Detection of human enteric viral genes in a non-native winter crane fly, Trichocera maculipennis (Diptera) in the sewage treatment facilities in Antarctic stations

Detection of human enteric viral genes in a non-native winter crane fly, Trichocera maculipennis (Diptera) in the sewage treatment facilities in Antarctic stations | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Detection of human enteric viral genes in a non-native winter crane fly, Trichocera maculipennis (Diptera) in the sewage treatment facilities in Antarctic stations Sook-Young Lee, Ji Hee Kim, Seunghyun Kang, Kye Chung Park, Sung Mi Cho, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4209981/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Nov, 2024 Read the published version in Parasites & Vectors → Version 1 posted 7 You are reading this latest preprint version Abstract Background The Antarctic environment is susceptible to the introduction of non-native species due to its unique ecosystem, which has evolved under geographical isolation and extreme climatic conditions over an extended period. The non-native winter crane fly, Trichocera maculipennis , which was recently introduced to maritime Antarctica, is likely to pose a serious threat to the Antarctic ecosystem. In this study, the possibility of the mechanical transmission of viruses was evaluated. Methods The possibility of the mechanical transmission of viruses was evaluated using next-generation sequencing (NGS), quantitative polymerase chain reaction (qPCR), and virus isolation methods from T. maculipennis (Tm)-related samples (Tm body-wash fluid and Tm homogenate) collected from habitats and sewage treatment facilities located at three research stations in Antarctica. Results Our findings revealed the presence of human adenovirus (AdV) and human endogenous retrovirus (HERV) in Tm-related samples through virome analysis. Notably, these viruses are commonly detected in human feces. In addition, we identified pepper mild mottle virus (PMMoV) and cucumber green motortle mosaic virus (CGMMV), which are known indicators of enteric viruses, in all Tm-related samples. It is postulated that these viruses originated from wastewater, as evidenced by their detection in the wastewater samples. However, the minute quantities of AdV and HERV genomes detected in Tm-related samples through qPCR, coupled with the observed non-viability of AdV, indicate that T. maculipennis has limited potential for mechanical transmission. Conclusions Our study represents the first evaluation of the potential risk of non-native species serving as vectors for pathogens in Antarctica despite the relatively low quantities of detected viruses. This study provides valuable insights for further risk assessments of non-native species that are newly introduced or are likely to be introduced to Antarctica due to climate change or increased human activity. Trichocera maculipennis Non-native winter crane fly Antarctica Virus Mechanical transmission Figures Figure 1 Figure 2 Introduction Antarctica is less exposed to non-native species than other continents due to its geographical isolation and extreme climatic conditions. For this reason, the Antarctic terrestrial ecosystem exhibits a notable scarcity of biodiversity owing to the survival of only a limited number of native species [ 1 ]. However, recent increases in human activity and climate change have led to the introduction of non-native species, some of which have managed to establish themselves within Antarctic communities [ 2 ]. The majority of non-native species introduced into Antarctica are primarily distributed in the sub-Antarctic region. Notably, the recently discovered Trichocera maculipennis (Diptera) in the Antarctic region serves as a prominent example of non-native species facing significant challenges in terms of complete eradication [ 1 , 3 ]. T. maculipennis , a non-native fly (NNF), was initially introduced to King George Island in the maritime Antarctic South Shetland Islands in 2006 and originated in the Northern Hemisphere [ 4 , 5 ]. Since then, it has been reported within or in the vicinity of several research stations, including the Uruguayan Artigas station in 2006 [ 4 ], Chilean Frei station in 2009/2010, Korean King Sejong station in 2013/2014 [ 6 ], Polish Arctowski station in 2017 [ 5 , 7 ], and Russian Bellingshausen station in 2018/2019 [ 3 , 8 ]. The populations settled at each research station exhibited two different lineages, implying that they had at least two geographic origins and were introduced by multiple events [ 9 ]. Although the probability of the introduction of non-native species into Antarctica is anticipated to increase, the associated risks to these species, such as mechanical transmission of pathogens by NNFs, have not yet been assessed. Arthropods, especially insects, are considered potential vectors for disease transmission, primarily because of their ability to fly. This capability facilitates mechanical transmission, wherein pathogens are passively acquired and temporarily carried by vectors, either internally or on their external surfaces, before spreading. Therefore, understanding the role of vectors in the pathogen transmission ecology is vital for assessing the potential risks to veterinary and public health. Recent studies have identified a range of pathogens, including viruses, bacteria, fungi, and parasites on the external surfaces of insects. These include influenza viruses, coronaviruses, Campylobacter , Salmonella , Cladosporum , Aspergillus , Ascaris , Entamoeba , and others [ 10 ]. Research has specifically focused on houseflies, assessing their potential as vectors of infectious diseases in livestock. Under laboratory conditions, the ability of houseflies to carry and transmit several subtypes of avian influenza viruses (AIVs), including H5, H7, and H9, has been confirmed [ 11 – 13 ]. Furthermore, laboratory studies have shown that porcine reproductive and respiratory syndrome virus (PRRSV), Newcastle disease virus, and Orf virus, which cause infectious diseases in livestock, may be mechanically transmitted through vectors [ 14 – 16 ]. In particular, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for the global pandemic, has been found to remain infectious in houseflies for up to 24h post-exposure [ 17 , 18 ]. This potential for mechanical transmission is further supported by the field studies, which have detected various animal infectious disease viruses, including SARS-CoV-2, in livestock farms and on flies collected outdoors [ 19 – 22 ]. Consequently, this study aims to evaluate the potential of the NNF, T. maculipennis , a species recently introduced to Antarctica that inhabits sewage treatment facilities at Antarctic research stations, for the mechanical transmission of pathogens. Materials and methods Sample preparation Trichocera maculipennis were collected using UV traps from sewage treatment facilities at three Antarctic research stations (Russian Bellingshausen station, Korean King Sejong station, and Uruguayan Artigas station) located in King George Island, Antarctica (Fig. 1 ). Ten flies were collected from each station and immediately washed in 3 mL of phosphate-buffered saline (PBS) containing 1% antibiotic-antimycotic solution (Corning, USA). The washing fluid samples from T. maculipennis (Tm) body surface (Tm body-wash fluid sample) were centrifugated at 3,000 rpm for 5 min at 4℃. Subsequently, the supernatant from each sample was filtered through a 0.45 µm membrane filter (Millipore, Germany) to remove bacteria and large particles. To validate the viral sequences identified in the Tm-related samples (Tm body-wash fluid and Tm samples), we utilized 2 L samples of both influent and effluent wastewater collected from the sewage treatment facilities at King Sejong station. All samples were stored at − 20℃ and shipped to a laboratory in South Korea by sea. Upon arrival, the fly samples were homogenized by TissueLyser Ⅱ (Qiagen, Germany), and the supernatant from ground flies (Tm samples) was then collected after centrifugation at 13,000 rpm for 5 min at 4℃. Both Tm body-wash fluid and samples were processed for viral gene detection following the methodology outlined by Wu et al. [ 23 ]. Naked nucleic acids were digested with a cocktail of DNase and RNase enzymes, and viral nucleic acids were extracted using the QIAmp MinElute Virus Spin Kit (Qiagen, Germany). Next-generation sequencing We synthesized cDNA and RNA libraries from the samples using a QIAseq FX Single Cell RNA Library Kit (Qiagen, Germany) following the manufacturer’s instructions. To measure the concentrations of the cDNA library and library length, we used LightCycle qPCR (Roche, Switzerland) and the Agilent High Sensitivity D5000 ScreenTape system (Santa Clara, USA), respectively. The quantified libraries were then sequenced to 3-Gb using an Illumina HiseqX 150PE instrument according to the manufacturer’s recommendations. Sequencing reads were quality-trimmed (Q ≥ 30 and length ≥ 50 bp) using Trim Galore (Babraham Bioinformatics, UK) and then classified with DeconSeq ver. 0.4.3 utilizing a database sourced from the National Center for Biotechnology Information (NCBI) with a coverage threshold of 70% and identity threshold of 90%. Contig assembly was performed using the SPAdes assembler ver. 3.14.1, followed by mapping with BWA-mem v0.7.17, and viral contig annotations were identified using BLASTn [ 24 – 26 ]. Concentration of wastewater samples in King Sejong station The wastewater samples stored at − 20℃ for approximately six months were thawed overnight