Ixodes uriae), is widely distributed around the circumpolar regions of the northern and southern hemispheres. It has been suggested that Ix. uriae spread from the southern to the northern circumpolar region millions of years ago and has remained isolated in these regions ever since. Such a profound biographic subdivision provides a unique opportunity to determine whether viruses associated with ticks exhibit the same evolutionary patterns as their hosts. To test this, we collected Ix. uriae specimens near a Gentoo penguin (Pygoscelis papua) colony at Neko harbour, Antarctica, and from migratory birds—the Razorbill (Alca torda) and the Common murre —on Bonden island, northern Sweden. Through meta-transcriptomic next-generation sequencing we identified 16 RNA viruses, seven of which were novel. Notably, we detected the same species, Ronne virus, and two closely related species, Bonden virus and Piguzov virus, in both hemispheres indicating that there have been at least two cross-circumpolar dispersal events. Similarly, we identified viruses discovered previously in other locations several decades ago, including Gadgets Gully virus, Taggert virus and Okhotskiy virus. By identifying the same or closely related viruses in geographically disjunct sampling locations we provide evidence for virus dispersal within and between the circumpolar regions. In marked contrast, our phylogenetic analysis revealed no movement of the Ix. uriae tick hosts between the same locations. Combined, these data suggest that migratory birds are responsible for the movement of viruses at both local and global scales.Ticks (order: Ixodida) are a highly diverse and ecologically important group of ectoparasitic blood-feeding organisms. One such species, the seabird tick ( Ixodes uriae, and their RNA viruses sampled from the far northern and southern hemispheres. Despite the huge geographic distance between them, phylogeographic analysis reveals that the same and closely related viruses were found both within and between the northern and southern circumpolar regions, most likely reflecting transfer by virus-infected migratory birds. In contrast, genomic data suggested that the Ix. uriae populations were phylogenetically distinct between the northern and southern hemispheres. This work emphasises the importance of migratory birds and ticks as vectors and sources of virus dispersal and introduction at both the local and global scales.As host populations diverge, so may those microorganisms, including viruses, that are dependent on those hosts. To examine this key issue in host-microbe evolution we compared the co-phylogenies of the seabird tick,  Following the physical separation of a population into geographically isolated sub-populations (i.e. vicariance) genetic changes unique to each sub-population will accumulate. Given a sufficient period of time, such process may result in marked genetic separation. By combining phylogenetic and geographical information—that is, phylogeography ,2—it is Puma concolor, that was not apparent in host genetic data [As host populations diverge, so will any microorganisms, including viruses, that are dependent on their hosts. Accordingly, analysis of genome sequence data from these microorganisms can provide additional, and sometimes more detailed, information about the evolutionary and epidemiological history of the host species ,4. Phylotic data .Ixodes is the most species rich group with nearly 250 species [Ix. uriae is the only known tick with a circumpolar distribution in both the northern and southern hemispheres. This species parasitizes close to 100 different vertebrate species, the majority of which are seabirds that breed in dense colonies [Alcidae and Laridae, and in the southern hemisphere they are mainly species of the Spheniciformes and Procellariiformes [Ix. uriae has three active life-stages  whose questing behaviour is most prevalent during the summer months with a peak during June–July in the northern hemisphere and December–January in the southern hemisphere [Ix. uriae can tolerate temperatures as low as -30°C and as high as +40°C [Ix. uriae is a well-known vector of multiple different viruses and bacteria, including Borrelia burgdorferi sensu lato [Ix. uriae, there is limited evidence for human disease associated with Ix. uriae transmission [Ticks (order Ixodida) are among the most diverse groups of ectoparasites. There are close to 900 species of both soft- and hard-bodied ticks within the Ixodida ,7, of wh species ,8–10. Wicolonies . In the iiformes –14. Likemisphere –18. Eachmisphere ,15,19,20as +40°C , formingas +40°C ,21. Ix. nsu lato ,23, the nsu lato ,25 amongnsu lato ). Howevesmission .Ix. uriae diverged from its most recent common ancestor, Ix. holocyclus, approximately 91 million years ago, and that the Ix. uriae species complex shared a common ancestor some 22 million years ago [Ix. uriae was introduced, possibly twice, into the northern hemisphere from the likely ancestral Australasian population approximately 10 million years ago [Ix. uriae sub-populations. Here, by comparing the viromes of Ix. uriae collected from seabirds–the Common murres’  and Razorbills’ (Alca torda)–from the northern hemisphere and from around a penguin (Pygoscelis papua) colony in the southern hemisphere, we investigated whether there has been virus dispersal either within or between the northern and southern hemispheres.Data from both mitochondrial and nuclear genes suggest that ears ago . Subsequears ago . Thereafears ago . HoweverIx. uriae individuals: 10 libraries using two tick individuals from the southern hemisphere, and six libraries from the northern hemisphere comprising five with two tick individuals and one with three tick individuals  sequence similarity. The viruses identified belong to the following orders/families: Bunyavirales (N = 5), Hepeviridae (N = 2), Flavivirus (genus) (N = 1), Mononegavirales (N = 1), Orthomyxoviridae (N = 2), Picornaviridae (N = 1), Reoviridae (N = 2) and Tombusviridae (N = 2)  .Ix. uriae, approximated via the host COX1 gene, varied between 1,359–8,101 reads per million (Flavivirus), Taggert virus , Neko harbour virus (Orthomyxoviridae) and Upmeje virus (Tombusviridae)—were highly abundant . Indeed, Gadgets Gully virus, Taggert virus and Upmeje virus were more abundant than the host COX1 gene  colony at Neko harbour, Antarctica, and seven were found from Ix. uriae ticks collected from Razorbill (Alca torda) and Common murre  seabirds on Bonden island in the Gulf of Bothnia, northern Sweden  in both Antarctica and northern Sweden  and Gadgets Gully virus (Flaviviridae) from the Neko harbour sampling site. Both these viruses have previously been found on Macquarie island, south-east of the Australian continent [Bunyaviridae) and Okhotskiy virus (Reoviridae), previously isolated from Ix. uriae from the Great island, Newfoundland, Canada, and from islands around the sea of Okhotsk, respectively [It was also notable that we identified both Taggert virus  and that sequenced here share 97.7% amino acid and 87.6% nucleotide similarity in the VP1 (RdRp) segment, also compatible with the idea that this virus has circulated in situ for more than 40 years. Regardless of how and when these viruses transferred to new locations, it is evident that there has been circumpolar dispersal of Ix. uriae associated viruses.Less clear is whether the divergence of these variants justifies their classification as new virus species. For example, the variant of Gadgets Gully virus identified here shares 92.7% amino acid similarity and 80.7% nucleotide similarity to the currently available genome (YP_009345034.1) originally isolated in 1976 . Given tIx. uriae was mined from all sequence libraries, and a phylogenetic analysis performed on two sets of representative outgroup and ingroup ixodid species: one utilising complete mitochondrial genome  was greater in the southern  than the northern population . Although more data is clearly needed, these patterns of genetic diversity are compatible with the idea that the southern circumpolar population represents the ancestral population.To better understand virus–tick co-evolution, the host mitochondrial genome of l genome  and a sel genome . For comispheres , S1 Fig Ix. uriae-associated viruses moves within the circumpolar regions inhabited by Ix. uriae. For example, Avalon virus, closely related to Paramushir virus [Ix. uriae [Ix. uriae, suggests that Ix. uriae associated viruses are being transported by migratory birds within and between the circumpolar regions. Hence, it is possible that these viruses may be present in all regions with permanent populations of Ix. uriae.The phylogenetic analysis of a subset of closely related bunyaviruses reveals several patterns of geographic dispersal . The clair virus , has beeir virus , along tir virus , and nowx. uriae , that hax. uriae  and coveHepeviridae) have previously been identified in Antarctic ticks [Mononegavirales) was discovered in several of the tick libraries from northern Sweden and was relatively abundant  in one library  was identified in several libraries from northern Sweden, although it was not abundant  that has previously been described in this region [Reoviridae) described above, we also identified Fennes virus in the Antarctic sequence libraries, with near identical sequence similarity  to the sequence of this virus identified previously [Ix. holocyclus ticks from Australia [In the Antarctic sequence libraries we observed Gerbovich virus  collected in the Republic of the Congo [Finally, we identified two novel and divergent viruses within the n Sweden , Table 1he Congo . Upmeje Ix. uriae, that is distributed across both circumpolar regions, as a means to understand virome composition, host–virus co-divergence and the long-distance dispersal of tick-borne viruses. In particular, we assessed whether we could infer dispersal events within and between the southern and northern circumpolar regions that are separated by a substantial geographic distances.Ticks are the most important blood-feeding arthropods in temperate and polar regions –54. TheiIx. uriae and some have been