Clematis vitalba L. is a climbing shrub and a pioneer plant in abandoned orchards or vineyards that are widespread in temperate climate zones. In past years, several viruses infecting the Clematis species have been identified, including different ilarviruses. Prunus virus I (PrVI) is a recently described ilarvirus, which has been shown to infect sweet cherries and peaches in Greece. Moreover, its presence has been detected in ornamental Clematis in Russia. In the present work, we analyzed the virome of wildly growing C. vitalba plants from Hungary, Slovakia and Croatia showing different kinds of symptoms using high-throughput sequencing (HTS) of small RNAs or ribodepleted RNAs. Applying HTS enabled us to identify the presence of PrVI in C. vitalba, and the bioinformatic analyses were further validated with RT-PCR using PrVI-specific primers and Sanger dideoxy sequencing. Nearly full genome sequences of all three viral RNAs of one Hungarian, two Slovak and one Croatian isolate were determined. Their phylogenetic analysis showed high similarity to each other and to other PrVI isolates described from Central Europe. As the sampled plants were co-infected with other viruses, it is not possible to determine a direct correlation between the infection with PrVI and the observed symptoms. Analyses of different Prunus species in stock collection showed infection of several peach and sweet cherry varieties in Hungary. Our results expand the knowledge on the natural host range of PrVI and highlight the necessity to evaluate alternative plant hosts (even non-Prunus) of PrVI and the role of the virus in the etiology of the potential diseases.  Clematis is a genus of about 380 species belonging to the Ranunculaceae family. They are deciduous, perennial plants growing in temperate regions in the Northern hemisphere and rarely in the tropics. Species and cultivars differing in color and shape of the flowers mainly originate from China and Japan and are used as ornamental plants. They have been constantly bred since 1862, maintained at collections and distributed worldwide, offering more than one hundred different cultivars. The native European version of the genus, Clematis vitalba L., is a fast-growing climbing shrub. It grows in and around forested areas, wet, riparian and recently disturbed areas, which is why it is widespread in the vicinity of vineyards and orchards. Although it grows very fast, it never grows as dense mass in this climate. In New Zealand, its invasiveness causes severe damage by blocking the sunlight from the lower vegetation and weighing down the branches of the trees. A biocontrol program to stop its spread was launched in 1989 [C. vitalba was completed, revealing that although it can be infected by several fungi and bacteria, only very few of them are pathogenic for this species. C. vitalba can also be infected by phytoplasmas [Clematis species can also be infected with viruses [Clematis species via serological methods and, only recently, using high-throughput sequencing (HTS), an unbiased detection method [ viruses . Eleven n method  , apple mosaic virus (ApMV) and PrVI  , Moroccaand PrVI .While TSV infection has been reported from the Croatian part of former Yugoslavia ,13 and fIlarvirus (family Bromoviridae). The genome of ilarviruses (genus Ilarvirus in the family Bromoviridae) consists of three positive-stranded RNA segments (RNA1–3) [Prunus species, PrVI is unique because it is the first and still only ilarvirus from subgroup 1 that infects the Prunus species [Clematis cultivars ; in Slovenia, it has been found growing in Picris echioides L. (Compositae) and sampled as a weed in the surrounding of tomato fields.PrVI was first described in 2021 from the Imathia region in Greece via HTS analysis of an asymptomatic sweet cherry, Ferrovia cultivar . Accordi(RNA1–3) . RNA1 is(RNA1–3) . RNA3 is species . An RT-P species  and Russ species . While iC. vitalba plants in Hungary, Slovakia and Croatia using high-throughput sequencing of small RNAs of the Hungarian samples and ribodepleted RNAs of the Slovak and Croatian samples. Nearly whole genome sequence data (lacking some of the UTR sequences) were used to investigate their phylogenetic relationship. Small-scale surveys testing C. vitalba plants and Prunus collections were also carried out in order to further investigate the incidence of the virus.In our work, the presence of PrVI was detected in the virome of C. vitalba were collected in July 2018 at Budakeszi (close vicinity to Budapest). RNA was extracted from 6 symptomatic leaves (3 showing line patterns and 3 chlorotic spots) using the phenol–chloroform method [Leaf samples from different branches of the same symptomatic m method  and werePeach and sweet cherry cultivars were sampled in 2017 and 2019, respectively. The peach trees  representing 34 cultivars were kept under an insect proof net and were the same plants whose viromes were characterized recently .C. vitalba were sampled in July 2021 (sample PlCv5) and June 2022 (sample PL622) around Bratislava, Slovakia. Total RNAs from leaves were extracted using a SpectrumTM Plant Total RNA Kit . Ribosomal RNA was removed using the Zymo-Seq RiboFree Universal cDNA Kit . Ribosome-depleted RNA preparations were used for double-stranded cDNA synthesis using the SuperScript II kit , and the samples were processed with the transposon-based chemistry library preparation kit  followed by HTS on an Illumina MiSeq platform . Leaf samples of C. vitalba from the vicinity of Zagreb with chlorotic mottling was used in 1989 to inoculate Chenopodium quinoa plants as described in Rana et al. [Ch. quinoa with local chlorotic and necrotic symptoms and systemic chlorotic mottling containing Cle-1 isolate of what was then considered to be TSV had been prepared and sent to DSMZ by Davor Miličić in 1991. The isolate was further propagated on Chenopodium quinoa and added to the DSMZ plant virus collection under accession No. PV-0309. Extracted total RNA (QIAGEN RNeasy) was sequenced using Illumina NextSeq in 2020, and the genomic sequences were deposited at GenBank (OL584348-50). Ch. quinoa containing various Cle isolates from the virus archives of the Department of Biology in Zagreb were used to mechanically inoculate [Nicotiana megalosiphon, N. glutinosa, Ch. amaranticolor and Ch. quinoa for the virus revival. Namely, Cle-1 isolate 680 from 1980 and 847 from 1985, as well as Cle-2 isolate 710 from 1981, respectively, were inoculated in 2021. Total nucleic acids (TNAs) were extracted using the CTAB-based method with buffer containing 2% PVP [The dried leaves of noculate  experimeg 2% PVP . TNAs weFor bioinformatics analysis of the small RNA reads, we used a CLC Genomic Workbench. After trimming and quality control, longer contigs were built de novo from the non-redundant reads employing an assembler of CLC (de novo assembly) using the default options: a word size of 20, a bubble size of 50 and simple contig sequences with a minimum length of 35 nt . To diaghttps://www.qiagenbioinformatics.com/ accessed on 1 September 2023) with automatic graph parameters set and with reads mapped back to contigs with the following parameters  and minimum contig length of 1000 bp. Contigs were aligned to the viral genomes database  using CLC Genomics Workbench v9.5.2. Alternatively, the reads were mapped against a selected full-length reference PrVI genome sequence using Geneious v.8.1.9.High-quality trimmed reads were used for de novo assembly using a CLC Genomics Workbench v9.5.2 . The purified products were cloned into a pJET vector system  and Sanger sequenced using several virus specific primers to cover the whole amplified and cloned viral part. Sequences were deposited into GenBank . For surValidation of the RNAseq results was performed via two-step RT-PCR using in-house designed PrVI-specific primers , spanninhttps://www.dsmz.de/fileadmin/_migrated/content_uploads/DAS-ELISA_01 accessed on 1 August 2023) using TSV antiserum AS-0903 available at DSMZ.A standard DAS–ELISA test was carried out  showing different leaf chlorosis were sampled in 2018, 2020 and 2021. Total nucleic acid was extracted using the phenol–chloroform method. cDNA was prepared using the Maxima H minus kit . Sweet cherry and peach samples, growing in a cultivar collection at Érd, were also tested for the presence of PrVI. From these plants RNA was extracted with the CTAB method [To survey the presence of PrVI in both B method . Clematis vitalba in Slovakia, leaf samples of five plants growing in different locations of Bratislava was harvested and analyzed with two-step RT-PCR as described in To survey the presence of PrVI in wild C. vitalba growing at the edge of a woodland area in Budakeszi  showed a nicely shaped line pattern