Apis mellifera) is critical for industrial fruit production, but bees can also vector viruses and other pathogens between individuals. Here, we utilized commercial honey bee pollination services in blueberry (Vaccinium corymbosum) farms for a metagenomics-based bee and plant virus monitoring system. Following RNA sequencing, viruses were identified by mapping reads to a reference sequence database through the bioinformatics portal Virtool. In total, 29 unique plant viral species were found at two blueberry farms in British Columbia (BC). Nine viruses were identified at one site in Ontario (ON), five of which were not identified in BC. Ilarviruses blueberry shock virus (BlShV) and prune dwarf virus (PDV) were the most frequently detected viruses in BC but absent in ON, while nepoviruses tomato ringspot virus and tobacco ringspot virus were common in ON but absent in BC. BlShV coat protein (CP) nucleotide sequences were nearly identical in all samples, while PDV CP sequences were more diverse, suggesting multiple strains of PDV circulating at this site. Ten bee-infecting viruses were identified, with black queen cell virus frequently detected in ON and BC. Area-wide bee-mediated pathogen monitoring can provide new insights into the diversity of viruses present in, and the health of, bee-pollination ecosystems. This approach can be limited by a short sampling season, biased towards pollen-transmitted viruses, and the plant material collected by bees can be very diverse. This can obscure the origin of some viruses, but bee-mediated virus monitoring can be an effective preliminary monitoring approach.Healthy agroecosystems are dependent on a complex web of factors and inter-species interactions. Flowers are hubs for pathogen transmission, including the horizontal or vertical transmission of plant-viruses and the horizontal transmission of bee-viruses. Pollination by the European honey bee ( Viruses are widespread in plant agricultural systems, but approaches towards virus detection are often biased for symptomatic plants and limited to serological or PCR-based detection assays of individual viruses ,2. RandoApis mellifera L., is the most widely used and important insect pollinator for global crop production [Many agricultural systems depend on bee pollination activities for food production, particularly long-lived perennial fruit production systems including tree fruit and small berry crops ,12. The oduction ,14. Honeoduction ,16,17. Foduction ,19. Durioduction .Vaccinium sect. Cyanococcus) is a major fruit crop in Canada pollenated by commercial honey bees. Highbush varieties (Vaccinium corymbosum) are cultivated primarily in Ontario (ON) and British Columbia (BC), and lowbush varieties (Vaccinum angustifolium) in Quebec and the Atlantic provinces [Bombus spp.) are used on blueberry farms to improve fruit set and production [Blueberry  and black queen cell virus  can be deposited on flowers for horizontal transmission between forager bees [Ilarvirus and Nepovirus [Ilarvirus), tomato ringspot virus  and tobacco ringspot virus  [Carlavirus) is a major issue in blueberry production systems, particularly the Pacific Northwest, but it is not known to be pollen transmitted [Flowers are central hubs for pathogen transmission between plants, and between pollinator species ,31,32,33ger bees . Many plepovirus ,41. Somepovirus) ,43,44,45Genomic sequencing technologies have advanced dramatically in recent years, but are still not widely used for pest monitoring in agricultural systems due to higher costs ,47. HoweVaccinium corymbosum) farms in BC (BC1 and BC2), and one in ON, in the summer of 2021, during blueberry bloom. Queen sources for the bee colonies were locally bred, and were mixtures of Italian and Carniolan-derived stocks. Blueberries were the primary crop grown at the ON site, but other small fruit crops were grown nearby including strawberry (Fragaria x ananassa) and raspberry (Rubus idaeus), while both BC sites were more monocultured. The BC sites were located in the Fraser valley, approximately 4.6 km apart, and both consisted of the variety “Hardy Blue” with BC1 having plants 30 years old, while BC2 had plants that were 20 years old. The four sample types consisted of the following: (1) 25 individual returning forager bees collected outside the hive and contained pollen in the corbicula; (2) approximately 10 mL of pollen collected using pollen traps ; (3) 25 individual hive bees (adult workers without visible signs of pollen on their bodies) collected from inside the hive; and, (4) approximately 20 mL of bee bread (stored pollen) collected from individual frames with a sterile spatula. Multiple hives were located at each farm site, and replicates were collected from separate colonies at each location. Four replicates of each sample type were collected in BC sites, while three replicates were collected in ON. In addition, two replicates of mixed flower and leaf samples (plant samples) were collected from 10–20 randomly selected blueberry plants at each site, located near the beehives. Sample names were created based on their location, plant species, and sample type and replicate . Forager bees were denoted by F, Pollen—P, Bee bread—B, and hive bee—H. Plant tissue samples were denoted by T). Total RNA (totRNA) was extracted from each hive-related sample using the spectrum total plant RNA extraction kit , while dsRNA was extracted from composite plant samples following Kesanakurti et al. (2016) [Four different sample types were collected from managed bee colonies temporarily located in two independent blueberry (. (2016) .TM Plant Kit for RNA-Seq  as per the manufacturer’s instructions. Ribo depleted totRNA and dsRNA HTS libraries were generated using the Illumina TruSeq Stranded mRNA Library Prep kit, following the manufacturer’s protocol, starting after mRNA selection steps [Extracted totRNA was treated with an rRNA depletion step using the RiboMinuson steps . An Illuwww.virtool.ca, accessed on 30 September 2021) for sample management, quality control (QC) and data analysis. Reads passing QC using FASTQC , were mapped to known virus species in plant and bee virus databases updated December 2021 using the Pathoscope 2 pipeline [HTS sample files were imported into Virtool  , and Betaflexivirus family (n = 3). Initial virus identification was carried out at the species level, with no strain distinctions. BlShV was the most commonly detected virus in BC, present in 78% of samples examined, followed by prune dwarf virus , strawberry necrotic shock virus , and cherry virus A . BlShV was present in 100% of samples collected from the BC2 site, and approximately 50% of samples collected from BC1. Other small berry-associated viruses including blackberry chlorotic ringspot virus , blueberry mosaic-associated virus , blueberry latent virus , and BlScV  were also detected. Average VRPM were highest for BlScV and PDV, while average genome coverage was greatest for privet leaf blotch-associated virus , prunus virus F , and CVA (63%)  .Ilarvirus), cherry virus F (genus Fabavirus), BLV, PLBaV, camellia ringspot-associated virus 1 and 2 (genus Prunevirus), and camellia ringspot-associated virus 3 (genus Capillovirus), CVA, apple stem grooving virus , and citrus tatter leaf virus (genus Capillovirus) were only detected in BC2, while brassica rapa virus 1 (unclassified Rhabdoviridae), cycas necrotic stunt virus (genus Nepovirus), pyrus pyrifolia cryptic virus (genus Deltapartitivirus), grapevine-associated ilarvirus , and actinidia virus X (genus Potexvirus) were only detected in BC1 samples  were detected in composite plant samples compared to bee-collected samples , but five viruses were detected in both bee and plant samples including BLV, BlMaV, BlScV, BlShV, and brassica campestris chrysovirus 1 (BCCV1) (Endornavirus), BrCaCV1, and bell pepper endornavirus  from BC2  a. Only tfrom BC2 . Averagefrom BC2 .Nepovirus, tomato ringspot virus (ToRSV) and tobacco ringspot virus (TRSV), were the most frequently detected viruses at 42 and 33%, respectively , and AHVd  (Vitivirus), BlMaV, BLV, and blueberry virus A , which were not observed in bee-collected samples  c. The pl ON: 8%) ; four vi samples b. BlMaV Iflavirus) and israeli acute paralysis virus  were common in BC samples , but were absent in the ON samples tested. In ON, only four viruses were identified, with BQCV being detected in 92% of the samples tested (Sinaivirus), Sacbrood virus (genus Iflavirus), and apis mellifera filamentous virus (unclassified dsDNA virus) were detected in both BC and ON samples. LSV was the only virus detected in plant tissue samples from BC , apis rhabdovirus 1 (unclassified Rhabdoviridae family), deformed wing virus (genus Iflavirus) and varroa destructor virus 3 (genus Iflavirus) were all present in BC samples, but absent in ON . While the trend was consistent in samples collected at the ON site, bee bread plant virus species richness was not significantly different from that of pollen or hive bees at this site (p < 0.05). In BC2, bee bread had an average of 9.25 viruses detected per sample, with other sample types ranging from 4.5–6.5  isolates formed a separate clade , cherry virus F (genus Fabavirus) and citrus virus A (genus Coguvirus) that may have originated from plants flowering concurrently, before or after blueberries. Bee bread represents a highly diverse collection of plant pollen samples, and could be useful for further study of plant virus diversity. Surprisingly, hive bees also showed an unexpected richness in plant virus species compared to the other sample types which in theory do not directly interact with plants, suggesting the potential for dissemination of plant viruses within a bee colony. BlScV and BlMaV were only identified in forager or hive bee samples, and could have been acquired through direct flower contacts, as opposed to pollen transmission.We expected the greatest diversity of plant viruses to be detected in plant tissues , and more bee viruses to be identified in bee samples , and for the most part this was consistent. Bee bread displayed the highest diversity of plant viruses, and also the highest sequence diversity of individual viruses. This is likely due to bee bread being collected over the seasonal flowering period of different plant species. Many of the viruses identified in bee bread are often associated with apple or tree fruit production systems such as ASGV, citrus concave gum-associated virus (genus Bee health and monitoring the viral load of bee colonies are other important aspects of ecosystem health monitoring ,67,68. BPrunus production systems [A large number of plant and bee viruses were detected widely in different sample types, sites, and regions. An arbitrary genome coverage threshold of 10% was established to remove low-confidence identifications. Other genomics-based studies have argued for genome coverage cut off levels between 10 and 15% for genomics, and 20% for metagenomics-based surveys ,51. Geno systems . MetagenAs evidenced from the wide diversity of plant viruses identified, pathogen monitoring through bee-mediated pollination activities is an excellent approach to understand the diversity of viruses present in an ecosystem. Previous studies of blueberry viruses have identified ~12 high priority viruses in the US and BC . Many of


Orthomyxoviridae, the RNA virus family that includes influenza virus. First, we use sequences of the orthomyxovirid Wǔhàn mosquito virus 6 to track the migrations of its host. We then look at orthomyxovirus genome evolution, finding gene gain and loss across members of the family, especially in the surface proteins responsible for cell and host tropism. We find that the surface protein of Wǔhàn mosquito virus 6 exhibits accelerated non-synonymous evolution suggestive of antigenic evolution, i.e., vertebrate infection, and belongs to a wider quaranjavirid group bearing highly diverged surface proteins. Finally, we quantify the progress of orthomyxovirus discovery and forecast that many diverged Orthomyxoviridae members remain to be found. We argue that continued metagenomic studies will be fruitful for understanding the dynamics, evolution, ecology of viruses, and their hosts, regardless of whether novel viruses are identified or not, as long as study designs allowing for the resolution of complete viral genomes are employed.Metagenomic studies have uncovered many novel viruses by looking beyond hosts of public health or economic interest. However, the resulting viral genomes are often incomplete, and analyses largely characterize the distribution of viruses over their dynamics. Here, we integrate accumulated data from metagenomic studies to reveal geographic and evolutionary dynamics in a case study of Orthomyxoviridae, with over 100 recently discovered viruses infecting hosts from humans to fish. We find that one virus called Wǔhàn mosquito virus 6, discovered in mosquitoes in China, has spread across the globe very recently. Surface proteins used to enter cells show signs of rapid evolution in Wǔhàn mosquito virus 6 and its relatives which suggests an ability to infect vertebrate animals. We compute the rate at which new orthomyxovirus species discovered add evolutionary history to the tree of life, predict that many viruses remain to be discovered, and discuss what appropriately designed future studies can teach us about how diseases cross between continents and species.The number of known virus species has increased dramatically through metagenomic studies, which search genetic material sampled from a host for non-host genes. Here, we focus on an important viral family that includes influenza viruses, the  Viruses that cause disease in humans and economically important organisms were the first to be isolated and characterized. Recently, cheap DNA sequencing has enabled a wave of metagenomic studies in a broader range of hosts, in which viruses are identified in a host sample by nucleic acid sequence alone and a new virus is said to be discovered if that sequence is sufficiently diverged. As a result, the number of known viruses has increased by more than an order of magnitude in the decade since 2012  and hostThis richer tree of viruses has provided some early success stories, such as jingmenvirids that are currently associated with human disease  but whicOrthomyxoviridae. Orthomyxoviridae is a family of enveloped segmented negative-sense single-stranded RNA viruses that infect vertebrates and arthropods. Traditionally, it is split into genera Thogotovirus, Quaranjavirus , Isavirus (found only in fish so far), and a genus for each of influenza A, B, C, and D viruses . Orthomyxovirus discovery has historically been driven by impact on human health  and livelihood , or association with known disease vectors . The metagenomic revolution has resulted in 10 times more orthomyxovirids being discovered over the last decade than in the previous 79 years since the first orthomyxovirus discovery, of influenza A virus, in 1933. The vast majority of known orthomyxoviruses use one of two surface protein classes, with vertebrate-infecting-only members (influenza and isavirids) using one or more class I membrane fusion proteins derived from hemagglutinin-esterase-fusion (HEF)  , sometimon (HEF) . OrthomyQuaranjavirus. WuMV-6 has since turned up in Culex mosquitoes across numerous metagenomic studies all around the world  complex for most of our analyses, since PB1 encodes the RdRp motif responsible for replicase activity (the two other proteins of the RdRp heterotrimer are PB2 and polymerase acidic (PA)). In looking at genome composition, we pay close attention to surface protein use across the PB1 tree, and focus particularly on gp64 proteins used by thogoto- and quaranjaviruses. We find surface proteins to be quite mobile across members of Orthomyxoviridae over evolutionary timescales and identify a clade of quaranjaviruses known to have acquired new segments using distinctly diverged gp64 proteins. Finally, we borrow methods from macroevolutionary research to quantitatively assess the pace at which orthomyxovirus