shown to have pathogenic properties to either birds and/or humans [Ix. uriae. These viruses could either be transported with the ticks carried by seabirds during migration or directly by infected birds. Indeed, several well-known bird-hosts of Ix. uriae have circumpolar migration patterns. For example, in the southern ocean, birds of the order Procellariiformes show circumpolar migration involving many stopover sites [Rissa tridactyla), Atlantic puffin (Fratercula arctica) and Thick billed murre (Uria lomvia) all show seasonal movements between the eastern and western north Atlantic [Overall, we identified 16 RNA viruses of which seven were novel. Of the nine viruses previously discovered, several have previously been documented in r humans ,57. For r humans ,58, agair humans , and nower sites ,60. In tAtlantic ,62.Ix. uriae population diverged into two sub-populations. In addition, as the northern and southern Ix. uriae populations are phylogenetically distinct [Sterna paradisaea) performs the longest migration of any avian species, traveling the distance between Antarctica to southern Greenland, approximately 24,000 km, in around 40 days [Ix. uriae on this species, it is likely that they can become infested as they breed in dense colonies near bird species that are well-known hosts of Ix. uriae. As noted above, Procellariiform birds are long distance migrators and a study using geolocators of the Short-tailed shearwater (Ardenna tenuirostris) revealed that this species migrated to south of the Antarctic Polar Front after their breeding period in Tasmania, and following a stopover migrated northward to spend the Arctic summer in a location as far north as the Bering sea [Ardenna grisea) also undertakes long distance trans-equatorial migrations [Thalassarche melanophris) and the South polar skua (Stercorarius maccormicki), have been occasionally observed in the Arctic region. As both the nymph and adult tick can feed for up to 12 days [Ix. uriae phylogeny suggests that there has been no movement of ticks among the southern and northern circumpolar regions, it cannot be excluded that there are as yet unsampled locations in either region where population admixture could occur.We also saw evidence of historic movement of viruses between the northern and southern circumpolar regions. In particular, our phylogenetic analysis revealed that Bonden virus, identified in ticks from northern Sweden, was closely related to Piguzov virus from Antarctica . Similardistinct , it seem 40 days . Althougring sea . Althouggrations , and oth 12 days ,66, thesUnder what circumstances could a tick then be transferred between the two polar-regions? Unless a journey is made directly between the polar-regions, which is theoretically possible in the case of Short-tailed shearwater, it would be necessary to occur sequentially both with respect to bird stop-over and tick life-stage development. For example, a nymph would initially latch onto the host shortly prior the bird migration, feed for the entire duration of the flight, and develop to the next life-stage during the stop-over. The adult tick would then need to find a new host to continue its journey. Given the long distances and time during which tick and virus have to survive, such events are unlikely to take place in one migration step. At the same time, that we could identify two clear cases of cross-circumpolar dispersal of closely related viruses from such a small sample of viruses indicates that cross-circumpolar transmission may not be infrequent.Ix. ricinus acts as both the virus reservoir and vector [Ix. uriae forming off-host aggregations, combined with the occasionally relatively short questing period for specific hosts [The finding of several previously discovered and novel viruses that are known or likely to be tick-associated raises interesting questions about how these viruses are maintained in nature. In the northern circumpolar distribution the tick life cycle can last up to seven years depending on host availability and temperature, and ticks may spend up to eleven months of the year off the host in aggregations formed in moist environments ,15,17.Fod vector ,68. In pic hosts ,69, suggic hosts ,67,70,71Ix. uriae harbours an extensive diversity of viruses belonging to several different families and orders of RNA viruses, and that there has been a transfer of viruses both within and between the northern and the southern circumpolar regions. As such, we stress the importance of the millions of birds that each year migrate across the globe and that have the capacity to transfer viruses to and from adjacent and distant geographical areas.In sum, we have shown that the seabird tick Ix. uriae ticks were collected during 2016–2017 from the Bonden island bird station in northern Sweden  and from the ground around a Gentoo penguin (Pygoscelis papua) colony at Neko harbour, Antarctica , during 2018. All ticks were morphologically keyed to species [Adult female  species  and werede novo using Trinity v.2.5.4 [Sequencing libraries, data generation and analysis was performed as previously described ,73. Brie v.2.5.4  with reade novo assembled contigs were initially screened against the complete non-redundant nucleotide and protein databases (NCBI GenBank) using blastn v.2.6.0+ [−5 for both methods. To further assess the data and to identify potential endogenous viral elements, all assemblies indicative of RNA virus origin were screened using the NCBI Conserved Domain Database (www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) with an expected value threshold of 1 × 10−3. Relative abundance of the identified viruses was determined by comparing the mapping results of the Ix. uriae mitochondrial cytochrome C oxidase I (COX1) gene  against all RdRp containing contigs using Bowtie2 v.2.3.4 [All v.2.6.0+  and Diamv.2.6.0+ , respect v.2.3.4 ,73. If tBunyavirales, Mononegavirales, Orthomyxovirales, the families Picornaviridae, Reoviridae, the Hepe-Nido-like and Tombus-Noda-like groups, and the genus Flavivirus. These sequences were then aligned using the E-INS-i algorithm in Mafft v.7.271 [To infer the evolutionary history of all the RNA viruses identified here they were combined with representative amino acid data sets of the RNA-dependent RNA-polymerase genes of viruses from the orders  v.7.271 . To redu v.7.271  under th v.7.271  via IQ-T v.7.271 . FinallyIx. uriae and a subset of closely related Bunyavirales found at both sampling sites (see Ix. uriae (NC_006078.1) was used as reference for mapping with Bowtie2 v.2.3.4 [Ixodidae reference genomes (N = 24), and (ii) complete genomes of Ix uriae and Ix holocyclus (N = 19) from the complete genome alignment combined with shorter mitochondrial gene sequences (N = 148) taken from two previously published data sets [https://github.com/rambaut/figtree/). Comparison of genetic diversity between the northern and southern populations was undertaken by computing the number of base differences per site averaged over all sequence pairs between the two populations (i.e. π) using Mega X v.10.1.1 [To compare the evolutionary history of ites see , the mite NC_0060.1 was usata sets ,33. Bothata sets . Followiata sets , model tv.10.1.1 .S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.


Apis mellifera simensis, using an unbiased metagenomic approach in which the next-generation sequencing step was preceded by an enrichment protocol for viral particles. Our study revealed five well-known bee viruses and 25 atypical virus species, most of which have never been found in A. mellifera before. The viruses belong to Iflaviridae, Dicistroviridae, Secoviridae, Partitiviridae, Parvoviridae, Potyviridae, and taxonomically unclassified families. Fifteen of these atypical viruses were most likely plant-specific, and the remaining ten were presumed to be insect-specific. Apis mellifera filamentous virus (AmFV) was found in one sampling site out of 10. Two samples contained high read counts of a virus similar to Diatraea saccharales densovirus (DsDNV), which is a virus that causes high mortality in the sugarcane borer. AmFV and the DsDNV-like virus were the only DNA viruses found. Three viruses that primarily infect Drosophila spp. were also discovered: La Jolla virus (LJV), Kilifi virus (KiV), and Thika virus. Our study suggests that phoretic varroa mites are involved in the transmission of LJV and KiV and that both viruses replicate in mites and adult bees. We also found an overwhelming dominance of the deformed wing virus type B variant, which fits well with the apparently harmless infestation by Varroa destructor. It was suggested that Ethiopian bees have developed tolerance against virus infections as the result of natural selection.Metagenomics studies have accelerated the discovery of novel or divergent viruses of the honey bee. However, most of these studies predominantly focused on RNA viruses, and many suffer from the relatively low abundance of viral nucleic acids in the samples . Here, we explored the virome of the Ethiopian honey bee,  Honey bees play a significant role in boosting crop production through pollination ,2. It isVarroa destructor mites are considered the most serious biotic threats to honey bees’ health [Globally, viral pathogens and ’ health ,8,9,10. ’ health , by weak’ health , by acti’ health ,14,15 an’ health , leading’ health . Moreove’ health . The cli’ health ,20,21. O’ health , to such’ health . Moreove’ health ,23.Picornavirales virome could be discovered [In Africa, the presence of common honey bee viruses such as DWV, Lake Sinai virus (LSV), acute bee paralyse virus (ABPV), Israeli acute paralysis virus (IAPV), chronic bee paralysis virus (CBPV), sacbrood virus (SBV), black queen cell virus (BQCV), and Apis mellifera filamentous virus (AmFV) have been confirmed in different parts of the continent, including Kenya, Uganda, and Sudan ,28,29,30scovered . Furtherscovered ,35. Pathscovered . Given tscovered , it seemApis mellifera [The advent of whole-community genome sequencing or metagenomics opened a new opportunity to understand the genetic diversity of the viral community without a priori knowledge of