As a major surface glycoprotein of enveloped viruses, the virus spike protein is a primary target for vaccines and anti-viral treatments. Current vaccines aiming at controlling the COVID-19 pandemic are mostly directed against the SARS-CoV-2 spike protein. To promote virus entry and facilitate immune evasion, spikes must be dynamic. Interactions with host receptors and coreceptors trigger a cascade of conformational changes/structural rearrangements in spikes, which bring virus and host membranes in proximity for membrane fusion required for virus entry. Spike-mediated viral membrane fusion is a dynamic, multi-step process, and understanding the structure–function-dynamics paradigm of virus spikes is essential to elucidate viral membrane fusion, with the ultimate goal of interventions. However, our understanding of this process primarily relies on individual structural snapshots of endpoints. How these endpoints are connected in a time-resolved manner, and the order and frequency of conformational events underlying virus entry, remain largely elusive. Single-molecule Förster resonance energy transfer (smFRET) has provided a powerful platform to connect structure–function in motion, revealing dynamic aspects of spikes for several viruses: SARS-CoV-2, HIV-1, influenza, and Ebola. This review focuses on how smFRET imaging has advanced our understanding of virus spikes’ dynamic nature, receptor-binding events, and mechanism of antibody neutralization, thereby informing therapeutic interventions. Virus spikes on the surface of enveloped viruses are often also viral fusion proteins that mediate the fusion between viral membranes and cellular membranes  essentiaAs spikes are highly exposed to our immune system, they are main targets of neutralizing antibodies and thus are critical for developing vaccines and anti-spike therapeutics. Most vaccines or vaccine candidates for HIV-1/AIDS and SARS-CoV-2/COVID-19 are based on their spike proteins to trigger the immune system to produce neutralizing antibodies. Interestingly, in the face of immune pressure, many viral spike proteins use conformational masking of vulnerable antibody-targeted epitopes. In addition to glycan shields and hypermutations, this strategy of conformational masking has been best-understood in HIV-1 ,35,36,37Collectively, virus spike proteins are structurally flexible and conformationally dynamic on the virus surface. The dynamic nature and conformational plasticity of spikes enables entry of enveloped viruses into host target cells through membrane fusion, while distracting the immune system from antibody recognition. Molecular understanding and modulation of spikes’ intrinsic dynamics can guide the rational design of spike-targeting interventions such as vaccines, small-molecule inhibitors, and antibodies to block virus entry. Single-molecule Förster resonance energy transfer (smFRET), a spectroscopic tool sensitive to inter-fluorophore molecular distances, has been well-positioned to capture the innate dynamics of virus spikes labeled with Förster resonance energy transfer (FRET)-paired fluorophores. This minireview covers recent work on single-molecule FRET imaging of class I fusion proteins of enveloped viruses, including SARS-CoV-2 , HIV-1 ,25,26,27Imaging macromolecules at the single-molecule/single-particle level has advanced our understanding of both static and dynamic aspects in virus–host interactions, merited by avoiding the averaging-out effect from traditional ensemble-level measurements. Those imaging techniques, such as single-particle cryoEM/cryoET, single-particle optical tracking, and super-resolution fluorescence microscopy exerted significant roles in addressing fundamental questions with regards to structures, dynamics, and functions of virus molecules underlying virus–host interactions ,42,43,44Single-molecule Förster resonance energy transfer (smFRET) imaging has been proven to be a reliable imaging tool for revealing the intrinsically dynamic, heterogeneous nature of biological systems ,46,47. IR/R0)6), where R is the inter-fluorophore distance and 0R is the Förster distance determining the range of sensitive measure of distance ..84]. TheEnv is also the target for neutralizing antibodies. In order to bind receptor and co-receptor, Env must open. However, these open Env conformations are extremely vulnerable to antibody recognition. As a consequence, HIV-1 Env changes conformations to conceal receptor and coreceptor binding sites by closing, and the easily elicited initial antibodies become non-neutralizing against the closed trimer ,37,95,96smFRET has permitted access to dynamic aspects and conformational profiles of HIV-1 Env in the context of intact virions ,25,26,27Moreover, smFRET imaging has provided mechanistic understandings of how Env trimer responds to anti-viral inhibitors ,25,27. O3 on the intact virus exhibits more open conformations of State 2 and State 3 in contrast to the mature Env . HA. HA29]. The most recent smFRET work of HA2 connected fusion-active states in time, extending our mechanistic understanding of how HA mediates membrane fusion . Das et Remarkably, long and transient exposure to acidic pH exert different conformational consequences on HA2, which uncover two different conformations behind the low FRET. Long exposure irreversibly shifted conformations of HA2 to the low-FRET dominance, believed to be the irreversible coiled-coil configuration—the post-fusion conformation. In the presence of receptor sialic acids containing liposomes, HA2 appeared to adopt the coiled-coil conformation even at neutral pH and rapidly transited to that conformation at low pH irreversibly. The role of accelerating the fusion process of receptors agreed with the rapid formation of the coiled-coil post-fusion conformation. On the other hand, transient exposure to acidic pH, followed by gradual reversing to neutral pH, reversed the HA conformational equilibrium back to that at neutral pH. The pH-modulated conformational reversibility implied the existence of a reversible fusion intermediate, in which the fusion peptide was released and distanced away from the HA base. The formation of this fusion intermediate was suggested to be before the insertion of fusion peptide into the target membrane (before the pre-hairpin intermediate). To sum up, smFRET imaging tackled long-standing questions regarding the timing, order, and frequency of conformational changes/transitions of HA2 during viral membrane fusion. Das et al.  showed h3 in which GP1 facilitates engagement with cells and the cellular receptor binding, and GP2 promotes fusion of viral membrane into endosomal membranes required for virus entry . They first observed that GP dynamically transited among multiple conformational states . On the basis of observations of the high-FRET destabilization in the presence of Ca2+, acidic pH, glycan cap removal, and NPC1 individually or together, the authors considered high FRET as a pre-fusion state with the fusion loop sequestered. Behind the low FRET was believed to be two different conformations. The low FRET that acidic pH and Ca2+ shifted the conformational equilibrium towards was assigned to a pre-fusion state in which GP2 N-terminus and the fusion loop were displaced to be distant. This assignment was further supported by the restoration of conformational landscapes after re-neutralizing the endosomal acid pH. In contrast, the low FRET that the receptor NPC1 irreversibly pushed the GP2 into was implied to be the post-fusion six-helix bundle. The nature of intermediate FRET remained unknown. Collectively, conformational modulations of GP2 by endosomal pH, Ca2+, NPC1, and glycan cap removal observed by smFRET imaging revealed roles of these factors in Ebola virus entry  low pH and Ca2+ cause reversible conformational shift of GP2 towards an intermediate, which facilitates subsequent receptor NPC1 binding; (3) GP1 with glycan cap removed renders conformational shifts of GP2 from a reversible intermediate to an irreversible post-fusion state, and thus glycan cap in GP1 likely plays a role in preventing premature activation of GP2. This study revealed two critical conformational states on the fusion pathway but inseparable in the FRET spectrum, one as a pre-fusion state, the other as the post-fusion state. Alternative labeling sites on GP2 could help the observation of clear separations among states and data interpretations.The two most recent smFRET studies used amber-click labeling and enzymatic labeling, and touched upon the critical questions above regarding GP-mediated viral membrane fusion ,30. Withus entry C: (1) loIn the second smFRET study of GP, Durham et al.  imaged pThe two discussed smFRET studies started to fill our knowledge gap on the conformational dynamics of Ebola glycoproteins (GPs). Several remaining questions are of interest, similar to the discussed fusion machines of influenza HA and HIV-1. How does the removal of GP1 modulate conformational changes in GP2? What is the molecular mechanism of switching between reversible and irreversible steps? Do cellular membrane compartments or other Ebola virus components besides GP play roles in Ebola viral membrane fusion? What is the molecular mechanism underlying GP antagonism by antibodies or inhibitors? Addressing such questions will facilitate a better understanding of Ebola virus entry governed by virus–host interactions.Virus spike–host interactions are complex and dynamic. This review covered recent fluorescence-based smFRET imaging studies on virus spike proteins of SARS-CoV-2 , HIV-1 ,27,112, Recent smFRET studies discussed in this review have advanced our understanding of viral membrane fusion; nevertheless, many critical questions regarding this process remain elusive. One of the long-standing problems is the stoichiometry of virus glycoproteins required for successful fusion pore formation and fusion pore expansion. Technically, smFRET cannot tackle this specific question, given that the aggregate of molecules is indistinguishable at the single-molecule level. According to other non-smFRET HIV-1 Env studies in the utilization of experimental analysis and mathematical modeling, the number of HIV-1 Env glycoproteins required for virus entry varies from one digit to double-digits, and it seems to be strain-dependent ,146,147.


