Since 2016, frequent outbreaks of egg-reducing syndromes caused by an unknown virus in duck farms have resulted in huge economic losses in China. The causative virus was isolated and identified as a novel species in Avihepatovirus of the picornavirus family according to the current guidelines of the International Committee on Taxonomy of Viruses (ICVT), and was named the duck egg-reducing syndrome virus (DERSV). The DERSV was most closely related to wild duck avihepatovirus-like virus (WDALV) with 64.0%, 76.8%, 77.5%, and 70.7% of amino acid identities of P1, 2C, 3C, and 3D proteins, respectively. The DERSV had a typical picornavirus-like genomic structure, but with the longest 2A region in the reported picornaviruses so far. Importantly, the clinical symptoms were successfully observed by artificially infecting ducks with DERSV, even in the contact exposed ducks, which suggested that DERSV transmitted among ducks by direct contact. The antibody levels of DERSV were correlated with the emergence of the egg-reducing syndromes in ducks in field. These results indicate that DERSV is a novel emerging picornavirus causing egg-reducing syndrome in ducks. Interestingly, the virus possesses the longest 2A region in all reported picornaviruses and might produce seven putative polypeptides. Our findings enriched the knowledge of picornaviruses and provided the key information for prevention and control of duck egg-reducing syndromes.Duck egg-reducing syndromes caused huge economic losses to the poultry industry in China recently, however, the causative agent had kept unknown since the disease broke out. In this study, the virus was isolated, identified, and named the duck egg-reducing syndrome virus, which was a novel virus species belonging to Dicipivirus that have two internal ribosome entry sites (IRES) and encode two ORFs  × 100. The serum was considered positive for DERSV reactivity when the PI value was ≥22.1%.5.5 EID50 of DERSV in a volume of 200 μL. One day later, eight naive laying shelducks were introduced into the same isolator to test the transmissibility of DERSV. The inoculated ducks were euthanized on 6 dpi, and the exposed ducks were euthanized one day later, and the liver, kidney, and follicles samples were collected. The serum samples were collected from the remaining inoculated ducks at 2, 4, and 6 dpi and from the contact ducks at 2, 4, and 6 dpc for antibody detection. All samples were collected for virus isolation by using 9-day-old embryonated duck eggs and detected by RT-PCR. Additionally, we identified the known avian viruses by PCR and RT-PCR again. For histopathological analyses, samples were fixed with 4% paraformaldehyde, sectioned, and pathological observation of tissue sections after staining with hematoxylin and eosin. Alternatively, immunohistochemical analysis was conducted with a Mab(3G11) against the DERSV.To determine the pathogenicity and transmissibility of DERSV, eight laying shelducks were inoculated intramuscularly with 10Since 2016, outbreaks of a duck egg-reduction syndrome caused by unknown pathogens were reported in AH, SD, JS, and GD provinces in China. Various antibiotics were used to mitigate the outbreak in ducks without any success, suggesting that the pathogen of the duck egg-reduction syndrome might be a viral pathogen. To identify the pathogen, the known avian viruses such as Tembusu virus and Adenovirus that cause egg drop symptoms in ducks, Avian influenza viruses, Newcastle disease virus, Duck reovirus, Muscovy duck reovirus, Duck hepatitis virus 1, Duck hepatitis virus 3, Infectious bursal disease virus, Avian leukosis virus, Duck circovirus, Duck virusenteritis, Duck astrovirus, Goose parvovirus, Muscovy duck parvovirus were detected by PCR or RT-PCR in the diseased duck samples, and all results were negative, which suggested that the pathogen might be a novel virus. To isolate this virus, a homogenate of the follicles, kidneys, and spleen samples collected from ducks with the egg-reduction syndrome were inoculated into the allantoic cavity of 14-day-old goose embryonated eggs. All the infected goose embryonated eggs died after 120 hpi. The allantoic fluid of the dead goose embryonated eggs was harvested. After three times of limited dilution purification in goose embryonated eggs, the purified virus was harvested and detected for the above-mentioned known viruses, which were all ruled out in the isolated virus. Therefore, the virus of the egg-reduction syndrome may be a novel unknown virus.In order to further characterize the novel virus, the virus was purified by sucrose density gradient centrifugation and observed under a transmission electron microscope. The virion was spherical and unenveloped with a diameter of approximately 25–30 nm. Moreover, the nucleic acids extracted from the purified virus were resistant to DNase but not to RNase, which suggested that this virus is an RNA virus.To determine the genome sequence of this RNA virus, the cDNA libraries were constructed by sequence-independent amplification using primer K-8N. A total of 100 positive clones were sequenced, and a continuous sequence (about 2700 bp) was obtained. The remaining sequence of the viral genome were amplified segmentally by RT-PCR using the primer walking process and by rapid amplification of the cDNA terminal ,15. The Ailurivirus, Anativirus, Boosepivirus, Bopivirus, Caecilivirus, Tropivirus, etc. in recent years , different members of a genus mean significant divergence (number of differences per site between sequences) of the orthologous proteins exceed 66% of P1 and 64% of 2C, 3C, and 3D. To further define the classification of the new virus, we analyzed the amino acid homology of P1, 2C, 3C, 3D between DERSV and DERSV-related picornavirus, such as WDALV, AalV-A and duck hepatitis A virus 1/2/3.Detailed analysis revealed that the new virus shared higher amino acid identities with WDALV , AalV-A , and duck hepatitis A virus 1   . Thus, teviously ,17. HoweThe complete polyadenylated RNA of the DERSV comprised 9026 nt excluding the poly(A) tail and had a single 8394 nt ORF flanked by 5′UTR (419 nt) and 3′UTR (303 nt), encoding a putative polyprotein precursor of 2768 amino acids A.The virus had the typical picornavirus-like genome organization. The possible cleavage sites of the polyprotein of DERSV were mapped based on (i) the amino acid alignment with the selected strains of the closest relatives WDALV, AalV-A, DHAV, and (ii) NetPicoRNA predictions . The Q/GDistinguishingly, the 2A region of the DERSV had six putative cleavage sites suggested that the virus might produce up to seven 2A peptides B. Five cPicornaviridae. Due to the insertion of 2A3, the 2A region of the DERSV is the longest in all the reported picornaviruses. The 2A2, 2A4, 2A5, 2A6, and 2A7 of DERSV possessed 44%, 69.6%, 58.9%, 51.9%, and 60.2% amino acid homology with that of WDALV. In addition, another predictive autocleavage motifs, GxExNPG788P, was found on 2A2, which suggested that more 2A peptides might be available in DERSV. Together with three characteristic sequence motifs (G1-G3) and GTP binding site of Ras-like-GTPase superfamily of small guanosine triphosphatases, the conserved motif of GxxGxGKS (x = any residue) potentially responsible for a 2C-like NTPase function was found in 2A6 , which revealed that the region from nt145 to nt424 showed structural similarity to hepacivirus/pestivirus (HP)-like type IV IRES with stem-loop domains II and III and the predicted initiation codon was mapped at position 417 that was surrounded by the optimal Kozak context (GxxAUGG)   [19,20,2Analysis of the capsid regions revealed that DERSV contained a potential myristylation sequence (GxxxxS/T) at positions 155–159. This location is not quite similar to those of AalV-A at positions 39–43, DHAV at positions 31–35 , canine To understand the relationship between DERSV and duck egg production decline in field, a total of 379 samples were collected from diseased ducks with egg decline syndromes in different duck farms distributed in AH, SD, JS, and GD for DERSV detection. The positivity rate of DERSV was 61.7% (234/379) detected by RT-PCR from diseased ducks’ tissues including, livers, spleens, pancreases, ovaries, intestines, oviducts, kidneys, which suggested that this virus has been widely spreading in the main duck-raising areas of China. The positive ratio of the virus was highest in tissues kidneys , ovaries . Then, the positive rate of the virus was also higher in livers , intestines , oviducts  spleens , pancreases . All these results show that DERSV is closely related to the decline of egg production in ducks. Eight tissue samples were selected from 234 of DERSV positive tissues from different regions and different times for virus isolation and sequencing, and compared with the early DERSV isolates. Sequence comparison showed that the nucleotide homology and amino acid homology between these DERSV isolates  were 99.9–100% and 99.7–100%, respectively, indicating that although the variation of duck egg reduction syndrome virus isolate was small, it was distributed in different duck breeds in many provinces.To further confirm whether DERSV caused the clinical symptoms of duck egg-reducing syndromes, the average egg production rate and the serological surveillance in 5 h in a breeding duck farm during the outbreaks of egg-reducing syndromes were monitored from October to December 2020. In the early stage, the egg-reducing syndromes were found only in house #16, whereas the egg production rates kept on a high level in the other four duck houses. The antibody against DERSV was detectable in a part of serum samples from ducks raised in the house where egg drop happened, whereas no positive samples were found in the other four duck houses. Twenty-five days later, the serum samples were collected when the egg-reducing syndromes happened in houses #13, #14. The antibody against DERSV turned positive in some serum samples collected in those two duck houses. At the same time, all of the serum samples collected in the house occurred the egg drop disease firstly convert positive to DERSV antibody, whereas the serum samples collected in the remaining two houses with normal egg production showed negative to DERSV antibody. Sixteen days later, the serum samples were collected when the egg drop disease happened in another duck house (8#). The DERSV antibody showed a similar covert pattern in the new outbreak house as in those three houses broke out early. At that time, the egg production rates in those three early outbreak houses kept at a lower level and showed no signs of recovery . The eme5.5 EID50 of DERSV in a volume of 200 μL. One day after inoculation, eight naive laying shelducks were introduced into the isolator where the i.m.