the virus type present in a sample . It hugeellifera ,32,40. Hellifera ,42. Moreellifera , althougellifera ,44. TherApis mellifera simensis) using an unbiased metagenomics approach. For the viral metagenomics analysis, we have employed the NetoVIR protocol: a reproducible protocol that enriches virus-like particles by removing non-viral nucleic acids, enabling the detection of both RNA and DNA viruses [A. mellifera before. Two of these atypical viruses were further investigated, aiming to show that they actually infect honey bees and how the transmission might occur.This study was designed to explore the diversity of viruses in Ethiopian honey bees  from the Tigray National Regional State of Ethiopia . Access Samples were collected (with the owners’ permission) between August and October 2017  from 20 bee colonies spread over 10 apiaries , and centrifuged at 17,000× g for 1 min.Samples derived from adult bees were used for metagenomics analysis. Two bees per hive were homogenized separately using a tissue homogenizer  at 3000 rpm for 1 min in the presence of 500 μL sterile phosphate-buffered saline (PBS) and three zirconium beads. After centrifugation at 17,000× 2 and 30 mM MgCl2, pH 8) and incubated at 37 °C for 2 h. Then, 7 μL of 10 nM EDTA was added to inactivate the enzymes. The total nucleic acids  were extracted using the QIAamp Viral RNA Mini preparation kits  according to the manufacturer’s instructions, without using carrier RNA.After filtration, samples were subjected to nuclease treatment to remove non-viral nucleic acids including host contaminants . Briefly, 130 μL of the filtrate was mixed with 2 μL of benzonase nuclease, 1 μL of micrococcal nuclease and 7 μL of 20× homemade buffer , according to manufacturer’s protocol. The concentration of the purified DNA was quantified using the Qubit dsDNA HS assay kit, according to the manufacturer’s protocol and then diluted to 1.2 ng/µL.cDNA synthesis and PCR amplification was performed according to the modified version of the Whole Transcriptome Amplification  kit . Amplico® AMPure® PCR purification protocol. The concentration of the purified library was quantified using a Qubit dsDNA HS assay kit, according to the manufacturer’s instructions. Then, the DNA library size distribution and quality were evaluated using an hsDNA chip on a Bioanalyzer platform . Sequencing was performed using the Illumina NextSeq platform for paired-end sequencing with a base length of 2 × 150 bp, and an average of 9,304,652 reads were retrieved per sample.DNA libraries were prepared using the Nextera XT DNA library preparation kit  as per the manufacturer’s instructions, and libraries were purified following the Agencourthttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/). After quality assessment, sequencing adapters and low-quality sequences with Phred scores below 20 were trimmed using Trimmomatic v0.38 [https://bitbucket.org/MAVERICLab/docker-clustergenomes). The resulting set of non-redundant scaffolds were compared to the NCBI non-redundant protein database (downloaded on 22 January 2018) using DIAMOND BLASTx [−3). Sequences that showed no homology to any sequences present in the non-redundant protein database were extracted and compared to the NCBI non-redundant nucleotide database (downloaded on 22 January 2018) using tBLASTx . BLAST output was visualized and taxonomies were assigned using the KronaTools software [The bioinformatics workflow is illustrated in ic v0.38 . Trimmedic v0.38  with kmeD BLASTx   under BioProject accession number PRJNA559070 and BioSample accession number SAMN12512743- SAMN12512752. La Jolla virus (LJV), Kilifi virus (KiV), and Thika virus (ThV) near-complete genome sequences were deposited in GenBank under accession numbers MT681680, MT681681, and MT681679, respectively. A multifasta file of the sequences retrieved in this study and used in the phylogenies of the known honey bee viruses is provided as a Quantification of the virus loads of adult honey bees in each honey bee colony under investigation was performed using RT-PCR for a selected set of five viruses: DWV, BQCV, SBV, LJV, and KiV. Later on, these analyses were extended with the detection of LJV and KiV in varroa mites and honey bee pupae  collected from the same colonies.n = 20) or ten pupae per apiary  were homogenized using a Bullet Blender Storm 5®/VISUM IDPBW instrument (at maximum speed) in 5 mL phosphate-buffered saline (PBS), 0.25 mL zirconia beads, and three metal beads. The homogenate (1 mL) was centrifuged at 1500 rpm for 10 min. Then, the supernatant was transferred to a new tube and centrifuged at maximum speed for 15 min. For mites, 10 mites per apiary  were crushed with a plastic pestle in a 1.5 mL Eppendorf tube containing 500 µL of Qiazol lysis reagent. After homogenization of the mite sample, 500 µL Qiazol lysis reagent was added, and the sample was incubated for 5 min. Next, 200 µL of chloroform was added, incubated for 2–3 min, and centrifuged at 12,000× g for 15 min. We used the RNeasy Lipid Tissue mini kit  to extract RNA from adult bees following the manufacturers’ instructions. RNA from pupa and mite samples was extracted using the QIAamp® viral RNA kit  and RNeasy Lipid Tissue mini kit , respectively. The cDNA was synthesized from 5 µL RNA template using the RevertAid H Minus First Strand cDNA Synthesis kit  with random hexamer primers, according to the manufacturer’s instructions.Ten adult bees per colony  was used. Each reaction consisted of 12.5 µL of Platinum® SYBR® Green qPCR SuperMix-UDG, 0.5 µL forward primer (10 µm), 0.5 µL reverse primer (10 µm), 10.5 µL DEPC-treated water, and 1 µL of cDNA template. To quantify the viral load in adult bees, we used 2 µL of cDNA template and 9.5 µL of DEPC-treated water, while the amount of the other reagents remained the same. The RT-qPCR reaction was carried out on the CFX96 Real-Time PCR Detection System  using the following program: 50 °C for 2 min, 95 °C for 2 min, and 40 cycles of a combined denaturation (15 sec at 95 °C) and annealing (30 sec at 60 °C) step. At the end of this program, a melt curve was generated by measuring fluorescence after each temperature increase of 0.5 °C for 5 sec over a range from 65 to 95 °C to verify the presence of the desired amplicon. PCR reactions to quantify viruses in adult bees was performed in triplicate, whereas the virus detection in mites and pupae was performed in duplicate. No template controls (water) were included in each assay. Primers for the detection of KiV and LJV were designed directly from the NGS sequencing data using Primer3plus software https://www.ncbi.nlm.nih.gov/tools/primer-blast/. All primers used are given in For the RT-qPCR assays, the PlatinumTo enable the absolute quantification of LJV and KiV, plasmid standards containing the viral of LJV and KiV were constructed using a TOPO TA Cloning Kit for sequencing, according to the manufacturer’s instructions. Then, the plasmids were isolated with the GeneJet plasmid Miniprep kit , according to the manufacturer’s instructions. The viral copy number per bee was obtained by multiplying the reported qPCR copy number values by a correction factor 300× copies/PCR. This conversion factor was calculated based on the volume used at each step of the RT-qPCR method .https://www.R-project.org).Retrieved, near-complete (redundant) genome sequences of DWV, BQCV, LSV, SBV, LJV, KiV, and ThV were used for phylogenetic analysis. Relevant reference sequences, retrieved from NCBI GenBank and ENA using BLASTn, were also included. For LJV, KiV, and ThV, BLASTp hits were used. Multiple sequence alignments were made on either nucleotide level  or on an amino acid level  using MAFFT  on the Lhttp://bioinfo.ut.ee/primer3/). They were chosen immediately adjacent to the MLPA probes, with no more than 15 nucleotides between the last nucleotide and the first nucleotide of the probe sequence. Negative strands were synthetically designed and used as a template for the positive control. All primers, probes, and synthetic templates were synthesized by Integrated DNA Technologies  and are listed in Multiplex Ligation Probe Amplification (MLPA) probes and Reverse Transcriptase (RT) primers were designed for KiV and LJV negative strands. The MLPA probes were designed as described previously . Custom RT-MLPA analysis was performed on pooled samples .Illumina sequencing generated a total of 93.05 million paired-end sequence reads of 150 bp in length (PRJNA559070). Around 63.5% (59.1 million reads) of the reads belonged to scaffolds that shared sequence homology with known taxa in the non-redundant protein and nucleotide databases (NCBI). The majority of the reads could be assigned to Eukaryota taxa (28.5 million reads) followed by virus (25.6 million reads) and bacteria (4.2 million reads). The viral reads mapped to members of several eukaryotic virus families including families . The majOur viral metagenomics analysis revealed both DNA and RNA viruses , althougOur study identified five known honey bee viruses: DWV, SBV, BQCV, AmFV, and LSV  in honeyWe identified 25 viruses, uncommon to honey bees, of which 15 showed homology to plant-specific viruses  and 10 sPartitiviridae (n = 10), Secoviridae (n = 3), Tombusviridae (n = 1), and Potyviridae (n = 1) (Iflaviridae family), KiV , Diatraea saccharalis densovirus (DsDNV) (Parvoviridae family), and eight unclassified/unassigned viruses (Thika virus (ThV), Wenzhou sobemo-like virus 4, Hubei mosquito virus 2, Hubei toti-like virus 2, Hubei partiti-like virus 51, Hubei partiti-like virus 34, Hubei mosquito virus 2, and Wuhan insect virus 27). LJV, KiV, and ThV were found in two, two, and one out of 10 sampling sites, respectively  and will be referred to as a DsDNV-like virus. Partitiviruses are dsRNA viruses and have been observed mainly in plants and fungi, but recently, they were also described as part of the bee virosphere [The plant-specific viruses belong to the families  (n = 1) . The ideectively , and theectively . The fourosphere . The HubDrosophila in Africa [We quantified the virus loads of adult bees from all colonies under investigation for a selected set of five