Saccharomyces cerevisiae (ScV-L-A), Penicillium chrysogenum virus (PcV), Penicillium stoloniferum virus F (PsV-F), and Rosellinia necatrix quadrivirus 1 (RnQV1). These capsids show structural variations of the same framework, with 60 asymmetric or symmetric homodimers for ScV-L-A and PsV-F, respectively, monomers with a duplicated similar domain for PcV, and heterodimers of two different proteins for RnQV1. Mycovirus capsid proteins (CP) share a conserved α-helical domain, although the latter may carry different peptides inserted at preferential hotspots. Insertions in the CP outer surface are likely associated with enzymatic activities. Within the capsid, fungal dsRNA viruses show a low degree of genome compaction compared to reoviruses, and contain one to two copies of the RNA-polymerase complex per virion.Most fungal, double-stranded (ds) RNA viruses lack an extracellular life cycle stage and are transmitted by cytoplasmic interchange. dsRNA mycovirus capsids are based on a 120-subunit T = 1 capsid, with a dimer as the asymmetric unit. These capsids, which remain structurally undisturbed throughout the viral cycle, nevertheless, are dynamic particles involved in the organization of the viral genome and the viral polymerase necessary for RNA synthesis. The atomic structure of the T = 1 capsids of four mycoviruses was resolved: the L-A virus of  Double-stranded RNA (dsRNA) viruses infect a diversity of host organisms, from bacteria to unicellular and simple eukaryotes (fungi and protozoa), through to plants and animals. No archaea-infecting dsRNA viruses have yet been reported . AlthougThe T = 1 capsids of dsRNA viruses are known to be critical for genome replication (minus-strand synthesis) and transcription (plus-strand synthesis), with the viral RNA-dependent RNA polymerase(s) (RdRp) frequently packaged as an integral component of the capsid. T = 1 capsids also function as molecular sieves, allowing the exit of single-stranded (ss) RNA transcripts for translation in the host cytoplasm, and the entrance of nucleotides for intra-capsid RNA synthesis. The pores are presumably small enough to exclude potentially degradative enzymes.T = 1 capsids remain structurally undisturbed throughout the viral cycle , isolatiSaccharomyces cerevisiae and the smut fungus Ustilago maydis respectively, were the first unambiguously described viruses with a T = 1 capsid formed by 12 decamers rather than 12 pentamers [The totiviruses ScV-L-A and UmV-P4, which infect the yeast entamers . The conentamers ,15 as thentamers ,17), as entamers ), or as entamers ).Reo- [Picobirnaviridae [Cystoviridae—bacteriophages that infect the prokaryote Pseudomonas syringae [The 120-subunit T = 1 capsids have been described for members of the Reo- ,22,23 anaviridae , which msyringae ,26. Membsyringae ,28,29, Psyringae ,31, Megasyringae , Chryso-syringae ,34,35, asyringae ,37 familsyringae ,39. Rathsyringae ,41.Orthoreovirus) [Cystoviridae, Gag of the yeast virus ScV-L-A [Penicillium chrysogenum virus  [Penicillium stoloniferum virus F  [Rosellinia necatrix quadrivirus 1  [Reoviridae members share an overall conformation [To date, 14 T = 1 capsid proteins have been resolved at the atomic level : VP3 of eovirus) , P3 of reovirus) , VP1 of eovirus) , VP2 of eovirus) , and VP3eovirus)  (these leovirus) , ϕ6 P1 [eovirus)  and ϕ8 Peovirus)  of the f ScV-L-A  (family viridae) , CP of Pviridae) , and theviridae) . The amiormation , and thoReoviridae, as well as evolutionary relationships among T = 1 capsid structural proteins. Whereas T = 1 capsids of reoviruses and cystoviruses share the same structural pattern, i.e., a 120-subunit capsid, most dsRNA mycoviruses exhibit high structural variability. ScV-L-A is built from 60 copies of a dimer of chemically identical subunits (as reo- and cystovirus), but the PcV T = 1 capsid is a variant of the 120-subunit capsid, as the CP has two motifs with the same fold, and the RnQV1 T = 1 capsid is composed of 60 dimers of two different proteins with a similar fold. In addition, the close relationship between the fungal dsRNA virus and its host probably place many constraints on the virus that it overcame by increasing CP complexity. In contrast to the plate-like protein found in reovirus and cystovirus T = 1 capsids, the 120-subunit capsid of fungal dsRNA viruses share a corrugated outer surface with domains rising above the continuous protein shell. In ScV-L-A virus, the CP has an extra domain with decapping activity, and the PcV and RnQV1 CP showed similar extra domains on the outer capsid surface with unknown functions. The present review focuses on the structure of dsRNA mycoviruses, and discusses how the lack of an extracellular phase has had unanticipated functional effects in their life cycles.Unlike their bacteria- and higher eukaryote-infecting counterparts, most mycoviruses are transmitted by cytoplasmic interchange; they never leave the host, and indeed have no strategy for entering host cells . Recent Saccharomyces cerevisiae (ScV-L-A) is the type species of the genus Totivirus (family Totiviridae) [The L-A virus of the yeast viridae) ,51. The viridae) ,53. Gag The structure of ScV-L-A was first examined by three-dimensional cryo-electron microscopy 3D cryo-EM) and later by X-ray crystallography (resolution 3.4 Å) . Dark-fiD cryo-EM7GMP) cap from the 5′ end of the cellular mRNA to the 5′ end of the viral RNA [Gag functions as an enzyme and has a major role in the sophisticated interaction between ScV-L-A and the host cell. The Gag segment Gln139-Ser182, in which His154 is the active site, contributes to the rough outer surface of the capsid, and is responsible for the cellular RNA decapping activity that transfers the 7-methyl-GMP , a prototype of the genus Victorivirus, family Totiviridae, infects the filamentous fungus H. victoriae, and has a similar capsid organization to that of ScV-L-A [Trichomonas vaginalis virus 1 (TTV1), a totivirus of the genus Trichomonasvirus that infects a human-hosted protozoan, has its RdRp fused to the CP, as in ScV-L-A, but by −2 ribosomal frameshifting [Giardia lamblia virus  [The  ScV-L-A ,57. The  ScV-L-A ,59. The  ScV-L-A ,57. Tricshifting . Notablyiavirus)  and the iavirus) ,61 sharePenicillium chrysogenum virus (PcV) is the prototype of the Chrysoviridae, a family of typically symptomless mycoviruses with a genome consisting of four monocistronic dsRNA segments (genome size 2.4–3.6 kbp). Each segment is encapsidated separately in a similar particle [Chrysoviruses are isometric virions characterized by a multipartite genome . Penicilparticle ,64, i.e.Cryphonectria nitschkei chrysovirus virus 1 (CnCV1) at subnanometer resolution [So far, the 3D structures of the capsids of two chrysoviruses have been determined by cryo-EM analysis, that of PcV at atomic resolution , and thasolution . Analytisolution ,34. The Superimposition of the PcV A and B αhelical domains identifies a single “hotspot” on the outer capsid surface where variation is introduced by insertion of 50–100 residue segments C,D. A prMembers of the family Partitiviridae have bisegmented, 1.4–2.4 kbp-long genomes. Each segment is encapsidated separately in a similar virus particle. dsRNA1 encodes RdRp (one copy per virion), whereas dsRNA2 encodes the CP. The partitiviruses that infect fungi are grouped into three genera: alpha-, beta-, and gamma-partitiviruses ,66. AlphPenicillium stoloniferum virus S (PsV-S) [Penicillium stoloniferum virus F (PsV-F)  [Fusarium poae virus 1 (FpV1) [Sclerotinia sclerotiorum partitivirus 1 (SsPV1) [Four fungal partitivirus structures have been resolved by 3D cryo-EM, including those of the gamma-partitiviruses  (PsV-S) ,68 and Polution) , and of 1 (FpV1)  and Scle (SsPV1) .The single-layered, 120-subunit capsids of these viruses are 35–42 nm in diameter and distinct in that they have “arch-like” surface features that protrude above the continuous capsid shell A. These Based on their capsid organization, partiti- and picobirnaviruses appear to be assembled from dimers of CP dimers . In contrast, the proposed assembly pathway for the 120-subunit capsids of Totiviridae and Reoviridae members is based on pentamers of CP dimers . Notably, the capsid protein P1 of bacteriophage ϕ8 (a cystovirus) appears as a soluble tetramer in an in vitro assembly system .Rosellinia necatrix quadrivirus 1 (RnQV1) is the type species of the genus Quadrivirus in the family Quadriviridae [Rosellinia necatrix, a pathogen of many plants, can be infected by dsRNA viruses belonging to at least six families [iviridae ,74. The families ,76. RnQVRnQV1 virus strains W1075 and W1118, isolated from different locations in Japan, have been analyzed by 3D cryo-EM and analytical ultracentrifugation . Their PDespite their low sequence similarity, the superimposition of P2 and P4 revealed their having a common α-helical domain D. As desStructural comparisons of CPs have been used to establish relatedness when sequence conservation is limited ,78,79, aParamecium bursaria chlorella virus 1 (PBCV1) [The similarity of the A and B α-helical domains of PcV CP, which have many well-matching secondary structural elements, indicate a common fold in both domains . Gene du (PBCV1) , and bac (PBCV1) , and the (PBCV1) . The con (PBCV1) ,47  or covalently joined folds (as in chrysoviruses). This event could direct the assembly of a T = 1 capsid with 120 subunits or domains with a dimer as the asymmetric unit—a necessary arrangement for dsRNA replication/transcription. Separate and joined folds are found in the CP of other virus families, such as picornaviruses  and comoThe CP of many-tailed dsDNA phages with the HK97-like fold has additional domains with specific functions related to capsomere and/or capsid stability . Human Victorivirus, family Totiviridae), such as HvV190S, except that the RdRp is expressed from the single genome segment of those viruses via a coupled termination-reinitiation mechanism [Totivirus), in contrast, the RdRp is expressed as a C-terminal fusion product with the CP . As a result, in ScV-L-A, the one or two RdRp domains per virion are covalently tethered to the capsid via the fused CP domain, which occupy one or two subunit positions in the capsid.Reovirus T = 1 cores have 10–12 RdRp complexes per virion, around which the dsRNA is densely coiled ,93. RdRpechanism ,59,96. FThe anchoring of RdRp at the five-fold axes on the reovirus capsid inner surface seems likely to occur in toti-, chryso-, partiti-, and quadriviruses too, with important consequences for the channeling of freshly synthesized transcripts into an exit pore.The mycovirus T = 1 capsid wall is perforated by many pores and channels, but none is large enough to pass an A-form 23-Å-diameter duplex A,B. Wher3, a spacing between dsRNA strands of 25–30 Å) [3, an interstrand spacing of ~40–45 Å) [With the exception of totiviruses, which have a single genomic segment, many fungal dsRNA viruses, including chryso-, partiti-, and quadriviruses, have multisegmented dsRNA genomes. In addition, the multisegmented viruses appear to be multiparticulate, i.e., segments are encapsidated separately . Fungal 25–30 Å) ,99,100, 40–45 Å) ,49,63. IMost mycovirus T = 1 capsids are negatively charged on their inner surface, a feature common to many such capsids of dsRNA viruses . This miComparative analysis of dsRNA packing densities in dsRNA virions have revealed two major tendencies among T = 1 capsids of dsRNA viruses: (1) those with 9–12 dsRNA segments densely packaged within the same particle and containing 9–12 RdRp complexes, as seen in reoviruses, and (2) those with a single-genomic dsRNA segment with less internal order and one or two copies of the RdRp complex per particle, as seen in mycoviruses.Structural studies of a limited number of fungal viruses have revealed them to conform to the basic concepts of dsRNA viruses, but also to have unexpected features that have contributed to a better understanding of their structure, function, and evolution. dsRNA mycovirus capsids, exemplified by ScV-L-A, PcV, PsV-F, and RnQV1, show structural variations of the same framework optimized for RNA metabolism; they possess 60 asymmetric or symmetric dimers of a single protein , dimers of similar domains (PcV), or dimers of two different proteins (RnQV1). Since mycoviruses are transmitted by cytoplasmic interchange and commonly confined to their hosts, their capsids incorporate polypeptides and domains on their outer surfaces for the acquisition of new functions without altering the structure and function of the CP. Such acquisitions would eventually lead to optimal viral-host interactions.Despite recent advances in understanding the structure of dsRNA mycoviruses, many aspects of several fungal (and protozoan) viruses remain unknown. Recent work has identified a positive-sense ssRNA virus—the yado-kari virus 1 (YkV1)—that hijacks the CP of a dsRNA virus that resembles totivirus—the yado-nushi virus 1 (YnV1) ,102. The