-inoculated ducks were housed. The inoculated ducks or contact exposed ducks showed a certain percentage of tissue damage including significant follicular hemorrhage and rupture , liver hemorrhage , and kidney   with 10d ducks) A. After d ducks) A. In add2.28 ± 0.22 EID50/g, followed by the ovaries (a titer of 101.33 ± 1.12 EID50/g) and liver (a titer of 101.14 ± 1.23 EID50/g) (2.18 ± 0.19 EID50/g), ovary (a titer of 101.58 ± 0.99 EID50/g) and liver (a titer of 101.39 ± 1.17 EID50/g) were similar to those in inoculated ducks  B. In theed ducks B. Moreoved ducks . All theAvihepatovirus or the genus Aalivirus. However, DERSV shared higher amino acid identities with WDALV, which belongs to the genus Avihepatovirus. As a result, we proposed that DERSV was a novel species in the genus Avihepatovirus. So far, both AalV-A and WDALV were failed to be isolated, although they were detected in ducks [A stunning increase in newly identified viruses in the past decade has shown that picornaviruses have an extremely wide range of hosts distributed globally ,28. Morein ducks , and theThe 2A protein is the least conserved non-structural protein in picornavirus, involved in the virus assembly and virulence ,31,32,33To date, five distinct types  of picornavirus IRES elements have been recognized. The picornavirus type IV IRES structures resemble HP IRESs in key respects, notably in comprising domains II and III, and containing a series of signature elements in domain III ,19,36. AAvihepatovirus genus. However, the main clinical signs and symptoms of the disease caused by the virus were the duck egg-reducing and follicular hemorrhage and rupture. To distinguish with duck hepatitis virus, we suggest naming the novel virus as duck egg-reducing syndrome virus according to the main clinical signs caused by the virus in ducks.The picornaviruses are important pathogens in humans and animals, causing diseases that affect the central nervous system, respiratory and gastrointestinal tract, heart, liver, pancreas, skin, and eyes . AlthougBoth DERSV and Duck egg drop syndrome virus (DEDSV) lead to a drop in egg production, but they are quite different: DERS is a disease caused by picornavirus infection. The main clinical symptoms are that the laying rate slowly decreases from a peak of 90% to 50%, and the laying rate will rise after a period of time, and infected ducks will not die. DEDS is a disease caused by DEDSV (flavivirus) infection, and its main clinical symptoms are anorexia, diarrhea, ataxia, paralysis and, most importantly, a significant decline in egg production, which rapidly dropped to less than 10% . Some ofAt present, in the absence of specific DERSV treatment, vaccination will be the most feasible strategy to control DERSV. In view of the successful experience of picornavirus vaccines such as foot-and-mouth disease virus vaccine , polioviTaken together, our results indicated that a novel virus named was DERSV was isolated and identified from egg-reducing syndrome ducks. This virus belongs to the avian hepatitis virus family of picornavirus and has a typical picornavirus-like genomic structure. In the field, the antibody level of DERSV was closely related to the egg-reducing syndrome.


Microbes drive myriad ecosystem processes, but under strong influence from viruses. Because studying viruses in complex systems requires different tools than those for microbes, they remain underexplored. To combat this, we previously aggregated double-stranded DNA (dsDNA) virus analysis capabilities and resources into ‘iVirus’ on the CyVerse collaborative cyberinfrastructure. Here we substantially expand iVirus’s functionality and accessibility, to iVirus 2.0, as follows. First, core iVirus apps were integrated into the Department of Energy’s Systems Biology KnowledgeBase (KBase) to provide an additional analytical platform. Second, at CyVerse, 20 software tools (apps) were upgraded or added as new tools and capabilities. Third, nearly 20-fold more sequence reads were aggregated to capture new data and environments. Finally, documentation, as “live” protocols, was updated to maximize user interaction with and contribution to infrastructure development. Together, iVirus 2.0 serves as a uniquely central and accessible analytical platform for studying how viruses, particularly dsDNA viruses, impact diverse microbial ecosystems. Microbiome researchers are revealing the power of microbes that live in, on, and around us to shape human health and Earth’s diverse ecosystems , 2. ThesProblematically, two key bottlenecks currently prevent researchers wanting to more broadly understand virus impacts in their ecosystem of choice: a) the toolkit for viruses is different to that of microbes, and b) even if a virus toolkit exists, it may not be as mature as for other ecosystems . The maiversion 1 iVirus effort offered 7 apps, numerous data projects , and 5 “live” protocols. Together this iVirus implementation provided researchers—from beginners to experts—the ability to process virus sequencing datasets from raw reads to taxonomically-classified virus genomes, and establish baseline ecological insights. Further, researchers have had unique and varied opportunity to grow and mature the field through feedback venues including (i) the “live protocols”, (ii) international viromics training workshops (https://u.osu.edu/viruslab/viromics-workshop/), and (iii) VERVE-net community networking efforts [To this end, we developed iVirus as a simplified, user-friendly, publicly accessible resource that is linked to “living” documentation allowing for community feedback and consensus-building . iVirus  efforts . Since i efforts , PhageWe efforts , and pha efforts ) or were efforts , Metavir efforts ). Thus, Not surprisingly, virus ecogenomics has advanced rapidly since iVirus’s introduction in 2017. Beyond new analytical capabilities and data resources, there are also additional cyberinfrastructures of relevance, in particular the Department of Energy’s (DOE) Systems Biology KnowledgeBase (“KBase”) whose goal is to “meet the grand challenge of systems biology—predicting and designing biological function on a range of scales, from the biomolecular to ecological” . While KHere we present iVirus 2.0, which offers the first integration of virus ecogenomics tools in KBase, as well as upgrading and expanding CyVerse capabilities with 20 new and updated apps, datasets, and protocols. We continue to provide extensive documentation and seek community feedback, as this motivates iVirus improvements, and helps us assist virus-interested researchers.We first implemented several core iVirus apps on KBase  to empowhttp://tinyurl.com/4ndkt4n2 and https://kbase.us/applist/, respectively). For iVirus development, we sought to complement these with virus-specific apps and resources to maximize virus inference in complex communities, with a focus to date on dsDNA viruses.When a researcher has new microbiome sequence data, whether virus-targeted or microbial, much of the front-end processing is identical. These include read quality control, assembly, gene calling and annotation, sequence alignments, and diverse file manipulations including compression, splitting paired-end reads, and converting between various file formats. As of March 2021, more than 1020 CyVerse and 220 KBase apps, respectively, can be leveraged with these goals in mind , along with a “v-score” to identify a diverse range of viruses, including dsDNA, ssDNA and RNA viruses. For sequences 1 kb or larger and with at least four genes, benchmarking [To offer users more choices in this critical first step in the virus ecogenomics workflow, we added several new apps. First, DeepVirFinder (DVF), a deep-learning based virus identification tool, which was the first such tool to employ deep learning . Unlike hmarking  showed VMetagenomic Archaeal Virus Detector [Because most genome-sequenced archaeal virus isolates derive from extreme environments, these viruses remain difficult to identify from “normal” environments—even when archaea are abundant . To helDetector , and itsDetector , are avaOnce virus contigs have been identified, researchers seek to annotate them to understand identifiable functional capacity. In the original iVirus release, no such tools were available beyond standard microbial tools that predicted open reading frames and searched databases one at a time. Since that time, however, two options have emerged with specific value for viruses – Cenote-Taker , 56 and The first, Cenote-Taker , was desThe second tool, DRAM, represents a different annotation strategy . SpecifiBeyond these two tools, as mentioned above, VIBRANT also includes AMG annotation capabilities. Specifically, VIBRANT uses KEGG annotations to identify novel functions in the new viruses it identifies, though no flagging features are implemented to evaluate certainty around the putative AMG being virus-encoded.Who Is the Host), which we have now integrated into CyVerse. Another host prediction tool incorporating WIsH, VirMatcher, is available as a KBase app (see below).Accurately predicting the host(s) of an uncultivated virus using only genome information remains a major challenge with several approaches currently used to link viruses to their hosts including tRNAs, the presence of prophage genes, shared genes between viruses and hosts , CRISPR spacer matches, and k-mer based signatures . Among tA long-standing problem in virus ecogenomics has been how to determine completeness and purity of newly discovered viruses. For this task, CheckV  was recehttps://bitbucket.org/MAVERICLab/stampede-clustergenomes). Complementarily, higher-level taxonomy can be resolved using gene-sharing network approaches that result in virus clusters (or VCs) that are remarkably concordant with the genera defined by the International Committee on the Taxonomy of Viruses. As such, unknown viruses can be classified with a relative confidence close to ICTV accuracy, at scale. The tool for this, vConTACT [Once new viruses are discovered, they need to be taxonomically classified, which is a major challenge for viruses since they lack any universal gene markers. Fortunately, a population genetic grounded biological species definition of 95% average nucleotide identity across shared genes and 85% coverage along the shorted contig was established and confirmed for several marine viruses , 61, andvConTACT , was impvConTACT , which oAs sequencing and assembly algorithms have advanced, virus ecogenomics researchers are now able to advance from gene- to population-based studies . With thTo increase the accessibility of virus ecogenomics analyses, we have added basic virus ecogenomic functionality to the KBase platform  as yet another means to disseminate information on protocols, workshops, research, apps and data resources. Complementing this, we maintain technical documentation at bitbucket (https://bitbucket.org/MAVERICLab/ivirus), and have established Singularity and/or Docker containers for all iVirus apps, which allows research labs to run analyses on their own resources, independent of either cyberinfrastructure if preferred.Finally, we revamped our website (Though we have sought to develop iVirus through an extensive community-engaged design cycle with specific capabilities for (dsDNA) virus researchers, there are other options available for analyzing virus sequencing datasets. For example, for researchers that have already identified virus contigs in their dataset, stand-alone or web-based platforms exist for analysis (summarized comparative feature sets in Table http://evbc.uni-jena.de/). Second, iVirus data repositories are currently “flat files” that will benefit from more sophisticated focus on best database management system practices to better serve these data and automate data acquisition from decentralized data repositories. Finally, as apps, data, and platforms grow, documentation and app integration, management and maintenance become challenging, which will undoubtedly require a distributed co-laboratory community effort moving forward [While iVirus 2.0 provides unique and state-of-the-art capabilities for virus ecogenomics analyses, it does carry some limitations. First, virtually all the analytics are for dsDNA viruses; future work will benefit from integrating ssDNA- and RNA- virus-specific analytics and workflows, such as those accumulating through efforts by the European Virus Bioinformatic Center , a centralized and modernized set of virus ecogenomic apps and data resources, and mechanisms for usage and community feedback (documentation via “live protocols” at protocols.io and workshops). Collectively, we hope that these efforts will empower dsDNA, and eventually all, virus research across diverse systems.Supplementary Table 1Supplementary Table 2