at 4℃. We employed a modified polyethylene glycol (PEG) precipitation concentration method as previously described by Sapula et al. [ 27 ]. Briefly, 250 mL of influent and effluent water samples were centrifuged for 30 min at 5,000 × g to precipitate large particles. Subsequently, 200 mL of the supernatant was transferred to 250 mL PPCO centrifuge bottles (Thermo Fisher Scientific, USA) followed by the addition of 7% PEG6000 with 0.4M NaCl. This mixture was gently agitated for 2 h at 4℃. Subsequently, the solution was transferred to 50 mL PPCO centrifuge tubes (Thermo Fisher Scientific, USA) and centrifuged for 90 min at 12,000 g and 4℃ to precipitate the pellet. After discarding the supernatant, the pellet was resuspended in 1 mL PBS. Finally, viral nucleic acids were extracted using an AllPrep PowerViral DNA/RNA kit (Qiagen, Germany). Quantification of viral genomes To quantify viral genes, we designed primers and probes based on the sequence of each viral contig (see Supplementary Table 1). For absolute quantitation by real-time (RT)-PCR, a standard curve was plotted using plasmids containing 123 bp and 218 bp fragments of adenovirus (AdV) and human endogenous retrovirus (HERV) genomes, respectively. The concentration of each viral genome was measured using a Nanodrop spectrophotometer (Thermo Scientific, USA), and the gene copy number in the standard was calculated using the following formula: (DNA amount (ng) × 6.0221 ×10 23 molecules/mole)/ ((DNA length × 660 g/mole) × 1 × 10 9 ng/g). A standard curve was generated using a decimal serial dilution of the plasmid, including each viral gene, and the threshold cycle values (Ct values) were plotted against the log concentrations of copy numbers. Calibration curves for AdV (y = -3.4618x + 51.128, R2 = 0.999) and HERV (y = -3.9411x + 52.644, R2 = 0.998) revealed a linear dynamic range between 4.51 × 10 3 and 4.51 × 10 8 . The copy number of each viral genome was measured from 5 µL of total nucleic acid extracted in Tm body-wash fluid and Tm samples using the commercial reagents TOPreal One-step qPCR/RT-qPCR Kit (Enzynomics, Korea) on the CFX Real-Time instruments (BIO-RAD, USA). For AdV, the PCR conditions were as follows: 95℃ for 10 min, 45 cycles at 95℃ for 5 sec, and 60℃ for 30 sec. For HERV, the PCR conditions were as follows: one cycle at 50℃ for 30 min, at 95℃ for 10 min, 45 cycles at 95℃ for 5 sec, and 60℃ for 30 sec. Copy numbers of the samples were calculated using the standard curve method. Propagation of viruses To determine the propagation of the detected viruses, we inoculated 200 µl samples of both Tm body-wash fluid and Tm samples into monolayer-cultured A549 cells (human lung carcinoma, CCL-185). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum (FBS) and 100 U/mL penicillin-streptomycin. The culture was then incubated at 37℃ in a humidified atmosphere containing 5% CO 2 [ 28 , 29 ]. After five to seven days, the propagation of the detected viruses in the inoculated cells was confirmed by quantifying each viral gene using the methods described in the above section. Results NGS data analysis We obtained six metagenomic datasets from Tm body-wash fluid and Tm samples collected from sewage treatment facilities at the Russian Bellingshausen, Korean King Sejong, and Uruguayan Artigas stations. The raw datasets were processed by trimming sequences with a base quality of ≥ 30 and a minimum length of 50 bp. Trimmed data ranging from 30,132,374 to 31,650,580 sequences were analyzed for viral sequences. From these, 3,966 to 143,369 viral reads were extracted based on coverage of ≥ 70% and identities of ≥ 90% using the NCBI virus database as a cut-off score. In each dataset, at least 16 contigs of ≥ 200 bp and one to three contigs of ≥ 1,000 bp were identified. Subsequent BLAST analysis using the NCBI VirDB confirmed 17 to 53 viral contigs (Rank1, e-value ≤ 1e-5), as detailed in Supplementary Table 2. Detection of various viruses by NGS In the Tm body-wash fluid and Tm samples (Tm-related samples) collected from the Bellingshausen station, we identified three insects and one mammalian viral genome. At King Sejong station, two mammalian and two insect viral sequences were detected in the Tm body-wash fluid and Tm samples, respectively. In the Artigas station samples, we identified one mammalian and two insect viral sequences (Fig. 2 , Supplementary Table 3). Among the viruses identified in the Tm-related samples were insect viruses, such as Choristoneura fumiferana granulovirus (ChfuGV) and Diolcogaster facetosa bracovirus (DfBV), as well as animal viruses, including human adenovirus (AdV) and human endogenous retroviruses (HERV). Additionally, plant viruses such as the pepper mild mottle virus (PMMoV) and cucumber green mottle mosaic virus (CGMMV) were found in these Tm-related samples (Supplementary Table 4). Detection of mammalian viruses Two mammalian viruses, AdV and HERV, were identified in Tm-related samples from sewage treatment facilities at Antarctic stations. AdV genomes were found in a Tm sample from Bellingshausen station and a Tm body-wash fluid sample from King Sejong station. Additionally, HERV genomes were detected in a Tm sample from King Sejong station and a Tm body-wash fluid sample from Artigas station. In the Tm sample from Bellingshausen station, 235 bp contigs showed approximately 80% coverage and 100% sequence identity with the E1B 55kDa protein gene of human AdV-1 (AC000017). Similarly, 381 bp adenoviral contigs in the Tm body-wash fluid sample from King Sejong station exhibited 99.7% sequence identity with the E1B 55kDa protein gene of human AdV-C (NC001405). Partial HERV sequences were also identified in the Tm sample from King Sejong station, showing 97.9% coverage (1,362/1,347 bp) and 92.7–95.7% sequence identity with the Pol and Env genes of HERV K113 (NC022518). Furthermore, a 434 bp HERV sequence in the Tm body-wash fluid sample from the Artigas station was 95.7% identical to the Gag gene sequence of HREV K113 (NC022518), as detailed in Table 1 and Supplementary Table 4. Table 1 Contig annotation of animal viruses detected from each sample Site Sample Virus Target accession no. Target gene Coverage (%) Length/matches (bp) Identity (%) Bellingshausen Tm # Human AdV 1 AC000017 E1B 55K 80.4 235/189 100 King Sejong Tm HERV NC022518 Pol, Env, and LTRs 97.9 1,362/1,347 92.7–95.7 Tm body-wash ¶ Human AdV C NC001405 E1B 55K 99.7 381/336 99.7 Artigas Tm body-wash HERV NC022518 Gag 95.9 434/416 95.7 # , T. maculipennis homogenate; ¶ , T. maculipennis body-wash fluid Quantification of viral genome in Tm-related and wastewater samples To quantify viral genomes in the Tm-related samples, primer/probe sets targeting the E1B and Pol genes of AdV and HERV were designed based on their respective contig sequences. In the Tm body-wash fluid sample from King Sejong station, the adenoviral genome was detected at a concentration of 1.35e + 2.36 copies/µL. However, it was not detected in the Tm sample from the Bellingshausen station. Similarly, the HERV genome was not found in the Tm and Tm body-wash fluid samples from the King Sejong and Artigas stations. In the concentrated influent and effluent samples from King Sejong station, the AdV genome was also detected with viral copy numbers of 1.35e + 2.16 copies/µL and 1.35e + 2.31 copies/µL, respectively (Table 2 ). Table 2 Adenoviral and retroviral quantification from each sample Site Sample NGS realtime-(RT)PCR Virus propagation AdV HERV Bellinsghausen Tm # Human AdV 1 - § - NT ‡ King Sejong Tm HERV - - - Tm body-wash ¶ Human AdV C 1.35e + 2.36 copies/µL - - Influent NT 1.35e + 2.16 copies/µL - NT effluent NT 1.35e + 2.31 copies/µL - NT Artigas Tm body-wash HERV - - NT # , T. maculipennis homogenate; ¶ , T. maculipennis body-wash fluid; § , not detected; ‡ , not tested The infectious potential of viruses detected in Tm-related samples and wastewater The inoculated cells were incubated for four to five days, during which their physical characteristics were monitored daily. However, no significant changes were observed in their shape or adhesion properties. Additionally, no viral genomes were detected in either the inoculated cell lysate or supernatant. Discussion Insects are often considered primary or intermediate hosts of human disease agents because of their ability to mechanically transmit diseases by harboring pathogens on their external surfaces and within their internal organs [ 5 , 7 ]. Recently discovered in Antarctica, T. maculipennis , which originated in the Northern Hemisphere, was introduced to Antarctic stations 20 years ago [ 4 , 5 , 9 ]. As prominent non-native species, they present challenges for eradication in Antarctica [ 6 , 8 ]. Given the uncertain impact of non-native species on the Antarctic ecosystem, this study aimed to assess the potential role of NNF as a vector for viral transmission. A variety of viruses, including insect, plant, and mammalian viruses, were identified in the virome analysis of Tm-related samples (Tm body-wash fluid and Tm samples). Insect viruses, such as Choristoneura fumiferana granulovirus (ChfuGV) and Diolcogaster facetosa bracovirus (DfBV), were detected along with the plant viruses pepper mild mottle virus (PMMoV) and cucumber green mottle mosaic virus (CGMMV). In addition, mammalian viruses such as human adenovirus (AdV) and human endogenous retrovirus (HERV) were identified. Among them, ChfuGV and DfBV, detected in at least one Tm-related sample, are recognized for their pathogenic and parasitic effects on insects, such as moths and wasps [ 30 , 31 ]. It is plausible that these