Phytophthora cactorum is an important oomycetous plant pathogen with numerous host plant species, including garden strawberry (Fragaria × ananassa) and silver birch (Betula pendula). P. cactorum also hosts mycoviruses, but their phenotypic effects on the host oomycete have not been studied earlier. In the present study, we tested polyethylene glycol (PEG)-induced water stress for virus curing and created an isogenic virus-free isolate for testing viral effects in pair with the original isolate. Phytophthora cactorum bunya-like viruses 1 and 2 (PcBV1 & 2) significantly reduced hyphal growth of the P. cactorum host isolate, as well as sporangia production and size. Transcriptomic and proteomic analyses revealed an increase in the production of elicitins due to bunyavirus infection. However, the presence of bunyaviruses did not seem to alter the pathogenicity of P. cactorum. Virus transmission through anastomosis was unsuccessful in vitro. Phytophthora includes notorious plant pathogens that severely threaten agricultural crops and forest trees worldwide. Phytophthora cactorum is an omnivorous pathogen that can infect a wide variety of plant species  in 90 mm diameter Petri dishes at 20 °C in the dark. To obtain zoospores, three experiments were conducted. In experiment 1, three replicates per isolate were used. For each of them, a mycelial plug containing mycelia (Ø 5 mm) was placed in the center of a Petri dish containing 11 mL clarified VA. After four and five weeks, cultures were flooded with 12 mL sterile demineralized water for 24 to 48 h under daylight at 20 °C. Water was decanted and replaced two to three times per day. For experiments 2 and 3, two 15 × 15 mm agar plugs from three-, five-, and six-day-old cultures  were cut from the same area for each replicate  were placed in an empty 90 mm diameter Petri dish and flooded with 12 mL of sterile demineralized water. In experiment 2, the water was replaced after 12 h, 16 h, 20 h, and 22 h with 10% pond water . In experiment 3, the water was replaced with sterile demineralized water at 3.5 h, with Volvic® mineral water  at 7 h and 21 h, and with Volvic® mineral water amended with approximately 1 mL of 2 days old non-sterile soil extract (250 mL of soil taken under asymptomatic Juglans regia L. trees flooded with 2 L of water) at 24 h. After the incubation period under daylight at 20 °C, zoospore formation and release were stimulated by cold shock (60 min in +4 °C). Zoospore suspension was prepared, and spore counts were determined, as in Harris & Webber [t-test in R (v 3.6.3).The isolates PhF66 and PhF66– were grown on clarified 11 mL vegetable juice agar [VA; 100 mL/L vegetable juice DM Bio Gemüse Saft , 3 g/L of CaCO& Webber . A longet-test in R (v 3.6.3).The dimensions of 50 randomly selected sporangia and 50 gametangia (oogonia and antheridia) were measured from the isolates PhF66 and PhF66–. Microscopic examinations and measurements were performed as in Jung et al. ,55, excePrior to total RNA extraction, the isolates were grown on MOS agar plates on cellophane membranes. The mycelia were harvested after two weeks, freeze-dried, and ground in liquid nitrogen, and total RNA was extracted using Spectrum Plant total RNA kit (Merck) from three technical replicates of PhF66 and PhF66– . These six samples were considered biological replicate 1. Furthermore, RNA from two more biological replicates of the isogenic strains  were extracted separately in the same way, resulting in four additional RNA-seq samples. The RNA of each sample was quantified with a NanoDrop 2000 Spectrophotometer . The samples were sent to a sequencing facility , where RNA integrity was assessed, eukaryotic strand-specific transcriptome libraries were built, and sequencing was performed using the Illumina NovaSeq6000 system, which generates stranded paired-end sequences.P. cactorum reference genome (GCA_010194725.1) with HISAT2. Reads were counted using HTSeq, and differential expression calls were made with edgeR and DESeq2 in Chipster v4 software. The raw reads are available in GenBank under BioProject PRJNA804427.The RNA-Seq data analysis was performed in Chipster software . Phred s20) for nine days, thoroughly washed in Tris-buffered saline (TBS)  to remove any residues of the cultivation medium, then frozen in liquid nitrogen and lyophilized. The proteome analysis was performed with liquid chromatography-mass spectrometry as previously described [P. cactorum [For proteome analysis, isolates were grown in liquid cultures  seedlings were inoculated with isolates PhF66 (15 seedlings), PhF66– (15 seedlings), Ph415 –another P. cactorum strain isolated from birch . Runner plugs of greenhouse-maintained strawberry plants of varieties Glima, Jonsok, Senga Sengana, Zefyr, and Honeoye were rooted in rockwool grow cubes  two weeks prior to inoculation. The well-rooted plug plants were then randomized and spaced into larger rockwool plates fitted in plastic containers, which were subsequently kept on a table in a greenhouse bench under +23 °C and long day (19 h) conditions. Six plants of each variety per each inoculation treatment were wounded in the stem (crown) base with a sterile blade tip, and a malt agar plug containing mycelium of PhF66, PhF66– or sterile agar plugs containing no hyphae (mock-inoculated control plants) was placed on the wound. The inoculum was covered with moist cotton and sealed with parafilm. The cotton was removed after five days. The experiment was monitored for 30 days, and repeated twice, two weeks apart. Strawberry test II was set up in June 2021 with strawberry plants  planted in the previous summer and growing outdoors in plastic growth bags filled with peat (Kekkilä Growth Sack Airboost 1 m). The plants were inoculated on 28 May 2021, as described above, with an agar plug containing PhF66 (four plants), PhF66– (four plants), PhF17/19 (six plants) –another rawberry – or no hAll isolates grew in all water matrix potentials applied in the experiment, but their growth was significantly reduced at −13 MPa and −15 MPa compared to growth rates at −11 MPa. The two replicates of each isolate  recovered from water potentials −13 MPa and −15 MPa were subjected to RNA extraction, reverse transcription, and virus-specific PCR. Amplification products of alphaendornaviruses PcAEV1, PcAEV2, and PcAEV3 were obtained from all replicates of the isolate hosting them (PhF9). The toti-like PcRV1 was also detected from all replicates of isolates PhF9 and PhF79. Phytophthora cactorum usti-like virus (PcUV1) as well as the bunyavirus PcBV2 were detected in all PhF79 replicates. However, the bunyavirus PcBV1 was not detected in PhF79, and both PcBV1 and PcBV2 were cured from all four analyzed replicates of PhF66. The result was confirmed by RNA-seq analysis of isolate PhF66–, where no reads of virus sequences were obtained.Mycelial samples taken from the recipient (the virus-free PhF66–) side of the dual cultures with virus hosting strains  were analyzed for the presence of transmitted viruses using RT-PCR with virus-specific primers [p < 0.001, N = 44).The mean lesion areas on apples were 10.0 cm2 (range 1.85–18.18 cm2) and 15.8 cm2 (range 1.97–31.14 cm2), respectively, which also differed significantly To test the phenotypic effects of bunya-like viruses in laboratory conditions, we conducted growth experiments using strains PhF66 and PhF66–, hosting two bunyaviruses and no viruses, respectively. The presence of these viruses significantly lowered the growth rate of the host isolate PhF66 in both growth substrates. There was a significant effect of the virus status on the growth rate of PhF66. The radial growth rate of the bunyavirus-containing PhF66 on MEA was 2.3 mm/day (range 2.20–2.51 mm/day) and that of PhF66– was 3.1 mm/day (range 2.89–3.24 mm/day), which differed significantly from each other  and smaller (p < 0.0001) sporangia compared to PhF66– (p < 0.001) in the virus-free PhF66– . All three biological replicates of PhF66 differentiated significantly from those of PhF66– in the principal component analysis and heatmap . A batchthan 1.5 , and in than 1.5 . Ten of than 1.5 . Notablythan 1.5 . The amithan 1.5 .Out of the 23 genes that were downregulated over 4× fold in the virus-containing isolate, four included a WD40 repeat-containing domain . WD40 dop < 0.05) in their abundance between PhF66 and PhF66–. The differentially abundant proteins included, e.g., two elicitins and various enzymes (p < 0.05)  .The virulence of isolates PhF66 and PhF66– to strawberry was tested in two occasions. In the strawberry test I which included five strawberry cultivars and two repetitions, no typical symptoms of crown rot appeared during the 30-day experiment. Cross-sections of crown stems of the strawberry plants were inspected, and re-isolation of the pathogen was attempted at 30 dpi. However, no lesions or browning were found in the internal tissues, and pathogen isolations were unsuccessful. In the strawberry test II, the first wilting of leaves was observed on two plants at 4 dpi, on plants inoculated with PhF17/19 (positive control), and the first dead plant was observed at 12 dpi. The last inventory was made at 20 dpi, when all plants inoculated with PhF17/19 were dying. No symptoms were found on plants inoculated with PhF66 or PhF66– a,b. Re-ip = 0.926, N = 41, In the pathogenicity test with silver birch, lesions in all inoculated plants were observed at 6 dpi c. HoweveP. condilina [P. castaneae [Halophytopththora spp. [Globisporangium polare [Phytophthora sp. from asparagus, but in contrast to the results of the current study, sporangia production was enhanced [Bunya-like viruses have been described from the oomycetes ondilina , P. castastaneae , Halophyora spp. , Globispm polare , as wellenhanced .Phytophthora and Pythium, during host plant interaction. They are apoplastic effectors that are utilized to suppress induced immunity responses in the host plant [Arabidopsis resistance to Phytophthora [Phytophthora species, which potentially have various roles in plant infection [P. infestans, P. sojae, and P. ramorum, coding for cytoplasmic effectors that trigger an immune response in the host [Both transcriptomic and proteomic analyses indicated that certain elicitins were differentially expressed in the virus-infected and virus-free isolate. The expressions of three elicitins were strongly upregulated at the RNA level, and one of them was also more abundant at the protein level in the virus-containing isolate . Elicitist plant ,72,73. Wst plant ,75. The ophthora ,77, it infection . The WD4the host ,80,81.Relatively few of the DEGs were found to be differentially abundant as proteins and vice versa. While RNA sequencing enables the profiling of slight changes in the transcript expression between two conditions, it does not capture the involvement of various post-transcriptional, post-translational, and degradation pathways that influence the relative quantities of expressed protein . Thus, RP. cactorum preferring birch as a host. However, we did not observe any differences between the virus-free and virus-containing isolates in pathogenicity to birch. Thus, understanding the roles and functions of the elicitins upregulated by these bunyaviruses requires further investigation.In the pathogenicity tests conducted with the bunyavirus-containing and virus-free isolates (PhF66 and PhF66–), both isolates failed to cause symptoms in strawberry. However, lesions were induced on silver birch indicating the isolate may belong to the population of P. infestans by the unclassified virus PiRV-2 [P. cactorum populations, Pánek et al. [P. cactorum isolates (representing woody species pathotype) to possibly be of hybrid origin. The isolates were also characterized by high oogonial abortion rates, suggesting that the putative hybrid status could be the reason for the relatively high oospore abortion rates detected in the present study. The higher oospore abortion rate in the virus-free isolate may also be related to stress caused by the virus-curing treatment in low water potential. Adverse conditions such as the long deep-freeze storage or