In the recent past, peste des petits ruminants (PPR) emerged in East Africa causing outbreaks in small livestock across different countries, with evidences of spillover to wildlife. In order to understand better PPR at the wildlife–livestock interface, we investigated patterns of peste des petits ruminants virus (PPRV) exposure, disease outbreaks, and viral sequences in the northern Albertine Rift. PPRV antibodies indicated a widespread exposure in apparently healthy wildlife from South Sudan (2013) and Uganda . African buffaloes and Uganda kobs <1-year-old from Queen Elizabeth National Park (2015) had antibodies against PPRV N-antigen and local serosurvey captured a subsequent spread of PPRV in livestock. Outbreaks with PPR-like syndrome in sheep and goats were recorded around the Greater Virunga Landscape in Kasese (2016), Kisoro and Kabale (2017) from western Uganda, and in North Kivu (2017) from eastern Democratic Republic of the Congo (DRC). This landscape would not be considered typical for PPR persistence as it is a mixed forest–savannah ecosystem with mostly sedentary livestock. PPRV sequences from DRC (2017) were identical to strains from Burundi (2018) and confirmed a transboundary spread of PPRV. Our results indicate an epidemiological linkage between epizootic cycles in livestock and exposure in wildlife, denoting the importance of PPR surveillance on wild artiodactyls for both conservation and eradication programs. Peste des petits ruminants virus (PPRV) is a small ruminant morbillivirus from the Paramyxoviridae family that groups with other notorious members like Rinderpest virus (RPV), measles virus, canine, or phocine distemper virus . The disClinical signs of PPR are associated to the lymphotropic and epitheliotropic nature of the virus and comprise of depression, conjunctivitis, ocular and nasal discharge, erosions/ulcers of the oral mucosa, respiratory distress, and diarrhea and may eventually lead to death ,8,9,10. Syncerus caffer) turned seropositive in analyses of convenience samples from 2004–2005 in different national parks across northern and western Uganda  in Dinder National Park in Sudan in 2017, but epidemiological and pathological information are lacking in these reports . Th. Th53]. No evidence of PPRV circulation was detected in the southwest Kisoro district of Uganda by June 2017, and the few seropositive animals detected may be explained by animals moved from other regions. These serum samples predated the first recorded PPR outbreak in Kisoro-Kabale districts in later June through August 2017. This outbreak in Uganda was likely related to the subsequent outbreak in the adjacent North Kivu province of DRC about two months later in 2017 . These aPPRV antibodies in buffalo and kobs from QENP did not show a clear spatial pattern. If African buffalo are relatively sedentary and Uganda kob at times territorial, these animals in QENP can be considered as one population whereas they may represent at least two epidemiological units divided by the extensive Maramagambo Forest . BuffaloPhacochoerus africanus), are highly spatially integrated with livestock even if direct physical contact is rare. Livestock is predominantly nomadic, with large seasonal movements including large flocks of small livestock at times and moving transboundary. These pastoral systems are extensive and show ample opportunity for spillover with frequent livestock infection cycles ) are not dissimilar to that experienced with RP in wildlife, historically with high morbidity and mortality, in the case of saiga approaching 100% and 80%, respectively [Eudorcas thomsoni), are well represented in Kenya and Tanzania, and no sign of disease in PPRV prevalent areas was seen or reported by wildlife or veterinary authorities, or even anecdotally by local communities. This apparent absence of disease in most populations of free-ranging African wildlife is consistent across all species shown to be exposed to PPRV by this study or previous serological studies [The absences of both virus isolation and observation of clinical disease in wildlife species in Africa is surprising given the increasingly visible epidemics in wildlife in Asia ,6,61,62.ectively . The clo studies , whilst  studies . A recen studies . A shift studies ,32. This studies , attempt


Further optimization may be needed for sediments with low biomass or high organic content. Overall, the new protocol enables fast and accurate quantification of marine sediment viruses, and opens up the options for virus sorting, targeted viromics, and single-virus sequencing.Viruses are highly abundant, diverse, and active components of marine environments. Flow cytometry has helped to increase the understanding of their impact on shaping microbial communities and biogeochemical cycles in the pelagic zone. However, to date, flow cytometric quantification of sediment viruses is still hindered by interference from the sediment matrix. Here, we developed a protocol for the enumeration of marine sediment viruses by flow cytometry based on separation of viruses from sediment particles using a Nycodenz density gradient. Results indicated that there was sufficient removal of background interference to allow for flow cytometric quantification. Applying this new protocol to deep-sea and tidal-flat samples, viral abundances enumerated by flow cytometry correlated well (R WIn general, viral abundance is a basic parameter for understanding virus-host interactions and the dynamics of viruses in sediments. Epifluorescence microscopy (EfM) has long been a standard technique for viral enumeration in aquatic environments ,8. RecenConventional sediment virus extraction protocols typically involve a combination of physical and/or chemical treatments, followed by centrifugation and filtration to remove cells and larger particles. With these protocols, very few cases of successful application of flow cytometry to sediments or soil have appeared in the literature. One study for example demonstrated the potential of flow cytometry for the enumeration of viruses from freshwater lake sediment . HoweverPreviously, a buoyant density-based method was developed for the extraction of microbial cells from sediments, which reduced interference associated with sediment particles ,23. The Here, viral particles were detected as SYBR Green I-stainable particles that pass through 0.2 µm pore size filters. This criterion excludes a certain fraction of viruses, for example large viruses, prophages or viruses that are not efficiently stained by SYBR Green I . On the other hand, non-viral particles such as gene transfer agents, membrane vesicles, cell debris or small cells that pass through 0.2 µm filters may also be erroneously detected as viral particles ,25. KeepA variety of sediment types, both deep-sea and tidal-flat sediments, from different biogeochemical and lithological backgrounds was chosen for analysis by FCM and EfM . The deeAdditionally, fresh tidal-flat sediments from the German Wadden Sea were sampled at the beach of Dangast . Two sample types with predominantly sandy and muddy sediment composition were collected near and below the low-water line of the beach to a depth of 10 cm using sterile spatulas. The samples were stored in the dark at an in situ temperature of 4 °C for two weeks until analysis. Before extraction, slurries were prepared by mixing the tidal-flat sediments 1:1 with 0.02µm-filtered 2.5% sodium chloride.Detailed descriptions of the geochemical analyses of the deep-sea sediments are found in Pohlner et al. . In brieg). This procedure limits the readsorption of viruses back onto sediment particles after separation. Larger and denser particles migrate to the denser Nycodenz layer, while the majority of viruses remains in the top and 30% Nycodenz layer. Some viruses still associated to particles may cross the 50% layer and are recovered in a second round of extraction. The virus-containing fraction was filtered through 0.2 µm filters  to further remove cells and particles. The extraction was performed twice for each sample and both extracts were combined. A subsequent DNase I treatment  reduced the background fluorescence of extracellular DNA as suggested by Danovaro and Middleboe [To remove particles that may interfere with FCM and EfM analysis, viruses were separated from the sediment matrix utilizing pyrophosphate, sonication, and a Nycodenz density gradient as described by Pan et al. . A schemiddleboe . For FCMiddleboe . The remExtracts were prepared and analyzed according to Brussaard et al. . First, To verify quantification by FCM, virus enumeration was also conducted using EfM, following Suttle and Fuhrman . BrieflyA set of marine sediments with a variety of different lithological characteristics was selected to test the method across different sediment types. The sediments can roughly be subdivided into shallow, tidal-flat sediments and deep-sea muds from different geographical areas. The geochemical analyses of the deep-sea sediments revealed differences in the productivity of the overlying water column, sedimentary TOC contents, as well as mineralogical features indicated by the Si/Al ratio . The sed7 virus particles per mL) compared to the conventional method (2.3 × 105 per mL) (The Nycodenz method has been shown to improve virus separation and recovery from sediment . For the per mL) . The flo8 to 5.7 × 109 per cm3 of sediment and from 1.4 × 108 to 3.4 × 109 in the sandy tidal-flat samples. Viral numbers in the deep-sea samples varied between 4.6 × 106 and 1.9 × 108 virus particles per cm3 of sediment for the South Pacific Gyre, between 2.9 × 107 and 5.3 × 108 for the Equatorial upwelling region and between 9.2 × 106 and 2.1 × 107 for the North Pacific Gyre.The Nycodenz separation method was tested on two sample types, covering a wide range of virus abundances. Tidal-flat sediments resulted in the highest virus counts, while deep-sea sediments contained the lowest numbers. Calculating the original virus numbers from FCM quantification, virus particles in the muddy tidal-flat samples ranged from 5.9 × 10Prior to analysis of the samples, gating of virus populations within the cytograms was decided based on comparing samples containing natural virus populations to blanks and autoclaved sediment controls. The virus population gate was close to the detection limit of the instrument for all samples . Autocla4 and 1.2 × 108 per ml of slurry, when determined by FCM and between 2.7 × 105 and 2.0 × 108 per ml of slurry, when enumerated by EfM. Linear regression analysis of all samples (n = 31) showed that results from both quantification methods were highly correlated , indicating that FCM is as suitable as EfM for the enumeration of sediment viruses over the range of tested marine sediments. Lab and instrument variation was low, as approximately two thirds of all data points fell inside the 99% confidence interval of the regression, irrespective of the use of different instruments in individual labs (2 = 0.92) than the Attune NxT at JAMSTEC (R2 = 0.71). However, this was likely due to a smaller sample set size and lower abundance range analyzed on the Attune NxT. Correlation between FCM and EfM was strong even among different types of samples and composition, suggesting the suitability of the extraction method for other sediment types.We assessed the FCM method against EfM using a variety of marine sediment samples from the deep sea and tidal flats . Viral aual labs . A stron6 virus particles per ml of slurry), counts of EfM tended to be higher than those determined by FCM. Within this