Heterozygosity at HLA class I loci is generally considered beneficial for host defense. We report here an element of HLA class I homozygosity that may or may not help preserve its existence in populations but which could indicate a new avenue for antiviral research.Lymphocytes from serologically HLA-homozygous or -heterozygous donors were examined for synthesis of influenza virus proteins and RNA after exposure to virus as peripheral blood mononuclear cells. The virus-exposed lymphocytes were also examined for internalization of the virus after exposure, and for susceptibility to virus-specific cytotoxic T lymphocytes in comparison with virus-exposed monocytes/macrophages and unseparated peripheral blood mononuclear cells. Results were compared using two-tailed Fisher’s exact test.Serologically-defined HLA-A2-homozygous lymphocytes, in contrast to heterozygous lymphocytes, did not synthesize detectable influenza virus RNA or protein after exposure to the virus. HLA-A2-homozygous lymphocytes, including both homozygous and heterozygous donors by genetic sequence subtyping, did internalize infectious virus but were not susceptible to lysis by autologous virus-specific cytotoxic T lymphocytes (“fratricide”). Similar intrinsic resistance to influenza virus infection was observed with HLA-A1- and HLA-A11-homozygous lymphocytes and with HLA-B-homozygous lymphocytes.A significant proportion of individuals within a population that is characterized by common expression of HLA class I alleles may possess lymphocytes that are not susceptible to influenza virus infection and thus to mutual virus-specific lysis. Further study may identify new approaches to limit influenza virus infection. Heterozygosity at HLA class I loci is generally considered beneficial by assuring that individuals, and therefore the population, can respond to a wide variety of infectious challenges –3. HowevThe current studies were undertaken in response to some surprising HLA-related results in studies of the requirement for monocyte/macrophage participation for influenza virus infection of human lymphocytes , 16. ThoPBMC were obtained from the peripheral blood of healthy adult volunteers by Ficoll-Hypaque sedimentation . InformeDonors were typed for HLA class I and class II antigens, using established serological techniques for class I antigens and commercially available typing reagents. For a subset of donors, cellular DNA was extracted using the Easy DNA kit for genomic DNA isolation  and HLA-A2 subtyping was performed using the Dynal HLA-A2 PCR sequence specific primers (SSP) subtyping kit  . All expPurified lymphocytes (> 99.5% purity and > 97% viability) were obtained by elutriation , 23, 24 Limited PBMC were donated by the HLA-B-homozygous donor. Therefore, T lymphocytes were separated from PBMC using Dynal T cell Negative Isolation Kit Ver II  rather than by elutriation.The PBMC or purified subpopulations of PBMC were exposed to infectious influenza A/Marton/43 (H1N1) at a multiplicity of infection (MOI) of three unless noted otherwise , 22. In In another subset of experiments, sham-exposed and influenza virus-exposed PBMC, monocytes-macrophages, or lymphocytes were treated with neuraminidase  which removes surface-bound virus , 29. Flo+ T lymphocytes (anti-Leu-4) and CD14+ monocytes/macrophages (anti-Leu-M1) (Becton Dickinson). Cells that were exposed to FITC-labeled virus were analyzed for bound versus internalized virus using resonance energy transfer techniques described previously [Phenotypes of separated populations were determined by direct immunofluorescent staining and flow cytometry  , 30, ideeviously , 31. ForLysates of pulse-labeled cells were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography , 32. ImmTotal cellular RNA was obtained from sham-exposed and influenza virus-exposed PBMC at varying times (0–24 h) after exposure and analyzed using Northern blots , 35. A cFor both viral protein and RNA synthesis assays, photographic reproductions of gels were prepared with relatively reduced exposure of lanes containing lysates of monocytes-macrophages, which were intense relative to lanes containing lysates of lymphocytes.HLA-A2-homozygous lymphocytes were exposed to the virus in the presence of monocytes/macrophages for 1 h before purification and treatment with neuraminidase. The lymphocytes were then layered over uninfected monocytes/macrophages for 1 h before removal from the monocytes/macrophages. The lysates of the monocytes/macrophages and lymphocytes purified after exposure to the virus, as well as the monocytes/macrophages that were exposed to the lymphocytes, were each collected after a subsequent 5 h of incubation. At the same time as they were layered over the uninfected monocytes/macrophages, lymphocyte samples were also layered over MDCK cell monolayers which were overlaid subsequently with 0.6% agarose, incubated at 37 °C for 48 h, then fixed and stained with methylene blue to facilitate plaque quantification. Based on previous studies, one plaque was assumed to be caused by one infected cell .51Cr-release assay [PBMC were infected with influenza A/Marton/43 (H1N1) and cultured for 6 days to generate CTL (effector cells) . Nonadhese assay .http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and DNAMan 6.0 (http://lynon.com) softwares: HA (AF494248.1) - Forward 5′-CCC AAACACAACACAACCAG-3′ and Reverse 5′-GCAAGGTCCAGTAAT AGTTCATCC-3′; NA (AY122326.1) -Forward 5′-GTC TGA ATG TGCCTGCGTAA-3′ and Reverse 5′-CAG TTG CCT TTT TCC ATC TTTG − 3′. All primers were synthesized by Sigma-Genosys (http://www.sigma-genosys.com/utmb/). Random primers and SYBR green PCR Master Mix were purchased from Roche  and Applied Biosystems , respectively. An annealing temperature of 60 °C was used to amplify cDNAs. For each different primer set, a no template control (NTC) was used to confirm amplification of specific PCR product in the post-amplification dissociation curve. After normalization to 18S rRNA  amplification, differential gene expression was measured by following the Cycle-threshold (ΔCt) method described in the manufacturer’s protocol. PCR products (~ 300 base pairs) were further verified by gel electrophoresis and sequencing.Differential expressions of the influenza A/Marton/43 hemagglutinin (HA) and neuraminidase (NA) genes  were conComparison of the responses of HLA-homozygous and HLA-heterozygous lymphocytes to exposure to influenza virus were performed using two-tailed Fisher’s exact test.We previously showed that human monocyte/macrophage and lymphocyte interaction after exposure to influenza virus, including H1N1, H2N2 and H3N2 strains, is required for synthesis of viral proteins by lymphocytes , 16. In Both virus-exposed purified monocytes-macrophages and lymphocytes from HLA-A2-heterozygous donors synthesized viral hemagglutinin, neuraminidase, matrix protein, and nucleoprotein, detected by pulse-labeling the cells 4–6 h after exposure to the virus Fig. a. The daThe time for pulse-labeling the cells in the above experiments was chosen, based on earlier kinetic experiments , for abiFurther analysis of PBMC from both HLA-A2-homozygous and HLA-A2-heterozygous individuals was performed using Northern blots for the detection of positive strand viral RNA indicating transcription of the viral neuraminidase gene product  were homozygous at the level of genetic sequence subtyping. The donors included both genotypically homozygous  and heterozygous  individuals: two were homozygous and three were heterozygous by genetic sequence subtyping. The donors were heterozygous for all the other HLA class I determinants. As described above, the data showed that none of the serologically homozygous HLA-A2 lymphocytes synthesized viral RNA or proteins after internalization of the virus whether the cells were genotypically homozygous or heterozygous.Lymphocytes were obtained from pairs of HLA-A2-homozygous and HLA-A2-heterozygous donors and exposed to FITC-labeled influenza virus in the presence of autologous monocytes/macrophages. The monocytes/macrophages and lymphocytes within the mixed cultures were identified and analyzed using flow cytometry, with addition of ethidium bromide to distinguish internalized from external cell-bound virus using fluorescence resonance energy transfer . As expeModified infectious focus assays were then used in several experiments to detect infectious virus present in HLA-A2-homozygous lymphocytes after exposure to virus in the presence of monocytes-macrophages. Lymphocytes were layered over influenza virus-exposed monocytes-macrophages which had been treated with neuraminidase to remove any potential extracellular virus . The lymFurther studies were performed to determine whether there was a correlation between decreased or absent synthesis of influenza viral proteins in lymphocytes of HLA-A2-homozygous donors and the results when such cells were used as targets in an autologous cytotoxic T lymphocyte (CTL) assay. Such an analysis could also be considered more sensitive for evidence of infection since major CTL targets like the viral nucleoprotein can lead to lysis despite an inability to detect them on the cell surface by other methods , 41. OthData are shown in Fig. Studies were next performed to determine whether the above-described lack of detectable viral protein synthesis by purified lymphocytes was specific to the homozygous expression of HLA-A2, or would be evident also in studies using lymphocytes from donors with homozygous expression of other HLA alleles. We were able to obtain cells from the three donors in our panel with homozygous expression of HLA-A1, and one donor with homozygous expression of HLA-A11 . Lymphocytes were exposed to virus in the presence of monocytes/macrophages, purified, and pulse-labeled at varying times after exposure as in the above experiments. All monocytes/macrophages synthesized viral proteins after exposure in these studies. In all experiments, lymphocytes from the donors with homozygous expression of the HLA-A alleles showed no detectable synthesis of influenza virus proteins Fig. a. In resWe then had the opportunity to study PBMC from a single donor whose cells were homozygous for HLA-B44 but heterozygous for all other HLA alleles, in comparison with HLA-B44-heterozygous cells tested concurrently. Cells from both donors took up the IAV-FITC Fig. b. Since P < 0.0001) in response to exposure to the virus.The different responses of the lymphocytes from HLA Class I homozygous and heterozygous donors were analyzed Table  and showMost individuals survive seasonal and even pandemic influenza and recover completely with establishment of homotypic immunity due to cell-mediated responses to the challenge. Murine models have been used to demonstrate rapid and substantial recruitment of PBMC to the lung after influenza virus challenge –49. ThesIt is well recognized that CD8+ CTL contribute greatly to the clearance of virus-infected cells and promote recovery from influenza virus infection . The curHLA polymorphism has generally been thought to be selected by the pressure of infectious challenges –3, 54. WIn studies perhaps most relevant to our in vitro observations, Falfán-Valencia and colleagues examined the frequencies of HLA Class I alleles and haplotypes in regard to genetic susceptibility to the pH1N1 virus, using 6-digit high resolution sequence-based typing . In infeAn extensive impact of influenza virus infection on cellular pathways, demonstrated using HeLa cells, is mediated by both replication-dependent and, at early stages, replication-independent events , with boThe current studies have certain limitations. They were limited in the number of subjects who could donate PBMC for the experiments, and the panel of donors that we could access had been HLA-serotyped rather than typed by genetic sequence. However, it should be noted that the ten individuals  included every one of the HLA class I homozygous subjects from our typed donor panel, and each was studied concomitantly with a heterozygous subject from the panel. When the five serologically HLA-A2-homozygous individuals were subsequently genotyped, they included both homozygous and heterozygous individuals by 4-digit genotyping. Representative autoradiograms and other analyses were provided in the results, but we note again that results were consistent for all of the homozygous lymphocytes which were all tested. In fact, the representative data provided for each figure, or section thereof, and for the tables were from different homozygous donors in order to demonstrate the consistency of the findings. None of the HLA class I homozygous lymphocytes synthesized influenza proteins even though they internalized the virus just as did heterozygous lymphocytes after exposure in the presence of monocytes/macrophages. Another limitation is that the current studies cannot establish that the protection of HLA-homozygous lymphocytes from infection works at a population level to ensure a proportion of HLA-homozygous donors in the population who are relatively protected from influenza virus infection, or perhaps other challenges, which might relate to natural selection for the species.Influenza virus binds to the majority of both human monocytes/macrophages and lymphocytes . The virLymphocytes are not the primary target cells for influenza virus and they only become infected when recruited to the respiratory tract to participate in immune defense. We do not know whether respiratory epithelial cells, the desired target for the virus, would show a similar effect of HLA homozygosity. It may be that such resistance to the infection is limited to lymphocytes since monocytes/macrophages from homozygous donors are infected by influenza virus to an extent equivalent to that of cells from heterozygous donors.It is puzzling that A1- and A2-homozygous lymphocytes show resistance to influenza virus infection whereas A1-A2-heterozygous lymphocytes do not. It is also puzzling that the lymphocyte phenotype is defined serologically but not genotypically. It is clear that the HLA-homozygous lymphocyte resistance to infection occurs at a stage after entry of the virus into the cell. The IFITM family of interferon-stimulated genes inhibit an early viral entry step  and mighThe results of the current studies delineate a unique HLA-associated lymphocyte defense against a major pathogen, influenza virus, that produces recurring epidemic and pandemic challenges. Further investigation offers an opportunity to elucidate a mechanism for cellular resistance to influenza virus infection that may provide direction for novel antiviral development. We hope that other investigators with access to larger panels of HLA-typed donors are able to extend these studies.