previous exposure to fungicides in the field can decreaseoospore viability .The PhF66 isolate containing the bunyaviruses also produced less and smaller sporangia. However, no effective zoospore release was documented. Stimulation of sporangia production was also reported in s PiRV-2 . The prek et al.  inferredP. cactorum are highly stable and resist curing attempts by osmotic stress, but the rarer infections of bunyaviruses seem less stable and more easily curable. In general, the curing and transmission of viruses in vitro has been challenging with the Phytophthora species [Phytophthora. We found that the P. cactorum isolates were very persistent during low water availability conditions and were able to grow in and were recoverable from all tested water potentials, down to −15 MPa. The method was originally developed and proved successful for the fungus Pseudogymnoascus destructans, whose growth ceased at −5 MPa, and an inhabiting partitivirus was cured already at −2 MPa [Phytophthora isolates and thus explain its viability in the very low water potentials during this study. All three alphaendornaviruses , a toti-like virus , and an ustivirus  remained in the host isolates during the treatment. However, bunyaviruses  were shown to be curable with this method. Previously, the unclassified dsRNA virus Phytophthora infestans RNA virus 2 (PiRV-2) and a member of “Fusagraviridae”, Phytophthora infestans RNA virus 3 (PiRV-3), were cured successfully after four and five hyphal tip transfers on Ribavirin-containing plates, respectively [Phytophthora may be subjects of further study. In conclusion, PEG-induced water stress is a potential method for curing Phytophthora viruses, but efficacy varies between viral species.In this study, we found that the most prevalent virus infections (endorna- and toti-like viruses) in  species ,85. In tt −2 MPa . In our ectively ,33. CuriP. cactorum [P. cactorum isolates in dual cultures was not successful. Virus transmission by hyphal anastomosis has been attempted but also proved relatively challenging for P. infestans [Phytophthora isolates in laboratory conditions. At a population level, P. cactorum was recently shown to host multiple viruses with an extremely high prevalence (99% of isolates hosted viruses), indicating a long co-evolution likely resulting in neutral or positive symbiotic relationships [P. cactorum along with host plants between continents [Phytophthora are prone to hybridization [Despite viruses being highly common in isolates of cactorum , we founnfestans . Howevernfestans  reportedionships . Furtherntinents . Howeverdization , which cP. cactorum by PEG-induced water stress, although transmission between different mycelia in laboratory conditions seems to be inefficient. Bunyaviruses were shown to cause phenotypic effects on the growth rate of P. cactorum on artificial media and in apple fruits, but not on the pathogenicity. Bunyaviruses also induced the upregulation of elicitin genes, one of them at both transcript and protein levels, in the virus-containing isolate. Upregulation of other apoplastic effectors  was also observed at the transcriptional level, indicating a putative viral effect on host pathogenesis.In summary, the present study showed that bunyaviruses, but not other tested viruses, may be cured from


The Czech Republic, a part of the former Czechoslovakia, has been at the forefront of several research directions in virology, genetics and physiology. For the latter disciplines, the names of Johan Gregor Mendel (1822–1884) and Jan Evangelista Purkynje (1787–1869), the two Austro-Hungarian scientists born in the territory of the contemporary Czech Republic, can be named. Modern times are proud of important Czech virologists, such as Karel Raška (1909–1987) who was a Director of the WHO’s Division of Communicable Disease Control since 1963. His new concept of eliminating smallpox was adopted by the WHO in 1967 and eventually led to the eradication of smallpox in 1977. Raška was also a strong promoter of the concept of epidemiological surveillance of diseases, which was adopted in 1968 and has since become a standard practice in epidemiology. In 1984, he received the Edward Jenner Medal awarded by the Royal Society of Medicine. Another brilliant scientist who must be named is Jan Svoboda (1934–2017), a pioneer of retrovirology, brilliant scientist, and, for many years, a living legend in the broader field of retroviruses, tumor viruses, and oncogenes. He created the hypothesis that retroviruses can transform healthy mammalian cells into cancerous ones. This had a direct impact on the discovery of reverse transcription described by Howard Temin, who won the Nobel Prize for this discovery. Last but not least, Antonín Holý (1936–2012) was a pioneering Czech organic chemist who specialized in the development of important antiretroviral drugs, especially nucleotide analogues used in the treatment of HIV-1 and hepatitis B. He was involved in the creation of numerous very effective and commercially successful drugs for the treatment of AIDS and other viral diseases.The current coronavirus pandemic has shown the importance of virological research. For historical reasons, the Czech Republic has no scientific institution where such research is concentrated. The former Institute of Virology of the Czechoslovak Academy of Sciences was established on 1 January 1953 in Bratislava (current Slovakia). The institute studied an important concept of the natural focality of diseases and the ecological problems of viruses and viral diseases. The Institute also participated in the discovery of tick-borne encephalitis virus transmission via goat milk and dealt with its structure, antigenic characteristics, and immunology. However, after the Dissolution of Czechoslovakia in 1 January 1993, resulting in split of the federal republic of Czechoslovakia into two independent countries, the Czech Republic and Slovakia, the Institute remained on its premises in the capital of Slovakia: Bratislava. This left the Czech Republic without any coordinated virological research. Only recently, due to the rising virological threats, has the discussion been initiated on establishing a virological institute in the Czech Republic. It became apparent that the understanding of viral properties and modes of spreading and treatment may not only help to bring significant discoveries leading to development of new antiviral strategies and compounds, but it may also facilitate communication between the scientific community and the institutions responsible for setting epidemiological rules. The concept of the currently emerging institute is based on the financial support of selected excellent virological groups established at several Universities and Institutes of the Academy of Sciences in the Czech Republic. Thus, the Institute of Virology will consist of 25 groups established at eight research institutions and coordinated by an executive director (Zdeněk Hostomský) and scientific director (Pavel Plevka). The concept of independent but mutually coordinated research groups at several institutions will profit from complementary disciplines and access to unique state-of-the-art facilities available at individual institutions. The integral part of the project is the sharing of know-how, intense student and staff communication and seminars. It should be mentioned that a minor part of the Institute is devoted to drug-resistant bacteria, which have recently become a similarly important issue as viruses. Moreover, both virology and bacteriology share many common methodological approaches. It is expected that the combination of these two scientific disciplines under the umbrella of one institution will be mutually inspiring.The research will be grouped into three major areas: (a) interactions between pathogens and hosts, (b) immunity against viruses and bacteria and (c) the development of drugs against viral and bacterial diseases The major focus areas of selected Czech virological laboratories, whether or not they are included in the Institute, are shown below .The Laboratory of Structural Biology at the Institute of Organic Chemistry and Biochemistry (head Evžen Bouřa) is focused on proteins essential for viral replication, including both host factors and viral enzymes . For insThe goal of the Klara Grantz Saskova group at the same Institute is to study the mechanisms of HBV escape from intrinsic and innate immunity for the discovery of new anti-viral drugs. To this end, they study (i) the effects of microenvironments of HBV-infected hepatocytes, including that of extracellular vesicles, on PBMCs; (ii) the interplay between promyelocytic leukemia protein nuclear bodies and HBV in the nucleus; and (iii) interactions of the HBV core (HBc) protein with the proteins of the host to inhibit virus replication. In vitro high-throughput screening assays are used to identify small compounds affecting the respective protein–protein interactions.Research in the Laboratory of Viral and Cellular Genetics at the Institute of Molecular Genetics in Prague (head Jiří Hejnar) is focused on retroviruses, the interactions of viruses with the host cell and the control of retrovirus replication by the host cell’s factors, whose presence or absence in the cell defines the cell’s permissiveness or resistance to retroviral infection. An example of such virus dependence factors are entry receptors in a model of avian leukosis virus. This virus diversified into several subgroups using different cell surface receptors  and is tIn the field of antiviral innate immunity, ALVs as a model virus and its natural host, chicken, aided in the discovery of “hidden” genes in microchromosomal regions extremely enriched in GC, including the restriction factor tetherin and the innate immunity factor TNFalpha . The anaThe Medical Zoology Laboratory of the Institute of Vertebrate Biology, Czech Academy of Sciences (head Zdeněk Hubálek), was originally founded by Bohumír Rosický as the Laboratory for research on natural focality of viral and bacterial diseases at the Parasitology Institute Prague, Czechoslovak Academy of Sciences, in 1972. The laboratory is active in the field of arboviruses. For instance, Ťahyna orthobunyavirus was isolated from mosquito larvae and from the blood of sick children in south Moravia by the first lab head Vojtech Bárdoš in 1975 , and ticThe main focus of the Jan Konvalinka group at the Institute of Organic Chemistry and Biochemistry, Academy of Science of the Czech Republic, is the identification, validation and characterization of traditional or novel therapeutic targets for the diagnosis and treatment of viral diseases and various tumors. They combine several approaches to achieve these goals, including synthetic chemistry, chemical biology, structural biology and biochemistry. The models studied involve HIV, influenza, Zika, Dengue and other human viruses, as well as prostate cancer and glioblastoma. The methods used span from organic synthesis to molecular modelling and medicinal chemistry, recombinant protein production in various organisms, enzymology and X-ray and NMR structure analysis to mammalian cell cultures and xenografts of human tumors and transgenic mice. Recently, the Konvalinka group developed novel chemical biology tools based on the conjugates of specific ligands with either biocompatible polymers or DNA oligonucleotides . They could be used for the identification, isolation, visualization and quantification of a variety of protein targets and for high-throughput testing of their inhibitors and ligands. Using these tools, they search for novel inhibitors of known target enzymes and try to identify new therapeutic targets.The Laboratory of Virology at the Institute of Experimental Botany (head Tomáš Moravec) focuses on the study of plant viruses and the management of viral diseases in plants. In addition to the development of tools for reliable virus detection, the main focus is on the area of molecular farming using viruses and their parts to express vaccines, enzymes and antibodies in plants. The programmable CRISPR/Cas9 nucleases in