Archaea have been identified. In contrast, 17 families of DNA viruses are known to infect archaea. In an attempt to uncover more of the elusive archaeal virosphere, we investigated the metatranscriptomes of hypersaline lakes that are a rich source of archaea. We sequenced RNA extracted from water filter samples of Lake Tyrrell  and cultures seeded from four lakes in Antarctica. To identify highly divergent viruses in these data, we employed a variety of search tools, including Hidden Markov models (HMMs) and position-specific scoring matrices (PSSMs). From this, we identified 12 highly divergent, RNA virus-like candidate sequences from the virus phyla Artverviricota, Duplornaviricota, Kitrinoviricota, Negarnaviricota, and Pisuviricota, including those with similarity to the RNA-dependent RNA polymerase (RdRp). An additional analysis with an artificial intelligence (AI)-based approach that utilises both sequence and structural information identified seven putative and highly divergent RdRp sequences of uncertain phylogenetic position. A sequence matching the Pisuviricota from Deep Lake in Antarctica had the strongest RNA virus signal. Analyses of the dinucleotide representation of the virus-like candidates in comparison to that of potential host species were in some cases compatible with an association to archaeal or bacterial hosts. Notably, however, the use of archaeal CRISPR spacers as a BLAST database failed to detect any RNA viruses. We also described DNA viruses from the families Pleolipoviridae, Sphaerolipoviridae, Halspiviridae, and the class Caudoviricetes. Although we were unable to provide definitive evidence the existence of an RNA virus of archaea in these hypersaline lakes, this study lays the foundations for further investigations of highly divergent RNA viruses in natural environments.Despite their widespread distribution and remarkable antiquity no RNA viruses definitively associated with the domain  Archaea (previously Archaebacteria) were originally distinguished from the Bacteria though the analysis of ribosomal RNA (rRNA) sequence data . Indeed, it is difficult to formally prove that a virus infects a particular host using metagenomic data alone. In the case of the RNA viruses of archaea, Ampullaviridae), spindle-shaped , coil-shaped (Spiraviridae), droplet-shaped (Guttaviridae), filamentous , pleomorphic morphology (Pleolipoviridae), and icosahedral with contractile tails (Caudoviricetes)  and Nanohaloarchaeota that inhabit hypersaline environments DNA viruses from the class Caudoviricetes and order Ligamenvirales have been documented. Importantly, DNA viruses are also key drivers of microbial evolution in archaea-dominated microbiomes  . In archrobiomes , and sucHerein, we attempt to offset some of the drawbacks of metagenomic virus discovery by searching for divergent RNA viruses using methods that do not only rely on the sequence similarity of putative new viruses to already known viruses. First, we screened metagenomic data for the canonical RdRp motifs that are broadly conserved across all the RNA viruses described to date . There aSecond, we searched for RNA viruses by matching candidate sequences to the spacers of the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) regions of host taxa . CRISPR/Finally, it might also be expected that archaeal RNA viruses would have biases in dinucleotide composition that reflect those of their archaeal hosts. Dinucleotide frequencies are not randomly distributed products of nucleotide frequencies, with some dinucleotides over- or under-represented in specific organisms For example, eukaryotes suppress CpG while this dinucleotide is overrepresented in some α-proteobacterial and halobacterial sequences . Hence, Fiersviridae (previously Leviviridae), are positive-sense single-stranded RNA (+ssRNA) viruses that fall within the phylum Lenarviricota that contains the largely microbial fungus-infecting families Narnaviridae and Mitoviridae . The only other RNA virus family currently confirmed to infect bacteria are the Cystoviridae that have a double-stranded RNA (dsRNA) genomes. Given their basal phylogenetic position and simple structure, which only comprises a replicase protein in the case of the Narnaviridae, it is a reasonable hypothesis that archaea-associated RNA viruses could have sequences and structures similar to those seen in the Lenarviricota. In addition, the Lenarviricota are associated with an extensive range of bacteria, and because these bacteria also often inhabit the same habitats as archaea, they clearly have opportunities to co-exist and transmit to other hosts in archaeal habitats. If archaea-associated RNA viruses are indeed akin to the Lenarviricota, we might expect them to possess relatively small genome sizes, from approximately 2,500 to 7,000 nucleotides, and simple icosahedral capsids , between 20th and 23rd of August 2018, with the cooperation of the land managers, Cheetham Salt. Based on previous research , we beliWe were also able to produce archaea-rich samples by growing cultures seeded from four enrichments of water filter samples taken from east Antarctica: (i) Rauer 3 Lake  collected on Filla Island in September 2014, (ii) Rauer 6 Lake  on Torckler Island in September 2014, (iii) Club Lake, Vestfold Hills  in November 2014, and (iv) Deep Lake, Vestfold Hills  in November 2014. Enrichment cultures were produced from these samples and were stored at −80°C. We re-enriched these cultures in August 2019, using two types of media: modified David Burns cultivation media 2 (DBCM2) and artificial deep lake vitamin broth (ADLVB) . CultureRNA was extracted from samples using the RNeasy PowerWater Kit , and quantified using a 2200 Tapestation system . Ribosomal RNA (rRNA) was not depleted as no effective means to remove archaeal rRNA was available. After isolation of RNA, read libraries were prepared, using a TruSeq Stranded mRNA Total Library Prep kit, and sequenced by the Australian Genome Research Facility using the NovaSeq 6000 platform . Sequence reads were prepared using FastQC v0.11.8;  and TrimThe overall analytical (bioinformatics) approach to identify candidate sequences is shown in Following these second-round searches, any remaining unidentified contigs that were >1,200 nt in length and had a >300 amino acid open reading frame (ORF)  were analysed using the Phyre2 server (v2.0) that examines homology between amino acid sequence queries and records in the Protein Data Bank (PDB) to build models of protein structure . BecauseP < 0.005) to non-RNA-virus sequences were not included in further analysis. We then created a BLAST database from these curated spacers, to which we queried our assembled contigs.We also attempted to detect the sequences of highly divergent RNA viruses using the CRISPR spacer regions of archaea. These regions comprise repeating motifs interspaced with short (∼12nt) sequences (spacers) that are complementary to virus genomes. We used Entrez Direct  to downlRNA viruses necessarily carry a replicase gene, either an RdRp or a reverse-transcriptase (RT), such that detecting these sequences is commonly used to identify RNA virus sequences in metagenomic data. To detect the canonical A, B, and C motifs within the viral RdRps, as well as sequence motifs associated with RTs, all sequence assemblies were searched using Palmscan  which coe-value cutoff of 5e−5. RdRp motif sequences for virus-like candidates are shown in As an additional method to detect highly divergent RNA viruses through the presence of RdRp signatures, we employed the recently developed RdRp-scan bioinformatic toolkit . This prTo better annotate putative RNA virus sequences, we collated 70 contigs with virus-related hits to the nucleotide (NCBI), protein , in-house RdRp, PROSITE, PHYRE2, RdRpscan profile, or Palmscan profile databases (see Results). Importantly, we examined contigs with any virus-related hit, including those that were not the top hit or considered significant using typical score thresholds, thereby increasing the scope of our investigation to detect extremely divergent RNA virus genomes. However, this procedure necessarily also increases the number of false-positive results. To compensate for this, we scored contigs by the quality and quantity of both their best search tool hits and any virus-related hits. The strength of a putative virus hit is scored individually, then summed across the different research tools (and databases) to obtain an overall score for the whole contig. Hits that are scored highly are those that match viruses, are of high confidence, and are the best hits from their respective search tool/database. Our simple scoring procedure is as follows:i an iS score was calculated, representing the combined confidence score for the ith contig being a sequence from an RNA virus. This score is determined by each search tool hit j for contig i, with hits scoring higher where they are the best hit returned from the tool and show high confidence similarity to an RNA virus. Variable ijA is scored 1 where hit j is to an RNA virus, 0.5 for a DNA virus, and −1 for a non-viral sequence. Variable ijB is scored 1 where hit j is the top hit of the hits provided by its respective search tool and 4 where it is not (this ensures that hits that are not the best recovered from a search tool are scored low). Variable ijC is scored 1 where hit j is a high confidence hit and 2 where it is not. Hits are considered high confidence when the given e-value is >0.05, or greater than 70 per cent confidence for Phyre2 hits.For each contig All contigs were submitted to LucaProt, which detects RdRp sequences using both sequence and structural information through an AI (i.e. machine-learning) approach . For allPxy = Fxy/FxFy, where F is the frequency of nucleotide x, y, or the dinucleotide xy , bacteria , and eukaryotes , which we then compared to the dinucleotide profiles of the putative virus contigs. We expect the profiles of viral sequences to be similar to those of their true host taxa and we used this property to make inferences on the most likely host group (i.e. determining which host reference sequence had a dinucleotide profile most similar to those of the virus contigs). Principal components analysis (PCA) was used to create an ordination of the dinucleotide odds ratios to visualise the variation in profiles.Profiles of dinucleotide odds ratios were calculated using the seqinr library in R , with ggotide xy . ProfileWe filtered BLASTx contig search results, collating the top three hits for each contig, to obtain sequence hits related to DNA viruses. These were then extracted, and the contigs sorted by type of virus protein  and by taxonomic group (at the level of virus family or genus). In addition, RPS-BLAST searches of these contigs were run to assist in inference of protein types, as many predicted proteins from archaeal DNA viruses are labelled