Culex annulirostris mosquitoes in Australia. Phylogenetic analysis of its RNA-dependent RNA polymerase sequence and that of other related viruses revealed 6 clades, two of which corresponded wholly or partly with existing genera in the family Nodaviridae. There was greater genetic diversity within the family than previously recognized prompting us to suggest that additional genera should be considered within the family.We describe a virus isolated from  Alphanodavirus (5 species) and Betanodavirus (4 species). The alphanodaviruses primarily infect insects, and betanodaviruses infect fish. Historically, the earliest isolations were of the alphanodaviruses in Australasia, initially with Nodamura virus (NoV) in Japan [Flock House virus (FHV), and then Black beetle virus (BBV), and Boolarra virus (BoV) [Pariacoto virus (PaV) [Barfin flounder nervous necrosis virus (BFNNV), Redspotted grouper nervous necrosis virus (RGNNV), Striped jack nervous necrosis virus (SJNNV), and Tiger puffer nervous necrosis virus (TPNNV) [Culex genus [Nodaviruses are positive-sense RNA viruses with bipartite genomes which are capped but not polyadenylated . There ain Japan ; Flock Hus (BoV) . More reodavirus  species. (TPNNV) . In 2014odavirus  species ex genus . This stThe two genomic RNAs of nodaviruses, a ~3.1 kb RNA1 and ~1.4 kb RNA2 respectively, encode for two open reading frames (ORFs) . InteresCulex annulirostris mosquitoes and which we have tentatively named culannivirus (CulV). Phylogenetic analysis of the RdRp sequence placed CulV distantly from any of the currently recognized genera. By contrast, phylogenetic analysis of the capsid protein placed CulV in a clade with the currently recognized alphanodaviruses. We suggest a re-organization of the family based on the RdRp sequence.In this work we describe a new species of nodavirus which was isolated from a sample of No specific permits were required for the described field studies, however permission was sought and obtained from the traditional land owners of Bradshaw Field Training Area to conduct mosquito trapping activities in the area. These field studies did not involve endangered or protected species.2-baited light traps from the Bradshaw Field Training Area (BFTA) in the Northern Territory of Australia in 2014, using previously described methods [Adult mosquitoes were collected using CO methods . Screeni methods . Successde novo assembly (GenBank accession number MH794143). To construct phylogenetic trees, global multiple protein sequence alignments of RNA1 and RNA2 ORFs were performed with the Geneious Alignment feature  [RNA extraction, first and second strand cDNA synthesis, library preparation and Illumina sequencing were as previously described . A virusalty, 3) . A mid-palty, 3)  from theCulex annulirostris mosquitoes collected from the Northern Territory of Australia in 2014. We tentatively named this virus culannivirus (CulV). Preliminary analysis of deep sequencing of extracted RNA identified the virus as belonging to the Nodaviridae family. We performed more detailed analysis using RdRp protein sequence as it has been previously suggested that it would be preferable for taxonomic determinations [Culex. This clade has been greatly expanded by recent sequencing studies, with material again originating from diverse arthropod hosts [Helicoverpa zea as well as a number of nodavirus sequences from diverse insect hosts [Alphanodavirus and Betanodavirus, or they may potentially reveal relationships based on biological characteristics which will be the basis of some future taxonomic delineation.An unidentified RNA virus was isolated during the screening of inations . Our aliinations . This mainations , 14, 15.od hosts . Clade 4od hosts , 17, perct hosts . Clade 6ct hosts , 20. TheMacrobrachium rosenbergii nodavirus and Penaeus vannamei nodavirus were also placed in different clades. Similar phylogenetic assignment differences with these viruses between RdRp and capsid were noted previously [The capsid protein sequence has been used as the major taxonomic determinant when assigning members of the nodaviruses . Analysieviously . These dThe outcome of nodavirus infection is influenced by both host and virus genetic determinants. Entry of the virus into the cell involves interaction between the virus capsid and cell membrane receptors. Studies on FHV have indicated that capsid protein gamma facilitates membrane penetration during host cell entry (reviewed in ). Once iCulex; the latter virus being the subject of this work. The phylogenetic analysis revealed 6 monophyletic clades, two of which were constituted either wholly or partly of the existing genera. Clade 1 corresponded to the genus Alphanodavirus. Clade 2 included members of the betanodaviruses and additional viruses that had been isolated from organisms other than fish. The genetic diversity of this genus is relatively small  and this possibly reflects either limited sampling or a genetic bottleneck in adapting to their common fish hosts. The internal diversity of clade 2  was larger than the alphanodaviruses , but smaller than the internal diversity of three of the other clades identified . Clade 2 includes viruses collected from a variety of arthropod hosts, similarly to most of the other nodaviruses, as well as fish. Virus genetic groupings generally reflect their biological characteristics such as replication strategy, genome structure, host tropism and others. The nodaviruses have a common genome structure and share the ability to infect a variety of mostly arthropod hosts, which makes it difficult to determine taxonomic groupings below the family level. Future research may reveal biological characteristics which reflect the genetic groupings identified in this study.The phylogenetic analysis of RdRp sequences has been reliably used for decades to determine the relationships of RNA viruses up to the family, and potentially higher, taxonomic level –31. Our Nodaviridae.In consideration of the greater genetic diversity revealed by this phylogenetic analysis, which includes metagenomics and other studies, there seems to be impetus for greater taxonomic division below the family level. On the basis of a characterized virus type strain, clear monophyletic groupings, which were well-separated from other clades and had good support values, were determined. Given the level of diversity identified, future biological characterization may reveal further genera within this family


Spodoptera littoralis) and one permissive host (S. exigua) were then selected and used to perform six serial passages involving a concentration corresponding to the ~25% lethal concentration for both viruses. Restriction endonuclease analysis showed fragment length polymorphisms in every host-virus system studied. In S. littoralis, serial passage of MbMNPV resulted in decreased pathogenicity and an increase in speed-of-kill, whereas no significant changes were detected for HearMNPV with respect to the initial inoculum. In contrast, both viruses showed a similar trend in S. exigua. These results highlight the low genetic diversity and a high phenotypic stability of HearMNPV with respect to the original inoculum after six successive passages in both insect hosts. This study concludes that host-baculovirus interactions during serial passage are complex and the process of adaptation to a novel semi-permissive host is far from predictable.Phylogenetic analyses suggest that Mamestra brassicae multiple nucleopolyhedrovirus (MbMNPV) and Helicoverpa armigera multiple nucleopolyhedrovirus (HearMNPV) may be strains of the same virus species. Most of the studies comparing their biological activities have been performed in their homologous hosts. A comparison of host range and stability in alternative hosts was performed. The host range of these viruses was compared using high concentrations of inoculum to inoculate second instars of six species of Lepidoptera. One semi-permissive host ( Baculoviridae comprises double-stranded DNA viruses that are specific to invertebrate hosts. Their insecticidal properties combined with high host specificity mean that they can be valuable agents for the biological control of insect pests of field and greenhouse crops and in forest ecosystems [Alphabaculovirus  show important variation in their host range, from viruses that are highly host specific, such as Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV), to viruses that can productively infect multiple species of hosts from different lepidopteran families, such as Autographa californica multiple nucleopolyhedrovirus (AcMNPV) [The family osystems ,2. Bacul(AcMNPV) .lef-8, lef-9 and polh/gran genes measured by the Kimura 2-parameter (K2P) value [Baculovirus classification based on the host from which the virus was isolated has obvious drawbacks for viruses that can productively infect multiple species of hosts. This can also create confusion when several baculoviruses are isolated from the same host species. To address this issue, the International Committee on Taxonomy of Viruses (ICTV) determined that the definition of species status should involve phylogenetic criteria for lepidopteran specific baculoviruses, based on the genetic distances of the P) value . For virP) value . For exaP) value ,6,7,8,9,P) value . NotwithM. brassicae and H. armigera [Both MbMNPV and HearMNPV can productively infect a relatively wide range of lepidopteran species ,12,13. Barmigera . ImportaHost range in baculoviruses is characterized by high variation in the susceptibility of different hosts to a particular virus , but is Natural baculovirus populations are characterized by high genetic variability that is likely to influence the host range and their ability to adapt to novel hosts . Serial Spodoptera littoralis and in the permissive host S. exigua was studied with the aim of identifying the variables  that most affect the genetic and phenotypic characteristics that contribute to improving the insecticidal properties of these viruses.In this study, MbMNPV and HearMNPV were selected in order to investigate the degree of similarity of their biological properties in alternative hosts. First, the degree of susceptibility to these viruses was studied in six different lepidopteran pest species. One semi-permissive and one permissive host were then selected for serial passage studies. The genetic variability and biological activity of each virus in the semi-permissive host S. littoralis was obtained from Israel, whereas the S. exigua colony was provided by Universitat