symptom, suggesting a potential virus infection  and PrVI in two clematis samples (PlCv5 and PL622) , from whTo obtain a genome-wide confirmation of the presence of PrVI, ORFs located on all three genomic RNA segments were specifically targeted using RT-PCR . In the PlCv5, amplicons representing the coding part for methyltransferase, helicase, a gap region between the two, RNA-dependent RNA polymerase (RdRp), movement protein (MP) and CP were successfully amplified , while tC. vitalba was found to be a host of a new strain of TSV in the 1980s [Ch. quinoa leaves, were dried and deposited as Cle-isolates in the virus archives of the Department of Biology in Zagreb. Essentially, two types of isolates named Cle-1 and Cle-2 were deposited over the 1980s. In the Cle-1 isolates, the symptomatology in the original host was characterized by chlorotic spots and in the Cle-2 by yellow netting [ netting . Both isVicia faba sample from Sudan (PV-0903). PV-0309 showed no positive reaction , whereas the homologous TSV isolate PV-0903 and PV-0615 (originating from sunflower/India) showed a strong reaction , indicating a serological differentiability. Out of four archival Cle-isolates mechanically inoculated here, three produced symptoms in Ch. quinoa and Nicotiana plants and thus were successfully revived. Cle-1 680 from 1980 and 847 from 1985 expectedly [Ch. quinoa, turning into necroses later during the course of infection, and systemic mottling. Yellow net appeared in N. megalosiphon and N. glutinosa in Cle-1 isolates, whilst Cle-2 isolate 710 from 1981 produced only yellow net in N. megalosiphon  were usually higher than the identity of isolates infecting other plant species (indicated with different colors).Investigation of the proteins encoded using the PrVI variants showed that RNA1-encoded proteins have higher than 98.4% identity, RNA2-encoded RdRp proteins have higher than 98.2% identity and RNA3-encoded MP protein have more than 98% identity between different strains, while amino acid identity between the RNA2-encoded 2b and RNA3-encoded CP is more variable, higher than 94.1 and 95.6%, respectively . The ideC. vitalba plants in the close vicinity of Budapest (Szada and Gödöllő) showed that some of them are infected with PrVI, but the presence of PrVI was not connected to the presence of the symptoms . Only one of these samples tested positive for the presence of PrVI, confirming there is no direct correlation between PrVI infection and the observed symptom manifestation.Moreover, we further tested five Prunus species in Hungary, we surveyed 89 peach and 106 sweet cherry trees at Erd, in a stock collection in a close vicinity of Budapest, representing 34 and 32 cultivars, respectively. All individuals of ten peach  and three sweet cherry cultivars  were found infected with PrVI, suggesting that the origin of the infection was the clonal propagation of the cultivar , having no reference in GenBank. Although we were not capable of its direct identification, the results showed hits to several different ilarviruses, suggesting the infection with a virus belonging to this genus but lacking a reference genome. This result could be validated, and during the validation, the identification of the virus was possible, showing that small RNA HTS is a potent diagnostic tool. Size distribution of the virus-derived siRNAs showed extreme bias of 21–22 nt long reads. This size range is typical of the products of antiviral DICERs: DCL4 and DCL2 . The higClematis plants have been mainly investigated using serological methods in the past. Although the infection of C. vitalba has been reported by TSV and ApMV, no sequence data are available for these strains. TSV was identified using serological methods [Clematis in Croatia, near Zagreb, showed that the archival material from 1991 used for obtaining this sequence  had been indeed infected by PrVI. This isolate also shows no serological reaction with the DSMZ TSV antiserum (AS-0903) in DAS–ELISA, indicating serological differentiability based on differences in the antigenic domains of the coat protein. The course of events reconstructed in this research showed that this sequence comes from the so-called Cle-1 isolates stored in the virus archive of the Department of Biology in Zagreb. Additional RT-PCR analyses of two Cle-1 and one Cle-2 isolates from the 1980s confirmed the presence of PrVI amplicons in the archival Ch. quinoa dried leaves from Zagreb  showed high identity (about 80%) in the amino acid sequence of the MP encoded by TSV dahlia strain [Clematis from today’s Croatia [C. vitalba in Italy [Clematis, this infection cannot be a falsely diagnosed PrVI as the amino acid identity of the Greek sweet cherry strain and the ApMV from Clematis in Turkey is low. Moreover, the primers that were used to amplify the partial RNA3 of ApMV could not anneal to the PrVI strains.Viromes of  methods ,10, whil methods . Cle str methods . It was m Zagreb b. Amazinm Zagreb a. It is a strain  or with a strain . The samPrunus spp. in Greece [Clematis spp. Whether it is a real bias or just a result of random and uneven sampling of the rural samples is a question that needs to be investigated later. Nonetheless, Clematis spp. could act as a potent natural PrVI host (in addition to Prunus spp.), thus making it quite an interesting target of epidemiological studies regarding the spread or persistence of this new ilarvirus species in the environment.PrVI has been described from n Greece , but latChirkov et al.  observedPrunus species identified the infection of several peach and sweet cherry cultivars in a small stock collection in Hungary. The peach trees, as potential plum pox virus (sharka) hosts, were kept under an insect proof net, while the sweet cherry trees were grown in an open field. We found 38% of the peach and 30% of the sweet cherry trees tested positive for PrVI, showing that similarly to Greece, the virus is present in the cultivated Prunus species in Hungary. The PrVI infection was restricted to some cultivars, and we could not detect the infection between neighboring different cultivars, suggesting that the infection in this case happened through propagation. Prunus species infected with the virus did not show any specific symptoms, while symptoms in Clematis could not be directly linked to the presence of the virus, as the sampled plants were co-infected with other viruses.Our small-scale survey of Picris sp. in Slovenia, suggesting a wider host range of the virus than just Prunus or Clematis genera members. PrVI has also been described from an herbaceous plant Ch. quinoa plants [Clematis could thus be acquired by pollen, and the same may be true in other cultivated or wild plants. Further research is needed to clarify these epidemiological scenarios. As an ilarvirus, it is highly possible that PrVI can be transmitted by pollen ,29. MoreClematis host range reported previously and suggests Clematis vitalba could be a natural reservoir of PrVI. However, the epidemiology of the virus, having apparently a broad host range, remains still to be elucidated. These data also highlight the necessity to reconsider alternative plant hosts of PrVI and their role in the etiology of the potential diseases.Our finding expands Prunus trees and cannot suggest at this stage a directive for its possible regulation. This question could be answered only after further research investigating the biology, symptomatology and transmission of PrVI in detail. Based on further information on how widespread PrVI is and if the virus has a negative impact on cultivated plants, decisions can be made regarding a necessary regulation or consideration in the certification of planting material.We do not know if PrVI can be harmful on the cultivated Before the rapid development of nucleic-acid-based research, plant virology relied mainly on biological characterization including symptom description, transmission experiments as well as serological and electron microscopical detection of viruses. The huge work of classical virologists enabled us to find correlations between viral diseases and sequence data of the later sequenced viruses. With the fast evolution of sequencing techniques, an unexpected number of plant-infecting viruses are currently being described, and their presence can easily be physically verified. Classical virologists took care of the virus isolates they characterized and deposited them in viral collections. Our research shows how recent and classical virus research can complement each other to explore and clarify the legacy of these great classical virologists—represented in our case by Professors Davor Miličić and Giovanni Paolo Martelli. We would like to honor their tremendous work and dedicate our research to them.