viruses. DWV, BQCV, and SBV were chosen because they were commonly found in bees in Africa ,26,27,30n Africa .12 virus copies per bee) followed by BQCV (6.1 × 105 virus copies per bee). Virus loads of SBV (2.1 × 104 virus copies per bee), LJV (5.3 × 104 virus copies per bee), and KiV (3.9 × 104 virus copies per bee) were the lowest.The virus loads of the five selected viruses varied among the 20 tested honey bee colonies . DWV hadPhylogenetic trees were constructed for DWV, BQCV, LSV, and SBV using the redundant, near-complete sequences retrieved in this study combined with their nearest BLASTn hits. DWV sequences obtained in this study showed an average nucleotide similarity of 97% with their respective GenBank hits. Retrieved sequences fell into the clade together with European DWV-B sequences. The Ethiopian strains were clustered relatively distant from established DWV-B strains, despite the overall high sequence similarity. The three retrieved LSV sequences were more divergent, showing an average nucleotide similarity of 75% with their respective GenBank hits. Phylogenetic analysis revealed the Ethiopian LSV strains paraphyletic to LSV-1 and LSV-2 species B. RetrieIflaviridae-like sequences were retrieved that were similar to Drosophila-infecting viruses, i.e., ThV, KiV and LJV. Phylogenetic reconstruction revealed these viruses clustering closely together with their nearest BLASTn hits   . The hig protein ) could iIn order to understand the routes of transmission of both LJV and KiV, we investigated their presence in varroa mites and in the pupal stage of the honey bee. RT-qPCR analysis confirmed the presence of these two viruses in six out of 10 varroa mite samples . However, LJV and KiV were not found in any of the pupa samples (infested by varroa or not).Then, we determined whether both viruses were actively propagating in the two samples types where they were found . This was done by the detection of their negative strand, the intermediate of viral replication, using the RT-MLPA technique. RT-PCR has been used this purpose in the past, but it was proven to be sensitive to the miss-priming and self-priming of RNA during reverse transcription . On the V. destructor in Ethiopia was confirmed for the first time in a survey of 2010 that was conducted after the first reports of colony losses [V. destructor in Ethiopian honey bees (A. m. simensis) [The occurrence of the y losses . Varroa y losses . In a foy losses . It seemimensis) . Here, wA. mellifera before. These viruses belong to Dicistroviridae, Iflaviridae, Partitiviridae, Parvoviridae, Potyviridae, Secoviridae, Tombusviridae, and taxonomically unclassified families . Nevertheless, it is noteworthy to study the impact of these plant-specific viruses associated with bees.We have also identified 15 viruses that were homologous to plant-specific viruses and belong to the families t plants ,86. Prevt plants ,72,87,88ntinents ,38,42,87ntinents . Cryptich pollen ,92,93,94 species . It has Drosophila spp. were discovered in this study: LJV, KiV, and ThV [A. mellifera. Remarkably, our study suggests that LJV and KiV replicate in both the varroa mite and adult bees, but not in pupae. This would implicate a function of the mite as a virus reservoir as seen elsewhere [Drosophila-infecting viruses and possibly denotes an unexpected inter-taxa transmission of viruses between Diptera and Hymenoptera.In addition, three viruses that previously were identified in  and ThV ,96,97. S and ThV . To the lsewhere , and thalsewhere . FurtherParvoviridae family [Diatraea saccharalis. Here, the sequences related to DsDNV were found in two sampling sites with high read counts . The presence of DsDNV-like virus with high read count and its virulence in its natural host may warrant further investigation on the impact of the virus on A. mellifera. Another densovirus was recently found in Bombus terrestris and Bombus cryptarum [DsDNV is a DNA virus that belongs to e family  and is aryptarum ,42. FinaNationwide health monitoring programs of honey bees often provide information of the prevalence of viruses and their importance in the context of bee health ,100,101.


The 2019 novel respiratory virus (SARS-CoV-2) causes COVID-19 with rapid global socioeconomic disruptions and disease burden to healthcare. The COVID-19 and previous emerging virus outbreaks highlight the urgent need for broad-spectrum antivirals. Here, we show that a defensin-like peptide P9R exhibited potent antiviral activity against pH-dependent viruses that require endosomal acidification for virus infection, including the enveloped pandemic A(H1N1)pdm09 virus, avian influenza A(H7N9) virus, coronaviruses , and the non-enveloped rhinovirus. P9R can significantly protect mice from lethal challenge by A(H1N1)pdm09 virus and shows low possibility to cause drug-resistant virus. Mechanistic studies indicate that the antiviral activity of P9R depends on the direct binding to viruses and the inhibition of virus-host endosomal acidification, which provides a proof of concept that virus-binding alkaline peptides can broadly inhibit pH-dependent viruses. These results suggest that the dual-functional virus- and host-targeting P9R can be a promising candidate for combating pH-dependent respiratory viruses. Here Zhao et al. report a promising broad-spectrum antiviral alkaline peptide—P9R—that is active against several respiratory, pH-dependent viruses, including Influenza and SARS-CoV-2. P9R interferes with virus internalization by binding to the virus and subsequent inhibition of endosomal acidification. Besides, the 2009 pandemic influenza A(H1N1)pdm09 virus had led to the first influenza pandemic in the 21st century, and the avian influenza virus A(H7N9) had caused a large zoonotic outbreak in mainland China7. Due to the lack of effective antivirals, especially for coronaviruses, these respiratory viruses are associated with significantly high morbidity and mortality. Furthermore, these emerging respiratory viruses have also caused severe economic and social disturbances.Novel respiratory viruses often cause severe respiratory tract infections and spread quickly due to the lack of pre-existing immunity. In the recent two decades, three highly pathogenic coronaviruses have crossed species barrier and caused human diseases, including the bat-related severe acute respiratory syndrome coronavirus (SARS-CoV) in 20038. Currently, there is no specific drug treatment for this new virus. An effective broad-spectrum antiviral will improve patients’ outcome and may reduce transmission in communities and hospitals even before the identification of the novel emerging virus and the specific antiviral drug.The COVID-19 outbreak has clearly illustrated the importance of broad-spectrum antivirals. While an outbreak of unusual pneumonia was reported from Wuhan of China in December 2019, SARS-CoV-2 was identified on 8 January 2020 by China CDC10. Most broad-spectrum antivirals are targeting host pathways which are utilized by virus replication cycle or by host antiviral immunity12. We have previously reported a broad-spectrum antiviral peptide P913, derived from mouse β-defensin-4, which showed antiviral activity against SARS-CoV, MERS-CoV, and influenza viruses through the inhibition of endosomal acidification. We have also reported different types of antiviral peptides which inhibit influenza virus by delivering defective interfering gene14, zika virus by targeting envelope protein15, MERS-CoV by blocking viral fusion16. Other groups have reported the broad-spectrum antiviral peptide BanLec which inhibits HIV, hepatitis C virus (HCV) and influenza virus17, the theta-defensin retrocylin 2, which inhibits influenza virus, Sindbis virus, and baculovirus18, the typical cationic and amphipathic tetradecapeptide mastoparan which inhibits enveloped viruses19, two scorpion venom peptide variants including mucroporin-M1 which inhibits measles, SARS-CoV and H5N1 virus20, and the Ev37 which inhibits dengue virus, HCV, zika virus, and herpes simplex virus21, and other host defense peptides which inhibit different enveloped and non-enveloped viruses with documented or unknown mechanism24. Since the first anti-HIV peptide enfuvirtide was approved by FDA for the treatment of HIV in 200325, other peptides with antiviral activity have been approved by FDA26.In past decades, researchers have devoted to the discovery of broad-spectrum antivirals targeting hosts or viruses13, which exhibits broad-spectrum antiviral activities against the enveloped SARS-CoV-2, MERS-CoV, SARS-CoV, A(H1N1)pdm09, A(H7N9) virus, and the non-enveloped rhinovirus. In vivo study indicates that P9R can efficiently protect mice from lethal A(H1N1)pdm09 virus challenge. Furthermore, the antiviral resistance study indicates that P9R does not induce the emergence of drug-resistant virus even after A(H1N1)pdm09 virus was passaged in the presence of P9R for 40 passages. Mechanistic studies show that positively charged P9R broadly inhibits viral replication by binding to different viruses and then inhibits virus–host endosomal acidification to prevent the endosomal release of pH-dependent viruses. We use P9R , PA1 (only binding to viruses) and P9RS  to identify and confirm the antiviral mechanism of alkaline peptides. The antiviral activity of alkaline peptide, requiring both of binding to viruses and inhibition of endosomal acidification, can be improved by increasing the positive charge of the peptide.Here, we report a potent antiviral peptide P9R, derived from mouse β-defensin-4 and our previous P927. Theoretically, an alkaline peptide with stronger net positive charge would reduce protons in the endosome, thereby inhibiting the endosomal acidification. Hence, to improve our previously antiviral peptide P913, we substituted the weakly positively charged amino acids (histidine and lysine) by arginine at positions 21 (H → R), 23 (K → R) and 28 (K → R)  , likely because endosomal acidification was not required in the viral life cycle of parainfluenza virus 3  Fig.  to incre01) Fig. . When P901) Fig. . Howevers 3 Fig. 28. In thCoV Fig. . For A could more significantly inhibit endosomal