Virus entry into a susceptible cell is the first step of infection and a significant point at which infection can be prevented. To enter effectively, viruses must sense the cellular environment and, when appropriate, initiate a series of changes that eventually jettison the protective shell and deposit virus genes into the cytoplasm. Many viruses sense pH, but how this happens and the events that follow are often poorly understood. Here, we address this question for a large multilayered bluetongue virus. We show key residues in outer capsid proteins, a pH-sensing histidine of a zinc finger within the receptor-binding VP2 protein, and certain histidine residues in the membrane-penetrating VP5 protein that detect cellular pH, leading to irreversible changes and propel the virus through the cell membrane. Our data reveal a novel mechanism of cell entry for a nonenveloped virus and highlight mechanisms which may also be used by other viruses. Reoviridae, is an insect-borne, double-capsid virus causing hemorrhagic disease in livestock around the world. Here, we elucidate how outer capsid proteins VP2 and VP5 coordinate cell entry of BTV. To identify key functional residues, we used atomic-level structural data to guide mutagenesis of VP2 and VP5 and a series of biological and biochemical approaches, including site-directed mutagenesis, reverse genetics-based virus recovery, expression and characterization of individual recombinant mutant proteins, and various in vitro and in vivo assays. We demonstrate the dynamic nature of the conformational change process, revealing that a unique zinc finger (CCCH) in VP2 acts as the major low pH sensor, coordinating VP2 detachment, subsequently allowing VP5 to sense low pH via specific histidine residues at key positions. We show that single substitution of only certain histidine residues has a lethal effect, indicating that the location of histidine in VP5 is critical to inducing changes in VP5 conformation that facilitates membrane penetration. Further, we show that the VP5 anchoring domain alone recapitulates sensing of low pH. Our data reveal a novel, multiconformational process that overcomes entry barriers faced by this multicapsid nonenveloped virus.Bluetongue virus (BTV), in the family IMPORTANCE Virus entry into a susceptible cell is the first step of infection and a significant point at which infection can be prevented. To enter effectively, viruses must sense the cellular environment and, when appropriate, initiate a series of changes that eventually jettison the protective shell and deposit virus genes into the cytoplasm. Many viruses sense pH, but how this happens and the events that follow are often poorly understood. Here, we address this question for a large multilayered bluetongue virus. We show key residues in outer capsid proteins, a pH-sensing histidine of a zinc finger within the receptor-binding VP2 protein, and certain histidine residues in the membrane-penetrating VP5 protein that detect cellular pH, leading to irreversible changes and propel the virus through the cell membrane. Our data reveal a novel mechanism of cell entry for a nonenveloped virus and highlight mechanisms which may also be used by other viruses. The host cell entry mechanism of viruses, particularly large (>500 Å in diameter), nonenveloped capsid viruses, is a highly coordinated process, often engaging more than one viral protein with multiple conformational stages. Unlike enveloped viruses and smaller nonenveloped viruses, the atomic details of these processes for large, complex, nonenveloped viruses are largely unknown. Recently, we determined the structure of bluetongue virus (BTV), an 85-nm triple-layered complex capsid virus at atomic resolution, revealing key features of the outer capsid proteins that may facilitate the viral entry process .Orbivirus genus of the family Reoviridae, is an agriculturally and economically significant insect-borne virus that causes serious illness and death in sheep and other domestic and wild ruminants in many parts of the world. Infection of mammalian cells by the BTV particle is established when the two-layered inner capsid, the “core” of the double-capsid particle, is translocated across the endosomal membrane following virus uptake  structure of BTV, obtained by cryo-electron microscopy, revealed an outer shell formed by 120 globular trimers of VP5 and 60 triskelion-like VP2 trimers , unfurling K69-F354), and anchoring (I355-A526). The unfurling domain is helix rich, with two long horizontal helices and a stem helix. Two parallel β strands connect the unfurling domain with the anchoring domain via a third antiparallel β strand. The anchoring domain has a cluster of histidine located within the four antiparallel β strands, and an N-terminal β strand tethers the dagger domain. Previous data demonstrated that VP2 detaches from the BTV particle when treated with acidic pH and VP5 undergoes conformational change  protein  and the Surprisingly, all mutants could be recovered by reverse genetics with very similar phenotypes to the wild-type virus except H640A, which showed marginal attenuation  and 2a. Our overall data indicate that none of the His residues that were targeted for mutagenesis analysis are critical for VP2 function as a virus entry mediator; therefore, the major pH sensor required for VP2 conformational change in the endosomal compartment must lie elsewhere.The high-resolution structure of VP2 identified a typical zinc-finger motif at the junction of the body and the hub domains of each monomer, formed by residues C162, C617, C851, and H164, which is highly conserved among all 25 BTV serotypes . In contin vitro, that chelation of zinc led to VP2 instability manifested by altered properties of thermal denaturation  and the anchoring domain (ANC) . Many arMutations H272A and H319A within the UNF and H365F and H412F located in the ANC domain failed to generate any viable virus . One mutTo ensure that failure to recover H272A and H365F viruses or the highly attenuated H465F virus was not due to a disruption of mutant protein expression and that VP5 was still processed correctly in the expressing cells, BSR cells were transfected with the mutant RNA segments together with the other nine BTV RNA segments, and VP5 distribution was assessed by immunofluorescence. This confirmed that expression levels were comparable to the wild-type VP5 . When thTo examine if the mutation affected the particle to PFU ratios of the highly attenuated mutant viruses, we determined the number of particle versus PFU of one representative mutant virus, H465F, which showed an average particle/PFU ratio of approximately 278. This was significantly higher than that of the wild-type virus which had an average particle/PFU ratio of 1.2 , indicatFurther, to ensure H385A did not impact the overall structure of VP5, which might have caused failure of virus recovery, the mutant was expressed using the baculovirus expression system, and while protein expression was lower , the abiImaging of the BTV particle in low pH by cryo-electron microscopy (cryo-EM) revealed that while the anchoring domain of VP5 remained attached to the underlying VP7 layer, both the dagger and unfurling domains refolded to form a flexible barb-like structure, potentially the membrane fusion form . To confThe high-resolution cryo-EM structure has made it possible to deduce how the outer capsid proteins VP2 and VP5 of BTV coordinate the process of cell entry. A comprehensive structure-function analysis of residues in VP2 and VP5, predicted to be key to function, revealed a novel role for the unique zinc finger found in VP2 and certain residues within a His cluster found in VP5. These roles relate to the disengagement of VP2 from the complete virion in the early endosome followed by the activation of the now exposed VP5 in the late endosome. This stepwise and concerted entry mechanism requires the ability to accurately sense the relative pH at each location. Despite apparently favorable locations at the interface of the VP2 hub domain with the top of VP7, no role was found for histidine in this location. This is somewhat surprising, as H866 is conserved across all BTV serotypes and had been previously proposed to play a role in the entry process . SimilarOur analysis also revealed how VP5 senses the late endosomal pH to trigger membrane permeabilization. VP5 contains a total of 19 His residues, 13 of which are clustered at the interface of the anchoring domain and the helices of the unfurling domain. This His cluster previously speculated as functional within the ANC domain was investigated. Most of the His residues in the ANC domain rendered the virus inviable or attenuated although the expression and integrity of VP5 are not significantly affected when mutated singly, indicating the location of each histidine might be more important than the cluster itself, as previously speculated , and expOur comprehensive molecular analysis reveals key amino acids of VP2 and VP5 required for detecting cellular pH, leading to an irreversible change that propels the virus through the cell membrane. Our data illustrate a novel function of zinc finger in sensing pH and identify for the first time the key residues essential for cell entry by BTV. Surprisingly, the amino acid residues that we identified as essential for BTV entry lie outside those predicted previously based on the virus structure. Our data further highlight the dynamic nature of the multiconformational process required for virus infection and indicate a potential mechanism that may be shared by other similar viruses, and may be targets for future therapies.Site-directed mutagenesis, as previously described , was perBSR cells were infected with wild-type or mutant virus at a multiplicity of infection (MOI) of 0.1 for 0, 24, 48, and 72 h in triplicate. Supernatant virus was collected at each time point and the titers were determined by plaque assays to generate the growth curve.2. It was then desalted with a PD-10 desalting column . VP5 ANC (P348-A526) in the pET28 backbone was expressed in BL21(DE3) pLysS cells (Invitrogen) induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 4 h. Cells were lysed in lysis buffer , and the lysate was applied to cobalt resin (Sigma) for His-tagged protein purification.Wild-type or mutant VP2 and VP5 proteins were expressed by infecting sf9 cells at an MOI of 5 with recombinant baculovirus for 48 h. Cells were lysed in the lysis buffer , and the S-tagged protein was purified using the S-protein agarose (Merck Millipore) and eluted with 3 M MgClA total of 20 μl of purified protein at 0.2 mg/ml was mixed with 5× SYPRO orange (Invitrogen). For the metal-binding experiments, VP2 was either untreated or incubated with Chelex-100 resin (Bio-Rad) and 1× DTT for 1 h. For the pH experiments, protein was acidified to the stated pH with 0.1 N HCl. The assay was performed on an MX3005P q-RT PCR system (Agilent Technologies) with the temperature ramped from 25 to 95°C at 45 s/°C as previously described  SDS gel (Invitrogen) using a guinea pig anti-VP5 antibody. The protein was prepared in NuPAGE lithium dodecyl sulfate (LDS) sample buffer (Thermo Fisher) with or without DTT and with or without heating at 100°C for 5 min. The bands were analyzed using GeneTools (SynGene) to determine the monomeric and trimeric VP5.A BSR monolayer transfected with 1 μg of wild-type or mutant S5 together with nine other BTV RNA segments using EndoFectin according to manufacturer’s instruction (GeneCopoeia) was fixed in 4% paraformaldehyde and then permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS). The guinea pig anti-VP5 antibody was used to detect VP5, using the relevant secondary fluorescent antibody. Nuclei were stained with Hoechst. Images were captured using the LSM510 inverted confocal microscope (Carl Zeiss Ltd.).2HPO4, and 2 mM KH2PO4) was prepared using a mini extruder with a 0.1-μm polycarbonate membrane (Avanti Polar Lipids). Unencapsulated calcein was removed by size exclusion chromatography. Purified WT or mutant VP5 or ANC was mixed with calcein-loaded liposomes at a final concentration of 0.1 mg/ml and incubated at room temperature for 10 min. The mixture was then acidified to the stated pH with 0.1 M HCl and incubated for 20 min at 37°C. Fluorescence was measured, and the percentage of calcein release was calculated as previously described  in 1 ml of the calcein buffer  in U-bottom 96-well plate and incubated for 1 h at room temperature as previously described . Then, tViral RNA was extracted using the GeneJET viral DNA and RNA purification kit (Thermo Fisher) following the manufacturer’s instruction. A total of 10 μl of eluent was used for cDNA synthesis using the RevertAid reverse transcriptase (Thermo Fisher) with a BTV S6 (NS1)-specific primer (GTAAGTTGAAAAGTTCTAGTAG). A total of 1 μl of 1:5 diluted cDNA was then used for quantitative PCR (qPCR) using the Maxima SYBR Green/ROX qPCR 2× master mix (Thermo Fisher) with primers  targeting the S6, following the manufacturer’s instruction. The viral particle number was estimated on the basis of a viral genome copy number determined by qRT-PCR.