Halorubrum lacusprofundi, a model organism for studying cold adaptation, exhibits one secondary chromosome and one megaplasmid that include a large arsenal of virus defense mechanisms. We isolated a virus  infecting Hrr. lacusprofundi, and present an in-depth characterization of the virus and its interactions with Hrr. lacusprofundi. While studying virus-host interactions between Hrr. lacusprofundi and HRTV-DL1, we uncover that the strain in use (ACAM34_UNSW) lost the entire megaplasmid and about 38% of the secondary chromosome. The loss included the majority of virus defense mechanisms, making the strain sensitive to HRTV-DL1 infection, while the type strain (ACAM34_DSMZ) appears to prevent virus replication. Comparing infection of the type strain ACAM34_DSMZ with infection of the laboratory derived strain ACAM34_UNSW allowed us to identify host responses to virus infection that were only activated in ACAM34_UNSW upon the loss of virus defense mechanisms. We identify one of two S-layer proteins as primary receptor for HRTV-DL1 and conclude that the presence of two different S-layer proteins in one strain provides a strong advantage in the arms race with viruses. Additionally, we identify archaeal homologs to eukaryotic proteins potentially being involved in the defense against virus infection.Halophilic archaea  are known to exhibit multiple chromosomes, with one main chromosome and one or several smaller secondary chromosomes or megaplasmids.  Hrr. lacusprofundi exhibits three replicons, the main chromosome CHR1, a secondary chromosome CHR2 and the megaplasmid pHLAC01. A metagenomics study revealed that the main chromosome of Hrr. lacusprofundi is highly conserved between strains, but the secondary chromosomes and megaplasmids are very diverse and responsible for the genetic diversity of the species  system, Toxin-Antitoxin (TA) systems, and Restriction Modification (RM) systems, additional to changes of cell surface proteins, to defend against viruses . The majHere we present an in-depth characterization of the interactions of an archaeal tailed virus and its host, including the characterization of the virus life cycle, determination of the virus host range, analysis of the host transcriptional response to virus infection and the characterization of virus escape mutants. We uncover that the host strain in use lost an entire megaplasmid and a part of its secondary chromosome while being grown under laboratory conditions. This loss included the majority of virus defense systems, including a CRISPR-Cas system, TA and RM systems and a BREX system, which allows us to discover new putative virus defense mechanisms and the host receptor for virus attachment.Halorubrum lacusprofundi strain, that was isolated from a sample taken in 2014 from Deep Lake, Antarctica  . Using HNatrialba virus PhiCh1 capsid proteins, the putative head-tail adaptor, the tail tape measure protein, and the tail terminator protein. ORF18 and ORF25 products, identified in the virus particle, did not show any similarities to other viral proteins, therefore, their role within the virus particle remains enigmatic. Two proteins that could be involved in host attachment were identified. ORF24 product, that is present in virus particle, exhibits a immunglobulin-like fold (fibronectin type III domain) at the N-terminus. This domain is commonly found in bacterial phage tails and has been shown to exhibit membrane binding activity (p = 99.06). ORF4 product, with significant similarities to DNA double-strand break repair protein MRE11, and ORF43 product, annotated as transcription initiation factor, were also detected in virus particles. Both could be associated with the virus genome. ORF4 product might facilitate circularization of the virus genome within the host, while ORF43 product could recruit the host RNA polymerase ensuring timely transcription of the viral genome after injection.Proteins building the virus particle were identified by mass spectrometry of purified virus particles and confirm our predictions . The majactivity . In the activity . It coulNC_021537) . Therefore, HRTV-DL1 should be classified into a different genus, that we propose to name Deelavirus in accordance with its origin (Deep Lake).Genome comparison with 63 arTV  and phyl_021537)  that havHaloarcula hispanica tailed virus 1 (HHTV-1), binds extremely slowly with virions adsorbed only after 3 h and binding of Halorubrum virus HRTV-1 was only detected after 25 min    . CRISPR ium DL1) . CRISPR impaired , showingimpaired , 2018.Hrr. lacusprofundi ACAM34 provided by the DSMZ. Surprisingly, while the virus successfully adsorbed to the host cell and injected its genome, HRTV-DL1 was not able to complete a lytic life cycle in the DSMZ strain. No lysis was observed in cultures and virus DNA could not be detected within the host at 56 h p.i.  and the HRTV-DL1 sensitive strain (ACAM34_UNSW), we re-sequenced the genome of both strains  representing the main chromosome (CHR1) with 99.99% identity to the published genome. Contig 2 is circular with a size of 525,899 bp , representing the secondary chromosome (CHR2) with 99.99% identity to the published sequence. Contig 3 is circular with a size of 431,344 bp , representing the plasmid pHLAC01 with 99.99% identity to the published sequence. Contig 4 is linear with the size of 6,176 bp matching a short region on CHR2 covering Hlac_3232 to Hlac_3234 with an insertion of a transposon within Hlac_3234. This contig has a lower coverage (432) than contig 2 and likely represents a variant of this region in the CH2 of the strain. In conclusion, the genome of ACAM34_DSMZ is to 99.99% identical with the published sequence.Halobacterium salinarium laboratory strain  and “Replication and repair” (102 out of 299), that are likely essential under virus infection or other stress condition influencing genome stability . HoweverWe analyzed the host response to HRTV-DL1 infection by determining transcriptional changes at two time points 32 h p.i.  and 56 h p.i.  for both strains ACAM34_DSMZ and ACAM34_UNSW.The ACAM34_DSMZ transcriptome recovered no reads for the virus genome in both samples, which is consistent with our results that the virus genome copy number is already very low at 32 h p.i. (time point 1 = T1) and not detectable at 56 h p.i. (time point 2 = T2) . None ofThe ACAM34_UNSW transcriptome recovered 0.2% of all mapped reads 32 h p.i. and 41% of all mapped reads at 56 h p.i. for the virus genome, consistent with the virus genome copy numbers increasing over time .The transcriptional profile at time point 1 is very similar to that of T1 in ACAM34_DSMZ with 371 genes upregulated (log2 > 1) and none downregulated, and functional categories transcription, replication and repair mechanisms, posttranslational modification, nucleotide metabolism and energy production and conversion being the most upregulated . AdditioHowever, T2 shows a different profile, with 201 genes upregulated and 333 genes downregulated. The most upregulated functional category is ‘replication and repair’, consistent with a takeover of HRTV-DL1 and the degradation of the host genome . Genes iWithin the 30 most downregulated genes at T2 we find genes encoding for ribosomal proteins, for proteins involved in amino acid metabolism, proteins involved in energy production and conversion and proteins involved in oxidative stress response , being aOnly one gene, predicted to be potentially involved in virus defense (ACAM34UNSW_01791), an Argonaut related nuclease [PADLOC ] that isACAM34UNSW_01107 (Hlac_1086), shows significant similarities  to Transposon-associated TnpB, that is guided by an RNA derived from a sequence upstream of the TnpB  and alsoTwo hypothetical proteins (ACAM34UNSW_01618 and ACAM34UNSW_01620) within the 30 most upregulated genes, enclose a predicted chromosome maintenance protein (SMC), ACM34UNSW_01619 . Additionally, another predicted SMC, ACM34UNSW_01796, is also slightly upregulated . SMC complexes were found to interfere with virus replication in humans . AdditioH. volcanii, however, Hlac_3088 is too small to be an S-layer protein. When comparing Hlac_3088 expression levels with those of the S-layer protein (Hlac_2976), Hlac_3088 has usually lower expression levels compared to the S-layer. However, at T2 it actually exceeds the expression levels of the S-layer gene . The gene is a passenger gene of a transposon with transposase ACAM34UNSW_02084 (Hlac_3078), and could be mobilized similarly to other virus exclusion mechanisms  or antibyer gene . We suggsorption . AlternaWhile none of the proposed mechanisms are fully effective against HRTV-DL1, because the virus is completing its lytic cycle, they might prevent the lysis of the entire population and allow virus escape mutants to evolve, and could be responsible for the long intracellular phase of the virus life cycle.Halobacterium salinarium a PATE has been shown to be active as ncRNA  in the N-terminus of the second S-layer gene . Since adsorption of HRTV-DL1 to UNSW_2.14 is fully abolished, we conclude that the S-layer represents the receptor for HRTV-DL1, as it has been proposed for another arTV (HFTV1) (Escape mutant ACAM34_UNSW_2.14 was found to be fully resistant to HRTV-DL1 infection . Infecti (HFTV1) . Variabi (HFTV1) , 2018. O (HFTV1) . Cell mo (HFTV1) , suggestAdsorption of HRTV-DL1 to escape mutant ACAM34_UNSW_3.3 is greatly reduced, but not fully impaired . The virorc1/cdc6 genes being upregulated in resistant ACAM34_DSMZ, five of seven remaining orc1/cdc6 genes are differentially regulated in sensitive ACAM34_UNSW . Studies have shown that Cdc6 can bind dsDNA without sequence specificity  isolated from Deep Lake, Antarctica. HRTV-DL1 belongs to the  viruses .Hrr. lacusprofundi strain (ACAM34_UNSW) grown in the laboratory for a time that is not retraceable anymore. However, the type strain of Hrr. lacusprofundi obtained from the DSMZ did not show susceptibility to HRTV-DL1 infection. Genome sequencing uncovered that ACAM34_UNSW had undergone a massive genomic rearrangement and lost about 18% of its genome, a phenomenon that has also been described for other haloarchaea , the supernatant of the culture (lysate) was filtered through 0.2 μm filters and used for infection of Halorubrum lacusprofundi ACAM34. Halorubrum lacusprofundi ACAM34 (DSM 5036), hereafter named ACAM34 _DSMZ, was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). The parental strain of Halorubrum lacusprofundi ACAM34_UNSW was provided by either Peter Franzmann or John Bowman (not traceable anymore) and subsequently cultured in the laboratory  from glycerol stocks stored at −80°C. The strain went through several rounds of culturing, plating and − 80°C storage in three different laboratories  prior to re-sequencing. The number