as ‘hypothetical’.4 amino acid substitution model chosen according to Bayesian information criterion, and with ultrafast bootstrap and approximate likelihood-ratio test (aLRT) used to measure clade confidence.We aligned the predicted amino acid sequences (aa) from these contigs with virus type species selected from the International Committee for Taxonomy of Viruses (ICTV) reports for their respective taxonomic groups. We used the L-INS-i algorithm in MAFFT v7.45;  to constWe used CCMetagen v1.1.3;  to classThe R packages ggplot2, ggtree, ape , and virWe assembled a total of 126 million sequence reads into 272,628 contigs from 10 samples representing five hypersaline lakes: Lake Tyrrell , Club Lake, Deep Lake, and two lakes in the Rauer Islands (Antarctica). Of these, the Australian samples were direct environmental samples, while those from Antarctic lakes were archaeal enrichments. The Antarctic lake samples were cultured in 2014 then stored at −80°C, before being re-enriched in 2019. After RNA extraction, we obtained 22 million reads that assembled into 148,998 contigs from the Lake Tyrrell samples , and a total of 104 million reads that assembled into 123,630 contigs from the Antarctic lake enrichments .Halorubraceae, particularly Halorubrum lacusprofundi, and Natrialbaceae, particularly Haloterrigena sp. BsC5, representing the major archaeal components. As well as the archaea, we found on average 4 per cent eukaryotes and 10 per cent bacteria from the phyla Actinomycetota and Proteobacteria. For the Lake Tyrrell libraries, the microbial community profiles were on average 73 per cent archaea, particularly from the order Halobacteriales (44 per cent). There were also an average of 16 per cent bacteria, primarily of the genus Salinibacter and family Ectothiorhodospiraceae, and 12 per cent eukaryotes of the species Dunaliella tertiolecta and Cladosporium herbarum.The samples utilised here were chosen for their archaea-dominated microbial communities which simplifies the attribution of RNA viruses to an archaeal host. To confirm this, we estimated host profiles for each library using CCMetagen . For theAfter confirming that our libraries represent archaea-dominated microbiomes, we searched for RNA virus sequences, particularly those that may have archaeal hosts. To do this, we attempted to identify all our contigs and in so doing separate host sequences from those that are likely to be viral . Using tP < 5e−5) hits to RNA viruses. They did, however, identify 44 contigs that had some similarity to virus genomes, which from here onwards are referred to as virus-like ‘candidates’. We also compiled a list of 177 contigs with no significant matches to any known protein, yet which had a length of >1,000 nt and an >300 aa ORF, suggestive of a viable protein; these are referred to as ‘ORFans’. We compared both candidate and ORFans contigs to available protein structures using Phyre2. Through this, we found that 26 of the ORFans from our candidate list were structurally similar to virus proteins. To increase sensitivity, contigs were considered candidates even with low confidence values or high e-values for their virus similarity.Strikingly, these analyses returned no significant (iS) for each contig (see Methods), allowing for low confidence results to collectively support identification of RNA viruses. This score summarises the results from each search tool/database described above: both the most significant result and any virus-related results for a candidate contig. Hence, the iS value for a contig is the sum of the scores calculated for each of its search tool results. A virus candidate is scored positively if it is linked to an RNA virus, less so if this link is to a DNA virus, and negatively if it links to a non-viral sequence.Combined, these analytical steps identified 70 virus-like candidates: 44 virus-like candidates and 26 ORFans. However, our low threshold to denote ‘significance’ necessarily meant that the false-positive rate will also have increased. To address this issue, we created a combined confidence score , Club-1 (Artverviricota), and Tyrrell-1 (Kitrinoviricota)—are shown in Deltaretrovirus, although the very short sequence meant that it was impossible to determine whether this was an endogenous or exogenous retrovirus sequence.Following this scoring exercise, 19 candidates were found to have a positive  viruses . These cPisuviricota, which are diverse and known to inhabit microbial environments, and the Duplornaviricota that are also known to infect microbial species. In contrast, the other phyla are usually associated with eukaryotes and hence perhaps less likely to be RNA viruses associated with archaea. However, all these putative sequences are so divergent that they could not be included in a viable phylogenetic analysis. Despite our hypothesis that the Lenarviricota was the viral phylum most likely to be associated with archaea, none of our top 12 RNA virus-like candidates were associated with this group.Of the 12 RNA virus-like candidates, nine exhibited similarities to viral RdRps. The virus-like candidates most plausibly associated with the archaea were from the Acinetobacter baumannii bacteriophage, bacteriophage T4, canine adenovirus, porcine adenovirus, fowl aviadenovirus 1, curtovirus (family Geminiviridae), and acidianus filamentous virus 1 (family Lipothrixviridae). The protein database identifiers for these viruses are provided in The final seven virus-like candidates were from the Lake Tyrrell libraries and have the closest hits to DNA viruses: bona fide RNA viruses and phylogenetic position requires additional verification. In addition, none of the nine RNA virus-like candidates with RdRp matches identified by the procedure described above  and FD (306 contigs), with 12 of these species falling as members of the Caudoviricetes. In total, 842 contigs matched viruses from the families Pleolipoviridae, Sphaerolipoviridae, and Halspiviridae , a virus of the Halomagnusviridae known to have a myo-like morphology: a long contractile tail and circularly permuted dsDNA genome. The virus contigs sampled from the Antarctic Filla Island libraries fell into three clades. A single contig fell as a sister lineage to Haloarcula californiae tailed virus 2 (species Samsavirus HCTV2) and Haloarcula hispanica tailed virus 2 , both of which are members of the Saparoviridae and have sipho-like morphology with long non-contractile tails and linear dsDNA genomes. Four contigs grouped in a clade sister to that containing Haloarcula vallismortis tailed virus 1 (species Tredecimvirus HVTV1), Haloarcula californiae tailed virus 5 (species Tredecimvirus HCTV5), and Haloarcula californiae tailed virus 1 (species Hacavirus HCTV1), all of which belong to the Druskaviridae. Finally, three contigs were related to Halorubrum sodomense tailed virus 2 (species Mincapvirus HSTV2) of the Hafunaviridae, and these contigs were ubiquitous and abundant across the Antarctic libraries.As members of the class onducted . This aniricetes . SpecifiPleolipoviridae. From these, we were able to extract 11 contigs that were most closely related to the major capsid of the Pleolipoviridae. However, a BLAST analysis revealed that these were more similar to the genomes of archaeal hosts than to those of archaeal viruses. Specifically, the Pleolipoviridae-related contigs had significant hits (average of 81 per cent nucleotide identity) to members of the genera Saliphagus and Natrinema . As these viruses are known to have members with a prophage stage , although the Natrialbaceae thrived on the conditions of our enrichments and was often a substantial presence in the communities, as also noted by Hqr. walsbyi, a member of the Halobacteriaceae.The archaea-enriched Antarctic lake cultures comprised an average of 96 per cent archaea, particularly those from the ubraceae . Researcubraceae  shows thLenarviricota that we hypothesised were the most likely group to have archaea-infecting members. It is unclear whether the absence of this group reflects the hypersaline environment, their true absence, or is due to the very high levels of sequence divergence.Through the use of a consecutive search protocol we determined that 89 per cent of our contigs were related to previously known sequences . FurtherH. litchfieldiae. Unfortunately, the short length of Deep-1 (459 nt) makes accurate host assignment impossible. The candidates identified through the Palmscan analysis of the Lake Tyrrell libraries (Tyrrell-1 to Tyrrell-13) were greater than 1,200 nt in length. The highest-ranking candidate of these, Tyrrell-1, has a kitrino-like C-motif, although the RdRp motifs in this sequence are not arranged in the usual order. Confirming the candidates as true archaeal viruses will ultimately require isolation and/or imaging of the virus. While it seems likely that the difficulty in identifying RNA viruses in our microbial communities is due to extreme divergence from known viruses, it is possible that the CRISPR-Cas pathway in archaea (Because our libraries are dominated by archaea we suspect that at least some of our virus-like candidates are likely to be associated with archaea, and most had similar dinucleotide representation to reference hypersaline bacteria and archaea. Critically, however, host assignment cannot be proven through metagenomic data alone and additional work is needed in this area. The highest ranking of our virus-like candidates—denoted Deep-1—exhibits similarity to a picornavirus coat protein with high confidence, although this may in part reflect the lack of more divergent structures in the PFAM database. In addition, it has a dinucleotide representation that is most similar to the archaeon  archaea  may alsoCaudoviricetes and the family Pleolipoviridae. The DNA viruses identified broadly accord with those described previously (Caudoviricetes and family Pleolipoviridae.It is striking how few, if any, RNA viruses were identified in these data compared to those viruses with DNA genomes. Other than the highly divergent sequences described above, we identified no representatives of any known group of RNA viruses, including those from the expansive data set recently generated by eviously , despiteeviously , 2013 arbona fide RNA viruses of archaea remain the lack of tools for archaeal rRNA removal, the lack of characterised archaeal genomes, and the extreme divergence of any potential archaeal RNA virus.In summary, we have identified candidates for possible RNA viruses in archaea based on analysis of Australian and Antarctic salt lake metagenomes. Although we have not described any definitive archaeal RNA viruses, we are arguably closer to breaking into this elusive RNA virosphere than ever before and the putative viruses identified here clearly merit additional investigation. The barriers to the identification of vead057_SuppClick here for additional data file.