Culex pipiens and Culex torrentium, collected in south-central Sweden. Following bulk RNA-sequencing (meta-transcriptomics) of 12 libraries, comprising 120 specimens of Cx. pipiens and 150 specimens of Cx. torrentium, we identified 40 viruses (representing 14 virus families) of which 28 were novel based on phylogenetic analysis of the RNA-dependent RNA polymerase (RdRp) protein. Hence, we documented similar levels of virome diversity as in mosquitoes sampled from the more biodiverse lower latitudes. Many viruses were also related to those sampled on other continents, indicative of a widespread global movement and/or long host–virus co-evolution. Although the two mosquito species investigated have overlapping geographical distributions and share many viruses, several viruses were only found at a specific location at this scale of sampling, such that local habitat and geography may play an important role in shaping viral diversity in Culex mosquitoes.Mosquitoes harbor an extensive diversity of ‘insect-specific’ RNA viruses in addition to those important to human and animal health. However, because most studies of the mosquito virome have been conducted at lower latitudes, little is known about the diversity and evolutionary history of RNA viruses sampled from mosquitoes in northerly regions. Here, we compared the RNA virome of two common northern mosquito species,  Culex  comprises more than a thousand species, with representatives found globally [Culex species are vectors of a number of important pathogens including West Nile virus (WNV) (Flaviviridae), Japanese encephalitis virus (JEV) (Flaviviridae), and Sindbis virus (SINV) (Togaviridae), as well as a variety of nematodes [Culex species is the Northern House mosquito, Cx. pipiens, which is distributed across the northern hemisphere. In Europe and the Middle East, it occurs together with Cx. torrentium, another Culex species with females and larvae that are morphologically identical to Cx. pipiens. These two species have overlapping distributions and share larval habitats. However, Cx. torrentium dominates in Northern Europe while Cx. pipiens is more abundant in the south [Cx. pipiens is one of the most common WNV vectors in both Southern Europe and North America, while Cx. torrentium is the main vector of SINV in Northern Europe [Cx. pipiens and Cx. torrentium due to the difficulties in species identification, which can only be reliably achieved through molecular means. Much of the biology of these species, such as their larval habitat and feeding preferences, is considered similar. However, one important difference between the two species is that while Cx. pipiens harbors a high prevalence of the intracellular bacteria Wolbachia pipientis, it is seemingly absent in Cx. torrentium [The mosquito genus globally . Culex sematodes ,2,3. Onehe south . Both sphe south . Cx. pipn Europe . Infectin Europe ,8. Despirrentium .Flaviviridae, Togaviridae, and the Bunyavirales [Cx. pipiens and Cx. torrentium are two common Culex species in Northern and Central Europe, and known vectors of SINV and WNV, they were chosen for RNA virome investigation and comparison by RNA-Seq.In recent years, studies utilizing RNA-sequencing  have revealed an enormous RNA virus diversity in both vertebrates and invertebrates ,11. Mosqavirales ,14,15,16avirales , and theavirales . A centrCx. pipiens/torrentium mosquitoes were removed to enable molecular identification to species [Mosquitoes were collected from two regions in Sweden: (i) from floodplains of the Dalälven River in central Sweden  in 2006, 2009, and 2011; and (ii) around the city of Kristianstad, in southern Sweden  in 2006 and 2007. Collections were performed using Centers for Disease Control and Prevention-light traps baited with carbon dioxide, and catches were sorted and identified to species on a chilled table, using keys by Becker et al. . In totaCx. torrentium (n = 150) and Cx. pipiens mosquitoes (n = 120)  following the manufacturer’s instructions. Three pools, L1 and L2 for Cx. torrentium and L3 for Cx. pipiens, respectively, were previously shown to be positive for SINV and were included as a reference for RNA viral diversity in the presence of a pathogen. The extracted RNA was subsequently DNased and purified using the NucleoSpin RNA Clean-up XS kit . Prior to library construction, ribosomal RNA (rRNA) was depleted from the purified total RNA using the Ribo-Zero Gold (human–mouse–rat) kit  following the manufacturer’s instructions. Sequencing libraries were then constructed for all rRNA-depleted RNA-samples using the TruSeq total RNA library reparation protocol (Illumina). All libraries were sequenced on a single lane  on an Illumina HiSeq X10 platform. Library preparation and sequencing was carried out by the Beijing Genomics Institute, Hong Kong. All 12 libraries were quality trimmed with Trimmomatic v.0.36 [Total RNA was extracted from 12 pools from the homogenate of individual n = 120) , using tc v.0.36 , using dc v.0.36 , employi−5 in both cases. To determine whether some of the assemblies represent potential endogenous viral elements (EVEs), all virus viral assemblies were blasted against the Culex quinquefasciatus reference genome (GCA_000209185.1). Assemblies identified as RNA viruses were screened against the NCBI Conserved Doman Database with an expected value threshold of 1 × 10−3 to identify viral sequence motifs. The mitochondrial cytochrome c oxidase I (COX1) gene, mined from the sequence data, and all contigs with RdRp-motifs was mapped back to all quality trimmed libraries to estimate abundance using Bowtie2 [Wolbachia bacteria in the libraries, published sequences of the Wolbachia Cx. pipiens wsp surface protein gene (DQ900650.1) and the mitochondrial COX1 gene (AM999887.1) were mapped backed against all libraries using the above criteria for abundance and presence/absence.Trinity assemblies were screened against the complete non-redundant NCBI GenBank nucleotide (nt) and protein (nr) databases using blastn, primarily to identify closely related RNA viruses and false-positive host-derived hits, as well as a diamond  blastx a Bowtie2 , employi Bowtie2 ,25. SuchAlphaviridae, (Order) Bunyavirales, Endornaviridae, Luteoviridae, (Order) Mononegavirales, Nido-like viruses, (Order) Orthomyxovirales, Partitiviridae, Picornaviridae, Qin-like viruses, Reoviridae, Totiviridae, Tymoviridae and Virgaviridae and Negev-like viruses. All RdRp amino acid sequence alignments were performed using the E-INS-i algorithm in Mafft [The evolutionary  history of the viruses discovered were inferred by aligning protein translated open reading frames with representative sequences from the in Mafft . Poorly in Mafft  employinTo help assess whether the novel viruses discovered were likely mosquito associated, that is, to distinguish those that actively replicate in the host from those present in diet mosquito or a co-infecting micro-organism, we considered four factors: (i) the abundance of viral contigs per total number of reads in a library ; (ii) if the abundance of the virus was higher in relation to the host COX1 gene; (iii) presence in the individual sequencing libraries ; and (iv) clear phylogenetic clustering with other mosquito-derived viruses . A mosquito association was tentatively assigned if a particular virus met two or more of the four criteria.The raw sequence data generated here have been deposited in the NCBI Sequence Read Archive (BioProject: PRJNA516782) and all viral contigs have been deposited on NCBI GenBank (accession numbers: MK440619–MK440659).Cx. pipiens and Cx. torrentium, collected from central and southern Sweden . For each viral family/order, between one and five virus species were identified and in total 28 novel RNA virus species were discovered here, which were named based on geographical location.We characterized the RNA viral transcriptome of two mosquito species, n Sweden . After hCx. torrentium were characterized by a higher number of viral reads compared to non-viral reads  was at low abundance, was not present in several libraries, and clustered with viruses derived from various environmental samples, suggesting that it is not mosquito associated. Similarly, although Gysinge virus  was abundant and present in several libraries , presence across multiple libraries, and phylogenetic relationship to other viruses . If a paectively . The hosibraries , its cloibraries  was a soibraries  was abunCx. torrentium carried four viruses of markedly higher abundance compared to Cx. pipiens: (i) Nam Dinh virus  reads, respectively, in library L2); (ii) Biggie virus ; as well as two newly identified viruses, (iii) Valmbacken virus ; and (iv) Jotan virus   . Cx. piprrentium , althougCx. pipiens this analysis revealed a total of 20 virus species from Kristianstad, 12 of which were unique to Cx. pipiens and five detected in Kristianstad only, all of which were unique to Cx. pipiens: Asum virus (Bunyaviridae), Eskilstorp virus (Chrysoviridae), Kristianstad virus (Bunyaviridae), Rinkaby virus (Virga–Negev virus), and Vittskovle virus (Qinvirus). A total of 28 viruses were found in Cx. pipiens from Dalälven. Eleven of these were unique to Cx. pipiens and four were unique to Cx. pipiens from Dalälven: Salari virus , Sonnbo virus (Partitiviridae), Culex mononega-like virus 1 , and Berrek virus (Luteoviridae)  . A similrrentium . Hence, Culex associated viruses within particular viral families, such as the Merida virus and Gysinge virus , and Tarnsjo virus   . In contn = 8), whereas the others fell within Nidovirales (n = 1), Luteoviridae (n = 4), Picornavirales (n = 2), and Togaviridae (n = 1), respectively , as well as those without clear host associations. For example, Biggie virus clusters in a distinct group of Biggie viruses (Virga/Endorna-viridae) sampled from other Culex mosquitoes [Endornaviridae—specifically the Kerstinbo virus and Hallsjon virus—that do not cluster with other arthropod-associated viruses , Mononegavirales (n = 5), Qin-like viruses (n = 4), and Orthomyxoviridae (n = 2)  and a number of novel viruses such as Anjon virus .In total, we identified 16 negative-sense RNA viruses, including nine novel viruses:  (n = 2) . As was Mononegavirales, Merida virus, Culex mononega-like virus 2, Culex mosquito virus 4, and Culex mononega-like virus 1 have previously been described in mosquitoes [Within the order squitoes ,15,29. AAedes and Culex mosquitoes in Australia [Cx. pipiens and Cx. torrentium libraries and was also more abundant than host non-RNA in the Cx. torrentium libraries , Reoviridae (n = 1), and Toti/Chrysoviridae (n = 5). For the family Reoviridae, Valmbacken virus clustered with Aedes camptorhynchus reo-like virus, previously discovered in mosquitoes [Culex associated reovirus  genes of Wolbachia pipientis. Although it is not possible to completely exclude the presence of other Wolbachia variants, our results suggest that differential presence/absence of Wolbachia has not affected the observed patterns of viral diversity and abundance.Finally, all sequencing libraries generated here were negative for Culex mosquitoes collected in Sweden we identified 40 viruses, including 28 that are novel. A virome comparison between the two vector species Cx. pipiens and Cx. torrentium revealed that although these mosquitoes are from the same genus and have overlapping geographical distribution, the virome family and species composition and abundance differed to some extent between the two species, and also by geographic location, at this scale of sampling , such that analyses of relative abundance and diversity are better conducted at the species level. Viewed at the level of species per region, we identified several viruses that were seemingly unique to their respective sampling location  was found in high abundance and in multiple libraries but did not cluster with any viruses that originated from mosquitoes, although it did group with other arthropods (Cx. torrentium. It is clear that many viruses are seemingly ubiquitous in mosquitoes, covering a wide variety of climates and habitats [Given their relatively close phylogenetic relationship , and thesociated , Table 3thropods . The fachabitats ,15,31, bhabitats  and havehabitats ,34. Relahabitats ,36, we cCx. torrentium and Cx. pipiens is interesting. It has previously been shown that Cx. pipiens is commonly infected with Wolbachia, while this bacterium is absent from Cx. torrentium [Wolbachia is well-known for its ability to block virus infection in some mosquito species, although it has mostly been studied in systems with pathogenic viruses such as dengue [Wolbachia pipientis, we found no compelling evidence for Wolbachia in any of the Culex samples studied here. Notably, our study reveals that pathogenic viruses such as SINV can sometimes have similar abundance levels to viruses not associated with human disease , in turnrrentium . Wolbachs dengue . AlthougCx. pipiens and Cx. torrentium has long been ignored, largely because of the need for molecular demarcation, so that it has been assumed that most of the biology of the two species is comparable. Our study indicates that these two species have differing virome compositions and also that Culex mosquitoes in northern temperate regions can harbor similar viral diversity as mosquitoes in tropical and sub-tropical regions. Further studies should consider the host range of these viruses, their potential interactions with pathogenic viruses, and how virome composition is determined by mosquito host structure and feeding preference. Isolating the viruses discovered here will also be a key priority to enable a better understand of mosquito–virus co-evolution. Ultimately, it will be essential to identify the key evolutionary, ecological, and environmental factors that determine virome composition, as well as the impact of virome composition on the mosquito.The species separation between