range, abundances assessed by both counting methods differed on average by a factor of 8.2 (range 4.2–16). Outside of these low abundance samples, the counts differed on average only by a factor of 1.8 (range 1–2.8). Samples measured via FCM exceeded those from EfM by 1 to 7.5 times (on average 2.5), a typical range reported in other studies [The counts determined by FCM and EfM differed on average by a factor of 2.7 range 1–16). For the majority of samples (65%), counts differed by a factor of less than 3. In lower ranges (<10. For the2 = 0.899) between virus counts conducted via FCM and EfM, indicating that the method proposed here is suitable for the quantification of marine sediment viruses. The correlation is in the same range as the FCM protocol optimized for aquatic samples [2 = 0.74) or activated sludge flocs [2 = 0.77). The use of the conventional method for extraction of sediment viruses [We measured a strong correlation  resemble those from a study on intertidal sand-flat sediments from the German Wadden Sea not far from our sampling station [6–1.9 × 108 virus particles per cm3) were similar to those counted from the same region by Engelhardt et al. [We tested the flow cytometry protocol on a variety of sediment types with different characteristics . Based o station , suggestt et al. . The dift et al. ,40. In ct et al. .For reliable comparisons of viral abundances between samples from different sites, an extraction protocol that works for a variety of sediment types is necessary. Sediment of different origins and compositions differ in physical and chemical structure as well as overall microbial activity. Adhesion forces between viruses and charged surfaces of sediment minerals affect the desorption of viruses ,18. Ther6 per cm3 [As viruses and organic matter share similar properties, it may be challenging to separate viruses from dissolved and particulate organics in some organic-rich sediments. Since tidal-flat sediments such as the ones used in this study are high in organic matter , this ma per cm3 . However per cm3 ,39, so f per cm3 ,42. Howe6 virus particles per mL of slurry). In aquatic studies, FCM counts are generally found to be higher than those obtained from EfM [In some of our samples, the viral abundances assessed by EfM exceeded FCM counts, especially in the lower abundance range . However, the sensitivity of virus detection may still vary among different flow cytometers as stained viruses are generally close to the detection limits of the instruments. On the Accuri C6, we were able to detect as few as 3.9 × 104 per mL of slurry. We did not test the lower limits for the Attune NxT due to lack of samples. The performance of other flow cytometers has not been tested, however many modern benchtop flow cytometers have been utilized for enumerating aquatic viruses, and we expect that the method could be translatable to other flow cytometer models and even cell sorters. More recently, sorting of virus particles coupled with multiple displacement amplification (MDA) has been successfully used to study both viral populations (targeted viromics) and/or single viral genomes [We were able to apply the described protocol in two independent labs, utilizing two different flow cytometers (Attune NxT and Accuri C6). This indicates that the method is reproducible for processing and analysis of sediment samples in different labs and on different instruments. Although the regression line correlating FCM and EfM was slightly different for both flow cytometers, the differences in the slopes and intercepts of the regression lines are statistically insignificant (analysis of covariance,  genomes ,53,54. TOverall, the sediment virus separation method using a Nycodenz gradient resulted in less background noise for a range of marine sediment samples, and enabled the fast and reliable quantification of marine sediment viruses via FCM. Since the removal of particles worked so well, the protocol could potentially be optimized for a broader range of sediment types that were not tested here, such as freshwater or estuarine sediments. As challenges with sediments containing a high organic matter load remain, the method should be further optimized before FCM is used for enumerating viruses in organic-rich matrices such as soils or highly eutrophic sediments. However, with the recent introduction of virus sorting and single-virus sequencing for aquatic environments, our method may contribute one step towards the application of these next generation flow cytometric methods to sediment samples.


An essential function of the host response is to protect the organism against invading pathogens. At present, a multiplicity of mechanisms has been described on how the host sense and response to virus infections. Viruses are intracellular pathogens. Both RNA and DNA viruses have evolved mechanisms to evade host detection and to blunt both the host innate and adaptive immune responses. Considering viruses as pathogens with a relatively fast evolutionary rate, particularly RNA viruses, the result of host-virus coevolution depends on the rapid recognition and response by the host as well as on the evasion mechanism by the virus as a continuous struggle for escape/spread and immunity/clearance of virus from the host.Picornaviridae includes some of the most important RNA viruses for human and veterinary diseases as poliovirus, rhinovirus, and foot-and-mouth-disease virus, which comprised pioneer studies on the structural aspects of viral components. In their minireview, Cifuente and Moratorio summarize genetic variation mechanisms used by picornaviruses on structural changes involved in binding receptor and capsid antibody evasion of enteroviruses to ensure adaptation, spread and survival. The Paramyxoviridae family includes several important human RNA virus as Measles, Mumps, and RSV. In their article, Bakre et al. hypothesize that viral quasi-species enable RNA viruses to modulate host gene expression by regulating miRNA function via sequence complementarity or identity with the miRNA seed sites and consequently they test the hypothesis by analyzing Paramyxovirus transcripts that mimic or bind to host miRNAs by seed sequence and found that complex molecular interactions likely occurred at this host-virus interface. The human RSV (hRSV) belongs to the Pneumoviridae subfamily and is the leading cause of severe acute lower respiratory tract infections in humans at all ages and is the main cause of hospitalization due to pneumonia, asthma, and bronchiolitis in infants. Vaccine development against this pathogen has been delayed after the detrimental effects observed in children by vaccination with a formalin-inactivated hRSV preparation, which caused enhanced disease upon natural viral infection. In this issue, Acevedo et al., discuss the eventual role of Fcγ receptor-mediated immunity underlying such disease exacerbation and immune response mechanisms involved in reinfections. Respiratory viral infections are associated to IgA nephropathy (IgAN). Here, Hu et al., using a RSV-induced IgAN exacerbation mouse model demonstrates that RSV activates C5a-C5aR1 axis and modulates the Th1, Th17, and Treg balance. C5aR1 inhibition alters both kidney damage and Th1, Th17, and Treg cell dysfunction supporting that blocking the C5a-C5aR1 axis might be a potential therapy for RSV-induced IgAN. The Dengue virus (DENV), a member of the Flaviviridae family, causes 400 million infections each year. Whereas, primary dengue infection by any of the four serotypes is asymptomatic or mild, secondary infection with a heterotypic serotype is associated with hemorrhagic fever suggesting that pre-existing immunity to DENV play a role for enhanced secondary infections. Ripoll et al. carried out molecular simulations guided by previous in vitro experiments and structural studies to explore the role of antibody fine-specificity, viral conformation, and maturation state on antibody dependent enhancement in the context of primary and secondary DENV infections. The Influenza viruses belong to the Orthomyxoviridae family and are major pathogens that affect both humans and animals causing severe respiratory illness, including pneumonia. Neutrophils and macrophages play essential roles in the clearance of influenza virus from lungs, before the onset of virus specific immunity but their uncontrolled recruitment and activation contribute to acute lung injury. In this special issue, Rudd et al. study chemokine receptors expression in a murine model of influenza-induced pneumonia and reported a new set of chemokine receptors that modulates several biological functions of neutrophils. On the other hand, Tao et al. performed a comparative analysis of whole-transcriptome RNA expression between two influenza viruses and discuss how differently expressed genes may be involved in host response and evasion mechanisms. The Herpesviridae family includes very important human pathogens as the herpes simplex virus (HSV), associated with mucosal lesions and encephalitis. Tognarelli et al. review and update several mechanisms used by HSV that have been described to evade the host antiviral response. The epithelial surfaces of the human body contain complex communities of microorganisms collectively referred to as microbiota. Since the discovery that gut microbiome instruct host immunity, great attention has been directed to this interaction. In their minireview, Domínguez-Díaz et al., provides a general overview of the pro- and antiviral effects of the microbiota to prevent viruses entry into host cells or to help them to evade the host antiviral immunity. Regulation of RNA homeostasis is a central step in eukaryotic gene expression. From transcription to decay, cellular messenger RNAs (mRNAs) associate with specific proteins in order to regulate their entire cycle, including mRNA localization, translation and degradation. The best characterized of such RNA-protein complexes are Stress Granules (SGs) and Processing Bodies (PBs), which are involved in RNA storage and RNA decay/storage, respectively, and are generally associated with repression of gene expression. Gaete-Argel et al. performed an exhaustive update about how viruses have evolved different mechanisms to counteract SGs and PBs assembly or to use them to his own benefit. Moreno-Altamirano et al. explore how viruses mimic, exploit, and/or interfere with host cell metabolic pathways and how, in doing so, they may evade immune responses. Programmed cell death protein (PD-1) and its ligands have received immense attention because of their role in the evasion of tumor cells from antitumor immunity. However, it has been less appreciated that the PD-1/PD-L1 axis also regulates antiviral immune responses and is therefore modulated by a number of viruses. Here, Schönrich and Raftery update and discuss the current literature regarding this important expanding field.In this Special Research Topics issue on the recent advances in Viral Evasion Mechanisms of the Host Response, we compiled a total of twelve research and review articles. The special issue includes five Original Research Articles, five Review Articles, and two Mini Review Articles. Meanwhile five articles were dedicated to viral general mechanisms, seven were specifically focused on picornavirus, respiratory syncytial virus (RSV), Dengue virus (DENV), herpes simplex virus (HSV), and Influenza virus (IAV). The family All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.SL is employed by Denka Life Innovation Research. The remaining 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.