combination with viruses is used to develop gene-editing strategies for marginal crops. Over the years, many plant viruses, and their infectious clones, namely, Tobacco mosaic virus strains U1 and Cg8, Potato virus X, Tobacco rattle virus, Apple latent spherical virus and Bean Yellow Mosaic Virus, were modified for the GoldenBraid convention. This greatly simplifies the exchange of parts and genes between viral vectors. A newly established joint laboratory with the Institute of crop protection will help to conduct both basic and applied research in the field of plant virology.The Laboratory of Structural Virology, headed by Pavel Plevka at CEITEC, Masaryk University, focuses on the cryo-electron microscopy of virus particles and the cryo-electron tomography of virus-infected cells. They aim to structurally characterize the replication cycle of the viruses and their interactions with components of the immune system. They determined the structures of several enteroviruses and described their genome release mechanism, which involves capsid opening ,17. FurtThe research of Iva Pichová laboratory of Viral proteins at the Institute of Organic Chemistry and Biochemistry in Prague has predominantly focused on the functional and structural characterization of proteins regulating the key steps in the lifecycle of different viruses, the identification of cellular proteins involved in virus replication and persistence development. The group investigated the maturation and assembly of retroviruses such as Mason-Pfizer monkey virus, HIV-1 and Mouse mammary tumor virus. Currently, the laboratory focuses on regulations of molecular mechanisms of hepatitis B virus proteins replication, cccDNA transcription activation and maturation of precore protein HBe during the HBV life cycle, with the goal to identify novel targets for drug development.The virology group at the University of Chemistry and Technology, Prague (head Michaela Rumlová), is focused on several aspects of the replication cycle of selected members of viruses from the families Retroviridae, Flaviviridae, and Coronaviridae. The research is focused on the process of assembly, during which viral proteins and viral genomic RNA form both mature and immature viral particles, as well as on the structure of viral proteins, the functional significance of their structural domains, their mutual interactions, interactions with RNA and membranes and the transport of viral proteins or whole intracytoplasmic viral particles in an infected cell . AttentiThe Laboratory of Arbovirology (head Daniel Růžek) is a joint research unit of the Institute of Parasitology, the Biology Centre of the Czech Academy of Sciences in Ceske Budejovice, and the Veterinary Research Institute in Brno, Czech Republic. The laboratory studies the molecular basis of diseases caused by tick-borne encephalitis virus and other flaviviruses, with particular emphasis on the interface between pathogenesis and host immunity and on the development and testing of new antiviral agents and vaccines. The laboratory contributed significantly to the characterization of tick-borne encephalitis virus structure  and the The Laboratory of Immunotherapy at the Faculty of Science, Charles University , combines the activation of innate and adaptive immunity to treat tumors induced by human papillomaviruses. To enhance the efficacy of cancer immunotherapy, mouse tumor models are developed and used for the examination of immunotherapy ,31,32. TThe Laboratory of Molecular and Tumor Virology at the Faculty of Science, Charles University, headed by Ruth Tachezy, is involved in the studies of small non-enveloped DNA viruses associated with tumors in humans. Apart from human papillomaviruses, the laboratory team focuses on human polyomaviruses and other small DNA viruses (anelloviruses or human bocavirus). The team focuses on molecular epidemiology of tumor viruses and developed several approaches to characterize the viral etiology of tumors ,36,37. RJan Weber’s laboratory is focused on interactions of the hepatitis B virus core protein with host cell proteins. In particular, they are interested in the characterization of proteins and cellular pathways involved in (i) the epigenetic regulation of transcription, (ii) ubiquitin–proteasome degradation and (iii) post-translational modifications ,41,42. FThe Laboratory of Trypanosomatid Biology at the Life Science Research Centre, University of Ostrava (head Vyacheslav Yurchenko) is focused on investigating trypanosomatids sensu lato. These unicellular flagellates are obligatory parasites, causing a number of human, domestic animal and plant diseases . BecausePublic Health Institute, Ostrava (PHIO), is a governmental institution providing services in the field of health promotion and protection. It comprises several National Reference Laboratories (NRL), including the NRL for arboviruses (head Hana Zelená). The NRL for arboviruses works as a part of the Department of Virology, providing laboratory diagnostics of a wide spectrum of human viral diseases, including infections with arboviruses and rodent-borne viruses , developing new in-house diagnostic tools and confirmatory assays. Their main research interests are in the diagnostics of arboviruses and rodent-borne viruses, imported and emergent viral diseases, viral infection of the nervous system and electron microscopy. Other professional activities are consulting activities in the field of clinical microbiology and antibiotic therapy. I sincerely thank the authors and reviewers for their efforts when contributing to the collection of the articles in this Special Ossue. I believe that the Special Issue has provided a fresh insight into current trends in virology in the Czech Republic and can be an impulse for establishing new collaborations.


Aedes mosquitoes that can (re)-emerge unexpectedly and cause severe viral infections in humans [In the twenty-first century, newly emerging viruses which are mostly zoonotic or vector-borne have continuously threatened public health and caused outbreaks of global concern. This has been highlighted by the recent COVID-19 pandemic which caused a catastrophic effect on the world’s healthcare system and the global economy [n humans ,5,6,7,8.n humans .The mosquito-borne flaviviruses such as dengue virus (DENV), Zika virus (ZIKV), yellow fever virus (YFV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) are responsible for significant human morbidity and mortality all over the world ,8,11,12.The alphaviruses are a genus of enveloped RNA viruses with medically important alphaviruses such as Chikungunya virus (CHIKV), Mayaro virus (MAYV), and Eastern equine encephalitis virus (EEEV) that can cause arthralgia (CHIKV and MAYV) or neuroinvasive disease (EEEV) ,9,10,23.Despite continued threats from emerging viral diseases, the COVID-19 pandemic has demonstrated that the twenty-first century has come with a new era in vaccinology in which recombinant genetic technologies allowed remarkably rapid development of vaccines against SARS-CoV-2 in 2020 . Over thThis Special Issue will feature the recent development of vaccines against flaviviruses and alphaviruses. Although licensed vaccines against flaviviruses exist such as DENV , JEV (inactivated), tick-borne encephalitis (inactivated), and YFV (attenuated), there are currently no vaccines against WNV and ZIKV . Moreove


Trichosanthes kirilowii is one of the most important perennial herbaceous vines that have been used in traditional Chinese medicine. In this study, a novel RNA virus was discovered in T. kirilowii plants showing leaf mottling and mosaic symptoms. The complete genome of this virus is 6,524 nucleotides long and encodes four open reading frames which are arranged in a manner typical of tobamoviruses. Phylogenetic analysis based on the complete genome sequence revealed that the virus was clustered into a branch with the tobamoviruses whose natural host are plants belonging to the family Cucurbitaceae. A full-length infectious cDNA clone was then constructed and demonstrated to establish a systemic infection with typical symptoms in Nicotiana benthamiana, T. kirilowii, and five other cucurbitaceous crops including Cucumis melo, C. lanatus, C. sativus, Luffa aegyptiaca, and Cucurbita pepo via agrobacterium-mediated infectivity assays. Further experiments provided evidence that the rod-shaped viral particles derived from the infectious clone could be mechanically transmitted and reproduce indistinguishable symptoms in the tested plants. Taken together, the mottle mosaic disease of T. kirilowii is caused by a distinct tobamovirus, for which the name Trichosanthes mottle mosaic virus (TrMMV) is proposed. As the infectious cDNA clone of TrMMV could also infect five other cucurbit crops, this distinct tobamovirus could be a potential threat to other cucurbitaceous crops. Trichosanthes kirilowii Maxim. is a vine in the family Cucurbitaceae that is mainly distributed in eastern and southern Asia, including China, South Korea, and Japan  is proposed.In this study, a novel tobamovirus was identified from T. kirilowii plants showing leaf mottling and mosaic symptoms  photoperiod.symptoms  were colT. kirilowii leaves using TRIzol reagent  according to the manufacturer’s instructions. The qualified total RNA was used to construct the small RNA (sRNA) library according to the instructions of the Small RNA Sample Pre-Kit   . The res States) . BLASTN ® FastPfu DNA Polymerase , and the amplified products were purified by Gel Extraction Kit  and ligated into pEASY-Blunt vector (TransGen) for sequencing. The exact 5′- and 3′-terminal sequences of viral genomic RNA were determined using the SMARTer® RACE 5′/3′ Kit . The sequences of 5′ and 3′ gene-specific primers  and nested primers  are shown in OL404963.To obtain the full genome sequence of the virus, three primer sets  were used for the linearization of the pCB301 vector. After gel purification, the PCR products of the two amplified virus fragments and the linearized pCB301 vector were assembled using a ClonExpress II one-step cloning kit  according to the manufacturer’s protocol. The recombinant plasmid pCB301-TrMMV was confirmed by Sanger sequencing.The infectious cDNA clone of TrMMV was constructed into the plant binary vector pCB301 using a one-step assembly strategy as described by Agrobacterium tumefaciens strain EHA105 via electroporation , and 100 μM acetosyringone with OD600 = 1.0. Approximately 400 μL of A. tumefaciens suspension was infiltrated into the abaxial side of the upper two leaves of 21-day-old T. kirilowii using a needleless syringe. For agroinoculation of N. benthamiana plants, three fully expanded upper leaves of 4-week-old plants were infiltrated. For agroinoculation of cucurbitaceous plants, fully expanded cotyledons were infiltrated with a needleless syringe. Infiltration experiments were conducted at least twice and five plants were used for each treatment. Plants mock-inoculated with A. tumefaciens harboring the pCB301 vector served as negative controls. All the inoculated plants were maintained in a growth chamber at 25°C with a 16 h light/10 h dark cycle.The recombinant pCB301-TrMMV binary plasmid was transformed into the poration . A. tumeescribed . After tN. benthamiana infiltrated with A. tumefaciens harboring pCB301-TrMMV were used as the source of inoculum. Approximately 1.0 g of N. benthamiana leaf samples was ground in 10 mL of 0.01 M phosphate buffer (pH 7.2) and used as crude sap . After centrifugation at 11,300 × g for 10 min, the supernatant was placed in a copper grid and a filter paper was used to remove impurities and moisture. The grids were covered with a drop of 2% tungsten phosphate staining solution (pH 7.0) and then dried at room temperature. Grids were observed and photographed with a transmission electron microscope  at a voltage of 80 kV.Transmission electron microscopy was used to observe the virus particles. Approximately 0.1 g of T. kirilowii plants showing mosaic and mottling symptoms were observed in Qianshan city of Anhui Province, China , kyuri green mottle mosaic virus (KGMMV) (accession number