We detected 2936 large and rare CNVs events . Case-control burden analysis and generalized linear regression models revealed significant association of CNVs with GSD in men, with the strongest effect observed with CNVs overlapping lipid metabolism genes . Our results indicate a clear link between CNVs and GSD in men and provides additional evidence that the genetic components of risk for GSD are complex, can be sex specific and include CNVs affecting genes involved in lipid metabolism.Gallstones Disease (GSD) is one of the most common digestive diseases requiring hospitalization and surgical procedures in the world. GSD has a high prevalence in populations with European or Amerindian ancestry (10–20%) and the influence of genetic factors is broadly acknowledged. However, known genetic variants do not entirely explain the disease heritability suggesting that additional genetic variants remain to be identified. Here, we examined the association of copy number variants (CNVs) with GSD in a sample of 4778 individuals (1929 GSD cases and 2849 controls) including two European cohorts from Germany ( Gallstones disease (GSD) is a multifactorial chronic metabolic disease characterized by the development of calculi inside the gallbladder composed mainly by cholesterol. GSD features a long silent progression where the accumulation of stones in the affected gallbladder can lead to clinical symptoms such as recurrent episodes of intense abdominal pain (biliary colic) and major complications, like obstruction and infection of the biliary tree (cholestasis and cholangitis), acute pancreatitis, and gallbladder cancer . At leasABCG5/8 gene  ATP8B1) gene [RB1) gene [HNF1A) gene [SULT2A1) gene [APOL1 and APOL2) involved in lipid transport. In addition, we note that the 23 candidate genes were enriched with 7 genes extremely intolerant to loss-of-function genetic variation  and variants within these genes are rarely found in the general population [The observed associations could not be attributed to one CNV nor to an specific gene and thus CNVs may confer risk to GSD through multiple highly rare and penetrants events. In an effort to identify genes with the strongest potential to be involved in the development of GSD by a CNV-related mechanism, we highlight 23 genes involved in lipid metabolic process, which are expressed in the small intestine and exclusively affected by CNV in cases. Direct functional links between the candidate genes and the etiology of GSD is available for: the lipid transporter ATPase phospholipid transporting 8B1, (B1) gene , the RB B1) gene , the hep1A) gene , the bilA1) gene , and thepulation . We hypoAs suggested by CNVs overlapping nonlipid metabolic process genes that are expressed in the small intestine Fig.  and the In summary, our study provides the first evidence of association of CNVs with GSD and will contribute to better understand the etiology of this complex and common metabolic disorder.Supplementary Material


Homo sapiens assures that much better translatability of research outcomes from model studies involving human diseases can be achieved than from those generated with other pre-clinical systems. Our group and others used during past two decades NHPs in research directed towards viral and autoimmune disorders of the gastrointestinal tract. This review summarizes progress made in the area of enteric viral infections including its applicability to human disease.There is an important role non-human primates (NHP) play in biomedical research. Phylogenetic proximity of any of the NHP species to  Since tChlorocebus pygerythrus), Rhesus Macaque (Macaca mulatta), and Pigtailed Macaque (Macaca nemestrina) [The main two groups of enteric viruses discussed and further specified in this section  were derived from the following NHP species: Vervet Monkey (estrina) ,9,10,11.estrina) ,12.Burkholderia psedomallei (http://www2.tulane.edu/tnprc/microbiology/resources/).To maintain the colonies of captive NHPs free of microbial pathogens, the following four “conventional” simian pathogens are being tested at the U.S. National Primate Research Centers to assure seronegative status: (1) Simian immunodeficiency virus (SIV); (2) Simian T lymphotropic virus type 1 (STLV); (3) Simian retrovirus type D (SRV); and (4) Herpes B virus. With continuous improvement of primate-specific diagnostic assays and increased demand for disease-free primates in biomedical research, some primate centers also raise expanded SPF colonies with voluntary diagnostic screening for following eight pathogens: (5) Simian foamy virus (SFV); (6) Primate cytomegalovirus (CMV); (7) Rhesus rhadinovirus (RRV); (8) Simian varicella virus (SVV); (9) Simian vacuolating virus 40 (SV40); (10) Lymphocytic choriomeningitis virus (LCV); (11) Measles virus; and (12) According to epidemiological surveys conducted during recent years by our group with participation of three National Primate Research Centers and three zoos in the U.S., a seasonal incidence of viral diarrhea-associated disease exists at captive NHP colonies ,8. DespiRotaviruses, along with noroviruses, are according to U.S. Centers for Disease Control and Prevention (CDC) the most common etiological agents of diarrhea in infants and young children worldwide. Both rotaviruses and noroviruses have also been isolated from monkeys, domestic mammals, plus rotaviruses also from birds and other species. It has been estimated that in Asia, Africa and Latin America more than 1 billion cases of diarrhea each year takes place with 2–3 million deaths. The majority of deaths occur in children less than five years of age. About half of all cases of severe diarrhea are caused by rotaviruses. As also reported by CDC, human noroviruses are annually worldwide responsible for more than one million hospitalizations and over 200,000 deaths in children less than five years of age. In the U.S. alone, an estimated 23 million cases of acute gastroenteritis, including 70,000 hospitalizations and 800 deaths, are attributed to human norovirus infections.The first rotavirus strain isolated from NHP species was the SA11 . SA11 waWhen intragastrically inoculated into serum antibody-negative juvenile (<6 months-old) macaques, with no evidence of previous rotavirus infection, TUCH rotavirus was shown to consistently produce asymptomatic intestinal infection associated with peripheral, cell-mediated and antibody responses, as well as high levels of virus shedding ,16. The RRV and TUCH rhesus-derived rotaviruses are currently being used in translational pathogenesis studies with murine model of biliary atresia, i.e., a neonatal obstructive cholangiopathy ,19,20. INo robust human-like models exist to study the pathogenesis and immunity of human noroviruses, although studies are being conducted with human volunteers, mice models and intestinal organoids ,27,28,29According to conventional pathogenesis paradigm concerning enteric viruses, small intestinal epithelium is the primary target of infection. Nonetheless, most of the attempts to demonstrate replication of enteric caliciviruses inside the enteric epithelium failed. The confocal microscopy imaging of infected intestinal tissues revealed that mouse noroviruses are crossing the intestinal epithelium while being carried by some bacterial species of gut microflora that express virus-binding histo-blood group antigens (HBGA) . It was As the example of such experiments, ReCV macaque model could be used to narrow down the taxa of primate gut microbiome that were suggested to facilitate transcytosis of human noroviruses across small intestinal epithelium . We haveClinical studies with direct enrollment of patients are the important part of biomedical research. However, these studies need to be conducted in concert with pre-clinical trials  that utiFrom the taxonomic groups of enteric viruses with capability to cause gastroenteritis in humans and animals, the following were confirmed in biological specimens obtained from captive or semi-wild NHPs: enteric adenoviruses, enteroviruses, picobirnaviruses, coronaviruses, noroviruses, sapoviruses, astroviruses, anelloviruses, smacoviruses and parvoviruses ,40,41,42Gorilla gorilla) and chimpanzees (Pan troglodytes), an abundance of viral families previously associated with gastrointestinal tract infections such as Herpesviridae and Reoviridae (gorillas), or Circoviridae and Adenoviridae (chimpanzees) were found increased [Several studies suggested that enteric virome might be in primates affected by immunodeficiency and/or autoimmunity in a putative gut dysbiosis fashion ,44,45,46


To understand the public sentiment regarding the Zika virus, social media can be leveraged to understand how positive, negative, and neutral sentiments are expressed in society. Specifically, understanding the characteristics of negative sentiment could help inform federal disease control agencies’ efforts to disseminate relevant information to the public about Zika-related issues.The purpose of this study was to analyze the public sentiment concerning Zika using posts on Twitter and determine the qualitative characteristics of positive, negative, and neutral sentiments expressed.Machine learning techniques and algorithms were used to analyze the sentiment of tweets concerning Zika. A supervised machine learning classifier was built to classify tweets into 3 sentiment categories: positive, neutral, and negative. Tweets in each category were then examined using a topic-modeling approach to determine the main topics for each category, with focus on the negative category.A total of 5303 tweets were manually annotated and used to train multiple classifiers. These performed moderately well (F1 score=0.48-0.68) with text-based feature extraction. All 48,734 tweets were then categorized into the sentiment categories. Overall, 10 topics for each sentiment category were identified using topic modeling, with a focus on the negative sentiment category.neural defects,Zika abnormalities, and reports and findings. These broad themes were based on domain expertise and from topics discussed in journals such as Morbidity and Mortality Weekly Report and Vaccine. As the majority of topics in the negative sentiment category concerned symptoms, officials should focus on spreading information about prevention and treatment research.Our study demonstrates how sentiment expressed within discussions of epidemics on Twitter can be discovered. This allows public health officials to understand public sentiment regarding an epidemic and enables them to address specific elements of negative sentiment in real time. Our negative sentiment classifier was able to identify tweets concerning Zika with 3 broad themes:  Zika was discovered in 1947 in Uganda . The firIn our previous exploratory study , we collIdentifying sentiment on a specific topic was pioneered by Chen et al ,6. SinceZika. Of those 500 images, only 342 were related to Zika. Of those 342 images, 193 were coded as health and 299 were coded as public interest. Of the health images, the majority related to transmission and prevention, which is similar to what we found in our previous study on Twitter . TheWithin the tweets with negative sentiment, the perplexity decreased rapidly until about 10 topics and then leveled off . The perThe results of the LDA are discussed below for the positive, neutral, and negative categories. Themes and topics for all 3 sentiment categories were determined by an epidemiology expert based on the words given for each theme and some sample tweets containing those words. First, the