Many viral infections can be prevented by immunizing with live, attenuated vaccines. Early methods of attenuation were hit-and-miss, now much improved by genetic engineering. However, even current methods operate on the principle of genetic harm, reducing the virus’s ability to grow. Reduced viral growth has the undesired side-effect of reducing the host immune response below that of infection with wild-type. Might some methods of attenuation instead lead to an increased immune response? We use mathematical models of the dynamics of virus with innate and adaptive immunity to explore the tradeoff between attenuation of virus pathology and immunity. We find that modification of some virus immune-evasion pathways can indeed reduce pathology yet enhance immunity. Thus, attenuated vaccines can, in principle, be directed to be safe yet create better immunity than is elicited by the wild-type virus. directed attenuation by interfering with (some) viral immune-evasion pathways can yield a mild infection but elicit higher levels of immunity than of the wild-type virus.Live attenuated virus vaccines are among the most effective interventions to combat viral infections. Historically, the mechanism of attenuation has involved genetically reducing the viral growth rate, often achieved by adapting the virus to grow in a novel condition. More recent attenuation methods use genetic engineering but also are thought to impair viral growth rate. These classical attenuations typically result in a tradeoff whereby attenuation depresses the within-host viral load and pathology , but reduces immunity . We use models to explore ways of directing the attenuation of a virus to avoid this tradeoff. We show that  Many highly successful viral vaccines use live attenuated viruses, which are typically variants of the wild-type virus that have a genetically reduced capacity to grow –3. AttenViral attenuation entered a new era with genetic engineering, when it became possible to achieve quantitative reductions in viral growth rate and to block evolutionary reversion of attenuation –10. MuchThe need for new methods of viral attenuation is not necessarily obvious. Many attenuated vaccines in use—created by old methods—have brought about dramatic reductions in the prevalence of infections with few side effects, so is there anything to be gained from changing their design? However, live attenuated vaccines typically do not elicit as much immunity as natural infection –16. ThisIt is not a trivial matter to engineer a live vaccine and knowingly vary the immune response. It has become practical to engineer a virus to knowingly reduce the pathology . HoweveThe first step in directing attenuation involves characterizing the different viral proteins and how they interact with the cells and molecules involved in the generation of immune responses—the subject of recent advances in molecular and cellular virology and immunology . YeThe approach to directed attenuation presented here is to develop a mathematical model that incorporates key elements of our current understanding of the replicating virus and the immune response it elicits. We then use the model to explore whether directed attenuation might simultaneously reduce pathology and enhance immunity, and if so, to identify candidate pathways for virus engineering.V equal the virus density and Z and X equal the magnitude of the innate and adaptive responses respectively; all are functions of time.We use a deliberately simple model for the dynamics of infection with a virus—whether wild-type or vaccine. Let r in the absence of innate and adaptive immunity. Virus is killed by innate and adaptive immunity at rates proportional to current immunity levels and the parameters kZ and kX respectively. Innate immunity, Z, is activated by infection and saturates at 1, so Z equals the fraction of the maximum possible value of innate immunity. Adaptive immunity, X, grows in proportion to innate immunity (Z). This representation is a modification of that usually used, where the stimulation of adaptive immunity depends only on the amount of virus V. It is justified on biological grounds that the generation of an adaptive immune response requires stimulation by activated innate immunity ; the observation that virus antigen is typically presented to antigen-specific immune cells in the lymph nodes for an extended period, potentially after live virus is cleared, also supports disconnecting adaptive immunity expansion from live virus  A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. 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If you believe any reviews to be missing, please contact [LINK]Reviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: This is a well-written paper presenting a novel and interesting idea on the more intelligent design of vaccines. The idea is illustrated by analyzing an elegantly simple model of the innate and adaptive immune system. This modeling is required as results are surprising: in retrospect most parameter combinations that work are intuitive, but I would not have been able to predict these particular combinations beforehand. I also like your figures: very intuitive and clear.I have only two major recommendations:1. One can test the generality of the main results, i.e., the 3 parameter combinations giving decreased pathology and increased immunity, by exploring a few alternative models. For instance, a model where the adaptive immune response is  limited by antigens.2. It would be good to see if the main mathematical results can be put into intuitive general conclusions like: prolonging innate immune is beneficial because it extends the period over which the adaptive immune response expands.Minor comments:Page 2: Historically, the mechanism of attenuation has been one of genetically reduced viral growth rate,=> Historically, the mechanism of attenuation has been a reduced viral growth rate, ?Page 3:  suggestedWrite our the theyFinally, although I agree that this needs to be worked our for every particular virus, filling in these details and modeling them in an intuitive manner will be a major challenge. Again, it would be good to have a better sense of the generality of the results.Reviewer #2: This is a very interesting paper, which proposes parameters of the interaction between a pathogen and the immune system which could be targeted in designing a vaccine. The authors suggest that making various changes to these parameters, such as extending the lifespan of innate immunity, could simultaneously lower the pathology of the vaccine and raise immunity to it. Standard vaccines involve a pathogen whose growth rate is diminished. This results in lower pathology, but also lower immunity. The effects of simultanously varying various pairs of parameters of the model of pathogen and immune response are attractively summarised in heat maps of the pathology and immunity. If it is found possible to vary the identified parameters biologically, this could be very useful in designing vaccines.Table 1 should be referred to in the text and authors should explain how their parameter estimates are derived.There are also a few tiny errors:p3 "This can potentially result in reinfection with circulating virus p10 "However if an individual was to" ("To" -> "to")p22 "As seen in Fig 3, in both these scenarios.." p 22 "As the adaptive immune response is needed for virus clearance we find that the duration of infection is not significantly affected and consequently the amount of adaptive immunity generated does not substantially decrease." 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For instructions see  18 Sep 2020AttachmentPlosResponseToReviewers.rtfSubmitted filename: Click here for additional data file. 2 Dec 2020Dear Prof. Antia,We are pleased to inform you that your manuscript 'Directed attenuation to enhance vaccine immunity' has been provisionally accepted for publication in PLOS Computational Biology.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. 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Although transport into the nucleus mediated by the importin (IMP) α/β1-heterodimer is central to viral infection, small molecule inhibitors of IMPα/β1-dependent nuclear import have only been described and shown to have antiviral activity in the last decade. Their robust antiviral activity is due to the strong reliance of many different viruses, including RNA viruses such as human immunodeficiency virus-1 (HIV-1), dengue (DENV), and Zika (ZIKV), on the IMPα/β1-virus interface. High-throughput compound screens have identified many agents that specifically target this interface. Of these, agents targeting IMPα/β1 directly include the FDA-approved macrocyclic lactone ivermectin, which has documented broad-spectrum activity against a whole range of viruses, including HIV-1, DENV1–4, ZIKV, West Nile virus (WNV), Venezuelan equine encephalitis virus, chikungunya, and most recently, SARS-CoV-2 (COVID-19). Ivermectin has thus far been tested in Phase III human clinical trials for DENV, while there are currently close to 80 trials in progress worldwide for SARS-CoV-2; preliminary results for randomised clinical trials (RCTs) as well as observational/retrospective studies are consistent with ivermectin affording clinical benefit. Agents that target the viral component of the IMPα/β1-virus interface include N-(4-hydroxyphenyl) retinamide (4-HPR), which specifically targets DENV/ZIKV/WNV non-structural protein 5 (NS5). 4-HPR has been shown to be a potent inhibitor of infection by DENV1–4, including in an antibody-dependent enhanced animal challenge model, as well as ZIKV, with Phase II clinical challenge trials planned. The results from rigorous RCTs will help determine the therapeutic potential of the IMPα/β1-virus interface as a target for antiviral development. Compartmentalisation is a key feature of the eukaryotic cell, with transport between the different compartments essential for cellular function . The douThe transport of proteins >45 kDa into and out of the nucleus is mediated by members of the importin (IMP) superfamily, of which there are multiple distinct α and β forms . The besMany viruses exploit the host cell nuclear trafficking machinery, and in particular, the pathway mediated by the host IMPα/β1 heterodimer, in order to enhance their ability to replicate or to subvert the host immune response . NuclearTo gain access to the nucleus, proteins generally require nuclear localisation signals (NLSs), which are recognised by either the IMPα subunit within the IMPα/β1 heterodimer, or by one of the IMPβs directly ,6,2,5. TThe host cell nuclear transport can be exploited by viral proteins that target nuclear processes to antagonise the host antiviral response. These include DENV NS5, which accesses the nucleus in order to impact many host nuclear processes ; this leIn the case of other proteins such as HIV-1 IN and influenza A NP, nuclear access is critical to the viral life cycle . DependeThe critical importance of the IMPα/β1-virus axis to viral infection has been formally demonstrated for many viruses, including DENV, where mutagenesis of the NS5 NLS nuclear targeting through impaired IMPα/β1 recognition results in an attenuated virus and markedly reduced infectious virus production . In addiStreptomyces avermitilis, which was discovered in 1975, and approved for use in humans as an antiparasitic agent 10 years later. It is US FDA-approved for treating a range of ectoparasitic and endoparasitic infestations, with an excellent safety profile and millions of people treated globally for onchocerciasis (river blindness) and lymphatic filariasis . Th. Th44]. in vitro and in vivo [In vitro antiviral properties have been demonstrated for a wide range of viruses, including Betacoronavirus SARS-CoV-2 (COVID19), the lentivirus HIV-1, alphainfluenzavirus (influenza A), flaviviruses , alphaviruses , the Hendra henipavirus, mastadenoviruses (human adenovirus — HAdV — B and C), the BK betapolyomavirus and PSV varicellovirus  [The IMPα-dependent host-targeted mode of action of ivermectin explains the wide range of viruses for which ivermectin has demonstrated antiviral effects both  in vivo . In vitrllovirus . In the  (PBMCs) ,44. It i (PBMCs) .). GW5074 was identified as an inhibitor of IMPα/β1-DENV NS5 binding [Compound library HTS has enabled the identification of small molecule IMPα-targeting inhibitors other than ivermectin see . GW5074  binding ,47. Rece binding  has been binding . GSP has binding .In silico structure-based-drug-design HTS has also been successfully applied to identify nuclear transport inhibitors [In silico screening of 1.5 million compounds for the ability to dock into the NLS-binding site of IMPα and thereby mimic binding of the VEEV CP NLS to IMPα identified a range of different small molecules that were subsequently confirmed experimentally as being able to inhibit binding of IMPα/β1 to VEEV CP. Several of the lead compounds, including 1111864 .The glucocorticoid budesonide was identified in the same HTS campaign as mifepristone (IC50 of c. 1 μM), subsequent characterisation using NMR confirming that budesonide also binds to the HIV-1 IN NLS region . SAR/NMRin vitro . Importain vitro . Both bua > 14 000 compound library for inhibitors of IMPα/β1:VEEV CP binding, followed by a nested counterscreen, many chemical scaffolds were identified that retained potent inhibitory activity [in silico-informed SAR analysis focussed on several of these, with compound G281–1564 confirmed to be a specific inhibitor of IMPα/β1:CP interaction. Experiments in transfected cells confirmed inhibition of CP but not SV-40T-Ag nuclear accumulation, with fluorescence recovery after photobleaching experiments demonstrating inhibitory effects on both the rate and extent of CP nuclear import. Importantly, G281–1564 was found to limit VEEV infection in cell culture (EC50 of 10.8 μM) [From HTS of activity . Subsequ10.8 μM) ; subsequ10.8 μM) .ex vivo model of severe antibody-dependent enhanced (ADE) human infection using PBMCs [One of the most promising inhibitors targeting the virus side of the IMPα/β1-virus axis is N-(4-hydroxyphenyl) retinamide , which was identified through HTS as a specific inhibitor of IMPα/β1:DENV NS5 interaction (IC50 of 0.8 μM) . 4-HPR wng PBMCs . 4-HPR ang PBMCs . TSA assng PBMCs , highligIt is of interest that 4-HPR has also been identified in HTS (see above) as an inhibitor of CHIKV , and repHost-directed agents that impact essential cellular activities like nuclear transport must be used with caution in a clinical setting. Of the small molecule inhibitor drugs targeted against the host IMPα/β1 side of the IMPα/β1:virus interface, ivermectin is the only one that has progressed from to human clinical trials for viral indications. Although it has an established safety profile in humans ,56, and Pre-clinical studies in a lethal mouse PRV challenge study showed that ivermectin administration (0.2 mg/kg) 12 h post-infection rescued 50% of mice, while the same dose at the time of infection rescued 60% of mice .50 indicated by in vitro experiments  worldwieriments  are likeResults from retrospective/observational trials for ivermectin  are also+ lymphocyte counts [Of the small molecule inhibitors targeting the virus side of the IMPα/β1:virus interface, only mifepristone has thus far been investigated clinically in any detail. Mifepristone was shown to reduce virus production and viral DNA copy number in HAdV-infected mice, concomitant with inhibition of HAdV genome nuclear import . Howevere counts .In the case of 4-HPR as a therapeutic for flavivirus infection, results are thus far restricted to mouse models. In AG129 mice, twice-daily dosage of 4-HPR at 180 mg/kg (resulting in ∼15 μM plasma concentration of 4-HPR), reduced viremia >50-fold in DENV2 infection . In a leIt is clear from the last decade that small molecules targeting the host IMPα/β1:virus interface can be found that have potent antiviral activity, and exciting potential to treat human infection . While hin silico HTS emerging as a complementary approach to identify promising leads, and facilitate focussed library selection  have pron e.g. ,52). Dru. Druin sNuclear import is essential to the infectious cycles of a range of viruses of medical significance. Inhibitors of nuclear import targeting either the host or virus side of the IMPα/β1:virus interface have been identified in recent years, and shown to possess potent antiviral activity.Host-targeted inhibitor of nuclear import ivermectin is showing exciting early results as a treatment for COVID-19 infection. Virus-targeted inhibitor of nuclear import of flavivirus NS5, 4-HPR, appears to be an exciting prospect for future clinical trials.The process of nuclear import, and the IMPα/β1:virus interface in particular, remains a viable target of interest for the future identification and development of antivirals to tackle existing and emerging viral disease.