of generations that the strain had undergone during this time is not traceable. Halohasta litchfieldiae tADL (DSM 22187) was provided by the DSMZ. Halophilic archaeon DL31 and Halobacterium DL were isolated previously  of 0.5. Cells were harvested , mixed with viral suspension with a multiplicity of infection (MOI) of 5–10 and incubated for 2 h at room temperature to allow viral adsorption. Treated cells were inoculated into 500 mL liquid cultures that were monitored by measuring optical density changes (OD600). After lysis occurred, cultures were centrifuged at 4,500 x g for 45 min to pellet the cells. The supernatant was recovered and viruses were subsequently precipitated with polyethylene glycol (PEG) 6,000  and incubation at 4°C overnight. Then, viral preparations were collected by centrifugation . Pellets were resuspended in DBCM2 salt solution , sterile filtered (pore size 0.2 μm) one time when used for plaque assay and twice when used in liquid culture infection assays. Virus solutions were stored at 4°C and were active for a minimum of 1 year. For downstream analyzes such as genome sequencing and mass spectrometry the virus particles were further purified by Cesium chloride (CsCl) density gradient centrifugation. The virus solution was treated with 200 U of DNase I and 50 μg/mL of RNase A to reduce host genomic contamination prior to loading on a CsCl density gradient  and centrifuged at 38,000 rpm for 22 h, 4°C . Bands containing virus particles were extracted with a syringe, diluted in three volumes of DBCM2 salt solution and re-precipitated with PEG . After centrifugation at 30,000 x g for 30 min the resulting pellet was washed twice with DBCM2 salt solution and stored at −20°C before further processing.For virus production, For Transmission electron microscopy (TEM), virus containing solution was adsorbed for 5 min to carbon coated copper grids and stained for 1 min with 2% uranyl acetate (w/v in water). Electron micrographs were generated using JEM2100 Plus at 200 kV acceleration voltage. For light microcopy, cells were fixed with 1% glutaraldehyde for 1 h at room temperature and then stored at 4°C until imaging with a Zeiss AxioPhot microscope with AxioCam MRm.® High-Fidelity DNA Polymerase (NEB) and contained 0.02 units/μL of DNA polymerase, primer concentration of 0.1 μM for both forward and reverse, 1X of Q5 Reaction Buffer and 1X Q5 High GC Enhancer. The following program was used: 5 min at 95°C, followed by 35 cycles of 30 s at 95°C, 30 s at 68°C for annealing and 30 s at 72°C for elongation. Digested DNA and PCR products were separated on 1% agarose gels in Tris-borate-EDTA buffer and stained with SYBR™ Safe DNA stain (Invitrogen). For HRTV-DL1, UNSW_2.14 and UNSW_3.3 library preparation  and sequencing  was performed at the Max Planck-Genome-Center Cologne . For PacBio sequencing of ACAM34_UNSW and ACAM34_DSMZ, DNA extraction, library preparation, sequencing  and assembly  according to the manufacturer’s instructions. Plasmid extraction was performed with ISOLATE II Plasmid Mini Kit  according to the manufacturer’s instructions. Nuclease digestions using specified restriction enzymes  or exonuclease III  were performed on 2–3 μg of DNA extracted from virus particles for 1 h at 37°C. PCR reactions were performed with the Q5ool v2.6  for ACAMhttps://victor.dsmz.de. Prediction of termini was done using PhageTerm  with the same settings searching customized databases Hrr. lacusprofundi ACAM34_UNSW and HRTV-DL1.Protein content analysis of HRTV-DL1 particles was performed by mass spectrometry on purified virus pellets as described previously for plasmid vesicles and membrane vesicles . Peak liHrr. lacusprofundi  were synchronized using an adaptation of the “Stationary phase method”  and cultures were then regrown up to OD600 ~ 1. Iterative dilution and growth of the culture were repeated twice before considering a culture synchronized. For infection with HRTV-DL1, cells from synchronized cultures (OD600 ~ 1) were collected by centrifugation, resuspended in 1 mL of fresh media and infected with HRTV-DL1 virus with a MOI of 5. After incubation , cells were transferred into liquid cultures and growth was monitored by optical density (OD600). Long-term cultures were established by continuously diluting infected cultures at OD600 ~ 1 to OD600 ~ 0.05. Infection was confirmed by PCR using primers HRTV-DLF and HRTV-DLR . Intracellular virus titers and host chromosome copy numbers were quantified by qPCR. Briefly, samples of 2 mL culture in biological replicates were collected and pelleted . Cell pellets were washed two times with 1 mL of fresh media and stored at −20°C upon DNA or RNA extraction. Quantification of Hrr. lacusprofundi and HRTV-DL1 genome copy number were carried out using a CFX96 Touch Real-Time PCR  and the software CFX Manager™ Software. Primers are listed in ™ Green Supermix (Bio-Rad) and primer concentrations as stated in 2 values ≥0.99 for all assays. The specificity of the qPCR was confirmed by unique signals in melting curves and gel electrophoresis of PCR products.To study the life cycle, cultures of  method” . The strHRTV-DLR . Viral t600 = 0.5 were harvested by centrifugation  and resuspended in 1 mL fresh medium. The cells were subsequently infected using a MOI of 5. At defined time intervals the adsorption was stopped by immediate centrifugation . The number of remaining free virus particles in the supernatant was determined by plaque assay. A cell-free control (only media) served as control. Three different strains of haloarchaea  Max Planck Society [Max Planck Research Group Archaeal Virology].The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


At 20 °C, this reduction was achieved after 19 days. These findings highlight the potential risk of aerosol and contact transmission of respiratory viruses, especially in enclosed environments, over extended periods. Studying surrogate viruses like FCoV provides important insights into the behavior of zoonotic viruses like SARS-CoV-2 in the environment.Enveloped respiratory viruses, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can be transmitted through aerosols and contact with contaminated surfaces. The stability of these viruses outside the host significantly impacts their transmission dynamics and the spread of diseases. In this study, we investigated the tenacity of Feline Coronavirus (FCoV) in aerosols and on surfaces under varying environmental conditions. We found that airborne FCoV showed different stability depending on relative humidity (RH), with higher stability observed at low and high RH. Medium RH conditions (50–60%) were associated with increased loss of infectivity. Furthermore, FCoV remained infectious in the airborne state over 7 h. On stainless-steel surfaces, FCoV remained infectious for several months, with stability influenced by organic material and temperature. The presence of yeast extract and a temperature of 4 °C resulted in the longest maintenance of infectivity, with a 5 log They have major medical, veterinary, and economic relevance, as they can infect various mammals and birds, depending on the genus. In these hosts CoVs can cause a wide spectrum of mild to serious diseases, often associated with high morbidity and mortality2. In our study, we used Feline Coronavirus (FCoV) as a surrogate virus for SARS-CoV-2. Another research group recently emphasized the importance of studying non-zoonotic animal CoVs to understand the behavior of zoonotic CoVs3. Respiratory viruses can be transmitted through three different routes: droplet, aerosol (droplet nuclei), and contact transmission6. At the beginning of the pandemic, the primary transmission routes of SARS-CoV-2 as a respiratory virus were a subject of frequent discussion7. While the focus was initially on droplet transmission, aerosol transmission was later confirmed as the predominant transmission route9. For a pathogen to be transmitted through these routes, it has to remain airborne and infectious outside the host for a certain duration. Respiratory activities such as breathing, talking, singing, coughing and sneezing generate particles of various sizes and quantities, which may contain the pathogen10. Larger respiratory droplets carrying infectious agents, predominantly expelled by symptomatic infected individuals through coughing or sneezing, sediment within 1–2 m of the source in seconds12. These droplets either directly reach the mucous membranes or conjunctiva of susceptible hosts or sediment on nearby surfaces13. If the virus remains infectious on these surfaces after drying for a certain duration, individuals may become infected through contact with the contaminated surface. Previous studies found that SARS-CoV-2 remained infectious for 4–7 days on stainless-steel surfaces at room temperature15. Additionally, another study reported a prolonged infectious period of SARS-CoV-2 on stainless-steel surfaces of 14 days when the virus was embedded in an organic matrix16. It is also likely for larger droplets to evaporate into droplet nuclei before reaching the ground. The particle diameter that distinguishes larger droplets from droplet nuclei is usually considered to be 5 µm, although some studies have used a cut-off size of 10 µm as these particles are able to deposit in the lower respiratory tract17. Previous studies have demonstrated that droplet nuclei can remain suspended in the air for hours or even days, travel longer distances from the source, and accumulate in enclosed environments12. SARS-CoV-2 has been found to remain infectious in aerosol particles for several hours18. Aerosol transmission occurs when these small infectious aerosol particles, produced by infected hosts during respiratory activities, whether symptomatic or asymptomatic, are inhaled19. Exhaled particles undergo evaporation at a rate, which depends on the particle surface, the ambient air temperature and relative humidity (RH)17. Evaporation changes the diameter, physical state and chemical composition of the particles, potentially influencing the stability of the enclosed virus, which may be harmed20. Therefore, virus stability is highly affected by environmental conditions, including ambient RH, temperature, as well as factors like UV radiation and oxidative stress. However, the exact mechanisms of viral inactivation are still not fully understood and are very specific to each virus21. The wide variability in physical and biological properties among viruses makes it challenging to generalize findings across different pathogens. Comprehensive analyses of numerous pathogens are necessary to understand the effects of environmental factors on their stability and to identify possible correlations. In this study, our focus was on determining the influence of RH on