topics for the positive and neutral categories will be briefly discussed. The tables, including the theme names, topic words, and example tweets for the positive and neutral topic models, are available online . Then, amagic bullet is actually a macromolecule that will attach to the surface of any virus and prevent it from attaching to a human cell [magic bullet is used [Within the positive sentiment themes, there were 4 broad qualitative topics within the 10 topics chosen using the perplexity measure with LDA: mosquito-killing methods, models to help understand the Zika virus, detection of the Zika virus in cells, and treatment and prevention discoveries . These bman cell . If the  is used . In the understand, develop, hope, discover, benefit, and reveal, among others. While themes in the positive sentiment category mainly addressed research to treat Zika and prevention methods, themes in the neutral category mostly comprised posts from news agencies stating facts.Overall, the broader themes in Within the neutral sentiment topics, there were 3 broader qualitative themes: public health messages, knowledge gaps, and Zika characteristics . In the In this case, the broader themes in neural defects caused by Zika infection, abnormalities because of Zika infection, and reports and findings concerning the Zika virus. Topics #1 brain defects, #2 neurological effects, #5 fetal effects, and #8 Guillain-Barré syndrome all concerned the nervous system. Topics #6 Zika abnormalities and #9 Zika effects were both related to abnormalities resulting from Zika infection. Topics #3 initial reports, #4 Zika impact, #7 ultrasounds, and #10 dengue association all concerned reports and findings concerning the Zika virus. There was significant overlap between topics #3 and #4 because they both addressed reports and findings concerning the Zika virus. However, topic #3 initial reports included tweets stating the locations where Zika is spreading, whereas topic #4 Zika impact included tweets concerning the BBC article that describes Zika as scarier than initially thought [Before data analysis, we had chosen to focus on the topics from the negative sentiment category specifically in the symptoms category from our previous paper  as that The broader themes in a? Evidence.”neural defects, Zika abnormalities, and reports and findings, will then be explored and discussed in more detail.In the discussion section, we will address one cause of tweets being misclassified with some examples. The 3 negative sentiment broader themes, active, saliva, feds, busted, beast, and prenatal, which were not seen by the model because the count of these words is less than the minimum count (set to 5) parameter given in the Word2vec model and hence were discarded. The minimum count was set to 5 (the default setting in Gensim) as words used fewer than 5 times do not add significant information to the analysis [As seen in analysis . Adding analysis . As thesIn this section, we focus on the negative sentiment topics of neural defects, Zika abnormalities, ultrasounds, and dengue association. These themes and topics were chosen for discussion because they were topics of public concern, have been addressed by the CDC or WHO ,40-44, abrain defects), #2 , #5 , and #8 (Guillain-Barré syndrome) all concern the neural system. For example, topic #1, brain defects, points to brain damage in babies because of microcephaly as seen in this tweet “scans show extent of brain damage in babies with microcephaly associated with zika….” Microcephaly has been a topic of concern for the CDC as babies born with microcephaly will require assistance throughout their lifetime [neurological effects (#2) includes tweets discussing the death of neural stem cells, which leads to neurological disorders in humans [fetal effects (#5) also addresses brain shrinking or brain damage but additionally the tweets discuss the destruction of the brain lobes that control thought, vision, and other functions in fetuses as seen in this tweet, “scans & autopsies show that zika eats away at the fetal brain. it shrinks or destroys lobes that control thought, vision & other functions.” Guillain-Barré syndrome (topic #8) is a sickness caused by damage to nerve cells. The tweet “human neural stem cells infected by #zika subsequently trigger an innate immune response that leads to cell death” includes information on how Zika can lead to damage of neural stem cells and causes a disease such as Guillain-Barré syndrome [Neural defects is a broader theme of concern for the public that needs to be addressed by public health officials to mitigate fear and concern because of the defects to the nervous system caused by Zika virus infection. For lifetime ,45. The n humans , as seensyndrome . The reasyndrome .Zika abnormalities) and #9 (Zika effects) are both related to abnormalities because of Zika infection but include diverse problems. The topic Zika abnormalities (#6) describes various anomalies associated with the fetus and babies born with Zika infection as seen in this tweet, “birth defects linked to #zika now also incl hearing loss, vision problems, impaired growth, abnormalities in limbs.” These types of abnormalities are termed as congenital Zika syndrome by the CDC and includes a collapsed skull, eye scarring, severe muscle tension, and brain calcification [Zika effects (#9) focuses on the stunt in growth and development of children. Again, both of these topics concern abnormalities because of Zika infection but focus on 2 different abnormalities and are therefore kept as 2 distinct topics. By pushing prevention such as the “Fight the Bite” campaign, officials can help ease fears concerning these abnormalities.Zika abnormalities is also an important broader theme to address because of the fear and concern of abnormalities and defects in infants because of Zika virus infection during pregnancy. In fication ,37. The ultrasounds is important to discuss because pregnant women may have a false sense of security after getting an ultrasound and Zika not being detected in their fetus in the early stages of pregnancy. The CDC states on their website that microcephaly is more readily detected late in the second trimester to early in the third trimester [Ultrasounds is another important topic to address because initial ultrasounds fail to reveal microcephaly and other birth defects, leading to a false sense of security for a couple ,42,48,49rimester . Researcdengue associations (#10), scientists suspected and are starting to confirm that earlier illness of dengue enhances the chances of Zika infection also because of ADE [Dengue association may explain why this Zika outbreak is associated with abnormalities and defects and previous infections were not, which is why it is an important topic to address ,50-52. De of ADE ,52. The Now that public health officials know what the public is concerned about, they can focus on addressing these concerns. When an incident occurs, the normal tendency is to seek more information on the topic of interest . This caComplications related to processing can include the accuracy of the information shared, as at times the media is quick to report information without having all the facts or the reader may interpret the facts incorrectly . TherefoThe authors understand all of these suggestions are already being followed at some capacity by public health officials. However, there is always room for improvement.The tweets in our analysis were limited to the English language, which limits the generalizability of the study. This is critical as South American countries were the first and hardest hit countries. Future studies can address this limitation by analyzing tweets in Portuguese and Spanish. The keywords used in data collection were Zika, Zika virus, Zika virus treatment, and Zika treatment. Therefore, tweets that refer to this disease in another language would be overlooked. Tweets that refer to the disease without mentioning it by name would also be overlooked.Without prosody, contextual, and spectral cues, sarcasm is difficult to detect , all 3 oDue to the short length of tweets and the large number of tweets collected, LDA has been previously shown to have some issues with overfitting, with the number of revealed topics exceeding the true number of topics . We atteOverall, the negative sentiment topics focused on neural defects and abnormalities caused by the Zika virus. As these tweets were categorized as negative sentiments, officials could see that the public was concerned with the symptoms caused by the Zika virus. As the public was concerned, officials could focus on spreading information encouraging prevention. Officials could also see that the top themes all concerned actual symptoms and defects and did not focus on misconceptions or misinformation that they needed to address. Moving forward, officials can also start informing the public that studies are providing evidence for the Zika-dengue interaction hypothesis. They should focus these messages in areas where dengue is endemic as they are the ones most at risk of the interaction causing more severe Zika infection.When another Zika outbreak occurs, we predict similar concerns  about the neurological defects will be expressed on social media. Although our current framework would still be applicable, the unsupervised topics within the tweets would change. Specifically, the relevancy and sentiment classifiers (the supervised part of the system) would still be effective in detecting tweets specific to Zika and specific to the particular topic such as symptoms. However, when a preventative vaccine for Zika virus infection is created and/or new symptoms arise that are associated with Zika virus infection, the topics of concern would change depending on the current issue of concern at a particular time. As of August 2018, no licensed vaccines were available; however, several candidates are in various stages of development, and clinical trials have begun . Once a On the contrary, if new symptoms for Zika were to develop or further complications for those born with neurological defects were discovered, the topics of concern in the negative sentiment category would change to reflect concerns specific to the new symptoms. During the most recent outbreak, scientists suspected that people who previously had a dengue infection experienced worse symptoms from Zika than those who had not been previously infected with dengue . Previoufight would typically have a negative connotation but has a positive one in this tweet. Some examples of other words that are typically considered negative but are actually positive when discussed under the context of epidemics are combat, prevent, and impair. If tweets containing these words were categorized using a sentiment word bank, they would have been incorrectly categorized as negative. This is an important issue because it does not correctly represent the public’s feelings and may cause experts to believe the public is not as concerned about Zika symptoms if some of the negative tweets were misclassified as positive/neutral. Therefore, we used a manual labeling process where an entire tweet was assigned to a sentiment category by 2 domain experts. We believe that this need for a combination of data science and domain expertise is what makes our study challenging and interesting.Our study is also useful for those that want to perform sentiment analysis with an epidemic, pandemic, or bioterrorism attack. Sentiment analysis is complex as most sentiment analysis tools just use the individual word polarities for measuring sentiment and generate an automated scoring mechanism based on these polarities to rate the sentiment levels of each tweet. This fails to incorporate the contextual information that needs to be incorporated for topic-specific sentiment analysis in this domain . ScientiThis is one of the first studies to address Zika sentiment classification using Twitter. Using such a system allows public health officials to ascertain public sentiment concerning disease outbreaks and address concerns in real time.Future studies could analyze the change in sentiment over time to see if the number of negative tweets decreases as the outbreak subsides and more advances in treatments are discovered. Studies could also look at sentiment by gender or geographic location. Both are prudent because of Zika’s effect on fetuses and its comparative prevalence in equatorial regions, respectively. We would also suggest future studies to leverage other sources of information, such as other social media sites, newspapers, and blogs. Similar methodologies could also be applied generally to future pandemics and epidemics to ascertain public sentiment.