The fields of extracellular vesicles (EV) and virus infections are marred in a debate on whether a particular mRNA or non-coding RNA  is packaged into a virus particle or copurifying EV and similarly, whether a particular mRNA or non-coding RNA is contained in meaningful numbers within an EV. Key in settling this debate, is whether the purification methods are adequate to separate virus particles, EV and contaminant soluble RNA and RNA:protein complexes. Differential centrifugation/ultracentrifugation and precipitating agents like polyethylene glycol are widely utilized for both EV and virus purifications. EV are known to co-sediment with virions and other particulates, such as defective interfering particles and protein aggregates. Here, we discuss how encased RNAs from a heterogeneous mixture of particles can be distinguished by different purification methods. This is particularly important for subsequent interpretation of whether the RNA associated phenotype is contributed solely by virus or EV particles or a mixture of both. We also discuss the discrepancy of miRNA abundance in EV from different input material. Virus particles have similar biophysical attributes to naturally occurring extracellular vesicles (EV) . Virus pFirst, how do RNAs co-purify with viruses and with EV by different purification methods?Second, what have we learned from different purification methods? Do we need standards and, if so, what criteria should be used to evaluate claims of RNA-EV and RNA-virion associations?Third, do DNA viruses package miRNAs or mRNAs into virus particles ? All vir3). Fick’s laws apply to all experiments conducted in culture dishes and represents a lower limit regarding experiments in animals, where inter-tissue diffusion, intra-tissue diffusion, clearance, and blood flow rapidly dilute bolus injections of EV. Viruses overcome physiological dilution by utilizing highly specific receptors and co-receptors, like cluster of differentiation 4 (CD4) .,27.18,27Ultracentrifugation—Centrifugation separates particles based on density and size. The standard equation for sedimentation velocity is:Here, e medium . When thgregates , a gradigregates . Often tgregates ,31,32,33- PEG has long been used to “precipitate” and purify viruses [Polyethylene glycol (PEG) Precipitation  viruses ,35. It i viruses ,35. The  viruses , and oft viruses ,35,37,38 viruses . This in-filtration is a size-based separation technique. Based on pore sizes of the membranes, filtration is divided into microfiltration (0.1–1 µm) and ultrafiltration (0.01–0.1 µm). Ultrafiltration membranes are referred to according to their molecular weight cutoff (MWCO). The technique can be performed either by normal flow filtration (NFF) or by tangential flow filtration (TFF), also called cross-flow filtration [Filtrationltration . It sepaltration ,40. Chromatography- Size-exclusion chromatography (SEC), ion-exchange chromatography (IEC), and affinity chromatography (AC) are commonly used chromatography methods for virus purification ,42,43. IAffinity chromatography (AC) offers a higher separation efficiency. AC relies on specific ligands, either low molecular weight or antibodies. For instance, heparin chromatography separated HIV-1 virus like particles (VLPs) from EV, after a Capto Core 700 polishing step . In beadUltracentrifugation is widely used to purify EV . This mePEG-based methods, such as ExoQuick  effectively precipitate EV out of solution. They also, invariably, precipitate contaminant soluble proteins and PEG concentrates almost all viruses. For high throughput diagnostics, this is completely acceptable as it maximizes analytic sensitivity at the cost of specificity; however, EV isolated by precipitation alone is not suitable for functional studies. Of note, PEG can change the osmolarity inside a vesicle by creating external osmotic pressure but PEG itself does not cross the lipid membrane .Proteinase digestion  can remoNucleic acid digestion removes molecules that are attached externally to the EV, such as circulating RNAs and cell-free DNA (cfDNA) fragments. RNase treatment represents a classic and essential step in separating externally associated RNAs from encased and protected RNAs. Of note, certain miRNAs that are associated with Ago proteins, but not part of EV are protected from RNase treatment . While eIn our hands, filtration by a larger-than-particle diameter is essential in removing aggregate EV, fused vesicles, and virions. For instance, filtration by 0.22 µm microfilter was essential in separating Kaposi’s sarcoma herpesvirus (KSHV) virion from KSHV–EV . The avaCommercially available SEC columns, such as qEV , outperform other precipitation-based commercial kits according to particle/protein ratios . RecentlA very recent example  used asyChemicals and proteins may be used to inhibit EV secretion (or virion maturation) and thereby reduce contamination of virion preparations or EV preparations. For example, the neutral sphingomyelinase (nSMase) inhibitor GW4869 ,62 and tAffinity-based tools and specialty column resins provide this capability and often out-perform traditional methods. Follow up experiments such as Western blot for virus or EV protein markers, virus plaque assay for infectivity or ELISA for EV marker acetylcholinesterase activity have been used to confirm separation of the virus from EV. The field standard , writtenIn sum, separating virions from EV is highly dependent on the intended use , the density, and the size of the specific EV populations and virus species that are being investigated. More than one technique is typically needed to ensure the separation of these two biologically different populations ,40,60. A virus particle acts as an extracellular vehicle to transfer the viral genome, virus-encoded proteins and enzymes, and some host factors. RNA viruses package RNA, in the form of the viral genome, but also for other purposes. For instance, HIV packages transfer RNA (tRNA) molecules in each virion to initiate reverse transcription ,66,67. TSeparating EV from virus particles, particularly exosomes and microvesicles, has proven to be a considerable hurdle in the field of host–pathogen interactions. Chugh et al.  and BessBreshnahan, et al.  was awarGreijer et al.  treated Since it is near impossible to separate EV from virions by biochemical methods, the absence of EV is typically demonstrated by the absence of EV protein markers. For instance, Cliffe et al.  checked The problem becomes more difficult when considering that as virus-infected cells not only release virions with virus-derived RNAs, they also release EV filled with virus-encoded RNAs at the same time, as well as various species of defective interfering particles. Hence, we would expect EV to contain viral RNA under most circumstances. EV emanating from cells infected with HIV, hepatitis C virus (HCV), and various human herpesvirus viruses (HHV) can have virus-encoded RNAs present within them ,84,85. IAnother open question in the field is how many copy numbers of nucleic acids are present in a single EV. On the one hand, functional studies clearly show distinct phenotypes that are mediated by miRNA transfer through EV ,86,87,88The miRNAs, mRNAs, and other RNAs enclosed within EV are transferred to other recipient cells to elicit functional impacts ,87,88. Tg spin for 70 min. Ultracentrifugation or filtration alone may not have been enough to produce clean exosome preparations in these studies. The exosome particle numbers could be overestimated since protein aggregates or cell debris may be present and counted by nanoparticle tracking analysis [It is difficult to establish the physiological significance of EV-transduced miRNAs, without knowing how many miRNAs are transduced in each experiment. On the one hand, stoichiometric analysis of exosomes isolated from healthy or cancer patient human biofluids found a very low number of copies (0.00825 ± 0.02) of any single miRNA per EV ,89. On tanalysis . Anotheranalysis . EV fromanalysis ,89, but analysis .For viral RNAs, we estimate ≥ 1 miRNA per EV, i.e., comparable to liposome-mediated transfection of small interfering RNAs (siRNAs). This enrichment is due to the fact that viruses reprogram the infected cells to preferentially expressed viral RNAs that are then packaged into EV. For instance, herpesviruses are unique in encoding and expressing multiple miRNAs ,96,97,988–109 particles/mL [11–1012 particles/mL [5–106 cells/mL. There are about 109 red blood cells, 108 platelets, and 106 white blood cells per mL in human blood. Roughly estimating, EV concentration is 100–1000 times higher than the nucleated cell concentration in culture and in the blood. In the meantime, EV is taken up by cells every hour [Unlike siRNA transfection in culture, which is a one-time event, EV mediated delivery of miRNA in vivo is a continuous and dynamic process over an extended period in a biological environment rich in EV. A single cell may be exposed to one million EV or more fusion events per cell division cycle. The precise rate of EV uptake per cell is unknown, but the EV concentration in conditioned media and bodily fluid is around 10icles/mL ,40 and 1icles/mL , respecticles/mL  by cellsery hour , so overery hour  or latenery hour —the cellery hour . Whereas the biological function of EV is still far from understood and requires further study, the diagnostic utility of EV is well established. Exosome enclosed miRNAs serve as potential biomarkers since miRNAs within the exosomes are protected from RNase digestion and are more stable in primary fluids than free circulating RNAs. They are especially useful for diagnosing viruses, which establish long-term low-level infections and chronic disease states that are dependent on the viral miRNAs ,46,100. The observation that nucleic acids and proteins can be encased and transferred by EV has prompted the discovery of new interplays between host cells, host organs, and viruses. Still, better and more carefully validated purification methods are necessary to prepare cleaner virion and EV preparations before many of the proposed biological functions that have been associated with EV can be accepted. Affinity reagents, in particular, reveal surprising heterogeneity amongst EV. Describing and limiting the increasing complexity of EV may seem burdensome, but it is essential for establishing biological relevance.