viruses originated from T. maculipennis itself. PMMoV and CGMMV, which were found in all Tm-related samples, infect plants and cause crop diseases [ 32 ]. These plant viruses, which are commonly detected in human feces and wastewater, serve as indicators of enteric viruses and microbial source tracking in wastewater treatment [ 33 – 36 ]. As these plant viruses possess exceptional stability in water and diverse environmental conditions, their presence in Tm-related samples is thought to originate from wastewater. This suggests that T. maculipennis harbors diverse pathogens from sewage. AdV and HERV, which have been detected in Tm body-wash fluid and Tm samples from Antarctic research stations, are important mammalian viruses. AdV, which is associated with gastroenteritis in young children, is often transmitted via the oral-fecal route and is frequently found in wastewater [ 37 – 39 ]. HERV, an endogenous retrovirus inserted into the human germ cell DNA, is commonly found in the human gut and feces [ 40 – 42 ]. Although no other animal RNA viruses have been detected, AdV and HERV, which are relatively more resistant to degradation than RNA viruses, seem to be more easily detected in T. maculipennis washed fluid or lysates because they can remain preserved in environmental samples for a prolonged period. A quantification test for adenoviral and retroviral genes ( E1B gene of AdV and Pol gene of HERV) to assess their abundance in Tm-related samples, indicated a very low quantity of viral genomes: 1.35e + 2.36 copies/µL in Tm body-wash fluid sample from King Sejong station. Although the concentrations of these viral genes were near the detection limits, we concluded that T. maculipennis carried these viral genes at very low concentrations. This conclusion is based on the consistently negative results in non-template controls and the fact that our analysis spanned a dynamic range of concentrations up to e + 3 in the standard curves [ 43 ]. The presence of a small amount of adenovirus in wastewater, 1.35e + 2.16 copies/µL (influent) and 1.35e + 2.31 copies/µL (effluent) suggested that the viral genes in T. maculipennis originated from the habitat at King Sejong station. However, the AdV gene was not detected in Tm samples from the Bellingshausen station, and HERV was not found in Tm or Tm body-wash fluid samples from the King Sejong and Artigas stations. Previous laboratory studies have shown that the concentration of live viruses and the duration of their external exposure are crucial factors that enable mechanical transmission by vectors [ 12 , 13 ]. In particular, research on the mechanical transmission of influenza virus by houseflies indicates that low viral concentrations, high incubation temperatures, and prolonged incubation can reduce the persistence of influenza viruses [ 11 – 13 ]. In these studies, although influenza virus genes were detected up to 96 h post-exposure, there was a significant decrease in virus isolation from flies as environmental exposure increased. In our study, while the duration of viral exposure on T. maculipennis was not precisely determined, the viral genomes in immediately collected samples were minimal, and the viability of the viruses was extremely low. However, given the number of flies used in our study and the uncertain environmental exposure time, we cannot completely rule out their potential as carriers of disease. The limited detection of AdV and the absence of HERV in the concentrated wastewater samples may be attributed to the long duration of transportation from Antarctica to the laboratory, despite being stored at − 20℃. Storage and repeated thawing of liquid wastewater can reduce the measurable concentrations of viral nucleic acids, especially RNA [ 44 , 45 ]. Recent studies suggest that storing wastewater solids, like RNA extract or concentrate, at − 20℃ for approximately four months yields more reliable viral RNA quantification compared to storing unprocessed raw wastewater at 4℃ or − 80℃ [ 46 , 47 ]. Therefore, for future research on Antarctic wastewater, it is crucial to preprocess or concentrate wastewater before shipment from Antarctica. Although our study detected only modest amounts of the virus in Tm-related and wastewater samples, it represents the first assessment of the potential risk posed by NNFs introduced into the Antarctic as a vector. Monitoring non-native species in Antarctica and evaluating the associated risks is essential, particularly given the increasing likelihood of introductions attributable to climate change and heightened human activity. Our study contributes to the establishment of risk assessment methods for non-native Antarctic species and offers a framework for diverse evaluations in future research. Conclusions This study explored the potential of the NNF, T. maculipennis , found in sewage treatment facilities at Antarctic research stations, to mechanically transmit viruses. A key finding was the detection of various viral genomes in T. maculipennis , including the human adenovirus (AdV) and human endogenous retrovirus (HERV), which are commonly found in human feces and wastewater. However, the detected viral quantities were minimal and nonviable, suggesting that T. maculipennis in Antarctica is unlikely to be an effective mechanical transmitter of these viruses, likely because of low viral loads. However, the possibility remains that T. maculipennis can potentially become a vector under conditions conducive to mechanical transmission. This is the first study to assess the risk posed by T. maculipennis as a vector of pathogens in Antarctica. This contributes to a broader evaluation of the risk of mechanical transmission of viruses by NNFs, providing insight into the potential implications of their future introduction. Abbreviations T.mculipennis Trichocera maculipennis Tm-related sample T. mculipennis -body wash fluid and T. maculipennis homogenate NNF non-native fly ChfuGV Choristoneura fumiferana granulovirus DfBV Diolcogaster facetosa bracovirus AdV adenovirus HERV Human endogenous retrovirus PMMoV pepper mild mottle virus CGMMV cucumber green mottle mosaic virus PEG polyethylene glycol DMEM Dulbecco’s modified Eagle’s medium FBS fetal bovine serum Declarations Acknowledgments We thank the Russian Bellingshausen station and the Uruguayan Artigas station for their collaboration and the generous provision of NNFs for analysis. We extend our thanks to the Korean overwintering crews for their invaluable cooperation in facilitating our research endeavors for several years. This research was supported by the Korea Polar Research Institute (PE24140, PE24170) and the Chilean National Research Program (FOVI220036). Funding Not applicable Availability of data and materials Illumina HiseqX 150PE data have been deposited in the GenBank Sequence Reads Archive (SRA) under the accession number PRJNA1052610. Author’s contributions SYL, JHK and SK conceptualized and designed this study. SYL, SK, KCP and SMC performed the experiments and analyzed the data. CXS, LR, HAB, TCM, AS and EJ collected and supplied fly samples. SYL, JHK, SK, KCP, SMC and SK contributed to the finalization of the manuscript. JHK and SK managed the project. All authors read and approved the final manuscript. Ethics approval and consent to participate Not applicable Consent for publication Not applicable Competing interests Not applicable References Hughes KA, Convey P. Alien invasions in Antarctica‒is anyone liable? Polar Research. 2014;33:22103. Matheson P, McGaughran A. How might climate change affect adaptive responses of polar arthropods? Diversity. 2023;15:47. León MR, Hughes KA, Morelli E, Convey P. 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Vogt VM. “Retroviral virions and genomes” in Retroviruses, Coffin J M, Hughes S H, Varmus HE, Eds. (Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY). 1997. Carding SR, Davis N, Holyes L. Review article: the human intestinal virome in health and disease. Aliment Pharmacol Ther. 2017;46:800–15. Cinek O, Kramna L, Odeh R, Alassaf A, Ibekwe MAU, Ahmadov G, et al. Eukaryotic viruses in the fecal virome at the onset of type 1 diabetes: A study from four geographically distant African and Asian countries. Pediatr Diabetes. 2021;22:558–66. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55:611–22. Hokajärvi AM, Rytkönen A, Tiwari A, Kauppinen A, Oikarinen S, Lehto K, et al. The detection and stability of the SARS-CoV-2 RNA biomarkers in wastewater influent in Helsinki, Finland. Sci Total Environ. 2021;770:145274. Weidhaas J, Aanderud ZT, Roper DK, VanDerslice J, Gaddis EB, Ostermiller J, et al. Correlation of SARS-CoV-2 RNA in wastewater with COVID-19 disease burden in sewersheds. Sci Total Environ. 2021;775:145790. Fernandez-Cassi X, Scheidegger A, Bänziger C, Cariti F, Corzon AT, Ganesanandamoorthy P, et al. Wastewater monitoring outperforms case numbers as a tool to track COVID-19 incidence dynamics when test positivity rates are high. Water Res. 2021;200:117252. Simpson A, Topol A, White BJ, Wolfe MK, Wigginton KR, Boehm AB. Effect of storage conditions on SARS-CoV-2 RNA quantification in wastewater solids. PeerJ. 