Caenorhabditis elegans is a laboratory model organism in biology. We study natural populations of this small animal and its relative, C. briggsae, and the viruses that infect them. We previously discovered three RNA viruses related to nodaviruses and here describe a fourth one, called the Mělník virus. These viruses have a genome composed of two RNA molecules. We find that two viruses may infect the same animal and the same cell. The two RNA molecules may be exchanged between variants of a given viral species. We study the diversity of each viral species and devise an assay of their infectivity and competitive ability. Using this assay, we show that the outcome of the competition also depends on the host.The roundworm  Caenorhabditis elegans and its relative, Caenorhabditis briggsae. Here, we report on a collection of more than 50 viral variants from wild-caught Caenorhabditis. We describe the discovery of a new related virus, the Mělník virus, infecting C. briggsae, which similarly infects intestinal cells. In France, a frequent pattern of coinfection of C. briggsae by the Santeuil virus and Le Blanc virus was observed at the level of an individual nematode and even a single cell. We do not find evidence of reassortment between the RNA1 and RNA2 molecules of Santeuil and Le Blanc viruses. However, by studying patterns of evolution of each virus, reassortments of RNA1 and RNA2 among variants of each virus were identified. We develop assays to test the relative infectivity and competitive ability of the viral variants and detect an interaction between host genotype and Santeuil virus genotype, such that the result depends on the host strain.Three RNA viruses related to nodaviruses were previously described to naturally infect the nematode IMPORTANCE The roundworm Caenorhabditis elegans is a laboratory model organism in biology. We study natural populations of this small animal and its relative, C. briggsae, and the viruses that infect them. We previously discovered three RNA viruses related to nodaviruses and here describe a fourth one, called the Mělník virus. These viruses have a genome composed of two RNA molecules. We find that two viruses may infect the same animal and the same cell. The two RNA molecules may be exchanged between variants of a given viral species. We study the diversity of each viral species and devise an assay of their infectivity and competitive ability. Using this assay, we show that the outcome of the competition also depends on the host. Caenorhabditis elegans and its relative, Caenorhabditis briggsae  was the first to be detected , which we show is most closely related to SANTV. We established a collection of variants of each virus, especially of the two C. briggsae viruses that were found most frequently, SANTV and LEBV. These viruses are often sympatric, coexisting even in the same individual worm and individual cell. Sequence analysis of the collection of viral isolates did not detect genetic exchange between these two viruses but did detect reassortment of the two RNA molecules in SANTV. Finally, we developed a phenotypic assay that allowed us to detect an interaction between the host genotype and the SANTV variant.Here, we aimed to collect  compost –16. We t(This article was submitted to an online preprint archive .)C. elegans and C. briggsae and searched for possible viral infections using host intestinal symptoms, in situ hybridization with probes corresponding to known viruses, and whole-RNA sequencing of infected cultures . Based on intestinal symptoms , was found in Mělník and Prague (Czech Republic). This virus causes the same intestinal symptoms as the other viruses  but do not infect C. briggsae JU1264.In our surveys in France and the occasional sampling elsewhere in Europe, C. elegans and C. briggsae were found at similar frequencies in France, viral infections of C. briggsae were more common than C. elegans infections  probe set to detect either SANTV or LEBV RNA1 molecules  , as shown in In order to assess the rate of d O1071) . The coid O1071) . In the C. briggsae animals . We did not find any evidence of genetic exchange between the different ing RDP4 . Indeed,Within each viral species, we found extensive genetic diversity among the strains. The highest genetic diversity was found for the SANTV RNA1 molecule, with a total of 21% of nucleotides (11.9% of amino acids) showing a polymorphism in the whole sequenced set. Mean pairwise diversity (± standard errors [SE]) for the entire sample was 0.086 ± 0.019 substitutions per site at the nucleotide level and 0.035 ± 0.005 at the amino acid level. The pattern of SANTV RNA1 diversity includes a deep split between two lineages, which we call A and B . The estThe SANTV RNA2 molecule displayed a lower level of variation than RNA1: mean diversity for the entire sample was 0.027 ± 0.002 substitutions per site at the nucleotide level and 0.011 ± 0.002 at the amino acid level. Interestingly, the phylogeny of RNA2 was not congruent with that of RNA1, suggesting reassortments . Using RFor LEBV, despite a similar sampling structure and geographic diversity, we found a much lower level of genetic diversity than that for the Santeuil virus on both RNA molecules. The RNA1 molecule displays 0.013 ± 0.001 substitutions per site at the nucleotide level and 0.007 ± 0.001 at the amino acid level, while RNA2 harbors 0.011 ± 0.001 substitutions per site at the nucleotide level and 0.007 ± 0.001 at the amino acid level. The topology of the phylogenetic relationships between LEBV variants for RNA1 and RNA2 was poorly supported, possibly because of the lower number of informative sites . Using RS) and the ratio of nonsynonymous to synonymous substitutions (dN/dS) along the RNA1 and RNA2 molecules are shown for the LEBV and SANTV variants in dN-dS] statistical test implemented in the HyPhy package  that is not also shared with the C. elegans virus (ORV). Remarkably, the N-terminal capsid domain is highly conserved between the two closely related viruses MELV and SANTV.Finally, the patterns of pairwise amino acid conservation between SANTV and MELV, ORV, or LEBV along the RNA1 and RNA2 molecules are shown in C. elegans wild isolates display a wide range of sensitivity to the original ORV strain, JUv1580  , 3 , JU1498 (by LEBV), JU1993 (by SANTV), and JU2160 . Consistent with the above-described results, the Santeuil virus genotypes JUv1551 and JUv1993 were comaintained in C. briggsae JU1264 in similar proportions of infected animals across the three generations, while more animals were infected by JUv1993 than by JUv1264 (P = 0.006) and even better in the host strain JU2160, where the JUv1264 variant was lost in all three replicates by the third generation .Third, we sought to compare the capabilities of these viruses in several periment , red. We JUv1264 . The samIn summary, these experiments indicate an interaction between host genotype and SANTV genotype, such that the result of the competition depends on the host strain.C. elegans isolate infected with ORV JUv2572 harbored stronger intestinal symptoms and slower growth than most ORV infections. Using the single infection protocol, the viral preparation from the JU2572 isolate was able to infect the strains N2 and MY10, which are more resistant to infection by JUv1580  in the laboratory could impact the survival of the Caenorhabditis animals, especially for the infected animals, and therefore lower the probability of recovering infected animals. Variation in host sensitivity  (MoNV RdRp shares 43% amino acid identity with Pariacoto virus) and a virus closely related to Lake Sinai virus 2 (MoNV capsid shares 16% amino acid with the LSV 2)  . In our C. briggsae host.Here, we also designed assays that allow us to study the interaction between the different viral variants and hosts. This allowed us to determine that the outcome of the competition between SANTV variants depended on the Caenorhabditis nematodes and their noda-like viruses provide an exciting model to investigate coevolution dynamics between an animal host and its natural viruses. First, C. briggsae and C. elegans are model organisms with a short life cycle, which eases multigenerational experiments and quantitative genetic analysis. Second, the natural combinations of one Caenorhabditis isolate with its natural viral variant can easily be isolated, maintained under laboratory conditions, kept frozen, and revived. Third, natural virus-nematode combinations can be dissociated by storing the viral isolate as a filtrate and by bleaching the Caenorhabditis isolate to remove viral infections .eviously , 20. We ecies 14–, 20, 21.ecies 14– and bactecies 14–, and conC. briggsae using in situ hybridization (see below) performed directly on a subset of worms coming out of the rotten fruits. The fixed worm population was split in two; half was hybridized with SANTV RNA1 plus RNA2 probes and the other half with LEBV RNA1 plus RNA2 probes . The cultures were propagated on agar plates seeded with Escherichia coli OP50 and frozen under standard conditions for C. elegans .In order to detect unknown viruses of usSeeker . Sequencin situ hybridization using fluorescently labeled oligonucleotides specific for each virus, as described in reference Each virus was detected by www.inkscape.org) software. Animals were considered infected when, for an exposure below or equal to 1,000 ms with the 40× objective, at least one intestinal cell was distinctly labeled at higher levels than background staining for that animal .Fluorescence microscopy was performed using an upright Zeiss AxioImager M1 equipped with 10× , 40× , and 63×  objectives. Images were acquired using a Pixis 1024B camera (Princeton instruments) and MetaVue imaging software. Image panels were assembled in ImageJ  and InksC. elegans infections with ORV, we used the custom Stellaris (Biosearch Technologies) probes labeled with Quasar 670 dyes for the ORV RNA1 molecule and with Cal Fluor red 610 dyes for the ORV RNA2 molecule described in reference To screen for the natural C. briggsae infections with SANTV and LEBV, we used custom Stellaris (Biosearch Technologies) probes labeled with Quasar 670 dyes for the SANTV RNA1 and LEBV RNA2 molecules and with Cal Fluor red 610 dyes for the SANTV RNA2 and LEBV RNA1 molecules. FISH was performed as described in reference To screen for the natural For the MELV, we developed a custom Eurofins 21-nt-long probe labeled with Texas Red dye for the MELV RNA1 molecule (Table S1c).For viral variant competition experiments, we developed custom Eurofins 22-nt-long probes specific for two divergent SANTV RNA1 variants, labeled with CFP-ATTO425 for JUv1993s and with Texas Red for JUv1264s and JUv1551s. The probe sequences are provided in Table S1c.C. briggsae animals were experimentally infected either with SANTV JUv1264 or with LEBV JUv1498 or left uninfected. The SANTV-infected, LEBV-infected, and noninfected animals were fixed, and we performed fluorescence in situ hybridization staining using SANTV RNA1  and LEBV RNA1 (Quasar 670) Biosearch probes (Table S1b).In order to test the specificity of the DNA probes used to detect LEBV and SANTV, JU1264 C. briggsae strain with either SANTV JUv1264 or with SANTV JUv1993 or left the animals uninfected. The SANTV JUv1264- or JUv1993-infected and the noninfected animals were fixed, and we performed fluorescence in situ hybridization staining using the JUv1993 RNA1 (Texas Red) and JUv1264/JUv1551 RNA1 (CFP) probes (Table S1c).In order to test the ability of our 22-nt-long single-stranded DNA (ssDNA) probes to distinguish between SANTV JUv1993 RNA1 and SANTV JUv1264 or JUv1551 RNA1 molecules, we infected the JU1264 C. briggsae coinfection with SANTV and LEBV at the scale of the fruit, we focused on two samples from the Le Blanc location, LB14-36 (plum) and LB14-37 (pear), and one from the Orsay orchard, apple O1071. Wild hermaphrodite L4 larvae were singled less than 4 h after the rotten vegetal matter was placed onto E. coli OP50-seeded nematode growth medium (NGM) plates maintained at 23°C. After 3 days, F1 progenies were fixed with ethanol. To monitor the presence of LEBV and SANTV, FISH was performed as described in reference To assess the rate of Caenorhabditis wild isolates were extracted using TRIzol-chloroform. Viral genomes were first reverse transcribed from the 3′ end, and from the middle of the segment when needed, using viral species-specific primers and Superscript III reverse transcriptase (Invitrogen) by following the manufacturer’s instructions. Reverse transcription was followed by PCR amplifications using the Q5 high-fidelity DNA polymerase (New England Biolabs) with primers amplifying two overlapping fragments per RNA segment. The PCR products were Sanger sequenced. To minimize sequence errors due to PCR, we performed two independent reverse transcription-PCR experiments but did not find any mismatches between replicates, and we were able to validate heterozygous single-nucleotide polymorphisms. All of the primers used in this study are listed in Table S1a. Sequences are available under the following GenBank accessions numbers: MN130962 to MN130984 (LEBV-RNA1), MN130985 to MN131007 (LEBV-RNA2), MN131008 to MN131027 (SANTV-RNA1), and MN131028 to MN131047 (SANTV-RNA2).RNAs of a subset of infected (i) Alignments and phylogenetic analyses. Nucleotide sequences were aligned using MUSCLE (g MUSCLE  with defg MUSCLE . When neg MUSCLE . The seqg MUSCLE  using thg MUSCLE  and testg MUSCLE  and estig MUSCLE .