Scientific advances have led to the development and production of numerous vaccines and antiviral drugs, but viruses, including re-emerging and emerging viruses, such as SARS-CoV-2, remain a major threat to human health. Many antiviral agents are rarely used in clinical treatment, however, because of their inefficacy and resistance. The toxicity of natural products may be lower, and some natural products have multiple targets, which means less resistance. Therefore, natural products may be an effective means to solve virus infection in the future. New techniques and ideas are currently being developed for the design and screening of antiviral drugs thanks to recent revelations about virus replication mechanisms and the advancement of molecular docking technology. This review will summarize recently discovered antiviral drugs, mechanisms of action, and screening and design strategies for novel antiviral agents. Viral infection has enormous social and economic impacts , but theA virus is a microorganism that is strictly dependent on the host body for reproduction. Viruses carry either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) as their genetic material, which can be single-stranded or double-stranded; the two structures represent different replication processes. Coronavirus infection is famous for the severe acute respiratory syndrome caused by severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003 . The corInfluenza viruses are classified into A, B, C, and D types, based on their antigenicity . The surThe main characteristic of the herpes simplex virus (HSV) is a latent infection, which can be reactivated from this latent state, giving rise to a recurrence of the disease. Symptoms caused by HSV infection mainly include oral and genital herpes, both of which are transmitted by contact . HSV hasIn addition to influenza viruses, which emerge every year, and HSV, which affects many adults, there is also a need to focus on coronaviruses, which may break through the interspecies barrier to infect people. Beyond developing safer and more effective vaccines, we also need to discover more antiviral drugs that can be used for treatment. Only by controlling the spread of a virus in these two ways can we better protect humans. Our laboratory focuses on the screening and mechanism studies of antiviral drugs. This review describes the pathogenic mechanism of some human viruses and the existing inhibitors in an effort to contribute to the on-going development of antiviral drugs.Coronavirus (CoV) is an enveloped virus belonging to the Coronaviridae family, Nidovirales order, with a 24.5–31.8 kb positive single-stranded RNA genome, the largest RNA virus genome ,13. The The influenza virus is an enveloped virus belonging to the Orthomyxoviridae family, with a genome consisting of 8 segments, single negative-strand RNAs . Unlike The transcription mechanism of the influenza virus is quite complicated. After the viral ribonucleoprotein (vRNP) complex, consisting of RNAs, nuclear protein, and RdRp, invades the cytoplasm, the whole complex enters the nucleus, mediated by nuclear localization sequences (NLS) carried by vRNP, where the virus completes transcription ,25. The The herpes simplex virus is an enveloped, double-stranded DNA virus belonging to the Herpesviridae family. Its genome is about 152 kb and encodes more than 80 different open reading frames . As a DNThe virus life cycle, in addition to viral proteins, also involves the regulation of a variety of cytokines. All of these involved proteins can serve as potential sites for antiviral drug action.CoV invasion is triggered by S protein binding with a receptor on the cell surface. The first therapeutic strategy is to block the receptors with antibodies or drugs. Several peptides are designed to target the receptor. MERS-5HB contains five copies of the S protein N-terminal, which can inhibit MERS-CoV infection by mimicking the binding of the S protein to its receptor dipeptidyl peptidase-4 (DPP4) . SimilarAs mentioned above, the CoV S protein binds to a specific receptor to initiate virus entry. After binding, transmembrane protease serine (TMPRSS) is activated to cleave the S protein, dividing it into S1 and S2, which is necessary for subsequent membrane fusion. In addition, TMPRSS activates the inflammatory reaction by regulating the NF-κB pathway. TMPRSS may, therefore, serve as a potential target where an antiviral could effectively inhibit viral entry. According to at least one report, inhibition of TMPRSS2 with camostat can reduce MERS-CoV and SARS-CoV production in vitro. Unfortunately, it may not effectively treat patients with COVID-19 . PropoliHerpes virus entry is quite different from that of an RNA virus. Herpes virus entry is facilitated by membrane protease activity, and the transport of viral capsids to the nuclear periphery is necessary for herpes virus entry . Lee et After binding, the virus enters the cytoplasm via membrane fusion or the endocytosis pathway. Most envelope viruses enter cells by these pathways, which involve cytoskeletal changes and lysosome acidification. Inhibitors targeting this process tend to have a broad spectrum of inhibition.2+ channel as a host cell surface receptor, mediates the entry of IAV [2+ channel inhibitor nisoldipine has been shown to inhibit the influenza A virus by impairing the internalization of the influenza virus into host cells [Zhao et al. demonstrated a broad inhibitor, frog-defensin-derived basic peptide (FBP), which can block low pH-induced HA-mediated fusion and antagonize endosomal acidification against virus infection. Moreover, FBP can also block coronavirus spike-mediated cell–cell fusion in 293T/ACE2 cell endocytosis . Lysosomy of IAV ,43. Indest cells   of mRNA for cap-dependent translation [All viruses need to hijack the cell translation system to perform viral protein translation after their genome is released into the cytoplasm. The eukaryotic translation mechanism consists of three steps: initiation, elongation, and termination ,56. Eukanslation ,61. Phosnslation  , including the Toll-like receptor (TLR) family, which recognize pathogen-associated molecular patterns (PAMPs), activating a series of responses, including nucleocytoplasmic transport of some proteins or complexes and activation of some cell receptors . BrieflyVitamin C is an essential nutrient deficiency that results in scurvy. Individuals with scurvy are highly susceptible to fatal infections, such as pneumonia . ClinicaCQ, as mentioned above, also has an immunomodulatory ability  downreguSome immunomodulators derived from natural products that treat tumors may be repurposed to regulate the immune response to viral infections. Curcumin has been shown to suppress breast cancer cell viability by downregulating expression levels of TLR4 and IRF3, which then inhibits expression of type I IFN (IFN-α/β) . ResearcIn recent years, to address the COVID-19 pandemic, researchers have accelerated the screening process for antiviral drugs. They found that Quercetin 3-O--beta-D-galactopyranoside, Tribuloside, and Rutin may be potential natural inhibitors for TMPRSS by molecular simulation and high-throughput screening methods. In addition, researchers have found that the natural products, including phyllaemblicin G7, neoandrographolide, and kouitchenside I, have the structural basis to inhibit the interaction of IRF3 with SARS-CoV-2 .As mentioned above, CoV encodes five structural proteins. N protein