Microcystis aeruginosa bloom in Lake Tai (Taihu) China to identify active infections by nucleocytoplasmic large DNA viruses (NCLDVs), RNA viruses, ssDNA viruses, bacteriophage, and virophage. Phylogenetic analyses revealed a diverse virus population with seasonal and spatial variability. We observed disproportionately high expression of markers associated with NCLDVs and ssRNA viruses (consistent with viruses that infect photosynthetic protists) relative to bacteriophage infecting heterotrophic bacteria or cyanobacteria during the height of the Microcystis bloom event. Under a modified kill-the-winner scheme, we hypothesize viruses infecting protists help suppress the photosynthetic eukaryotic community and allow for the proliferation of cyanobacteria such as Microcystis. Our observations provide a foundation for a little considered factor promoting algal blooms.Drivers of algal bloom dynamics remain poorly understood, but viruses have been implicated as important players. Research addressing bloom dynamics has generally been restricted to the virus-infection of the numerically dominant (i.e. bloom forming) taxa. Yet this approach neglects a broad diversity of viral groups, limiting our knowledge of viral interactions and constraints within these systems. We examined hallmark virus marker genes in metatranscriptomic libraries from a seasonal and spatial survey of a  Harmful algal blooms (HABs) are extreme biological events that have detrimental environmental and socioeconomic effects on both fresh and salt waters. In particular, freshwater HAB events during the last two decades have posed threats to potable water supplies and socioeconomic resources for population centers . FreshwaResearch over the past two decades has made it clear that viruses are central to ecosystem function . There aTaihu in Mandarin) bloom event during 2014. Lake Tai experiences yearly successions from photosynthetic protists to cyanobacteria (dominated by Microcystis) and therefore presents an opportunity to study the impact of viruses during and after that transition . In addition to observations of active eukaryotic viruses, determinations of putative host diversity and statistical co-occurrence analyses of “who infects whom” reveal potential advantages for Microcystis that account for part of its ability to form large bloom events in fresh waters.To explore the role of the “neglected” viruses, we examined metatranscriptomic data from a Lake Tai  and immediately treated with ∼2 mL of RNAlater (Qiagen) for preservation. Samples were collected in a manner consistent with the separation of bacteria and phytoplankton from dissolved materials: free virus particles generally pass through these filters. Environmental parameters and nutrient data were collected with each sample and have been reported elsewhere  as previously described (Samples were collected monthly (June to October) from a eviously . Biomasslsewhere . Total Rescribed . Raw seqescribed .Taihu samples . A custom python script was used to identify the coordinates of the aligned sequence within the query contig and extract only this region: these extracted components of the contigs were subsequently considered “viral candidates” . Contigs containing both structural and non-structural components were considered “near complete genomes” (data not shown) .Mavirus, Sputnik, and Zamilon remain the only virophage that have been isolated, purified and sequenced to date  have been isolated but not purified beyond unialgal lysates  amongst the identified viral groups, with 457 candidates assigned to families . The majne algae . The mostebrates . OverallWe also observed virophage signatures in our dataset, although with less richness (30 unique candidates) than the NCLDVs . Most caMicrocystis phage. Of the RNA virus candidates, 72% of the expression was assigned to ssRNA viruses and 28% to dsRNA viruses. The distribution between the number of single-stranded and double-stranded candidates was coincidentally identical, with 193 ssRNA virus candidates representing 72% of the richness and 75 dsRNA virus candidates representing 28% of the richness  populations allowed us to detect infections by both DNA and RNA viruses, the latter of which should primarily be cell-associated. We detected 268 unique RNA virus candidates . Overallrichness . The dsR diatoms . In our ct hosts , comprisMarnaviridae or Nodaviridae. Each meta-assembled virus contained both an RNA-dependent RNA polymerase and a structural gene, most commonly a capsid protein specific to the phylogenetic group.We took the opportunity to examine the RNA virus contigs to determine if any represented near or full-length virus genomes. Of our original 268 contigs, 16 were determined to contain a “near full-length” genome based on our parameters . All of Microcystis spp. (“other” phage). However, neither group contributed much to the overall transcript abundance. The ssDNA viruses had only 17 unique candidates, which comprised only 0.42% of the total viral reads or <0.0001% of the total reads  over the course of the 5-month bloom. e sample . Accordie sample  by pH ane sample .Co-occurrence networks described putative virus and host interactions across our 35 samples. We established 20 significant co-occurrence clusters that ranged in membership from 2 to 11 members, with each network containing at least one putative virus and putative host. Twelve clusters contained a bacterial host with an RNA virus or a NCLDV, while four clusters contained a eukaryotic host with an RNA virus, a NCLDV, or ssDNA virus . Three cSphagnum peat bogs   .Microcystis blooms have focused on the Microcystis phage  in selection during transient phytoplankton blooms. Bloom forming species are often thought of as opportunists, able to take advantage of ephemeral increases in resource availability, predominately through rapid cell division . In claspothesis , the potMicrocystis depends on an ability to both consume the available nutrients and itself resist viral infection. One mechanism of Microcystis resistance is through high number of restriction modification systems  by other phage, ultimately prolonging the existence of the host  the significant top down pressure from free Microcystis-infecting phage.The latter season dominance of  systems , but Micic state . In othethe host . In the Microcystis phage, which was not a surprise, given the dominance of the Microcystis host. However, Microcystis blooms are commonly accompanied by an abundant heterotrophic bacterial microbiome, particularly proteobacteria, who can play a large role in nitrogen cycling in this system  in similar manners over the course of the bloom. We resolved 23 clusters that contained both host and virus transcripts with a statistically significant co-occurrence of expression across all 35 samples. While this is not directly indicative of a viral infection of the host in the cluster, it does imply these groups were responding to similar pressures in the system, either abiotic or biotic, and to some degree, their ecologies are thus “linked”. For example, we discovered several virophage that clustered significantly with RNA viruses and ssDNA viruses with no obvious NCLDV candidate. As virophage have only ever been identified co-infecting a host with a NCLDV, the observed clusters likely do not represent active co-infections, but hypothetically two different viruses benefiting from similar  scenarios. We also observe clusters that contain only bacterial hosts and NCLDVs, the latter of which are to date only known to infect eukaryotes. Again, these co-occurrence observations are likely describing community dynamics (i.e. microbiomes) as opposed to direct infections. These smaller clusters of viruses and potential hosts can be used to generate hypotheses regarding predator-prey cascades in a community and interspecies viral interactions.Microcystis viruses were involved in the establishment and/or maintenance of the Microcystis bloom. We observed two statically significant co-occurrences including cyanobacterial species and a Marnaviridae-like virus (consistent with viruses that infect C. tenuissimus and R. setigera)  . By consTaihu_metatranscriptome_project.” Sequencing information and quality control details have been previously reported (Taihu 2014 MegaHit Assembly 1.”Raw sequencing data is publicly available in the MG-RAST database  under threported . AssemblHP and SW established the project direction. LK and XT performed the environmental sampling and subsequent RNA extractions. HP, EG, MH, and MS were responsible for computational processing. HP, DT, and SW were responsible for interpreting the data and composing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Potyviridae: Turnip mosaic virus (TuMV), Papaya ringspot virus (PRSV) and Wheat streak mosaic virus (WSMV). For TuMV in Arabidopsis thaliana, profiles were obtained for mechanically inoculated rosette leaves and systemically infected cauline leaves and inflorescence. Results are consistent with selection pressure on the viral genome imposed by local and systemic movement. By genetically removing gene silencing in the plant and silencing suppression in the virus, our results showed that antiviral gene silencing imposes selection in viral populations. Mutations in siRNAs derived from a PRSV coat protein transgene in the absence of virus replication showed the contribution of cellular RNA-dependent RNA polymerases to the generation of mutations in virus-derived siRNAs. Collectively, results are consistent with two sources of mutations in virus-derived siRNAs: viral RNA-dependent RNA polymerases responsible for virus replication and cellular RNA-dependent RNA polymerases responsible for gene silencing amplification.RNA viruses exist as populations of genome variants. Virus-infected plants accumulate 21–24 nucleotide small interfering RNAs (siRNAs) derived from viral RNA (virus-derived siRNAs) through gene silencing. This paper describes the profile of mutations in virus-derived siRNAs for three members of the family  Virus-derived siRNAs are generated by dicer-like proteins (DCL), mainly DCL4 (21-nt) and DCL2 (22-nt), using dsRNA substrates that include virus replication intermediates and fold-back structures in single stranded RNA7. Additionally, siRNAs