(ii) Detection of reassortments. To detect events of RNA molecule reassortment between variants of the same viral species, we concatenated, for each variant, their RNA1 and RNA2 nucleotide sequences. We detected putative reassortment and recombination events using the RDP, GENECONV, Bootscan, Maxchi, Chimera, SiSscan, and 3Seq methods implemented in RDP4  Analysis of polymorphisms along the ORFs within the set of LEBV and SANTV variants. Mean evolutionary diversities for the entire populations and mean interpopulational evolutionary diversities were estimated using MEGA7  and the ratios of nonsynonymous to synonymous substitutions (dN/dS), where dS is the number of synonymous substitutions per site (s/S) and dN is the number of nonsynonymous substitutions per site (n/N), were calculated for the RNA1 (RdRp) and RNA2 (capsid-delta fusion protein) molecules of SANTV and LEBV in a sliding window, with a window of 20 codons and a step of 5 codons. In order to describe the distribution of the total, synonymous, and nonsynonymous polymorphisms, we used the maximum likelihood analysis of natural selection codon by codon using the HyPhy software package (s) and nonsynonymous (n) substitutions and the numbers of sites that are estimated to be synonymous (S) and nonsynymous (N) were produced using the joint maximum likelihood reconstructions of ancestral states under a Muse-Gaut model  in favor of an alternative hypothesis (dN > dS = 1.00 or dN < dS = 1.00). The variance of the difference was computed using the bootstrap method . Analyses were conducted using the Nei-Gojobori method  Infections of four  We established C. elegans infections with two ORV variants. Five L4 larvae were infected with 50 μl of viral preparation. Infected C. elegans populations were propagated onto OP50-seeded NGM plates at 20°C for 2 to 3 host generations so that the final titer is a function of the host-pathogen interaction rather than of the initial titer . The number of infected cells was counted manually.al titer , 3. The  orchard . F2/F3 pC. briggsae hosts.(ii) Competition between SANTV and LEBV in different  We first performed a two-step infection experiment to test the effect of the initial viral titer  on infection rate after 2 to 3 host generations. We infected C. briggsae JU1264 animals with the JUv1264 viral preparation in three replicates. We visually assessed symptoms on 50 adult hermaphrodites and checked that after 7 days more than 80% of the nematodes were infected. We then mixed i sick  adults with j adult animals from a noninfected culture in four different proportions (i + j), 6 + 0, 4 + 2, 2 + 4, and 0 + 6, and let them reproduce for 7 days at 23°C before fixing the animals for in situ hybridization using 22-nt specific probes specified in Table S1c. The numbers of infected animals and cells were counted manually.C. briggsae JU1264. We independently infected C. briggsae JU1264 animals with the tested viral preparations  in three replicates. We visually assessed symptoms on 50 adult hermaphrodites and checked that, after 7 days, more than 80% of the nematodes were infected. We then mixed 6  sick adult animals  from the originally infected populations with either of two viruses and let them reproduce for 3 days at 23°C in three replicates. For each replicate, we passed at each generation (every 3 days) 20 L4 larvae to a new plate and fixed each generation for in situ hybridization. The number of (co-)infected animals and cells were counted manually. (Co-)infection levels of SANTV- versus LEBV-infected animals were scored using FISH staining with SANTV RNA1  and LEBV RNA1 (Quasar 670) Biosearch probes (Table S1b). (Co-)infection levels of JUv1551- versus JUv1993-infected animals were scored using FISH staining using JUv1264/JU1551 RNA1 (CFP/ATTO425) and JUv1993 RNA1 (Texas Red) 22-nt-long probes (Table S1c).Second, we used a two-step protocol to compare virus infectivity  in single infections and in competition in the host, C. briggsae JU1264 animals with the tested viral preparations  in three replicates. We visually assessed symptoms on 50 adult hermaphrodites and checked that after 7 days, more than 80% of the nematodes were infected. We then mixed 20 (10 + 10) sick adult animals  from the originally infected populations with either of two viruses and let them reproduce for 3 days at 23°C in three replicates. For each replicate, we passed at each generation (every 3 days) 20 L4 larvae to a new plate and fixed each generation for in situ hybridization using 22-nt specific probes specified in Table S1c. The numbers of (co-)infected animals and cells were counted manually.Finally, we used a two-step protocol to perform pairwise competition assays between SANTV JUv1993 and two other SANTV variants, JUv1264 and JUv1551. We independently infected C. briggsae wild isolates . We then passed at each generation (every 3 days) 20 L4 larvae to a new plate and fixed each generation for in situ hybridization using 22-nt specific probes specified in Table S1c.A third step was added to the above-described experiment. We prepared a virus filtrate in the first generation of coinfection and reinfected several MK774657 to MK774660 and MN130962 to MN131047.Sequences were deposited in GenBank under accession numbers


The deeper we dig, the more we realize that there is much more to a virus infection than is initially apparent. Historically, major research efforts have been invested in understanding the biology and impact of the standard fully replicative virus during infection. This is the virus that spreads, infects additional individuals, and evolves to become increasingly more successful in the process. Because of its ability to propagate the infection, this is also the version of the virus that we target with antivirals and therapeutics. However, we now appreciate that virus variants and additional secondary products of virus replication play a critical role in determining the quality of the host immune response and the outcome of the infection, including the survival of both the virus and its host. Importantly, increased appreciation of the role of incomplete viral replication products in shaping the infection outcome is revealing new mechanisms that could be targeted to diminish virus pathogenesis.My laboratory studies the effect of defective viral genomes in modulating virus pathogenesis. In addition, we are interested in understanding the role of these types of genomes in shaping the long-term virus-host interaction. Defective viral genomes were first observed in the 1940s as incomplete forms of influenza virus, and since then, they have been found in every scrutinized virus. However, after decades in the spotlight, the scientific community lost interest in defective viral genomes, in part because it became a nonfundable subject due to lack of evidence for their role in natural infections. Regrettably, basic research to study how defective viral genomes contribute to shaping infection dynamics and outcomes was delayed, and the potential to harness defective viral genomes to improve human and animal health was not appreciated until recently.So, how did I end studying what others had dismissed as unimportant and artifactual byproducts of virus replication?Before moving to the United States, I trained in Chile as a biochemist and immunologist. As an undergraduate student, I worked in an immunology laboratory studying contact dermatitis to urushiols, the compound in poison ivy that causes a severe allergic reaction in many people. During that time, I became more and more intrigued by viruses, as I realized how spectacularly efficient and resourceful they are. In graduate school, I settled in a lab that studied antiviral immunity. I remember nervously approaching Tom Moran to ask about joining his group before I had even set foot in the lab for my rotation. To my surprise, he immediately said yes. After working for months on testing a hypothesis that proved incorrect, I began to study how dendritic cells from the immune system responded to viral infections. This was a hot topic at the time, because dendritic cells were being recognized as pivotal players at the onset of the immune response, and Toll-like receptors were identified as the primary cellular sensors of microbes. Our data comparing the immune response to infection with two strains of a mouse virus (Sendai virus [SeV]) revealed that Toll-like receptors were not the main initiators of the immune response against this virus (we now know that SeV is sensed by retinoic acid-inducible gene-I [RIG I]-like receptors) and that an unknown viral component conferred strong ability to activate dendritic cells and initiate the immune response to one strain of the virus but not the other. I wanted to figure out what was driving differences in the ability of these two viral strains to initiate the antiviral immune response, and there was no better place within my geographical limitations to continue with this work, so I decided to continue in Tom’s lab for my postdoc  and then as an assistant professor on the research track. Together with Jacob Yount, then a graduate student, we tested whether mutations in the virus or differences in the activity of viral-encoded molecules that antagonize the cellular immune response explained our observations. The exciting result only came when we evaluated the role of defective particles that contain defective viral genomes. Adding defective particles to the poorly immunostimulatory virus turned it into one with strong immunostimulatory activity. Similarly, removing these particles from the highly immunostimulatory strain crippled its ability to activate dendritic cells. This result was one of the most exciting and memorable of my career so far. I remember thinking: how cool! No one cares about these types of viral genomes anymore and yet these data show that we can learn so much about how viruses trigger effective antiviral immunity by studying them!I founded my laboratory in September 2010 based on that initial experiment. Since then, we have demonstrated that defective viral genomes have an essential role in determining the infection outcome during experimental infection with a number of respiratory viruses. Additionally, we have shown that defective viral genomes are present in humans when infected with respiratory syncytial virus, a common natural human respiratory viral infection. Through our work, we have enhanced our understanding of the short- and long-term impact that defective viral genomes have in infected cells and the molecular mechanisms involved. Most importantly, the field is now reinvigorated with a number of laboratories around the globe studying defective viral genomes of many different viruses. It is my hope that these studies, which were founded on curiosity, lead to a much more accurate understanding of the interaction between viruses and their host and eventually open new avenues for controlling pathogenic viral infections.