BMC Biology entitled “A tissue level atlas of the healthy human virome” by Kumata et al. describes a meta-transcriptomic analysis of RNA-sequencing datasets from the Genotype-Tissue Expression (GTEx) Project. Using a workflow that maps the GTEx sequences to the human genome, then screens unmapped sequences to detect viral transcripts, the authors present a quantitative analysis of the presence of different viruses in the non-diseased tissues of over 500 individuals and assess the impact of these viruses on host gene expression. Here we draw attention to an issue not acknowledged in this study. Namely, by relying solely on GTEx datasets, which are enriched for transcripts with poly(A) tails, the analysis will have missed non-poly(A) viral transcripts, rendering this tissue level atlas of the virome incomplete.A recent paper in A commentary on Kumata et al. . Viruses are obligate parasites and require a living cell to complete their life cycles. Like mRNAs in the eukaryotic host cell, RNAs of many DNA and RNA viruses generate polyadenylated transcripts  tails) that are synthesized post-transcriptionally , and in Flaviviridae family [Many viruses, however, generate transcripts without poly(A) tails, a feature that has been maintained over evolution, especially in positive-strand RNA viruses as for instance are dengue virus, West Nile virus, Japanese encephalitis virus, yellow fever virus, Zika virus, bovine viral diarrhea virus, and hepatitis C virus in the e family –6. Othere family . Viral-ee family .BMC Biology article by Kumata et al. presented the first tissue level atlas of the human virome by analyzing the RNA-seq data from the GTEx database [Next-generation sequencing offers high sensitivity, specificity, and reproducibility in detection of low levels of transcripts thereby serving as a sensitive and reliable tool to qualify and quantify viruses at DNA and RNA levels . Neverthdatabase . GTEx usdatabase . We belidatabase . Furtherdatabase . Indeed,database , 6, whicBefore other researchers are motivated to apply their meta-transcriptomic study approach  to other Correspondence: ksato@ims.u-tokyo.ac.jpBMC Biology titled “A tissue level atlas of the healthy human virome” [In our recent paper in  virome” , we perfAs described in the first sentence of the “Method” section of our paper , we usedSome aspects of the technical limitations of our study were described in the “Discussion” and “Conclusion” sections of our study , and it


Giardia intestinalis is an intestinal protozoan most commonly found in humans. It has been grouped into 8 assemblages (A-H). Markers such as the glutamate dehydrogenase gene, triose phosphate isomerase and beta-giardin (β-giardin) have been widely used for genotyping. In addition, different genetic targets have been proposed as a valuable alternative to assess diversity and genetics of this microorganism. Thus, our objective was to evaluate new markers for the study of the diversity and intra-taxa genetic structure of G. intestinalis in silico and in DNA obtained from stool samples. We analysed nine constitutive genes in 80 complete genome sequences and in a group of 24 stool samples from Colombia. Allelic diversity was evaluated by locus and for the concatenated sequence of nine loci that could discriminate up to 53 alleles. Phylogenetic reconstructions allowed us to identify AI, AII and B assemblages. We found evidence of intra- and inter-assemblage recombination events. Population structure analysis showed genetic differentiation among the assemblages analysed.  Giardia intestinalis , a single-celled eukaryotic protozoan, is the most common cause of parasitic diarrhoea in humans worldwide [orldwide . It infeorldwide . Transmiorldwide  and wildorldwide . The freorldwide . Still, G. intestinalis [Different molecular tools ,7,8 and stinalis ,13, basestinalis ,15. Thesstinalis ,17,18,19stinalis . Assemblstinalis ,22,23. Sstinalis ,25, havestinalis  or low rstinalis  when ideSome authors have proposed the study of new genes , which, G. intestinalis is needed to identify new markers for understanding its diversity and evolution. Such markers should possess sufficient discriminatory power to establish groupings related to epidemiological factors. Thus, investigation of new markers should focus on detection and typing, and allow additional inference on reproduction, evolution, zoonotic potential and population structure [Evaluation of additional regions of the genome of tructure ,31,32. Structure . Initialtructure ,34, and tructure ,36,37,38tructure ,40,41. TG. intestinalis are available that sought to address additional genetic markers. Yet, generating multilocus analyses is essential for understanding the genetic characteristics of circulating strains in different geographical regions and monitoring their evolution and adaptation. Such analysis will encourage the design of strategies to decrease infection incidence [G. intestinalis tested in publicly available whole-genome sequences (WGS) and subsequently analyse these markers in DNA from stool samples from some regions of Colombia. Our proposed new markers are capable of elucidating diversity, population structure and possible recombination events between and within G. intestinalis assemblages.Few studies on ncidence . In the Pyrophosphate-fructose 6-phosphate 1-phosphotransferase alpha subunit (PFP-ALPHA1), Fructose-bisphosphate aldolase (FBA), Phosphoglycerate kinase (PGK), enolase, Acetyl-CoA synthetase (ACS), NADP-dependent malic enzyme (NADP-ME), Serine palmitoyltransferase 2 (SPT), Glutamate dehydrogenase (GDH) and Triose phosphate isomerase (TPI) genes in 80 available WGS from G. intestinalis. , AII (DH), B (GS and GS_B) and E (P15)  for varitinalis. . We mappNADP-ME and SPT genes displayed the highest values for both nucleotide diversity (Pi: 0.107 and 0.108. respectively), Theta (per site) from Eta (0.060 and 0.059. respectively) and numbers of segregating polymorphic sites . The SPT showed the fewest haplotypes (h: 11) and haplotypic diversity (Hd: 0.686). In contrast, genes such as GDH, commonly used to type G. intestinalis, showed a nucleotide diversity value, Pi of 0.058 and a Theta (per site) from Eta of 0.034, both being the lowest among analysed loci . However, when analysing assemblages, results for all loci in AI and B assemblages were negative  and discriminatory power (DP), using MLSTest software  . Numbers alleles . We alsoS, 0.815 .G. intestinalis assemblages most commonly found in humans, the AI, AII and B  , for all evaluated alignments, using Dnasp software . The higctively) . This re, both by gene and concatenated sequence. . The preStatistics used for genetic differentiation between populations are shown in G. intestinalis, collected in different regions of Colombia, from Amazonas [We randomly chose 24 samples positive for Amazonas , CasanarAmazonas .G. intestinalis axenic culture. All primers adequately amplified corresponding regions with each locus. A single band of the expected size was obtained for each marker (n = 24) of positive samples. Of the 24 samples, 95.8% (n = 23) amplified GDH; 83.3% (n = 20) amplified TPI; 66.7% (n = 16), ACS; 29.2% (n = 7) SPT, and 50.0% (n = 12), enolase. Many other samples amplified the target genes, and a band of the expected size was evident. However, other bands of different sizes were also observed. Also, concentrations of products obtained after the polymerase chain reaction (PCR) were low in some cases, showing bands so thin that it was impossible to obtain results from these sequences or no amplification occurred. Poor quality electropherograms were obtained for the NADP-ME gene, and sequences for this gene were ignored. Finally, five genes  were evaluated with DNA from the 24 stool samples. At least three markers were amplified in each sample, and all five loci were amplified in a few samples. , compared to other loci, and the TPI locus the lowest number (S = 133), even though it displays the largest number of sites analysed. Also, nucleotide diversity, Pi, for the ACS gene was 0.210, followed by SPT and TPI loci with estimates of 0.114 and 0.109, respectively. Similarly, the ACS locus presented the highest value for Theta (per site) from Eta. Thus, locus that shows the greatest diversity is ACS. It is important to highlight that calculation of D Tajima as a neutrality test produced almost universally negative values. Not all values statistically significant, but still may indicate population expansion, at least for those loci, D Tajima for the TPI locus was positive, suggesting balancing selection. Another index calculated for this data set, corresponding to Rm, indicates a high value (Rm = 52) for the TPI locus compared to other target genes  [G. intestinalis [When comparing ML trees results with phylogenetic networks, each assemblage shows important divergence with respect to AII. Such divergences in notable for A and B, and to a lesser extent, AI. Further, we observed reticulation signals and possible genetic exchange among assemblages, mainly between A and E, and for AI and AII . InconsissurRNA) . Other gssurRNA) , based issurRNA) ,26,33,64stinalis , considestinalis . Also, cstinalis ,68.TPI gene from different continents. Genetic differentiation is reported between assemblage A and populations in Asia, Australia, and America. Moderate genetic differentiation is also seen with comparison using assemblages B and E. The latter case shows the dispersion of the same population of G. intestinalis [G. intestinalis, and for investigating diversity, evolution, genetic structure and plausible genetic exchange events.Further, admixture was observed among assemblages with the contribution of E assemblage alleles to AI, AII and B assemblages . Mixturestinalis . We consG. intestinalis in the EuPathDB database: The Eukaryotic Pathogen Genomics Resource (GiardiaDB) (https://giardiadb.org/) , Pyrophosphate-fructose 6-phosphate 1-phosphotransferase alpha subunit (PFP-ALPHA1), Fructose-bisphosphate aldolase (FBA), Phosphoglycerate kinase (PGK) and enolase. We chose Acetyl-CoA synthetase (ACS) from the end of the alcoholic fermentation process, NADP-dependent malic enzyme (NADP-ME) from the TCA cycle and Serine palmitoyltransferase 2 (SPT) from lipid synthesis  , we searorganism . Other eorganism . Specifiynthesis .https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and were chosen considering: (1) primers were located in conserved regions for all assemblages, (2) the final product had a size between 300 and 700 bp, (3) selected markers were single-copy genes in the G. intestinalis genome, and (4) information on