are silencing signals and move cell-to-cell, root to shoot, and in some cases between plants and fungi8.In plants virus infection triggers antiviral gene silencing that results in the accumulation of virus-derived small interfering RNAs (siRNAs) that are normally 21–24 nucleotides long and associate with argonaute proteins to form RNA-induced silencing complexes and guide translational repression or cleave of viral RNA targets based on sequence complementarity9. Genetic heterogeneity in combination with genetic robustness allows viruses to adapt to diverse hosts, vectors, and environments12. In infected plants, viruses exists as diverse populations of genomes forming a quasispecies12. A critical source of variation that drives virus evolution is the viral RNA-dependent RNA polymerases which lack proof-reading activity and introduce mutations  during virus replication. As a result, each replication cycle might produce new variants containing nucleotide substitutions, insertions, or deletions15. Selection eliminates unfit variants and favors variants with a competitive advantage, resulting in one or few dominant genetic variants surrounded by a large number of low-frequency, less-fit, or emerging variants16.Plant virus infection depends on the ability of the virus to replicate in infected cells, move cell-to-cell via plasmodesmata and systemically24. Using computational approaches, complete genomes of DNA or RNA viruses have been assembled de novo using virus-derived siRNAs26, and viral variants were detected25, suggesting that variation in virus-derived siRNAs reflect variation in genomic RNA. However, nucleotide variation in virus-derived siRNA has not been characterized. Here, we profiled virus-derived siRNA variation in three members of the Potyviridae: Turnip mosaic virus (TuMV), Papaya ringspot virus (PRSV) and Wheat streak mosaic virus (WSMV).High-throughput sequencing has expanded our knowledge of virus-derived siRNA biogenesis, diversity, virus-host interactions, their effect on host gene expression, virus discovery, and diagnosticsArabidopsis thaliana (Arabidopsis) ecotype Colombia, and comparisons were made between the mechanically inoculated rosette leaves, and systemically infected cauline leaves, or inflorescence. As they move across tissues, viruses face several selection pressures. In the collection of Arabidopsis single mutants available, only ago2-1 meets the requirements of being systemically infected by suppressor-deficient TuMV-AS9 and not preventing the biogenesis of virus-derived siRNAs27. Thus, to determine the effect of antiviral gene silencing on the accumulation of mutations, we partially removed gene silencing in the host by using single ago2–1 Arabidopsis mutant plants. In the virus, we genetically removed silencing suppression by using suppressor deficient TuMV-AS927. Accumulation of mutations in virus-derived siRNAs is in agreement with antiviral gene silencing imposing selection pressure in viral populations.Virus-derived siRNAs measuring 21-to 24-nt were used to profile mutations in several tissues or in cultivars with contrasting tolerance to virus infection. Small RNA profiles were generated for TuMV-infected 29. A subset of the viral RNA population targeted by siRNAs is used as a template by cellular by RNA-dependent RNA polymerases to synthesize dsRNA that is processed into secondary virus-derived siRNAs by DCL proteins29. This biogenesis pathway predicts that cellular RNA-dependent RNA polymerases contribute to the generation of mutations in virus-derived siRNAs. To test this hypothesis, we profiled siRNAs derived from a PRSV coat protein transgene, in the absence of PRSV replication. Results support the contribution of cellular RNA-dependent RNA polymerases to the generation of mutations in virus-derived siRNAs.In plants, amplification of gene silencing is necessary to establish an antiviral state and a significant proportion of virus-derived siRNA is amplification dependent31, TuMV-derived siRNA are predominantly 21- to 24-nt long. With respect to the TuMV reference genome, virus-derived siRNAs were classified into two groups: perfect match (zero mutations), and one or two mismatches . Sequences with more than two mismatches represented less than 1% and were not included  was detected. Abundance was normalized to reads per millionror Fig. . AccordiUnique virus-derived siRNAs with no mutations were less frequent (2 to 4-fold) than unique siRNAs with one or two mutations. In contrast, reads of siRNAs with no mutations were more abundant (14 to 16-fold) than siRNAs with mutations Fig. . This pa37.For positive-strand RNA viruses, in infected cells, the positive strand RNA is 10 to 100 times more abundant than negative-strand RNA. Such discrepancy is mainly due to positive-strand RNA in virions. In the absence of virion formation, positive-strand RNA accumulates two to four times more than the negative-strand RNA38. Abundance of TuMV genomic RNA predicts that virus-derived siRNAs of positive polarity are more abundant than virus-derived siRNAs of negative polarity. However, consistent with results previously described for TuMV27 and other positive-strand RNA viruses39, in the analyses describe here, TuMV-derived siRNAs of positive and negative polarity were equally abundant  in systematically infected inflorescence tissue was 2.03 times higher than in inoculated rosette leaves (27.1%) while the common mutations were only 18.7% and their abundance represented 73% of the observe in inflorescences  in TuMV-derived siRNAs from mechanically inoculated rosette leaves and systemically infected inflorescence Fig. . The totues Fig. .Together, these results support the hypothesis that a high proportion of mutations that originate early in infection are lost in the population that establishes systemic infection, only a small proportion of mutations might be fixed in the virus genome, and that new mutations arise in systemically infected tissue.Virus-derived siRNAs originate from the entire genome in both polarities Fig. . Howeverago2–1 single mutant Arabidopsis plants and silencing suppressor-deficient TuMV-AS927. TuMV-AS9-derived siRNAs were profiled from mechanically inoculated rosette leaves, and systemically infected cauline leaves  than siRNAs with mutations (0.07) Fig. . Interes27, the total number of siRNAs with mutations was higher in cauline leaves than in inoculated rosette leaves  infected with Wheatstreak mosaic virus . Profiles were generated from small RNA libraries made from cultivar Arapahoe at 18 °C and 27 °C and one from Mace at 27 °C40 that were mechanically inoculated with SWMV. Cultivars Arapahoe and Mace have contrasting tolerance to WSMV. Arapahoe is susceptible at 18 °C and at 27 °C while Mace is resistant to WSMV at temperatures below 19 °C41.We profiled siRNAs in WSMV-derived siRNAs were predominantly 21- to 24-nt long. On average, unique siRNAs with mutations were more frequent (2.77 fold) than unique siRNAs with no mutations. However, siRNAs without mutations were more abundant (31.4 -fold) than siRNAs with mutations and no strand bias was detected . PRSV-derived siRNAs, predominantly 21- to 24-nt, with no mutations and with one or two mutations were detected from the entire viral genome in both polarities  mechanically inoculated with ies Fig. . Virus-dies Fig. . Unique ies Fig. .Figure 8A larger number of siRNAs with mutations was detected in leaf samples than in flowers of non-transgenic cultivar AU9 infected with PRSV  as a transgene. Compared to PRSV-derived siRNAs detected in PRSV-infected plants, there were several key differences. Transgene-derived siRNAs exclusively mapped to the coat protein Fig. , siRNAs 10. In TuMV and WSMV, mutations in virus-derived siRNAs did not overlap with hypervariable genomic areas  and transversions (66.7%). However, the A to G transition was the most abundant (12.1%) nucleotide substitution and was detected at a frequency higher than the expected randomly (8.3%) . However, some viral silencing suppressors, such as the turnip  crinkle virus coat protein, compromise DCL proteins42.Genome-wide variation detected using full-length RNA sequences, was not a predictor of variation observed using virus-derived siRNAs Fig. . This diSeveral features of the virus-derived siRNA profiles were similar in TuMV, WSMV, and PRSV: lack of strand bias, higher accumulation of virus-derived siRNAs in the CI and 6K1 cistrons, higher frequency of the A to G transition and the C to A transversion. These observations support the model that the mechanisms that introduce mutations in virus-derived siRNAs is conserved across monocot and dicot plants, and possibly across viruses. Additionally, there are virus intrinsic properties that contribute to the distribution of virus-derived siRNAs per cistron, which might interact with the host and environmental factors, as observed in cultivars with a genetic difference in temperature-dependent difference in susceptibility to WSMV . Custom scripts are available upon request.Computational analyses were done on high-performance computing nodes at the University of Nebraska-Lincoln Holland Computing Center (Turnip mosaic virus (TuMV)-derived siRNAs were profiled from mechanically inoculated rosette leaves and systemically infected inflorescence of wild type Arabidopsis thaliana (Arabidopsis). Data sets correspond to accession number GSE64911. Inoculated rosette leaves (libraries 77 and 78) were collected at 5 days post-inoculation (dpi), and systemically infected inflorescence (libraries 69 and 70) were collected at 10 dpi. Single ago2-1 Arabidopsis mutants were inoculated with suppressor deficient TuMV-AS9. Samples from inoculated rosette leaves (libraries 21 and 22) and systemically infected cauline leaves (libraries 29 and 30 in GSE64911) were collected at 7 dpi and 15 dpi, respectively. As related to antiviral gene silencing response, these libraries were described before27.Small RNA sequence data sets used in this study are available in Gene Expression Omnibus. Carica papaya were described in43. In non-transgenic cultivar AU9 infected with Papaya ringspot virus (PRSV) samples were taken from flowers of female plants (accession GSM712526) or leaves from male plants (accession GSM712527). Transgenic