Co-infection of plant hosts by two or more viruses is common in agricultural crops and natural plant communities. A variety of models have been used to investigate the dynamics of co-infection which track only the disease status of infected and co-infected plants, and which do not explicitly track the density of inoculative vectors. Much less attention has been paid to the role of vector transmission in co-infection, that is, acquisition and inoculation and their synergistic and antagonistic interactions. In this investigation, a general epidemiological model is formulated for one vector species and one plant species with potential co-infection in the host plant by two viruses. The basic reproduction number provides conditions for successful invasion of a single virus. We derive a new invasion threshold which provides conditions for successful invasion of a second virus. These two thresholds highlight some key epidemiological parameters important in vector transmission. To illustrate the flexibility of our model, we examine numerically two special cases of viral invasion. In the first case, one virus species depends on an autonomous virus for its successful transmission and in the second case, both viruses are unable to invade alone but can co-infect the host plant when prevalence is high. Transmission is a key element in understanding the epidemiology of plant virus diseases, particularly those transmitted by arthropod vectors ,2,3,4. ICo-infection of hosts by two or more plant viruses is common in both agricultural crops ,9 and naMost experimental studies on the relationship between co-infection and transmission have been done for viruses with non-persistent transmission by aphids. Syller  reviewedCo-infection can cover scenarios ranging from two viruses (or virus strains)/one vector through to many viruses/many vectors, but with some nuances. There is an extensive literature on co-infection across this range. Some representative but not exhaustive publications are noted in In this paper, we formulate a general epidemiological model for one vector species and one plant species that allows for co-infection of the host plant by two virus species or strains. The model is used to investigate the role of vector transmission on co-infection, specifically acquisition and inoculation, as well as antagonistic and synergistic interactions. The basic reproduction number provides a condition for invasion of a single virus infection. For co-infection, we derive a new invasion threshold. Given that a single virus can persist in the host plant, the invasion threshold highlights some key epidemiological parameters for successful co-infection. In addition, we investigate the roles of the vector acquisition and inoculation parameters when one virus depends on an autonomous virus for its successful transmission, or when both viruses are unable to invade alone but can facilitate co-infection if they occur in high enough prevalences. We also explicitly test when the simplifications—which are almost always left implicit—in models which do not explicitly include the infection status of vectors lead to potentially misleading results.A and virus B. We make several simplifying assumptions. The two viruses are not transmitted vertically in the plant population  nor in the vector . The plants can be infected by a single virus or co-infected by both viruses. The vectors are only inoculative with a single virus; namely, we assume that acquisition of the first virus precludes inoculation by a second virus by the same vector for as long as the vector retains the first virus. This assumption applies to both modes of transmission. For non-persistent transmission, it may arise because stylet receptor sites are saturated by the first virus. For persistent-circulative transmission, it may be due to a phenomenon similar to “super-infection exclusion” taking place in the vector [The general epidemiological model for vectors and plants consists of a system of differential equations with either infection by a single virus, or co-infection by two viruses (species or strains). For simplicity, we refer to the two viruses as virus e vector , or simpA and B . The total vector density is A or B are acquired, given that acquisition is from a co-infected plant, P being the total plant population density, the vector model takes the following form: The vector model has three stages, A, virus B or both viruses, respectively. The total plant population density is i in a co-infected plant The plant model consists of four stages, The parameters nfection ,25,30. TTwo important disease threshold parameters are derived from models (1) and (2), the basic reproduction number and the invasion reproduction number. The density of non-infective vectors at the disease-free equilibrium (DFE) in the vector model (1) is approach ,34,35,36A or virus B, A exceeds the value of one, A can invade the disease-free vector-plant system and if B can invade. An epidemiological interpretation of A is introduced into a healthy vector-plant system, it will inoculate and infect plants at a rate of A or B can invade the vector-plant system.The two terms in the preceding definition are basic reproduction numbers corresponding to infection with either virus B can invade when the system is at the virus A equilibrium, i.e., A are An invasion reproduction number can be derived if the system is already infected with a single virus. We consider whether virus B can invade, we apply the next generation matrix approach to derive an invasion matrix A equilibrium, defined in (4), with the remaining states B, A. If B can invade and if B cannot invade. Each element in matrix A has already invaded).To determine whether virus computed . We assuB increases so does the invasion reproduction number. The direction of change for the invasion reproduction number is not as straightforward if the vector visitation rate A are changed, as the equilibrium values Since the invasion matrix (5) has non-negative entries, an increase (or a decrease) in any matrix entry also increases (or decreases) the invasion reproduction number . In partThe vector-plant system can be reduced to a simplified plant model, where the vectors are not explicitly included . If the The vector-implicit model retains the inoculation and acquisition terms. At the DFE, the basic reproduction number is the same as in (3). But the equilibrium for virus A B  . InvasioThe effect of vector transmission on virus establishment in the vector-explicit model is examined in several numerical examples. In addition, the conditions for invasion of a second virus in the vector-explicit model are compared to the vector-implicit model.A can invade and persist in the vector-explicit model but not virus B. In particular, the acquisition probability of virus B is set to zero, A  keeping track of co-infections makes the models less tractable mathematically, (ii) modeling vector dynamics explicitly may not be necessary relative to the research question addressed, and (iii) the biological knowledge or the data may be too scarce to reasonably increase the model’s complexity . MoreoveHowever, we have showed that vector-implicit and vector-explicit models may yield qualitatively different results . The defA and B:iA is able to complete a full infection cycle, invade a disease-free population and settle at an endemic equilibrium in the absence of virus B. By contrast, virus B can systematically infect a plant but cannot be acquired by a vector in the absence of co-infection in the host plant. Successful invasion of virus B depends on a complex relation between vector acquisition and inoculation rates, the relative inoculation success of viruses A and B and the prevalence of infected hosts and infective vectors carrying virus A.Virus iiA and virus B can both complete their infection cycles but are unable to invade a disease-free population in the absence of the other virus. The only biologically feasible endemic equilibrium is a co-infection equilibrium. The system will approach the co-infection equilibrium if the initial prevalences of virus A and B are sufficiently large, i.e., if the initial condition is in the basin of attraction of the co-infection equilibrium. Otherwise, if the initial virus prevalences are so low that the initial condition is in the basin of attraction of the disease-free equilibrium, both viruses will disappear from the system. The separatrix between these two different outcomes represents a curve of tipping points. On either side of these tipping points, we have contrasting dynamics, namely a disease-free versus a co-infected system. We have seen that increased vector mortality rates  moves the separatrix by making the co-infection equilibrium less resilient. This could lead to an abrupt  extinction of co-infection.Virus To illustrate the implications of our model, we considered two distinct special cases with two viruses (or virus strains) denoted A equilibrium when the invading virus B species/strain is at low prevalence levels. The dynamics of the models away from the virus A equilibrium can be quite complex and exhibit bistability, where both the virus A equilibrium and the co-infection equilibrium are stable. In this case, the initial prevalences of viruses A and B determine whether virus B can successfully invade. We also investigated the potential for co-infection and the conditions that need to be satisfied when one virus depends on an autonomous virus for its successful transmission or when both viruses are unable to invade alone. For persistent-circulative transmission, competition between viruses/virus strains, either direct or indirect, as they move through the vector is identified as a key and challenging area for further research that would improve modeling attempts to predict the epidemiological consequences of co-infection.In this paper, we have formulated a general epidemiological model for potential co-infection in a host plant by two virus species or strains. We used the model to investigate the effects of vector transmission on co-infection, specifically acquisition and inoculation, as well as antagonistic and synergistic interactions between viruses. We showed that reducing the model to a vector-implicit model can lose some of the key features gained when the vector is included explicitly (such as competition for vector). We derived a new invasion threshold that determines whether or not a second virus can invade a host population in which a first virus had successfully established. The invasion threshold highlights the key epidemiological parameters that are important for successful co-infection. However, the invasion threshold only applies near the virus