acidification in live cells than that of P9 Fig. , which wIn order to investigate whether P9R could have antiviral action before endosomal acidification, we showed that P9R did not disrupt viral particles under TEM analysis  which had the same positive charge as P9R (+5.6), but P9RS differed from P9R by 11 of 30 amino acids. P9RS efficiently inhibited host endosomal acidification to the similar degree as P9R in live cells Fig. . However−1 Fig. .To investigate why P9RS failed to inhibit viral replication despite potent inhibition of host endosomal acidification, we studied the binding between the peptide and virus. Using ELISA and RT-qPCR assay, P9R and PA1 could efficiently bind to SARS-CoV-2 and A(H1N1)pdm09 virus but P9RS did not bind to SARS-CoV-2 and A(H1N1)pdm09 virus Fig. . The obsIn the above experiments, we had demonstrated that P9R and P9RS can inhibit the acidification of the no-virus endosomes Fig. . HoweverTo further confirm that the broad-spectrum antiviral activity of P9R was due to the broad spectrum of binding of P9R to different viruses and viral proteins, we demonstrated that P9R but not P9RS could also bind to MERS-CoV, A(H7N9) virus, rhinovirus, SARS-CoV and viral proteins Fig. . This reNext, we determined the structure of P9R using NMR spectroscopy Fig. . The resP < 0.05 for 12.5μg/dose), which was consistent with the significantly better antiviral activity of P9R than P9 in vitro.We demonstrated that the efficient antiviral activity of P9R in vitro is reliant on binding to viruses and the positive charge of P9R to inhibit virus–host endosomal acidification. To further investigate the antiviral activity of P9R in vivo, we treated A(H1N1)pdm09-infected mice at 6 h post infection and gave additional two doses to mice in the following one day. In this model, 70% of P9R-treated mice and 80% of zanamivir-treated mice survived, which was significantly better than PBS-treated group and PA1-treated group Fig. . The pro14, especially with the new polymerase inhibitor baloxavir29. To determine whether P9R treatment induces viral resistance, A(H1N1)pdm09 virus was serially passaged 40 times in the presence of P9R in MDCK cells pdm09 virus (P0) was 35 nM  of P9R for 40 passages, P9R (5.0 μg ml−1) could efficiently inhibit P30 and P40 virus replication lls Fig. . The IC5 nM Fig. . After 1 nM Fig. . These iion Fig. . No obviIn this study, we identified a broad-spectrum antiviral peptide P9R with potent antiviral activity against enveloped coronaviruses , influenza virus, and non-enveloped rhinovirus. First, we demonstrated that the antiviral activity of P9R could be significantly enhanced by increasing the net positive charge for more efficient inhibition of endosomal acidification. Second, antiviral mechanistic studies further demonstrated that positively charged P9R could bind to different respiratory viruses to inhibit virus–host endosomal acidification, while PA1 (only binding to viruses) or P9RS  did not show antiviral activity. Third, the in vivo antiviral activity of P9R was demonstrated by protecting mice from lethal influenza virus challenge. Fourth, there was no reduced susceptibility of serially passaged viruses (40 passages) against P9R.9. However, there is an urgent need for broad-spectrum antivirals for combating emerging and re-emerging new virus outbreaks, such as the SARS-CoV-2, before the new virus is identified or specific antiviral drug is available.The “one bug-one drug” approach to antiviral drug is successful for HIV, hepatitis C virus and influenza virus9. In this study, with the increased positive charge in P9R, it could more efficiently inhibit pH-dependent viruses than that of P9. The more positive charge in P9R allowed the peptide to more efficiently reduce protons inside endosomes, and thereby inhibiting the endosomal acidification. In previous studies, the clinically approved anti-malarial drug chloroquine and hydroxychloroquine with activity of inhibiting endosomal acidification had been demonstrated to inhibit enterovirus-A7130, zika virus31 and SARS-CoV-233. The anti-parasitic drug niclosamide also inhibited influenza virus, rhinovirus, and dengue virus by interfering endosomal acidification35. However, researchers demonstrated the lack of protection of chloroquine in vivo for treating influenza virus and Ebola virus37. Differing from these drugs by interfering host endosomal acidification without targeting viruses, P9R inhibits viral replication by binding to viruses and then inhibiting host acidification of the endosomes containing the virus bounded by P9R, which allows P9R to selectively and efficiently inhibit the replication cycle of endosomal viruses. The protection of P9R on A(H1N1)-infected mice further confirmed the antiviral efficiency in vivo.Endosomal acidification is a key step in the life cycle of many pH-dependent viruses, which is one of the broad-spectrum antiviral targets39, and the positive charge of P9R may play a role for binding to viruses with negatively charged surface41. The five cysteines in P9R may also affect the structure-based binding because previous studies indicated that cysteine substitutions could affect defensin-peptide structure and biological activity43.The antiviral activity of P9R required both the binding to viruses and the inhibition of endosomal acidification. PA1 with less positive charge could not inhibit SARS-CoV-2 and H1N1 virus even though it had the similar binding ability and binding sites to viruses as P9R Fig. . When weIn addition, comparing with zanamivir which caused significant drug-resistant virus after 10-virus passages in the presence of zanamivir, P9R showed very low possibility to cause drug-resistant virus even A(H1N1)pdm09 virus was passaged in the presence of P9R for 40 passages. The low risk of resistance induction by P9R may be partially due to the dual targeting abilities which broadly bind to viruses and target host endosomes. More co-binding structure analysis will be needed to illustrate the broadly binding mechanism and help to understand the low drug resistance mechanism in future study.In summary, most highly pathogenic emerging viruses are endosomal pH-dependent viruses. The emerging and re-emerging virus outbreaks remind us of the urgent need of broad-spectrum antivirals. Continual studies on similar kind of antivirals, which can broadly bind to different viruses and inhibit pH-dependent viruses by preventing virus–host endosomal acidification with low possibility of inducing drug resistance, will give us more armamentarium to combat novel emerging virus outbreaks in future.−1 penicillin and 100 μg ml−1 streptomycin. The virus strains used in this study included 2019 new coronavirus (SARS-CoV-2)44, SARS-CoV, MERS-CoV (hCoV-EMC/2012), A/Hong Kong/415742/2009, A/Hong Kong/415742Md/2009 (H1N1) (a highly virulent mouse-adapted strain), A/Anhui/1/2013 (H7N9)13, rhinovirus45 and human parainfluenza 3 (ATCC-C243). For in vitro experiments, viruses were cultured in MDCK, VERO-E6, RD and LLC-MK2 cells. For animal experiments, H1N1 virus was cultured in eggs46.Madin Darby canine kidney , Vero-E6 (CRL-1586), RD (CCL136), LLC-MK2 (CCL-7), A549 (CCL-185) cells obtained from ATCC  were cultured in Dulbecco minimal essential medium (DMEM) or MEM supplemented with 10% fetal bovine serum (FBS), 100 IU mlP9, P9R, PA1 and P9RS were designed as shown in Fig. 14. Briefly, peptides were dissolved in 30 mM phosphate buffer (PB) containing 24.6 mM Na2HPO4 and 5.6 mM KH2PO4 at a pH of 7.4. Peptides or bovine serum albumin  were premixed with 50 PFU of coronaviruses , influenza viruses (H1N1 virus and H7N9 virus), rhinovirus, or parainfluenza 3 in PB at room temperature. After 45–60 min of incubation, peptide-virus mixture was transferred to VERO-E6, MDCK, RD, or LLC-MK2 cells, correspondingly. At 1 h post infection, infectious media were removed and 1% low melting agar was added to cells. Cells were fixed using 4% formalin at 2–4 day post infection. Crystal blue (0.1%) was added for staining, and the number of plaques was counted.Antiviral activity of peptides was measured using a plaque reduction assay−1) in PB for 1 h and then infected VERO-E6, MDCK, and RD cells, correspondingly. After 1 h infection, infectious media were removed and fresh media with supplemented P9R or BSA (50–100 μg ml−1) were added to infected cells for virus and cell culture. At 24–30 h post infection, the supernatants of cells were collected for plaque assay.Coronaviruses , influenza viruses (H1N1 and H7N9 virus) and rhinovirus (0.005 MOI) were premixed with P9R or BSA (50–100 μg ml50) using a tetrazolium-based colorimetric MTT assay13. Briefly, cells were seeded in 96-well cell culture plate at an initial density of 2 × 104 cells per well in MEM or DMEM supplemented with 10% FBS and incubated for overnight. Cell culture media were removed and then DMEM supplemented with various concentrations of peptides and 1% FBS were added to each well. After 24 h incubation at 37 °C, MTT solution  was added to each well for incubation at 37 °C for 4 h. Then, 100 μl of 10% SDS in 0.01 M HCl was added to each well. After further incubation at room temperature with shaking overnight, the plates were read at OD570 using VictorTM X3 Multilabel Reader . Cell culture wells without peptides were used as the experimental control and medium only served as a blank control.Cytotoxicity of peptides was determined by the detection of 50% cytotoxic concentration  or Arbidol (100 μg ml−1) were mixed with or without same volume of H1N1 virus (HA titer >128) for 1 h, and then 60 μL of 2% chicken red blood cells was added for 15 min. PBS and Triton-100 (0.1%) were included as the negative and positive control of hemolysis. The precipitated erythrocytes were incubated with sodium citrate solution (pH of 4.9) for 25 min. The hemoglobin release in supernatants was detected at 450 nm.Serially diluted peptides in PBS were incubated with chicken red blood cells for 1 h at 37 °C. PBS was used as a 0% lysis control and 0.1% Triton X-100 as 100% lysis control. Plates were centrifuged at 350 × 2O were coated onto ELISA plates and incubated at 4 °C overnight. Then, 2% BSA was