AJ295948), and cucumber fruit mottle mosaic virus (CFMMV) (accession number JN226146). The longest contig (245 nts) was closely related to ZGMMV (accession number MF066176), with 98% coverage and 90.08% identity at the nucleotide level, indicating the presence of a tobamovirus infection in the samples. To validate the presence of potential tobamovirus in each T. kirilowii sample, a pair of specific primers . ORF1 terminates at position 3,571 with UAG as the termination codon and encodes a 131-kDa replicase protein including viral RNA helicase  and viral methyltransferase . ORF2, which terminates at position 5,083 by reading through the leaky termination codon, encodes a 189-kDa replicase protein that includes viral methyltransferase , viral RNA helicase , and RNA-dependent RNA polymerase  domains. ORF3 and ORF4 encode a 28-kDa MP  and a 17-kDa CP , respectively. The 5′ untranslated region (UTR) and 3′ UTR of the virus are 70 and 163 nts in length, respectively . Pairwise genome similarity comparison of the tobamovirus to known tobamoviruses showed that the full-length virus genome shared 71.9–87.8% nucleotide similarity with known tobamoviruses (Tobamovirus (a threshold of 10% nucleotide variability) .BLASTN search using the assembled 6,524-nt viral genome suggested that the viral sequences shared the highest nucleotide identity (87.8%) with ZGMMV-ZT-1 (Accession number oviruses . Accordiability) , we propComparison of the nucleotide sequence of ORF1, ORF2, ORF3, and ORF4 of TrMMV to those of other tobamoviruses revealed that TrMMV is most closely relative to ZGMMV-ZT-1. The four ORFs of TrMMV shared nucleotide sequence identity of 86.6, 87.4, 87.4, and 90.5% to those of ZGMMV-ZT-1, respectively . The amiTobamovirus using the complete genomes of tobacco rattle virus and Indian peanut clump virus as outgroups. As shown in To better understand the relationship of TrMMV to other known tobamoviruses, a phylogenetic tree was constructed based on the complete genomes of TrMMV and 43 species of the genus N. benthamiana using agrobacterium-mediated infiltration assays. At 10 days post-inoculation (dpi), typical symptoms such as leaf curling and chlorosis began to appear in the non-inoculated systemic leaves of all the 15 inoculated N. benthamiana plants  to yield pCB301-TrMMV . The infa plants . The prea plants . In conta plants . Electro virions . These rT. kirilowii. At 17 dpi, typical symptoms such as leaf mosaic and mottling symptoms appeared in the non-inoculated systemic leaves of all the 10 inoculated T. kirilowii plants . At 18 dpi, the systemic leaves of C. melo seedlings showed symptoms of yellowing, crinkle, and chlorosis ” is proposed. Koch’s postulates assay demonstrated that TrMMV is the causal agent associated with the T. kirilowii mottle mosaic disease.r leaves . A new Tirilowii . In thisHQ667979) shares 94.2, 94.4, and 91.7% with those of turnip vein-clearing virus (accession number U03387), respectively. The high similarity of amino acid sequences encoded by proteins among different species is due to the degeneracy of codons (The complete genome of TrMMV shares the highest nucleotide sequence identity (87.8%) with ZGMMV and has a genome organization resembling those of other known tobamoviruses . Sequencf codons . Host adf codons . Previouf codons . It woulin vivo and were able to infect N. benthamiana, T. kirilowii, and other five tested cucurbitaceous crops, indicating that TrMMV could recruit similar host factors of these species to establish a successful infection. Compared with the mild mosaic and mottling symptoms of T. kirilowii, TrMMV infection on the other five cucurbitaceous crops produced more obvious symptoms, suggesting that TrMMV could be a potential threat to a wide range of cucurbit crops.Infectious clone of plant viruses provides a useful tool for studying the function of viral proteins and the interactions between virus and host. Infectivity experiments demonstrated that the infectious clone of TrMMV constructed in this study could form biologically active rod-shaped viral particles N. benthamiana, and other five tested cucurbit plants. Although it is not known whether TrMMV is seed-borne at this stage, the availability of the infectious clone developed here would allow us to address this issue in the near future.Tobamoviruses have extremely stable virus particles and accumulate to high titer in susceptible host plants. They are easily transmitted by mechanical means. Some tobamoviruses such as tobacco mosaic virus, cucumber green mottle mosaic virus, and tomato brown rugose fruit virus have posed a serious threat to agriculture . AlthougThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/XY and XZ conceived and designed the research. CC, MD, DP, WL, and JX performed the experiments. CC and XY analyzed the data. CC, XY, and XZ wrote and reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.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.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.


Viral infections can be fatal and consequently, they are a serious threat to human health. Therefore, the development of vaccines and appropriate antiviral therapeutic agents is essential. Depending on the virus, it can cause an acute or a chronic infection. The characteristics of viruses can act as inhibiting factors for the development of appropriate treatment methods. Genome editing technology, including the use of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) proteins, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), is a technology that can directly target and modify genomic sequences in almost all eukaryotic cells. The development of this technology has greatly expanded its applicability in life science research and gene therapy development. Research on the use of this technology to develop therapeutics for viral diseases is being conducted for various purposes, such as eliminating latent infections or providing resistance to new infections. In this review, we will look at the current status of the development of viral therapeutic agents using genome editing technology and discuss how this technology can be used as a new treatment approach for viral diseases. Viruses are considered to be on the borderline of living and non-living things, as they display characteristics of both the living and non-living. They are generally composed of genetic materials surrounded by a protein shell. Viruses cannot reproduce by themselves and must infect living organisms and replicate through them. In addition, viruses use the molecular machinery of the infected host cell for multiplication. A new virus is created in the infected host cell through a series of processes and continues to infect other host cells. Consequently, the virus causes acute or chronic disease, depending on how long its host cell remains intact. Some viruses cause simple diseases, such as a cold, while others, such as SARS-Cov-2 COVID-19), acquired immune deficiency syndrome (AIDS), and hepatitis can cause serious diseases. In addition, some viruses are highly infectious and can cause fatal diseases of the human body, posing a great threat to human health. Hence, the development of effective antiviral treatments is of great interest to the global health community [9, acquirGenome editing technology involves the use of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) proteins, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs) to precisely manipulate specific genomic sequences. This technology allows various manipulations, such as deleting a specific DNA or RNA sequence of the genomic locus or adding a specific sequence to a DNA locus. When genome editing is performed on a target DNA, a nuclease-induced double-stranded break (DSB) generally occurs. Homology-directed repair (HDR) or nonhomologous end-joining (NHEJ) processes then follow to repair the DSB . The devSo far, several genome editing-based antiviral agents have been developed for various purposes, such as eliminating latent infections or providing resistance to new infections . This reRepresentative tools for genome editing include ZFN, TALEN, and the CRISPR/Cas system , all of Xanthomonas [After ZFN, TALEN was designed as a novel genome editing tool . Similarthomonas ,13. Eachthomonas . In genethomonas . Howeverthomonas ,15,16.Streptococcus pyogenes is the most commonly used system for gene editing. The type 2 CIRSPR/Cas system utilizes Cas9 as an endonuclease. Cas9 is guided to a target by a chimeric CRISPR RNA (crRNA); trans-activating RNA (tracrRNA) also called guide RNA (gRNA) [The CRISPR locus was first discovered in E. coli in 1987 . Since tA (gRNA) . In genoA (gRNA) . The sgRA (gRNA) ,26. The A (gRNA) . While tA (gRNA) . The fleHuman papillomavirus (HPV) is a non-enveloped double-stranded DNA virus of the family Papilomaviridae. Most HPV infections show no symptoms. Patients can recover within two years. However, in some cases, the infection can cause warts or precancerous lesions, with an increased risk of the cervical, vulval, vaginal, penile, and anal cancers . In partVarious groups have developed genome editing-based therapeutic agents for HPV infection or cancer caused by HPV infection, and the development of therapeutic agents that target viral genes or host factors known to interact with viruses is in progress. Most studies have been conducted with the HPV E6/E7 oncogene as a target. Shankar et al.  designedIn some studies, additional methods were used to increase the efficiency of genome editing. Fan et al.  developeStudies on safe delivery of the developed system in vitro or in vivo have also been conducted. Jubair et al. ,40 develThere have been several attempts to obtain therapeutic effects by modulating host cellular factors involved in HPV tumorigenesis. Sterile alpha motif and histidine-aspartic domain HD-containing protein 1 (SAMHD1) is a deoxynucleotide triphosphate triphosphohydrolase (dNTPase) enzyme that can regulate intracellular levels of dinucleotide triphosphates (dNTPs) and act as an intrinsic immune response factor . James eHerpes simplex viruses 1 and 2 (HSV-1 and HSV-2) are linear double-stranded DNA enveloped viruses that belong to the Herpesviridae family. They can infect epithelial tissues and invade the nervous system, where they enter a latent stage of infection. Both viruses persist in the human body by hiding in the cell bodies of neurons. When activated, they can move from neurons to the skin, where virus replication and shedding occur . Most HSResearch to develop HSV infection therapeutic agents using genome editing mainly targets the HSV genome. Roehm et al.  developeAnother strategy is to target cellular factors related to HSV-1 infection. Li et al.  developeEpstein–Barr Virus (EBV) is a linear double-stranded DNA enveloped virus that belong to the Herpesviridae family. EBV infects B cells and epithelial cells of the immune system. After the initial lytic infection is properly controlled by the immune system, it remains dormant in B cells for life . EBV is Various groups have developed genome editing-based therapeutic agents for EBV infection. Yuen et al.  developeAnother strategy is to target cellular factors related to EBV infection. EBV infection and nasopharyngeal carcinoma (NPC) have been widely recognized in recent decades . In partHCV is a small, enveloped virus of the family Flaviviridae. It has a positive-sense single-stranded RNA as a genome. This genome consists of an untranslated (UTR) region (at 5′ and 3′ ends) and a region coding for a viral polyprotein. HCV causes hepatitis C, liver fibrosis, cirrhosis, and liver cancer . Thus, HAs with other general virus-targeting genome editing technologies, the development of HCV therapeutic agents through genome editing also targets the HCV genome or host cellular factors related to HCV infection. Clement et al.  developeZika virus (ZIKV) is a single-stranded positive-sense RNA enveloped virus of the family Flaviviridae. The ZIKV genome is directly translated into a viral polyprotein that encodes three structural proteins and seven non-structural proteins. The ZIKV RNA genome’s