the stability of airborne FCoV. Additionally, we investigated time-dependent loss of infectivity of airborne FCoV in an enclosed environment under moderate climatic conditions. Furthermore, we examined the stability of FCoV, embedded and dried in different organic matrices, on stainless-steel surfaces under different environmental conditions.The importance of understanding the tenacity of respiratory viruses, particularly coronaviruses, in the environment has been highlighted by the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. The stability of these viruses in the environment has a direct influence on their transmission and spread of diseases. Coronaviruses (CoVs) are enveloped positive single-stranded RNA viruses22. The cells were grown in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum  and 1% of a solution containing 100 IU/mL penicillin G, 100 µg/mL streptomycin  and 25 µg/mL Amphotericin B . Virus quantification was performed as described recently22. In brief: we performed endpoint dilution assays. The tissue culture infectious dose 50 (TCID50/mL) was calculated using the Spearman-Karber method24 after virus titration in 96-well plates  in an eightfold approach. Every well was investigated for a cytopathic effect five days after infection.An FCoV I strain, biotype FECV (isolate “München”), provided by the Friedrich Loeffler Institute , was used for all experiments. FCoV was propagated and titrated using Crandell-Rees Feline Kidney Cells  as described previously3. Further technical details concerning the aerosol chamber were published previously25. In brief, the bioaerosol was generated by an ultrasonic nebulizer , producing particles with an average initial size of 18 µm. A virus suspension containing FCoV in DMEM and 10% FBS was transported to the ultrasonic nebulizer using a perfusor pump at a flow rate of 36 mL/h. The concentration of the original FCoV suspension was calculated after each experiment and based on this the theoretically possible concentration of viruses per m3 air was calculated. The chamber can be operated in different modes, resulting in dynamic or static aerosols. In the dynamic aerosol, there was continuous air exchange at a rate of 100 m3/h, and a fan installed in the ceiling of the chamber distributed the aerosol with a rotation speed of 3770 rpm. On the other hand, in the static aerosol, there was no air exchange, and the fan was inactive. Climate parameters such as RH and temperature could be individually regulated in the dynamic aerosol, as they were set in the chamber supply air.The aerosol experiments were performed in an airtight walk-in aerosol chamber with a volume of 7 mWe used the Coriolis µ cyclone air sampler  for air sampling. The total sample volume was 3000 L, using an air flow rate of 300 L/min over a sampling time of 10 min. Depending on the type of aerosol, the air samplers were either placed inside the chamber (static aerosol) or connected to the chamber from outside using a stainless-steel tube, enabling air collection from the center of the chamber (dynamic aerosol). In the case of static aerosol, the samplers were placed inside the chamber to avoid the generation of negative pressure that would result from air extraction without incoming airflow. We conducted preliminary tests to compare collection efficiencies, and both setups achieved similar results, ensuring accurate air sampling. The Coriolis µ cones were filled with 10 mL of supplemented DMEM. Unlike the cell culture medium, we added only 1% of the FBS. In addition, 0.3% autoclaved linseed oil was added to prevent foam formation. Following sampling, the air samples and the remaining original virus suspension, which had been aerosolized, were stored on ice until quantification using an endpoint dilution assay, as described previously.3/h, while introducing the virus aerosol into the chamber at a perfusor pump flow rate of 36 mL/h throughout the entire experiment. The ultrasonic nebulizer, air exchange and the fan were started 8 min before air sampling to establish a homogeneous viral aerosol in the chamber. They continued to run for the entire sampling period, in total 18 min, enabling a more precise assessment of the impact of RH levels and minimizing sedimentation losses. To confirm a good distribution of the aerosol in the chamber, we collected two simultaneous air samples using two Coriolis µ samplers connected to the chamber at different heights. The heights were 1.6 m and 0.8 m. All samples were quantified using an endpoint dilution assay, as described previously.In the first part of this study, we examined the stability of aerosolized FCoV under varying RH conditions. Specifically, we investigated the stability at 30%, 40%, 50%, 60%, and 70% RH in a fivefold approach. The temperature was adjusted to 24 °C throughout each trial. We generated a dynamic aerosol in the chamber with continuous air exchange at a rate of 100 mIn the second part of this study, we focused on investigating the natural loss of infectivity of aerosolized FCoV over time. Our aim was to produce a static aerosol to simulate an enclosed environment without any air exchange where a virus emitter was present and to investigate how the viral load in the air changes over time. The ultrasonic nebulizer operated for 10 min at a perfusor pump flow rate of 36 mL/h to generate a concentrated viral aerosol within the chamber. After the initial aerosolization, the virus supply was stopped, and the aerosol remained in the chamber for several hours without any further airflow. Air samples were collected after 10, 60, 100, 180, 300, and 420 min in a fivefold approach, respectively. A new experiment was conducted for each time point to prevent previous air sampling from affecting the results. We recorded the RH and temperature in the chamber using a Testo 400 probe . As there was no supply air, we were unable to regulate the climate parameters individually. The average RH in the chamber was 33%, and the temperature was 23 °C throughout all experiments. Furthermore, the particle concentration in the aerosol was measured by an aerosol spectrometer . Additionally, as part of our investigation, we implemented sedimentation plates to collect any virus particles that settled over time. Therefore a 12 × 12 cm plate , filled with 20 mL of supplemented DMEM, was placed at the center of the chamber for each trial. All samples were quantified using an endpoint dilution assay, as described previously.50/germ carrier), triplicate germ carriers were investigated immediately after drying. Subsequently, triplicate germ carriers were examined at each designated time point. The dried virus suspension was resuspended in 1 mL of DMEM with the addition of 3% FBS and 0.3% autoclaved linseed oil by adding glass beads  and vortexing for 1 min. An endpoint dilution assay was then performed, following previously described methods. For the yeast extract/BSA organic load, the time points were as follows: daily on days 1, 2, 3, and 4; every second day on days 6, 8, and 10; every third day on days 13, 16, 19, 22, and 25; and approximately weekly on days 31, 40, 50, and 60. After 60 days, a second series of trials was started because we were out of germ carriers as we underestimated the stability of FCoV. The new germ carriers were examined at approximately weekly to every second-week intervals on days 6, 25, 60, 85, 98, 105, 111, 125, 134, 140, 147, 162, 168, and 174, or until no infectious virus was detectable. For the sheep blood/BSA organic load, the time points were as follows: daily on days 1, 2, 3, and 4; every second day on days 6, 8, and 10; every third day on days 13, 16, 19, 22, and 25; and approximately weekly on days 31, 35, 40, 50, and 55, or until no infectious virus was detectable.In a further experimental series, we investigated the stability of FCoV when dried in organic matrices on stainless-steel germ carriers, subjected to different environmental conditions. The round stainless-steel germ carriers  were manufactured from 1.4301-2B (X5CrNi1810) steel, with a diameter of 2 cm and a thickness of 1 mm and have a flat surface. To load the germ carriers, we combined 100 µL of FCoV suspension with 11 µL of two different organic loads in a 10:1 ratio. The first organic load consisted of 100 g/L yeast extract  and 100 g/L Bovine Serum Albumin , resulting in a final concentration of 10 g/L per germ carrier. The second organic load consisted of 30 g/L defibrinated sheep blood  and 30 g/L BSA, resulting in a final concentration of 3 g/L per germ carrier. Both organic load solutions were previously sterilized and mixed. Following a drying period of 45 min in a desiccator, the carriers were stored at either 20 °C or 4 °C until further use. To determine the initial virus concentration on day 1 . Since viral TCID50/m3 air we used a mixed count regression with log link. Due to overdispersion we choose a negative binomial distribution. The TCID50/m3 was modelled depending on RH and adjusted for Coriolis height, both variables were included as categorical effects. Post-hoc comparisons between all RH were adjusted for multiple comparisons using the Bonferroni method. Estimated marginal means and multiple comparison post-hoc tests were performed using the emmeans R package (version 1.8.6). For visualization we modelled the TCID50/m3 depending in the relative humidity as a continuous effect using a restricted cubic spline with the actually measured RH with the package mgcv (version 1.8-42).To model the effect of the RH on the recovered FCoV TCID50/m3 air was modelled as an exponential decay (loss of infectivity) using a Bayesian loglinear model.The influence of time on the airborne FCoV TCIDinitial conc (intercept) is the estimated aerosolized log10 TCID50/m3 concentration of FCoV at 0 min and time is the reduction in log10 TCID50/m3 per min . For the stability of FCoV on surfaces we had to add a loglog linear effect to account for the longer survival of FCoV due to the organic matrix/load (yeast extract/BSA or sheep blood/BSA). One model was fitted for each temperature and organic load combination.The log(time). This effect represents the longer survival of virus which is protected from environmental factors by being embedded in the organic matrix. Both models were fitted using brms (version 2.19.)26 and rstan (version 2.26.13). Time until log reduction was calculated based on these models.The slowing rate of decay was modelled with 10 TCID50/ml for each trial. After a stabilization time of the aerosol of 8 min, air samples were simultaneously collected at two different heights (1.6 m and 0.8 m) in each trial. Overall, there was no significant difference in the amount of collected infectious viruses between the two heights (p = 0.789). FCoV was most robust at 