dimers, formation of forks, melting temperature (Tm), per cent GC, size of initiator, size of the amplified region, and specificity were available. Single-copy genes were needed to estimate intra- and inter-assemblage allelic diversity, avoiding bias by recombination between copies of the genes. The fourth criterion was verified using Basic Local Alignment Search Tool (BLASTn).For bioinformatic design of primers, CDS sequences of orthologous and synthetic genes for these enzymes were downloaded for AI (isolate WB), AII (isolate DH), B (isolates GS and GS_B) and E (isolate P15) assemblages of eight genes . We alighttps://orthomcl.org/, was queried. The corresponding group of orthologs was searched for each protein to identify any alternate AA sequences. In the second, we used the CD-HIT comparative analysis algorithm, a tool for grouping and comparing biological sequences [Two approaches were used to verify single-copy genes. First, the Ortholog Groups of Protein Sequences (OrthoMCL DB) database, available at equences . SequencG. intestinalis genomes available online. Among these loci, we included genes commonly used for typing of G. intestinalis, glutamate dehydrogenase (GDH) and triose phosphate isomerase (TPI). Genes were used to evaluate genetic diversity, recombination, typing capacity, and discrimination of G. intestinalis in whole-genome sequencing (WGS) data available in the public database of The European Nucleotide Archive (ENA)  .G. intestinalis. All other genomes were discarded  tool  to extraInitially, consensus sequences of each gene and their concatenation were aligned using the multiple sequence alignment programme MAFFT v7  . Subsequhttp://itol.embl.de) [Phylogenetic trees were constructed from alignment of consensus sequences from WGS data . Subsequembl.de) . Additioembl.de) , with thOnce assemblages were established for each consensus sequence with each loci, an allelic plot schema was constructed with concatenated sequences and by gene. Each assemblage was represented in a different colour, the AI in blue, the AII in red, the B in turquoise, the E in green, and sequences that did not correspond to any assemblage were left in black. The scheme compares assemblages assigned to each consensus sequence with each gene, so that assemblage agreement among loci is represented. This scheme represents alleles found, such that the number of colours in the allelic plot represents the number of clusters discriminated by each marker, as reported in other studies .To verify the existence of recombination events, we performed an additional analysis with the Recombination Detection Programme version 4 (RDP4) , using thttp://www.ub.edu/dnasp) were sequences aligned for each marker. Indices used included nucleotide diversity (Pi)—the average number of nucleotide differences per site between a pair of DNA sequences; Theta (per site) from the total number of mutations (Eta); numbers of polymorphic (segregating) sites (S); numbers of haplotypes (h); and haplotypic diversity (Hd). The latter index indicates the probability that two random haplotypes are different. Tajima D was calculated to determine if sequences evaluated reflected neutral variation or were involved in a selection process. This index indicates a balancing selection for positive values and a purifying selection for negative values [All sequences in WGS data were used to calculate diversity indices, for each gene and concatenated sequence. Indices were also calculated with sequences grouped by assemblage. Input data in the DnaSP v.5  programme values . The minhttp://www.ub.edu/dnasp).Separate statistics for genetic differentiation among assemblages for each gene and concatenated sequence were also calculated. Assemblages that could not be defined for some sequences in some genes were assessed, as was the concatenated sequence from the E assemblage. Genetic differences were estimated using statistics based on haplotypes (Hs), nucleotide sequences (Ks), and several others that reflect gene flow from nucleotide sequences, including Wright’s F (Fst), Delta ST, Gamma ST, and Nst. Average number of nucleotide differences in pairs (Kxy), nucleotide substitutions per site (Dxy), net nucleotide substitutions per site (Da), and gene flow from haplotypes (Gst) were then calculated. DnaSP v.5 software  was usedAll subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the National University of Colombia  and the ethics committee of the Universidad del Rosario (registered in Act No. 394 of the CEI-UR). This project was conducted under the contract number RGE131 of access to genetic resources granted by the “Ministerio de Medio ambiente y Desarrollo sostenible”. G. intestinalis was performed as described in Sánchez et al. [G. intestinalis positive samples were taken at random to evaluate markers assessed in silico. The number of samples by location were: 12 from Amazonas, six from Bolívar, three from Casanare and three from Córdoba. Convenience sampling of human faeces samples was performed in the departments of Amazonas, Bolívar, Casanare, and Córdoba in Colombia. The collection, extraction and typing of samples positive for z et al.  for sampz et al.  for sampG. intestinalis axenic cultures. PCR was performed in a final volume of 25 µL, containing 2 µL of quenched DNA, 12.5 µL of Go Taq Master Mix Green (Promega) (cat. No. M7122) . at a final concentration of 1X and primers at a concentration of 1 µM each. Primers used for PCR are shown in G. intestinalis positive stool sample was amplified using the above conditions. All PCR products were verified by observation on 2% agarose gel, stained with SYBR Safe, Thermo Fisher Scientific (cat No. S33102) . Each PCR product was purified with ExoSAP-IT®, Affymetrix ™ (cat. No. 15513687)  following the manufacturer’s recommendations. Both chains of each product were sequenced with the Sanger method. Sequences were edited in MEGA 7.0 [We amplified each locus from DNA extracted from MEGA 7.0  to extraTrees and phylogenetic networks were constructed with sequences obtained for each locus. Consensus sequences from the SRST2 tool output were concatenated with sequences obtained from human faeces of Colombian origin. This process used the same procedures described above for phylogenetic reconstructions. Diversity indices were also calculated by gene with the Dnasp v.5 programme .G. intestinalis. These loci can also be used as alternatives to and supplements for genomic studies of Giardia diversity. We demonstrate intra-taxa diversity and show both genetic structures for established assemblages, and admixture among populations due to genetic exchange, apparently by recombination among individuals. Analysis of proposed loci should extend to future studies that include genomic sequences of additional assemblages to define their diversity and population structure. Further, future studies might focus on increasing the number of samples to evaluate markers on a large scale and extend sampling and analysis to other hosts and water sources that may be sources/reservoirs of infection. Such investigations will help elucidate transmission dynamics of the pathogen. Finally, future studies should assess a broader set of stool samples using nested PCR to examine the usefulness of genetic markers developed in the present study.We highlight loci useful of an MLST scheme for typing of


Utilizing a combination of 1H NMR, NOESY, DOSY, DLS, SEM, and viscosity measurements, the formation of polypseudorotaxane was shown to be dependent on the concentration of [2]pseudorotaxanes/[PdCl2(PhCN)2] and temperature. Furthermore, a temperature-responsive supramolecular gel with reversibly gel–sol transformation was obtained via spontaneous assembly of the polypseudorotaxanes at high concentrations.Self-assembly of polypseudorotaxanes in high-polar organic solvents is difficult due to remarkably weak interactions between macrocycles and axles. Reported here is a novel metal-coordinated poly[2]pseudorotaxane constructed by pillar[5]arene, 1,4-bis(4-pyridyl pyridinium)butane, and [PdCl Over the past 20 years, supramolecular architectures of (pseudo)rotaxanes and catenanes have played a significant role in supramolecular topology and the fabrication of mechanically interlocked molecules Loeb, . Polyps.Macrocycles are the basic building blocks in the construction of pseudorotaxanes because of the strong binding ability between macrocyclic hosts and guests. Therefore, there is no doubt that introducing new macrocycles and novel non-covalent interactions into polypseudorotaxanes will expand the applications of polypseudorotaxanes. Furthermore, variations in supramolecular structures allow them to show unique responsivity to stimuli. Pillar[n]arenes, the fifth generation of host macrocycles, have been applied to the formation of various functional supramolecular materials, owing to their rigid pillar architecture, easy functionalization, and outstanding binding properties in host–guest chemistry dicationic guest in DMSO is surprisingly high, leading to the formation of a [2]psdudorotaxane-type complex -containing coordination interactions [PdCl2(PhCN)2]. Therefore, a linear polypseudorotaxane was constructed by [2]pseudorotaxanes making up of P5A and bis(pyridinium)dicationic (1) via metal–coordination interactions in DMSO   at 298 K in DMSO. Dynamic light scattering (DLS) was analyzed on a Malvern Zetasizer 3000HSA at 298 K. Scanning electron microscopy (SEM) images were recorded on SHIMADZU SSX-550.All reagents and solvents were commercially available and used without further purification, unless otherwise noted. Compound  on guest 1 could be observed in the presence of P5A owing to the shielding effects in the cavity, while no apparent change was observed in the proton signals of 1. When comparing to the corresponding signals of the uncomplexed P5A and 1, new peaks were observed, demonstrating a slow exchange on the NMR timescale for this binding process. The results are in agreement with the spatial structure that the host P5A as a wheel was fully threaded by the axle of guest 1 and left pyridine moiety outside its cavity, indicating the formation of a [2]pseudorotaxane between P5A and 1 in DMSO 2] into 60 mM premixed solution of P5A and 1, all of the main peaks broadened remarkably, and the signals of 1 clearly shifted downfield (2(PhCN)2] and 1 was investigated. To a mixture of 1 and P5A ([1]: [P5A] = 1:5) in DMSO-d6, [PdCl2(PhCN)2] was added in different ratios and 1H NMR spectra were recorded. As shown in 2(PhCN)2], both the proton signals of 1 shifted downfield significantly, suggesting the