de Valencia, Valencia, Spain. Due to the detection of a SeMNPV infection during serial passage in this species, a new S. exigua colony was established from eggs provided by Entomotech . These insects were tested for the presence of persistent SeMNPV infections by qPCR before establishing the new colony.The larvae of lepidopteran species were obtained from laboratory colonies reared at the Universidad Pública de Navarra (UPNA) and maintained at 25 ± 1 °C, 75% relative humidity and 16 h light: 8 h dark photoperiod on a wheat germ-based semi-synthetic diet . A colon® . In contrast, HearMNPV, originally from the former USSR [M. brassicae and H. armigera, at a concentration that resulted in approximately 90% mortality, to obtain the passage zero population of each virus, named MbMNPV-P0 and HearMNPV-P0.The MbMNPV strain was isolated from the microbial insecticide Mamestrinmer USSR , was kinH. armigera, Lobesia botrana, M. brassicae, Ostrinia nubilalis, S. exigua and S. littoralis). For each species, groups of 28 newly-molted second instars that had been starved for 12 h were inoculated with a single high concentration (5 × 106 OBs/mL) of MbMNPV OBs or HearMNPV OBs using the droplet feeding method [8 OBs by mixing 20 μL of OB suspension with 50 μL of 0.5 M Na2CO3, 25 μL of 10% (w/v) SDS and 155 μL of sterile double-distilled water followed by incubation at 60 °C during 10 min. The undissolved OBs were removed by centrifugation at 2300× g for 5 min. The supernatant was treated with 15 μL of proteinase K (20 mg/mL) and incubated at 50 °C for 30 min. Protein was then precipitated by the addition of 150 μL of MPC Protein Precipitation Reagent . Viral DNA was isolated by alcohol precipitation. The resulting pellet was re-suspended in 60 μL of 0.1 × TE buffer . Approximately 1 μg of DNA was digested with 2 µL PstI and HindIII  in a total volume of 20 µL at 37 °C for 1 h. The restriction fragments were separated by electrophoresis in 1% agarose gels in TAE buffer  at 16V for 15 h. The fragments were visualized on a UV transiluminator after GelRed® (Biotium) staining.In order to compare the host range of each virus, susceptibility tests were performed on six lepidopteran species  was used to infect three batches of larvae of each species. Then, following death by polyhedrosis, dead infected insects of each lineage were pooled, purified and the resulting OBs were used for the next passage. At each passage, three groups of 36 larvae in each lineage were orally inoculated and were reared individually on semi-synthetic diet at 25 ± 1 °C until death or pupation. For every lineage and passage, groups of 28 larvae were mock-infected as controls. The serial passages were performed using a 25% lethal concentration (LC25). This was undertaken as low inoculum concentrations are likely to favor the transmission of virus genotypes that are prevalent in the inocula due to efficient replication in the previous heterologous host, and to hinder the transmission of defective genotypes that can arise during serial passage [6, 2 × 107 and 2 × 108 OBs/mL for both viruses in S. littoralis and 6 × 102, 3 × 103 and 1.5 × 104 OBs/mL for both viruses in S. exigua. Once the SeMNPV infection was detected in lineage 2 of S. exigua-MbMNPV system, the whole S. exigua population was replaced with free-virus insects from southern Spain. The new insect colony was subjected to qPCR screening to ensure the absence of persistent SeMNPV infections, as described previously [S. exigua population was found to be more resistant to infection, and inoculum concentrations were increased to 3 × 103, 1.5 × 104 and 7.5 × 104 OBs/mL for passages four to six. Virus-killed cadavers of the treatment that resulted in ~25% mortality were pooled within each lineage, purified, counted twice using a Neubauer hemocytometer and used as oral inoculum for the next passage. This same methodology was performed for every passage  analysis of DNA from OBs obtained at each passage was performed and compared with the MbMNPV-Pectively . For thi0 and HearMNPV-P0) were used as reference treatments in every bioassay. To examine changes in the biological activity of MbMNPV and HearMNPV after serial passage in the selected permissive and semi-permissive hosts, bioassays were performed on the OB suspensions obtained at passages three and six of each host-virus combination and for each lineage to determine OB pathogenicity and speed-of-kill . The OBs50 and LC90), was estimated in newly-molted second instars of each host species using the droplet feeding method [S. exigua and one of six OB concentrations for S. littoralis. The same OB concentrations were used for both viruses in S. littoralis  and in S. exigua . In both cases, the range of concentrations was estimated to kill between 5 and 95% of inoculated insects. The larvae that ingested the inoculum suspension in a 10 min period were individually transferred to 28-well plates containing semi-synthetic diet and were incubated at 25 ± 1 °C. The control insects were treated identically but did not consume OBs. Virus-induced mortality was recorded at 24 h intervals until death or pupation. The bioassays were performed in triplicate using different batches of insects. Virus-induced mortality results were subjected to probit analysis using POLO-PLUS software [Pathogenicity, expressed as 50% and 90% lethal concentrations (LCg method . Furthersoftware .90), or a sucrose solution free of OBs as a control treatment. Larvae that drank the inoculum within 10 min were reared individually at 25 ± 1 °C and virus-induced mortality was recorded at 8 h intervals until all the larvae had died or pupated. The experiment was performed on three occasions. The time-mortality data were subjected to Kaplan–Meier survival analysis using SPSS Statistics v.25.0 software [To determine the speed-of-kill responses (median time to death), groups of 28 newly-molted second instars of each host species were treated with a single OB concentration estimated to result in ~90% mortality , one semi-permissive host  and three permissive hosts   . As a reIn every host-virus combination, three different concentrations  was performed using PstI and HindIII in order to compare broadly the genetic characteristics of these viruses and to establish the initial identities of the starting inocula  of 2.88 × 10−6 ± 1.50 × 10−6 ng SeMNPV DNA/ng total DNA , which were consistent with the presence of very low level SeMNPV replication in the persistently infected S. exigua colony.The submolar PstI fragments of 7.5 and 9 Kb observed in MbMNPV-Pineage 3 . In line0 was detected from the first passage to the last one in lineages 1 and 2 . This is consistent with a very low level persistent infection in the S. exigua colony.For HearMNPV, an intensification of the 12 kb submolar HindIII fragment present in HearMNPV-P 1 and 2 . In cont50 value of MbMNPV-P0 OBs varied from 2.6 × 107 to 4.1 × 106 OBs/mL in the semi-permissive host S. littoralis, depending on the bioassay, whereas LC90 values varied from 5.4 × 108 to 1.3 × 108 OBs/mL  for bioassays of the pathogenicity of OBs from subsequent passages. After the third passage, all the lineages of MbMNPV OBs showed a 3- to 10-fold decrease in pathogenicity, whereas no marked changes were observed in the HearMNPV lineages compared to the P0 inoculum   , or the sixth passage compared to the HearMNPV-P0 inoculum   A. These = 0.003) C. For He= 0.731) B,D.50 values of MbMNPV-P0 OBs varied from 1.1 × 105 to 5.3 × 104 OBs/mL in the permissive host, whereas LC90 values varied from 1.6 × 106 to 3.6 × 106 OBs/mL  and of 17 h in lineage 2 for HearMNPV  compared to the P0 inoculum  and of 12 h in lineage 1 for HearMNPV   of 18 h was observed in lineage 1 for MbMNPV  C,D.S. littoralis [S. littoralis was a semi-permissive host for both viruses, at lower inoculum concentrations than used in a previous study [S. littoralis inoculated with heterologous viruses.In the present study, MbMNPV and HearMNPV had a similar host range when used to inoculate heterologous lepidopteran species. The same lepidopteran species were considered as permissive, semi-permissive and non-permissive for both viruses. These species had been previously described as susceptible species for both viruses with the exception of ttoralis ,14,33,34us study . As restH. armigera larvae resulted in an increase in genetic diversity [The OB concentration selected for the serial passage experiment was based on the desire to maintain a near-constant 25% prevalence of virus-induced mortality. Virus entry to midgut cells is the main barrier to the establishment of a successive infection , but onciversity . Baillieiversity  suggesteiversity ,39,40.Despite the fact that all lineages were treated identically to increase the reproducibility of the technique, genetic analysis revealed differences in the REN profiles among the lineages in each host-virus combination, indicating that host-virus interactions did not follow a common genetic trajectory. This variability among lineages has been previously reported following serial passage of lineages established from a single common inoculum of HearNPV OBs . DespiteChanges in submolar bands were detected during serial passage in every host-virus system. Minor changes in restriction profiles may be associated with significant changes in the phenotypic characteristics of the virus ,48,49. TModifications in REN profiles could also be due to the proliferation of minority genotypes or because new genotypes appeared. Genotypic variants can be generated by several mechanisms, such as recombination events, insertion or deletions of viral sequences, point mutations, horizontal gene transfer, as well as alterations in the relative abundance of genotype variants ,21,55,56S. exigua were identified by qPCR as SeMNPV and a MbMNPV contamination event, respectively. The activation of persistent infections in lepidopteran larvae by alphabaculoviruses is well documented [S. exigua, lineage 2 of MbMNPV had to be discarded and the other lineages were continued using a new population of S. exigua. After passage four, the presence of MbMNPV clearly decreased in HearMNPV lineage 3, so the presence of MbMNPV in the HearMNPV-inoculum was considered to be a transient contamination event and the experiment was continued. The REN profile variations detected in MbMNPV lineage 2 and HearMNPV lineage 3 after the third passage in the permissive host cumented ,59,60,61M. brassicae and H. armigera. In the present study, differences were observed between MbMNPV and HearMNPV in the insecticidal characteristics of the OBs following serial passage in S. littoralis. After the sixth passage, the insecticidal properties of HearMNPV did not show any alteration in pathogenicity or speed-of-kill compared to passage zero inoculum, whereas a significant decrease in MbMNPV pathogenicity was detected. The speed-of-kill of MbMNPV varied in each lineage but recovered the speed-of-kill of the original passage zero inoculum after six passages in two of the lineages. Several studies have demonstrated an increase in the insecticidal activity of a baculovirus in a particular semi-permissive host through serial passage [S. littoralis showed an increase in its speed-of-kill, which was the only significant improvement in the insecticidal properties detected after the last passage in all the host-virus systems in this study.In the biological activity analyses, some small differences were observed among the lineages of each host-virus system. Although pathogenicity characteristics were similar among the lineages in each of the host-virus combinations, small differences in the speed-of-kill were detected in four out of the eight host-virus systems studied. Rovesti et al.  reported passage ,20,21,62 passage ,63,64. TS. exigua, the only alteration in insecticidal activity observed at the sixth passage compared to passage zero, was a significant decrease in the speed-of-kill in lineage 1 of both MbMNPV and HearMNPV, whereas the other lineages remained unchanged at the final passage. However, a significant improvement in biological activity, both in pathogenicity and speed-of-kill in lineage 2 of MbMNPV was detected after three passages, which was probably related to the presence of SeMNPV in the OB inoculum, as explained before. It is important to note that the activation of latent infections should not be excluded as a possible reason for marked improvements in insecticidal activity in serial passage host-adaptation studies in which no genetic characterization was performed on the progeny OBs [S. exigua with respect to passage zero and the other lineages, which was associated with a transient contamination by MbMNPV that subsequently disappeared, possibly due to competitive exclusion by the HearMNPV population.In the permissive host geny OBs . As no iNo clear correlation was observed between genetic and biological changes in any of the host-virus combinations analyzed. Indeed, lineages with REN profiles that seemed to be very similar elicited different biological responses. Others have reported changes in REN profiles with no marked effects on pathogenicity after passages in homologous and heterologous hosts ,66.Most studies on baculovirus adaptation to heterologous hosts have involved performing a high number of passages, often between 12 and 20. In some cases, an increase in the speed-of-kill and mortality has been detected after fewer than ten passages or even after a single passage ,67. HoweThis study highlighted similarities in the host range of HearMNPV and MbMNPV, but also indicated that HearMNPV appeared to be more phenotypically-stable than MbMNPV following serial passage in a semi-permissive host. However, host-baculovirus interactions are highly complex and differences among the three lineages in our study demonstrate that the process of adaptation to a novel host is far from predictable.


Although rubella is epidemic in Indonesia, the phylogenetic profile of circulating rubella virus strains has not been clarified. In 2017, rubella virus was detected in 2 travelers who returned from Indonesia to Japan. These strains were classified into genotype 1E lineage 2, which may be an indigenous strain in Indonesia. Rubivirus, family Togaviridae  ; it reveTo verify rubella elimination, interruptions in transmission of indigenous or imported rubella virus strains must be confirmed through effective surveillance systems (Japan has a high risk for subsequent rubella epidemics because the proportion of persons susceptible to rubella virus (≈9.0%) has not changed since 2013. In addition, an epidemic can occur when rubella virus is transported from rubella-endemic countries and the infection occurs in susceptible populations, as happened in Japan in 2013. Of the 11 imported cases of rubella to Japan reported in 2017, 4 were from Indonesia, according to the NESID of Japan.. In the case we describe, we identified the rubella-exporting country and clarified the genetic information of the strain, which may contribute to countermeasures for worldwide importation of rubella virus. Rubella control by 2020 is the flagship goal of the World Health Organization South-East Asia region. Indonesia is conducting rubella immunization campaigns targeting ≈70 million children in 2017–2018. Therefore, constructing effective surveillance systems, accumulating genetic information, and promoting immunization in rubella-endemic countries are steps toward the global elimination of rubella.


Rhipicephalus, Amblyomma, Dermacentor, Hyalomma, and Haemaphysalis genera identified numerous viruses, among which several viruses could be candidates for future emergence as regards to their phylogenetic relatedness with known tick-borne arboviruses. Luciferase immunoprecipitation system targeting external viral proteins of viruses identified among the Orthomyxoviridae, Phenuiviridae, Flaviviridae, Rhabdoviridae, and Chuviridae families was used to screen human and cattle Thai populations highly exposed to tick bites. Although no positive serum was detected for any of the six viruses selected, suggesting that these viruses are not infecting these vertebrates, or at very low prevalence , the virome of Thai ticks presents an extremely rich viral diversity, among which novel tick-borne arboviruses are probably hidden and could pose a public health concern if they emerge. The strategy developed in this pilot study, starting from the inventory of viral communities of hematophagous arthropods to end by the identification of viruses able (or likely unable) to infect vertebrates, is the first step in the prediction of putative new emergences and could easily be transposed to other reservoirs/vectors/susceptible hosts interfaces.Emerging zoonoses caused by previously unknown agents are one of the most important challenges for human health because of their inherent inability to be predictable, conversely to emergences caused by previously known agents that could be targeted by routine surveillance programs. Emerging zoonotic infections either originate from increasing contacts between wildlife and human populations, or from the geographical expansion of hematophagous arthropods that act as vectors, this latter being more capable to impact large-scale human populations. While characterizing the viral communities from candidate vectors in high-risk geographical areas is a necessary initial step, the need to identify which viruses are able to spill over and those restricted to their hosts has recently emerged. We hypothesized that currently unknown tick-borne arboviruses could silently circulate in specific biotopes where mammals are highly exposed to tick bites, and implemented a strategy that combined high-throughput sequencing with broad-range serological techniques to both identify novel arboviruses and tick-specific viruses in a ticks/mammals interface in Thailand. The virome of Thai ticks belonging to the  Among the list of human-infecting pathogens, two-thirds are zoonotic agents , tick-borne encephalitis , and spotted fever    were primarily associated with Hyalomma asiaticum and Rh. sanguineus ticks ]  Moyer, . The sam) Moyer, , they ca) Moyer, . The pro) Moyer, . For exaWe hypothesized that currently unknown tick-borne arboviruses could silently circulate in specific biotopes where mammals (including humans) are highly exposed to tick bites and used wide range identification techniques to track them in a tick/mammal interface in Thailand. Despite the fact that the description of the virome of ticks is a prerequisite to the evaluation of the risk of spillover, few studies have tried to go further and characterize, among the viral communities infecting ticks, which viruses would more likely be transmissible to vertebrates. Starting from the inventory of viruses infecting tick vectors, the first step in the understanding of the mechanisms of viral emergence is therefore to identify which viruses can cross the species barrier and infect vertebrates, even without any reported clinical signs. Serological techniques are useful tools for getting insights into arbovirus exposure history of new hosts without the limits of genomic tests, which need to collect biological samples in the course of a viremic phase. However, the recognition of the antigen (Ag) by its specific antibodies (Ab) requires good conservation of epitopes conformation, a problem frequently encountered in solid phase Ab/Ag assays  and bacteria  are carried by ticks. Viruses belonging to the Reoviridae, Togaviridae, Flaviviridae, Nairoviridae, and Orthomyxoviridae were also reported  database, de novo assembled after extraction of mapped reads, and submitted to the BOLD Identification System. Boophilus sp. ticks were identified as Rh. microplus, and Haemaphysalis sp. were identified as Haemaphysalis hystricis.A total of 266 ticks  were collected in Ban Huay Muang village  in November 2012. Ticks were morphologically identified at least at the genus level: 40 Amblyomma ticks were collected on wild boars and rodents; Rh. microplus ticks were collected on cattle (except for two ticks collected by flagging); D. marginatus ticks were collected on wild boars (except for one tick collected by flagging); Ha. hystricis ticks were collected either on wild boars (N = 11), on dogs (N = 2), and by flagging (N = 3); Hyalomma ticks were either collected on dogs (N = 3) or on wild boars (N = 15); and Rh. sanguineus ticks were either collected on dogs (N = 12) or on the floor of houses were dogs lived (N = 4).All of the genera collected here contain several tick species that can either bite wildlife or domestic animals (including cattle) and even humans , document no 0517.1116/661.Public health personnel made appointment with the villager participants in Saen Thong subdistrict, Nan province. Voluntary participation in the research project was obtained after explanation of the purposes of the study and by signing the informed consent/assent form. Blood samples were collected once by local nurses from a primary care unit, as previously described  was used for library preparations and sequenced on an Illumina HiSeq2000 sequencer in a single-read 100-bp format outsourced to DNAVision Company.in-house bioinformatics pipeline comprising quality check and trimming [based on AlienTrimmer package ], de novo assembly . A BLAST-based similarity search was performed for all contigs and singletons against the protein Reference Viral database  of all sequences being associated to this taxon instead of summing the raw number of sequences, to take into account the identification of long viral contigs. In addition, to take into account that contigs are the results of the assembly of numerous reads, we weighted the identification of viral hits coming from contigs by multiplying the length (in amino acids) of contigs with their respective http://www.atgc-montpellier.fr/phyml-sms/ using the corrected Akaike information criterion. Phylogenetic trees were constructed using maximum likelihood method implemented through RAxML program under the CIPRES Science Gateway portal  gene. Complete ORFs were aligned along with other representative sequences of viral orders/families using MAFFT (Multiple Alignment using Fast Fourier Transform) aligner under the L-INS-i or G-INS-i parameters , and cDNA was subsequently used to fill the gaps in the genomes using the Phusion High Fidelity DNA polymerase . Positive PCR products were further purified and sequenced by Sanger sequencing on the Eurofins Segenic Cochin platform. When start and stop codons were lacking, rapid amplification of cDNA ends PCR were performed using the 5′/3′ RACE kit, second generation .For prevalence study, individual RNA extracts were reverse transcribed into cDNA using the qScript cDNA Supermix kit according to the manufacturer's instructions . Briefly, the reaction was performed in a final volume of 5 μl containing 1 μl of qScript cDNA Supermix 5×, 1 μl of RNA, and 3 μl of RNase-free water. Cycling condition was as follows: one cycle at 25°C for 5 min, one cycle at 42°C for 30 min, and one final cycle at 85°C for 5 min. Specific Taqman qPCR systems targeting the polymerase gene were designed for six relevant viruses (the sequences of primers and probes are available upon request). Real-time Taqman qPCR assays were performed in a final volume of 12 μl using the LightCycler® 480 Probe Mastermix (Roche Applied Science) at 1× final concentration, with primers and probes at 200 nM, and 2 μl of cDNA. Negative (water) control