40% RH, with a concentration of M = 4.72 (SD 0.30) log10 TCID50/m3 air. Conversely, FCoV was most unstable at 50% RH, with a concentration of M = 3.99 (SD 0.50) log10 TCID50/m3 air. Significant differences were observed between 40 and 50% RH (p = 0.009) as well as between 40 and 60% RH (p = 0.020). Additionally, we calculated recovery rates of FCoV in the aerosol by dividing the concentration of collected FCoV per m3 of air by the theoretical concentration of viruses per m3 of air, which was previously calculated from the original FCoV concentration in the suspension. The maximum average recovery rate of FCoV in the aerosol was 13.3% at 40% RH, while the minimum average recovery rate was 2.5% at 50% RH.The stability of aerosolized FCoV as a function of RH (30–70%) in dynamic aerosols is shown in Fig. . The average RH in the chamber was 33%, and the temperature remained constant at an average of 23 °C throughout all experiments. The concentration of the original FCoV suspension was M = 6.15 (SD 0.31) log10 TCID50/mL for each trial. After 10 minutes (min) of aerosolization (t0), we detected an initial FCoV concentration of M = 5.20 log10 TCID50/m3, resulting in a recovery rate of 13% at t0. Afterwards, the initial concentration decreased over time linearly with a rate of − 0.009 log10 TCID50/m3 per min  or − 2% per min, which corresponds to a half-life of 34.8 min . After median (Md) = 117 min , the initial concentration decreased by 1 log10 level. After Md = 349 min , the initial FCoV concentration decreased by 3 log10 levels, corresponding to a recovery rate of only 0.01%. The experiment was terminated after 420 min, as we had almost reached the detection limit of the assay of 0.8 log10 TCID50/m3. According to the model, a 4 log10 reduction of the initial concentration was predicted after Md = 464 min . At the same time, we conducted measurements of the aerosol particles throughout the entire experiment to assess any potential loss of particles, including viruses, from the air. The aerosol spectrometer detected the highest concentration of total particles at M = 7.8 log10 particles/m3 after 15 min. The particle count remained relatively stable, with only a 1 log10 level decrease over the 420 min duration. Additionally, we used sedimentation plates to collect infectious virus particles that sedimented over time. Table 10 TCID50/mL at various time points. The concentration averaged M = 3.02 (SD 0.09) log10 TCID50/mL and remained relatively constant at every time point. To estimate the proportion of infectious virus particles that sedimented on the base area of the chamber, we extrapolated the concentration per ml to the entire chamber base area and divided it by the theoretical concentration of viruses in the chamber calculated from the original FCoV suspension. We found that M = 31.4% of infectious virus particles immediately sedimented and were no longer available for the air sampler. Notably, this value remained consistent across all time points.Furthermore, we investigated the stability of airborne FCoV in a static aerosol over time without any additional external influences. The loss of infectivity of FCoV over time and the corresponding total particle count is shown in Fig. 210 level has been achieved are presented in Table 10 TCID50/germ carrier after drying, respectively. The trials were terminated when the concentration of infectious FCoV reached the detection limit of the assay of 1.1 log10 TCID50/germ carrier. Generally, the loss of infectivity was initially higher and gradually slowed over time. FCoV was most unstable with the yeast extract/BSA load at 20 °C, with a 5 log10 TCID50 reduction achieved after Md = 19 days . In contrast, with the sheep blood/BSA load at 20 °C, a 5 log10 TCID50 reduction would be reached after Md = 58 days . A similar decrease in FCoV concentration was observed with sheep blood/BSA at 4 °C, where a 5 log10 TCID50 reduction was projected after Md = 54 days . Notably, FCoV was most robust with yeast extract/BSA at 4 °C, as the initial concentration decreased significantly slower. It decreased by only 3 log10 levels after Md = 68 days , remaining infectious three times longer than with sheep blood/BSA at 4 °C. Finally, a 5 log10 TCID50 reduction was achieved after Md = 167 days .The stability of FCoV when dried in organic matrices on surfaces was assessed using stainless-steel germ carriers with four different combinations of organic loads and temperatures. Its loss of infectivity over time at 4 °C and 20 °C with either a 10 g/L yeast extract and BSA (yeast extract/BSA) load or a 3 g/L sheep blood and BSA (sheep blood/BSA) load is shown in Fig. The investigation of virus tenacity in the environment plays a crucial role in enhancing our understanding of potential transmission routes for infectious diseases. Our study focused on assessing the tenacity of airborne FCoV and FCoV in dried organic matrices on surfaces. Overall, airborne FCoV showed a remarkable level of stability over a wide range of RH conditions. However, it is important to note that relative humidity has an impact on FCoV stability. The virus showed higher stability at both low and high RH levels, whereas medium RH conditions (around 50–60%) were associated with a higher probability of decay. Remarkably, FCoV remained infectious for over 7 h in the airborne state at medium RH levels. Moreover, on surfaces, FCoV showed the ability to remain infectious for extended periods, even up to several months. The stability on surfaces was influenced by factors such as temperature and the presence of organic material.3, previous research has shown that non-zoonotic animal coronaviruses like FCoV, canine coronavirus (CCV), transmissible gastroenteritis virus (TGEV) or mouse hepatitis virus (MHV) could be a suitable surrogate for survival of zoonotic SARS-CoVs29. To the best of our knowledge, this is the first study investigating the tenacity of FCoV in aerosols and on surfaces.FCoV was used as a surrogate for SARS-CoV-2 in our study. Working with infectious pathogens of biosafety level 3 (SARS-CoV-2) is only possible in a limited number of laboratories, and especially with virus aerosols, it is very challenging. FCoV, on the other hand, can be studied under biosafety level 2 (BSL2) conditions, making it a safer and more cost-efficient option. FCoV belongs to the genus Alphacoronavirus, while SARS-CoV-2 is a Betacoronavirus. Although they share only 44.0–44.5% similarity at their nucleotide level3, allowing individual airflows and climatic conditions. The chamber has been previously utilized in a study investigating the stability of Escherichia coli in aerosols30. Other studies investigating pathogen stability in aerosols, including SARS-CoV-2, have utilized a rotating drum, as described by Goldberg et al.31, to generate a dynamic aerosol. Our chamber offers a good opportunity to create a more realistic setting for exploring pathogen behavior within a room. To investigate FCoV stability, we worked with both dynamic (air exchange) and static (no air exchange) aerosol setups.Our aerosol experiments were conducted in an aerosol chamber with a volume of 7 m33. However, studies on human coronaviruses have shown varying results. For example, Sars-CoV and MERS have been found to be more stable at medium humidity levels35. Oswin et al. demonstrated that at low humidity levels, the initial stability decreases significantly but then remains relatively stable compared to higher humidity levels. If this initial decrease is neglected, a U-shaped pattern could also be observed21. Overall, coronaviruses in the aerosol state appear to be more stable than influenza or filoviruses at medium humidity levels34. When comparing studies on stability of viruses at different humidity levels, it is important to consider the medium used, as significant differences in stability can arise due to this factor. The most important fact to take into consideration when talking about the relationship between stability of viruses and RH is the microenvironment of the droplet and therefore the medium in which it resides36. Simulating human respiratory fluids accurately is still challenging due to the unknown exact components and concentrations. Therefore, many studies used cell culture medium such as DMEM as a model medium. One previous study compared DMEM with porcine respiratory fluid (PRF) and found that they differed greatly in the NA:K ratio. In addition, PRF contained significantly more protein37. It is important to note that studies using simulated respiratory fluids or real respiratory fluids instead of model medium have shown differences in virus stability38. These studies suggest that virus stability might be underestimated in most cases40. To make studies more representative, changes should be made to the virus suspension medium in further aerovirology studies37.We observed a slight U-shaped trend in the stability of FCoV in dynamic aerosols at different RH levels, indicating that FCoV was more likely to decay at medium RH levels, ranging from 50 to 60%. This U-shaped pattern has also been observed for other enveloped viruses, including TGEV and influenza virus42. Yang et al. investigated the influence of different model media on the stability of Influenza A viruses in droplets, comparing DMEM and PBS, each with or without the addition of 5% FBS as a protein source. In general, they found better viability in DMEM than in PBS especially at medium and low RH36. Notably, the addition of FBS significantly affected virus stability at medium RH levels, suggesting a protective effect of proteins44. When a droplet leaves the respiratory tract, it evaporates by approximately half its original size depending on the ambient RH. This leads to a high concentration of substances within the droplet, such as salts, which are usually harmless but can become toxic to the virus. This effect is only relevant at medium RH levels just before the salts crystallize36. The exact RH at which the salts crystallize (efflorescence RH) depends on the droplet's composition and medium45. These findings support our own observations, as we observed a slight decrease in stability at medium RHs in the aerosol. Overall, we observed a high stability of FCoV in the aerosol, likely due to the presence of 10% FBS. The dynamic aerosol setup aimed to simulate a ventilated room where a virus emitter is present. The results indicate that the ambient RH in a room can significantly impact the stability of the emitted virus in the aerosol and thus its transmission potential. To minimize the risk of infection, it is advisable to keep the relative humidity at medium levels in indoor places.In our study, we modified DMEM by supplementing it with 10% FBS as a protein source, as respiratory droplets contain