Multiple sclerosis imposes a heavy burden on the person who suffers from it and on the relatives, due to the caregiving load involved. The objective was to analyse whether the inclusion of social costs in economic evaluations of multiple sclerosis-related interventions changed results and/or conclusions.A systematic review was launched using Medline and the Cost-Effectiveness Analysis Registry of Tufts University (2000–2019). Included studies should: (1) be an original study published in a scientific journal, (2) be an economic evaluation of any multiple sclerosis-related intervention, (3) include productivity losses and/or informal care costs , (4) be written in English, (5) use quality-adjusted life years as outcome, and (6) separate the results according to the perspective applied.Twenty-nine articles were selected, resulting in 67 economic evaluation estimations. Social costs were included in 47% of the studies. Productivity losses were assessed in 90% of the estimations , whereas informal care costs were included in nearly two-thirds of the estimations . The inclusion of social costs modified the figures for incremental costs in 15 estimations, leading to a change in the conclusions in 10 estimations, 6 of them changing from not recommended from the healthcare perspective to implemented from the societal perspective. The inclusion of social costs also altered the results from cost-effective to dominant in five additional estimations.The inclusion of social costs affected the results/conclusions in multiple sclerosis-related interventions, helping to identify the most appropriate interventions for reducing its economic burden from a broader perspective. A shift towards a societal perspective in the economic assessment of healthcare technologies, including not only the payer’s or the provider’s point of view (direct costs), but also the impact on patients and their families and the public/societal expenditure (indirect costs), has been observed over the last decade . The incMultiple sclerosis (MS) is an autoimmune and neurodegenerative condition that affects the brain and spinal cord, causing a disfunction in part of the nervous system’s ability to transmit signals due to damage to the insulating covers of the nerve cells . Some ofMS is particularly interesting to study since it is a disease associated with considerable healthcare and other social costs, due to early onset of symptoms which commonly manifest themselves in childhood and early adulthood (20 s and 30 s), and with a debilitating pathogenesis, making the disease one of the most common causes of disability in younger adults. Even though most people with MS are diagnosed at 20 to 50 years old, a recent study has shown that individuals with a diagnosis of MS at younger ages (during childhood) may develop a more severe stage of the disease after longer periods of time, even as long as 32 years after the diagnosis, than those individuals with a later diagnosis, who might worsen after 18 years, and who also take longer to reach disability milestones . The onsApart from studies which focus entirely on the estimation of healthcare costs, the literature in the field of cost-of-illness studies reveals that non-healthcare costs, mainly those resulting from the loss of work and the cost of care associated with the loss of patients’ autonomy due to disability, represent a very high cost for society, being even higher than healthcare costs . FurtherHowever, and although several non-medical costs related to MS have been identified , which aDespite the efforts developed in the field of burden and cost-of-illness studies, to the best of our knowledge, there is no evidence on the influence of considering the perspective of the society in economic evaluations of healthcare interventions on MS. The aim of the present work was, therefore, to study the effects of considering a broader perspective  instead of the healthcare funder’s perspective in the economic assessment of MS-related healthcare interventions. Therefore, we tried to discover whether a consideration of society’s perspective would significantly modify the results and the recommendations of the economic evaluations performed.We performed a systematic literature review, using the methodological framework outlined in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement . A searcThis review focuses on the cost-utility analysis, which measures the health outcomes in QALYs and the costs of multiple sclerosis-related interventions. Therefore, to reflect the true range of costs and outcomes of multiple sclerosis interventions, the analysis examines the interventions from both the healthcare perspective and the societal perspective.For the purpose of the study, the concept of healthcare costs has been defined in accordance with the OECD report of 2000 (revised in 2011) “System of Health Accounts methodology proposed by the Organisation for Economic Cooperation and Development (OECD)” . It is iTo eliminate bias and errors in the methodology, the process of study selection and data extraction was double-blinded and was conducted using peer review. We followed a three-stage selection method based on the inclusion and exclusion criteria described above. First, BRS screened the lists of selected titles and abstracts identified in the electronic literature searches, and full texts were retrieved if the abstract indicated that the study was an economic evaluation of multiple sclerosis or sclerosis and if it was written in English. Second, based on the inclusion criteria, two investigators (SD and BRS) independently conducted a full-text review and assessed each paper as included, excluded, or unsure. The individual screening results were compared, and discrepancies were resolved by a third reviewer (LMPL or IAR) conducting a full-text review. Final agreement was achieved through discussion among the research team members to achieve consensus. Third, relevant data from the selected articles were extracted using an Excel-based data extraction table specifying the following information: author’s full name, year of publication, journal, title, intervention type, country and currency, discount rate (costs/outcomes), time horizon, perspective applied , costs and QALYs as a consequence of the intervention against its comparator from both perspectives, the threshold assumed for the economic evaluation, costs included , and the method used for calculating social costs. The data-extracting process was also double-blinded, using peer review with two independent researchers (BRS and SD) extracting the data. Disagreements in the data-extracting phase were settled by introducing a third researcher (LMPL or IAR).Following the data extraction, a narrative synthesis of the results from the included studies was performed. To assess the influence of the inclusion of social costs  on the result and conclusion of the economic evaluations of total costs of MS interventions, information about the incremental cost-utility ratios (ICURs) was collected or calculated  from both perspectives. The ICURs were then compared to determine whether the inclusion of social costs in the analyses had affected the conclusion or results of the study based on the threshold value assumed by the authors. In this sense, two options could produce changes in results or in the conclusions. We recorded a change in the conclusions when the decision about the adoption of a new technology was changed as a result of the inclusion of the social costs. For instance, from the healthcare perspective, the ICUR was above the threshold value, so the assessed technology was not recommended. However, when social costs were included, the ICUR was below the corresponding threshold. On the other hand, a change in results was identified when social costs were introduced and (1) the ICUR fell below the threshold  or (2) the intervention became cost-saving . It is important to stress that a significant change in the results may not change the conclusions of the analysis, as explained in the latter case. For example, an intervention assessed with a favourable ICUR from the healthcare perspective would already be recommended. If the inclusion of social costs made the ratio significantly more favourable (or even dominant), there would have been a change in the results of the economic evaluation without leading to a change in the conclusions, as the evaluated intervention would have already been recommended from the healthcare perspective.Based on the literature search in the 2 databases, 421 records were retrieved, after dropping duplicates , without specifying the type of MS –54, 57, Of the 29 studies included in the analysis, 8 were performed in the United Kingdom , 53, 57;Most often, the assessed interventions (22) were pharmaceutical treatments –56, 58. Lifetime was the most frequently used time horizon for the evaluations , 53, 54 Only three studies exclusively included informal care costs , 32, 39,In the case of labour productivity losses, the human capital approach was used in 15 studies , 56, 57 Table The inclusion of social costs changed the incremental costs of the assessed intervention versus its comparator in 15 economic evaluation estimations. Although still positive (having higher costs than the comparator) when social costs were considered, the incremental costs were reduced so as to make the corresponding ICUR fall below the comparative threshold in three cases. In another one, the opposite pattern was observed. Moreover, in eight additional estimations, from the societal perspective the incremental costs of the assessed intervention changed from being positive to negative, implying that the evaluated intervention was cost-saving against its comparator. Three additional estimations showed the contrary trend: positive incremental costs from the societal perspective, but cost-saving from the perspective of the healthcare payer/provider.In view of the aforementioned changes in incremental costs due to the inclusion of social costs , the conclusions were modified in ten EEs . Economic evaluations 35, 36 and 37 showed that, when informal care and labour productivity losses were included, the procedure that was the subject of analysis became cost-effective, as its cost per additional QALY lay below the €30,000 threshold set by the authors, compared to its lack of cost-effectiveness from the healthcare payer’s perspective. Moreover, in three EEs, the inclusion of social costs  resulted in a more dramatic change: the assessed interventions became dominant from the societal