evolutionary history is being uncovered, finding that despite their already transformative effect, metagenomic discovery efforts are likely to continue to find substantially diverged members of Orthomyxoviridae for some time.We start by showing how closely related virus sequences observed across numerous studies can reveal host spatial dynamics and virus microevolution, using the orthomyxovirid Wǔhàn mosquito virus 6 (WuMV-6). WuMV-6 was discovered in China in 2013 from a single polymerase basic 1 (PB1) sequence . WuMV-6 he world –16. ThQuaranjavirus and has two extra segments compared to other quaranjavirids  Californian lineages   ] is the segments , indicatuch gaps  in underOrthomyxoviridae are a moderately sized virus family, their members make use of a diverse and evolving set of surface proteins.The phylogenetic tree of PB1 with surface protein classes indicated also demonstrates the plasticity in orthomyxovirus genome composition—regardless of rooting, the PB1 tree requires at least two switches in viral membrane fusion protein class to explain the current distribution of HEF-like (class I) and gp64-like (class III) proteins. Even within gp64-using orthomyxoviruses, changes between different gp64 lineages are apparent, e.g., Hùběi orthomyxo-like virus 2 carries gp64 related to the Asto-Ūsinis clade yet does not belong to it in PB1 ; Fig. S9Orthomyxoviridae members by quantifying the evolutionary contribution (branch length in amino acid substitutions per site) contributed by each new orthomyxovirid taxon. There are two clear phases of discovery: before 2015, public health investigations of pathogens infecting humans and farmed animals or vectored by ticks led to the discovery of 14 viruses. Since 2015, when lower costs of sequencing enabled large metagenomic surveys in arthopods and vertebrates of little immediate economic value . In contrast, the average PD contributed per virus does decrease with time , as shared evolutionary history is attributed to earlier discoveries. While the orthomyxoviruses discovered each year are, on average, less distinctive, the increased host breadth and rapid pace of current studies result in evolutionarily highly distinctive viruses.We find that distinctive viruses, those contributing significant PD, have continued to be discovered each year . For exaA = 46.3 and B = 62.4], indicated with a red line in Orthomyxoviridae tree of life undersampled, would manifest in a future PD curve higher than the extrapolation of the one observed so far. There is, regardless, an eventual limit, in which the PD gain of a new discovery is less than the threshold of difference used to define a new taxon . If current trends continue, there would remain at least hundreds of additional taxa to be discovered.Orthomyxoviridae, synthesizing data across numerous studies to analyze geographic, evolutionary, and diversity discovery trends.In this work, we endeavored to show how the accumulation of metagenomic data can lead to a new stage in viral studies. We focused on the family We first focus on a single recently discovered mosquito RNA virus—Wǔhàn mosquito virus 6 —whose frDrosophila research, to date, has uncovered the full spectrum of dynamics—a suspected sweep of a Sigma virus in British Drosophila obscura over roughly a decade  were derived from a previous publication . AssemblSRR3168916, SRR3168920, SRR3168922, and SRR3168925  entries R3168925  to segmeR3168925  but most13450231  using th13450231  were simAll successfully assembled segments  were aligned using multiple alignment using fast Fourier transform (MAFFT)  and trimTo confirm sufficient molecular clock signal in WuMV-6, maximum likelihood phylogenies  were infWe also ran molecular clock analyses on all available segment sequences individually using BEAST v.1.10.4  with tipTwenty-seven complete WuMV-6 genomes  were analyzed using the reassortment network method  implemenIn our personal experience ReassortmentNetworkSummarizer is overly conservative when summarizing reassortment networks due to reassortant edges requiring conditioning on both the origin and destination clades. As such, we removed reassortant edges with ≤0.1 posterior support and summarized posterior supports by first extracting the embedding of each segment from the summarized network by carrying out the subtree prune-and-regraft procedures implied by reassortant edges and then finding how many of the same clades are found in posterior summaries of segment embeddings and how many of those are supported with posterior probability ≥0.95 to produce https://github.com/evogytis/baltic) and matplotlib  was used to infer a maximum likelihood tree using PhyML v.3.3.2 which was rooted on isavirids. For each protein, the date of either its publication in literature or on GenBank was noted. For each year of discovery available, tree branches were marked with the evolutionary path uncovered that year, starting from oldest published sequences. The sum of branch lengths contributed by any given sequence to the tree is what we call phylogenetic diversity. As well as the relationship between year of discovery and maximum PD contributed in


However, the presence of viral immune escape mutations may limit the effect of CD8+ T cells on viral rebound. Here, we studied the impact of CD8 immune pressure on post-treatment rebound of barcoded SIVmac293M in 14 Mamu-A*01 positive rhesus macaques that initiated ART on day 14, and subsequently underwent two analytic treatment interruptions (ATIs). Rebound following the first ATI (seven months after ART initiation) was dominated by virus that retained the wild-type sequence at the Mamu-A*01 restricted Tat-SL8 epitope. By the end of the two-month treatment interruption, the replicating virus was predominantly escaped at the Tat-SL8 epitope. Animals reinitiated ART for 3 months prior to a second treatment interruption. Time-to-rebound and viral reactivation rate were significantly slower during the second treatment interruption compared to the first. Tat-SL8 escape mutants dominated early rebound during the second treatment interruption, despite the dominance of wild-type virus in the proviral reservoir. Furthermore, the escape mutations detected early in the second treatment interruption were well predicted by those replicating at the end of the first, indicating that escape mutant virus in the second interruption originated from the latent reservoir as opposed to evolving de novo post rebound. SL8-specific CD8+ T cell levels in blood prior to the second interruption were marginally, but significantly, higher . CD8+ T cell depletion approximately 95 days after the second treatment interruption led to the reappearance of wild-type virus. This work suggests that CD8+ T cells can actively suppress the rebound of wild-type virus, leading to the dominance of escape mutant virus after treatment interruption.HIV rapidly rebounds after interruption of antiretroviral therapy (ART). HIV-specific CD8 + (‘killer’) T cells target and apply selective pressure on the HIV virus. Unfortunately, immune escape mutation may occur, allowing HIV to evade immune recognition. HIV may be more susceptible to immune pressure when there are low levels of virus, such as during initial viral rebound following treatment interruption. However, it is unknown if the immune system can target and suppress the growth of rebounding virus. In this study, we demonstrate that CD8+ T cells can inhibit the reactivation of susceptible virus following treatment interruption. However, escaped virus can circumvent CD8 pressure and reactivate if it is present in the latent viral reservoir.During untreated HIV infection, CD8 HIV viral replication can be suppressed by ART. However, interruption of treatment leads to rebound of replicating virus in most individuals. This rebound arises from reactivation of individual ‘reactivation founder’ viral strains, and the frequency of reactivation from latency has been estimated to range from around once a week in humans, to several times a day in SIV-infected macaques –3. This + T cells drive immune escape during both the acute and chronic phases of HIV and SIV infection  A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Robert F. SilicianoAcademic EditorPLOS PathogensSusan RossSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogens  orcid.org/0000-0002-7699-2064  ***********************Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: This paper examines the role of the cellular immune system in the kinetics of viral rebound in SIV-infected macaques. The main claim of the paper is that it provides evidence that the CD8 T cell (CTL) response provides a selection pressure on viruses reactivating and exiting the latent reservoir, and that in the presence of such a response latent virus with CTL escape mutations preferentially are about to reactivate and contribute to rebound. The study involves 14 animals who are infected then treated with combination ART after 14 days, then after ~7 months treatment is stopped and viral rebound is allowed to occur . ART is restarted after ~8 weeks, then interrupted AGAIN after about 3 months and rebound allowed to occur AGAIN. Finally, some animals are then subjected to CD8 T cell depletion after the second ATI. The authors are able to dissect rebound kinetics and immune escape because of two important innovations of the study design - a) the virus animals are infected with is barcoded, which allows some understanding of the number of unique viral reactivation events that contribute to rebound, and b) the animals are breed that has a specific HLA background that leads to a CTL response that predominantly targets a single known epitope . The main finding was that even though the viral reservoir  was predominantly non-escape virus, escape virus was selected during later rebounds. During the first ATI/viral rebound, early rebounding virus had the same mainly-non-escape genotype as the reservoir, but then escape virus increased in frequency before ART was restarted. During the second rebound, fewer unique reactivating lineages were compared to the first, and, escape virus was at a much higher frequency.Interestingly, the number of measured peripheral CTL cells specific to the epitope of interest did not actually change from before the first ATI (containing predominantly non-escape virus) to before the second ATI (predominantly escape virus), which suggested that it was some aspect of the "quality" of this response, not just "quantity", that exerted the strong selection pressure on reactivating virus.In addition, when the authors conducted a CD8 depletion during the second ATI, they found an increase in viral linages without escape mutations, again providing support that CTL pressure was preventing rebound of non-escape strains.At first the study design seemed a bit odd and convoluted to me , but it seems originally these animals were used to test an immunotherapy that turned out not to work and then the data was repurposed for this study .Overall I think this is an interesting and rigorous study and the paper is really clearly written with nice graphics etc. It is properly placed in the context of previous literature. I did not find the results to be particularly surprising, and while interesting scientifically, it is unclear how they will push the HIV cure field forward, or what relevance they might have outside it. We know that CTL responses are important for (even imperfect) control of HIV/SIV so naturally they will have some impact on viral rebound. I don't think it's surprising that CTLs have some effect immediately after viral reactivation  and immediately exert a selective pressure. Just because these responses are not strong enough fully prevent rebound, or prevent superinfection, does not mean they do not have any effect.Reviewer #2: In this report by Docken et al, the authors use the SIV infected Mamu-A01 rhesus macaque model to examine the interaction between CD8 T cells and virus during ART and multiple ART interruptions. The study is highly and narrowly focused on CD8s that target the Tat SL8 epitope, which has been studied extensively for years and has a well defined pattern of viral escape as well as available reagents (tetramers) for focused examination of CD8s that target this epitope. They use a series of two ATIs, and a final CD8 depletion and ultimately find that breakthrough virus during the second ATI is almost entirely comprised of escape variants, despite the majority of sequenceable proviruses remaining wild type. They conclude that CD8s against this epitope created a sieve effect wherein the only virus that could outgrow contained escape mutations. This conclusion was backed by a subsequent CD8 depletion, and the clever use of a barcoded virus that allowed tracking of individual clones and linking them to specific sequences within the SL8 epitope. The study is excellently done and clearly presented. Every question or caveat that occurred to me while reading was either addressed by the next experiment, or clearly discussed as a caveat. There are indeed caveats – notably the early ART initiation, the focus on one epitope, and the lack of tissue sampling, but these are all addressed and the story remains intact and important. I have no significant suggestions for improvement.Reviewer #3: The authors studied the impact of CD8 T cells on the characteristics of rebounding virus following treatment interruptions in a non-human primate model. Using viral barcode lineages, the authors assessed the evolution of immune escape at the Tat-SL8 epitope following two separate ATIs.While in the first ATI, wt virus dominated the rebound, following the second ATI, viral rebound was dominated by Tat-SL8 escape mutants. Interestingly, the authors speculate that CD8 T cells act as a “sieve” of the latent reservoir and suppress the reactivation of wt virus in the second ATI. Interestingly, CD8 depletion led to greater wt reactivation highlighting the importance of CD8 immune pressure in reservoir activation. Furthermore, the authors speculate that boosted CD8 T cell function during viral replication in the first ATI may lead to enhanced immune control of wt virus in the second ATI. However, the viral replication of wt in the first ATI may result in viral escape from the CD8 immune response in the second ATI.The analysis is very interesting, and the manuscript is very well written. Methods are described clearly. I have only minor comments.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: NoneReviewer #2: (No Response)Reviewer #3: None**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: I found that the calculation of "viral reactivation rate" was confusing and unnecessary for the message of the paper. When I tried to understand this calculation I found the authors mainly referred to a previous paper, which is very annoying for a reader. Importantly, they did not even summarize the assumptions that went into the calculation (which definitely should be included in a revision). I tried to briefly look at the referenced papers and it seems the calculation requires assuming that reactivation events are evenly spaced, and that the growth rates of all strains identical . Both these seem unlikely to be true, but the second especially ... the whole point of this paper is showing that growth rate of some non-escape strains is so much lower than that of escape strains that none are detected at all! So naturally this would not be a binary thing but likely continuous and there could be a while distribution of rates. I thought the paper would be stronger if the authors used some measure of number of reactivating virus or other quantity extracted from the barcodes that was more data-driven and less reliant on an unclear and perhaps overly restrictive model.In a similar vein, I also thought it would be more clear if the authors reminded readers somewhere that their measures of # of reactivating lineages were only those surviving, and that many others could have died out by stochastic extinction before detection.In addition I thought it was an interesting observation that