a variety of salts and proteins46. However, re-aerosolization of these sedimented infectious virus particles may also occur. In these experiments, the RH averaged 33%. Previous studies on related viruses have found that SARS-CoV-1 and SARS-CoV-2 remain stable in aerosols for over 3 h, with respective half-lives of 1.1 and 1.2 h, at an RH of 65%14. Similarly, MERS-CoV was found to be infectious in aerosols for over 3 h35. In our study, we observed that FCoV has a half-life of 34.8 min in aerosols and was detectable for over 7 h. There was one other research group that investigated the stability of SARS-CoV-2 in aerosols over a longer period and found infectious virus after 16 h at an average RH of 53%. However, this was a single observation without replication18. Comparing the half-lives of SARS-CoV-2 and FCoV indicates that both exhibit relatively short durations, suggesting similar behavior in aerosol stability. Observed differences may be more likely attributed to different aerosol generation processes and sampling methods. When considering influenza A viruses, their infectivity in aerosols varies lasting from 1 to 24 h, depending on RH levels. Furthermore, influenza A viruses adapted to animals tend to demonstrate longer stability compared to human influenza A viruses48. It is important to note that comparisons between these studies are challenging due to variations in RH levels and medium used, as both factors strongly influence the stability of airborne viruses, as mentioned earlier. In general, our findings underscore the potential risk of aerosol transmission of enveloped respiratory viruses, especially in enclosed and unventilated environments over an extended period. This aligns with previous studies that have demonstrated aerosol transmission of SARS-CoV-2 between animals using hamsters as an animal model50.Furthermore, our study demonstrated that FCoV remained infectious in static aerosols for over 7 h with a half-life of 34.8 min. The static aerosol setup aimed to simulate an enclosed room without regular air exchange, where a virus was released for a specific duration. During these experiments, we considered the possible natural loss of virus due to sedimentation. We observed a 31.4% loss of infectious virus through sedimentation, which occurred within the initial 10 min and was than constant over the subsequent 7 h. Moreover, the particle count remained stable throughout the entire experiment, indicating that virus-containing particles relevant for aerosol transmission remained suspended in the aerosol. It is known that aerosol particles < 5 µm, which are relevant for inhalation, remain suspended as droplet nuclei in the air for hours, while larger droplets > 10 µm settle to the ground within minutes due to gravity32. Döhla et al. emphasized the importance of selecting an appropriate sampling method, as it can influence the stability of viruses in the sample. Since there is no generally recommended virus air sampling method, the choice of air sampler needs to be individually determined based on the specific experimental setup51. Most commonly used air samplers for collecting SARS-CoV-2 include filters, impactors, cyclone samplers and impingers52. For our experiments we chose the Coriolis µ cyclone air sampler. Previous studies aiming to detect SARS-CoV-2 in hospitals or healthcare settings have also utilized cyclone samplers due to their high collection volume56. While SARS-CoV-2 RNA has been detected in these studies, the identification of infectious SARS-CoV-2 was reported in only a few cases. It should be noted that cyclone samplers may be less efficient in detecting low levels of viruses compared to other air samplers, as the centrifugal forces affecting the viruses during collection could potentially cause stress57. However, in our study, we worked with high concentrations of viruses in a controlled environment, which made the Coriolis µ sampler suitable for our purposes, and we were able to detect infectious viruses.At optimal environmental conditions the recovery rate of airborne FCoV was approximately 13% in our study. Several factors may have an influence on recovery rates of airborne viruses, including inactivation during aerosolization, loss through sedimentation, as well as sampling losses. We assume that our ultrasonic nebulizer and the aerosilization settings used resulted in the production of a suitable viral aerosol. In a study by Kim et al. various nebulizers and settings like pressure and nebulization time were tested to evaluate their impact on the stability of TGEV, and it was concluded that the stability of TGEV was not significantly affected59. Furthermore, viruses tend to be more stable at lower humidity levels and temperatures59. In our study, we demonstrated that FCoV remained infectious for 19–58 days at 20 °C and low RH, with the organic load significantly influencing the virus's stability. Comparatively, SARS-CoV-2 remained infectious on stainless steel surfaces for 4–7 days at room temperature, while MERS and Sars-CoV-1 remained infectious for 2 days61. TGEV and MHV, other non-zoonotic CoVs, remained infectious at room temperature for 3 days at 50% RH and up to 28 days at 20% RH27. It is important to note that differences in the results of various studies may occur due to varying medium used. While most of these studies were conducted using cell culture medium, we enriched our medium with 10 g/L yeast extract/BSA or 3 g/L sheep blood/BSA, representing a high organic load according to the guidelines for virus inactivation studies on nonporous surfaces62. Exhaled droplets that would sediment on surfaces consist of respiratory tract residues, saliva and organic material from the environment, resulting in a high organic load. Other studies added a tripartite soil load  following international standard ASTM to the medium and found increased stability of SARS-CoV-2 on stain-less steel surfaces at 20 °C for 14–28 days, indicating a protective effect of the organic load63. Therefore, we would suggest using a high organic load, such as ASTM International’s standardized tripartite soil load64, for further studies to avoid underestimating the stability of these viruses in the environment. However, it should be taken into consideration that stability may differ in dried human respiratory fluids. Regarding the influence of temperature, we found that infectious FCoV was detectable at 4 °C and 50% RH for 54–167 days, depending on the organic load. Only few studies have investigated CoVs stability at temperatures below 20°. Notably, Onianwa et al. observed a reduction in infectiousness of the Delta variant of SARS-CoV-2 at 24 °C and 65% RH in the first 2.5 h, while no reduction was observed at 4 °C and 85% RH within 2.5 h65. TGEV and MHV also remained infectious at 4 °C for over 28 days at all tested RHs, with the lowest losses observed at 20% RH27. Interestingly, we observed prolonged infectivity with yeast extract at 4 °C, although the reason for this difference remains unclear.We observed that 31.4% of the infectious virus sedimented onto the ground or surfaces within the first 10 min in the static aerosol. This finding highlights the potential risk of contact transmission and the importance of studying virus infectivity on commonly encountered surfaces. We focused on stainless steel surfaces, which are frequently found in public buildings and clinical settings and are frequently touched. Previous studies have shown that CoVs exhibit greater stability on non-porous surfaces like metal, glass or plastic compared to porous surfaces, such as paper or fabrics66. Studies investigating stability of CoVs on surfaces, including our study, all used larger droplet volumes, which are not in line with realistically expelled droplet volumes (< 0.5 µL) and may lead to different conclusions about virus stability. Another limitation of our study design is that we could not regulate the RH at the storage place and therefore could not distinguish between the influence of temperature and RH after drying. However, French et al. found that that viral decay during the wet phase was higher than during the dry phase regardless of RH66. In our experiment, all germ carriers were dried under controlled conditions for 45 min, allowing us to neglect the influence of RH during the wet phase. Thus, the observed differences in stability may be primarily attributed to temperature and variations in organic load.Like in aerosols, evaporation, and thus RH, plays an important role in terms of virus stability in droplets that sediment. French et al. studied the interplay of droplet volume and RH on surfaces and found that loss of infectivity was slower and more affected by RH in larger droplets (50 µL) than in small droplets (1 µL)In summary, our study demonstrated that FCoV could remain infectious in the airborne state for hours and on surfaces up to months, with the duration depending on environmental conditions. Factors such as RH, temperature, and the presence of organic material significantly impact the pathogen's infectivity outside the host. Comparing studies on virus stability is challenging due to the lack of standardized experimental setups and medium used in these investigations. Additionally, reproducing respiratory fluids in the laboratory is difficult as their exact composition is still unknown. However, existing evidence suggests that viruses may exhibit even greater stability in respiratory fluids. It can be stated that aerosol transmission as well as droplet and contact transmission are possible transmission routes for coronaviruses under various environmental conditions over an extended period. Whether an infection occurs depends on many other factors, such as the viral load in the environment, the minimum infection dose, and the immune state of individuals. Especially enclosed, poorly ventilated rooms and low RH environments may pose a higher risk of infection due to the accumulation and better stability of these enveloped viruses. Given that, different pathogens respond uniquely to environmental conditions based on their biological and physical properties, it is essential to study a wide range of viruses to identify and understand potential correlations. The exact mechanisms that lead to the inactivation or protection of enveloped viruses by environmental components remain unknown and require further research. Our study suggests that FCoV could be a valuable surrogate for studying the behavior of zoonotic coronaviruses like SARS-CoV-2 in the environment. Although surrogates could offer valuable insights into the stability and persistence of these viruses outside the host, enhancing our understanding of zoonotic transmission dynamics, it remains crucial to directly investigate the actual virus.