cultivar SunUp expressed the PRSV coat protein as a transgene and small RNA libraries were made from female leaf samples (accession GSM712525).Small RNA libraries from wild type and transgenic Wheat streak mosaic virus (WSMV). Mace is resistant to WSMV at 18°C and susceptible at 27 °C. Arapahoe is susceptible both at 18°C and at 27°C. Plants were mechanically inoculated, and small RNA libraries from systemically infected leaves were made for both cultivars at both temperatures. Accession numbers were as follows: Arapahoe at 27°C (GSM1306038), Arapahoe at 18°C (GSM1306039), and Mace at 27°C (GSM1306040), as described40.Wheat cultivars Arapahoe and Mace have a contrasting response to 32. Read quality of the fastq files were checked with FastQC. In libraries from wheat, adaptors were removed using adapt tool44 (version 0.9.5) with the following parameters: a maximum error rate of 15% (-e 15), base trimming when the Phred-like quality score was lower than 45 (-q 45), and iterating up to three times (-n 3). The PRSV data was available as in text format and libraries were normalized to reads per million43.Raw data for TuMV and WSMV was in fastq format. TuMV libraries were demultiplexed using Libparse.pl45. Downstream analyses were performed using custom bash and Perl scripts.High quality reads from each of the samples were mapped to their corresponding reference genome: TuMV (NC_002509.2), PRSV (NC_001785.1) and WSMV(NC_001886.1) using Bowtie2 with two mismatches allowed32. Unique sequences measuring 21- to 24-nt were captured in a fasta file. Reads containing ambiguous bases were discarded.From Bowtie2, the resulting SAM files were used to filter virus-derived siRNAs into fasta files based on the following criteria: match class , polarity (sense or antisense), and size class (18 to 30 nt). Unique sequences and their abundance (number of reads) were determined for each size class and mismatch group. Per library, abundance of siRNAs was normalized to Reads Per Million (RPM)http://bio-bwa.sourceforge.net/)46, allowing one or two mismatches. The alignment file generated by BWA (SAM) was used as input for variant discovery using SAMtools47 and recorded in Variant calling format (VCF)48. High-confident variant sites were filtered based on the following criteria: mapping quality >50 , base quality/base alignment quality >20 ,>10 reads (-d in Samtools filter) covering each site. In parallel, Sam2Tsv (http://lindenb.github.io/jvarkit/Sam2Tsv.html) tool was used to characterize the variants where the.SAM files (from BWA output) of each libraries was used as input along with the virus reference genome49. The resulting output (as tsv file) was parsed and examined to categorize the variants as Transitions (Ts) or Transversions (Tv). To maximize specificity, high-quality variants were selected from the intersection of those identified both by Samtools and Sam2Tsv. The frequency of each transition and transversion was determined using in-house scripts. A heat map was generated and analyzed using the frequency (Ts /Tv) across the tissues/experiments of virus infected plants employing hierarchical clustering using the ClustVis package in R .To characterize mutations in virus-derived small RNAs, single nucleotide variants were determined in sequences that were 21- to 24-nt long. The fasta file generated by parsing the SAM file was used as input. Sequences were mapped the genome using the Burrows-Wheeler Aligner (BWA) (Virus-derived siRNAs (21 to 24-nt long) from the fasta file were separated into perfect matches (no mutations) and mismatches  for both sense and anti-sense polarity. The abundance of those siRNAs was determined on a 50-nt window using a shell script and maps to the virus genome, generating a genome-wide distribution.de-novo genome assembly using Velvet 0.7.31 (https://www.ebi.ac.uk/~zerbino/velvet/)50. Different kmer options  were optimized, where k-mer of 15 was found to produce larger contigs with best assembly outcomes. The quality of the genome assembly (% coverage) was checked with the QUAST (http://quast.bioinf.spbau.ru/)51. Viral contigs were mapped to the reference genome.Small RNAs of 21- to 24-nt with no mutations, from two biological replicates, were combined as a single fasta file, and used as input for 52. In Arabidopsis, siRNAs (fasta file) from rosette leaves were compared to siRNAs from cauline leaves or inflorescence. Subsequently, abundance (number of reads per million) of siRNAs for common and unique sequences between tissues were captured by parsing the SAM files of the corresponding libraries using in house bash scripts.Virus-derived siRNAs (21- to 24-nt siRNAs) with or without mutations were analyzed separately, and classified as tissue-specific or common using Venny toolsAccumulation of virus-derived siRNAs (21- to 24-nt) was determined for each cistron, the 5′UTR, and 3′UTR using the SAM file. For each region in the viral genome, siRNAs were binned using a custom bash script. The number of unique sequences and their abundance (reads per million) determined for perfect matches and mismatches, separately. The number of unique sequences and their abundance was normalized to the length of the cistron or UTR.Supplemental information.Supplemental information 2.Supplemental information 3.Supplemental information 4.Supplemental information 5.Supplemental information 6.Supplemental information 7.Supplemental information 8.Supplemental information 9.


Apis mellifera, and honey production are compromised by high levels of annual colony losses globally. The majority are associated with disease caused by deformed wing virus (DWV), a positive-strand RNA virus, exacerbated by the ectoparasitic mite Varroa destructor. To improve honey bee health, a better understanding of virus transmission and pathogenesis is needed which requires the development of tools to study virus replication, transmission, and localisation. We report the use of reverse genetic (RG) systems for the predominant genetically distinct variants of DWV to address these questions. All RG-recovered viruses replicate within 24 h post-inoculation of pupae and could recapitulate the characteristic symptoms of DWV disease upon eclosion. Larvae were significantly less susceptible but could be infected orally and subsequently developed disease. Using genetically tagged RG DWV and an in vitro Varroa feeding system, we demonstrate virus replication in the mite by accumulation of tagged negative-strand viral replication intermediates. We additionally apply a modified DWV genome expressing a fluorescent reporter protein for direct in vivo observation of virus distribution in injected pupae or fed larvae. Using this, we demonstrate extensive sites of virus replication in a range of pupal tissues and organs and in the nascent wing buds in larvae fed high levels of virus, indicative of a direct association between virus replication and pathogenesis. These studies provide insights into virus replication kinetics, tropism, transmission, and pathogenesis, and produce new tools to help develop the understanding needed to control DWV-mediated colony losses.Environmental and agricultural pollination services by honey bees,  Varroa destructor—the most important pathogen of the European honey bee (Apis mellifera). DWV has a near-global distribution  is arguably—in concert with its vector the ectoparasitic mite smission ) and, inthogenic . In contthogenic ,4,5,6, athogenic . Evidencthogenic ,9, selecvariants , and synvariants  have beevariants  and othevariants . AlthougIflaviridae family [Varroa and named Varroa Destructor Virus type 1 (VDV-1) [Varroa to naïve colonies with a diverse virus population was interpreted as an indication that this variant was more virulent [DVD (GenBank HM067437) and VDV-1VVD , both bearing the DWV A capsid proteins coding region and DWV B non-structural coding region [Varroa transmission toward the emergence of variants with enhanced virulence [DWV is a picorna-like virus from the e family ,14. The e family  exhibit  (VDV-1) . As a co (VDV-1)  and its  (VDV-1) ,18. A th (VDV-1) ,20,21,22virulent ,22. Convvirulent . In addivirulent ,25,26. Firulence . Therefode facto standard approach to address questions about virus replication, virulence and pathogenesis [Varroa destructor. Finally, by introducing a reporter encoding sequence to the virus genome we visualise the in vivo tissue distribution of DWV in infected honey bee brood. The results of this study provide new insights into understanding the nature of DWV infection and introduce new molecular tools for honey bee research.In virology, RG involves the manipulation of the genotype, the recovery of the virus, and the investigation of the phenotype. Over almost four decades, it has become the ogenesis ,28. To fogenesis . Moleculogenesis ,30. In tVarroa-infested honey bee colony from Warwick-HRI apiary (2014), and a cloned full-length cDNA was incorporated into a plasmid vector containing all required elements for the transcription of the viral RNA. VVD RG clone is identical to the source VDV-1-DWV-No-9, with the exception of two nucleotide substitutions , resulting in creation of an HpaI restriction site. In order to obtain VDD and VVV constructs bases 2727–4888 (capsid proteins encoding region) and 4885-9783  were replaced with corresponding DWV A and B fragments respectively (HpaI (5275-5280 nt in VVD variant), Kpn2I and AvrII (2751-2756 and 4884-4889 nt in VDD variant), and AvrII, PflFI and BglII .RG constructs used in this study were based on the cDNA clone of a recombinant DWV variant VDV-1-DWV-No-9 (GenBank HM067438.1). VDV-1-DWV-No-9 sequence was rescued using samples from a ectively . Insertsapiary 20, and a cE) were obtained by NEBuilder Hifi assembly  reaction applied to previously made RG constructs. EGFP sequence flanked by 32 additional nucleotides (ATGGATAACCCT from 5’ and GCAAAACCAGAG from 3’ end) resulting in duplication of the protease cleavage site (amino acid composition of the site is AKPEMDNP [in silico cloning simulation. Full cDNA sequences of DWV clones used in this study are available in EGFP-encoding RG constructs (DWVAKPEMDNP ) was insPme I (cutting at the end of the sequence encoding the poly-A tail) or Nru I  respectively. Linearized DNA was purified by