even though their was more evidence of CTL escape at the second ATI, there was still lower peak and set point viral load than the first ATI, suggesting that overall control of viremia was higher, perhaps due to the combined effect of a fitness cost of the escape mutation(s) and the residual CTL control. This is important because people might  assume that using future immunotherapies to control rebound could make things worse than no treatment if they select for escape (like people sometimes think about drug resistance). Of course this might be case specific but it clearly doesn't seem like the case here.I thought the authors could give a little bit more background on this particular macaque model they were using and why they feel they can assume escape vs non-escape strain based on a single epitope, as readers unfamiliar with that literature would be a little lost here.This is probably a better fit for future work, but I also thought the study would be a good opportunity to do some modeling to provide some quantitative estimate of the cost of escape/strength of CTL response, rate of CTL expansion, etc.Finally, in this day and age I don't think it's acceptable to submit a paper for review with a data/code availability statement saying it will be shared upon publication. This should be part of the review process. When I was looking back at the authors' previous paper in this same journal to see the methods about the rebound virus (Fennessy 2017) I noticed there they mistakenly claim their data is available in the SI but it is not, so these resources need to be confirmed by reviewers/editors pre-publication.Reviewer #2: The abstract is unnecessarily short for this journal, and leaves out important details of the study. This is a minor criticism at best.Reviewer #3: Figure 1: Sub-figures C, E, and G are of a poor quality. Please enhance the quality of the light and dark green dots and lines.Figure 1B caption 1: There is a typo in line 125.Line 146: Since FTY720 had no effect on the rebound characteristics, was there any difference between FTY720 and control group? Has it been previously shown that the FTY720 had a limited or no effect? Maybe you could add a reference.Figure 2: It seems that the mutant increases in two phases: initially fast and later slow. What do you think drives the two-phasic increase in mutant virus? Maybe you can write one or two sentences in the manuscript explaining the phenomenon.Line 513/514: You could refer to Figure 1B.Line 562/563: closing bracket is missing.**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, Data Requirements:http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. 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Gait-stabilizing devices (GSDs) are effective at preventing falls, but people are often reluctant to use them until after experiencing a fall. Inexpensive, convenient, and effective methods for predicting which patients need GSDs could help improve adoption. The purpose of this study was to determine if a Wii Balance Board (WBB) can be used to determine whether or not patients use a GSD. We prospectively recruited participants ages 70–100, some who used GSDs and some who did not. Participants first answered questions from the Modified Vulnerable Elders Survey, and then completed a grip-strength test using a handgrip dynamometer. Finally, they were asked to complete a series of four 30-second balance tests on a WBB in random order: (1) eyes open, feet apart; (2) eyes open, feet together; (3) eyes closed, feet apart; and (4) eyes closed, feet together. The four-test series was repeated a second time in the same random order. The resulting data, represented as 25 features extracted from the questionnaires and the grip test, and data from the eight balance tests, were used to predict a subject’s GSD use using generalized functional linear models based on the Bernoulli distribution. 268 participants were consented; 62 were missing data elements and were removed from analysis; 109 were not GSD users and 97 were GSD users. The use of velocity and acceleration information from the WBB improved upon predictions based solely on grip strength, demographic, and survey variables. The WBB is a convenient, inexpensive, and easy-to-use device that can be used to recommend whether or not patients should be using a GSD. Falls are the leading cause of injury among older adults , 2. Neargait-stabilizing devices (GSDs) such as canes or walkers may also help prevent falls [Several successful programs for preventing falls exist, such as exercise programs –9, medicnt falls . GSDs hant falls . Unfortunt falls . Given tSimple survey-based risk assessment scores are available to help determine the risk of future falls for seniors , 16. UnfThe goal of this project was to build an accurate and inexpensive system to determine if an individual uses GSDs. To do this, we used the output  from an inexpensive and readily available device, the Wii Balance Board (WBB), to measure postural stability among older adults who use GSDs well as those who do not use GSDs. Then, after combining these data with measurements of grip strength and a few survey questions, we used logistic regression to determine GSD use. The resulting classification system could potentially, in the future, be used to recommend GSD use to new individuals explicitly based on their overall similarity to GSD users in the model. Because people are often reluctant to use a GSD, the ability to quickly and reliably screen non-GSD users whose stability profile aligns more with GSD users identifies a promising target population for a wide range of fall-reduction interventions.In our community, we estimated that approximately 10,000 people were eligible for this study. Two hundred and sixty-eight participants between 70 and 100 years of age were recruited: sixty-two had missing data elements and were excluded, but both GSD  users and non-GSD users were included in the final pool. Participants were recruited from multiple retirement communities and nursing homes across the state of Iowa; the Internal Medicine Clinic at the University of Iowa Hospitals and Clinics; a mass email sent to all University of Iowa students, faculty, and staff; and the Seniors Together in Aging Research (STAR) Registry. All participants provided written informed consent. The study was approved by the University of Iowa Institutional Review Board.The study consisted of one appointment lasting 20 to 30 minutes. Each participant was asked to self-identify as a GSD user (GSD+) or a GSD non-user (GSD-). Participants completed a short questionnaire that consisted of the Vulnerable Elders Survey , the parNext, participants completed a grip-strength test using a handgrip dynamometer . Each participant was instructed to sit in an armless chair preferably without leaning back against the chair. With elbows flexed at a 90° angle and not touching the side of the body, participants were then asked to first inhale and then exhale while squeezing the dynamometer as firmly and quickly as possible. Grip strength was measured twice for each hand, alternating between hands to allow for recovery time.Finally, participants were asked to complete a series of four balance tests while standing as still as possible on a WBB. Each test consisted of holding one of four specified poses for 30 seconds: (1) eyes open, feet apart; (2) eyes open, feet together; (3) eyes closed, feet apart; and (4) eyes closed, feet together see . Pose orEach participant was labeled GSD+ or GSD- depending on how they self-identified: this is the “ground truth” value we are interested in learning to predict. Survey responses were encoded using a Likert scale from 0 (‘Poor’) to 4 (‘Excellent’) or 0 (‘No Difficulty’) to 4 (‘Unable to do’) as appropriate to each individual question, and handgrip scores were measured in kilograms (kgs) of pressure. Together with age and sex, this provided a total of 25 non-WBB features per participant . Seven subjects were missing 4 or fewer values in the demographics, survey, and grip variables. For