used to block plates at 4 °C overnight. For virus binding to peptides, viruses were diluted in PB and then were added to ELISA plate for binding to the coated peptides at room temperature for 1 h. After washing the unbinding viruses, the binding viruses were lysed by RLT buffer of RNeasy Mini Kit  for viral RNA extraction. Viral RNA copies of binding viruses were measured by RT-qPCR.Peptides (1.0 μg per well) dissolved in H14, Peptides (1.0 μg per well) dissolved in H2O were coated onto ELISA plates and incubated at 4 °C overnight. Then, 2% BSA was used to block plates at 4 °C overnight. For HA and S binding, 150 ng HA1 or S in solution I buffer  was incubated with peptides at 37 °C for 1 h. The binding abilities of peptides to HA1 or S proteins were determined by incubation with rabbit anti-His-HRP  at room temperature for 30 min. The reaction was developed by adding 50 μl of TMB single solution  for 15 min at 37 °C and stopped with 50 μl of 1 M H2SO4. Readings were obtained in an ELISA plate reader  at 450 nm.For ELISA assay# 52906, USA) according to the manufacturer’s instructions. Real-time RT-qPCR was performed as we described previously14. Extracted RNA was reverse transcribed to cDNA using PrimeScript II 1st Strand cDNA synthesis Kit  with GeneAmp® PCR system 9700 . The cDNA was then amplified using specific primers .Viral RNA was extracted by Viral RNA Mini Kit  according to the manufacturer’s instructions as previously described but with slight modification14. First, MDCK cells were treated with BSA (25.0 μg ml−1), P9 (25.0 μg ml−1), P9R (25.0 μg ml−1), PA1 (25.0 μg ml−1), or P9RS (25.0 μg ml−1) at 4 °C for 15 min. Second, MDCK cells were added with 100 μg ml−1 of pH-sensitive dye and DAPI and then incubated at 4 °C for 15 min. Before taking images, cells were further incubated at 37 °C for 15 min and then cells were washed twice with PBS. Finally, PBS was added to cells and images were taken immediately with confocal microscope .Endosomal acidification was detected with a pH-sensitive dye  according to the manufacture introduction. DIO-labeled virus was treated by TAMRA-labeled P9R and TAMRA-labeled P9RS for 1 h at room temperature. Pre-cool MDCK cells were infected by the peptide-treated virus on ice for 15 min and then moved to 37 °C for incubation for 15 min. Cells were washed twice by PBS and then fixed by 4% formalin for 1 h. Nuclei were stained by DAPI for taking images by confocal microscope .H1N1 virus was labeled by green Dio dye pdm09 virus at 1 MOI pretreated with BSA (25.0 μg ml−1), bafilomycin A1 (50.0 nM) or P9R (25.0 μg ml−1). After 3.5 h post infection, cells were fixed with 4% formalin for 1 h and then permeabilized with 0.2% Triton X-100 in PBS for 5 min. Cells were washed by PBS and then blocked by 5% BSA at room temperature for 1 h. Cells were incubated with mouse IgG anti-NP  at room temperature for 1 h and then washed by PBS for next incubation with goat anti-mouse IgG Alexa-488  at room temperature for 1 h. Finally, cells were washed by PBS and stained with DAPI. Images were taken by confocal microscope .According to our previous experiment−1 (0.29 mM) of P9R in 0.5 ml solvent was used for the NMR study. Data were collected in H2O/D2O (19:1 v/v), as well as 99.996% D2O, with the internal reference trimethylsilylpropanoic acid. All NMR spectra were acquired on either a Bruker AVANCE III 600 MHz spectrometer  or a Bruker AVANCE III 700 MHz spectrometer at 25 ˚C. 2D 1H-1H correlation spectroscopy (COSY), total correlated spectroscopy (TOCSY) and nuclear Overhauser effect spectroscopy (NOESY) spectra were recorded for resonance assignments. Supplementary Fig. 48 and served as inputs for the Aria program. Dihedral angle is predicted from the chemical shifts using the program DANGLE49. The NMR solution structure of P9R was calculated iteratively using Aria 2.3 program50. One hundred random conformers were annealed using distance restraints in each of the eight iteratively cycles of the combined automated NOE assignments and structure calculation algorithm. The final upper limit distance constraints output from the last iteration cycle were subjected to a thorough manual cross-checking and final water solvent structural refinement cycle. The 20 lowest energy conformers were retained from these refined 100 structures for statistical analysis. The convergence of the calculated structures was evaluated using root-mean-square deviations (RMSDs) analyses. The distributions of the backbone dihedral angles  of the final converged structures were evaluated by representation of the Ramachandran dihedral pattern using PROCHECK-NMR51. Visualization of three-dimensional structures and electrostatic surface potential of P9R were achieved using UCSF Chimera 1.13.152. The final water solvent refined structures were deposited at Protein Data Bank (https://www.rcsb.org/) under the code of 6M56 and the resonance assignments of P9R were deposited at Biological Magnetic Resonance Bank (http://www.bmrb.wisc.edu) under the accession number of 36321.Freshly prepared 1 mg ml46. The mouse adapted H1N1 virus was used for lethal challenge of mice. To evaluate the therapeutic effect, mice were challenged with 3 LD50 of the virus and then intranasally inoculated with PBS, P9, P9R, PA1 or zanamivir at six hours after the viral inoculation. Two more doses were given to H1N1-challenged mice at the following one day. Survival and general conditions were monitored for 16 days or until death. Viral loads in mouse lungs were measured at day 4 post infection.BALB/c female mice, 10–12 weeks old, were kept in biosafety level 2 laboratory (housing temperature between 22 °C and 25 °C with dark/light cycle) and given access to standard pellet feed and water ad libitum. All experimental protocols followed the standard operating procedures of the approved biosafety level 2 animal facilities. Animal ethical regulations were approved by the Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kongt test. Results were considered significant at P < 0.05.Survival of mice and the statistical significance were analyzed by Gehan–Breslow–Wilcoxon test. The statistical significance of the other results was calculated by the two-tailed Student Further information on research design is available in the Supplementary informationPeer Review FileReporting Summary


Respiratory viruses are a common cause of respiratory tract infection (RTI), particularly in neonates and children. Rapid and accurate diagnosis of viral infections could improve clinical outcomes and reduce the use of antibiotics and treatment sessions. Advances in diagnostic technology contribute to the accurate detection of viruses. We performed a multiplex real-time polymerase chain reaction (PCR) to investigate the viral etiology in pediatric patients and compared the detection rates with those determined using traditional antigen tests and virus cultures. Fifteen respiratory viruses were included in our investigation: respiratory syncytial virus A/B (RSV), influenza virus A (FluA) and influenza virus B (FluB), human metapneumovirus (MPV), enterovirus (EV), human parainfluenza virus (PIV) types 1–4, human rhinovirus (RV), human coronavirus OC43, NL63, and 229E, human adenovirus (ADV), and human bocavirus (Boca). In total, 474 specimens were collected and tested. Respiratory viruses were detected more frequently by PCR  than they were by traditional tests . The leading pathogens were RSV , RV , PIV3 , FluA , and ADV . For children younger than 5 years, RSV and RV were most prevalent; for children older than 5 years, FluA and ADV were the most frequently detected. Of the specimens, 25.8% (92/357) were coinfected with two or more viruses. RV, Boca, PIV2, FluB, and PIV4 had higher rates of coinfection; MPV and PIV1 had the lowest rates of coinfection (9.1% and 5.3%). To conclude, the detection power of PCR was better than that of traditional antigen tests and virus cultures when considering the detection of respiratory viruses. RSV and RV were the leading viral pathogens identified in the respiratory specimens. One-quarter of the positive specimens were coinfected with two or more viruses. In the future, further application of PCR may contribute to the rapid and accurate diagnosis of respiratory viruses and could improve patient outcomes. Respiratory viruses are ubiquitous and cause a large variety of clinical symptoms. Respiratory tract infection (RTI) is undoubtedly common, and the recognition of a causative pathogen contributes to the appropriate management . In addiA timely and accurate diagnosis of viral infection can be challenging. Rapid antigen tests are used to detect influenza virus infection worldwide, but there are some concerns regarding the sensitivity of currently available viral antigen tests ,15. Tech® CCL-171TM), human epithelial cell line type 2 , human adenocarcinomic alveolar basal epithelial cell line , Madin–Darby canine kidney cell line , and human rhabdomyosarcoma cell line . Additionally, viruses commonly isolated were adenovirus, MPV, RSV, influenza virus type A (FluA) and B (FluB), parainfluenza virus types 1, 2, and 3; echovirus 4, 6, 9, 11, and 30; coxsackievirus B1–B6; enterovirus 70; enterovirus 71; pan-enterovirus; herpes simplex virus types 1 and 2; cytomegalovirus; coxsackievirus A9, A16, and A24. The residual specimens were stored, and then present multiplex PCR was performed for RTI viruses.This study was approved by the Institutional Review Board of the MacKay Memorial Hospital, Taipei, Taiwan . For children with respiratory symptoms and with a clinical suspicion of virus infection, a test for RSV antigen test, human parainfluenza virus (PIV) type 3 antigen test, viral PCR for enterovirus, or viral cultures was prescribed by the judgment of pediatricians. A nasopharyngeal swab or aspiration was performed by pediatricians using a small swab that was inserted into the nostril. The cotton swab was then inserted and mixed in a 2.5 mL viral transport medium. After testing original tests, the residual specimens were stored at −20 °C in the clinical viral laboratory at the Department of Laboratory Medicine, Mackay Memorial Hospital. Within