forms nucleocapsid, which participates in viral genome transcription and protein translation. The M protein determines the shape of the viral envelope and drives the formation of the viral envelope. The E protein may be involved in virus assembly and budding. In addition, CoV encodes 16 non-structural proteins, most of them key proteins in viral replication and replication regulation, such as Nsp3 (papain-like protein), Nsp5 (3CLpro), and Nsp12 (RdRp). Nsp1 is involved in the regulation of the innate immune response . Nsp4 anIn addition to their role in hijacking cellular proteins, viral proteins are equally important to the virus’ own life cycle. Unfortunately, drugs targeting viral proteins require highly specific design and screening processes. Although they may ultimately face drug resistance, they should have a good therapeutic effect in the short term.Nsp1 is unique to alpha- and beta-CoV and has been found to inhibit interferon response in PEDV and SARS-CoV-2 by promoting the degradation of the CREB-binding protein (CBP) . PEDV NsAs proteases, Nsp3 (PLpro) and Nsp5 (3CLpro) are mainly responsible for cutting and modifying other non-structural proteins. These non-structural proteins have biological functions only after being cleaved and modified. Therefore, both Nsp3 and Nsp5 have been considered potential targets for antiviral agents. Hesperidin, a flavonoid, can inhibit the cleavage activity of 3CLpro in a dose-dependent manner . QuercetRNA virus genomic RNA can be used with RdRp as a template to synthesize progeny RNA. Therefore, RdRp is an important target for antiviral agents. Murphy et al., for example, reported that the nucleoside analogue GS-441524 inhibits feline coronavirus replication by blocking the binding of RdRp with viral RNA . SubsequInterestingly, the amentoflavone, a bioflavonoid, seems to bind with a variety of non-structural proteins of CoV, including Nsp3, Nsp5, Nsp12, and Nsp15 . It has Influenza virus proteins have many functions, from ion channels (M2) to nuclear localization signals (NS2), and proteases. RNA synthesis of the influenza virus is accomplished by multiple RNA polymerases . As mentThe glycoproteins HA and NA play important roles in virus entry and release, which are the first targets for restricting viral infection. Indeed, previous specific drugs against the influenza virus include NA inhibitors, such as zanamivir and oseltamivir, which inhibit the cleavage of NA and limit the release of influenza viruses. However, resistance to zanamivir and oseltamivir develops rapidly in clinical settings because of virus variation ,115. SimThe diversity and high mutation rate of HA and NA make them prone to drug resistance, but the conservation of RNA polymerase may be a target to solve the problem of drug resistance. The influenza virus polymerase system, which includes PB1, performs RNA synthesis; PB2 recognizes cap structures, and PA-PB1 interactions mediate polymerase entry into the nucleus. Therefore, the RNA polymerase of the influenza virus is a potential target for exploring antiviral agents.To inhibit polymerase activity, it is a good strategy to block the interaction between PA and PB1. Mélin, et al. reported that the lead compound LM146 displays a strong affinity with PB1 . Huang eChemical compound DAAs easily lose their inhibitory effect with the mutation of viral proteins; thus, it is necessary to constantly screen new drugs or search for natural products that target multiple sites, which are thus less vulnerable to resistance.HSV is a large DNA virus that encodes a large number of viral proteins with different functions. The most important viral proteins include origin-binding protein UL9, single-stranded DNA binding protein ICP8, DNA polymerase complex UL30/42 , and theHSV invades cells by using surface glycoproteins, which are important targets for screening the antiviral agents. Majmudar et al. found that sulfated pentagalloylglucoside (SPGG) may be an effective inhibitor of HSV-1 entry by interacting with HSV-1 glycoprotein . In addiThe classic HSV inhibitor is acyclovir, which is the basis of 2′-deoxiguanosin, targeting HSV DNA polymerase to restrict viral DNA replication. It has been reported that psoromic acid, a bioactive lichen-derived compound, could act against HSV-1 and HSV-2 by blocking HSV DNA polymerase . Wald etDifferent kinds of viruses have different kinds of viral proteins. Antiviral drugs that target individual viral proteins are more specific, which means lower cytotoxicity and better therapeutic efficacy. Different viruses invade cells through different receptors. Based on this, antiviral drugs have high specificity and can effectively inhibit viral entry. After entering, all viruses need to release their genomes into the cytoplasm, and then use the host system for their own replication. In this process, the translation of viral proteins needs to hijack the host translation system. In addition, different viruses induce innate immunity in different ways, but they use the cellular proteins that are used in immune regulation. These are common to different viruses, which means they are potential targets for broad-spectrum antiviral drugs. Therefore, antiviral agents should be screened and used specificity to make them more effective.Developers of antiviral drugs face a dilemma. On the one hand, DAAs show resistance due to viral mutations, and on the other hand, HTAs have greater cytotoxicity. Our review considers natural products targeting both viral proteins and cellular proteins, which is one of the characteristics of natural products. CQ, for example, not only inhibits viruses that are dependent on low pH, but also regulates the immune system. Some natural products of bases or acids can kill the virus directly. Other purified natural products are compounds with a well-defined structure that have the structural basis of targeted drugs. Natural products offer a potentially rich field for future drug screening.High-throughput screening (HTS) methods have become an important tool for discovering antiviral drugs. Unfortunately, HTS is very costly and time consuming; it requires the development of specific assays for selected pharmaceutical targets, and a compound library must first be synthesized. With the development of virtual screening (VS) methods, the fast and cheap identification of lead and hit structures of target proteins is possible. Swamy et al. described how available antiviral candidates can be reused as a potential SARS-CoV-2 3CLpro inhibitor, using molecular docking studies. They found that some types of compounds from the antiviral library, such as Mitoxantrone and Leucovorin, are potential inhibitors of SARS-CoV-2. In addition, glutamic acid (Glu166) of 3CLpro is a key residue holding and forming a stable complex of these compounds by forming hydrogen bonds and salt bridge . In addiThis screening technique could break through the limits of the drug library to screen for more natural compounds, which offers the possibility of high resistance to viral mutation and lower cytotoxicity. In subsequent studies, this screening technology can be combined with biological experiments and clinical trials, and safer and more effective antiviral drugs may be screened. In addition, by better understanding the mechanism of virus-host cell interaction, natural products from the library can be found more efficiently that share structural similarities with antiviral compounds, thereby reducing toxicity and expense.