these subjects we imputed the missing values using predictive mean matching via multivariate imputation by chained equations [velocity and acceleration metrics, where velocity is defined as change in X and Y location per timestep, and acceleration is defined as the rate of change in velocity per unit of time. To ensure data from different subjects and different trials were comparable, we considered the velocity/acceleration every 1/10th second, so that each test contains 300 (= 30/0·1) velocity measurements and 300 acceleration measurements. For each test within each trial, we created a feature by taking the sum of the square of these 300 velocity measurements and treated this feature as a functional covariate; we treated acceleration similarly. We also aggregated these data across all four tests in trial 1 as well as over all eight tests in both trials. This yielded 20 functional variables per subject .Data from the balance tests were encoded as sequences of X and Y center of balance locations in roughly 0.02 second intervals. From these data, we computed the sequences of We fit generalized functional linear models based on the Bernoulli distribution, using the functional covariates as well as the survey responses and grip strength measurements with the goal of determining the added value of the WBB data. Each predictive model had either zero or one functional covariate, as there were insufficient data to estimate more than one functional covariate at a time.curves for functional variables in addition to regression coefficients for non-WBB variables. These regression curves are integrated against the functional variable to determine the predicted probability of needing a GSD. Some subjects were unable to complete any of their balance trials without assistance. Under the logic that such an individual is more likely to need a GSD than even the least stable subjects who were able to complete their balance trials without assistance, we automatically set their predicted value to be GSD+. That is, if a subject were able to complete their balance trials without assistance, we used the generalized functional linear models to predict GSD+/GSD-, and if a subject were not able to complete their balance trials without assistance we predicted GSD+. The out-of-sample predictive performance of these models was evaluated using area under the ROC curve (AUC), accuracy, sensitivity, and specificity. The threshold for making precise predictions from predicted probabilities was set in such a way to ensure a specificity of 0·75 or greater. All analyses were conducted using R version 4.0.4 [Model performance was evaluated through 10-fold cross validation, where participants were randomly assorted into 10 like-sized subgroups and the models were trained on either 185 or 186 participants (90%) from 9 of these groups and then tested on the remaining 20 or 21 participants (10%) in the last group. Within each fold, we used backward selection with AIC as the selection criterion on the 25 non-WBB variables. For each functional covariate, we then used functional data analysis (FA) to combine the selected non-WBB variables with the functional variable. FA estimates regression on 4.0.4 .Two-hundred sixty-eight people participated in this study. Twenty-one were removed because of equipment error, 39 subjects were missing survey data, and 2 participants were missing WBB data. Of the remaining 206 participants, 109 self-identified as GSD-, and 97 self-identified as GSD+. Characteristics of the participants can be found in Inspecting the FA regression curves provides insight into the time period during which the balance board data helps to predict GSD. Our results demonstrate that we can accurately identify people who currently use a GSD based on their answers to a few simple questions and their performance on a brief balance test using only inexpensive equipment. Given the accuracy of our results, our system could be used to recommend the use of GSDs to people who are currently not using one but that would likely benefit from a GSD and other fall-prevention interventions. Our system also produces a convincing comparative visualization of each participant’s postural stability, which can be used to better communicate a subject’s relative instability, possibly helping to overcome device- adoption barriers.The risk for falls is often multi-factorial. Our approach quickly measures two important risk factors including postural stability (via the WBB) and grip strength (via a simple hand-grip dynamometer). Our best performing model included velocity aggregated over the first 4 tests in trial 1. Given the ease of our approach, it provides a few distinct advantages. First, our approach could be used quickly and efficiently (about 5 minutes per patient) in a clinical setting without the use of specialized or expensive equipment (WBB are out of production but used WBB can easily be purchased for approximately $25). Also, the WBB is small, portable and could be placed next to a scale. It does not need personnel with specific training or a large, designated space both of which may be difficult to obtain in many clinical settings. Second, our approach produces an objective measurement. While the Get-Up-and-Go test, as well as other functional tests geared toward older adults/seniors , have beDespite the effectiveness of GSDs, possession of a GSD does not ensure its use. Furthermore, it is difficult to monitor adherence to such devices. Indeed, people who do fall and own a GSD are often not using it at the time of their fall . In receOur approach, with its clear visualization of postural stability could be used to help encourage GSD use among people who have such a device, but who do not use it regularly. Our approach could also be used to screen patients who would likely benefit from a wide range of other fall-prevention efforts. In addition, given the granularity of our postural stability measurements, we might be able to build new models in future applications to determine if we can detect improvements in postural stability. For example, while our FA model was successful at prediction, the number of subjects in our study was sufficiently small as to preclude the use of multiple functional covariates in the same model. Indeed, while we built a similar FA model using squared acceleration rather than velocity with very similar results, there were insufficient data to include both velocity and acceleration functional covariates simultaneously. We believe it likely that given a larger number of subjects we would be able to train FA models using more nuanced functional covariates, thereby enabling us to build a more accurate and comprehensive picture of patients’ postural stability.The WBB has previously been used to assess postural stability, a well described risk factor for falls. In general, the WBB compares favorably to research-grade force plates, which are much more expensive . WBBs haOur results are subject to several limitations. First, our results are based on a limited sample of only 206 participants, albeit one with a relatively balanced training set (49% of subjects used GSDs). While our population is limited, in general, increasing our sample should increase and not decrease our model’s performance. Second, it may be that some of the people in our study were assigned to either the GSD+ or GSD- group incorrectly. Third, recommendations alone may not be convincing enough to change user behavior and increase GSD use: future work will be needed to show if our more objective approach can be used to change adherence to GSDs. Fourth, the signals from the WBB are noisy, but we were still able to predict GSD use. Finally, for the purposes of this initial work, we did not distinguish between different types of GSDs . Future work including larger samples and ways to include patient and family preference are needed. In conclusion, despite these limitations, we demonstrate a convenient, inexpensive and easy-to-use approach for identifying patients who are currently using a GSD.