2021;9:e11933. Additional Declarations No competing interests reported. Supplementary Files SupplementaryTable.xlsx Supplementary information Additional file 1: Table S1. Primer information for quantification of viral genes (AdV and HERV). Table S2. The results of NGS data analysis from each data set. Table S3. Number of viral contigues detected from each site. Table S4. Information of the sequences of various viruses (insect, mammalian, and plant virus). Cite Share Download PDF Status: Published Journal Publication published 24 Nov, 2024 Read the published version in Parasites & Vectors → Version 1 posted Editorial decision: Revision requested 04 Jun, 2024 Reviews received at journal 04 Jun, 2024 Reviewers agreed at journal 14 May, 2024 Reviewers invited by journal 24 Apr, 2024 Editor assigned by journal 03 Apr, 2024 Submission checks completed at journal 03 Apr, 2024 First submitted to journal 03 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4209981","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":286920776,"identity":"28a8b45c-7f52-432e-9130-48d02a15b0a2","order_by":0,"name":"Sook-Young Lee","email":"","orcid":"","institution":"Korea Polar Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Sook-Young","middleName":"","lastName":"Lee","suffix":""},{"id":286920777,"identity":"bc82d6b9-eefa-4e78-b8d5-2bbad3758a04","order_by":1,"name":"Ji Hee Kim","email":"","orcid":"","institution":"Korea Polar Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Ji","middleName":"Hee","lastName":"Kim","suffix":""},{"id":286920778,"identity":"e06b6ca8-1ce0-46fd-9ef7-1c5bfe3e77ae","order_by":2,"name":"Seunghyun Kang","email":"","orcid":"","institution":"Korea Polar Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Seunghyun","middleName":"","lastName":"Kang","suffix":""},{"id":286920779,"identity":"e9592109-edb9-47e2-9798-41e50c98b27f","order_by":3,"name":"Kye Chung Park","email":"","orcid":"","institution":"The New Zealand Institute for Plant and Food Research Ltd","correspondingAuthor":false,"prefix":"","firstName":"Kye","middleName":"Chung","lastName":"Park","suffix":""},{"id":286920780,"identity":"790a8d7f-5bd7-4913-a093-e4a2520668cb","order_by":4,"name":"Sung Mi Cho","email":"","orcid":"","institution":"Korea Polar Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Sung","middleName":"Mi","lastName":"Cho","suffix":""},{"id":286920781,"identity":"5b1388e9-663a-4aec-8148-0837c5d73deb","order_by":5,"name":"Carla Ximena Salinas","email":"","orcid":"","institution":"Instituto Antártico Chileno","correspondingAuthor":false,"prefix":"","firstName":"Carla","middleName":"Ximena","lastName":"Salinas","suffix":""},{"id":286920782,"identity":"65ac0402-a936-45a9-995f-404659f0f5ad","order_by":6,"name":"Lorena Rebolledo","email":"","orcid":"","institution":"Instituto Antártico Chileno","correspondingAuthor":false,"prefix":"","firstName":"Lorena","middleName":"","lastName":"Rebolledo","suffix":""},{"id":286920783,"identity":"dc43b4bf-3ff7-46aa-b7e9-7e1e03047c10","order_by":7,"name":"Hugo A. Benítez","email":"","orcid":"","institution":"Universidad de Magallanes","correspondingAuthor":false,"prefix":"","firstName":"Hugo","middleName":"A.","lastName":"Benítez","suffix":""},{"id":286920784,"identity":"c4e95407-f0fa-482f-bdc8-7cbcfc8391e3","order_by":8,"name":"Tamara Contador Mejías","email":"","orcid":"","institution":"Universidad Católica del Maule","correspondingAuthor":false,"prefix":"","firstName":"Tamara","middleName":"Contador","lastName":"Mejías","suffix":""},{"id":286920785,"identity":"7d2bf8b4-6104-4c90-8020-f98e1ef178e0","order_by":9,"name":"Alvaro Soutullo","email":"","orcid":"","institution":"Universidad de la República","correspondingAuthor":false,"prefix":"","firstName":"Alvaro","middleName":"","lastName":"Soutullo","suffix":""},{"id":286920786,"identity":"bb925a5e-d714-4564-99c7-5efde1f53a1c","order_by":10,"name":"Eduardo Juri","email":"","orcid":"","institution":"Instituto Antáretico Uruguayo","correspondingAuthor":false,"prefix":"","firstName":"Eduardo","middleName":"","lastName":"Juri","suffix":""},{"id":286920787,"identity":"5f4e3ee8-3aba-4669-a3ad-09a4b475db42","order_by":11,"name":"Sanghee Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYJACZiA2YGNgPgzi8IAIxgbitLAlk6gFqNgYLoJXi3z72cOvCypsjPnEznw2+LjjngyD9OEDjDP34NZicCYvzXrGmTQzNunczYkzzxTzMPClJTBueIZHC0OOmTFv22EbkJbDvG0JPAw8PAaMDw7gcVj/G6CWf/+BWnIeQ7Xwf8CrheFGjvFj3oYDQIflMCdDbWFg3IBHi8GNN2bMPMeSjdmk04wNZ55J4GHjYTM4OAOvw3KMP/PU2BnOn538WOLjjgR7fh7mhw978DmMgYFNAs4ExQcbkMavARiTH1C0jIJRMApGwShABwDvnkmdBbKo7gAAAABJRU5ErkJggg==","orcid":"","institution":"Korea Polar Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Sanghee","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2024-04-03 05:03:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4209981/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4209981/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13071-024-06555-4","type":"published","date":"2024-11-24T15:58:06+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54274360,"identity":"e0294013-da20-4860-a0ce-b5b3975ca62c","added_by":"auto","created_at":"2024-04-08 07:30:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1233608,"visible":true,"origin":"","legend":"\u003cp\u003eCollection sites of non-native winter crane flies. The three research stations of Antarctica are marked with an asterisk. From the left, Bellingshausen station of Russia, Artigas station of Uruguay, and King Sejong station of South Korea.\u003c/p\u003e","description":"","filename":"Figure1.Collectionsitesofnonnativeflies.png","url":"https://assets-eu.researchsquare.com/files/rs-4209981/v1/25f321edf9c7a36e41680c5c.png"},{"id":54274361,"identity":"89dc648e-c664-4f05-910e-753b33c0739b","added_by":"auto","created_at":"2024-04-08 07:30:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":103843,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of virus, viral contigs, and viral reads detected in Tm-related samples collected from each Antarctic station. \u003cstrong\u003ea\u003c/strong\u003eRussian Bellingshausen station, b Korean King Sejong station, and \u003cstrong\u003ec\u003c/strong\u003e Uruguayan Artigas station.\u003c/p\u003e","description":"","filename":"Figure2.Numberofviralcotiguesdetectedfromeachsite.png","url":"https://assets-eu.researchsquare.com/files/rs-4209981/v1/9f1625a68e239cc5283deba4.png"},{"id":69835510,"identity":"8c4cc16f-2407-43f4-b03e-f0c0a0f873e6","added_by":"auto","created_at":"2024-11-25 16:13:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1873270,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4209981/v1/d39a97fb-53fd-4fb1-959c-ab11af3aaf40.pdf"},{"id":54274359,"identity":"06b36353-35fb-4599-bd3f-2164dac84ba5","added_by":"auto","created_at":"2024-04-08 07:30:45","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":22117,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 1: Table S1. \u003c/strong\u003ePrimer information for quantification of viral genes (AdV and HERV). \u003cstrong\u003eTable S2.\u003c/strong\u003e The results of NGS data analysis from each data set. \u003cstrong\u003eTable S3.\u003c/strong\u003e Number of viral contigues detected from each site. \u003cstrong\u003eTable S4.\u003c/strong\u003e Information of the sequences of various viruses (insect, mammalian, and plant virus).\u003c/p\u003e","description":"","filename":"SupplementaryTable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4209981/v1/666b025fedf8fcd1ee5be7be.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Detection of human enteric viral genes in a non-native winter crane fly, Trichocera maculipennis (Diptera) in the sewage treatment facilities in Antarctic stations","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAntarctica is less exposed to non-native species than other continents due to its geographical isolation and extreme climatic conditions. For this reason, the Antarctic terrestrial ecosystem exhibits a notable scarcity of biodiversity owing to the survival of only a limited number of native species [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, recent increases in human activity and climate change have led to the introduction of non-native species, some of which have managed to establish themselves within Antarctic communities [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The majority of non-native species introduced into Antarctica are primarily distributed in the sub-Antarctic region. Notably, the recently discovered \u003cem\u003eTrichocera maculipennis\u003c/em\u003e (Diptera) in the Antarctic region serves as a prominent example of non-native species facing significant challenges in terms of complete eradication [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. \u003cem\u003eT. maculipennis\u003c/em\u003e, a non-native fly (NNF), was initially introduced to King George Island in the maritime Antarctic South Shetland Islands in 2006 and originated in the Northern Hemisphere [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Since then, it has been reported within or in the vicinity of several research stations, including the Uruguayan Artigas station in 2006 [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], Chilean Frei station in 2009/2010, Korean King Sejong station in 2013/2014 [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], Polish Arctowski station in 2017 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and Russian Bellingshausen station in 2018/2019 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The populations settled at each research station exhibited two different lineages, implying that they had at least two geographic origins and were introduced by multiple events [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Although the probability of the introduction of non-native species into Antarctica is anticipated to increase, the associated risks to these species, such as mechanical transmission of pathogens by NNFs, have not yet been assessed.