The lime plant is a horticultural plant that grows in tropical regions. One of the cultivation maintenances to increase the production of lime fruits is pruning. However, the pruning technique of lime requires high production costs. In addition, phytohormones such as ethylene and abscisic acid have regulation to help drop leaves and branches. The study aimed to identify genes in lime involved in the self-pruning process during ethephon and abscisic acid treatments. Total RNA was extracted and subjected to long-read sequencing using a PCR-cDNA sequencing kit, Oxford Nanopore Technologies. The transcripts were produced 5,914 using the RATTLE program and ranged from 201 – 8,156 bp, and N50 was 1,292 bp. The RNA-seq dataset is available as a raw sequence read that scientists can further process and analyze, and this data can be helpful for lime breeding programs that can shed branches and leaves. Specifications Table•Citrus aurantifolia (Christm.) Swingle transcriptome reference using Oxford Nanopore Technologies of long-read sequencing on ethephon and abscisic acid treatment.This data provides •Citrus aurantifolia (Christm.) to ethephon and abscisic acid treatment.The presented dataset could help researchers to determine the responses of •Citrus aurantifolia (Christm.) under the effects of ethephon and abscisic acid treatment.The data is beneficial for researchers involved in identifying the self-pruning-related genes on 1citrus sinensis) Lime is a small citrus with a diameter ranging from 4-6 cm and green color with a sour and fresh taste. Lime fruit has anti-inflammatory and anti-bacterial effects 2Citrus aurantifolia(Christm.) Swingle using long-read sequencing. The total RNA was extracted from the leaves of the seedling stage. The full length as clean-reads was obtained, and de novo assembly was constructed using the RATTLE program. All statistics of reads and assembled transcripts were analyzed (Citrus aurantifolia Swingle Gene Ontology (GO) classification is presented in  Swingle leaves were collected from a first-year-old lime plant grown in the Leuwikopo Experimental Station, Bogor, West Java, Indonesia (6°33′51.5"S 106°43′29.4"). To conduct RNA sequencing, three types of samples were prepared (−5 using the filtered-UNIPROT database (Magnoliopsida (TaxID: 3398), downloaded on 19 October 2021) prepared . First, No animal or human experiments were performed in this study.Elda Kristiani Paisey: Conceptualization, Methodology, Writing – original draft, Funding acquisition. Edi Santosa: Conceptualization, Validation, Investigation, Writing – review & editing, Funding acquisition. Ani Kurniawati: Supervision, Writing – review & editing. : Supervision, Writing – review & editing. Deden Derajat Matra: Supervision, Writing – review & editing, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Hepacivirus, and the delta-like virus group. Of particular note was the identification of a marsupial-specific clade of delta-like viruses that may indicate an association of deltaviruses with marsupial species. In addition, we identified a highly divergent hepacivirus in a numbat liver transcriptome that falls outside of the larger mammalian clade. We also detect what may be the first Jingchuvirales virus in a mammalian host—a chu-like virus in Tasmanian devils—thereby expanding the host range beyond invertebrates and ectothermic vertebrates. As many of these Dasyuromorphia species are currently being used in translocation efforts to reseed populations across Australia, understanding their virome is of key importance to prevent the spread of viruses to naive populations.Although Australian marsupials are characterised by unique biology and geographic isolation, little is known about the viruses present in these iconic wildlife species. The Dasyuromorphia are an order of marsupial carnivores found only in Australia that include both the extinct Tasmanian tiger (thylacine) and the highly threatened Tasmanian devil. Several other members of the order are similarly under threat of extinction due to habitat loss, hunting, disease, and competition and predation by introduced species such as feral cats. We utilised publicly available RNA-seq data from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database to document the viral diversity within four Dasyuromorph species. Accordingly, we identified fifteen novel virus sequences from five DNA virus families  and three RNA virus taxa: the order Jingchuvirales, the genus  Through a combination of hunting, habitat loss, competition for resources with introduced species and, potentially, the introduction of exotic pathogens, the last known thylacine died in 1936 and the species was declared officially extinct in 1986   and are  in 1986 . The Tas in 1986 .Myrmecobius fasciatus) is the last remaining member of the family Myrmecobiidae, one of the few diurnal marsupials, and the only one that feeds exclusively on termites (Sminthopsis crassicaudata), have recently been listed as threatened by the state of Victoria due to population decline (Antechinus flavipes) has been proposed as a candidate species for translocation as part of a rewilding project in an area near Canberra, Australia  Sequence Read Archive (SRA), followed by genomic and phylogenetic analysis.2.2.1.https://github.com/wwood/kingfisher-download). Sequencing reads first underwent quality trimming and adapter removal using Trimmomatic (v0.38) with parameters SLIDINGWINDOW:4:5, LEADING:5, TRAILING:5, and MINLEN:25, prior to assembly  RNA-seq datasets available on the SRA, excluding faecal samples. Raw FASTQ files for all libraries were downloaded using Kingfisher (assembly .De novo assembly was conducted using MEGAHIT with default parameters (v1.2.9)  and removed manually. NCBI Web BLAST (https://www.ncbi.nlm.nih.gov/BLAST) was then used to check for false positives, disrupted open reading frames (ORFs), and to manually assess alignment to virus motifs.(v1.2.9) . The ass(v1.2.9)  and the (v1.2.9)  using Di2.9) (−5 . To remo2.9)  program . The exp2.4.Sequences representing novel virus species, defined as those with <95 per cent nucleotide sequence identity to their closest relative, were assigned to a taxonomic family based on their identity to previously characterised virus species. For each family, a reference dataset was downloaded from NCBI Virus  and the 2.5.As only one reference sequence for a single gene (DNA-dependent DNA polymerase) was available for the species of Antechinus herpesvirus previously identified, Bowtie2 (v2.2.5)  was used2.6.https://researchdata.edu.au/indexed-reference-databases-kma-ccmetagen/1371207) using the KMA aligner (v1.3.9a) and the CCMetagen program (v1.1.3) (https://www.adobe.com).To assess the taxonomic composition of each library, contigs were aligned to a custom NCBI nucleotide database without environmental and artificial sequences ((v1.1.3) . The abu3.3.1.S. crassicaudata (fat-tailed dunnart) (21 libraries), A. flavipes (yellow-footed antechinus) (113 libraries), A. stuartii (brown antechinus) (10 libraries), S. harrisii (Tasmanian devil) (292 libraries), T. cynocephalus (Tasmanian tiger) (6 libraries), M. fasciatus (numbat) (3 libraries), and Pseudantechinus macdonnellensis  (1 library).As of March 2023, the NCBI SRA database contained 446 RNA-seq libraries from Dasyuromorphia , comprisyellow-footed antechinus, numbat, and fat-tailed dunnart. Following secondary assembly and removal of contigs with hits to endogenous viruses, twenty-two partial or full virus genomes were identified in forty-three libraries including four host species  appeared to contain viruses identified as likely contaminants. Specifically, the human-associated viruses Gammapapillomavirus 19 and Betapapillomavirus 1 were identified with over 99 per cent nucleotide sequence identity, and human reads were found in three of these libraries , one in antechinus and one in a numbat, and a chu-like virus  here named Tasmanian devil chu-like virus. The chu-like virus, which has an unsegmented, non-circular genome, was identified in a DFTD cell line that was cultured from primary tissue . The genuvirales . The higuvirales .Hepacivirus. The full virus genome was assembled from library SRR11306639, a library of male liver tissue and the sequence identity across the other five individuals varied between 93 and 98 per cent. The virus was also identified in spleen, kidney, stomach, and cerebrum. Based on the NS5 (RdRp) protein, this virus was most closely related to a hepacivirus identified in an Ixodes holocyclus tick engorged with the blood of a long-nosed bandicoot in Australia (Antechinus hepacivirus was detected in multiple libraries from a single BioProject (PRJNA565840) in which multiple tissues of thirteen antechinus individuals were sequenced. The assembled virus genome was 9,094 nucleotides in length and was detected in five of the thirteen individuals and in at least two tissue types from each positive individual, with libraries of liver tissue consistently having the highest abundance . The genome structure was a single polyprotein, consistent with the genus ustralia . In turnustralia .hepacivirus was identified in a transcriptome of numbat liver tissue  contained two short contigs that included the glycoprotein B gene , which could be utilised in phylogenetic analysis, here named Dasyurid herpesvirus 5. The other, a library of prostate tissue, contained twenty-four contigs with sequence identity to gammaherpesvirus genes, also including the glycoprotein B sequence, here named Dasyurid herpesvirus 4. A comparison of these two glycoprotein B sequences revealed that they shared only 25 per cent amino acid sequence identity. We used Bowtie2 to align reads from the herpesvirus-positive libraries to the Dasyurid herpesvirus 1 reference sequence (MF576269.1), for which only the DNA-dependent DNA polymerase is currently available. A single read from SRR11306664 (containing Dasyurid herpesvirus 4) aligned with 92 per cent nucleotide sequence identity to the Dasyurid herpesvirus 1 reference, while no reads aligned from the other three Antechinus libraries containing herpesvirus contigs. Based on phylogenetic