perspective , when, from the healthcare payer’s perspective, they were not cost-effective. Conversely, in EEs numbers 9, 10 and 12, the assessed intervention was cost-effective when the analysis was performed from the healthcare payer’s perspective, but it became dominated by the comparator when social costs were included, owing to higher incremental costs. Economic evaluation 65 showed that once labour productivity losses were considered, the intervention was no longer cost-effective, as it was from the perspective of the healthcare payer/provider.Moreover, and although not involving any change in the conclusions as in the ten estimations described above, the inclusion of social costs did show a change in results in five additional EEs : the EEs numbered 17, 20, 33, 45 and 58 became not only cost-effective, as they were already from the healthcare payer/provider’s perspective, but also dominant after the inclusion of social costs, because, in addition to being better in terms of health outcomes, they were cost- saving.From the healthcare payer/provider’s perspective, 12 EEs (17.91%) reported negative incremental costs , pointing towards the assessed intervention being cost-saving against the comparator. However, only eight (11.94%) of them led to the conclusion that the intervention was dominant . On the other hand, if the societal perspective was applied, 17 estimations (25.37%) showed negative incremental costs , all of which, apart from 1 EE, proved to be dominant, leading to lower costs and higher gains in QALY. Another remarkable result is the one obtained in the two EEs (number 21 and 22) from the study carried out by Hettle et al. (2018)  and in eWith regard to the incremental cost-utility ratios (ICURs), from the healthcare perspective, 18 (26.87%) EEs  proved to be cost-effective . From the societal perspective, 12 ICURs were below the threshold and were thus cost-effective .Figure In this review, we sought to explore changes that might occur due to the application of different costing perspectives  to economic evaluations of MS interventions. To fill the gap in the existing literature, the objective of this review was twofold. First, to identify the number of economic evaluations, carried out from a societal perspective, of the treatments related to multiple sclerosis. Second, to investigate the effect that the choice of perspective  has on the results and conclusions of the economic evaluations implemented in this area. Our analysis shows that the results and possible recommendations for decision-makers can differ depending on the perspective selected.In relation to the first objective, the proportion of articles about economic evaluations that include the societal perspective in the field of multiple sclerosis is noteworthy. Almost half of the articles  used the societal perspective. This proportion is notably higher than that found in the area of treatments for rare diseases, where a review found that only 11% of the studies included a societal perspective, a ratioIn the economic evaluations which include a societal perspective, a further difference was found between the types of social costs that were included in the estimations for MS and those included in the economic evaluations of other diseases. In the case of MS, the majority of EEs (about 90%) that considered the societal perspective analysed costs associated with labour productivity losses. This finding is quite similar to that for diseases such as depression  and rare diseases (about 80%). However, in other diseases such as Alzheimer’s, this figure barely reaches 3% –61. The Finding the reason for these differences in the weight and/or the presence of informal care costs and productivity losses, depending on the diseases considered, is neither clear nor intuitive. They could be explained by the nature of the disease . Thus, in the case of Alzheimer’s, rare diseases or multiple sclerosis, the costs may be shared between the patients (through productivity losses) and the family , whereas in the case of depression, the burden could be mainly supported by individuals (through loss of work). In case of the latter disease, it would seem that the burden is still usually considered as a problem for the patient as an individual (through loss of work), and the financial strains on the affected family are not taken into consideration. As has been previously stated, multiple sclerosis is often diagnosed at early ages, even during childhood or early adulthood , and leaIt is not easy to know why the societal perspective is more evident in connection with some diseases compared to others since there is evidence that the social costs associated with them are very substantial. In the case of MS, two studies conducted as long as 20 years ago highlighted the importance of informal care costs in some European countries , especiaWith regard to the second objective of the review, the inclusion of social costs modified the incremental costs of the assessed interventions versus their comparators in 15 economic evaluation estimations. In 10 of them , the use of the societal perspective could modify the recommendations/conclusions of the evaluations. In six cases, the consideration of social costs made the evaluated intervention cost-effective compared to its comparator, but in four other cases the effect was the opposite, and the evaluated intervention was no longer cost-effective against its comparator. Even though the inclusion of social costs did not affect the adoption of the assessed intervention in all the estimations, it is also worth mentioning that it did produce a change in results in 7.5% of the economic evaluations analysed, which changed from having a good cost-effectiveness ratio from a healthcare perspective to being dominant from a societal point of view. When comparing these results with other diseases , it was observed that, even though consideration of the societal perspective had a positive influence by changing the interventions in those diseases to dominant (cost-saving) –61, thisFrom a methodological point of view, the lack of transparency in many of the studies analysed is particularly worrying. In two-thirds of the articles which included the costs of informal care as part of the costs analysed from the societal perspective, the method of assessing informal care time was not made explicit by the original authors of the study. Of the six articles which specified the method used, three used the opportunity-cost approach and in the other three, the replacement-cost approach was used. Of the 26 articles that included productivity losses, 10 of them (38.5%) did not specify the method used. Of the 16 studies that indicated the approach, 15 used the human capital as the method, while only one study used the alternative approach of friction costs. It should be noted that high heterogeneity was identified in the items valued as productivity losses. Some articles only included absenteeism, while other papers included presenteeism, permanent sick leave, early retirement, and premature mortality.Some limitations should also be taken into account when interpreting our findings. However, most of these limitations were due to the lack of homogeneity in the information provided by the original authors. First, since no homogeneous methodology was observed among the studies included , the comparability between studies might be compromised. Second, we did not aim to reassess the evaluated healthcare interventions in each original study, but to review whether the inclusion of the societal perspective could modify the conclusion. However, the authors considered different time horizons, discount rates, healthcare costs and cost-effectiveness thresholds, which may also limit the comparability of results. Moreover, the stage of the disease might be a relevant factor behind the economic burden of the condition, but there is no consistency in relation to this question, since 10 studies did not specify the degree of severity –54, 57, To conclude, the systematic review performed indicates that the adoption of a societal perspective would modify the results of economic evaluations of MS-related interventions, as well as the conclusions about their implementation. Therefore, consideration of the perspective used in the economic evaluations carried out in the field of MS, far from being neutral, can lead to important consequences in relation to the information generated for decision-makers. Excluding the societal perspective when performing economic evaluations in diseases such as MS could lead to the omission of relevant information for decision-makers, and could even result in a misguided recommendation about whether a new and available treatment should be adopted or not. In addition, to be truly social, the societal perspective should also include the effect on caregivers’ health status. It is remarkable that, although almost two-thirds of the selected economic evaluations included informal care costs, only two studies , 56 alsoFor ease of comparison, results are shown in additional euros per additional QALY. For this, the euro-currency exchange rates of the year of each article were applied. The values were not updated to any base year since the efficiency thresholds applied as a usual reference are usually kept constant over several years. In this sense, and to facilitate the interpretation of the results, two vectors were drawn with the values of €30,000/QALY and €50,000/QALY. These values were adopted as they are frequently cited thresholds in the economic evaluation literature.


Potato Virus Y (PVY) occupies the top position. PVY can be transmitted mechanically and by sap-feeding aphid vectors. The application of insecticide causes an increase in the resistant vector population along with detrimental effects on the environment; genetic resistance and vector-virus control are the two core components for controlling the deadly PVY. Using transcriptomic tools together with differential gene expression and gene discovery, several loci and genes associated with PVY resistance have been widely identified. To combat this virus we must increase our understanding on the molecular response of the PVY-potato plant-aphid interaction and knowledge of genome organization, as well as the function of PVY encoded proteins, genetic diversity, the molecular aspects of PVY transmission by aphids, and transcriptome profiling of PVY infected potato cultivars. Techniques such as molecular and bioinformatics tools can identify and monitor virus transmission. Several studies have been conducted to understand the molecular basis of PVY resistance/susceptibility interactions and their impact on PVY epidemiology by studying the interrelationship between the virus, its vector, and the host plant. This review presents current knowledge of PVY transmission, epidemiology, genome organization, molecular to bioinformatics responses, and its effective management.Potato, the world's most popular crop is reported to provide a food source for nearly a billion people. It is prone to a number of biotic stressors that affect yield and quality, out of which  Solanum tuberosum L.) is a fundamental source of nutrient-providing food crops across the world. It occupies the third position globally in terms of consumption after rice and wheat , Potato Virus Y (PVY), Potato Virus A (PVA), Potato Virus