a week, the samples were transported to a deep freezer (−70 °C) at the Department of Medical Research, Mackay Memorial Hospital, for present multiplex PCR tests. Commercialized antigen diagnostic kits were used for antigen tests  and tube cultures were used for virus cultures. The cell lines for virus cultures were medical research council cell line strain 5  following the manufacturer’s instructions. Extracted nucleic acids were eluted in 100 μL elution buffer and stored at −70 °C. Reverse transcription (RT) was carried out using High-Capacity Complementary DNA (cDNA) Reverse Transcription Kit (Applied Biosystems Part Number: 4375575 Rev.C). The total volume of RT mix was 40 μL per reaction, containing 4 μL RT buffer (10×), 1.6 μL dNTP mixture (25 mM of each dNTP), 4 μL random primers (10×), 2 μL RNase inhibitor (20 U/μL), 2 μL MultiScribe Reverse Transcriptase (50 U/μL), and 26.4 μL template, whereby the RT reagent mix was prepared on ice. The thermal profile of the RT program consisted of 10 min incubation at 25 °C, 120 min RT at 37 °C, 5 min RT inactivation at 85 °C, and cooling down to 4 °C and was performed in a 96-well GeneAmp PCR System 9700. The resulting cDNA was stored at −20 °C.The following multiplex PCR assays were performed for each sample to detect RNA/DNA of 15 respiratory viruses, including RSV A or B, FluA, FluB, human enterovirus (EV), MPV, human parainfluenza virus types 1–4, human rhinovirus (RV), coronavirus OC43/NL63/229E, human adenovirus (ADV), and human bocavirus (Boca). In the present study, previously published primers and PCR assays were used for multiplex RT-PCR and the details of primers are summarized in t-test and chi-square test were used to analyze and compare the categorical demographic characteristics including clinical manifestations and laboratory tests. Kappa statistic was used to evaluate the consistency between PCR and original tests  and Cohen’s kappa coefficient (κ) was regarded as poor to fair consistency if κ ≤ 0.4; moderate consistency if 0.41 ≤ κ ≤ 0.60; and good consistency if 0.61 < κ. A two-sided p < 0.05 was considered statistically significant. Statistical analyses were performed using the SPSS software version 23.0 .Student’s k: 0.72~0.961, p < 0.01, In total, 474 residual specimens for detecting respiratory viruses were obtained, including 156 specimens for RSV antigen tests, 58 for parainfluenza virus antigen tests, and 260 for viral cultures. The seasonality of virus detection is shown in In this study, we found that PCR had higher detection rates compared with traditional antigen tests and viral cultures (75.3% vs. 48.3%). RSV, RV, and PIV3 were the leading pathogens detected in pediatric RTI patients. However, FluA, ADV, and EV were more prevalent in children older than 5 years. Knowledge of epidemiology contributes to the awareness of pathogen, accurate diagnosis, and prompt management. We also found that approximately one-quarter of specimens were coinfected with two or more viruses. However, no obvious differences in clinical manifestations and laboratory tests were found in individual virus infection or between single infection and coinfection; the clinical significance of coinfection was not fully elucidated.A rapid and accurate diagnosis of respiratory viruses is increasingly important in clinical settings. The availability of rapid diagnostic assays is essential for optimizing the efforts of infection control teams to reduce the transmission of virulent or resistant pathogens in hospitals . NucleicViral infections are ubiquitous and may present with fever and respiratory symptoms. It is sometimes difficult to differentiate between bacterial infections and viral infections, and thus the use of unnecessary antibiotics is common. Antimicrobial resistance (AMR) has been increasing worldwide, resulting in poor treatment responses and deplorable clinical outcomes . The proThe incidence of etiologic pathogens differs between adults and children. It has been reported by Jain et al. that, among the hospitalized adults with CAP, pathogens were detected in 38% of patients, and the leading pathogens were RV (9%) and influenza viruses 6%) [% [6]. ByOne-quarter of the positive specimens were coinfected with other respiratory viruses in our study. A similar prevalence was found in previous studies ,41,42. TMycoplasma pneumoniae and Streptococcus pneumoniae also play an important role in respiratory infections and commonly cause coinfections with other pathogens [The strength of our study lies in the comprehensive detection of respiratory viruses and further comparison of the clinical manifestations and laboratory tests in single and coinfection. Our study is subject to some limitations that warrant discussion. Firstly, although our findings were consistent with those of previous studies, respiratory specimens were not collected in all patients with respiratory symptoms. The prevalence of EV was underestimated because the clinical diagnosis of EV infection relies mainly on the presence of oral vesicles. Further virus culture might not be performed when vesicles over oropharynx were found. Secondly, we did not include bacteria in our detection spectra. Some bacteria such as athogens . Furtherathogens .The use of PCR resulted in greater detection of respiratory viruses than the use of traditional rapid antigen tests or viral cultures. More than half of the respiratory specimens that showed negative detection in the original tests were positive for the PCR-based detection method. Further application of PCR has great potential for rapid and accurate diagnosis and will be beneficial for primary pediatricians. Furthermore, RSV and RV were the leading pathogens identified in our pediatric respiratory specimens; in children older than 5 years, FluA, ADV, and EV were more prevalent. Approximately one-quarter of the positive respiratory specimens were coinfected with two or more viruses, but no obvious differences in clinical manifestations and laboratory tests were observed between single infection and coinfection. Further studies are warranted to investigate the accuracy, feasibility, accessibility, and cost of PCR in detecting respiratory viruses, and to clarify the clinical significance of coinfection.


Over the last 30 years, diseases caused by emerging swine viruses (ESV) have acquired great relevance, more than in other species. Diseases caused by porcine reproductive and respiratory syndrome virus (PRRSv), high pathogenicity porcine epidemic diarrhea virus (PEDv), porcine circovirus type 2 (PCV-2), and influenza virus H1N1pdm09 had great economic impact. Others, however, such as porcine enteroviruses, porcine toroviruses (PToV), porcine sapelovirus (PSV), porcine bocavirus (PBoV), porcine kobuvirus (PKBV), and porcine Torque teno sus virus (TTSuV) are mostly subclinical in swine herds. Furthermore, novel emerging viruses, such as SENECA virus, atypical porcine pestivirus (APPV), PCV-3, SADS-CoV, influenza D, and others with regional or worldwide distribution constitute a new challenge for researchers and practicing veterinarians.Emerging viruses should be considered to occur when there are changes in the relationship between the agent, the host and the enviroment. The response to how and why the ESV have emerged can be explained through several factors.First, interspecies transmission means the presence of a potentially pathogenic agent into a new host, such as between aquatic migratory birds and human beings for influenza A. Bats are the source of Nipah virus, and swine acute diarrhea syndrome (SADS coronavirus). Both have limited distribution to Asia or TGE and PED. Currently PCV-3 have been found with high homology with bats PCV-1.Secondly, changes in the virulence  of the agents in the same host, particularly the RNA and single strand DNA viruses that have a high mutation rate (10–4/10–5 nucleotides per replication cycle), that facilitate its adaptation to the innate immune response. The absence of enzymes (transcriptase) in infected cells that correct errors in reading RNA synthesis and segmented RNA chains favor reassortant. A population of RNA viruses does not consist of a single genotype, but a “set or cloud” of related viruses that interact with each other called “quasispecies”. Relevant examples are HP PRRSv, influenza A H1N1pdm09 and PEDv.Next, the viruses have been present for a long period of time as subclinical infections and have been discovered with the development of metagenomic techniques , or exogenous factors as most emerging viruses do not grow in traditional culture media. Viruses such PCV-3, SADS-CoV, and LINDA virus have been characterized by the aforementioned techniques.Now we come to change in the production system. The presence of farms of good health, large size and homogeneous genetics favors the fitness, and the ability of a particular population of viruses to multiply and spread in a specific environment.Our fifth point is the recognition and sensitization of practicing veterinaries of “abnormal” cases or syndromes through the routine postmortem monitoring of pigs that die “unexceptionally” on the farm, as well as the syndromes surveillance at the slaughterhouse.Finally, Specialized diagnostic laboratories that offer new and accessible diagnostic tools for the diagnoses of known and unknown emerging viruses.in situ Hybridization (ISH); porcine hemagglutinating encephalomyelitis coronavirus (PHE-CoV); porcine circovirus type 3 (PCV-3); classical swine fever virus (CSF); porcine torovirus (PToV); porcine respiratory reproductive syndrome virus (PRRSV); human influenza A; Nipah virus in humans and pigs swine; atypical porcine pestivirus (APPV); porcine epidemic diarrhea coronavirus (PEDV); and porcine delta coronavirus (PDCoV) pathogenesis. These subjects provide a discussion on the broad field of emerging swine viruses infections and its control.This Research Topics comprises ten review articles are related by: Next Generation Sequencing (NGS) plus Three original research articles about phylogeny and genome composition of novel Seneca virus (SVA) isolated in China; estimation of time to porcine epidemic diarrhea