\u003c/p\u003e \u003cp\u003eArthropods, especially insects, are considered potential vectors for disease transmission, primarily because of their ability to fly. This capability facilitates mechanical transmission, wherein pathogens are passively acquired and temporarily carried by vectors, either internally or on their external surfaces, before spreading. Therefore, understanding the role of vectors in the pathogen transmission ecology is vital for assessing the potential risks to veterinary and public health. Recent studies have identified a range of pathogens, including viruses, bacteria, fungi, and parasites on the external surfaces of insects. These include influenza viruses, coronaviruses, \u003cem\u003eCampylobacter\u003c/em\u003e, \u003cem\u003eSalmonella\u003c/em\u003e, \u003cem\u003eCladosporum\u003c/em\u003e, \u003cem\u003eAspergillus\u003c/em\u003e, \u003cem\u003eAscaris\u003c/em\u003e, \u003cem\u003eEntamoeba\u003c/em\u003e, and others [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Research has specifically focused on houseflies, assessing their potential as vectors of infectious diseases in livestock. Under laboratory conditions, the ability of houseflies to carry and transmit several subtypes of avian influenza viruses (AIVs), including H5, H7, and H9, has been confirmed [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, laboratory studies have shown that porcine reproductive and respiratory syndrome virus (PRRSV), Newcastle disease virus, and Orf virus, which cause infectious diseases in livestock, may be mechanically transmitted through vectors [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In particular, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for the global pandemic, has been found to remain infectious in houseflies for up to 24h post-exposure [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This potential for mechanical transmission is further supported by the field studies, which have detected various animal infectious disease viruses, including SARS-CoV-2, in livestock farms and on flies collected outdoors [\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Consequently, this study aims to evaluate the potential of the NNF, \u003cem\u003eT. maculipennis\u003c/em\u003e, a species recently introduced to Antarctica that inhabits sewage treatment facilities at Antarctic research stations, for the mechanical transmission of pathogens.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample preparation\u003c/h2\u003e \u003cp\u003e \u003cem\u003eTrichocera maculipennis\u003c/em\u003e were collected using UV traps from sewage treatment facilities at three Antarctic research stations (Russian Bellingshausen station, Korean King Sejong station, and Uruguayan Artigas station) located in King George Island, Antarctica (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Ten flies were collected from each station and immediately washed in 3 mL of phosphate-buffered saline (PBS) containing 1% antibiotic-antimycotic solution (Corning, USA). The washing fluid samples from \u003cem\u003eT. maculipennis\u003c/em\u003e (Tm) body surface (Tm body-wash fluid sample) were centrifugated at 3,000 rpm for 5 min at 4℃. Subsequently, the supernatant from each sample was filtered through a 0.45 \u0026micro;m membrane filter (Millipore, Germany) to remove bacteria and large particles. To validate the viral sequences identified in the Tm-related samples (Tm body-wash fluid and Tm samples), we utilized 2 L samples of both influent and effluent wastewater collected from the sewage treatment facilities at King Sejong station. All samples were stored at \u0026minus;\u0026thinsp;20℃ and shipped to a laboratory in South Korea by sea. Upon arrival, the fly samples were homogenized by TissueLyser Ⅱ (Qiagen, Germany), and the supernatant from ground flies (Tm samples) was then collected after centrifugation at 13,000 rpm for 5 min at 4℃. Both Tm body-wash fluid and samples were processed for viral gene detection following the methodology outlined by Wu et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Naked nucleic acids were digested with a cocktail of DNase and RNase enzymes, and viral nucleic acids were extracted using the QIAmp MinElute Virus Spin Kit (Qiagen, Germany).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eNext-generation sequencing\u003c/h2\u003e \u003cp\u003eWe synthesized cDNA and RNA libraries from the samples using a QIAseq FX Single Cell RNA Library Kit (Qiagen, Germany) following the manufacturer\u0026rsquo;s instructions. To measure the concentrations of the cDNA library and library length, we used LightCycle qPCR (Roche, Switzerland) and the Agilent High Sensitivity D5000 ScreenTape system (Santa Clara, USA), respectively. The quantified libraries were then sequenced to 3-Gb using an Illumina HiseqX 150PE instrument according to the manufacturer\u0026rsquo;s recommendations. Sequencing reads were quality-trimmed (Q\u0026thinsp;\u0026ge;\u0026thinsp;30 and length\u0026thinsp;\u0026ge;\u0026thinsp;50 bp) using Trim Galore (Babraham Bioinformatics, UK) and then classified with DeconSeq ver. 0.4.3 utilizing a database sourced from the National Center for Biotechnology Information (NCBI) with a coverage threshold of 70% and identity threshold of 90%. Contig assembly was performed using the SPAdes assembler ver. 3.14.1, followed by mapping with BWA-mem v0.7.17, and viral contig annotations were identified using BLASTn [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eConcentration of wastewater samples in King Sejong station\u003c/h2\u003e \u003cp\u003eThe wastewater samples stored at \u0026minus;\u0026thinsp;20℃ for approximately six months were thawed overnight at 4℃. We employed a modified polyethylene glycol (PEG) precipitation concentration method as previously described by Sapula et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Briefly, 250 mL of influent and effluent water samples were centrifuged for 30 min at 5,000 \u0026times; g to precipitate large particles. Subsequently, 200 mL of the supernatant was transferred to 250 mL PPCO centrifuge bottles (Thermo Fisher Scientific, USA) followed by the addition of 7% PEG6000 with 0.4M NaCl. This mixture was gently agitated for 2 h at 4℃. Subsequently, the solution was transferred to 50 mL PPCO centrifuge tubes (Thermo Fisher Scientific, USA) and centrifuged for 90 min at 12,000 g and 4℃ to precipitate the pellet. After discarding the supernatant, the pellet was resuspended in 1 mL PBS. Finally, viral nucleic acids were extracted using an AllPrep PowerViral DNA/RNA kit (Qiagen, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of viral genomes\u003c/h2\u003e \u003cp\u003eTo quantify viral genes, we designed primers and probes based on the sequence of each viral contig (see Supplementary Table\u0026nbsp;1). For absolute quantitation by real-time (RT)-PCR, a standard curve was plotted using plasmids containing 123 bp and 218 bp fragments of adenovirus (AdV) and human endogenous retrovirus (HERV) genomes, respectively. The concentration of each viral genome was measured using a Nanodrop spectrophotometer (Thermo Scientific, USA), and the gene copy number in the standard was calculated using the following formula: (DNA amount (ng) \u0026times; 6.0221 \u0026times;10\u003csup\u003e23\u003c/sup\u003e molecules/mole)/ ((DNA length \u0026times; 660 g/mole) \u0026times; 1 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e ng/g). A standard curve was generated using a decimal serial dilution of the plasmid, including each viral gene, and the threshold cycle values (Ct values) were plotted against the log concentrations of copy numbers. Calibration curves for AdV (y = -3.4618x\u0026thinsp;+\u0026thinsp;51.128, R2\u0026thinsp;=\u0026thinsp;0.999) and HERV (y = -3.9411x\u0026thinsp;+\u0026thinsp;52.644, R2\u0026thinsp;=\u0026thinsp;0.998) revealed a linear dynamic range between 4.51 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e and 4.51 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe copy number of each viral genome was measured from 5 \u0026micro;L of total nucleic acid extracted in Tm body-wash fluid and Tm samples using the commercial reagents TOPreal One-step qPCR/RT-qPCR Kit (Enzynomics, Korea) on the CFX Real-Time instruments (BIO-RAD, USA). For AdV, the PCR conditions were as follows: 95℃ for 10 min, 45 cycles at 95℃ for 5 sec, and 60℃ for 30 sec. For HERV, the PCR conditions were as follows: one cycle at 50℃ for 30 min, at 95℃ for 10 min, 45 cycles at 95℃ for 5 sec, and 60℃ for 30 sec. Copy numbers of the samples were calculated using the standard curve method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePropagation of viruses\u003c/h2\u003e \u003cp\u003eTo determine the propagation of the detected viruses, we inoculated 200 \u0026micro;l samples of both Tm body-wash fluid and Tm samples into monolayer-cultured A549 cells (human lung carcinoma, CCL-185). Cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) supplemented with 5% fetal bovine serum (FBS) and 100 U/mL penicillin-streptomycin. The culture was then incubated at 37℃ in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. After five to seven days, the propagation of the detected viruses in the inoculated cells was confirmed by quantifying each viral gene using the methods described in the above section.