S (PVS), Potato Virus M (PVM), Potato Virus X (PVX), Potato aucuba mosaic virus, Potato mop-top virus, and Alfalfa mosaic virus (AMV) were reported to have significant damage potential. Different aphid species, PVY strain, and infesting host range is detailed in Potato  , inoculation accession period (IAP), and virus retention inside the vector body. Starving before feeding and inoculation increase the non-persistent viral transmission efficiency in aphids Powell, . StarvinA wide diversity of plants have been listed as natural PVY hosts and comprise 495 species of plants from 72 genera belonging to 31 families including edible crops such as potato, tomato, pepper, and eggplant, in addition to 211 species within 9 genera of the Solanaceae family. Additionally, Amaranthaceae, Fabaceae, Chenopodiaceae, Compositae, and Brassicaceae were also reported to be host species of PVY  and a single-stranded positive sensed RNA genome (+ssRNA), encapsulated with the coat protein of 2,000 units of the same monomer (30 kDa). The RNA genome contains 9,700 nucleotides ending with a viral protein genome-linked (VPg) at the 5′ end and a poly-A tail at the 3′ end. Furthermore, VPg is a multifunctional protein at the 5′ terminus of +RNA and acts mainly as a primer during RNA synthesis in a variety of +ssRNA viruses, including potyviridae where the poly-A in 3′ of RNA terminus (mRNA and +RNA viruses) that has only adenine bases and is involved in gene expression. The PVY genome is reported to contain two open reading frames (ORF), including the main ORF that is translated to a large polyprotein which ultimately terminates in 10 functional proteins, and the small ORF, otherwise known as Pretty Interesting Potyviridae ORF (P3N-PIPO or PIPO), that is a +2 frameshift within the main ORF and is translated into another protein of smaller size  from the monotypic genus Roymovirus was reported to lack the DAG motif in the CP, and the substituted HC-Pro motifs PTK and KITC by the C-2x-C motif at the N-terminus, favors transmission by the eriophyid mite , the DTG motif similar to DAG is found near the N-terminus of CP, but it lacks the PTK and KITC motifs, so it is non-transmissible by the aphid vectors , which confers ER response to PVY in potato, was identified and functionally described    viral regulation of host components essential for effective infection, (2) plant defensive mechanisms, and (3) the counter-defense mechanisms of viruses to evade plant defenses. The inter-relationship among these factors is a suitable determinant of reactions among the compatible interaction of virus or incompatible host interaction. In response to aphid infestation, susceptible plants develop a diverse local and systemic infection, while leaves of tolerant plants undergo mild symptoms , PVYC (C strains), and PVYZ (Z strains), respectively . This HR was introduced into potato from wild relatives, Ny, Nc, and Nz, imparting resistance to PVYN and PVYNTN (cvs. Igor and Nadine)  for both endogenous genes and targeted genes were used to calculate the log2-fold changes . By GO enrichment analysis the altered processes and functions of the vector in response to the virus are defined. The numbers of DEGs were reported to be involved in biological-cellular-molecular functions, and biological processes can be estimated by conducting the GO enrichment analysis. Cellular components involve different organelle membranes, protein complexes, and the connecting synapses; while biological processes include DEGs governing diverse cellular processes like a response to diverse stimuli, signaling, bio-metabolic processes, biological regulation, and signal transduction. Furthermore, in the molecular functions category, DEGs are associated with diverse catalytic activity, binding, transduction, antioxidant, and transporter activities.Gene Ontology (GO) is a bioinformatic initiative undertaken to unify the representation of gene and gene product attributes across species that utilizes different gene annotations against the UniProt GO database  sheds light on the virulence genes. In all, 16,610 genes out of 22,021 predicted genes had putative roles associated with other aphids, primarily Myzus persicae, Acyrthosiphon pisum, and Diuraphis noxia. Insecticide resistant genes, virus transmission genes, transcription factors, and mitochondrial genes were among the virulence genes they discovered. Other virulence genes included those for defense and detoxification, salivary genes, and chemoreceptors. Additionally, analysis of the GO and KEGG pathways revealed that genes were enriched mostly for molecular function and signal transduction, respectively. A phylogenetic examination of 12 aphid species demonstrated genetic divergence, and A. solani is closely linked to M. persicae.Deepti et al.  investigM. persicae individual stylets, which is similar to PVY transmission by M. persicae, however, the information on transmission efficiency came from another study by Moreno et al.  and Solanum habrochaites plants, respectively, was also documented     Ross, , S. tube) Asama, . Three g) Asama, .® series of cultivars are based on known cultivars like as “Russet Burbank” and Shepody, which have been reworked for “NatureMark.” Pesticide resistant insects (Colorado beetle) and disease resistant pathogens (PLRV and PVY) can be found in their genomes  study of the 08H1 cross, the 05H1 and 08H1 populations were found to be resistant to PVY due to the presence of a single dominant gene located on chromosome 9  with large effects. Thus, resistance breeding approaches can be followed to adopt an eco-friendly management strategy.Regarding genetically engineered resistance based on RNA silencing, the basal antiviral defense system of plants along with CRISPR-Cas13a can provide protection to potatoes against PVY 4E)], that interact with viruses to assist viral infection, and vacuolar invertase, a core enzyme in CIS (Miroshnichenko et al., Altering host plant-insect vector-virus interaction is the most effective way to achieve potential success in PVY control. Physio-morphological changes were reported as a result of the virus spreading to nearby cells that lack active defense systems. Although a variety of passive and active defense systems against plant pathogens were detected earlier, physio-chemical and molecular analyses of the plant-pathogen interaction are still in infancy (Whitham et al., Thus, the current review emphasizes a holistic approach involving the adoption of suitable identification, resistance breeding approaches including new breeding techniques, identifying the active defensive pathways and aphid elicitors involved in pathogen transmission, which will ultimately be helpful for solanaceous growers.TB, IS, DM, JK, PM, and AP: conceptualization, writing—original draft preparation, preparation of tables, and supervision. DM, VS, MN, MAA, PB, and MA: preparation of figures, supervision, review, and editing. All authors have read and approved 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Aphis gossypii, Aphis forbesi and Chaetosiphon fragaefolii species. Single-aphid transmission trials are performed under experimental conditions. Both different viruses and individual virus strains show varying performances in single aphid acquisition. The obtained data suggests that numerous individual transmission events lead to the establishment of multistrain infections. These data will be important for the development of epidemiological models in plant virology.Advances in high-throughput sequencing methods have boosted the discovery of multistrain viral infections in diverse plant systems. This phenomenon appears to be pervasive for certain viral species. However, our knowledge of the transmission aspects leading to the establishment of such mixed infections is limited. Recently, we reported a mixed infection of a single strawberry plant with strawberry mottle virus (SMoV), strawberry crinkle virus (SCV) and strawberry virus 1 (StrV-1). While SCV and StrV-1 are represented by two and three molecular variants, respectively, SmoV has three different RNA1 and RNA2 segments. In this study, we focus on virus acquisition by individual adult aphids of the  Plant viruses enlist a diverse group of viral species that often rely on transmission via invertebrate vectors . While fClosteroviridae) [Potyviridae) [So far, our current knowledge about multistrain infections is based on studies of citrus tristeza virus  ,12,15 anviridae) ,17. Althviridae) ,10.Modes of arthropod-mediated transmission of plant viruses might be non-persistent or semi–persistent ,18. UponBromoviridae), a non-persistent virus with a tripartite genome, were transmitted by aphids [Nevertheless, the establishment of a viral infection in a susceptible host must overcome numerous barriers ,22,23. Dy aphids ,26.Cytorhabdovirus, family Rhabdoviridae, negative-sense ssRNA genome) and strawberry mottle virus  are strawberry viruses known for decades [Cytorhabdovirus) was reported in China and in the Czech Republic in 2019 [Nearly 30 different viruses have been reported to infect strawberries, which are predominantly transmitted by different aphid species ,29,30,31 decades ,32, whil in 2019 . All thr in 2019 . While S in 2019 . The sym in 2019 ,35.Fragaria × ananassa (Weston) Duchesne plant [In this study, we examined the transmission peculiarities of three different viral species, SCV, SMoV and StrV-1, and their genetic variants (genotypes) previously reported to infect a single strawberry, ne plant ,36. BothF. ananassa, cultivar Cacanska rana, was used as a virus source. The CRM3 clones, or daughter plants, were produced using stolons. The CRM3 plant was reported previously to be infected with the following three viruses: SCV, SMoV and StrV-1 [A, SCVB; SMoV–SMoV RNA1A, RNA1B, RNA1C, RNA2A, RNA2B, and RNA2C; StrV-1–StrV-1A, StrV-1B, StrV-1C [A–RNA2A was not investigated. The plants were maintained in a custom-built mesh cage. Recipient plants were Alpine strawberry, Fragaria vesca L. v. f. semperflorens, raised from seeds. All plants were maintained in an insect-proof greenhouse with a controlled temperature and a 16-h day/8-h night period.The CRM3 isolate of d StrV-1 ,36. Each StrV-1C ,36. NoteAphis forbesi Weed, the melon aphid Aphis gossypii Glover and the strawberry aphid Chaetosiphon fragaefolii Cockerell. Isoclonal aphid cultures were started from the field-collected aphids. A single 1st instar nymph was isolated and transferred to a new F. vesca plant. This step was repeated three times. The colonies were maintained by transferring unwinged adult individuals to a new F. vesca plants every month.The following three aphid species were used in this work: the strawberry root aphid cox and cytb genes, cox gene sequences were deposited at the NCBI repository under accession numbers OK181865.1, ON756037 and MN420510.1 for A. gossypii, A. forbesi and C. fragaefolii, respectively. The partial cytb gene sequences have accession numbers ON756035, ON756036, and ON756034 for A. gossypii, A. forbesi and C. fragaefolii, respectively.Their taxonomic identification was performed using molecular barcoding  and transferred to the greenhouse at 25 °C 16-h/8-h day. The plant samples were collected and analyzed thirty days post inoculation.Graft-mediated virus transmission was performed via petiole wedge grafting technique using two trifoliate leaves of