analysis of glycoprotein B, Dasyurid herpesvirus 4 was most closely related to an elephant herpesvirus . DasyuriDyolambdapapillomavirus  also contained a contig showing sequence identity to the large T antigen of a novel polyomavirus. A second library, SRR13765794, contained an identical polyomavirus large T antigen but no other virus sequences. No other polyomavirus genes were detected in these libraries. As this large T antigen sequence was related to but distinct from Tasmanian devil polyomavirus 1 and 2 , we nameTranscripts of two novel anelloviruses were detected, although only one of these—here named Antechinus anellovirus—contained the ORF1 sequence and hence was analysed further. Phylogenetically, Antechinus anellovirus was extremely divergent in the ORF1 protein from the current diversity of Anelloviridae and was closely related to a group of anelloviruses detected in seals, cats, and a giant panda . AntechiMastadenovirus, but due to the high degree of divergence, the phylogenetic position of this virus is still unclear  where it was the only virus identified. Across libraries, the virus sequences were 99 per cent similar at the nucleotide level. The virus was mainly found in libraries of kidney tissue (8/10), as well as in one library of spleen and one of prostate tissue. Antechinus adenovirus was most closely related to members of the genus  unclear .4.We identified twenty-two full or partial virus genomes from eight virus taxa within 446 SRA transcriptome libraries of tissue samples from species within the order Dasyuromorphia. Of these virus genomes, fifteen were subjected to phylogenetic analysis. The remaining seven virus contigs either were viruses identified as contaminants (in four cases discussed later) or were too short for meaningful analysis; this was the case with two herpesviruses identified in a fat-tailed dunnart library and a Tasmanian devil library, as well as the ORF2 segment of a novel anellovirus in a Tasmanian devil. Human papillomavirus sequences were identified in four antechinus libraries, three of which also contained contaminating human sequences. Although this is a risk when analysing SRA data, the use of taxonomic classification software such as CCMetagen  can be uUntil 2018, deltaviruses were believed to be exclusive to humans. However, this was disproven by the identification of delta-like viruses in birds and snakes . Since tTwo distinct marsupial carnivore hepaciviruses were also identified in this study. Hepaciviruses are associated with liver disease in humans, although in most animals their pathogenicity is unknown. Three marsupial hepaciviruses have previously been identified: Koala hepacivirus, Possum hepacivirus, and Collins beach virus, all of which were identified metagenomically . AntechiPiscichuvirus) within the Chuviridae. More recently, three novel Piscichuvirus species were identified and associated with encephalitis in turtles (A highly divergent chu-like virus was identified in a transcriptome of a DFTD cell line (SRR6380970) grown from primary tissues . The Chu turtles , suggest turtles , a cell Transcripts from five families of DNA viruses were identified in these samples. Papilloma-, polyoma-, and herpesviruses are often associated with disease and, in some cases cancer, while anellovirus and adenovirus have less definitive links to disease. We identified a novel anellovirus in antechinus spleen transcriptomes, which co-occurred with Antechinus hepacivirus in one library. These viruses are ubiquitous and their disease association is still debated, although it has been suggested that they may play a role as co-infecting viruses . SimilarAntechinus in 2014 (Sequences from two distinct species of the family Gammaherpesvirinae were identified in antechinus transcriptomes of stomach and prostate tissue, along with partial herpesvirus sequences in Tasmanian devil peripheral blood mononuclear cells and fat-tailed dunnart mitogen-stimulated splenocytes. Herpesviruses are relatively well characterised in marsupials, with Dasyurid herpesvirus 1 identified in  in 2014  and subs in 2014 . However in 2014 . Given tBettongia penicillata papillomavirus 1, isolated from papillomatous lesions on a brush-tailed bettong in Australia (We identified five distinct but related species of papillomavirus in Tasmanian devil lip tissue transcriptomes within the libraries of a single SRA BioProject. The DFTD status of the sampled individual did not appear to be associated with the presence of papillomavirus, nor did the DFTD status correlate with any clustering in the phylogeny of these species. Two Tasmanian devil–associated papillomaviruses were previously identified in a faecal meta-transcriptome, but as these animals are carnivores, it was impossible to determine their true host . Phylogeustralia , and morustralia . InteresAntechinus libraries, with 10/13 sequenced individuals being associated with at least one virus. Antechinus hepacivirus was particularly prevalent, and as these species are being proposed for species reintroduction programmes, it is of particular importance that the viromes of these individuals are investigated to prevent the spread of these viruses to naive populations. From the data provided here, PCR primers could be designed to screen for these viruses in individuals being used in translocation programmes and in cases of undiagnosed disease in marsupial species to determine if there is some disease association.Although our study identified evidence of a relatively high diversity of DNA viruses within transcriptomic data, the lack of complete genomes limits the value of these sequences in phylogenetic comparison and genomic characterisation of these species. In contrast, there was a relatively limited diversity of RNA viruses in these data, which may be a product of the sequencing methodology used. For example, poly-A selection during library preparation will remove many virus sequences . HoweverIn light of the diversity of marsupial carnivore viruses identified in this small study of transcriptomic data not generated for the purposes of virus discovery, it is evident that considerable viral diversity exists in these species and that further study is required to understand the viromes and viral evolutionary history of these unique species. Given the threat that these species face as a result of human activity, it is imperative that we understand the risk that viral disease poses to what are often small, isolated populations.vead061_SuppClick here for additional data file.


Arthropod-borne virus (arbovirus) and arthropod-specific virus (ASV) are viruses circulating amongst hematophagous arthropods that are broadly transmitted in ecological systems. Arbovirus may replicate in both vertebrates and invertebrates and some are known to be pathogenic to animals or humans. ASV only replicate in invertebrate arthropods yet they are basal to many types of arboviruses. We built a comprehensive dataset of arbovirus and ASV by curating globally available data from the Arbovirus Catalog, the arbovirus list in Section VIII-F of the Biosafety in Microbiological and Biomedical Laboratories 6th edition, Virus Metadata Resource of International Committee on Taxonomy of Viruses, and GenBank. Revealing the diversity, distribution and biosafety recommendation of arbovirus and ASV at a global scale is essential to the understanding of potential interactions, evolution, and risks associated with these viruses. Moreover, the genomic sequences associated with the dataset will enable the investigation of genetic patterns distinguishing the two groups, as well as aid in predicting the vector/host relationships of the newly discovered viruses. Generally, there are two major groups of viruses that circulate in hematophagous arthropods: arthropod-borne virus (arbovirus) and arthropod-specific virus (ASV) (or arthropod-only virus). These viruses are diverse in taxonomy and may have unsegmented or segmented (two to twelve) genomes.1.Arbovirus are transmitted by hematophagous arthropods such as mosquitoes, ticks, sandflies, and other vectors. Arbovirus can be pathogenic to either animals or humans, such as the mosquito-borne dengue virus, tick-borne Crimean-Congo haemorrhagic fever virus, and midge-borne bluetongue virus. These pathogenic viruses replicate in both vertebrates and invertebrates. With the progression of factors including climate change, urbanization, increased international travel and trade, arbovirus continue to emerge and re-emerge worldwide, which poses a serious challenge to global public healthin vivo and in vitro in these arthropods. However, they are inherently unable to replicate in the vertebrates and their respective cells1. Some of these ASV have been classified as members of classical viral families that are traditionally associated with arbovirus, including but not limited to the families Flaviviridae, Togaviridae, Reoviridae, Peribunyaviridae, Nairoviridae, and Phleboviridae. It is important to note that viruses identified as ASV may in some cases originate from arthropod commensal fungi/bacteria, and this condition is currently difficult to distinguish2. There are two views on the relationship between ASV and arbovirus: (1) ASV are basal to many types of arbovirus based on phylogenetic analysis5, and (2) ASV are interleaved within arbovirus phylogenies6. Moreover, both in vitro and in vivo studies have indicated that ASV may affect the vector competence of arthropods by direct competition with arbovirus or indirectly affecting arthropod physiology, a strategy that can be applied in developing novel approaches for arboviral disease or vector control3.Studies over the past decade have established that arthropods do harbor a rich and diverse group of ASV. These ASV naturally infect hematophagous arthropods and replicate both 7. The 3rd edition was published in 1985, and it was the last printed version. Currently, the Arbovirus Catalog is maintained by the Centers for Disease Control and Prevention (CDC) on its website (https://wwwn.cdc.gov/arbocat/), and it includes around 537 distinct viruses consisting of both arbovirus and other