phenol-chloroform extraction and ethanol precipitation and T7 transcription was performed with T7 RiboMAXTM Express Large Scale RNA Production System  according to the manufacturer’s protocol. In order to account for the stabilization effect of the poly-A tail truncated transcripts were subjected to an additional polyadenylation step with poly(A) Tailing Kit . RNA transcripts were purified with GeneJet RNA Purification Kit  using clean up protocol and eluted in RNAse free H2O. All transcripts were analysed for integrity by gel electrophoresis and stored at −80 °C.DWV RNA was synthesized using linearized plasmid templates. Full length and truncated templates were linearised with in vitro transcribed RNA. Homogenized tissue was diluted with sterile PBS in 1:1 (w:v) ratio and centrifuged at 13 000× g, 4 °C for 10 min. The supernatant was sterilized by passing through 0.22 μM PES filter  and treated with RNase A to destroy all non-encapsidated RNA. RNA was extracted from 100 μL of the virus stock using RNeasy kit  and analysed by RT-qPCR.DWV stocks were prepared from honey bee pupae injected with Apis mellifera) brood in this study was obtained from the research apiary of the University of St Andrews. Hives used for sampling were regularly treated for Varroa with an appropriate miticide and routinely screened for DWV. DWV level in bees obtained from these hives was found to range within 102–106 genome equivalents (GE) per 1 μg of total RNA. No phenotypic evidence for other honey bee viruses being present was found within the colonies throughout the course of the studies. Pupae and larvae of the required age were collected from the comb and transferred to the incubator set at 34.5 °C and 90% relative humidity. Pupae were kept on folded sheets of filter paper, and larvae were transferred into 96-well plates with round bottom wells containing feeding diet (6% (w/v) glucose, 6% (w/v) fructose, 1% (w/v) yeast extract, and 50% (w/v) royal jelly in H2O).All honey bee . On the next day, feeding packets containing artificial feeding diet and supplemented with DWV stock or equal amount of PBS were prepared. The feeding packets were prepared by wrapping a 175 μL drop of the diet containing RNase A treated DWV inoculate (prepared as described in Locusta migratoria) haemolymph was frozen-thawed, heat-treated, and centrifuged for 30 s at 7500× g.Mites were placed in groups of 5–10 mites per feeding packet with four replicates for each treatment and kept in the incubator for four days. At the end of incubation live mites were collected, snap-frozen in liquid nitrogen and stored at −80 °C until further analysis. Total RNA was extracted from pooled mite samples using the standard TriReagent protocol .RNA and virus injections into pupae were performed with insulin syringes ). 5 μL injections were introduced by inserting the syringe needle between the second and third abdominal segments of a pupa. All pupae were maintained on a warm heating plate during the injections.10 and 8.7 × 1011 of DWV RNA copies) of in vitro transcribed RNA was injected individually into white-eyed honey bee pupae. Truncated VVD transcript injections were used as a negative control in RNA transcript injections. RNA-injected pupae were analysed at 72 h post-injection. 0.5 and 5 μg . Total RNA was extracted with GeneJet RNA Purification Kit  using a protocol adapted for vacuum manifold application. cDNA was prepared with qScript cDNA Synthesis Kit  from 1 μg of total RNA following the manufacturer’s protocol with both oligo dT and random hexamer primers included in the reaction mixture in the reaction volume of 20 μL.Total RNA was extracted individually from all bee samples, actin, used as an internal RNA quality control, was carried out by end-point PCR with Taq DNA polymerase  and 2 μL of cDNA. DWV_RTPCR primers were designed to detect all three DWV variants under study. To amplify the cDNA regions containing restriction site tags in VDD and VVV virus variants Kpn2I_F/R and PflFI_F/R primer sets were used respectively. PCR cycling conditions were 30 cycles of 95 °C (15 s), 55 °C (15 s), 68 °C (2 min) with an initial 95 °C step (30 s), and a final extension at 68 °C (5 min). PCR samples were analysed on a 1% agarose gel stained with ethidium bromide. When required, DWV PCR products were subjected to restriction digest prior to loading on the gel.All sequences of primers used in this study are shown in E RNA was assayed by a set of primers amplifying a junction region between EGFP and viral sequence (primers EGFP_qPCR_F and VDD_VP2qPCR_RP or VVV_VP2qPCR_RP depending on the virus variant used). DWV and DWVE titers were calculated by relating the resulting Ct value to the standard curve generated by performing qPCR from a serial dilution of the cDNA obtained from 1 μg of VVD or DWVE RNA transcript respectively.The quantification of DWV genome copies was performed by SYBR-Green Real-Time Quantitative PCR (qPCR). Reactions were carried out in a C1000 Thermal Cycler  using Luna Universal qPCR master mix , 0.25 μM forward and reverse DWV_qPCR primers, and 2 μL of cDNA with the following thermal profile: 1 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 30 s at 60 °C with a final post-amplification melting curve analysis step. Accumulation of DWVTaq DNA polymerase  using a forward primer identical to the adapter sequence (primer 388) and DWV(-RNA)_RT R primer. PCR was run for 35 cycles in the same conditions as described above.Strand-specific detection of DWV RNA was performed as described earlier . BrieflyE was done using Leica TCS SP8 confocal microscope with 10× HC PL FLUOTAR objective . All imaging to detect EGFP signal in samples infected with DWVLive larvae were washed with increasing concentrations of aqueous ethanol solution and fixed in 4% paraformaldehyde in PBS for 2 h at 4 °C. Fixed larvae were allowed to sink in 30% sucrose in PBS, mounted in NEG-50 Frozen Section Medium  and subjected to microsectioning on CM1860 Cryostat . Sections of 50–80 µm thickness were placed on Superfrost Plus microscope slides , mounted in ProLong Gold antifade medium, and analysed by microscopy.in vitro transcribed viral RNA from relevant cDNAs. All RG templates were derived from an infectious cDNA of a recombinant DWV variant (GenBank HM067438.1) [To examine the biology and pathogenesis of different strains of DWV we prepared near-clonal virus stocks from honey bee pupae injected with 67438.1) . The 5′ 67438.1) . Each cDin vitro synthesised RNA enabled recovery of genetically tagged virus at high efficiency. Using qPCR analysis, the average viral load in RNA-injected pupae was shown to be 1.5 × 1010 DWV GE per 1 μg of total RNA , but there was no discernible difference in yield of the three variants when ≥10 GE were inoculated (p > 0.05). Incubation of pupae for a further 24 h resulted in virus yields of ~1010 GE/μg of RNA with no notable difference between the three DWV variants tested. This shows that RG-generated DWV is infectious and the kinetics of virus replication is very rapid, with the genome being amplified ~108–1013 times within 48 h of injection of pupae (minimum yield of RNA/pupa was 200 μg).We investigated the infectivity of RG-recovered VDD, VVV and VVD variants by inoculation of white-eyed honey bee pupae using serial dilutions of virus stocks and analysed all samples individually 24 h post-inoculation by RT-qPCR . Under tVarroa feed throughout the development of the pupa and possibly during the phoretic phase of the life cycle. We investigated whether pupal development phases were partially or totally refractory to DWV replication by inoculating morphologically distinct stages with 102 GE of VVD DWV . High DWV levels (up to 1010 GE/μg of RNA) were detected in 5 × 106 GE fed groups for VVV and VVD inoculates and for all three variants in 5 × 107 GE fed larvae, clearly indicating virus replication. It was notable that a proportion of larvae showed little or no amplification of DWV over the fed amount suggesting, in comparison to inoculation via injection, a significant barrier to the development of a productive infection may exist for virus acquired per os. Having demonstrated pupal susceptibility to directly inoculated virus we then examined virus transmission to developing larvae via the oral route. First instar larvae were individually fed a diet containing dilutions of RG-derived DWV, and the virus was detected and quantified after 48 h. Although RG-derived DWV was detectable when 5 × 10s tested c. UninocVarroa. A confounding issue in some studies is the pre-existing presence of DWV within the mite, and the potential presence of contaminating DWV genomic negative strands (a marker of replication) in either the mite or the inoculum. To address this, we investigated the replication of RG-derived genetically tagged VVD in Varroa using an in vitro feeding system [There are conflicting reports on the replication of DWV in g system  containiVarroa were maintained on artificial feed packets supplemented with 1.75 × 109 GE VVD (107 GE/μl). VVD RG-derived virus stock was amplified in honey bee pupae, extracted and treated with ribonuclease A to remove any non-encapsidated viral or cellular RNA. Varroa, and consequently high DWV, levels. Upon digestion with the relevant restriction enzyme (HpaI), this product was partially cleaved in the VVD groups only, indicating that at least some of it originated from the negative-strand replication intermediate of VVD  results in predominantly asymptomatic  infection, with no apparent phenotypic deformities. We tested the morbidity in honey bees infected orally with clonal DWV by feeding larvae with a virus-supplemented diet, allowing pupation and eclosion and scoring viability and the presence of overt symptoms. Although lower levels of virus (≤105 GE) in the diet again failed to establish robust replication in the experimental groups, the feeding of 2 × 107 GE of RG-derived DWV to honey bee larvae  resulted in high morbidity rates (12 GE/μg of RNA (In the field non-ty rates . In larvg of RNA . Notablyg of RNA . It is kE), analogous to that previously reported for DWV A [pro proteolytic cleavage site [The tropism and pathogenesis of DWV remains poorly understood. Relatively little is known about the sites of virus replication and