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eNGS data analysis\u003c/h2\u003e \u003cp\u003eWe obtained six metagenomic datasets from Tm body-wash fluid and Tm samples collected from sewage treatment facilities at the Russian Bellingshausen, Korean King Sejong, and Uruguayan Artigas stations. The raw datasets were processed by trimming sequences with a base quality of \u0026ge;\u0026thinsp;30 and a minimum length of 50 bp. Trimmed data ranging from 30,132,374 to 31,650,580 sequences were analyzed for viral sequences. From these, 3,966 to 143,369 viral reads were extracted based on coverage of \u0026ge;\u0026thinsp;70% and identities of \u0026ge;\u0026thinsp;90% using the NCBI virus database as a cut-off score. In each dataset, at least 16 contigs of \u0026ge;\u0026thinsp;200 bp and one to three contigs of \u0026ge;\u0026thinsp;1,000 bp were identified. Subsequent BLAST analysis using the NCBI VirDB confirmed 17 to 53 viral contigs (Rank1, e-value\u0026thinsp;\u0026le;\u0026thinsp;1e-5), as detailed in Supplementary Table\u0026nbsp;2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eDetection of various viruses by NGS\u003c/h2\u003e \u003cp\u003eIn the Tm body-wash fluid and Tm samples (Tm-related samples) collected from the Bellingshausen station, we identified three insects and one mammalian viral genome. At King Sejong station, two mammalian and two insect viral sequences were detected in the Tm body-wash fluid and Tm samples, respectively. In the Artigas station samples, we identified one mammalian and two insect viral sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Supplementary Table\u0026nbsp;3). Among the viruses identified in the Tm-related samples were insect viruses, such as \u003cem\u003eChoristoneura fumiferana\u003c/em\u003e granulovirus (ChfuGV) and \u003cem\u003eDiolcogaster facetosa\u003c/em\u003e bracovirus (DfBV), as well as animal viruses, including human adenovirus (AdV) and human endogenous retroviruses (HERV). Additionally, plant viruses such as the pepper mild mottle virus (PMMoV) and cucumber green mottle mosaic virus (CGMMV) were found in these Tm-related samples (Supplementary Table\u0026nbsp;4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDetection of mammalian viruses\u003c/h2\u003e \u003cp\u003eTwo mammalian viruses, AdV and HERV, were identified in Tm-related samples from sewage treatment facilities at Antarctic stations. AdV genomes were found in a Tm sample from Bellingshausen station and a Tm body-wash fluid sample from King Sejong station. Additionally, HERV genomes were detected in a Tm sample from King Sejong station and a Tm body-wash fluid sample from Artigas station. In the Tm sample from Bellingshausen station, 235 bp contigs showed approximately 80% coverage and 100% sequence identity with the \u003cem\u003eE1B\u003c/em\u003e 55kDa protein gene of human AdV-1 (AC000017). Similarly, 381 bp adenoviral contigs in the Tm body-wash fluid sample from King Sejong station exhibited 99.7% sequence identity with the \u003cem\u003eE1B\u003c/em\u003e 55kDa protein gene of human AdV-C (NC001405). Partial HERV sequences were also identified in the Tm sample from King Sejong station, showing 97.9% coverage (1,362/1,347 bp) and 92.7\u0026ndash;95.7% sequence identity with the \u003cem\u003ePol\u003c/em\u003e and \u003cem\u003eEnv\u003c/em\u003e genes of HERV K113 (NC022518). Furthermore, a 434 bp HERV sequence in the Tm body-wash fluid sample from the Artigas station was 95.7% identical to the \u003cem\u003eGag\u003c/em\u003e gene sequence of HREV K113 (NC022518), as detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplementary Table\u0026nbsp;4.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eContig annotation of animal viruses detected from each sample\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSite\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVirus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTarget accession no.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTarget gene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCoverage (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eLength/matches (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIdentity (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBellingshausen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTm\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHuman AdV 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAC000017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eE1B 55K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e80.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e235/189\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eKing Sejong\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHERV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC022518\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePol, Env, and LTRs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e97.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1,362/1,347\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e92.7\u0026ndash;95.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTm body-wash\u003csup\u003e\u0026para;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHuman AdV C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC001405\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eE1B 55K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e99.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e381/336\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e99.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArtigas\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTm body-wash\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHERV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC022518\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGag\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e95.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e434/416\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e95.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u003csup\u003e#\u003c/sup\u003e, \u003cem\u003eT. maculipennis\u003c/em\u003e homogenate; \u003csup\u003e\u0026para;\u003c/sup\u003e, \u003cem\u003eT. maculipennis\u003c/em\u003e body-wash fluid\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of viral genome in Tm-related and wastewater samples\u003c/h2\u003e \u003cp\u003eTo quantify viral genomes in the Tm-related samples, primer/probe sets targeting the \u003cem\u003eE1B\u003c/em\u003e and \u003cem\u003ePol\u003c/em\u003e genes of AdV and HERV were designed based on their respective contig sequences. In the Tm body-wash fluid sample from King Sejong station, the adenoviral genome was detected at a concentration of 1.35e\u0026thinsp;+\u0026thinsp;2.36 copies/\u0026micro;L. However, it was not detected in the Tm sample from the Bellingshausen station. Similarly, the HERV genome was not found in the Tm and Tm body-wash fluid samples from the King Sejong and Artigas stations. In the concentrated influent and effluent samples from King Sejong station, the AdV genome was also detected with viral copy numbers of 1.35e\u0026thinsp;+\u0026thinsp;2.16 copies/\u0026micro;L and 1.35e\u0026thinsp;+\u0026thinsp;2.31 copies/\u0026micro;L, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAdenoviral and retroviral quantification from each sample\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSite\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNGS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003erealtime-(RT)PCR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVirus propagation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAdV\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHERV\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBellinsghausen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTm\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHuman AdV 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003csup\u003e\u0026sect;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNT\u003csup\u003e\u0026Dagger;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eKing Sejong\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHERV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTm body-wash\u003csup\u003e\u0026para;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHuman AdV C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.35e\u0026thinsp;+\u0026thinsp;2.36 copies/\u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInfluent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.35e\u0026thinsp;+\u0026thinsp;2.16 copies/\u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eeffluent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.35e\u0026thinsp;+\u0026thinsp;2.31 copies/\u0026micro;L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArtigas\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTm body-wash\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHERV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003e#\u003c/sup\u003e, \u003cem\u003eT. maculipennis\u003c/em\u003e homogenate; \u003csup\u003e\u0026para;\u003c/sup\u003e, \u003cem\u003eT. maculipennis\u003c/em\u003e body-wash fluid; \u003csup\u003e\u0026sect;\u003c/sup\u003e, not detected; \u003csup\u003e\u0026Dagger;\u003c/sup\u003e, not tested\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eThe infectious potential of viruses detected in Tm-related samples and wastewater\u003c/h2\u003e \u003cp\u003eThe inoculated cells were incubated for four to five days, during which their physical characteristics were monitored daily. However, no significant changes were observed in their shape or adhesion properties. Additionally, no viral genomes were detected in either the inoculated cell lysate or supernatant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eInsects are often considered primary or intermediate hosts of human disease agents because of their ability to mechanically transmit diseases by harboring pathogens on their external surfaces and within their internal organs [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Recently discovered in Antarctica, \u003cem\u003eT. maculipennis\u003c/em\u003e, which originated in the Northern Hemisphere, was introduced to Antarctic stations 20 years ago [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. As prominent non-native species, they present challenges for eradication in Antarctica [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Given the uncertain impact of non-native species on the Antarctic ecosystem, this study aimed to assess the potential role of NNF as a vector for viral transmission.