the CRM3 plant per a seed-raised Alpine strawberry plant. The plant samples were collected and analyzed sixty days post grafting.Total plant RNA extraction was performed from 50 mg of plant fresh material snap-frozen in liquid nitrogen using the Thermo Scientific GeneJET Plant RNA Purification Mini Kit (Thermo Scientific) following the manufacturers’ recommendations. Total aphid RNA extraction was performed using TRI Reagent  or TRIzol  kits from a single aphid individual. Quantification and quality control of RNA extracts were performed using Nanodrop 1000 UV-Vis spectrophotometer and Qubit HS RNA and IQ assays (Invitrogen).Total RNA  was reverse transcribed in cDNA using Maxima H Minus First Strand cDNA Synthesis Kit with dsDNase (Thermo Scientific) in 20 µL reactions following manufacturers’ recommendations. The cDNA was then diluted 1:10 with milliQ-grade water and proceeded for qPCR assays. Each reaction was run in duplicate. No template controls for all primer sets and no reverse transcriptase controls for the aphids’ endogenous transcripts  were incRT-qPCR assays were conducted on the CFX96 real-time PCR detection system . The 10 µL reaction was prepared from 5 µL of tenfold diluted cDNA, 0.25 µL of forward and reverse primers  to verify the specificity of primer amplification and the presence of potential primer dimers based on the presence of a single peak. No template, positive, and, if necessary, no reverse transcriptase controls were included to check the potential cross-contamination and presence of genomic DNA. As an internal endogenous control for levels of plant material presence, the NADH mRNA was used  and corrP gene, where the qPCR primers target, P gene in addition to the antigenomic positive RNA strand.Total RNA was reverse-transcribed with virus-specific primer  and 1:10As nearly all the aphids previously showed positivity in qPCR, generic cDNA primers, i.e., those targeting all the virus variants and the variant-specific primers , were usqC values were used to calculate the ratio between negative and positive strands of viral RNAs using the formula A—1.98, SCVB—1.95, StrV-1A—2.01, StrV-1B—1.96).The obtained F. ananassa and the F. vesca cv. Alpine strawberry plant 4 from the graft-transmission experiment were analyzed. Non-degenerate primers were designed in conserved regions  and CLC Genomics Workbench 9.5.1 . All RT-qPCR data were processed with Bio-Rad CFX Maestro 1.1, version 4.1 (Bio-Rad), and further data analyses were conducted in R version 4.1.0  and the To assess the RT-qPCR results, we performed recombination analyses for each virus. Such events might jeopardize virus variant identification that was based on a single region. A series of PCRs were performed to amplify nearly full-length sequences of viral genomes that were deeply sequenced on the Illumina platform.The data analysis involved several mapping steps. In order to filter out non-chimeric viral sequences, stringent mapping of quality and adapter-trimmed reads to viral references was performed . Between 81% and 90% of the reads were totally mapped , which oIn order to estimate the plant-wide distribution of viruses and their variants, the following CRM3 plant samples were collected: young and old trifoliate leaves from the crown part, and the short stem, leaves of the daughter plant and terminal leaves from runners. All three viruses, including their variants, were detected.F. vesca plants. Sixty days post-grafting (dpg), one plant was tested as virus-free, one was infected only with SMoV, one with SMoV and some of the SCV and StrV-1 variants  and tested at 30 dpg. After confirmation of the infection, the plants were used for graft transmission with the other variant . Both StrV-1A and B were detected in the grafted plants at 30 dpg.Two trifoliate leaves of the F. vesca plant infected with either RNA1A:RNA2C or RNA1BC:RNA2AC of SMoV were separately graft-transmitted to F. vesca plants (n = 3) and tested at 30 dpg for the virus variants. After confirmation of the infection, the plants were used for the second graft transmission with the opposite variant and were tested at 30 dpg. The plants initially infected with RNA1A:RNA2C were not protected against SMoV variants RNA1BC or RNA2A and, otherwise, RNA1BC:RNA2AC plants were subsequently infected with RNA1A. A similar experiment with plants (n = 3) initially infected with SMoV RNA1BC:RNA2AC resulted in successful infection with RNA1A:RNA2B by graft transmission from the SMoV RNA1ABC:RNA2ABC donor.Two leaves of an A. forbesi, A. gossypii and C. fragaefolii. As the endogenous control, aphids’ 16S RNA was employed and its qC was subtracted from the qC values of viral targets. The obtained data showed a broad range of the ∆qC values for rhabdoviruses—from −1 to 20  and antigenomic (positive) strands should be present, we estimated the ratios between negative and positive strands and compared the ratios against the corresponding ∆q values  in eightqC = 12.5 as a cutoff, the majority of SCV and StrV-1 values for A. gossypii and C. fragaefolii were filtered out  in comparison to A. gossypii and C. fragaefolii . Furthermore, only A. forbesi individuals contained several variants of either SCV and/or StrV-1, while only one A. gossypii individual  and with viral species . On average, the viral prevalence was higher in A. forbesi (68.6%) and relatively similar and lower for A. gossypii (33.3%) and C. fragaefolii, respectively. Among viruses, the highest prevalence was found for SMoV (94.5%), and it was much lower and similar for the two cytorhabdoviruses (17.2% for SCV and 18.0% for StrV-1). Interestingly, the interaction term between virus and aphid species was not significant , suggesting that the observed differences among aphids were independent of the virus being analyzed.To gain additional quantitative insights into the prevalence of the different viruses and variants in the three aphid species, we fitted the data in p-values to account for multiple comparisons of the same null hypothesis of no association between viruses. The results from this analysis are shown in A and SCVB were found together in the same individual aphids significantly more than expected by shared chance . StrV-1A and StrV-1B were also significantly correlated  among them, but not with StrV-1C. Likewise, the three SMoV RNA1 variants were significantly associated among them . Moving now to the associations among variants of different viruses, only some significant associations were found for the cytorhabdoviruses; most remarkably, SCVA showed a significant association with the three StrV-1 variants , while SCVB was significantly associated only with StrV-1A and StrV-1B . No other interspecific significant association was obtained.Next, we sought to evaluate whether the presence or not of a given viral variant was dependent upon the presence of other variants of the same or another virus. To do so, we computed pairwise partial (controlling for aphid species) correlation coefficients and applied a stringent false discovery rate correction to the A. forbesi (n = 28), A. gossypii (n = 30) and C. fragaefolii (n = 28) were individually transferred from the CRM3 plant onto a leaf of a recipient F. vesca plant and left for 24 h. The plants were analyzed for the presence of virus infection at 30 dpi and the positive results are shown in Wingless adults of A. forbesi, A. gossypii and C. fragaefolii, respectively. There were 12 unique combinations of viruses and their variants out of 13 virus-positive plants . Interestingly, there were no cases of SCV transmission by any of the three used aphid species, while StrV-1 was transmitted only by A. forbesi. Cases of special interest include plants 9 and 10, where A. forbesi transmitted StrV-1, but no SMoV infection was detected.Levels of successful transmission were 18%, 13% and 14% for Mixed infections diagnosed by RT-qPCR were further confirmed by Sanger sequencing of other genomic regions .Error-prone RNA viral polymerases create substantial genetic variability that might feed virus evolution. During the progression of a viral infection, a mutant cloud is formed that encompasses a wide range of closely related entities, usually differing by a few nucleotides ,42. AddiWhen a CRM3 strawberry plant was subjected to HTS, a complex mixture of coinfecting viruses and genetic variants was found. Firstly, two SCV genotypes coexisted (sharing 84% of nucleotide identity ). SecondA, SCVB and StrV-1C assemblies, which were then completed with Sanger sequencing and additional HTS runs [Secoviridae family as SMoV, we have not detected such in SMoV. In addition, the genotype-specific RT-qPCR was verified with Sanger sequencing of StrV-1- and SMoV-positive plants after the single aphid transmission.As initial genomic sequences were derived using HTS, the coverage of some genomes was rather low and there were multiple gaps in SCVHTS runs ,36. In tHTS runs ,39,42. SHTS runs ,47 as thHTS runs . Both SMHTS runs , a membeSuccessful (88%) graft transmission of SCV, SMoV and StrV-1 and all their variants showed that both the viruses and their variants were able to systemically colonize new recipient hosts, which confirms the absence of strong bottlenecks during this type of transmission. Interestingly, symptomatic infections were observed only in SMoV-positive plants, irrespective of SCV and/or StrV-1 infection.The original CRM3 strawberry plant was kept for over 20 years , and theWorthy of note is the heterogeneous prevalence of viruses and, in particular, their variants in individual aphids, although each studied aphid population consisted of genetically identical individuals. While both cytorhabdoviruses, due to their propagative mode of transmission, have a much closer relationship with the aphid and must overcome multiple barriers to reach the viruliferous status of the vector, SMoV is transmitted in a non-persistent manner. Nevertheless, the obtained data showed significant differences in virus acquisition for the three tested aphid species regardless of the transmission mode. Quite interesting is the observation that aphid individuals were detected to have only the RNA2 part of the SMoV genome. It should be investigated as to whether they efficiently transmit such incomplete genomes to already SMoV-infected plants, thus increasing the complexity of the existing SMoV infection.C. fragaefolii, a well-known strawberry pest, appeared to not be efficient in vectoring StrV-1, as well as the melon aphid A. gossypii. The latter is known for its broad range of host plants and transmitted viruses [A. forbesi showed statistically better virus acquisition than the other two species, which corresponded with higher percentages of rhabdovirus-positive individuals . Interestingly, some genetic variants of the same virus appear to be found in aphids together in a statistically significant manner. Furthermore, most, but not all, variants of the two cytorhabdoviruses were also found together more often than expected by chance. Future studies may focus on understanding the differences in rhabdoviral levels of accumulation among individual aphids.Interestingly, the strawberry aphid  viruses . HoweverA, RNA2A and RNA2C were predominantly found in positive plants, although ∆qC values for at least RNA1B were higher than for RNA1A in aphids  variants did not cross-protect infected plants against each other.