Medicago truncatula (for both RNS and AMS), Oryza sativa (for AMS but not RNS), and Arabidopsis thaliana (for neither RNS or AMS) were investigated using domain swap approach in response to rhizobia in Lotus japonicus. Full-length SYMRK from rice and Medicago but not from Arabidopsis could complement Lotus symrk-409 mutant plants to contribute RNS. The chimeric protein with the extracellular domain (ED) of LjSYMRK and cytoplasmic domains (CD) of SYMRK from both Medicago and rice but not Arabidopsis could contribute to RNS in Lotus, suggesting that the CD of SYMRK is required for symbiotic signaling. The chimeric receptors containing the CD of LjSYMRK (SYMRKCD) and the EDs of MtDMI2 (MtDMI2ED), OsSYMRK (OsSYMRKED), AtSYMRK (AtSYMRKED), NFR1 (NFR1ED), and NFR5 (NFR5ED) could complement Lotus symrk-409 mutant plants to develop nodules. However, MtDMI2 could partially complement Lotus symrk-409 mutants to form both effective nodules and ineffective bumps, which is similar to the complementation results from MtDMI2ED-LjSYMRKCD and LjSYMRKGDLC in Lotus symrk-409 mutants, suggesting that ED of SYMRK has a very fine-tune regulation for RNS in Lotus. The deletion of either MLD or LRR on SYMRKGDLC (a mutant version of SYMRK with GDPC motif replaced by GDLC) could contribute to RNS when overexpressed in Lotus symrk-409 mutants, suggesting that MLD and LRR domains might work together to be involved in symbiotic signaling and the LRR domain might play a negative role in LjSYMRKGDLC-mediated RNS. By mutagenizing the conserved amino acids on LRR domain, five serine residues were found to be required for the function of LjSYMRKGDLC in RNS. These finding precisely refine the molecular mechanisms of SYMRK function in symbiotic signaling in L. japonicus.Symbiosis receptor kinase (SYMRK) is a cell membrane-localized protein kinase containing extracellular malectin-like domain (MLD) and leucine-rich repeat (LRR) domains, which is critically required for both root nodule symbiosis (RNS) and arbuscular mycorrhizal symbiosis (AMS). SYMRK is widely distributed in the genomes of different plant species; however, the contribution of different domains of SYMRK and its homologs from other plant species to RNS is largely unclear. In this study, SYMRK and its homologs from three typical plant species including  In barren soils, leguminous plants form symbiosis with rhizobia leading to the development of a new organ called a nodule, where rhizobia reside and subsequently reduce nitrogen into ammonium for the plant host in exchange for nutrients. In comparison to the root nodule symbiosis (RNS), which is mostly restricted in legumes, the arbuscular mycorrhizal symbiosis (AMS) is widespread in most landed plants, with a few exceptions (for example cruciferous plants). It is estimated that AMS originated about 400 million years ago, while RNS originated about 60 million years ago   was shown to be able to interact with NFR5 and transfer the symbiotic signal to downstream targets  could also induce spontaneous nodule formation in the absence of rhizobia  and OsSYMRK but not AtSYMRK could complement Lotus SYMRK−/− plants. In addition, multiple chimeric SYMRK proteins were developed to analyze the function of different domains of SYMRK in RNS. The data refine the molecular mechanisms of each domain of SYMRK in RNS in Lotus.In this study, we demonstrate that MtDMI2 , and 18 SYMRK homologs were identified. A phylogenetic tree based on various SYMRK proteins was made using the MEGA 5.1 software  and AMS. To study its function involved in RNS, SYMRK homologs were identified based on sequence similarity with Lotus, an LORE1-insertion symrk mutant were identified from the Centre for Carbohydrate Recognition and Signaling   contains an LORE1-insertion between exon 4 and intron 4, leading to the early termination of translation of SYMRK . In response to rhizobia treatment, infection threads and nodules were not detected in the symrk-409 mutant plants when compared with Lotus B-129 Gifu 7- and 21-day post-inoculated (DPI) with rhizobia , indicating that the symrk-409 is another knock-out mutant that could be used for future study.To study the function of SYMRK involved in RNS in Medicago that could have both RNS and AMS, OsSYMRK from rice that could only have AMS, and AtSYMRK from Arabidopsis that could have neither RNS or AMS.To analyze the symbiotic function of various SYMRK proteins, one SYMRK protein from each clade-based phylogenetic tree, respectively, was chosen for study, i.e., MtDMI2 from symrk-409 mutant plants under the control of the Ljubiquitin promoter using hairy root transformation. The Ljubiquitin promoter could induce a strong expression of multiple genes in previous publications . Both LjSYMRK and MtDMI2 and control vector were introduced into dmi2-1 using hairy root transformation. In comparison with no infection threads and nodules observed on the dmi2-1 mutant transformed with empty vector , a significant number of nodules and infected nodule primordium was detected on roots transformed with either MtDMI2 or LjSYMRK , which is consistent with nitrogenase activity measured in these two transgenic nodules , LjSYMRKMLDΔ (LjSYMRK without MLD), LjSYMRKLRRΔ (LjSYMRK without LRR domain and “GDPC” motif), and LjSYMRKGDLC (Proline replaced with leucine). LjSYMRK deleted with MLD, or LRR, or ED could rescue the defects on nodulation phenotype in symrk-409 mutant plants  whose MLD was not detected to be cleaved during symbiosis could not completely complement symrk-409 mutant plants with only ineffective bumps developed  in LjSYMRKGDLC induced the formation of nodules in symrk-409 mutant plants, some of which are rhizobia-infected nodules with measurable nitrogenase activity  between MLD and LRR showed strong inhibition on bacterial infection but slight suppression on the development of nodules  in RNS was exaggerated with only ineffective nodules formed in Lotus when SYMRKGDLC was overexpressed. However, overexpression of SYMRKGDLC with deletion of either MLD or LRR domain only produced effective nodules, suggesting that both MLD and LRR domain of SYMRK might play a negative rule but might work together in SYMRKGDLC-mediated nodule organogenesis in Lotus. The same phenotype with effective nodule formation was also observed in Lotus when LjSYMRKGDLC with its LRR replaced by the LRR domains from OsSYMRK or AtSYMRK but not from MtDMI2 was overexpressed, suggesting that LRR domains from OsSYMRK or AtSYMRK might be not functional in mediating RNS in Lotus. The negative role of LRR domain in SYMRKGDLC-mediated response is further confirmed by point mutations at five conserved serine residues, leaving us a possibility that the negative function of LRR might be regulated by an unknown phosphorylation.The ED of SYMRK contains two important domains, i.e., MLD and LRR linked by a GDPC motif. The well-established model about SYMRK reveals that cleavage of MLD is required for its association with NFR5 to mediate symbiotic signaling transduction in Lotus. Replacement ED with EDs from OsSYMRK or AtSYMRK proteins or even with NFR1 and NFR5 could contribute symbiotic response when overexpressed in Lotus plants. Since these proteins located far from LjSYMRK based on the phylogenetic tree, it is possible that these chimeric proteins with nonfunctional EDs still keep some function as LjSYMRKEDΔ to induce symbiotic response. However, the chimeric protein MtDMI2ED-LjSYMRKCD could not completely complement Lotus SYMRK−/− plants, indicating that the functional difference between Lotus and Medicago possibly due to that different nodule types formed on these two plants. The abovementioned hypothesis is further confirmed by the finding that LjSYMRKGDLC-MtLRR but not LjSYMRKGDLC-AtLRR or LjSYMRKGDLC-OsLRR is not functional to mediate RNS in Lotus. The whole ED from LjSYMRK might be nonfunctional when LRR domain was replaced with LRR from AtSYMRK or OsSYMRK. However, since the LRR from MtDMI2 might have difference to mediate RNS, the MLD of LjSYMRK seems not to match with the LRR from MtDMI2 which finally made the chimeric protein LjSYMRKGDLC-MtLRR lose its function in RNS. The data indicated that MLD and LRR must have a fine-tune regulation on the activation of symbiotic response.The ED of SYMRK seems to have a fine-tune regulation on the activation of symbiotic signaling in Lotus. GDPC motif that connects MLD and LRR domain required for MLD cleavage in response to RNS is responsible for nodule organogenesis. Both MLD and LRR domains might have a fine-tune regulation of SYMRK-mediated nodule organogenesis in Lotus. This progress in defining the molecular functions of SYMRK in RNS. However, identification of the direct targets of SYMRK and crystal structure of SYMRK are the future topics for understanding the molecular mechanisms mediated by SYMRK in NRS.In conclusion, the intracellular domain of SYMRK is critical while the ED of SYMRK plays a relatively minor role in mediating RNS in Lotus SYMRK with the protein blast suite at the National Center for Biotechnology Information database (https://blast.ncbi.nlm.nih.gov) used. The phylogenetic tree was built by MEGA 5.1 software  and hair root transgenic seedlings were grown in vermiculite and perlite mixed (2:1 volume ratio) supplied a 1/2 B&D medium and placed in a growth chamber set at 22°C for 16-h-light/8-h-dark cycle. Plants were inoculated with Mesorhizobium loti strain NZP2235 expressing beta-galactosidase (LacZ) after 5 days. Rhizobia grown in liquid TY medium adding tetracycline at OD600 of 1.0. They were then pelleted and resuspended in 1/2 B&D medium containing 0.5 mM KNO3 at OD600 of 0.02.Medicago hair root transgenic seedlings were grown in vermiculite and perlite mixed (2:1 volume ratio) supplied a 1/2 FM medium and placed in a growth chamber set at 22°C for 16-h-light/8-h-dark cycle. Plants were inoculated with Sinorhizobium meliloti strain Sm2011 expressing LacZ after 5 days. Rhizobia grown in liquid TY medium adding tetracycline at OD600 of 1.0. They were then pelleted and resuspended in 1/2 FM medium containing 0.5 mM KNO3 at OD600 of 0.02.symrk-409 insertion alleles are provided in Table Genomic DNA was extracted from putative mutant plants and used for PCR amplification of 2 min soak at 95°C, followed by 35 cycles of 30 s at 94°C, 30 s at 58°C, and 50 s at 72°C, followed by 5 min soak at 72°C. Primers for genotyping of TM7 Real-Time PCR System  using One-Step SYBR PrimeScript RT-PCR kit II . Lotus ATP synthase (Genbank ID: AW719841) and ubiquitin (Genbank ID: AW720576) were used as reference genes, which are stably expressed in all plant tissues. Primers for qRT-PCR are listed in Table Total RNA was extracted from roots at 7 DPI using the EASYspin Plant RNA Kit . Primescript RT Reagent Kit  was used to remove gDNA of RNA samples and reverse transcribe RNA into cDNA. Quantitative real-time PCR was performed on an ABI ViiAXbaI and StuI, then linked to a fragment or multi-fragments by Gibson assembly , and placed at room temperature for 40 min, washed twice 10 min in 0.1 M potassium phosphate buffer. Roots were vacuum infiltrated for 5 min with X-Gal staining solution  and kept overnight at 28°C in the dark. Roots were washed twice in 0.1 M potassium phosphate buffer for 5 min, then rinsed twice in ddH2O for 5 min. Stained roots were examined using a fluorescence stereo microscope  and a fluorescence microscope .Plants were inoculated with rhizobia carrying a Acetylene reduction activity (ARA) was used to detect nitrogenase activity of root nodules at 21 DPI. Four nodulated seedlings were transferred into test tubes with 2 ml acetylene added for additional growth at 28°C for 2 h. At least three repeats of each experiment were analyzed. Acetylene was surveyed using a GC-4000A gas chromatograph .AAV88623.1 for SrSYMRK, XP_004512550.1 for CaSYMRK, CAD22013.1 for MaSYMRK, CAD10812.1 for PsSYMRK, XP_003517193.1 for GmSYMRK, CAD10811.1 for MtSYMRK, AAM67418.1 for LjSYMRK, NP_001234869.1 for SlSYMRK, NP_001105860.1 for ZmSYMRK, XP_015646949.1 for OsSYMRK, XP_014752741.1 for BdSYMRK, XP_002460874.1 for SbSYMRK, XP_020677592.1 for DcSYMRK, NP_564904.1 for AtSYMRK, XP_020890981.1 for AlSYMRK, XP_010511724.1 for CsSYMRK, XP_009105317.1 for BrSYMRK, and XP_018445268.1 for RsSYMRK.Sequence data from this work can be found under the following GenBank accession numbers: HL, YC, and ZZ designed the experiments and analyzed the results. HL, MC, TZ, and LD performed the experiments and analyzed the data. HL, YC, and ZZ wrote the paper.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.