\u003c/p\u003e \u003cp\u003eA variety of viruses, including insect, plant, and mammalian viruses, were identified in the virome analysis of Tm-related samples (Tm body-wash fluid and Tm samples). Insect viruses, such as \u003cem\u003eChoristoneura fumiferana\u003c/em\u003e granulovirus (ChfuGV) and \u003cem\u003eDiolcogaster facetosa\u003c/em\u003e bracovirus (DfBV), were detected along with the plant viruses pepper mild mottle virus (PMMoV) and cucumber green mottle mosaic virus (CGMMV). In addition, mammalian viruses such as human adenovirus (AdV) and human endogenous retrovirus (HERV) were identified. Among them, ChfuGV and DfBV, detected in at least one Tm-related sample, are recognized for their pathogenic and parasitic effects on insects, such as moths and wasps [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. It is plausible that these viruses originated from \u003cem\u003eT. maculipennis\u003c/em\u003e itself. PMMoV and CGMMV, which were found in all Tm-related samples, infect plants and cause crop diseases [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. These plant viruses, which are commonly detected in human feces and wastewater, serve as indicators of enteric viruses and microbial source tracking in wastewater treatment [\u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. As these plant viruses possess exceptional stability in water and diverse environmental conditions, their presence in Tm-related samples is thought to originate from wastewater. This suggests that \u003cem\u003eT. maculipennis\u003c/em\u003e harbors diverse pathogens from sewage.\u003c/p\u003e \u003cp\u003eAdV and HERV, which have been detected in Tm body-wash fluid and Tm samples from Antarctic research stations, are important mammalian viruses. AdV, which is associated with gastroenteritis in young children, is often transmitted via the oral-fecal route and is frequently found in wastewater [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. HERV, an endogenous retrovirus inserted into the human germ cell DNA, is commonly found in the human gut and feces [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Although no other animal RNA viruses have been detected, AdV and HERV, which are relatively more resistant to degradation than RNA viruses, seem to be more easily detected in \u003cem\u003eT. maculipennis\u003c/em\u003e washed fluid or lysates because they can remain preserved in environmental samples for a prolonged period.\u003c/p\u003e \u003cp\u003eA quantification test for adenoviral and retroviral genes (\u003cem\u003eE1B\u003c/em\u003e gene of AdV and \u003cem\u003ePol\u003c/em\u003e gene of HERV) to assess their abundance in Tm-related samples, indicated a very low quantity of viral genomes: 1.35e\u0026thinsp;+\u0026thinsp;2.36 copies/\u0026micro;L in Tm body-wash fluid sample from King Sejong station. Although the concentrations of these viral genes were near the detection limits, we concluded that \u003cem\u003eT. maculipennis\u003c/em\u003e carried these viral genes at very low concentrations. This conclusion is based on the consistently negative results in non-template controls and the fact that our analysis spanned a dynamic range of concentrations up to e\u0026thinsp;+\u0026thinsp;3 in the standard curves [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The presence of a small amount of adenovirus in wastewater, 1.35e\u0026thinsp;+\u0026thinsp;2.16 copies/\u0026micro;L (influent) and 1.35e\u0026thinsp;+\u0026thinsp;2.31 copies/\u0026micro;L (effluent) suggested that the viral genes in \u003cem\u003eT. maculipennis\u003c/em\u003e originated from the habitat at King Sejong station. However, the AdV gene was not detected in Tm samples from the Bellingshausen station, and HERV was not found in Tm or Tm body-wash fluid samples from the King Sejong and Artigas stations. Previous laboratory studies have shown that the concentration of live viruses and the duration of their external exposure are crucial factors that enable mechanical transmission by vectors [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In particular, research on the mechanical transmission of influenza virus by houseflies indicates that low viral concentrations, high incubation temperatures, and prolonged incubation can reduce the persistence of influenza viruses [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In these studies, although influenza virus genes were detected up to 96 h post-exposure, there was a significant decrease in virus isolation from flies as environmental exposure increased. In our study, while the duration of viral exposure on \u003cem\u003eT. maculipennis\u003c/em\u003e was not precisely determined, the viral genomes in immediately collected samples were minimal, and the viability of the viruses was extremely low. However, given the number of flies used in our study and the uncertain environmental exposure time, we cannot completely rule out their potential as carriers of disease.\u003c/p\u003e \u003cp\u003eThe limited detection of AdV and the absence of HERV in the concentrated wastewater samples may be attributed to the long duration of transportation from Antarctica to the laboratory, despite being stored at \u0026minus;\u0026thinsp;20℃. Storage and repeated thawing of liquid wastewater can reduce the measurable concentrations of viral nucleic acids, especially RNA [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Recent studies suggest that storing wastewater solids, like RNA extract or concentrate, at \u0026minus;\u0026thinsp;20℃ for approximately four months yields more reliable viral RNA quantification compared to storing unprocessed raw wastewater at 4℃ or \u0026minus;\u0026thinsp;80℃ [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Therefore, for future research on Antarctic wastewater, it is crucial to preprocess or concentrate wastewater before shipment from Antarctica.\u003c/p\u003e \u003cp\u003eAlthough our study detected only modest amounts of the virus in Tm-related and wastewater samples, it represents the first assessment of the potential risk posed by NNFs introduced into the Antarctic as a vector. Monitoring non-native species in Antarctica and evaluating the associated risks is essential, particularly given the increasing likelihood of introductions attributable to climate change and heightened human activity. Our study contributes to the establishment of risk assessment methods for non-native Antarctic species and offers a framework for diverse evaluations in future research.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study explored the potential of the NNF, \u003cem\u003eT. maculipennis\u003c/em\u003e, found in sewage treatment facilities at Antarctic research stations, to mechanically transmit viruses. A key finding was the detection of various viral genomes in \u003cem\u003eT. maculipennis\u003c/em\u003e, including the human adenovirus (AdV) and human endogenous retrovirus (HERV), which are commonly found in human feces and wastewater. However, the detected viral quantities were minimal and nonviable, suggesting that \u003cem\u003eT. maculipennis\u003c/em\u003e in Antarctica is unlikely to be an effective mechanical transmitter of these viruses, likely because of low viral loads. However, the possibility remains that \u003cem\u003eT. maculipennis\u003c/em\u003e can potentially become a vector under conditions conducive to mechanical transmission. This is the first study to assess the risk posed by \u003cem\u003eT. maculipennis\u003c/em\u003e as a vector of pathogens in Antarctica. This contributes to a broader evaluation of the risk of mechanical transmission of viruses by NNFs, providing insight into the potential implications of their future introduction.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eT.mculipennis\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eTrichocera maculipennis\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTm-related sample\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eT. mculipennis\u003c/em\u003e-body wash fluid and \u003cem\u003eT. maculipennis\u003c/em\u003e homogenate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNNF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enon-native fly\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eChfuGV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eChoristoneura fumiferana\u003c/em\u003e granulovirus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDfBV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eDiolcogaster facetosa\u003c/em\u003e bracovirus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAdV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eadenovirus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHERV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHuman endogenous retrovirus\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePMMoV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epepper mild mottle virus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCGMMV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecucumber green mottle mosaic virus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePEG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epolyethylene glycol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efetal bovine serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank\u0026nbsp;the Russian Bellingshausen station\u0026nbsp;and\u0026nbsp;the Uruguayan Artigas station\u0026nbsp;for their collaboration and the generous provision of NNFs for analysis. We extend our thanks to the Korean overwintering crews for their invaluable cooperation in facilitating our research endeavors for several years. This research was supported by\u0026nbsp;the Korea Polar Research Institute (PE24140, PE24170) and the Chilean National Research Program (FOVI220036).