Old World monkeys (OWM), simians inhabiting Africa and Asia, are currently affected by at least four infectious retroviruses, namely, simian foamy virus (SFV), simian immunodeficiency virus (SIV), simian T-lymphotropic virus (STLV), and simian type D retrovirus (SRV). OWM also show chromosomal evidence of having been infected in the past with four more retroviral species, baboon endogenous virus (BaEV), Papio cynocephalus endogenous virus (PcEV), simian endogenous retrovirus (SERV), and Rhesus endogenous retrovirus-K (RhERV-K/SERV-K1). For some of the viruses, transmission to other primates still occurs, resulting, for instance, in the HIV pandemic. Retroviruses are intimately connected with their host as they are normally spread by close contact. In this review, an attempt to reconstruct the distribution and history of OWM retroviruses will be made. A literature overview of the species infected by any of the eight retroviruses as well as an age estimation of the pathogens will be given. In addition, primate genomes from databases have been re-analyzed for the presence of endogenous retrovirus integrations. Results suggest that some of the oldest retroviruses, SERV and PcEV, have travelled with their hosts to Asia during the Miocene, when a higher global temperature allowed simian expansions. In contrast, younger viruses, such as SIV and SRV, probably due to the lack of a primate continuum between the continents in later times, have been restricted to Africa and Asia, respectively. Indeeol) gene .At present, two retrovirus species infect humans, namely the lentivirus human immunodeficiency virus (HIV) and the deltaretrovirus human T-lymphotropic virus (HTLV). HIV has been transmitted from African non-human primates quite recently, probably around the beginning of the 20th century ,10,11. SFor the existence of other simian retroviruses, namely, baboon endogenous virus (BaEV), Papio cynocephalus endogenous virus (PcEV), simian endogenous retrovirus (SERV) and rhesus endogenous retrovirus (RhERV-K/SERV-K1), all evidence we have comes from the “fossil” record. These viruses have disappeared from circulation but have left their proviral genomes in OWM chromosomes. All four appear to be OWM specific, as their sequences have not been found in hominoid genomes, and neither have novel infections in humans or apes been documented. Summarizing, the eight retroviral species listed here spread among African primates, belong to different retroviral families, and have various tendencies to target the germ-cell line. The exogenous primate retroviruses can all be transmitted to humans. This review and hypothesis paper will discuss, partly on the basis of new research, the epidemiology  and putative age of primate retroviruses from the Oligocene till present, including both the extant and the extinct members of the family, to deduce probable OWM retrovirus dispersal routes.www.ncbi.nlm.nih.gov/pubmed) and Google Scholar (scholar.google.nl) were manually searched using the name of the virus ”simian immunodeficiency virus”, “simian foamy virus”, “simian T-lymphotropic virus”, “Papio cynocephalus endogenous virus”, “simian endogenous retrovirus”, “baboon endogenous virus”, or their abbreviation  and a second term appropriate to the topic, such as “age”, “prevalence”, “phylogeny”, “evolution”, “dating”, “presence”, “detection”, “monkey”, etc. Abstracts were inspected for relevance; from the selected abstracts, only those of which a full-length publication could be retrieved were read and included. In addition, references in the retrieved papers were inspected and used when appropriate. The PubMed database  chromosomal DNA library, were used to identify endogenous PcEV and SERV nucleotide sequences in OWM genomes from GenBank (www.ncbi.nlm.nih.gov/genome/) and Ensembl (www.ensembl.org/index.html) databases by similarity search using the BLASTn/BLAT algorithm provided. In addition, BaEV, another full-length provirus  and SER D10032; ), was usCercocebus atys, Cercopithecus mona, Cercopithecus neglectus, Chlorocebus sabaeus (formerly known as Cercopithecus aethiops sabaeus), Colobus angolensis, Erythrocebus patas, Macaca fascicularis, Macaca fuscata, Macaca mulatta, Macaca nemestrina, Mandrillus leucophaeus, Mandrillus sphinx, Papio anubis, Piliocolobus tephrosceles, Rhinopithecus bieti, Rhinopithecus roxellana, Theropithecus gelada, and Trachypithecus francoisi.Default settings were used in the NCBI BLASTn search, as the aim of the research was to retrieve and describe specifically PcEV, SERV and BaEV integrations, and not more distantly related endogenous viruses, searches were optimized for “highly similar sequences” (megablast). Only when no results were retrieved with megablast, the discontiguous megablast option (“more dissimilar sequences”) was used. In Ensembl BLAST searches, search sensitivity was the default  and filtering low complexity regions or filtering query sequences with RepeatMasker was disabled. When no full-length proviruses were retrieved, virus presence was defined by detecting fragments with >90% homology to both coding and non-coding  fragments of the specific virus. Primate genomes queried were: BLAST results were downloaded from the databases and aligned using ClustalW as implemented in BioEdit (bioedit.software.informer.com). Alignments were optimized through visual inspection. Sequence distances were calculated with the Kimura-2-parameter method and evolutionary relationships were inferred using the Neighbor-joining method with bootstrapping as implemented in MEGA6 ,41. GapsFour retrovirus species are currently circulating in OWM, namely SFV, SIV, SRV, and STLV. SFV is also widespread in apes and New World monkeys (NWM). STLV is present in both Asian and African monkey species and in apes plus humans, while SIV is solely found in African primate species (including African apes), and recently, in humans .Sampling is not systematic; some species remain untested because they may be difficult to reach.Sampling may not be optimal: it is done at the wrong age , the wrong type of sample is taken, or the viral load is below the detection level.The distinction between an exogenous and endogenous retrovirus is not always clear . Moreover, every type of retrovirus has the capacity to enter the mammalian germ line, so Mendelian inheritance is not a distinguishing characteristic ,43,44.Several limitations apply when reconstructing the history of exogenous retroviruses from epidemiological data:Spumaviruses, also known as foamy viruses due to the effect they induce in cell cultures, are a widely spread, distinct type of retrovirus, which share some similarities with hepadnaviruses with regard to their replication cycle . InfectiAn overview of the estimated contemporary distribution of OWM retroviruses and the deduced prehistoric virus migration routes, is given in SIV infection has been detected in African, but not Asian primates or NWM, suggesting that origin and spread of the virus occurred after the separation of OWM and NWM, and also after the major migrations of African primates to Asia in the Miocene period.Two independent integrations of an endogenous lentivirus, termed pSIV, with a phylogenetic position in between the feline lentivirus feline immunodeficiency virus (FIV) cluster and modern simian SIV strains, have been detected in the genome of Malagasy lemur species, suggesting that SIV precursors infecting prosimian primates already existed ~4.3 mya ,84,85,86Chlorocebus females test positive for SIVagm infection [Chlorocebus) subspecies each harbor a distinct lineage of SIVagm. This suggests a long-time presence of the virus, with an origin dating to somewhere between AGM subspeciation, estimated at 1.5–3 mya, and subsequent monkey migrations during the Plio-Pleistocene [Theropithecus species did disperse to Europe in the early Pleistocene [Theropithecus is not infected by SIV, nor are baboons, which currently still inhabit Saudi Arabia and Yemen. Papio hamadryas likely migrated from Africa to Arabia in the late Pleistocene and remained there ever since [Erythrocebus patas), another ground-dwelling species living among AGMs, was likewise reported to carry a SIVagm strain [SIV is remarkably widespread, having infected over 40 African primate species and at least 9 genera . Prevalenfection ,88. SIV nfection ,89. Initnfection ,91. Subsnfection . Confusiistocene . Theropiistocene . Howeverer since ,96. Looker since ,80,81. Am strain .Concluding, a relatively young age has been calculated for SIV based on nucleotide sequences and from geographical separation. Such a young age is supported by its absence in Asian primates, suggesting a post-Miocene origin. Probably, the virus originates from the tropical forests of sub-Saharan Africa and is now spreading to ground-dwelling primates through AGMs, which are widespread in Africa, have high SIV prevalence, and thrive in many habitats. AGMs themselves have probably obtained SIV from cross-species transmission to one of their subspecies with subsequent onwards transmission to the other AGM subspecies in the past . Macaca nemestrina, Macaca fascicularis, and Macaca mulatta [Semnopithecus entellus, from India [pol, shows high homology to SERV pol from baboons , it could also be a reactivation of such a provirus [SRV, a primate betaretrovirus, which can induce considerable pathology in captive macaques, was found to have natural reservoirs in the Asian macaque species  mulatta ,64,66,98om India , suggest and see ). Jayashprovirus . PO-1-Luprovirus ,102. TheMacaca, and have the ability to induce severe disease. Such characteristics are typical of a recently evolved virus species, making the putative recombinant SRV-types 6 and 7 detected in Indian langurs a distinct viral species. Increasing the search for both infectious and endogenous SRV-like retroviruses in Asian monkeys should help gain a better understanding of the distribution and evolutionary connections within this virus family.Little is known about the origin and age of SRV, except for its spread in primate centers since at least the 1970s ,103. SRVHomo-specific variants that arose after cross-species transmission) [The deltaretrovirus STLV comprises multiple types and subtypes and has a broad distribution in OWM and apes, but is not found in NWM . Phylogemission) ,106. Thrmission) . HTLV-1 mission) . A fourtmission) .env gene sequences [tax and env genes, respectively, suggested that HTLV-4 diverged from HTLV-2/STLV-2 approximately 49,800–378,000 years ago [tax gene) and 385,100 (env gene) years ago [Pathogenicity in humans and non-human primates has only convincingly been documented for PTLV-1 . PTequences . Howeverequences ), wherebequences . In factequences ,111. Anaears ago . In thisears ago . Using aears ago . A thirdears ago .Overall, it is clear that PTLV comprises a clade of ancient, genetically diverse viruses, where repeated interspecies transmissions as well as viruses migrating between Asia and Africa pose a challenge for those trying to reconstruct the history of the virus. Despite some variation in estimated divergence dates, the above studies all agree on a late Pleistocene origin for PTLV, with the three main types likely arising in the last 200,000 years.Endogenous retrovirus genomes and parts of such proviruses abound in vertebrates. Most have entered the germ line long before the rise of the order of primates. The primate germ line was thus already seeded with many of such proviral remains before the diversification of extant OWM