougulations .Wolbachia-mediated virus protection in terms of virus abundance levels reflected in RPM values. This provides insights into virus resistance, but not into tolerance or host survival. Thus, it is still possible that Wolbachia is increasing tolerance to virus infection as has been documented for DCV [Wolbachia density, results of previous studies have indicated that wAu is maintained at high-density in D. simulans and has a role in virus blocking [As well as the small sample size, an important caveat of our work is that we explored the  for DCV . In addiblocking . FurtherwAu infected and uninfected D. simulans revealed the presence of natural and relatively highly abundant Drosophila-associated viruses in both groups [D. simulans [Tombusviridae and Bunyaviridae were related to viruses in trypanosomatid protozoa (Leptomonas and Leishmania). Similarly, given their normal host range distribution, the novel viruses from the family Narnaviridae might be associated with fungal hosts. Evidence of trypanosomatids and fungi in the gut of several species of Drosophila, with effects on larvae eclosion and pupation times [D. simulans can be parasitized in nature [Collectively, comparisons of the virome composition in h groups . Consisth groups . In addisimulans . For inson times , has been nature .wAu strain on the virome of D. simulans, using a meta-transcriptomic analysis of individual wAu-infected and uninfected flies. In doing so we identified Drosophila-associated viruses along with five novel viruses likely to be associated with fly diet or microbiome. Although our sample size is small, we saw no detectable Wolbachia-associated antiviral effect on virus composition and abundance, although the approach taken prevented us from drawing conclusions on virus tolerance. Further research employing larger sample sizes over broad spatial scales, including different Wolbachia–Drosophila combinations, will enable a more nuanced understanding of Wolbachia–virus dynamics in wild Drosophila populations.In sum, we provide a preliminary framework for assessing the effect of the Click here for additional data file.Click here for additional data file.