was included in each run. Thermal cycling conditions were as follows: 95°C for 5 min, 45 cycles at 95°C for 10 s, then 60°C for 15 s, with a final cooling step at 40°C for 10 s.To identify possible endogenous viral elements (EVEs) originating from arthropod hosts that could have been sequenced during the process, nested qPCRs were performed on tick-borne total DNA without any RT step targeting the polymerase gene of the six selected as relevant viruses. Positive or suspicious results were further analyzed by Sanger sequencing.https://swissmodel.expasy.org/interactive)   or nucleoproteins (NP) when GP were not identified. The annotation of the genes and the consecutive identification of surface proteins were first performed by position-specific iterated Blast (Psi-Blast). For viral ORFs for which we could not determine the function of the gene by Psi-Blast, Swiss Model was used to model the structure of the viral protein and to compare it to known structures deposited onto the PDB database  to identify extracellular regions of the proteins. These regions were used to produce viral antigens. The coordinates of the extracellular regions expressed for relevant viruses are presented in Complete extracellular regions of viral glycoproteins and/or nucleoproteins were cloned into pFC32K vector , at the N-terminal end of the NanoLuc luciferase gene, using the Gibson Assembly (GA) kit , according to the manufacturer's instructions. First, the Kosak sequence  was used to clone by GA the target fragments at a ratio of 1:3 to 1:10 (depending on the constructs) in XL1 competent cells . Positive clones were screened by PCR with primers designed in the vector and flanking the inserts and verified by Sanger sequencing. PureLink HiPure midiprep kit (Invitrogen) was used to extract plasmids from 100 ml of bacterial overnight cultures grown in LB medium supplemented with 25 μg/ml of kanamycin.5 per well) were plated on a six-well plate the day before transfection, and 5 μg of plasmid DNA was transfected with 20 μl of 1 mg/ml PEI. Two days post-transfection, fusion proteins were harvested as previously described  by adding the NanoLuc substrate  to 10-fold dilutions of the lysates. Titers (in light unit LU/ml) were corrected for background by subtracting LU values produced by negative controls constituted of cells transfected with the same amount of PEI without plasmid DNA.A modified version of Burbelo's protocol was developed for expression of fusion proteins: HEK-293A cells  were transfected with polyethylenimine  instead of Cos-1 cells and Fugene-6 transfecting reagent . The maximal prevalence of viral infection in each exposed group was estimated assuming a sensitivity of 90% and a specificity of 99% of the test and the Blaker confidence interval calculation using the True Prevalence calculator implemented in the EpiTools portal .Significant differences between exposed and non-exposed groups of human sera tested by LIPS were calculated using the Student MN095535 to MN095546.Complete coding regions of the six viruses characterized were deposited into the GenBank database under the numbers 6 reads. Most sequences were assigned to the Eukaryota domain while bacteria and viruses represented 0.09 and 0.001% of validated taxonomic assignation of reads, respectively. Of these, more than 99% of viral sequences were composed of RNA viruses, while few other related viral sequences were identified  presented a mean amino-acid identity of 93% with the polyprotein of Trinbago virus , or to the Luteoviridae family . More than 98% of sequences were taxonomically related to Trinbago virus, a BLTV4-related virus identified in Trinidad and Tobago go virus . Other pe family .Mononegavirales or Jingchuvirales orders. For example, sequences assigned to the Rhabdoviridae family mainly mapped onto a novel strain of Wuhan tick virus 1 (WhTV1), with amino-acid identities ranging from 93 to 97%, depending on the gene ; while other unclassified Rhabdoviridae-related sequences were detected (as for example a distant Tacheng tick virus 3 virus or a new strain of Nayun tick rhabdovirus). Few sequences, were assigned to the Sprivivirus, the Vesiculovirus, and the Ledantevirus Rhabdoviridae genera. Viruses belonging to the Chuviridae mapped onto Wuhan tick virus 2 (WhTV2) and Brown dog mivirus 1 (a new strain of Changping tick virus 2 [CpTV2]), with amino-acid identities ranging from 89 to 99% . Finally, a novel unclassified mononegavirus genome presented distant homologies with Norway mononegavirus 1 .The negative-strand ssRNA viruses constituted more than 97% of viral sequences . Among tBunyavirales, and more precisely to the Phenuiviridae family. More than 92% of sequences were related to Rhipicephalus-associated phlebovirus 1, a novel strain of Lihan tick phlebovirus (LTPV) primarily associated with Rh. microplus Chinese ticks. Tentatively named LTPV-Thailand virus, this viral genome presented amino-acid identities ranging from 97 to 100% with its closest viral relative, depending on the gene (accession no MN095537-38). Several sequences close to Tick phlebovirus Anatolia 1 were also detected.Up to 23% of ssRNA viruses were assigned to the Orthomyxoviridae-related sequences , either belonging to the Thogotovirus or the Quaranjavirus genera were detected, but Oz virus-related viral sequences were the most abundant. Tentatively named Thailand tick thogotovirus (accession no MN095539-44), this virus presented amino-acid identities ranging from 70 to 82% depending on the genes .Rhabdoviridae is a family of RNA viruses infecting a large spectrum of hosts, ranging from plants to invertebrates and vertebrates. Tick-borne rhabdoviruses include viruses belonging to the recognized Ledantevirus and Ephemerovirus, and the putative Sawgravirus genera /P (phosphoprotein)/M (matrix)/G (glycoprotein)/L (RNA-dependent RNA polymerase). WhTV1-Thailand virus presents a similar gene organization, although only four ORFs were identified (Rhabdoviridae phosphoproteins), the absence of transmembrane domains, and its richness in acidic amino acids (PI = 4.89) suggest that ORF2 could code for the putative P protein of WhTV1-Thailand virus. ORF3 codes for a small protein of 205 aa corresponding to the putative matrix protein of the virus. The last ORF codes for a large protein of 2,183 aa corresponding to the RNA polymerase of the virus. WhTV1-Thailand virus present therefore an N–P–M–L gene organization, suggesting that a major evolution event in its emergence was the loss of G gene. Walker et al. noted that gain and/or loss of genes was common and a major driver of rhabdoviruses evolution  and Ixodidae ticks are infected by tick-borne chuviruses . Chuviridae viruses present a large variety of genome organization, from a circular L–G–N order of genes . CpTV2-Thailand virus presents similar genome organization as WhTV2-Thailand virus, with a circular ssRNA genome, except that its genome is shorter (10 kb) and that the G ORF overlaps the L ORF on 61 nucleotides (this junction was confirmed by specific amplification followed by Sanger sequencing). The L gene code similarly for the RdRP of 2,164 aa, the G gene codes for the glycoprotein of 710 aa and the N gene codes for the nucleoprotein of 374 aa , Tenuivirus (plant arboviruses), and Phlebovirus  genera. This latter is the unique known viral genus among the family able to infect vertebrates, including humans and domestic animals. Recognized tick-borne phleboviruses (TBPVs) are currently clustered into three main serogroups: SFTS virus, Bhanja virus, and Uukuniemi virus serogroups  or migratory birds  ticks from USA and Norway , Pegivirus , Pestivirus , and Hepacivirus (human Hepatitis C virus). Tick-borne flaviviruses (TBFVs) are divided into mammalian- and seabird-infecting viruses  virus primarily identified in Hy. asiaticum Chinese ticks and later in Rh. sanguineus ticks from Trinidad and Tobago (Trinbago virus)   the six segments genome architecture  constituting the PB1 protein complex; segment 3 for the PA protein of 637 aa; segment 4 for the GP protein of 510 aa; segment 5 for the NP protein of 465 aa; and segment 6 for the M protein of 252 aa); and (ii) the structural conformation of Thailand tick thogotovirus GP close to the baculovirus fusion protein gp64 (data not shown)  and in one Rh. microplus tick. Interestingly, these two viruses are genetically close but presented only 82% of nucleotide identity in the RdRP gene. Thogotovirus-related virus was identified at low prevalence (<3%) in adult Rh. microplus ticks, while WhTV2-Thailand chuvirus was more prevalent (18%). CpTV2-Thailand virus was only detected in Rh. sanguineus ticks at a global prevalence of nearly 19%. Finally, WhTV1-Thailand virus was only detected in more than 21% of Rh. microplus ticks .Few coinfections of ticks were detected. The presence of two viruses (most frequently WhTV1-Thailand virus associated with LTPV-Thailand virus or WhTV2-Thailand virus) in the same individual tick was identified in 13 us ticks . Four coTicks harbor numerous EVEs within their genome  highly exposed to tick bites, and therefore could constitute putative novel tick-borne arboviruses, we developed LIPS-based serological screening against Thailand tick thogotovirus and LTPV-, BLTV4-, WhTV1-, WhTV2-, and CpTV2-Thailand viruses. Results are presented in p = 0.0003), while none of the sera in the exposed nor non-exposed groups exceeded the positivity threshold. Similarly, none of the cattle sera presented a luciferase activity higher than the positivity threshold (p = 0.05) in humans and cattle, respectively, meaning that these infections of vertebrates, if they occur, are very rare.For the tick/human interface, no significant difference was observed between exposed and non-exposed groups , and no hreshold , showingOrthomyxoviridae, Phenuiviridae, Rhabdoviridae, and Flaviviridae families) and a recently recognized viral family for which the zoonotic potential was still unknown (Chuviridae).The increasing accessibility of HTS technology and the resulting description of viral communities in various environmental samples pave the way for a better understanding of viral spillovers. However, identifying a list of viruses at higher risk of emergence requires first the characterization of the ecological cycle of the virus, including its putative reservoir hosts, hematophagous arthropod vectors (in the case of arboviruses), and vertebrate recipient hosts , which could suggest that they are tick endosymbionts (Bouquet et al., Rh. microplus ticks was inferior to 3%, suggesting that this virus possibly infects vertebrate hosts, according to Bouquet hypothesis linking prevalence of a given virus in ticks with its putative infectivity in vertebrates (Bouquet et al., The prevalence of Thailand tick thogotovirus in Although identifying novel zoonotic viruses is of great importance and a prerequisite to monitor silent viral emergences, it is of equal importance to determine, among the huge number of viruses composing the virome of vectors and reservoirs, which viruses are not able to cross the species because it could lead to a better understanding of the mechanisms underlying viral emergence, and especially to understand why some viruses that are phylogenetically close to zoonotic viruses are not able to spill over. The strategy developed in this pilot study starting from the inventory of viral communities infecting a given vector by HTS to finally identify reactive antibodies possibly present in exposed vertebrate populations by luciferase immunoprecipitation is well-adapted to such considerations and could easily be transposable to other vectors/mammals interfaces.MN095535 to MN095546.The datasets generated for this study can be found in the NCBI GenBank database The studies involving human participants were reviewed and approved by Ethical Committee of the Faculty of Tropical Medicine, Mahidol University, Thailand. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Department of Livestock Development, Thailand.ST, MDu, J-FC, MV-T, SMou, and ME designed and conceived the study. ST, DC, EDu, MDu, EDe, MDe, LY, and LG performed the experiments. ST and TB performed the bioinformatics analyses. MDe, SJ, AK, KC, J-FC, MV-T, and SMor realized fieldwork in Thailand. ST wrote the manuscript. ST, DC, TB, SP, MDu, SMor, SMou, MDe, and ME reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Viruses of wild and domestic animals can infect humans in a process called zoonosis, and these events can give rise to explosive epidemics such as those caused by the HIV and Ebola viruses. While humans are constantly exposed to animal viruses, those that can successfully infect and transmit between humans are exceedingly rare. The key event in zoonosis is when an animal virus begins to replicate (one virion making many) in the first human subject. Only at this point will the animal virus first experience the selective environment of the human body, rendering possible viral adaptation and refinement for humans. In addition, appreciable viral titers in this first human may enable infection of a second, thus initiating selection for viral variants with increased capacity for spread. We assert that host genetics plays a critical role in defining which animal viruses in nature will achieve this key event of replication in a first human host. This is because animal viruses that pose the greatest risk to humans will have few (or no) genetic barriers to replicating themselves in human cells, thus requiring minimal mutations to make this jump. Only experimental virology provides a path to identifying animal viruses with the potential to replicate themselves in humans because this information will not be evident from viral sequencing data alone. Virologists and epidemiologists view the emergence of new viral diseases in very different ways; this Essay describes two virologists' view of how animal viruses come to infect humans. We are constantly exposed to animal viruses through the food that we eat, the pets that we keep, and our interactions with nature. The vast majority of viruses that enter our bodies pass harmlessly through our gastrointestinal tracts or are destroyed by our immune systems. However, on rare occasions, an animal virus encounters a human host and begins to replicate itself, executing its entire lifecycle within human cells and expanding one virion into a population of many. Replication of an animal virus within the body of this first human subject is the key moment in the zoonotic process because it renders possible two things. First, the virus will now mutate and evolve under the selective constraints of the human body for the first time, adapting and improving itself for replication in this new host. Second, high virus titers produced by viral replication mean that spread to a second human is now possible, initiating selection for variants with increased capacity to spread in the human population.Significant effort has been put into understanding the factors that contribute to the spread of zoonotic viruses through the human population once an animal virus has begun the process described above. Factors that facilitate spread of viruses through populations can include high population density, the presence of viral vectors, and many others ,2. HowevFig 1) .–5.Fig 1)Fig 1) . AssuminFig 1) , there aFig 1) ,9. In coFig 1) ,10. ThisFig 1) ,11. A moBox 1). Viruses replicate themselves inside the cells of their hosts, and to do so, they must correctly execute tens to hundreds of protein–protein interactions. For instance, HIV is thought to interact with as many as 400 proteins in human T cells [Work in molecular virology has revealed why virus replication tends to be so specific for particular host species . Likewise, many species have been explored as models for human dengue virus infection, including pigs, monkeys, and rodents [Table 1). This explains one reason why mice are such a popular model organism: when no species can be found that will support the replication of a human virus, mice afford an opportunity to knock out host immunity genes in order to create an animal that will support viral replication. Additionally, mice can be modified to express human versions of proteins that support virus infection. For instance, creation of transgenic mice expressing human versions of the cellular receptors used by poliovirus and hepatitis C viruses leads to enhanced viral infection because these human viruses are not compatible with the mouse orthologs of their receptors (Table 1). In other cases, blocks to human viruses in animals are extreme, and it has been more practical to study related animal viruses in their natural hosts (Table 1). If we assume that the success of reverse zoonosis reflects the success of zoonosis, then decades of work modeling human viruses in animals would suggest that zoonosis is exceedingly difficult to achieve.Experimental virologists know all too well how difficult it is to get viruses to infect alternate hosts. Virologists attempt “reverse zoonosis” all the time in the process of establishing animal models for human viruses, with very poor success. It is almost always impossible to get human viruses to replicate unaltered in any laboratory animal precisely because most viruses are exquisitely adapted to their natural host. HIV is a prime example. For 30 years, scientists have tried to get HIV to replicate in laboratory animals , including numerous species of monkeys, rodents, shrews, dogs, cats, rabbits, pigs, and cows –18. In t rodents . Yet, ag rodents . In mice rodents  , then we would expect that zoonosis would occur more commonly from the animals that we domesticate, and indeed this does appear to be true [Alternately, perhaps humans have historically been infected by very few animal viruses  simply because the opportunity for animal viruses to infect humans is very limited. Certainly, an animal virus that never comes into contact with humans would have no path for infecting them. If opportunity is key to understanding the main bottleneck in zoonosis  harbors memory of past infections and ensures a robust and specific response upon secondary exposure. Adaptive immunity is likely important for limiting human disease caused by animal virus infection. However, infection with a new animal virus represents a first exposure, and the adaptive immune system will take several days to activate and mediate an effective response. By the time the adaptive immune response kicks in, a swarm of viral variants will exist in the newly infected human, and this swarm may contain variants with improved intracellular replication or even immune escape. Certainly, more work is needed in understanding the role of adaptive immunity in overcoming zoonotic infections. Since this essay especially focuses on the key event of initial viral replication cycles in a first human host, we can at least speculate that adaptive immunity is unlikely to be relevant here. Only after high virus titers have been reached will an adaptive immune response be mounted.Box 2). The result is that HIV is now insensitive to these factors in humans [Nonspecific immune barriers, including skin, mucosa, and chemical defenses , play a key role in blocking animal viruses at portals of entry into the human body. However, even if these barriers are penetrated and an animal virus gains access to human cells of its target tissue type, productive replication is still far from guaranteed. To launch even a first round of viral replication, animal viruses will need to avoid interaction with a suite of constitutively expressed human innate immunity proteins present inside of human cells, namely restriction factors and pathogen sensors that can activate the interferon response. If the animal virus is successfully recognized by any of these human proteins, this will potently block viral replication and will limit even initial rounds of animal virus replication. For example, several human restriction factors are efficient at blocking replication of primate-derived simian immunodeficiency viruses (SIVs), and this was important in the emergence of HIV in humans. SIV from chimpanzees (SIVcpz) adapted to overcome the human restriction factors APOBEC3H and Tetherin, giving rise to pandemic HIV-1 group M . Because the viral capsid protein interacts with RanBP2, we found that the viral adaptive mutations acquired during transmission to and between different ape species mapped to capsid [In addition to avoiding adaptive and innate immune defenses, zoonotic animal viruses must also successfully interact with and recruit tens to hundreds of host proteins that the virus needs in order to replicate itself in human cells . If the human version of even one of these essential host factors cannot be recruited by an animal virus because of differences in protein sequence between the human and animal version of that protein, then this, too, can impede viral replication. This is probably the main way that immunity to animal viruses differs from immunity to human viruses. Something as simple as a human transcription factor, mismatched to the animal virus attempting to use it, can be as potent in blocking viral replication as an immunity protein would be. For instance, the host transcriptional activator acidic nuclear phosphoprotein 32 family member A (ANP32A) is a key cofactor required for influenza virus replication ,56. AviaTable 2; ). WithouTable 2; –60. We ho capsid . Table 2Table 3), it is straightforward to identify the predominant virus families on which these efforts should be focused. Identifying genetic blocks to animal virus replication in humans will require significant laboratory experimentation. Furthermore, the human blocks will be unique for each animal virus of interest. While it may sound laborious, virologists are already well on their way to identifying genetic barriers that protect humans from key families of animal viruses (Table 2).The key to identifying the animal viruses that are most likely to harm humans boils down to understanding “thick” versus “thin” barriers to zoonosis. We must identify the human proteins  that are currently protecting us from each class of animal virus with high zoonotic potential. Such blocks may include restriction factors like APOBEC3H or Tetherin, or incompatible host proteins like ANP32A or RanBP2, all of which were discussed above. Based on this, we can then determine for which animal viruses our resistances are few (a thin barrier to zoonosis) versus many (a thick barrier to zoonosis). Based on historical zoonoses . But when coronoviruses are sampled from wild bats, a small number of them already encode spike variants compatible with human ACE2 [Dangerous animal viruses are those that require zero or only a few mutations in order to begin replicating themselves in human cells. They are dangerous because these mutational combinations might arise in the animal reservoir or in an intermediate host species, creating alive-on-arrival viral variants that can begin replicating upon encountering a human host. It is critical to note that these mutational combinations must arise without the advantage of being selected for within the human body, because selection can only occur after replication in humans has begun. Further, all necessary mutations for replication in humans would need to appear together in the same viral variant arising in nature. With these criteria in mind, it is easy to see why animal viruses are highly unlikely to randomly