whether differences in tissue tropism after oral or mite-mediated transmission account for the appearance of symptomatic and asymptomatic forms of disease. To facilitate virus localization in experimentally inoculated bees, we developed an EGFP-expressing chimeric DWV , but was largely absent from the Malphigian tubules and thoracic muscles c. Since s before , all hadlication .7 GE of DWVE purified from previously inoculated pupae and analysed larval fluorescence 4 and 6 days later. Samples infected with DWVE exhibited extensive fluorescence; external observation localized this to the larval head, two thoracic segments containing wing rudiments, the caudal part of the body and to cells surrounding the spiracle openings  and a recombinant that has previously been reported to predominate in colonies ,26. We ain vitro gene synthesis, we constructed infectious cDNAs for type A (protein coding sequence), type B and a recombinant variant of DWV, recovered molecularly tagged virus after RNA inoculation and investigated the kinetics of virus replication in honey bee pupae. Replication was rapid, amplifying to ~108 GE/μg of RNA within 24 h, and plateauing at ~1010 GE per μg of total cellular RNA within 48 h. There were no major differences between the replication rates of the three DWV variants under study, and all stages of pupal development tested appeared equally susceptible to virus infection , any virus replication in the mite would likely guarantee the transmitted dose would significantly exceed the ID50. The viral load in mites has been reported to be high [per se. Previous analysis of virus replication in Varroa has produced conflicting results. Mass spectroscopy studies failed to detect viral non-structural proteins implying that DWV may not replicate in the mite [in vitro on a diet containing no honey bee-derived components. The only DWV virus  present would therefore be derived from the last pupa the mites had fed on, together with any subsequent replication in the mite. We supplemented the diet with the RG-derived VVD variant of DWV carrying a unique genetic tag allowing its unambiguous identification. The presence of genetically tagged negative strand RNA of DWV in the pooled mites samples fed on this diet demonstrates that VVD does replicate in Varroa and that newly acquired virus can replicate in the presence of a pre-existing virus population in the mite  is probably critical. Analysis of whether infection of all stages of pupal development are as likely to result in wing deformities may be informative in this regard, though Varroa-mediated virus transmission will initially occur when the foundress mite feeds on the just-capped pre-pupa. One of the factors which can potentially influence the development of overt disease are the sites of virus replication in the developing honey bee. This may result in direct cytopathicity and tissue damage or indirectly by dysregulation leading to damage at remote locations. To address the tissue tropism of DWV, we constructed a modified virus genome co-translationally expressing the green fluorescent protein. Inoculation of pupae, or ingestion by larvae, resulted in distinct fluorescence in a range of organs and tissues. In fed larvae, the EGFP signal accumulated in thoracic segments where the developing wing buds are located [It remains unclear what determines whether virus exposure results in overt disease or asymptomatic infection  although located , and inf located , would pVarroa as a vector the oral route no longer plays the decisive role in DWV spread, but the observed tropism of DWV infection indicates the initial evolutionary trait used by the virus. Robust virus replication in the primary tissues that enable subsequent transmission often results in spillover to secondary sites that are permissive for virus infection, but that offer no further route to a new host, e.g., the neurotropism of the faecal-orally transmitted poliovirus [in vivo time course studies of virus dissemination and facilitate host-vector transmission studies. More generally, the ability to introduce a ‘payload’ to the virus genome may also be exploited by using genetically modified DWV as a virus-based gene delivery system [Varroa or Bombus [Our observations indicate that DWV selectively targets organs most favourable for its oral and vertical transmission. Sites of DWV replication colocalize with exocrine glands, which are responsible for the secretion of larval diet components, and digestive tract tissues, from which it can presumably be transmitted in faeces  and, folliovirus . It is ty system . Our resr Bombus  or, morein vitro. With no system for propagating DWV in vitro these studies have had to be conducted in vivo. We show that the availability of a reverse genetic system allows the kinetics of virus replication in larvae and pupae to be examined. Comparative studies demonstrate that the two predominant variants of DWV essentially replicate equivalently, and that the symptomatic outcome of DWV infection is not linked to a particular genotype of the virus nor to the route of transmission. Unique genetic tags introduced to the genome enabled discrimination of input from the endogenous virus population and provide unequivocal evidence for virus replication in Varroa following detection of de novo synthesised negative-sense viral RNA. A viable GFP-tagged DWV genome supplies the basis for a better understanding of virus tropism and pathogenesis in infected brood and will provide a useful tool for elucidating the mechanisms behind symptomatic DWV infection in developing honey bees.In many single stranded positive sense RNA viruses, the development of reverse genetic systems was facilitated by a good understanding of virus replication


We tested serum samples for the immunoglobulin G (IgG) antibody against WNV using competitive Enzyme-Linked Immunosorbent Assay (c-ELISA), whereas tracheal and cloacal swabs were subjected to consensus Polymerase Chain Reaction (c-PCR) for the detection of the flavivirus RNA. Overall, we detected 11.9%  samples were seropositive, including 15.9% in the migratory wild birds and 10.7% in the resident wild birds. The migratory wild Tufted duck showed 28.5% seropositivity, whereas the resident wild house crows showed 12.5% seropositivity. None of the swab samples was positive for flavivirus RNA infection . These study findings recommend continued surveillance for early detection and to better understand the epidemiology of WNV and other flavivirus circulation in both birds and mosquitoes in Bangladesh.West Nile Virus (WNV) is a vector-borne zoonotic disease maintained in a sylvatic cycle involving mosquito vectors and birds. To detect WNV and other  Flaviviridae in the genus Flavivirus [Culex spp. mosquitoes are competent vectors of the virus [Culex spp. and Aedes spp. are mainly responsible for the transmission of the virus [West Nile Virus (WNV) is an emerging zoonotic arbovirus, having enveloped positive-sense single-stranded RNA belonging to the family avivirus . WNV wasavivirus . The detavivirus , and witavivirus . WNV is avivirus . WNV nathe virus . The virhe virus . In the he virus . Dependihe virus , thus plhe virus . When mihe virus ,11,12,13he virus . These che virus . The virhe virus . WNV serhe virus  to 14.5%he virus  in humanhe virus  in Indiahe virus ,20. Recen = 184) from December 2012 to February 2016 at  following the protocol described by [Serum samples were then transferred into cryovials and preserved at −80 °C . We evalribed by . The c-Eribed by . The plaribed by . The Opt5 gene, which contains short amino acid motifs that are 100% identical in all known flaviviruses. We used the universal control plasmid (synthetic gene) as a positive control in consensus PCR, and the sequence included in the universal control for this flavivirus assay is WNV, as described by [We also collected cloacal and oropharyngeal swabs using sterile polyester swabs with plastic shafts (Fisher) along with blood samples, from each bird. Swab samples were obtained from birds by inserting swab sticks into the vent  for cloacal swabs and oropharyngeal airway and wall of oropharynx for oropharyngeal swabs. Each of the cloacal and oropharyngeal swab samples was placed independently into a cryovial containing one mL of sterile viral transport media . We testribed by ,31. The n), percentage (%), and 95% confidence interval (CI). Fisher’s exact test was done to compare the variables and their significance. The p value < 0.05 was considered a statistically significant difference.We used Microsoft Office Excel 2013 for data management and STATA/IC- 13  for performing data analysis. We analyzed the collected data (both demographic and laboratory) and expressed the results as frequency . Handling and sampling of birds were approved by the Chattogram Veterinary and Animal Sciences University Animal Experimentation Ethics Committee (permit ref. no. CVASU/Dir (R and E) AEEC/2015/02), Bangladesh. Birds were released without injury or harm into their cages after sampling, and all efforts were made to minimize animal suffering throughout our research.n = 184; 95% CI: 0.07–0.16) and 0% , respectively. This result was higher than the findings of a study conducted in Spain (1.96%) using the ELISA test [The overall sero- and viral prevalence of WNV in wild birds of Bangladesh was 11.9% , followed by lesser whistling duck (22.7%), Asian pied starling (20.9%), and crow (12.5%). Contrarily, none of the northern pintail, white-throated kingfisher, rock pigeon, seagull, house sparrow, common moorhen, common myna, and barn owl were positive for WNV . We sampThe virus is prevalent in birds and humans of countries like India ,18,39 anThe detection of WNV-reactive antibodies for the first time in resident and migratory wild birds of different areas of Bangladesh indicates likely exposure to WNV or other closely related flaviviruses. However, no WNV-genomic RNA was detected, and we, therefore, cannot exclude the possibility that the birds were seropositive for related flaviviruses. Future studies should be directed toward the continuous surveillance of the virus using a serum neutralization test (SNT) in humans and birds to inform and appropriate control measures and enable them to be implemented.