coronavirus (PED-CoV) removal in Ontario herds and a longitudinal serological and RT-PCR fecal studies of hepatitis E virus (HEV) were published.Resende et al. provide comprehensive information about the results of the combination of NGS-ISH for the diagnosis of known and unknown emerging pathogens in tissues by NGS, and its relationship with specific lesions where it is visualized in active infection through detection of mRNA by ISH. Results on the application of PCV-2, PPV-2, Seneca virus, and Mycoplasma hyorhinis are comments.Mora-Díaz et al. review the disease produced by porcine hemagglutinating encephalomyelitis coronavirus (PHE-CoV), a neurotropic virus affecting piglets <4 weeks old. Subjects such as: characteristic of the virus, history of the emergence of PHE-CoV, global distribution, clinical signs, pathogenesis, lesions, and diagnosis are discussed. As the infection is endemic in most swine herds, and no current vaccines are available, early exposure to old or young sows to induced maternal immunity is the only way to prevent the disease.Klaumann et al. discuss the current knowledge on a new circovirus named porcine circovirus type 3 (PCV-3). Originally this was identified by metagenomic analyses from an outbreak of PDNS in sows associated with reproductive failure, myocarditis, and multisystemic inflammation. Thereafter it was found associated with respiratory, digestive and nervous signs in healthy pigs and wild boars. Retrospective studies detected PCV-3 as early as the 1990s. Coinfection with several virus and bacteria were reported. The authors emphasize the need of studies related to pathogenesis, the role of coinfections and their association or not with certain clinically pathological entities.Zhou reviews classical swine fever (CSF) in China. The author discusses the epidemiology, and the geographical distribution of genotypes where 2.1, 2.1b, and 2.1c are currently dominant in China. The first one persists in an immune population by natural infection due to the high mutation rate of the enveloped glycoprotein E2. For eradication of CSF it is necessary to distinguish between the naturally infected and vaccinated animals by live attenuated marked vaccine. An experimental E2 subunit vaccine was developed in China. Besides preventive vaccination, we need culling strategies, skilled veterinarians, up-to-date diagnostic and monitoring technology, and biosecurity.Hu et al. review the progress in the knowledge on porcine torovirus (PToV) a single-stranded RNA enteric virus found in piglets with diarrhea in North America, Africa, Asia, and Europe. The authors describe the virus morphology, the genomic structure and genotypes division, although chimeric strains with genes from porcine and bovine ToV has been identified as well as recombination with enterovirus. For epidemiological studies an indirect ELISA based in recombinant N protein expressed in baculovirus is available. Other methods included RT-PCR; qRT-PCR; and nested PCR. Prevalence of PToV is quite variable according with the country.Montaner-Tarbes et al. analyze numerous gaps in PRSSv knowledge. Related with the biology, the scarce whole genome sequencing from different geographical origins hinder understanding the virus evolution/mutation. The function and the complex interaction of viral non-structural proteins with the target cells are reviewed. The mechanisms of the virus to avoid the innate and acquired immune response through recognition and antibody neutralization are reviewed. The new known mechanisms of dissemination mediated by cell to cell connected nanotubules and extracellular vesicles are thoroughly discussed. Later on, the development of exosomes, as a novel vaccine is analyzed.Rajao et al. review the role of pigs in the interspecies transmission and how their susceptibility to different viruses can affect the overall epidemiology of swine influenza. The factors that have been implicated in the interspecies transmission of influenza such as receptor-binding specificity/affinity, balance between HA and NA content, host temperature and host-specific immune factors are analyzed. Surveillance of IAV in swine has shown that human viruses are transmitted to pigs more frequently than from pigs to humans. The result is the establishment of several human-origin virus lineages, antigenic diversity and failure of current swine vaccines.McLean and Graham provide an update about Nipah virus (NiV), an RNA paramyxovirus that causes a severe neurological disease in humans. In suckling pigs, NiV infection causes high mortality and in older pigs, respiratory and neurological signs. The natural host of NiV is a fruit bat of the genus Pteropus, and pigs act as an “amplifying host”. The disease has been found in Asia in people with close contact to pigs. Recombinant NiV mutant, attenuated and subunit vaccine using several viral vectors have been studied. Currently, neither a human nor a pig vaccine has been licensed.Gatto et al. review the information around a new RNA Pestivirus named atypical porcine pestivirus (APPV), detected in pigs with congenital tremors (CT) type AII, and splayed legs in offspring from sows by NGS technology. Viral genomes were detected in semen, preputial swabs and fluids highlighting the importance of AI in APPV epidemiology. Horizontal transmission can be made by surviving CT in healthy new-born boars, piglets and adult pigs. The virus exhibits high genetic diversity. Recently, APPV was detected in wild boars. Until the development of a vaccine, the authors recommend feedback on reproductive management in sows with CT cases.Koonpaew et al. review the emergence of highly pathogenic porcine epidemic diarrhea (PED) and porcine delta coronavirus (PDCoV) as agents of watery diarrhea in suckling piglets. The authors describe aspects related to the biology pathogenesis and the host innate immune response of gastrointestinal tracts against those enteric coronaviruses. The agents evade recognition by host pattern recognition receptors (PRRs), present in resident antigen present cells (APCs) and located in gut associated lymphoid tissue (GALT), through the inhibition or blocking of interferon (IFN) induction and the signaling cascade, respectively. This knowledge will profit the development of immune modulators as well as effective vaccines.Sun et al. analyze the phylogeny and genome compositions of 17 novel Seneca virus (SVA) isolated in China in 2017 and compare them with the genomic sequences deposited in the GenBank. SVA is a single stranded positive-sense RNA virus associated with porcine idiopathic vesicular disease (PIVD), and sudden neonatal dead reported in six countries in Asia and America. The isolated strain clustered into three distinct groups: A, B, and C, not related with the previously SVA identified in China and different from SVA identified in other countries. More effort should be directed to SVA monitoring, rapid and specific diagnosis and vaccination strategies.Perri et al. studied the estimation of time to eliminate porcine epidemic diarrhea coronavirus (PED-CoV) in Ontario herds based in large-scale disease control program database (DCP). The analysis takes into consideration the time between the initial infection, and the confirmation of PED-CoV freedom at the minimum level of 10%. The median time to elimination varied from 23 weeks in nursery herds, to 43 weeks in farrow-to-feeder herds. Farrow-to-wean herds had the highest hazard of PED-CoV elimination. Type of herds, season and year of original diagnosis were associated with the time of negativity and reflect the complexity of the infection control practices.Krog et al. work to determine the dynamics of infection of hepatitis E virus (HEV) by carrying out a longitudinal serological and RT-PCR fecal studies. Sows and their progeny from 2 weeks to slaughter were sampled. Antibodies were only detected in offspring born from sows with high levels of maternal antibodies (MAbs) and a few of them became shedders. All pigs seroconverted at 13–17-week-olds. By PCR 65.5% of pigs were positive at least one time during the weeks 13, 15, and 17. In 3 out of 10 slaughter pigs, HEV was detected in feces and organs. As MAbs reduced the shedder of HEV, sow's vaccination might be an option.As a summary, this Research Topic provides a comprehensive review about the results of the combination of NGS-ISH for the diagnosis of known and unknown emerging pathogens. It's also an important discussion of potentially emerging viruses such as PCV-3, torovirus, atypical pestivirus, reemerging viruses such as PHECoV and transboundary viruses such as classical swine fever. The mechanisms used by PRRS to circumvent the host's innate immune and vaccine immune response are updated along with the development of vaccines to exosomes. The immune response against enteric coronaviruses such as PED and PDCoV and innovative vaccines for both viruses are analyzed. The role of pigs as an amplifier of the Nipah virus is reviewed, as well as the importance of vaccination to pigs for the prevention of this infection in man. The repeated transmission of human seasonal viruses to pigs has resulted in the establishment of several human-origin virus lineages globally and the failure of the current pig vaccines. A research study indicated that isolated strain Seneca virus from vesicular fluid of sows, clustered into three distinct groups, A, B, and C not related with the previously SVA identified in China, and highlight that different genotypes of SVA co-exist and spread. A study of the estimation of time to porcine epidemic diarrhea coronavirus (PED-CoV) elimination in Ontario Type of herds was carried out. The year and season of original diagnoses are associated with the time of negativity and reflect the complexity of the infection control practices. Finally, a study shows that hepatitis E virus infection is widespread in the herd, and pigs spread virus during the final stages of life, and there's a strong chance to find infected pigs at slaughter. As maternal antibodies reduce, the shedder of virus vaccinating sows might be an option.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.