acquire compatibility with humans when there are many different host blocks to overcome. This is, in fact, the basis of successful treatment of HIV infections with multidrug cocktails; while HIV can easily mutate to escape one drug, no single viral genome acquires the multiple drug escape mutations required for viral replication in a multiple-drug–treated host. Alarmingly, some of the H5N1 influenza viruses that are currently circulating in the avian reservoir already harbor PB2 mutations that would make them compatible with the human ANP32A ,73. Likeman ACE2 ,75. The Within the vast constellation of host–virus interactions described herein, how do we find the human proteins that block replication of a particular animal virus of concern? Here, we have one critical advantage: only proteins that bind viruses differentially between the old  and new (human) hosts are relevant to zoonosis. A certain host transcription factor may be critically important to the replication of a virus, but if the virus can engage that transcription factor equally in its animal host and in humans, then it is not relevant to zoonosis. Indeed, the vast majority of mammalian proteins are highly conserved in sequence because they perform important functions that constrain their evolution. Viruses are expected to interact with animal and human versions of most host proteins equivalently. Thus, the vast majority of critical host–virus interactions will not be relevant to zoonosis.Table 2) are evolving under positive selection [However, in every host genome, some genes get trapped into a different mode of evolution in which they are selected for continual sequence change at the protein level. This occurs in genes that are engaged with ever-changing forces in the external world, with classic examples being genes that help us to discriminate new food sources, evade new predators, or resist new pathogens . These telection ,95,96,98election ). The beFig 2). Because of sequence divergence on the surface of cellular receptors, animal viruses often require compensatory mutations in their surface proteins before they can utilize the ortholog of their entry receptor in a new host species . Similarly, Ebola virus, presumably transmitting from bats, has also mutated to refine its interaction with the human ortholog of its NPC1 receptor after it entered the human population (Box 3) [Viral receptors serve as an excellent demonstration of this approach. One of the predominant themes that has arisen in the study of zoonosis is that cell-surface receptors are commonly protecting humans from infection by animal viruses. To identify and enter their target cells, viruses interact with receptors  expressed on the surface of host cells. We and others have observed that many host receptors for virus entry have evolved under intense selective pressure to modify the protein sequence on their virus-binding surface ,95–97,99ple, see ). Such aple, see ,102, andple, see  (Table 2 (Box 3) –71. But Kids and bats meet in a hollowed-out tree. In the 2013–2016 Ebola virus epidemic, the index case was a 2-year-old boy in the village of Meliandou, Guinea, who died on December 28, 2013 [Mops condylurus; [28, 2013 . Epidemidylurus; ). Althoudylurus;  and alsodylurus; . The tredylurus; .Did a viral variant arise in this tree that was capable of replicating in humans? Villagers report that both kids and bats had frequented this tree for many years. So, what exactly happened in December of 2013, when the toddler became infected with Ebola virus? Perhaps the bats in this tree had, themselves, only become infected recently or were shedding higher-than-normal levels of virus for some reason. But there is another possibility. Viruses exist as swarms of related genotypes in nature. These swarms are the pools from which variants with unique abilities can arise. Perhaps one of these bats was harboring a unique viral variant that was poised for success in a human host. We will never know for sure if the bats in this tree harbored Ebola virus or if the virus that they harbored was special in its sequence because the tree was burned before it could be properly investigated [stigated .Was Ebola virus perfecting itself for humans as the epidemic progressed? About 3 months after the first toddler died, sequence data from the epidemic revealed the appearance of a new mutation in Ebola virus genomes recovered from infected patients. This mutation caused a single amino-acid change, A82V, in the surface glycoprotein of Ebola virus, which increased viral fusion with human cells via the endosomal receptor NPC1 [tor NPC1 –71. Prestor NPC1 . TherefoIn summary, receptors and innate immunity proteins are prominent examples of proteins that block the replication of animal viruses in humans. But any cellular protein important to virus biology that has diverged significantly in protein sequence between the animal host and humans has the potential to act as a barrier to zoonosis. Other, unexpected types of host proteins have been identified that are important for viruses but that don’t translate from one host species to the next, such as DNA repair proteins , nuclear0) depends on two important factors: 1) how well the virus is adapted for transmission in the new host species and 2) the external  factors that facilitate spread in the new host. For instance, a less pathogenic influenza virus variant that causes lower levels of inflammation and cytopathic effects in the respiratory tract may not be transmitted well. This is because inflammation and cytopathology contribute to increased airway congestion, resulting in coughing fits, sneezing, and increased nasal secretions, which serve as major routes for influenza virus transmission. However, factors external to the virus are also integral to transmission. If a poorly spreading virus, like the influenza variant just mentioned, emerged in a densely populated megacity, then it might be capable of continued spread and further refinement in humans. On the other hand, if an influenza virus variant arises with characteristics that make it excellent at spreading in humans, but this event occurs in a remote part of the world with low population density, then the virus may falter very quickly.Once an animal virus has achieved measurable titers in its first human host, it is possible that a second human will become infected through contact with the first. Ultimately, this virus might go on to infect only a small handful of people or millions of people at the other extreme. Once epidemics have begun, the spread of viral pathogens through populations is an elongated and complex process, with many factors at play. At this point, the virus population will experience selection for variants with increased capacity to spread through the human population. As Don Ganem put it, “What evolution is operating on is not disease, disease is incidental. It operates on spread” . How welFig 3). These examples show how parameters extrinsic to the virus itself are critical to the spread of zoonotic viruses in humans [Unique human situations and behaviors are often factors in defining the scale of epidemics. The strong human desire to care for sick family members has promoted the spread of Ebola virus whenever it strikes, and it has been heartbreakingly stated that “one of the tragedies of Ebola is that it spreads through love” . The 191n humans . If we dn humans ,117.In summary, dangerous animal viruses are those that require few mutations in order to begin replicating themselves in human cells. These viruses are dangerous because the required combination of mutations might randomly arise in the natural reservoir. To identify these dangerous viruses, we must identify the human proteins  that are currently protecting us from each class of animal virus with high zoonotic potential and determine whether those are few or many. Dangerous viruses will be separated from us by only one or a few host blocks. Identifying human blocks to animal virus replication will require significant work, including the integration of viral surveillance with virologic experimentation in the lab. These efforts are worth it, not only because disease emergence is an important public health topic, but because zoonosis provides a unique example of evolution in action that teaches us about biology more generally.


In most epithelia, a series of Cl-/HCO3- exchangers and Cl- channels that mediate Cl- absorption and HCO3- secretion have been detected in the luminal and basolateral membranes. Slc26a9 belongs to the solute carrier 26 (Slc26) family of anion transporters expressed in the epithelia of multiple organs. This review summarizes the expression pattern and functional diversity of Slc26a9 in different systems based on all investigations performed thus far. Furthermore, the physical and functional interactions between Slc26a9 and cystic fibrosis transmembrane conductance regulator (CFTR) are discussed due to their overlapping expression pattern in multiple organs. Finally, we focus on the relationship between slc26a9 mutations and disease onset. An understanding of the physiological and pathophysiological relevance of Slc26a9 in multiple organs offers new possibilities for disease therapy.Transepithelial Cl Slc26a9 was cloned on the basis of its homology to other Slc26 isoforms. In humans, SLC26A9 maps to chromosome 1 and encodes a 791-amino-acid protein . SLC26A9us cells . Loriol trations . Second, channel , Cl-/HCOxchanger  and Na+ nsporter . Additiouctances .- secretion in cultured human bronchial epithelial (HBE) cells, and Slc26a9 has been suggested to functionally interact with CFTR in vitro  suggested that Slc26a9 can function as a Cl/HCO3 exchanger in gastric surface cells to support HCO3- secretion in the stomach, which can be inhibited by NH4+in vivo  infection due to the production of ammonia (NH3)/NH4+ through its urease activity, which may predispose patients to acidic injury and peptic ulcers by impairing Slc26a9-mediated gastric HCO3- secretion. However, a subsequent study showed that the mRNA and protein expression levels of Slc26a9 are not altered in gastric surface epithelial cells in Hp-infected mice, which might be a compensatory response to overcome the chronic inhibition of Slc26a9-mediated HCO3- secretion by the Hp infection  in infants with cystic fibrosis (CF)  treatments strongly reduced HCO3- secretion in the absence of Slc26a9 expression, suggesting that Slc26a9 plays an important role in orchestrating the acid-induced duodenal HCO3- secretory response  , which asis (CF) . Howeverresponse . Subsequresponse . The losan level . The ass3- secretion, in young female mice. This finding demonstrates that Slc26a9 plays a role in pancreatic ductal physiology despite the fairly low expression levels compared to those of CFTR and other electrolyte transporters. Furthermore, a slight reduction in the normalization of blood glucose levels was observed after an intravenous glucose bolus in older female mice. The female preponderance for an Slc26a9-related loss of ductal function is interesting in the context of the higher incidence of diabetes in female CF patients , the initial portion of the inner medullary collecting duct (IMCD), and principal cells . Functiopressure . HoweverHR cells . TherefoXenopus oocytes  database, Romero’s group found that slc26a9 polymorphisms cause several functional modifications, including increased or decreased Cl- current and Cl-/HCO3- exchanger activity and altered protein expression, which could lead to a spectrum of pathophysiologies. However, SLC26A9 is not directly associated with any disease  in the slc26a9 gene were significantly associated with increases in MI in CF infants  in patients with CF and mutations, resulting in