vertebrate virus . This website records the meta information of each virus including name, original source, method of isolation, virus properties, antigenic relationship, and other factors, but without genetic information such as viral sequence, and whether the genome is segmented or not. Moreover, with classic virus isolation methods and the widespread utilization of deep sequencing and metagenomic analysis techniques, many novel viruses have been discovered and isolated from arthropods over the recent past, but have not been registered in the Arbovirus Catalog9.The American Committee on Arthropod-Borne Viruses (ACAV) published the first edition of “Catalogue of Arthropod-borne Viruses of the World ” in 1967https://www.cdc.gov/labs/BMBL.html), provides safety guidelines to those working with arbovirus, as well as ASV that are closely related to arboviral counterparts10. Table 3 and 4 in Section VIII-F of BMBL 6th provides an alphabetical listing of the recognized arbovirus and ASV at the time of publication (by the year of 2019) and includes the common name, acronym, virus family or genus, Biosafety level (BSL) recommendation etc., separately.Section VIII-F of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) 6th published by CDC in 2020  is aiming at categorizing viruses using a single classification method by their evolutionary relationships. The ICTV Virus Metadata Resource  provides virus meta information almost all classified viruses especially about the viruses classified after the year of 2019, but no specific information about if the virus belongs to arbovirus or ASV.International Committee on Taxonomy of Viruses  is a regularly updated public nucleotide sequence database, enlists many meta information of the virus as well as the nucleotide/amino acid sequences. However, the isolate, segment and host information of GenBank is complex and non-uniformly formatted due to the different standards adopted by the numerous submitters. In addition, it does not include categorical information on whether a virus is belonging to arbovirus or ASV.GenBankCurrently, there is no comprehensive dataset containing both arbovirus and ASV in a globally accessible scale. To address this issue, we collected, extracted, cleaned, and sorted information from the Arbovirus Catalog, Section VIII-F of the BMBL, ICTV and GenBank, that includes a complete information set on viral taxonomy, biological characteristics, vectors and vertebrate hosts, distribution, recommended biosafety levels, nucleotide/amino acid sequences, and genome segment. This dataset will be useful and beneficial to the larger community of scientists/researchers that study arbovirus and ASV, specifically in the fields of viral vector/host prediction through deep learning, disease outbreak risk warning, arbovirus/ASV interactions, phylogenetic and evolutionary relationships, as well as in biosafety risk assessment studies.th and Virus Metadata Resource_(18-191021_MSL36) from ICTV were extracted, then the records containing artificial  or short sequences (length <100 bp) were removed, to generate tempdata 1 and tempdata 2 separately. The corresponding submit/release date, taxonomy, isolate source were further extracted from the NCBI Taxonomy (https://ftp.ncbi.nih.gov/pub/taxonomy/) and NCBI Virus-Host (https://www.genome.jp/virushostdb/).In the first step, the virus names from Arbovirus Catalog/Table 3 and 4 of Section VIII-F of BMBL 6GenBank records belonging to the vertebrate virus (host source by vertebrate) in the tempdata 1 were excluded to generate tempdata 3.To process the arbovirus and ASV which were not present in tempdata 3, we first extracted records with hosts derived from invertebrates only or from both invertebrates and vertebrates from tempdata 2 and removed records that were redundant from tempdata 3.25 to ascertain if it was a true “arbovirus”. If the host source belonged to an invertebrate, a further check was done to see if the isolation source was hematophagous arthropods ’, ‘argas’, ‘midge’, ‘sandfly’), then identified as ‘ASV’. GenBank records identified as arbovirus and ASV in these steps were combined to generate tempdata 4.If the host source of them belonged to invertebrates or vertebrates, the virus associated with these records was selected and rechecked by relevant literatureth January 2023) according to viral meta information data by GenBank ID to generate the viral nucleotide sequences file  in the fna (FASTA nucleic acid) format and amino acid sequences file in the faa (FASTA amino acid) format.Finally, tempdata 3 and 4 were combined to generate the viral meta information data of arbovirus and ASV with comprehensive information in Microsoft Excel 2019 spreadsheet (.xlsx), virus segment, biosafety recommendation, host, and other relevant information were subsequently added to the virus information file. Nucleotide and amino acid sequences were extracted from GenBank (downloaded on 28https://pandas.pydata.org/).The final global dataset of viral sequence, diversity, distribution, and biosafety recommendation of arbovirus and ASV consist of one virus meta information file (.xlsx), one virus nucleotide sequence file (.fna), and one amino acid sequence file (.faa). The data screening and integration process was accomplished using software Pandas v2.0.0  and 711,624 (tempdata 2) GenBank records, respectively. After removing records that were neither arbovirus nor ASV from tempdata1 and tempdata 2, 97,949 records (tempdata 3) and 3,145 (tempdata 4) records were used for dataset merging. Finally, a total of 101,094 eligible records (640 virus species/805 viruses) were generated, comprising 98,994 records (460 virus species/615 viruses) of arbovirus and 2,100 records (180 virus species/190 viruses) of ASV. The corresponding 101,094 viral nucleic acid sequences and 139,338 viral amino acid sequences were also extracted from the NCBI GenBank database Fig. . All recThe most essential information extracted from the Arbovirus Catalog/BMBL, ICTV and NCBI database included (i) Locus ID and GenBank accession number, (ii) Taxonomy of viruses and their isolate sources, (iii) Segment number of the genome, (iv) Biosafety recommended level, and (v) Global geographical coordinates. All data was extracted and integrated into a final dataset including one excel sheet and two FASTA files, which were examined independently and thoroughly by a five-member team to avoid possible errors and redundancies.https://data.apps.fao.org/map/catalog/srv/eng/catalog.search#/metadata/9c35ba10-5649-41c8-bdfc-eb78e9e65654), with records without corresponding information populated with acronym ‘NAV’. The original latitude and longitude records and the centroids of countries/regions or states/provinces were used for the subsequent cartography. GeoPandas v0.12.2 (https://geopandas.org/en/stable/) and Matplotlib v3.6.0 (https://matplotlib.org/) were used for data processing and visualization of geographic information.The geographical locations of all collected records were extracted from the NCBI GenBank database. This included countries or regions, states or provinces, latitude and longitude, and any other geographical information. All acquired geographical information was standardized to the countries or regions and state/province levels based on the digital map of the World Food and Agriculture Organization (FAO) , a nucleic acid sequences file (.fna) and amino acid sequences file (.faa), as accessible from figshareThe column details of the viral meta information file (.xlsx) are as follows :Taxonomy InformationVirus_Group: (customized field) viruses in the database are divided into two groups: arbovirus and ASV. The former has both vertebrate and arthropod hosts, the latter has only arthropod hosts.Name: (source from GenBank) the virus name, each name represents a distinct virus.Acronym: (source from BMBL) acronym of virus name.NCBI_Taxonomy_ID: (source from GenBank) taxonomy identifier of virus from NCBI Taxonomy Database.Isolate: (source from GenBank) Isolate of virus from NCBI GenBank.Unified_Isolate_Number: (customized field) renumbering of the field Isolate. Each isolate of the same virus is numbered.Species: (source from ICTV) species that the virus belongs to. Species of the viruses are normally different with their names.Genus: (source from ICTV) genus that the virus belongs to.Family: (source from ICTV) family that the virus belongs to.Genome InformationSegmented: (customized field) whether the genome of the virus is unsegmented (recorded as “no”) or segmented virus (recorded as “yes”). Virus with an unknown number of segments (recorded as “NAV”).Number_of_Segments: (source from GenBank) the theoretical number of segments of the virus.Molecule_Type: (source from GenBank) molecule types of the virus genome which are divided into ssRNA(+), ssRNA(−), ssRNA(+/−), dsRNA, RNA, ssDNA(+/−), dsDNA and etc.Sequence InformationAccession: (source from GenBank) NCBI GenBank Accession of the nucleotide sequence.Locus: (source from GenBank) the locus name of the nucleotide sequence.SRA_Accession: (source from GenBank) NCBI SRA Accession of the nucleotide sequence.Submitters: (source from GenBank) submitters of the nucleotide sequence.Sequence_Type: (source from GenBank) whether the nucleotide sequence is a reference sequence (recorded as “RefSeq”) or a non-reference sequence (recorded as “GenBank”).BioSample: (source from GenBank) NCBI BioSample Accession of the nucleotide sequence.GenBank_Title: (source from GenBank) the field “DEFINITION” of NCBI GenBank database of the sequence.Genotype: (source from GenBank) genotype of the nucleotide sequence.Segment: (source from GenBank) segment identifier of the nucleotide sequence.Unified_Segment_Number: (customized field) renumbering of the field Segment. Each segment is assigned a new number from 1. Segment of the unsegmented virus is assigned as 1.Host InformationHost_Species: (customized field) the species of the dead-end host of the virus.Host_Genus: (customized field) the genus of the dead-end host of the virus.Host_Family: (customized field) the family of the dead-end host of the virus.Host: (source from GenBank) the