species. Examples of ancient proviruses, with their calculated time of integration, are human endogenous retrovirus-H , HERV-W (±63 mya), HERV-S (±43 mya), HERV-R (±33 mya), HERV-I (±33 mya), and HERV-E (10.7–41.3 mya) ,117,118.The situation for the supergroup of primate betaretroviruses, represented by HERV-K viruses in humans, and CERV-K in chimpanzees, is more complex ,120. Seqenv mRNA expression has been verified [Four endogenous retroviruses are exclusively present in OWM genomes, namely, BaEV, PcEV, SERV, and SERV-K1/RhERV-K. Of these viruses, full-length proviral genomes can be detected in at least some OWM species ,122,123.Not all infected species may still roam the earthNot all infected species may contain germ line integrationsNot all species have had their genomes sequencedNot all proviral sequences are likely due to bona fide viral infection and integration; they may for instance be acquired by hybridization between speciesProviruses may have been completely or partially lost from the germ line, making identification challengingThe quality of a genome assembly may be insufficient for provirus detectionHeterozygous proviral insertions (ERV insertion polymorphism) are not included in genome assemblies ,128The distinction between endogenous and exogenous retroviruses is not always clear Several limitations apply when reconstructing the history of endogenous retroviruses from genome assemblies:Chlorocebus kidney epithelial tissue [Theropithecus gelada showed a single (baboon) or no (gelada) substitutions between the 5′ and 3′ LTR, respectively (unpublished observation). As retroviral LTRs are identical at the time of integration, after which they start to diverge with the host mutation rate, little or no LTR variation is indicative of a relatively recent origin, probably at most ~300,000 years ago for proviruses with identical LTRs [BaEV was discovered as an integrated provirus in baboon DNA in 1978 and was completely sequenced in 1987 ,134. Nonl tissue . BaEV isl tissue . Most Bal tissue ,125,134.l tissue . In phyll tissue . The BaEl tissue . Analyzical LTRs .Papio cynocephalus) genomic library [Colobus: Colobus guereza and Colobus angolensis (The full-length endogenous gammaretrovirus PcEV was found by screening a baboon ( library . Additio library ). IntereColobus, but not in Cercopithecini, the exogenous period of PcEV can be estimated to have started around the macaque/papionin split, 7.6 ± 1.3 mya, until about 150,000–300,000 years ago. An ancient cross-species transmission to an ancestor of present-day Colobus species could explain its presence there [So, as PcEV is present in ce there .Chlorocebus sabaeus, which suggests that many are so recent that they have not had sufficient time to become fixed in the population [Similar to PcEV, SERV, a full-length integrated type D betaretrovirus was first described from a baboon genomic library . Subsequpulation . Fixatiopulation , suggestpulation . SERV papulation .Theropithecus and Chlorocebus there are also a few proviruses with only 1–2 nt substitutions between the LTRs (unpublished observation), indeed pointing to a lengthy period of germ line activity. Rhinopithecus roxellana SERV integrations with open reading frames were found to be significantly younger than the ones with frame-shift mutations [Full-length proviruses, intact reading frames, virus expression and heterozygous insertions do suggest a young age, but SERV has been calculated to have integrated on average 6.16 ± 3.41 (range 0‒21.62) mya in Asian colobines, which, however, carry a distinct variant of SERV, and somewhat later, 3.42 ± 2.20 (range 0.27–14.09) mya, in cercopithecine species; that is, monkeys of the subfamily Cercopithecinae; thus, both Cercopithecini and Papionini . It was utations . FurtherHomo–Pan divergence [The primate non-D type betaretrovirus superfamily, to which all ERV-K viruses belong, has a complex history. Deep-rooted proviral integrations are present in all OWM and hominoids, but there is also evidence of later activity. In both humans and chimpanzees, the possession of open reading frames combined with low LTR diversity in some proviruses as well as the existence of polymorphic insertions, are illustrative of such recent activity . In casevergence . Anothervergence . SERV-K1vergence . Most liMacaca mulatta and Macaca fascicularis proviruses did contain both LTRs, while those in Macaca fuscata lack them. Furthermore, SERV-K1 proviruses are incomplete in Macaca nemestrina, although the particular LTR sequences are present (unpublished observation). Asian Trachypithecus francoisi and African Colobus angolensis, but not other colobine species, do contain integrations with LTR sequences that are largely homologous to SERV-K1 LTRs. Considering its age and spread, SERV-K1 may have migrated with its colobine and papionin hosts to Asia in the late Miocene. Comparing SERV-K1 LTR sequences from rhesus macaque full-length proviruses shows that the 5′ and 3′ LTRs have no, or very low, nucleotide diversity , are present in all OWM . HoweverColobus/Trachypithecus, is thus an interesting subject for further study. The uneven distribution of SERV-K1 complete proviruses suggests that either the infectious virus did not always reach the OWM germ line, that the integrations were lost, or that they are somehow not in the genome assemblies. Overall, due its apparently highly infectious nature over long periods of time, and a tendency to recombine, the epidemiological trail of the ERV-K virus family is not easy to follow. The recent spread, on two continents, of two related SERV-K viruses in two very different monkey genera, papionins and Infectious period estimates of the eight OWM retroviruses in relation to the geological time scale are summarized in Papio and Theropithecus were the only species then trekking to Eurasia through Arabia [Macaca sylvanus, is now left in (northern) Africa; European macaques have gone extinct in the late Pleistocene [The order of primates probably dates to the Paleocene (56–65 mya). Primate fossils appeared simultaneously in Europe, Asia, and Africa in the early Eocene (55–50 mya), suggesting that by then, the order was firmly established. Since primates are relatively intolerant of cooler climates, their history is largely determined by climatic variations, which in their turn are linked to tectonic and cosmic events . The raph Arabia . Macaqueh Arabia . Only a istocene . Modern istocene .With the above described primate evolution in mind, an attempt to retrace the history of one of their successful parasites, the retrovirus family, was made. Retroviruses are generally transmitted by close contact, for instance by the exchange of saliva or blood (biting), through sexual encounters, or from mother-to child, implying that these pathogens are closely associated with their host species. Infections by aerosols, the oral–fecal route or by insect vectors are relatively rare or are unlikely; only equine infectious anemia virus, EIAV, and possibly bovine leukemia virus, BLV, can sometimes be transmitted by flies from viremic hosts ,142. So,Investigating the spread and estimated age of eight OWM-infecting retroviral species suggests that SFV, the most widely spread virus of all, with a distribution over three continents, is also the oldest virus in the group. Ancestral primates most likely migrated, possibly by rafting, to South America during the Paleogene when that continent was relatively close to Africa . As the The next oldest viruses, SERV and SERV-K1 (RhERV-K), are now known only in an endogenous form. Their putative Miocene origin, together with a wide distribution in OWM species in both Africa and Asia, suggests that SERV and SERV-K1 have been travelling the land bridge(s) between the continents established after the final closure of the Tethys Seaway ~14 mya . The relMacaca sylvanus carry PcEV and/or BaEV integrations.Another virus that likely migrated with its host from Africa to Asia is PcEV, which is found in African papionins as well as in Asian macaques. Such a distribution suggests that PcEV is at least 5.5–7 mya old, but not much older as the virus is not found in the germ line of the Cercopithecini, implying that migration with its macaque host took place in the late Miocene or early Pliocene. Recombination of PcEV with SERV, when at least one of the two viruses was actively replicating, gave rise to BaEV, which, by definition, should be younger than its parents. Indeed, BaEV is not found in Asian macaques, suggesting its infectious period postdates the divergence of macaques from the other papionins. The putative Pleistocene origin of BaEV prohibited its spread to Asia, as by then the climate was no longer favorable for monkey expansions. BaEV may have been present in European baboons, having gone extinct there together with its host. Moreover, it would be interesting to investigate whether or not African Mandrillus, Cercocebus, and Lophocebus. An exception here are AGMs (Chlorocebus spp.), found just about everywhere in sub-Saharan Africa, which carry a great diversity of SIV strains and have high SIV prevalence. They may have facilitated SIV cross-species transmissions in the past, similar to modern AGM transmitting SIVagm to individual baboons and patas monkeys. An example of another retrovirus shared between AGMs and papionins is BaEV.Much younger OWM retroviruses appear to be SIV and SRV; SIV has an exclusively African distribution while SRV is seemingly only found in Asian macaques. The uninhabitable nature after the Miocene period of areas in key corridors for primates between Africa and Asia, with subsequent isolation of monkey species on both continents, likely impeded virus transfer between continents for these recently emerged viruses ,151,152.For endogenous viruses that have gone extinct, once infected species may be missed because no germ line introductions occurred or were lost in subsequent generations. Still, the available data on endogenous viruses can be used to infer a plausible evolutionary trajectory. Retroviruses that use the neutral amino acid transporter ASCT2 as a cellular receptor, which is expressed on oocytes, spermatozoa, and pre-implantation embryos, have a tendency to enter the germ line ,154,155.Interestingly, all four now endogenous OWM retroviruses have disappeared from the circulation, suggesting that virus endogenization ultimately means the end of the contagious variant. SIR could be such a possible mechanism to limit virus spread. Thus, opting for germ line transmission seems to be an indicator for extinction of infectious retrovirus lineages, while old age, as exemplified by the current infectivity of SFV, does not need to be.Current distribution of primates, prehistoric primate evolution and expansion together with virus prevalence and sequence information have been used to infer the history of primate retroviruses. African and Asian monkey species are, or have been, infected by at least eight distinct retroviral species. Four of those have now become part of the germ line while four others are currently in the infectious stage, including the most ancient retrovirus, SFV. Evidence suggests that the older retroviruses—PcEV, SERV, SERV-K1—have migrated with their hosts to Asia ~5 to 10 mya, while the more recent ones—BaEV, SIV, and SRV—are limited to their respective continents of origin. For STLV, the reconstruction of its history proved more difficult, as its relatively recent age and widespread distribution does not correspond with Miocene migration.