coordination of metal to the pyridine rings. No obvious change was observed for the signals of 1]: [PdCl2(PhCN)2] was increased to 1:1, indicating that the binding ratio between [PdCl2(PhCN)2] and 1 was 1:1 or n:n, which fitted well with the coordination characteristics between pyridine and [PdCl2(PhCN)2] 2] and [2]pseudorotaxane.Subsequently, after dissolving 1.0 equiv of [PdClownfield . These o3 was employed to bind palladium(II) ions to investigate the phase transition between polypseudorotaxane and [2]pseudorotaxane. Upon adding 1 equiv of PPh3 to the polypseudorotaxane, a precipitate formed at the bottom of the mixed system solution. As shown in 1H NMR spectrum was almost the same with the spectrum of [2]pseudorotaxane  ions, resulting in the disassembly of the polypseudorotaxane 2] was added to the solution, and the peaks of protons on guest 1 returned to their original positions 2] increased from 0.5 to 200 mM, the weight average diffusion coefficient (D) decreased significantly from 4.59 × 10−10 to 0.88 × 10−11 m2 s−1, suggesting an increase in the average size of the polymeric structure owing to the generation of polypseudorotaxane from the small oligomers  were adopted to explore the polypseudorotaxane. When the concentration of [2]pseudorotaxane/[PdClligomers .2(PhCN)2] were carried out in DMSO at 298 K. A double-logarithmic plot of specific viscosity vs. the initial concentrations of [2]pseudorotaxanes is shown in 2(PhCN)2]/[2]pseudorotaxanes), implying that no linear polypseudorotaxane formed 2] increased above the critical polymerization concentration , a sharp increase in the viscosity was obtained (slope = 3.02), which indicated the formation of linear polypseudorotaxane resulting from strong interactions between [2]pseudorotaxanes and [PdCl2(PhCN)2] 2], metallosupramolecular polypseudorotaxane transformed into a supramolecular gel. Notably, the metal-coordinated polypseudorotaxane gel was sensitive to temperature and could transform into sol reversibly by heating to 60°C and cooling to room temperature (25°C) (2(PhCN)2]. Heating decreased that interaction and decomposed the polypseudorotaxane, and therefore, gel changed to sol. However, upon cooling, the intermolecular entanglement restored, resulting in the recovery of the supramolecular gel. The morphology of polypseudorotaxane xerogels prepared by freeze-drying was investigated by scanning electron microscopy (SEM). Regular long and fine fiber structures were observed, and the diameter was determined to be 0.2–0.3 μm  . A possi2–0.3 μm . These o2(PhCN)2] in a highly polar solvent of DMSO, which was comprehensively confirmed by various techniques, such as 1H NMR, NOESY, DOSY, DLS, Viscometry, and SEM. The formation of polypseudorotaxane was shown to be dependent on the concentration of [2]pseudorotaxanes/[PdCl2(PhCN)2] and temperature. Moreover, the reversal transition between polypseudorotaxane and [2]pseudorotaxanes can be realized by the successive addition of metal linker [PdCl2(PhCN)2] and competitive ligand PPh3. Significantly, the metal polypseudorotaxane could transform into supramolecular gel when the concentration was above 500 mM, which showed reversibly temperature-induced gel–sol transformation. This study provides a new insight into the construction of macrocycles-based polypseudorotaxane in highly polar organic solvent and benefits to the fabrication of smart materials.In summary, a novel metallosupramolecular polypseudorotaxane was successfully fabricated from pillar[5]arene-based [2]pseudorotaxanes and [PdClThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.CL, BL, and LL conceived of this project and designed the experiments. HS and WC contributed to most of the experimental work. All authors analyzed the data. CL, WC, and Z-YZ co-wrote the paper. All authors discussed and commented on 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.


Soybean mosaic virus (SMV), which belongs to the Potyviridae, causes significant reductions in soybean yield and seed quality. In this study, both tag-free and reporter gene green fluorescent protein (GFP)-containing infectious clones for the SMV N1 strain were constructed by Gibson assembly and with the yeast homologous recombination system, respectively. Both infectious clones are suitable for agroinfiltration on the model host N. benthamiana and show strong infectivity for the natural host soybean and several other legume species. Both infectious clones were seed transmitted and caused typical virus symptoms on seeds and progeny plants. We used the SMV-GFP infectious clone to further investigate the role of key amino acids in the silencing suppressor helper component-proteinase (Hc-Pro). Among twelve amino acid substitution mutants, the co-expression of mutant 2—with an Asparagine→Leucine substitution at position 182 of the FRNK (Phe-Arg-Asn-Lys) motif—attenuated viral symptoms and alleviated the host growth retardation caused by SMV. Moreover, the Hc-Prom2 mutant showed stronger oligomerization than wild-type Hc-Pro. Taken together, the SMV infectious clones will be useful for studies of host–SMV interactions and functional gene characterization in soybeans and related legume species, especially in terms of seed transmission properties. Furthermore, the SMV-GFP infectious clone will also facilitate functional studies of both virus and host genes in an N. benthamiana transient expression system. Potyviridae represents one of the largest families of plant viruses that cause severe yield losses in many major crops [Soybean mosaic virus (SMV) is a monopartite positive-sense ssRNA virus in the Potyviridae with a genome of approximately 10 kb. SMV encodes 11 putative proteins, including potyvirus 1 (P1), helper component-proteinase (Hc-Pro), potyvirus 3 (P3), N-terminal half of P3 fused to PIPO (P3N-PIPO), 6 kinase 1 (6K1), cylindrical inclusion (CI), 6 kinase 2 (6K2), Viral genome-linked Protein (VPg), nuclear inclusion a proteinase (NIa-Pro), nuclear inclusion b (NIb) and coat protein (CP) [The or crops ,2,3. Soyein (CP) ,5. The pein (CP) .I locus, which regulate the accumulation of chalcone synthase (CHS) mRNA levels by RNA silencing [I locus allele contains inverted repeats of the CHS gene that produce dsRNAs and lead to the tissue-specific silencing of CHS expression, resulting in normal yellow seed coats. SMV-infected soybean seeds show brown-mottled symptoms, with elevated CHS mRNA levels, which is attributed to the partial suppression of the RNA silencing of the CHS mRNAs by SMV Hc-Pro [Tobacco Ring Spot Virus (TRSV) remained on soybean seeds at 2 °C for 5 years [As for the SMV isolates, seven strains (G1–G7) were reported in the United States and five strains (A-E) were identified in Japan ,9,10. SMilencing . In healV Hc-Pro . In addiV Hc-Pro . The seeV Hc-Pro . The vir 5 years .Brome mosaic virus (BMV) [E.coli growth and unexpected deletions [Saccharomyces cerevisiae) is a simple, rapid and efficient way to obtain infectious clones of potyviruses [Infectious clones are useful reverse genetic manipulation tools for studies of host–virus interactions and understanding virus infection cycles and pathogenesis . Since tus (BMV) , a varieus (BMV) ,22. Conveletions ,24,25. Tyviruses ,27. Tobacco etch virus (TEV) hinders the assembly of siRNAs into RISC complexes by the direct binding to and sequestration of siRNAs [Zucchini yellow mosaic virus (ZYMV) Hc-Pro has a high RSS activity, while the Potato virus Y (PVY) and Wheat streak mosaic virus (WSMV) Hc-Pro shows weak RSS activity [Plants evolved sophisticated antiviral mechanisms, among which RNA interference (RNAi) is a common strategy for resisting RNA viruses in a sequence-specific manner ,29. To ef siRNAs . Hc-Pro f siRNAs , or diref siRNAs . Hc-Pro f siRNAs . Many mof siRNAs ,37,38,39f siRNAs . Howeverf siRNAs . There aactivity . Hc-Pro activity . Tobacco vein mottling virus (TVMV) and Turnip mosaic virus (TuMV) Hc-Pro behaves as dimers or trimers [Lettuce mosaic virus (LMV) and PVY; however, other research identified interaction sites in both the N-terminal and the C-terminal domains in Potato virus A (PVA) Hc-Pro [For the potyvirus Hc-Pro, the active form is dimers or multimers, which can be attributed to Hc-Pro self-interactions . Size ex trimers . Some re) Hc-Pro ,46. In o) Hc-Pro . Yeast t) Hc-Pro . CollectN. benthamiana, soybean and several other legume species, and were confirmed to be seed transmitted in soybeans. Hc-Pro from SMV was shown to form oligomers, and Y2H and BiFC assays demonstrated that the specific region of oligomerization was located in the central and C-terminal domains. Twelve mutants were generated for key amino acids associated with RSS activity in Hc-Pro by site-directed mutation. The co-expression of mutant 2, which contains an Asn182Leu replacement in a FRNK motif, can cause attenuated symptoms and growth recovery in SMV-GFP infected plants, and it appears to enhance Hc-Pro self-interactions. In the present study, we constructed a functional full-length cDNA infectious clone of SMV-GFP using the yeast HR system for the first time. A tag-free SMV infectious clone was also obtained by Gibson assembly. Both infectious clones showed strong infection ability in ® GXL DNA Polymerase . The five fragments were gel-purified using the Thermo Scientific™ Gene JET™ GeL Extraction Kit, and Gibson assembly was performed using the NEBuilder HiFi DNA assembly master mix . The ligate was transformed into the E. coli strain Trans2 Blue, and the positive colonies were confirmed by restriction endonuclease digestion, PCR amplification and Sanger sequencing. The correct assembled recombinant virus  was transformed into Agrobacterium tumefaciens strain GV3101 for agroinfiltration. SMV stocks were provided by Prof. Qingshan Chen from Northeast Agricultural University in China. A mild isolate of SMV-N1 was propagated in the Hefeng 25 soybean and was harvested and maintained at −80 °C as stocks. Total RNAs were isolated from virus-infected seedlings and reverse transcribed with the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) according to the manufacturer’s instructions. To build infections clones of SMV, the SMV genome was split into four overlapping fragments: 1# , 2# , 3#  and #4  by RT-PCR amplification using the primers listed in GFP ORF sequence (792 bp) was amplified from pJL24 (a TMV-GFP infectious clone) [NIb and CP genes of SMV. The SMV genome was divided into five overlapping fragments: A , B , C , D  and F , respectively. The vector backbone pCB301–2µ-HDV (7838 bp) was linearized between the CaMV 35S promoter and the tNOS sequence by PCR