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIllumina HiseqX 150PE data have been deposited in the GenBank Sequence Reads Archive (SRA) under the accession number PRJNA1052610.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor’s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSYL, JHK and SK conceptualized and designed this study. SYL, SK, KCP and SMC performed the experiments and analyzed the data. CXS, LR, HAB, TCM, AS and EJ collected and supplied fly samples. SYL, JHK, SK, KCP, SMC and SK contributed to the finalization of the manuscript. JHK and SK managed the project. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHughes KA, Convey P. Alien invasions in Antarctica‒is anyone liable? Polar Research. 2014;33:22103.\u003c/li\u003e\n\u003cli\u003eMatheson P, McGaughran A. How might climate change affect adaptive responses of polar arthropods? Diversity. 2023;15:47.\u003c/li\u003e\n\u003cli\u003eLe\u0026oacute;n MR, Hughes KA, Morelli E, Convey P. International response under the Antarctic treaty system to the establishment of a non-native fly in Antarctica. Environ Manage. 2021;67:1043\u0026ndash;59.\u003c/li\u003e\n\u003cli\u003eVolonterio O, Le\u0026oacute;n MR, Convey P, Krzemińska E. First record of Trichoceridae (Diptera) in the maritime Antarcitc. Polar Biol. 2013;36:1125\u0026ndash;31.\u003c/li\u003e\n\u003cli\u003ePotocka M, Krzemińska E. \u003cem\u003eTrichocera maculipennis\u003c/em\u003e (Diptera)-an invasive species in Maritime Antarctica. PeerJ. 2018;14:e5408. \u003c/li\u003e\n\u003cli\u003eRep. of Korea. Non-native flies in sewage treatment plants on King George Island, South Shetland Islands. Information Paper 52. Antarctic Treaty Consultative Meeting ⅩXXIX. 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Environ Res. 2023;10:117636.\u003c/li\u003e\n\u003cli\u003eKhamesipour F, Lankarani KB, Honarvar B, Kwenti TM. A systematic review of human pathogens carried by the housefly (Musca domestica L.). BMC Public Health. 2018;18:1049.\u003c/li\u003e\n\u003cli\u003eNielsen AA, Skovg\u0026aring;rd H, Stockmarr A, Handberg KJ, J\u0026oslash;rgensen PH. Persistence of low-pathogenic avian influenza H5N7 and H7N1 subtypes in hose flies (Diptera: Muscidae). J Med Entomol. 2011;48:608\u0026ndash;14. \u003c/li\u003e\n\u003cli\u003eWanaratana S, Panyim S, Pakpinyo S. The potential of house flies to act as a vector of avian influenza subtype H5N1 under experimental conditions. Med Vet Entomol. 2011;25:58\u0026ndash;63.\u003c/li\u003e\n\u003cli\u003eSalamatian I, Moshaverinia A, Razmyar J, Ghaemi M. In vitro acquisition and retention of low-pathogenic avian influenza H9N2 by Musca domestica (Diptera: Muscidae). 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Sci Total Environ. 2020;713:136604.\u003c/li\u003e\n\u003cli\u003eVogt VM. \u0026ldquo;Retroviral virions and genomes\u0026rdquo; in Retroviruses, Coffin J M, Hughes S H, Varmus HE, Eds. (Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY). 1997.\u003c/li\u003e\n\u003cli\u003eCarding SR, Davis N, Holyes L. Review article: the human intestinal virome in health and disease. Aliment Pharmacol Ther. 2017;46:800\u0026ndash;15.\u003c/li\u003e\n\u003cli\u003eCinek O, Kramna L, Odeh R, Alassaf A, Ibekwe MAU, Ahmadov G, et al. Eukaryotic viruses in the fecal virome at the onset of type 1 diabetes: A study from four geographically distant African and Asian countries. Pediatr Diabetes. 2021;22:558\u0026ndash;66. \u003c/li\u003e\n\u003cli\u003eBustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55:611\u0026ndash;22. \u003c/li\u003e\n\u003cli\u003eHokaj\u0026auml;rvi AM, Rytk\u0026ouml;nen A, Tiwari A, Kauppinen A, Oikarinen S, Lehto K, et al. The detection and stability of the SARS-CoV-2 RNA biomarkers in wastewater influent in Helsinki, Finland. Sci Total Environ. 2021;770:145274.\u003c/li\u003e\n\u003cli\u003eWeidhaas J, Aanderud ZT, Roper DK, VanDerslice J, Gaddis EB, Ostermiller J, et al. Correlation of SARS-CoV-2 RNA in wastewater with COVID-19 disease burden in sewersheds. Sci Total Environ. 2021;775:145790.\u003c/li\u003e\n\u003cli\u003eFernandez-Cassi X, Scheidegger A, B\u0026auml;nziger C, Cariti F, Corzon AT, Ganesanandamoorthy P, et al. Wastewater monitoring outperforms case numbers as a tool to track COVID-19 incidence dynamics when test positivity rates are high. Water Res. 2021;200:117252. \u003c/li\u003e\n\u003cli\u003eSimpson A, Topol A, White BJ, Wolfe MK, Wigginton KR, Boehm AB. Effect of storage conditions on SARS-CoV-2 RNA quantification in wastewater solids. PeerJ. 2021;9:e11933.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Trichocera maculipennis, Non-native winter crane fly, Antarctica, Virus, Mechanical transmission","lastPublishedDoi":"10.21203/rs.3.rs-4209981/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4209981/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe Antarctic environment is susceptible to the introduction of non-native species due to its unique ecosystem, which has evolved under geographical isolation and extreme climatic conditions over an extended period. The non-native winter crane fly, \u003cem\u003eTrichocera maculipennis\u003c/em\u003e, which was recently introduced to maritime Antarctica, is likely to pose a serious threat to the Antarctic ecosystem. In this study, the possibility of the mechanical transmission of viruses was evaluated.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe possibility of the mechanical transmission of viruses was evaluated using next-generation sequencing (NGS), quantitative polymerase chain reaction (qPCR), and virus isolation methods from \u003cem\u003eT. maculipennis\u003c/em\u003e (Tm)-related samples (Tm body-wash fluid and Tm homogenate) collected from habitats and sewage treatment facilities located at three research stations in Antarctica.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOur findings revealed the presence of human adenovirus (AdV) and human endogenous retrovirus (HERV) in Tm-related samples through virome analysis. Notably, these viruses are commonly detected in human feces. In addition, we identified pepper mild mottle virus (PMMoV) and cucumber green motortle mosaic virus (CGMMV), which are known indicators of enteric viruses, in all Tm-related samples. It is postulated that these viruses originated from wastewater, as evidenced by their detection in the wastewater samples. However, the minute quantities of AdV and HERV genomes detected in Tm-related samples through qPCR, coupled with the observed non-viability of AdV, indicate that \u003cem\u003eT. maculipennis\u003c/em\u003e has limited potential for mechanical transmission.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur study represents the first evaluation of the potential risk of non-native species serving as vectors for pathogens in Antarctica despite the relatively low quantities of detected viruses. This study provides valuable insights for further risk assessments of non-native species that are newly introduced or are likely to be introduced to Antarctica due to climate change or increased human activity.\u003c/p\u003e","manuscriptTitle":"Detection of human enteric viral genes in a non-native winter crane fly, Trichocera maculipennis (Diptera) in the sewage treatment facilities in Antarctic stations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-08 07:30:40","doi":"10.21203/rs.3.rs-4209981/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-05T03:45:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-04T18:50:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"45331410813817120970974764504562225402","date":"2024-05-14T18:36:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-24T10:43:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-03T05:42:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-03T05:41:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2024-04-03T05:01:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"efec8ec4-598f-4644-93cc-efef73c016b7","owner":[],"postedDate":"April 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-25T16:08:22+00:00","versionOfRecord":{"articleIdentity":"rs-4209981","link":"https://doi.org/10.1186/s13071-024-06555-4","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2024-11-24 15:58:06","publishedOnDateReadable":"November 24th, 2024"},"versionCreatedAt":"2024-04-08 07:30:40","video":"","vorDoi":"10.1186/s13071-024-06555-4","vorDoiUrl":"https://doi.org/10.1186/s13071-024-06555-4","workflowStages":[]},"version":"v1","identity":"rs-4209981","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4209981","identity":"rs-4209981","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}