Cultural competency has been identified as a barrier to lesbian, gay, bisexual and transgender (LGBT) populations seeking care. Mystery shopping has been widely employed in the formal health care sector as a quality improvement (QI) tool to address specific client needs. The approach has had limited use in community-based organizations due in part to lack of knowledge and resource requirement concerns. Several mystery shopping initiatives are now being implemented which focus on the LGBT population with the goal of reducing barriers to accessing care. One subset targets men who have sex with men (MSM) to increase uptake of human immunodeficiency virus (HIV) testing. No study investigates the costs of these initiatives. Get Connected was a randomized control trial with the objective of increasing uptake of HIV-prevention services among young men who have sex with men (YMSM) through use of a resource-locator application (App). The initial phase of the trial employed peer-led mystery shopping to identify culturally competent HIV testing sites for inclusion in the App. The second phase of the trial randomized YMSM to test the efficacy of the App. Our objective was to determine the resource inputs and costs of peer-led mystery shopping to identify clinics for inclusion in the App as costs would be critical in informing possible adoption by organizations and sustainability of this model.Through consultation with study staff, we created a resource inventory for undertaking the community-based, peer-led mystery shopping program. We used activity-based costing to price each of the inputs. We classified inputs as start-up and those for on-going implementation. We calculated costs for each category, total costs and cost per mystery shopper visit for the four-month trial and annually to reflect standard budgeting periods for data collected from September of 2019 through September of 2020.Recruitment and training of peer mystery shoppers were the most expensive tasks. Average start-up costs were $10,001 (SD $39.8). Four-month average implementation costs per visit were $228 (SD $1.97). Average annual implementation costs per visit were 33% lower at $151 (SD $5.60).Peer-led, mystery shopping of HIV-testing sites is feasible, and is likely affordable for medium to large public health departments. While HIV incidence has decreased in the US population, it has remained constant or increased among men who have sex with men (MSM), particularly among younger age groups . ImproviExtensive research and intervention efforts have been aimed at increasing test uptake among young men who have sex with men (YMSM). Lack of cultural competency among providers has been identified as a significant barrier to care engagement for this group . StudiesPublic health department quality improvement (QI) has been increasingly promoted to improve service delivery, efficiency and responsiveness to public needs . The NatMystery shopping, derived from business marketing, is a tool used in a wide range of settings to evaluate and monitor service performance, and inform QI initiatives to increase customer satisfaction . MysteryThe price of mystery shopping is determined by the content and mode of the visit. Costs to undertake mystery shopping include the start-up costs to initiate the program, such as script creation and training in its execution . On-goinIn the formal health care arena, mystery shopping has been used to improve patient experiences in areas such as appointment referral, lab testing, receipt of mammograms, pharmacy delivery, physician waiting rooms and in family planning clinics –18.Recent work in the US and internationally has begun to explore the feasibility of using mystery shopping as a QI tool in community-based HIV-testing sites. Several studies have investigated the level of cultural competency and the nature of provider/client interactions at community-based organizations with the goal of improving elements, such as site friendliness and provider knowledge, as a means to increase test uptake among the YMSM –21. A guThe Get Connected trial focused on YMSM at-risk of contracting HIV and employed a QI intervention. The trial objective was to increase uptake of HIV-prevention services in this group through use of a resource-locator App populated with LGBT-friendly clinics. The initial phase of the trial identified culturally competent HIV testing sites for inclusion in the App. The second phase of the trial randomized YMSM to test the efficacy of the App.In the first phase, high-intensity, peer-led mystery shopping was conducted in clinics recruited from three cities with high-HIV incidence: Atlanta, Houston, and Philadelphia, serving as study sites. Clinics determined to be YMSM-friendly HIV testing sites, based on visits, were included as locations in the mobile App . ResearcThe objective of this paper is to investigate the financial sustainability and resource requirements for possible adoption of the mystery shopping model. We documented the resource inputs and used activity-based costing to estimate start-up, implementation and total costs, along with cost per shopper visit, for four-months, an arbitrary period determined by the trial, and annually to reflect expenses over a standard budgeting period .We focused on non-research-related tasks. Based on input from site staff, we created an inventory of activities, materials and equipment required to develop and implement mystery shopping. We used program budgets and activity-based costing to determine program costs for the period September 2019 through September 2020 , 25.Staff reported hours spent on each activity. Labor costs were calculated based on reported hours spent on activities, multiplied by the median hourly wage for each staff category. Activities that were handled centrally due to RCT site responsibilities, such as clinic identification, were allocated equally across sites. Cost values for materials were drawn from program budgets. Peer shoppers visited each clinic twice and were paid $50 per visit, including de-briefing.For each site we calculated start-up costs or those incurred prior to initiation of mystery shopping which cannot be recouped. We also calculated implementation costs which become a recurrent budget expense if the program is adopted . We thenUsing published data on visit numbers, we calculated implementation and total cost per mystery shopper visit for the four-month duration of the trial. We then estimated annual costs by adding eight-month on-going implementation costs, consisting of visit coordination and visits, to the four-month values. We estimated annual cost per shopper visit by assuming the rate of average visits for the four-month period applied through the remainder of the year . Data arMystery shopping activities, inputs and their associated costs were allocated to start-up and implementation, shown in Table Implementation began with identifying clinics to serve as shopping sites. This also handled centrally by one site. With the cooperation of the local departments of public health, study staff used AIDSVu.org and Google to collect clinic data. Elements included: information on location, services offered, opening hours and contact information. Costs for clinic identification totaled $1398 of which one-third, $466, was allocated to each site.Other implementation activities included peer recruitment and training of shoppers and coordinating and executing shopper visits, which together