Advanced therapy medicinal products (ATMPs) are the new frontier of medicine. Advanced therapy medicinal products are set out to satisfy unmet medical needs and provide new innovative, cutting-edge therapies for serious or life-threatening diseases, thus providing new therapeutic options for people with few or no possibility of treatment. They are divided into four groups including gene therapy medicinal products, cell-based therapy medicinal products, tissue-engineered products, and combined ATMPs, which in Europe refer to products that incorporate one or more medical devices with any of the previously mentioned ATMPs as part of the advanced medicine product . Advance Advanced therapy medicinal products (ATMPs) have unique attributes which differentiate them from standard pharmaceuticals and biologics. Indeed, ATMPs have curative potential as they address underlying genetic or cellular mechanisms of disease, which means that they can have a dramatic and long-lasting positive impact on health. They act through multiple mechanisms and on different cellular targets.ATMPs are often administered just once or a handful of times within a short period. However, as they are typically paid as a one-time treatment, they have a high up-front cost. They are complex products and thus, have also difficult manufacturing processes, often requiring highly specialized manufacturing equipment, processes, and skills. Many cell-based gene therapies, for example CAR-T therapy used to treat some blood cancers, are individually manufactured for each patient: cells are collected from the patient’s blood using a process called apheresis, they are modified and expanded in the laboratory, and then re-infused into the patient several hours later. These processes are usually carried out by trained individuals in specialized centers.Overall, ATMPs can also have a positive impact on patient quality of life, caregivers and on the whole society. In fact, the use of ATMPs decreases hospitalization, avoids continuous drug administration, and reduces nursing. Notably, this approach speeds up the patient productivity, enabling quick return to work with no burden on the society.Thus, ATMPs appear to have the extraordinary potential to offer durable, life-changing solutions for the society. These highly complex treatments rely on current surgical practice and could not prescind from it, but differ from traditional medicines, both in terms of how they are made/administered and by the type of benefits they may provide.In particular, the 3.3% of total number of ATMP clinical trials worldwide are in the field of ophthalmology . GloballAdvanced therapy medicinal products can be classified into three main types:•Gene therapy medicinal products: consist of a vector or delivery formulation containing genes that lead to a therapeutic, prophylactic, or diagnostic effect. The genetic construct is engineered to express a specific transgene. The ‘recombinant’ genes are inserted into the body and by using such gene therapy constructs, in vivo genetic regulation or genetic modification of somatic cells can be achieved. A recombinant gene is a stretch of DNA that is created in the laboratory, bringing together DNA from different sources  in the recipient body. They can be used to cure, diagnose, or prevent diseases.•Tissue-engineered products: these contain engineered cells or tissues that have been modified so they can be administered with the aim of regenerating, repairing, or replacing human tissue.In addition, there are ATMPs that consist of one of the first three categories combined with one or more medical devices as an integral part of the product, which are referred to as combined ATMPs .Up to June 2019, a total of 14 ATMPs have been granted marketing authorization in Europe: seven gene therapies, four cell therapies, and three tissue engineered products. However, four ATMPs have been withdrawn from the market because they did not obtain any reimbursement .®, the first ATMP ensuring a specific number of stem cells, for the treatment of moderate and severe Limbal Stem Cell Deficiency (LSCD). In February 2015, Holoclar® received conditional approval by the European Medicines Agency (EMA) for the use in the European Union  recommended Holoclarnion EU; .®, approved in 2018 as the first gene therapy to restore vision in people with rare inherited retinal disease, caused by mutations in the RPE65 gene. This therapy can be provided to patients with enough residual cells in the retina. Ten years of evidence of its safety were proposed and its use has been studied in patients with ages ranging between 4 and 44 years old.The second was Luxturna® has not enough data available to assess the product’s impact. It is still too early for a comparison of outcomes of patients treated with Luxturna® versus traditional therapies, after authorization  adequate time from approval is available to evaluate economic consequences, (ii) significant follow-up of patient outcomes is collected with the approved technique, and (iii) routine treatments are available as comparator of ATMP effects.®, approved in 2016, has long term follow-up data, but no comparators for economic analysis are available.For example Strimvelis®) for the treatment of blindness and visual impairment. This product was launched in 2015, therefore, extensive information are available on more than 100 patient outcomes with several years of follow up. In addition, alternative approaches are available as comparators, as described below.As a consequence, we analyzed the use of the first CLET  and cataracts (33%). Other causes are glaucoma (2%); and age-related macular degeneration (AMD), trachoma, diabetic retinopathy, and corneal opacities (1%). Furthermore, a large proportion of cases (18%) have an undetermined cause.Meanwhile, the main causes of blindness are cataracts (51%), glaucoma (8%), AMD (5%), corneal opacities (4%), uncorrected refractive errors and trachoma (3%), and diabetic retinopathy (1%), while 21% of cases have undetermined causes .Visual impairment and blindness have considerable socio-economic consequences attributable to the following:-Direct healthcare costs incurred within the healthcare system by the government and/or other payers. These include, for example, general ophthalmic services, hospitalizations, treatment, expenditure associated with injurious falls due to visual impairment and blindness, and rehabilitation.-Direct non-healthcare costs caused by the illness but not imputable to medical treatment: e.g., home improvements , technical assistance such as sticks, guide dogs or computer interface, mobility, home care.-Indirect costs include the economic impacts of this condition outside the healthcare system and on the wider society. These include:-Reduced productivity of the patients and their caregivers due to absenteeism, limited efficiency at work (presenteeism), part-time employment or loss of work,-Informal cares ,-Social security costs .-Intangible costs reflecting the burden of the disease in terms of worsening of the patient’s quality of life due to, for example, pain and other aspects such as the stress felt by the caregiver. Indeed, these can be tangible to some extent, such as in cases of costs related to depression, anxiety and further excess of morbidity. These can be traced back to other types of direct healthcare costs that can be easily calculated , the inclusion of marginal cost in the evaluation is emphasized, as it should not be a “on/off” decision, but rather a “more/less” decision.An economic calculation may reveal, despite an apparent level of spending, that the additional  costs can change the final result of the cost evaluation .Investments on ATMP that could cure patients with visual impairment, will lead to a healthier population, which in turn, could result in a more affordable medical budget for governments, a healthier tax-paying workforce, and lower productivity losses, improving the wellbeing and quality of life of patients and their caregivers.Here, we analyzed the annual costs of blindness and visual impairment reported in three western countries (from EU) in order to compare direct healthcare costs, direct non-healthcare costs, and indirect costs . Indeed,The aim of the treatment for LSCD is to restore the surface of the eye, achieve corneal clarity and improve visual acuity in monolateral or partial bilateral blindness. Current treatment practices usually start with supportive care treatments such as lubrication, autologous serum eye drops, antibiotics, anti-inflammatory drugs, and therapeutic soft or scleral contact lenses. Conservative surgery such as corneal scraping may also be offered before attempting limbal stem cell transplantation. The latter includes several types of invasive surgical options, the aim of which is to transplant stem cells to the affected eye. The surgical options differ in terms of where the cells come from and how they are transferred, specifically the following:•Conjunctival limbal autograft (CLAu), in which stem cells are taken from the contralateral uninjured limbal tissue from the patient’s healthy eye.•Conjunctival limbal allograft (CLAL), in which stem cells are taken from a living, related donor or dead donor and transplanted into the diseased eye of the recipient.•Keratolimbal allograft (KLAL), transplants the entire limbus from a dead donor using the corneoscleral carrier to deliver a large number of stem cells to the recipient.•Simple limbal epithelial transplantation (SLET), reduces the tissue withdrawal of CLAu, but it can treat milder severity  than CLET. In the SLET procedure, re-epithelialization is slower than in some of the other therapies (it takes about 5–6 weeks) .•2), minimizing the risk of injury to the healthy eye. In addition, CLET does not require lifelong immunosuppression and in cases of failure, the treatment can be repeated multiple times.Cultured limbal epithelial transplantation (CLET) procedure  has some®, has been conditionally approved in 2015 by the EMA as the first stem cell−based therapy . This increases the risk of damage to the donor eye and the treatment cannot be repeated in case of failure. The CLAu technique leads complete corneal epithelialization from day 18 up to several weeks after surgery .®) has several advantages, such as the absence of immunological rejection (autologous cells do not require immunosuppression), the use of a small limbal biopsy (1–2 mm2), and the standardization of each preparation of the product made individually from the donor’s cells for a single treatment. It is important to note that treatments can be repeated multiple times if both eyes need to be cured , unknown long-term efficacy, and absence of pharmacovigilance on adverse events. Altogether, these missing points produce uncontrolled long-term increase/variability of costs (of the procedure) as calculated in Another advantage of the ATMP is that it uses new autologous healthy tissue grown in the laboratory that can be implanted onto the damaged cornea, healing the receiving eye in a few days with a rapid decrease in the patient’s symptoms .Results in Current clinical practices mainly rely on day surgeries, with a decrease on in-hospital patient average length of stay (days) in EU countries over the past few years, although the pace of diffusion has varied widely across countries.Indeed, Therapies not following this trend, require an extension of the recovery and associated medical evaluations; they create cost-related problems and, above all, organizational problems to the entire healthcare system.It is worth noting that patients with a less severe condition and an acceptable quality of life are less likely to suffer co-morbidities or adverse events requiring further, potentially expensive, therapies and support. Different studies have showed that visually impaired patients suffer from increased levels of depression, psychological stress, anxiety, and mental fatigue. It is estimated that depression occurs in patients with visual impairment more often (about 17%) than in patients with no vision damage . Thus, dA careful scientific and economic evaluation of the additional costs of each therapy should drive the selection of affordable medical solutions. As the number of ATMPs is increasing, the high prices associated with them have become the topic of many debates. Here, we have focused on sight recovery as it is a critical issue worldwide.Beyond the value of ATMPs for healthcare and considering the total cost for a single patient, it is important to consider healthcare-related costs as well as non-healthcare and indirect costs to perform an appropriate evaluation. It is necessary to have a holistic view on expenses for the government, especially in the case of therapies with high up-front costs.Hospitalization, home care, medical evaluations and adverse events are the most relevant costs for a blind or visual impaired patient. However, costs are not only related to the length of stay in hospital, but also to the hospital’s logistic expenses, to the cost of physicians and nurses that take care of patients for months and to other expenditures. Such costs are widely reduced with the new innovative therapies.Nevertheless, it is also important to consider the global experience of the patient and the possible correlated illnesses such as infections, pain and depression caused by prolonged and non-resolutive therapies. Patients often undergo long-term treatments with drugs in order to reduce symptoms but without any sight restoration. They live with pain and related depression and are not productive, representing often a burden for the society .Supportive care or surgical approaches can appear, at first, as cheaper choices but, afterward, they are often less effective than new therapies. These last ones potentially bring more significant benefits, especially in the long-term, not only for the success of the treatment but also for the caretakers, families, and for the society as a whole.Therefore, the main aim of each therapy and treatment is patient’s healthcare, its satisfaction and self-sufficiency, all in the frame of economic sustainability.Finally, the costs of ATMPs include GMP production costs. The apparently high up-front costs of ATMPs are compensated by the high levels of therapy standardization and safety, ensuring a cost/benefit ratio. Concerning R&D, its related costs are compensated by the usefulness of R&D in assessing public policies and stimulating drug development and innovation.Overall, our analysis highlights that, globally, there is not increase in the costs of ATMPs versus surgery, due to their guarantee of success and short duration.FM and AM provided the acquisition. GP, FM, and AM analysis and interpretation of data for the work, drafted the work and revised it and approved for the publication of the content. All authors are accountable for all aspects of the work.FM and AM have a training employment by the company Holostem Terapie Avanzate, author GP is a member of the Board of Directors and R&D Director of Holostem Terapie Avanzate.