In this review, we provide updates on Sin Nombre virus (SNV) and other New World hantaviruses. We discuss the history of SNV, its biology and relatedness to other emerging New World hantaviruses, its ecology, and its disease in humans. This review highlights an important emerging virus pathogen from North America.Peromyscus maniculatus), and the virus is transmitted primarily through aerosolized rodent excreta and secreta. Here, we review the history of SNV emergence and its virus biology and relationship to other New World hantaviruses, disease, treatment, and prevention options.Sin Nombre virus (SNV) is an emerging virus that was first discovered in the Four Corners region of the United States in 1993. The virus causes a disease known as Hantavirus Pulmonary Syndrome (HPS), sometimes called Hantavirus Cardiopulmonary Syndrome (HCPS), a life-threatening illness named for the predominance of infection of pulmonary endothelial cells. SNV is one of several rodent-borne hantaviruses found in the western hemisphere with the capability of causing this disease. The primary reservoir of SNV is the deer mouse ( Bunyavirales and family Hantaviridae. Hantaviruses are found across the globe, but they are divided into Old World  and New World (North and South America) viruses that are associated with different diseases. Old World hantaviruses, like Hantaan virus, Puumala virus, Dobrava virus, and Seoul virus, cause Hantavirus Hemorrhagic Fever with Renal Syndrome (HFRS). Due to the availability of vaccines and therapeutics, HFRS has a case fatality rate of less than 1% [Sin Nombre virus , is a negative-sense single-stranded RNA (-ssRNA) virus of the order  than 1% .Peromyscus maniculatus. Hantaviruses are transmitted to humans through exposure to infected rodent saliva, urine, or feces. This review discusses the emergence, biology, host range, transmission, clinical presentation, treatment, and prevention of SNV.The presence of New World hantaviruses was first detected in the United States in 1993, when there was an SNV outbreak in the Four Corners region. Most cases were identified in New Mexico and Arizona. Subsequently, SNV and other hantaviruses have been identified across the U.S. The geographical distribution of these hantaviruses is determined by the rodent vectors that carry each type of virus. For SNV, the carrier is the common deer mouse, In the spring of 1993, an outbreak of an enigmatic respiratory illness began in the Four Corners region of the United States; it was characterized by severe pulmonary symptoms primarily affecting young, previously healthy individuals . As caseSince 1993, the annual reported cases of SNV in the United States have consistently ranged between 10 and 50 cases per year, accounting for approximately 850 reported cases across 39 states to date . ResearcHantaviruses form mostly spherical or pleiomorphic enveloped virions covered in tetrameric spikes composed of the heterodimers of the two viral glycoproteins, Gn and Gc . SNV hasLike other pathogenic hantaviruses, SNV enters into endothelial cells using integrins as the receptor—specifically, the β3-integrins . The virOnce in the host cell cytoplasm, it has been shown that the three SNV genome segments get transcribed at different times post infection. First, the N gene is transcribed, and those transcripts can be detected starting at four hours (h) post infection (p.i.) in Vero E6 cells. GPC mRNA starts to be detected at about 32 h p.i, and, finally, L protein is detected at 48 h p.i. Transcription is thought to occur using a prime and realign strategy for at least some transcripts. Once transcripts are made, they snatch caps from host transcripts that are in the cytoplasm, possibly at cytoplasmic processing bodies ,16,18,19SNV has been observed to produce both granular and filamentous inclusion bodies in the cytoplasm of infected cells, and it has demonstrated a type of budding unique to hantaviruses. Unlike Old World hantaviruses that bud at intracytoplasmic membranes, like the Golgi, SNV virions bud at the plasma membrane . The virN and GC structure has been solved through high resolution X-ray crystallography [N and GC) arrange into a heterodimer where the GN forms the stalk and GC forms the globular head. These heterodimers then form a tetrameric spike [The structure of the Tula virus virion, an apathogenic Old World hantavirus, was determined through cryoelectron microscopy . It showlography . The glyic spike ,23. Thesic spike . The spiic spike .During SNV replication, the GPC protein is co-translationally cleaved in the endoplasmic reticulum by the host signal peptidase, producing an N-terminal fragment (Gn) and a C-terminal fragment (Gc) . For NY-Hantaviridae family members that use arthropod vectors, hantaviruses usually infect rodents, as is seen with SNV and other HPS-causing hantaviruses in the western hemisphere , which were shown to be prone to SNV infection in a 1999 study [Unlike other misphere . Sigmodom of HPS . Many ro99 study . The levP. maniculatus), is located. The range of this species includes almost the entire populated area of North America, with the exception of parts of the southeastern United States, Alaska, and the most northern parts of Canada  . During While no correlation was found between the duration of symptoms at the time of admission and survival during the Four Corners outbreak, it remains highly recommended that patients be admitted as early as possible after the onset of symptoms for the best chances of survival and recovery ,37. AnalSince the initial outbreak in 1993, there have been over 700 cases of HPS to date ,3. OtherDiagnosis of HPS and, more specifically, SNV infection, can be accomplished through a combination of immunologic assays and clinical presentation . AccordiDue to the low occurrence and the timeline for diagnosing hantavirus infections, treatment options are limited if not started prior to the onset of viremia  3,39,40,4039,40.Several different therapeutic options have been explored experimentally. A study evaluating ribavirin use in a deer mouse model showed that ribavirin may be able to reduce infection in concert with human plasma from a seroconverted SNV patient . Use of Other treatments explored for treating New World hantavirus infections include favipiravir, a pyrazine derivative, and vandetanib, a tyrosine-kinase inhibitor of the vascular endothelial growth factor receptor 2 (VEGF2). Favipiravir has been evaluated for efficacy against both SNV and ANDV infection models in hamsters . AgainstStudies evaluating both monoclonal and polyclonal antibodies derived from SNV and ANDV infections have demonstrated protection in a hamster model ,49,50. TWhile there remain no FDA- or WHO-approved vaccines for New World hantaviruses and commercial development seems unlikely currently due to the low incidence of HPS disease, several studies are currently underway in attempt to develop vaccines against both SNV and ANDV. A recent viral-vectored vaccine employing a vesicular stomatitis virus (VSV) backbone and expressing either the SNV GPC or ANDV GPC was able to induce cross-reactive antibody responses and protect against lethal challenge in an ANDV model . This stThe CDC notes on their website that people should avoid or minimize contact with rodents in the home, workplace, or while camping. Spaces for rodents to enter human habitats should be sealed and kept clean and clear of any food that might attract rodents, and traps should be placed around areas where there are signs of rodent infestations.Globally, there continues to be an increasing incidence of emerging viral pathogens. Since the discovery of SNV in 1993, there have been several additional hantaviruses discovered across North and South America. New World hantaviruses, such as SNV and ANDV, are associated with HPS and are of considerable health concern due to their high mortality. Currently, there remain limited therapeutic options for treatment and no approved vaccines. Fortunately, cases of HPS remain low. As of the end of 2021, the CDC reported a total of 850 cases of HPS in the United States since reporting began in 1993. However, cases of HPS have been identified in 39 states as well as the District of Columbia, thus stretching from coast to coast in the United States. The prevalence of the rodent vectors for New World hantaviruses currently appears stable. Yet, it is unclear how climate change and demographic shifts, such as the continued migration of people from rural to urban settings, will impact both rodent populations and the potential for transmission to people. As evidenced by the Four Corners outbreak, favorable crop yields tend to be associated with increases in vector populations. In addition, there remains low awareness of hantaviruses or their diseases among people living within the geographic ranges of these pathogens. Lack of awareness and familiarity with the ecology of hantaviruses may inadvertently lead to conditions that might favor explosive growth in vector populations and increase the potential for transmissibility. Furthermore, due to antigenic similarities between SNV and ANDV, it remains unclear whether there remains a potential for New World hantaviruses to recombine if a co-infection event occurs. While there does not appear to be considerable overlap in the known vectors of New World hantaviruses, there is a need for continued ecological study to track changes in both vector and virus incidence.More research is needed to better understand the biology of New World hantaviruses, the ecology of their vectors, and their pathogenesis in humans. The availability of rodent models for infections of both SNV and ANDV has proven helpful for better understanding pathogenesis within these vectors as well as evaluating the potential for both therapeutics and vaccines to mitigate disease. However, ecological studies investigating the abundance of SNV and related hantaviruses in nature and additional potential vectors for transmission would be beneficial to identify hotspots or areas with the greatest likelihood for exposure. Collectively, advances in both understanding viral antigenicity and vaccine design suggest that targeted antibody-based therapeutics and vaccines are possible, and that they may contain broadly neutralizing potential against heterologous New World hantaviruses. Recent advances in the development of both artificial intelligence and protein dynamics mapping combined with recent discoveries pertaining to the structure of hantavirus virions may permit better models for understanding viral replication, targets for therapeutic design, and vaccine development. While there remains a low likelihood of commercial development of vaccines for SNV and ANDV currently based on the low incidence of HPS cases, these studies provide promise for improved clinical outcomes for future cases of hantavirus infections.SNV and other New World hantaviruses possess the potential for severe disease. Due to their transmission by different rodent vectors, it remains unclear how changes in both human demographics and climate change will impact viral abundance and exposure events in the future. While our understanding of hantaviruses has greatly expanded over the last few decades, future research is needed to better understand the biology, ecology, and pathogenesis of these viruses.