amplification. All of the six PCR insert products and the linearized vector were designed to share a 20 bp homologous sequence at their borders. To assemble the plasmid with the yeast HR system, equivalent molar ratios of linearized vector, the five viral cDNA fragments and GFP fragment were co-transformed into yeast cells (Y2H Gold). Positive colonies were screened on tryptophan-minus media at 30 °C for at least 2–4 days, and the recombinant plasmids were isolated from the yeast cells. Positive colonies were confirmed by restriction endonuclease digestion and sequencing. The correct assembled recombinant virus  was transformed into Agrobacterium tumefaciens strain GV3101 for agroinfiltration.In order to generate the GFP-tagged SMV infectious clone by yeast HR, the s clone)  and was N. benthamiana infection was performed according to Diao et al. [N. benthamiana plants were leaf infiltrated with GV3101 containing SMV infectious clones at OD600 = 1.0 in infiltration buffer . The N. benthamiana leaves with leaf rolling and mosaic symptoms were collected at 5 dpi (days post infection) and used for mechanical inoculation assays on soybean and other legume species including the vegetable soybean, Vigna unguiculata, Vigna angularis, Vigna radiata, Phaseolus v1, Phaseolus v2, Vicia faba, Astragalus sinicus, Medicago falcata, Glycine max v1, Pisum sativum, Phaseolus v3 and Lablab purpureus. Five grams of leaf tissue was ground in 20 mL of 0.02 M potassium phosphate buffer (PH 7.0) with 10 mg/L silicon carbide. Seedlings at the three-leaf stage were rub inoculated with the above inoculum, and inoculation buffer without SMV was used as a mock treatment. The seedlings were grown in a greenhouse at 22–25 °C with a photoperiod of 14:10 (light/dark) and relative humidity of 70%. Symptom development was observed after infection at desired time points. Successful infection was also verified by the RT-PCR amplification of virus CP fragments or GFP mRNA via specific primers listed in N. benthamiana leaf samples. The same samples were also used for detecting SMV CP protein expression by Western blotting using a CP-specific antibody. o et al. , and soyo et al. . N. bent®II One Step Cloning Kit . All the constructs were confirmed by SalI/kpnI restriction analysis and Sanger sequencing. The full-length ORF sequence of Hc-Pro was PCR amplified from SMV infectious clone DNA and cloned into the pMD19-T vector via TA cloning (Takara). Twelve RSS-activity-related mutants for Hc-Pro were obtained using this recombinant vector as a template with the Mut Express MultiS Fast Mutagenesis Kit  according to the manufacturer’s instructions. The twelve individual mutations are R181I, N182L, K183A, CDNQLD198-203AAAAAA, N200S, RKN240-243AAA, RE247-248AA, GS252-253DE, IGS251-253RAP, C344S, DE360-361AA and DH411-412AA, and the mutants are named as Hc-Prom1-m12. Wild-type Hc-Pro and the mutants were ligated into the binary vector pCambia-1300 with the ClonExpressAgrobacterium tumefaciens strain GV3101 by electroporation. A. tumefaciens was grown at 28 °C, centrifuged for collection, and diluted to OD600 = 1.0 or the indicated concentrations in infiltration buffer , and stored at room temperature for three hours. The inoculum was infiltrated into the abaxial side of full-grown leaves of N. benthamiana plants using a 1 mL syringe without a needle. For the mutant assay, the A. tumefaciens carrying the constructs of SMV-GFP and twelve Hc-Pro mutants (Hc-Prom1-m12) were mixed at a ratio of 1:1 for inoculation. GFP fluorescence was detected with a handheld long-wave (352 nm) UV lamp. The recombinant vectors and SMV infectious clones were transformed into In order to construct plasmids for Y2H analysis, the full-length ORF sequences of wild-type Hc-Pro and mutant 2 (N182L) were PCR amplified and ligated into the pGADT7(AD) and pGBKT7(BD) vectors, respectively. To determine the homodimerization region for Hc-Pro, the central and C-terminal fragment (amino acid residues 287 to 457) of Hc-Pro were also ligated into the AD and BD vectors. The AD and BD recombinant vectors were co-transformed into the Y2H Gold yeast strain, and the transformants grown on SD/-Try/-Leu medium were confirmed by colony PCR. The yeast cells were transferred to SD/-Try/-Leu/-His/X-α-Gal media for 3–7 days at 30 °C. The fully grown colonies as well as blue-colored colonies were photographed. All experiments were repeated at least three times.Agrobacterium tumefaciens strain GV3101 and infiltrated into N. benthamiana leaves. Infected leaves were analyzed 72 h after infiltration, and the YFP and DAPI fluorescence were detected using a confocal laser-scanning microscope.For the BiFC assays, the wild-type, mutant 2 (N182L) and truncated Hc-Pro sequences were cloned into the pDEST-nYFP and pDEST-cYFP vectors, fused with the yellow fluorescent protein (YFP) at the N-terminal region (nYFP) or the C-terminal region (cYFP), by Gateway cloning. Recombinant plasmids were transformed into N. benthamiana or soybean β-actin were used as internal controls. The ratio of mRNA abundance between the treatment and mock samples was calculated by the 2−ΔΔCT method for each gene. Three biological replicates were performed, and a T-test was performed between the treatment and the control mock treatment groups. The experimental procedures followed the literature . BrieflyE. coli and recombinant colonies were recovered, they showed the expected size  upon SalΙ digestion , and systemic infection occurred in all of infected plants in the newly grown leaves at 10 dpi  to construct the SMV-GFP infectious clone via yeast homologous recombination. To this end, the SMV genome was amplified into five fragments with 20 nt homologous termini. Meanwhile, the ORF sequence for GFP (green fluorescent protein) was cloned from the TMV-GFP vector (pJL24) and inserted in-frame between the CP genes . To mainigestion  and Sangperiment . N. benthamiana showed typical SMV phenotypes such as curling, mottling and mosaic , showed SMV symptoms ranging from typical mosaic to local necrotic lesions and leaf malformation , Pisum sativum, Phaseolus vulgaris v3 and Lablab purpureus showed no viral symptoms. The results demonstrated the infectivity and utility of the infectious clones in some legume species, although a limited host range was observed.RT-PCR analysis also confirmed the presence of SMV in the leaves of these plant species B. By conIn all of the above plant species, SMV induced more severe virus symptoms than SMV-GFP. Therefore, we quantified the virus titers in these plant species via RT-qPCR experiments. Slightly higher virus accumulation was observed for SMV than SMV-GFP, which might partly explain the more serious virus symptoms in SMV than SMV-GFP C. 170 displayed both stronger self-interactions and inter-molecular interactions with the full-length Hc-Pro. The results demonstrated that the interaction sites of SMV-Hc-Pro are mainly located in the central and C-terminal domains  and co-inoculated into N. benthamiana leaves, strong YFP fluorescence could be detected in the cytoplasm and, especially, in the nucleus  strongly increased the local GFP fluorescence intensity in agroinfiltrated N. benthamiana leaves at 5 dpi. However, the GFP fluorescence intensity was not improved by Hc-Pro. Interestingly, Hc-Prom2 resulted in the disappearance of GFP fluorescence on all of inoculated plants among the twelve Hc-Pro mutants  in Pisum sativum, Yellow mosaic virus (YMV) in Vigna mungo, Beet curly top virus (BCTV) in Petunia hybrida, Cucumber mosaic virus (CMV) in Capsicum annuum, Sweet potato leaf curl virus (SPLCV) in Ipomoea batatas, and Zucchini yellow mosaic virus (ZYMV) in Cucurbita pepo ssp. Texana were reported [Soybean vein necrosis virus (SVNV) and oTmato yellow leaf curl virus (TYLCV) were also reported to be seed transmitted in soybeans [SMV can be transmitted through seeds and causes seed mottle on the epidermis ,15. Prevpression . In the reported . Besidessoybeans ,77,78,79G. max (cultivated soybean) and G. soja (wild soybean). Apart from soybean, most strains only systematically infect several leguminous plants such as Vigna angularis, Phaseolus vulgaris, Vigna radiata, Pisum sativum, Vicia faba and Astragalus sinicus. In addition, some non-leguminous plants including Chenopodium album, Senna occidentalis and Pinellia ternate, Passiflora spp. could also be infected by SMV through mechanical or natural inoculation [Phaseolus vulgaris v1, Vigna angularis, Vigna unguiculata, Phaseolus vulgaris v2 and Glycine max v1 (vegetable soybean), can be systemically infected by SMV derived from our infectious clones. They showed similar symptoms to cultivated soybeans on the leaves, including mosaic, rugosity and leaf curling. By contrast, eight other legumes including Vigna radiata, Vicia faba, Astragalus sinicus, Medicago falcata, Glycine max v2 (black bean), Pisum sativum, Phaseolus vulgaris v3 and Lablab purpureus showed no viral symptoms. The results demonstrated the selective infectivity and usefulness of the infectious clones in some legume species, although a limited host range was observed.SMV’s host range is reported to be relatively narrow, and the natural host range of SMV is mainly restricted to legumes such as culation ,84,85,86Papaya leaf distortion mosaic virus (PLDMV) and ZYMV R→I mutation significantly reduced the viral symptoms, and the same mutation in Hc-Pro from PVY abolished its RSS activity [Tobacco vein banding mosaic virus (TVBMV) [Hc-Pro is one of the most widely studied multifunctional proteins in potyviruses ,87, whicactivity . In addi (TVBMV) . In the N. benthamiana transient expression system. Furthermore, we identified a key amino acid for SMV infectivity and symptom development in the FRNK motif of the silencing suppressor Hc-Pro. Taken together, our data will help to expedite the understanding of molecular mechanisms for soybean–SMV virus interactions. Soybean mosaic virus (SMV) is a severe soybean pathogen posing threats to soybean cultivation, and the molecular mechanisms underlying SMV–soybean interactions are poorly known. In view of this, we developed two infectious clones for SMV—in particular, SMV-GFP—and we investigated their infectivity in plant hosts and their utility in functional gene studies. The infectivity of SMV derived from both infectious clones for the natural host soybean and several other legume species makes them useful tools for studying host–SMV interactions. Especially, the infectivity and efficient GFP expression of the SMV-GFP infectious clone might facilitate functional studies of both virus and host genes using the