replication depends on the synthesis of double-stranded RNA from its single-stranded positive-sense RNA genome, after which the replication of new single-stranded positive-sense RNA proceeds . ZIKV caThe family of adenosine deaminases acting on dsRNA (ADARs) is a human host factor important for the genetic diversity and evolution of ZIKV. Zhou et al.  developeCoronavirus (CoV) is a positive-sense single-stranded RNA virus of the family Coronaviridae, with a genome of about 30 kilobases in size. CoV’s genome is organized as 5′-leader-UTR-replicase (ORF1ab)—spike (S)—envelope (E)—membrane (M)—nucleocapsid (N)—3′UTR-poly (A)—tail. Thus, it can act like a messenger RNA (mRNA) . CoV relIn 2003, severe acute respiratory syndrome (SARS) caused by severe acute respiratory syndrome coronavirus (SARS-CoV) began in Asia and spread rapidly. So far, SARS-CoV has infected more than 8000 people, killing approximately 10% of them . In SeptCRISPR-Cas13 is an RNA-guided RNA-targeting CRISPR system. The characteristic of this system may be used to target the RNA of SARS-CoV-2 and inhibit its replication. Abbott et al.  developeThe influenza virus is an enveloped virus with a segmented negative single-stranded RNA as its genome. It is a member of the family Orthomyxoviridae. There are four types of this virus: type A, type B, type C, and type D. However, only types A and B are clinically relevant to humans . The infIt seems that the CRISPR/Cas13-based genome editing system can play a role in the development of influenza virus treatment, similar to other RNA viruses. Abbott et al.  developeHIV is a lentivirus (a subgroup of retroviruses) that causes acquired immunodeficiency syndrome (AIDS) . It is aThere is no complete cure for HIV/AIDS yet. However, current treatments, the antiretroviral therapies, slow the progression of the disease and reduce the risk of death. They include a combination of three or more antiretroviral drugs (cART) from several different drug classes. Unfortunately, cART has numerous shortcomings, such as severe side effects and high cost. Therefore, genome editing-based systems might be a potential therapeutic approach to overcome the disadvantages of conventional HIV treatment.The development of HIV therapeutic agents based on genome editing technology has mainly targeted the HIV genome, provirus, or related host factors. In particular, many studies have been conducted targeting CCR5 and CXCR4, which are co-receptors for HIV entry. Perez et al.  generateTargeting the HIV genome, such as the HIV proviral DNA, is another strategy for developing HIV therapeutic agents. Qu et al.  and Ji eResearch on the development of HIV treatment based on TALEN has also been conducted in several studies. Shi et al.  designedStudies targeting the HIV genome have also been conducted. Ebina et al.  designedThe genome editing technology based on CRISPR/Cas is one of the most popular platforms for HIV treatment development. Like ZFN and TALEN, the CRISPR/Cas system has also been studied for co-receptors such as CCR5 and CXCR4. Wang et al.  developeIn addition to targeting CCR5, studies targeting CXCR4 or both have also been conducted. Wang et al.  designedHIV remains incurable due to the permanent integration of the viral genome to the host chromosome, which increases the risk of viral reactivation even after antiviral therapy. Therefore, another way to use the CRISPR/Cas system for the treatment of HIV is to target the genome, such as the HIV provirus. Hu et al.  used theHowever, this system is associated with viral escape caused by the off-target effect. Consequently, several groups have tried to use various sgRNAs to target various regions of the genome. Wang et al.  developeDirectly targeting the HIV RNA genome is another approach for developing therapeutic agents for HIV. Since the CRISPR/Cas13 system, an RNA-guided RNA cleavage system, can target ssRNA, it can be a powerful tool for developing therapeutic agents for viruses that have ssRNA as their genome. Nguyen et al.  designedHepatitis B virus (HBV) is a partially double-stranded DNA virus that belongs to the Hepadnaviridae family. It is classified into eight genotypes from A to H. The HBV viral DNA genome is rendered to fully double-stranded DNA by cellular DNA polymerases and transcribed by cellular RNA polymerases. Transcribed viral RNAs are translated into viral proteins essential for the virus’s life cycle. HBV causes hepatitis B, which can be either acute or chronic, and may lead to liver cirrhosis and hepatocellular carcinoma. Despite an effective HBV vaccine, it is still a global health concern due to the lack of adequate treatments.The virus persists in infected hepatocytes, because covalently closed circular DNA (cccDNA), the template for the transcription of viral RNAs, is stable in nondividing cells. Major treatments for HBV infection include the use of interferon-α and nucleotide analogs. However, they cannot eradicate cccDNA. Therefore, many studies targeting HBV cccDNA have been conducted. Dong et al.  designedThe off-target effect is one of the biggest challenges in the development of HBV treatment. Therefore, there have been efforts to develop therapeutic agents to prevent such off-target effects. Induction of a double-strand break (DSB) on the targeted genome by Cas9 risks an unwanted off-target effect on the host genome. Cas9-nickase can cleave a single strand of DNA. Therefore, two sgRNAs are required to induce DSBs using the Cas9-nickase, lessening the unwanted off-target effects. Kurihara et al.  examinedThe genome editing technology is a relatively recently developed technology. However, the convenience and potential of this technology have made great strides in many fields of life science. Efforts have been made to use this technology to develop therapeutic agents for viral diseases, and several groups have used the technology in clinical practice.Perez et al.  generatehttps://clinicaltrials.gov/ct2/show/NCT05144386, 15 September 2021). EBT-101 is an AAV9-based CRISPR/Cas9 system targeting the proviral DNA of HIV-1 and composed of Cas9 and three gRNAs. Many other clinical studies based on genome editing for antiviral therapy are expected to proceed in the near future.Excision Bio Therapeutics recently received U.S. Food and Drug Administration approval for the clinical trial of EBT-101 for HIV-1-infected patients . Nonetheless, there are several systems that have been devised to deliver them. In particular, many studies have been conducted to deliver genome editing tools to in vitro or in vivo models through viral delivery systems such as adenovirus, AAV, and lentivirus ,43,105. With the advancement of genome editing technology, ethical issues are also becoming major concerns . The dev


The circular replication‐associated protein (Rep)‐encoding single‐stranded (CRESS) DNA virus emergence in diverse host has been associated with severe disease. Porcine circovirus‐like virus (Po‐Circo‐like [PCL] virus) is a CRESS DNA virus, the prevalence and pathogenicity of which are rarely studied.We obtained two blood samples, four faecal samples, and two intestinal samples from a pig farm suffered from diarrheal disease in the delivery room in September 2020 and attempted to isolate and identify a causative pathogen. Subsequently, only PCL virus was positive, and qRT‐PCR was designed to detect the loading titre of PCL virus. We then initiated a heightened surveillance program on the pathogenicity and epidemiology of PCL virus.Six PCL virus strains, with severe diarrhoea and haemorrhagic enteritis, have been found in six different pig farms in Guangdong province, China. A multiple sequence alignment of these PCL viruses and bovine circovirus‐like virus/CH showed a similarity of 92.5‐94.8% for the Rep protein, indicating these PCL viruses are highly homologous to Bo‐Circo‐like virus associated with calf diarrhoea. There were striking similarities between the PCL virus and bovine circovirus‐like virus outbreaks in aetiological settings and Genomic sequence. We found that 11.2% (20/178) of diarrhoea samples and 13.3% (6/45) of pig farms were positive for PCL virus, suggesting that PCL virus may have spread widely in Pig farms. Moreover, this article underscores the risk of PCL virus spilling over and adapting to new species.Porcine circovirus‐like virus was found to be associated with porcine diarrheal disease in China. Detailed information on gene primers used in qRT‐PCR is listed . The res2.5The complete gene sequences of PCL viruses obtained in this study have been uploaded to Genbank with the accession numbers . The gen3CRESS viruses can infect a wide range of animals, even plants and mosquitoes. These viruses are found in pigs include PCV1, PCV2, PCV3, PCV4, porcine circovirus‐like virus P1 and PCL virus Meng, . PCV2 haIn this study, PCL virus was detected from six pig farms in different areas Figure . This stIn the six farms, intestinal contents did not identify any pathogenic bacteria causing diarrhoea in pigs. In farm B, the PCL virus isolate CQY09 were detected from newborn piglets (1 week to 4 weeks) associated with severe diarrhoea, haemorrhagic enteritis, and vomiting in Qingyuan, China. The swine enterovirus  have been retested on PCL positive samples as shown in Further studies shown that PCL positive sample CMM06 in farm A was coinfected with PBOV. Enteritis and diarrhoea primarily occurred in piglets, but severe diarrhoea, loss of appetite, and significant reduction in average daily weight gain occurred in fat pigs, speculating the cause of coinfection of these viruses. Besides, three farms of the six PCL virus‐positive farms were coinfected with porcine epidemic diarrhoea virus. Although piglets with diarrheal diseases in the six farms have higher morbidity (80‐100%) and mortality (20‐70%), this may only be caused by PEDV, PBoV or PCL virus, or PCL virus may aggravate PEDV or PBoV infection. The detection of PCL virus was closely associated with diarrhoeal disease in pigs in this study, the presence of the virus in healthy pigs was not assessed. The PCL virus has not been successfully isolated from cells, so animal model tests on the pathogenicity of PCL virus cannot be carried out. In conclusion, more studies on the pathogenicity of PCL virus are needed.GGGCAATTCTGCCC, GGGCAAGTCTGCCC, and GGGCAAATCTGCCC (Subsequently, the full genomes of six strains were sequenced, and their gene characteristics were further analysed Figure . The genTCTGCCC . MoreoveThe genome‐wide pairwise identities between the six PCL virus strains and the Bo‐Cir‐like virus were about 89%. Besides, a multiple sequence alignment of these strains and Bo‐Circo‐like virus CH, showed a sequence similarity of 86.2‐94.4% for the rep gene lines and sequence similarity of 89.4‐97.7% for the Rep protein arrangements. Phylogenetic trees based on amino acid sequences of the Rep protein have been constructed Figure , indicatThe strains of PCL virus had a close relationship to strains of Bo‐Circo‐like virus CH and the strains of GX14, GX15, GX19, 21 and 22 of the PCL viruses on amino acid sequences of the Rep protein Figures  and 4, iThe striking similarities between the PCL virus and bovine circovirus‐like virus outbreaks in aetiological settings and Genomic sequence cannot be ignored (Guo et al., These findings have helped us to understand the status of intestinal infection in the Chinese pig population and also prompted us to accelerate research into pathogenesis and epidemiology of the PCL virus.The authors declare no conflict of interest.The data that support the findings of this study are available from the corresponding author upon reasonable request.The authors show that it is available.This work is approved.The authors approve that the study is suitable for participation.The authors agree that the study is publishable.Conceived and designed the experiments: CX S. Performed the experiments:XH L, XM Z, G X, Z W, HQ S, and KQ L. Analysed the data: XH L, JH Z, YH L, PY L, LY Z and YL L. Contributed reagents/materials/analysis tools: CX S. Worte the paper: XH L and CX S.SUPPORTING INFORMATIONClick here for additional data file.