field from the NCBI GenBank database that represents dead-end host or vectors.Biosafety InformationRecommended_BSL: (customized field) recommended biosafety level of laboratory to research the virus .BMBL_Recommended_BSL: (source from BMBL) BMBL recommended biosafety level of laboratory to research the virus .Basis_of_Rating: (source from BMBL) risk assessment of the virus .Antigenic_Group: (source from BMBL) the antigenic group of the virus.Isolated: (customized field) whether the virus has been isolated (“Yes” or “No”).Source InformationLatitude_and_Longitude: (source from GenBank) longitude and latitude of the virus isolation source.State_or_Province: (customized field) state or provincial administrative unit of the virus source.Geo_Location: (source from GenBank) geographical position of the virus source.Country_or_Region: (customized field) the country or region of the virus source.Isolation_Source: (source from GenBank) the organism which the virus was collected from.Collection_Date: (source from GenBank) the date that the virus was collected.Submit_Date: (source from GenBank) the date that the virus was submitted.Release_Date: (source from GenBank) the date that the virus was released or last modified.ReferencesPublications: (customized field) the number of publications and literature covering the specific virus research.Accession_URL: (customized field) the DOI leading directly to the GenBank source.Locus: (source from GenBank) NCBI GenBank LOCUS ID of the nucleotide sequence.Accession: (source from GenBank) NCBI GenBank Accession of the nucleotide sequence.Protein_ID: (source from GenBank) a protein sequence identification number (for amino acid sequences file).The nucleotide sequences file and amino acid sequences file are standard FASTA files. Each sequence information consists of two lines, header and content. The header contains two types of information, locus and accession, split by ‘|’. Content is a specific nucleic acid or amino acid sequence. The detailed definitions of the fields in the header are as follows:This dataset collected 101,094 records (805 viruses) from 615 arboviruses and 190 ASVs derived from 849,062 GenBank records , that were submitted and released from 1988 to 2022. All records were collected and processed by two members of the team with the other three members tasked with cross-checking and confirming, and all uncertain or discrepant records were discussed separately.th and Arbovirus Catalog, a reliable source for record arbovirus and ASV records. Other records that were not queried in the Arbovirus Catalog/BMBL 6th were manually inspected against relevant literature and isolate sources. To ensure the accuracy and reliability of manual inspection, the following strategies were adopted: (i) If the isolate source contained both vertebrates and invertebrates, the specific host information reported in the relevant literature for the record was used to determine the virus type , (ii) If the isolate source contains invertebrates only and belongs to hematophagous arthropods , the record was labelled as ASV. The taxonomy at the family and genus level of all included arbovirus and ASV, as well as the correspondence between virus taxonomy and isolate sources, were shown in Figs. The accuracy of virus group classification (arbovirus/ASV) is critical, therefore the virus groups of records in this database were first defined by referring to Tables 3 and 4 in Section VIII-F of the BMBL 6Emerging and re-emerging viral diseases transmitted by arthropods remain one of the most serious threats to humans and animals, with potentially devastating social and economic consequences. Knowledge of the classification, molecular characteristics of their genomes, abundance and diversity, worldwide distribution, vector and vertebrate host, and biosafety risk of arbovirus and ASV is crucial in supporting the development of policies and directing the necessary actions for preventing and managing relevant diseases, and developing novel biological control strategies. This dataset serves as the foremost comprehensive compilation of both arbovirus and ASV on a global scale, which can be used in biosafety risk assessment, to infer arbovirus and ASV evolutionary relationships, model arboviral diseases’ ecological risks, and infer vectors and hosts of the newly discovered arbovirus.


Background: Pain is a leading cause of admission to the emergency department (ED) and moderate-to-severe acute pain in medically supervised settings is often treated with intravenous (IV) opioids. With novel noninvasive analgesic products in development for this indication, it is important to assess the costs associated with IV administration of opioids.Materials and Methods: A retrospective observational study of data derived from the Premier database was conducted. All ED encounters of adult patients treated with IV opioids during a 2-year time period, who were charged for at least one IV opioid administration in the ED were included. Hospital reported costs were used to estimate the costs to administer IV opioids.Results: Over a 24 month-period, 7.3 million encounters, which included the administration of IV opioids took place in 614 US EDs. The mean cost per encounter of IV administration of an initial dose of the three most frequently prescribed opioids were: morphine $145, hydromorphone $146, and fentanyl $147. The main driver of the total costs is the cost of nursing time and equipment cost to set up and maintain an IV infusion ($140 ± 60). Adding a second dose of opioid, brings the average costs to $151-$154. If costs associated with the management of opioid-related adverse events and IV-related complications are also added, the total costs can amount to $269-$273. Of these 7.3 million encounters, 4.3 million (58%) did not lead to hospital admission of the patient and, therefore, the patient may have only required an IV catheter for opioid administration.Conclusions: IV opioid use in the ED is indicated for moderate-to-severe pain but is associated with significant costs. In subjects who are discharged from the ED and may not have required an IV for reasons other than opioid administration, rapid-onset analgesics for moderate-to-severe pain that do not require IV administration could lead to direct cost reductions and improved care.


We report the autopsy pathology findings of a 21-week stillborn fetus with congenital mpox syndrome that occurred in the Democratic Republic of the Congo in 2008. The fetus acquired mpox from the mother after intrauterine transplacental monkeypox virus transmission. We confirmed monkeypox virus infection in the mother, fetus, and placenta by using a monkeypox virus–specific quantitative PCR. Subtyping of the virus was not performed, but the mother and fetus were almost certainly infected with the clade I variant that was endemic in the Democratic Republic of the Congo at the time. Risk for intrauterine infection appears to differ between virus clades, but clinicians should be aware of potential for intrauterine monkeypox virus transmission among pregnant persons during ongoing and future mpox outbreaks. Orthopoxvirus. Monkeypox virus is related to the variola virus, the etiologic agent of smallpox before its eradication. Mpox has caused illness and death in endemic countries of Central and West Africa, where it infects thousands of persons annually. In 2022, a global outbreak of mpox occurred that has resulted in >87,000 infections, most of which have occurred in nonendemic countries  and the US Army Medical Research Institute of Infectious Diseases jointly designed and conducted a prospective study at the Kole Hospital in Kole, located in the Sankuru District of Kasaï-Oriental Province in DRC . She had become febrile at 18 weeks’ gestation and had mpox viremia confirmed by virus-specific qPCR. Two weeks after enrollment, she noted absence of fetal movement, which was confirmed both clinically and by 2 sequential obstetrical ultrasounds, leading to diagnosis of intrauterine fetal death. 2 to 106 viral copies/mL over a 3-day period after cessation of fetal movement. Transcutaneous amniocentesis was positive for mpox virus. Labor was induced by using oxytocin. After membrane rupture, a 21-week gestation stillborn female fetus was delivered vaginally. Umbilical vein blood was positive for mpox virus by qPCR. Oral and written autopsy consent was obtained in the native language from the mother, per the study protocol, which included photography and use of all body specimens, including tissue samples, for further study and publication. After the delivery, the mother was monitored in the hospital; although depressed, she recovered from the mpox infection and was discharged to home in good health. She was well when she returned for her 75-day postpartum follow-up visit.Maternal viremia rose rapidly from 10https://explore.omsystem.com).The fetal autopsy was performed in a surgical operating suite at the Hôpital General du Référence in Kinshasa. The lighting was suboptimal for photography, and equipment for measuring organ weights and taking dictation was not available. Photographs of the fetus were taken by using a digital Stylus 790 SW camera  involving the right nostril, upper mid-lip, forehead, right cheek, and temporoparietal regions of the scalp. The lesions on the head were not as well developed as lesions on other parts of the body. Many maculopapular lesions, varying from 0.3–0.5 cm in size, were noted on the skin of the extremities, including the soles of the feet and palms of the hands bilaterally, back, chest, shoulders, abdomen, and buttocks  virus and that it had sufficient novel mutations to be classified as a new subclade, clade IIb (In conclusion, we report a case of fetal death after placental infection and intrauterine transmission of monkeypox virus clade I in the Democratic Republic of the Congo in 2008. Risk for intrauterine infection appears to differ between virus clades. Nonetheless, clinicians should be aware of potential for placental infection and intrauterine transmission of monkeypox virus among pregnant persons during ongoing and future mpox outbreaks.