improvements in pulmonary function  onset  onset , accordiS) onset . AlthougS) onset . The rolSlc26a9 is a member of a relatively new Slc26a gene family with multiple functions. Although extensive work has been performed to investigate the expression pattern, functional diversity, regulatory mechanism and pathophysiology of this novel protein in different organs by various systems, the exact physiological and pathophysiological role of Slc26a9 remains a mystery in many fields. Future work should not only further investigate its regulatory role in ion transport but also elucidate the characteristics of its non-ionic transport, i.e., Slc26a9 is possibly involved in volume control/apoptosis/migration/differentiation. Importantly, previous studies mainly focused on the role of Slc26a9 as a disease modifier of CF-related diseases, based on the overlapping expression pattern and function of the two proteins in some organs. However, an increasing number of functional studies have shown that Slc26a9 may also be involved in some non-CF-related diseases, such as Sjögren’s syndrome, schizophrenia and unreported diseases, including duodenal ulcers, Type 2 diabetes and hypertension. Therefore, basic and genetic research is required in the future to determine whether Slc26a9 can be a clinically relevant disease modifier or promising therapeutic target.XL and TL performed the work and contributed equally. All authors wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Heterobasidion spp. root rot fungi are highly destructive forest pathogens of the northern boreal forests, and are known to host a diverse community of partitiviruses. The transmission of these mycoviruses occurs horizontally among host strains via mycelial anastomoses. We revealed using dual cultures that virus transmission rates are affected by pre-existing virus infections among two strains of H. annosum. The transmission efficacy of mycovirus HetPV15-pa1 to a pre-infected host was elevated from zero to 50% by the presence of HetPV13-an1, and a double infection of these viruses in the donor resulted in an overall transmission rate of 90% to a partitivirus-free recipient. On contrary, pre-existing virus infections of two closely related strains of HetPV11 hindered each other's transmission, but had unexpectedly dissimilar effects on the transmission of more distantly related viruses. The co-infection of HetPV13-an1 and HetPV15-pa1 significantly reduced host growth, whereas double infections including HetPV11 strains had variable effects. Moreover, the results showed that RdRp transcripts are generally more abundant than capsid protein (CP) transcripts and the four different virus strains express unique transcripts ratios of RdRp and CP. Taken together, the results show that the interplay between co-infecting viruses and their host is extremely complex and highly unpredictable. Cryphonectria parasitica, by hypoviruses in Europe  and a capsid protein (CP)  of both strains have been characterized previously , and two moderately related partitiviruses, HetRV13-an1 and Heterobasidion partitivirus 15 strain pa1 (HetPV15-pa1) sharing 68% (aa) identity, being only distantly related (with 31% aa identity) to the other two viruses . HetPV11H. australe and H. parviporum hosting HetPV11-au1 and HetPV11-pa1 were cultivated on MOS agar plates. The dsRNA was isolated by cellulose affinity chromatography and agarose gel electrophoresis followed by purification  were used as viral hosts in this investigation   received viruses from 94233 infected by either HetPV11-pa1, HetPV11-au1, or HetPV13-an1  was tested using HetPV15-pa1 in co-infection with either HetPV13-an1, HetPV11-au1, or HetPV11-pa1 in donor 03021, and using 94233-32D as a recipient. This experiment was conducted using 20 replicates.In all experiments on MEA plates, mycelia was picked from recipient side of the culture using a sterilized Pasteur pipette with a diameter of 5 mm from 3 sampling points, unless the area occupied by the slowly growing recipient strain allowed sampling from only one spot. The three subcultures were then combined, cultured on modified orange serum (MOS) agar covered with a cellophane membrane and analyzed for the transmission of viruses using RT-PCR with both RdRp and CP primers . The sigH. annosum 03021 and 94233, three or four independently infected recipient subcultures from all obtained virus-host combinations were selected randomly for single hyphal tip isolations. The hyphal tips were collected under a binocular microscope with a modified Pasteur pipette using 1–3 day old cultures grown on 2% MEA as described by Vainio et al.  from each host/virus combination (either one or two viruses in the host) and partitivirus-free controls (94233-32D and 03021) as well as the original 94233 isolate with a natural infection of HetPV13-an1. The tested virus hosting isolates were obtained from the transmission experiments, and selected based on their apparent growth rates on single plates  to cover the range of variation for each host/virus combination. The inoculum size was 0.5 cm in diameter and picked as circular agar plug one centimeter from the edge of the fresh mycelium and placed at the center of a 2% MEA plate. Twelve independent biological replicates were used for each isolate.t-test in Microsoft Excel 2010  and statistical difference was tested using cel 2010 .Total RNA was extracted from fungal mycelia after one  or 2 weeks  growth on MOS agar plates  was made from 2 μg of DNase I treated total RNA using RevertAid First Strand cDNA Synthesis Kit  and random hexamer primers  as recommended by the manufacturer.® TA Cloning Kit  according to instructions by the manufacturer. The purified plasmids were then used in absolute quantification as shown by Jurvansuu et al. . EvaGreen® dye  was used in qPCR on Rotor-GeneQ  according to manufacturer's recommendations. PCR primers were used as follows: RNA polymerase II transcription factor (RNA pol2 TF) was used as a reference gene and specific primers based on RdRp and CP genomic segments of four virus strains including HetPV1-13an1, HetPV15-pa1, HetPV11-pa1, and HetPV11-au1 were used for virus transcripts . The CP u et al. . The noru et al.  and the MG948857 and MG948858. The CP encoding genome segments of HetPV11-au1 and HetPV11-pa1 share 97.2% nt and 99.8% aa sequence identity. Only one out of the total of 40 single nucleotide polymorphisms located in the CP ORF region results in an amino acid substitution, while the remaining ones are silent. As a comparison, the RdRp encoding genome segments of these virus strains share 97.7% nt sequence identity with 38 sequence polymorphisms in the ORF region causing five aa substitutions (99.2% aa sequence identity)  , and significantly lower similarity (27% or less) with other viruses such as Diuris pendunculata cryptic virus , Rhizoctonia fumigata partitivirus , Amasya cherry disease-associated mycovirus , Ceratobasidium partitivirus CP-b1 , and Cherry chlorotic rusty spot associated partitivirus . The RdRp aa identity between HetPV11 and HetPV1 was ~75% , HetPV15-pa1 to the hosts pre-infected with HetPV13-an1 (P < 0.001) or HetPV11-au1 (P < 0.001), HetPV11-au1 to the hosts with pre-existing viruses HetPV15-pa1 (P = 0.041) or HetPV11-pa1 (P < 0.001), and HetPV11-pa1 to the host containing HetPV11-au1 infection (P < 0.001).Statistical analysis on the differences between transmission rates to virus-free and pre-infected recipients indicated that six out of the 12 viral interactions were affected significantly by pre-infected viruses including transmission of HetPV13-an1 to pre-infected host with HetPV11-au1  compared to transmissions from the single infected strains. No transmission (0/20) was observed from donors with double infections by HetPV15-pa1 and HetPV11-pa1 (P < 0.001) or HetPV15-pa1, and HetPV11-au1 (P < 0.001)  .The transmission of both genomic segments RdRp and CP for all the replicates were screened by RT-PCR which consistently showed the transfer of both segments (and thus both particles) of each virus strain. Moreover, pairing tests and genotyping was conducted to confirm the recipient strain of all replicates of successful transmission.This part of the research, as a whole, showed that HetPV15-pa1 had a significant positive effect on the transmission frequency of HetPV13-an1 and vice versa, whereas coinfecting HetPV15-pa1 prevented completely the transmission of HetPV11 strains.The presence of HetPV13-an1 with or without co-infecting viruses caused an unusual hyphal morphology characterized by dense, stunted, and copiously branched hyphae, whereas the occurrence of other viruses or virus combinations was associated with normal hyphal morphology .H. annosum 94233 strains with 12 parallel measurements for each host/virus combination. H. annosum 94233-32D derivatives with single infection of HetPV13-an1 had 87 and 89% reduction in their growth rates  strains of 94233-32D had 84 and 95% reduction in their growth rates. The growth rates of these two strains differed significantly from each other and also from the controls and all other isolates  were studied using RT-qPCR for absolute quantification using the same fungal strains as in growth rate experiments. The amounts of transcripts of HetPV15-pa1 remain, on average, almost the same in single and coinfection with HetPV13-an1, although there was variation between the two independently created isolates . HoweverIndependent double infections by each of the highly similar strains of HetPV11 with HetPV13-an1 showed variable effects when compared with single infections of each of the three virus strains. Comparing the amounts of transcripts in coinfection to single infection, the quantity of viral transcripts for HetPV11-pa1 reduced drastically up to 9.6 and 40 times for CP and RdRp, respectively, whereas the CP and RdRp transcript levels of HetPV13-an1 were reduced only by 2 and 5 times, respectively , only RdConversely, the overall amounts of viral transcripts of HetPV11-au1 were reduced only by 4 times (but significantly) in both viral segments in coinfection, whereas HetPV13-an1 showed 1.3- and 2.5-fold reduction in CP and RdRp transcript quantities in coinfection, only the RdRp change being significant . No cleaOverall, the quantity of viral transcripts of the virus strains was affected by coinfecting viruses except in the case of HetPV15-pa1.H. annosum when present in the recipient  mutually hampered each other's transmission between two mycelia of Heterobasidion strains in laboratory and in nature, and even between related species  with 97% nt RdRp/CP similarity to HetPV13-an1 , whereas HetPV15-pa1 had similar amounts of viral transcripts in single and coinfection (with HetPV13-an1) situations. The RdRp/CP ratio, however, remained very similar for both HetPV13-an1 and HetPV15-pa1 as in single infection. Very interestingly, Wu et al.  showed tHeterobasidion spp. are complicated. Furthermore, especially the effects of HetPV13-an1 and HetPV15-pa1 on each other's transmission frequency and host phenotype makes them highly promising in terms of biocontrol against Heterobasidion spp. due to their simultaneous high transmission frequency and negative phenotypic effects.In conclusion, the interactions between partitiviruses of MG948857, and MG948858.The datasets generated for this study can be found in NCBI Genbank, MK, EV, and JH conceived and designed the experiments. MK and JJ performed the experiments. MK, JJ, EV, and JH analyzed the data and wrote the paper.MK, EV, and JH are employed by the Natural Resources Institute Finland which has a pending patent application on the combined use of two mycoviruses for biological control based on this work. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.