Sulfolobus islandicusLAL14/1 CRISPR arrays. Spacer acquisition was observed as early as 30 minutes post infection, with the newly acquired spacers uniformly distributed across the genome of the virus. Although the newly acquired spacers were predominantly effective only against the CRISPR-Cas susceptible mutant virus, we were able to isolate a host mutant with a novel spacer which provides immunity against the multiple Acr encoding wildtype virus,Sulfolobus islandicusrod-shaped virus 2 (SIRV2).Spacer acquisition, the first step in CRISPR-Cas adaptive immunity, plays a critical role in establishing and strengthening host defense against mobile genetic elements (MGEs). Here we present a host-virus system, where an increase in the multiplicity of infection (MOI), of a CRISPR-Cas susceptible virus, forces rapid spacer acquisition in the Sulfolobusmonocaudavirus (SMV1) inSulfolobus. Spacer acquisition was also observed upon overexpression of the transcriptional regulator, Csa3a, predominantly from theSulfolobusgenome . Here we present a system with accelerated spacer uptake that could be an ideal choice for the study of acquisition inSulfolobus.CRISPR-Cas adaptation or spacer acquisition involves the integration of a DNA fragment from an invading MGE into the leader-repeat junction of the CRISPR array. Transcription of the array and subsequent processing provides the template, crRNA, necessary for the identification and targeting of the invader. In archaea, spacer acquisition from conjugative plasmids and viruses was observed 3-12 days post coinfection withS. islandicusLAL14/1. This feature allowed us to study possible spacer uptake from SIRV2M in the wildtype host. Upon infection with a range of MOIs, we observed spacer uptake 2 hours post infection within the CRISPRarray_2at MOIs greater than 5 .S. islandicus LAL14/1 encodes 5 CRISPR arrays classified into subtype I-A leader-repeat (array_1andarray_2) and subtype I-D leader-repeat  . Spacer uptake was observed in at least one of the subtype I-A and one of the subtype I-D arrays, indicating that both acquisition modules inS. islandicusLAL14/1 were activated upon high MOI infection . Furthermore, sampling at earlier time points showed that spacer uptake was initiated as early as 30 minutes post infection and acquisition intensity increased at later time points . SIRV2 specific spacer uptake was also seen on a plasmid engineered to encode the subtype I-A (array_2) leader-repeat sequence .Previously, we isolated a mutant SIRV2 virus with a 3.9 kbp deletion on the left terminus of the wildtype genome upon propagation in a CRISPR deficient host . The mutant virus, SIRV2M, lacking the subtype I-D inhibitor AcrID1, is sensitive to targeting by the CRISPR-Cas systems of the wildtype hostS. islandicusLAL14/1 strains with new spacers in the CRISPR arrays. To achieve this, cells infected with SIRV2M at high-MOI for ~20 hours were washed to remove any extracellular virus, plated and screened for spacer acquisition in single colonies. Four positive colonies with single spacer acquisition and one colony with double spacer acquisition were isolated. As with the WT host, all the five colonies were immune to infection by the mutant virus whereas one of the five isolates,S. islandicusLAL14/1 CR1Y433 gained immunity to the wildtype virus, SIRV2. Interestingly, the new spacer, Y433 (Extended Data), of the isolate CR1Y433 matched in sequence the transcript of the SIRV2 early genegp48i.e.,acrIIIB1. In order to verify the role of Y433 in CR1Y433 immunity against SIRV2, we constructed a plasmid based mini-CRISPR array carrying Y433 under the control of an arabinose promoter (pSpcY433).S. islandicus LAL14/1 Δarrays pSpcY433showed complete resistance to the wildtype virus SIRV2 confirming that the resistance gained in the isolate CR1Y433 was solely due to the presence of the new spacer Y433 . In total, we sequenced 16 novel spacers acquired on the CRISPR arrays either on the host genome or on the plasmid encodingarray_2leader-repeat (Extended Data). The newly acquired spacers were evenly distributed across the genome of the SIRV2M virus with no strand bias, implying a naive spacer acquisition . On average, the spacers were 41 bps in length, with the protospacer adjacent motif (PAM) predominantly CCN for subtype I-A spacers (Extended Data).Next, we tried to isolateSulfolobusCRISPR I-A loci , the accelerated spacer uptake observed here could be due to potential activation of the corresponding HEPN regulatory proteins of the I-A and I-D systems  upon continuous SIRV2M infection at high MOIs.Despite encoding 13 spacers matching SIRV2 genome , the wild-type host is compelled to reinforce its CRISPR-Cas immune system with additional spacers to overcome continuous virus infection. Previously, we have also demonstrated that early gene targeting by subtype III-B systems is immune to inhibitory activity of AcrIIIB1 . Here, isolation ofS. islandicusLAL14/1 CR1Y433 demonstrates that acquisition of a spacer targeting early viral gene(s) can indiscriminately protect the host from viruses carrying multiple Acrs, including an inhibitor of type III system. Similar occurrences in a natural environment would lead to complete eradication of viruses. Therefore, a lack of spacers targeting early SIRV2 genes inS. islandicusLAL14/1 could be either due to rapid mutations within the protospacer regions or the presence of an inhibitor of spacer acquisition among SIRV2 early genes, which are absent in the mutant virus, SIRV2M.Previously, naive spacer acquisition was proposed to occur from inactivated viruses as demonstrated with a replication deficient bacteriophage . In accordance with this work, we propose here that naturally occurring Acr deficient viruses, generated upon infection of CRISPR-deficient hosts, could be targets for naive spacer uptake in CRISPR-active hosts. As an overexpression of the HEPN domain containing transcription regulator Csa3a was found to induce spacer acquisition inS. islandicusLAL14/1 and Δarraysliquid cultures were grown at 78℃, 200 rotations per minute. TheE. coli/Sulfolobus shuttle vector pEXA was used for cloning of thearray_2leader repeat sequence intoS. islandicusLAL14/1. Electroporation of plasmid intoSulfolobusand virus titre estimations were performed as described earlier . The mini-CRISPR loci plasmid, pSpcY433transcribing the spacer Y433 targetingSIRV2gp48was constructed as described earlier  using primers Y433_Spc_For and Y433_Spc_Rev.AllSpacer acquisition assaySulfolobuscultures were transferred to fresh medium at OD600= 0.05 and allowed to grow until the OD600reached between 0.1 and 0.2. Specified amount of virus supernatant was transferred into the cultures to achieve the desired MOI. At the specified time intervals 10 ml of cell culture was withdrawn and centrifuged at 6300 x g for 6 minutes. The pellet was washed twice with medium salts and resuspended in TL buffer. Total genomic DNA was extracted using the E.Z.N.A Tissue DNA kit (Omega BIO-TEK) following the manufacturer instructions. The extracted genomic DNAs were utilized as templates in the proceeding PCR reactions. For sequencing of novel spacers,array_2was amplified with primers, Array2_LR_SphI_For and Array2_LR_NotI_Rev, the expanded bands of size larger than the wildtype arrays were gel extracted, restriction digested and cloned into the pEXA plasmid. Plasmids carrying new spacers were sequenced using plasmid specific sequencing primers.OvernightSingle colony isolationS. islandicusLAL14/1 was performed as defined in the spacer acquisition assay. The withdrawn samples were washed thrice with either medium salts orSulfolobusmedium  to remove any virus present in the supernatant. Serial dilutions of the infected cells were plated onto a 2X SCV/gelrite plate and incubated for 7-10 days at 78℃. The single colonies were resuspended in medium salts, spotted onto new 2X SCV/gelrite plates and incubated for 3 days at 78℃. The spots were then transferred into liquid SCV medium. Genomic DNA, extracted from the single colonies, was used as templates in PCR to detect clones with new spacers acquired into the CRISPR arrays.High MOI infection ofTable 1: primers used in this studyarray_2.Italicised nucleotides correspond to restriction sites utilised for cloning and sequencing of10.22002/che7n-2dw90Description: List of spacer sequences newly acquired upon infection with SIRV2M (column 1) and their PAM sequence (column 2). The CRISPR-Cas subtypes capable of utilizing each new spacer is specified (column 3) along with the location of the spacer in the SIRV2 genome (column 4). Spacer complementarity to sense strand (column 1) is especially important for the transcription dependent targeting of subtype III-B CRISPR-Cas systems.. Resource Type: Text. DOI:


Bunyavirales of the Orthobunyavirus genus in the Peribunyaviridae family. Bunyamwera is a negative-sense RNA virus composed of three segments S, M, and L. Genetic recombination is possible between members of this order as it is already documented. Additionally, it can lead to pathogenic or host range improvement, if it occurs with viruses of public health and agricultural importance such as Rift Valley fever virus and Crimea–Congo hemorrhagic fever virus. Here, we characterize five African Orthobunyavirus viruses from different geographical regions. Our results suggest that the five newly characterized strains are identified as Bunyamwera virus strains. Furthermore, two of the five strains sequenced in this study are recombinant strains, as fragments of their segments are carried by Ngari and Bunyamwera strains. Further investigations are needed to understand the functional impact of these recombinations.Bunyamwera virus is the prototype of the Bunyamwera serogroup, which belongs to the order  Orthobunyaviruses include a heterogeneous viral group, which Bunyamwera serogroup is one of the largest. The Bunyamwera serogroup regroups arthropod-borne virus members, Bunyamwera virus (BUNV), Ngari virus (NRIV), and Batai virus (BATV), are some of the most isolated members of this serogroup in Africa [The Bunyavirales are one of the largest viral orders, containing more than 350 viruses including several viruses of public health and agricultural importance such as Rift Valley fever virus (RVF) and Crimean–Congo hemorrhagic fever virus . As Bunyn Africa .BUNV, BATV, and NRIV have a tri-segmented RNA genome: small (S), medium (M), and large (L). The S segment encodes the nucleocapsid protein (N) and a non-structural protein (NSs), which are translated from overlapping open reading frames (ORF). The M encodes glycoproteins (Gn and Gc) and a non-structural protein (NSm), and the L mRNA encodes the RNA-dependent RNA polymerase (RDRP) .Aedes mosquitoes and Amblyomma and Hyalomma ticks [Bunyamwera virus (BUNV) was first isolated in Uganda in 1943. Since that, BUNV was isolated from many arthropods such as ma ticks . BUNV isma ticks ,7. BUNV ma ticks . Anopheles maculipennis in Czechoslovakia in 1950, the Batai virus (BATV) is nowadays the Bunyamwera serogroup member with the largest geographical distribution. BATV was isolated in Asia , Europe , and Africa (Uganda) [Firstly, isolated from (Uganda) . In live(Uganda) , and in Bunyamwera orthobunyavirus spp. [Bunyamwera orthobunyavirus sp. [Genetic reassortment and genetic recombination are the driving forces in the evolution of viruses from the Bunyamwera serogroup ,11. Recorus spp. . They arirus sp. . As the irus sp. ,17,18. Firus sp. ,19.orthobunyavirus members use the same hosts and are located in the same area showed enormous potential for the emergence of reassortment and recombinant bunyaviruses with pathogenic potential improvement or host range [A recent study shows that this segment reassortment between Batai and Bunyamwera probably occurred in co-infected mammalian cells  and thatst range .orthobunyavirus isolates from the Institut Pasteur de Dakar (IPD) biobank using Next Generation Sequencing (NGS) was aimed in this study.In Senegal, few are known about Orthobunyavirus, so a genetic characterization of The viral strains used in this study were provided by the WHO collaborating center for reference and research for arboviruses and hemorrhagic fever viruses (WHOCC) in the Virology Department of the Institut Pasteur de Dakar (IPD). Those strains are described in Orthobunyavirus S segment by RT-PCR using TITAN one-tube RT-PCR kit  reagents and the primers previously described [2O, 5 μL of buffer, 2.5 μL of each primer, and 0.5 μL of the enzyme. The PCR was performed following the manufacturer’s recommendations. PCR products were visualized on 2% ethidium bromide-stained agarose gel.RNA was extracted from supernatants using the QIAamp RNA Viral Kit  following the manufacturer’s recommendations. Using the RNA extracts, we tested the presence of the escribed . For theRNA extract was used for Illumina sequencing. Briefly, the host’s background RNA was depleted as previously described using mammal ribosomal RNA-specific primers and RNAseH enzyme (NEB). Purified viral RNA was reverse transcribed into first-stranded cDNA using the Invitrogen SuperScript IV Reverse Transcriptase kit . Then, amplicons were tagged using the “index tags” incorporated into the Nextera kit XT DNA Library Preparation kit . The obtained libraries were quantified, normalized, pooled, and loaded onto an Illumina MiSeq platform with 151 bp paired-end reads using the Miseq reagents kit v3, according to the manufacturer’s protocol . Demultiplexing, removal of sequencing adapters, and genome mapping were performed by a homemade package incorporated into the IPD server. Genetic identity was determined by a nucleotide Basic Local Alignment Search Tool (nBLAST).The MUSCLE alignment algorithm was used for multiple nucleotides sequences alignments in VIPR software  using nuTo fulfill the gap of the lack of data about the genetic characterization of the Bunyamwera viruses isolated in Senegal, we aim to genetically characterize Orthobunyavirus strains that are available in the WHO collaborating center for arboviruses and viral hemorrhagic fever (WHOCC) located in IPD. After sequencing, five complete genomes were obtained and nucleotide BLAST showed that the segments of the ArB218, ArN31, ArMsam263, ArY380, and ArY52 strains were identified as Bunyamwera viruses with high nucleotide similarities (>95%). Except for the strain ArB218 which showed 98.12% of nucleotide identity for the segment S of NRIV strain 9800535 (JX857328.1) for the S segment. An analysis of the genetic distances shows that our isolates are closer to Bunyamwera than Ngari and Batai .The fixation indices (FST) measured between populations also show that these newly sequenced strains are closer to the Bunyamwera virus .Analyses of the amino acid similarities of the L segments between our strains and the sampled NRIV, BATV, and BUNV sequences reveal 98.82% of similarities with BUNV .p-value < 0.001)   result from recombination with the S and L segments of the newly characterized strain ArN31, with a recombination signal detected by all the seven detections methods (< 0.001) . Moreove< 0.001) .Phylogenetic analysis shows that all the strains characterized in this study had clustered with Bunyamwera virus strains for all segments, except for the S and L segments of ArB218, which had clustered in the base of the node of the S and L segments of the NRIV strains group A–C with In this study, we have genetically characterized five Bunyamwera strains provided by the WHOCC in IPD. The results of BLAST show that our five orthobunyavirus strains were identified as belonging to the Bunyamwera serogroup as all their three segments are identified as a Bunyamwera virus (BUNV) except for the S Segment of ArB 218, which was identified as an Ngari virus (NRIV). This result is strengthened by an analysis of the genetic distance, amino acid similarities of the L segments, and the differentiation index (FST), which indicate that these newly sequenced strains are the Bunyamwera virus. Otherwise, two recombination events were detected during our analysis. Ngari strains  result from recombination with S and L segments of our strain ArN31. Additionally, Bunyamwera virus strain ATCC_R_VR_87 results from recombination with S, M, and L segments of the ArB218 strain. Natural genetic segments exchange between Bunyamwera serogroup members has already occurred. Genomic data showed that NRIV carried the M segment of BATV and the L and S segments of BUNV ,28. ThisFurthermore, among the strains  that fall in the Bunyamwera genetic cluster for all the three segments, the newly sequenced Ar Ms am 263 and Ar Na 31 from Kenya were grouped for the S, M, and L segments in a BUNV cluster, even if they were isolated 10 years apart. This could be explained by the fact that they were isolated from hosts that belong to the same genus. Besides the strains from Cameroon, Ar YM 52 and Ar Y 380/69 did not group by two except for the S segments. Additionally, the L segment is close to some of the Bunyamwera isolates from Kenya GSA/S4/11232_WT, MGD/S1/12060_WT, and 46A-122.Finally, the genetic analyses of five newly characterized strains revealed that these strains are characterized by the Bunyamwera virus and that two of them are recombined with the Ngari and Bunyamwera strains. Further investigations are needed to elucidate the impact of these recombinations on the biology of this virus, such as growth kinetics, pathogenesis analysis, and vector competence, which are needed to understand the role of this genetic recombination.


There is currently a need for new rapid viral diagnostic electron microscopy methods. Although the gold standard remains the transmission electron microscopy (TEM) negative staining method for electron microscopic examination of samples containing a virus, difficulties can arise when the virus particle content of the sample that has to be examined is poor. Such samples include supernatants of virus-infected cells that can be difficult to examine, as sometimes only a few virus particles are released in the culture medium upon infection. In addition to TEM, scanning electron microscopy (SEM) can also be used for visualizing virus particles. One advantage of SEM over TEM is its ability to rapidly screen several large specimens, such as microscopy slides. In this study, we investigated this possibility and tested different coating molecules as well as the effect of centrifugation for analyzing SARS-CoV-2-virus-infected cell culture supernatants deposited on microscopy glass slides by SEM. We found that centrifugation of 25XConcanavalinA-coated microscopy glass slides in shell vials provided an improved method for concentrating SARS-CoV-2-virus-infected cell supernatants for virus-like particle detection by SEM. There is currently a need for new rapid virus screening methods using microscopy , especiaSuch samples that can be difficult to examine include the supernatants of virus-infected cells  as sometIn addition to TEM, scanning electron microscopy (SEM) can also be used to visualize virus particles ,27,28,29Here, we used SEM to analyze microscopy slides and studied different concentration methods for analyzing virus-infected cell supernatants. In order to concentrate the virus-infected cell culture supernatants, we chose SARS-CoV-2 as model virus and tested a panel of coating molecules for microscopy slides as well as the effect of centrifugation. We found that the use of 25X ConcanavalinA lectin coating of 12 mm microscopy glass slides in glass shell vials coupled with ultracentrifugation provided the best method for concentrating SARS-CoV-2-virus-infected cell supernatants for SEM routine examination during viral diagnosis.2. The medium was completed to 2 mL and supernatants were collected after further incubation of the inoculated cells for 48 h at 37 °C with 5% CO2 corresponding to the appearance of a cytopathic effect. Before being deposited on glass slides, cell culture supernatants were filtrated at 0.2 µm. Polymerase Chain Reaction (PCR) was performed in parallel on cell culture supernatants. The mean Ct value was 13.7 ± 1.3 (n = 7).The Delta variant of the SARS-CoV-2 IHUMI-3630 strain was used in this study as a model virus. After thawing from −80 °C, the virus sample was diluted to 1/10th in M4 medium consisting of MEM medium supplemented with 4% fetal calf serum and 1% L-glutamine. Vero-E6 cells in 12-well plate were inoculated with 500 µL of this virus solution and incubated for 1 h at 37 °C with 5% COMicroscopy Lab-Tek 8-well chamber slides  and 12 mm glass shell vials  were coated with a panel of both positively charged and negatively charged molecules by depositing 100 µL (Lab-Tek) and 200 µL  of these solutions on top of the slides for 60 min at room temperature (RT) in a moist chamber prior to depositing the supernatants. The concentrations of the different coating molecules used in this study were as follows: polyethylene glycol  at 1X; poly- D-Lysine  at 0.01 mg/mL; poly-L-Lysine  at 0.01 mg/mL; ApoH  at 0.1 mg/mL; Polybrene  at 0.1 µg/mL; Sulfatides  at 0.1 mg/mL; ConcanavalinA  at concentrations of 0.1X, 1X, 5X, 10X, 25X, 75X and 100X, corresponding to concentrations of 250 ng/mL, 2.5 µg/mL, 12.5 µg/mL, 25 µg/mL, 62.5 µg/mL, 187.5 µg/mL and 250 µg/mL, respectively. After being deposited, these coating solutions were discarded and replaced by 100µL (Lab-Tek) and 200 µL  of cell culture supernatants for 10 min at RT. Centrifugation of the shell vials was performed for 30 min at 3452 g using a Heraeus Multifuge 3 S-R ultracentrifuge .For scanning electron microscopy (SEM), deposited glass slides  were fixed with glutaraldehyde 2.5% in 0.1 M of sodium cacodylate buffer for 60 min. Slides were rinsed with 0.1 M sodium cacodylate buffer and distilled water for 1 minute each. Slides were dehydrated with increasing ethanol solutions  for 2 minutes and with 100% ethanol for 5 minutes. Slides were incubated with 100% ethanol/100% hexamethyldisilazane (HDMS) in a 1:2 ratio for 5 minutes. Slides were incubated with 100% of HDMS for 5 minutes and air-dried for 30 min. We checked that no particles had been lost after this virus preparation (dehydration and drying) for SEM (not shown). Finally, the slides were platinum sputter-coated for 20 s at 10 mA (Hitachi MC1000). The observation was made using a SU5000  SEM with an SE detector in high-vacuum mode at 1 kV acceleration voltage, observation mode (spot size 30). The working distance ranged between 1 mm and 5 mm. For quantification, automatic 6 × 6 mosaic tiled images at ×5k magnification were acquired at a random position with auto contrast/brightness using the microscope zig-zag function after manual adjustment of the focus and no further auto-focus. Pixel size was 3.96 nm. Images were 1280 × 960 pixels corresponding to 5078 × 3808 nm fields of view.For immunocytochemistry, supernatant-deposited slides were fixed with 4% paraformaldehyde for at least 20 min at RT alongside slides fixed for SEM from the same experiments. After a single rinse in Phosphate Buffer Saline (PBS), slides were saturated with 10% Fetal Bovine Serum (FBS) and 0.1% Tween 20 in PBS for 10 min at 37 °C. Slides were incubated for 1 hour at 37 °C with a polyclonal rabbit anti-SARS Coronavirus Spike protein  The slides were washed twice with PBS and incubated for 30 min at 37 °C with an Alexa Fluor 488-Goat anti-Rabbit IgG (H + L) secondary antibody . The slides were rinsed twice in PBS then mounted with Moviol for observation. Images were acquired using a LSM800 confocal laser scanning microscope (Zeiss) with a Plan-Apochromat 63X/1.4 oil objective. Scan zoom was 2.4. Pixel dwell time was 4.12 µs. Averaging was 2. Image dimensions were 512 × 512 pixels, 42.26 × 42.26 µm. Pixel size was 83 nm. Mosaics of 6 × 6 tiled images were randomly acquired on each slide, representing 2816 × 2816 pixels and 232.4 × 232.4 µm fields of view.Quantification of the abundance of SARS-CoV-2-like virus particles in the SEM x25k images was performed manually upon visual inspection. The number of deposited SARS-CoV-2-like particles was counted and was considered as the 100% deposition amount for comparisons. Counting was also carried out in parallel with a macro created with Fiji  in orderThe use of SEM for imaging whole virus particles is comparable to the TEM negative staining technique, providing morphological features and measurable dimensions . As a result of preliminary experiments on SARS-CoV-2-infected cell monolayers cultured in Lab-Tek chambers A–C, we cAs potential adhesive molecules for capturing SARS-CoV-2 viruses, we chose both theoretically negatively and positively charged coating molecules. The negatively charged molecules were (1) polyethylene glycol (PEG) ; (2) sult-test). Next, we asked whether we could further improve the efficiency of the deposit with different concentrations of the ConA solution. We compared 0.1X, 1X, 25X, 50X, 75X and 100X concentrated ConA coating solutions to no-coated slides and we found a peak increase of 154% in particle deposition with ConA at 25X concentration , we quantified the amount of SARS-CoV-2-virus-like particles deposited on non-coated slides compared to ApoH-, PEG-, polybrene- and sulfatide-coated slides A. In thentration C . Because the device was found to be too complex to handle for routine SEM observation of the use of TEM grids, we chose to perform centrifugation of shell vials, glass tubes classically used in our NSB3 laboratory for culturing virus-infected cells, containing 12 mm microscopy glass slides.t-test). When adding centrifugation to the 25XConA-coating, we obtained a 1145% increase in the deposit of SARS-CoV-2-virus-like particles, with 6.44 ± 1.80 versus 0.77 ± 0.23 particles per field of view, respectively (t-test).We observed a significant 125% increase in the amount of deposited SARS-CoV-2-like particles on non-coated 12 mm glass slides after performing centrifugation, with 0.68 ± 0.30 versus 0.30 ± 0.35 particles per field of view, respectively A,B1,B2  for SEM and (ii) for fluorescence microscopy after anti-SARS-CoV-2 fluorescent staining . Figure t-test)  B.Thus, CLSM datasets confirmed that SARS-CoV-2 virus-like particles in our SEM images that we counted in our experiments were indeed likely to be SARS-CoV-2 virus particles.Our SEM images of SARS-CoV-2 virus particles from infected cell culture supernatants provide very suitable images for automatic particle analysis due to the high contrast. One potential issue would be the aggregation of particles, which may pose difficulties for depicting single virus particles. In our experiments, we did not notice a substantial aggregation of Delta SARS-CoV-2-virus-like particles originating from cell supernatants, which was shown for retroviral particles pseudotyped with the Delta variant SARS-CoV-2 Spike protein .We used a targeted Fiji macro dedicated to highlighting and counting SARS-CoV-2 particles for preliminary quantifications of our SEM images folders see . We alsoSuch macros may be adapted and used for estimating non-viral background materials from cell culture supernatants after 0.2 µm filtration as well as for discovering the presence of a known or unknown virus in a suspected sample. Finally, in the present study, we chose 512 × 512 pixels at x63/zoom 2.4 magnification (80 nm pixel size) for quantifying fluorescence signal of anti-SARS-CoV-2 stained supernatants by CLSM. For counting single SARS-CoV-2 virus particles, 4096 × 4096 pixel images at ×63/zoom 2.4 magnification (10 nm pixel size) would be more accurate but more time-consuming to acquire.In the following scheme , we summThe total duration of our method with ready to use coated glass-slides represents around 2 h and 20 min in total for assessing virus-like particle presence: 10 min for cell culture supernatant deposition, 30 min for ultracentrifugation, 30 min for fixation, 1 h for washes, dehydration, drying, slides mounting and metallization and 10 min for SEM examination.Our method was primarily designed for bringing a yes/no answer to the question of the presence of virus-like infectious particles, especially for poor cell culture supernatants. It also opens perspectives for quantitative considerations about the viral charge of a cell culture supernatant, which may be of interest when performing pharmacological studies or comparing variants. Compared to real-time polymerase chain reaction (PCR) experiments, our method does not bring advantages for monitoring viral charge in case of known viruses. However, in the case of unsuspected or unknown new viruses, our method can provide a monitoring of the amount of virus-like particles located in an inoculated cell culture, making our method of interest for emergent viral infections, even with low-replicating viruses, thus contributing to DEM .Regarding the question of the qualitative morphological characterization of virus-like particles, i.e., virus identification, our method is limited by several points: (i) the metallization process that adds thickness to nano-objects, thus deforming them and limiting observation of fine details, such as Sars-Cov-2 spikes; (ii) our method would also benefit from critical-point-drying that would better preserve particles morphology; and (iii) higher magnifications would improve morphological depiction. Nevertheless, our imaging setup in our method can be used to discard potential viruses according to shape and diameter and refine virus class. Our method may thus be used as a routine first-line strategy for EM viral diagnosis of suspects samples, with more in-depth second-line morphological analysis using TEM, for example. Potential improvements and greater efficiency when performing targeted LM immunostainings may arise from super-resolution light microscopy  and the Machine learning would also be of high interest for automating detection and quantification of virus-like particles in SEM ±CLSM images ,46 as we