constituted two-thirds of four-month average implementation costs of $9995 (SD $2522.8). Costs generally increased with site size with Philadelphia, having the highest costs due to increased costs of shopper recruitment and visit coordination.Average total four-month costs were $19,995 (SD $2517.8) and were evenly split in percentage terms between start-up and implementation. Implementation costs per visit averaged $228 (SD $2.00) and decreased with site size as did total costs per visit at $469 (SD $55.9), reflecting the costs of increasing visit numbers.Annual costs are shown at the bottom of Table We identified the activities and resources required to undertake high-intensity, peer-conducted mystery shopping programs to increase cultural competency among community-based clinics offering HIV testing. Our findings indicate that instituting peer mystery shopping to increase HIV-testing among YMSM is feasible given that public health department staff are highly likely to be equipped to undertake mystery shopping activities. Programs were run centrally at study sites in three areas to model conditions for medium to large public health department or community-based organizations. Based on the cost of resource inputs, annual average cost per visit was approximately $150. Costs declined with site size, reflecting economies of scale whereby costs decline as start-up costs are spread over an increasing number of visits. Costs varied significantly based on the duration of the program, with costs decreasing as the implementation period increased from four months to one year. Annual values are most relevant for public health departments and community-based organizations, given standard annual budgeting periods. Average total costs fell by over-half from the four-month period to the annual period, while average implementation costs fell by a third, primarily as visit numbers increased, both demonstrating the economies of scale of the mystery shopping programs.Start-up and implementation costs are important to separate as one indicates the initial investment requirement and the other the on-going cost of the program once it has started. Average start-up costs were around $10,000 per site. These costs appear minimal in comparison with the size of many public health department budgets. For example, in Georgia, location of the Atlanta site, the current state public health department proposed budget for fiscal year 2022, was $298 million dollars . Given iAverage annual implementation cost per visit of $151 (SD $5.6) were at the lower end of published estimates ($50 - $5000) . The intCosts presented here reflect the context of the Get Connected trial, conducted in high incidence urban setting and thus may not be generalizable to dissimilar locations. Some values may also have been overestimated. Staff were asked to identify and report time spent on activities related to undertaking mystery shopping only and not research-related costs.The difficulty of separating these two, particularly in terms of project management hours, may have led overestimation of costs. Salaries to value time reflect those of academic research settings and staff had to have skills related to research in addition to those required to conduct mystery shopping. Thus, wage rates may be higher than those for community staff not engaging in research.Costs generally increased with site size, with Philadelphia having higher costs in terms of recruitment and training of peer shoppers, coordinating shopper visits, and shopper payments. It should be noted, however, that Philadelphia region was the first site within the clinical trial. As a result, its increased costs may also be linked to unexpected costs associated with being the first site brought on-line.Other assumptions may have led to an underestimation of costs. We assumed no additional equipment was involved as the App was loaded onto mobile phones already in use by peer shoppers. Reported material costs were minimal reflecting that the intervention relied on mobile devices. No costs were included for acquisition of the App given it is available through open-source software. Annual implementation costs did not account for staff turnover, as none occurred during the four-month period.Other possible biases are of unknown direction. Costs reported here also reflect the geographical area and features of the local market under investigation, such as ease of access to shopping targets and of shopper recruitment. Staff hours were based on recall which may have led to an over or under-estimate of costs.Peer-navigator findings from mystery shopping visits were communicated to clinics with the goal of promoting service modification to reduce access barriers to YMSM. This feedback, however, was not communicated formally nor was the degree to which changes were made. This shortcoming needs to be addressed and efforts expanded to help clinics address the cultural barriers identified and subsequent confirmation that service modifications were made.In addition, user feedback was initially explored through focus groups that provided input during App development. The trial assessed willingness to use and length of use once the App was deployed. Soliciting end-user feedback, however, could provide important information for continuous quality improvement. Feedback could range from including affirmation that the clinic was LBGT-friendly, such as through a “like”, a user clinic rating or qualitative feedback to the App developers. All of which would likely increase value and promote sustainability.Considering future adoption, it may be possible to institute a lower intensity protocol to identify LGBT-friendly clinics, such as reducing visits to one, or changing the intensity of the visit. Creation of a peer-led, detailed mystery shopper training toolkit, with sample scenarios included, may reduce start-up costs. In addition, the model could be easily modified to include other groups, such as transgender youth, which would further increase its value.Lack of cultural competency in HIV testing clinics is a known barrier to YMSM, and the wider LGBT population, accessing services in the US –6. ImpleHere we investigated the resource and financial requirements to inform possible sustainability for peer-led, high intensity mystery shopping programs aimed at YMSM. Public Health Departments, in high incidence urban area, would be suited to launch such programs. The initiatives appear to be highly affordable and a relatively minor expense for public health department and community-based organizations of this type. However, the approach may not be viable for individual clinics or small public health departments to initiate on their own given centralization is necessary to reap economies of scale or declining average costs per visit.While the US health care delivery and structure of community-based clinic system is unique, the methodological approach and use of activity-based costing is replicable in other countries.Given the public health goals of promoting health and responding to community needs, peer-led, mystery shopping offers a promising tool to improve service delivery to an underserved, vulnerable population. Increases in testing can reduce cases of HIV thereby helping achieve the CDC goal of eliminating HIV. In addition, establishing linkage to care can increase the overall health of YMSM.


Myzus persicae. The genotypes ‘DK Temptation’ and ‘Rescator’ had the lowest and highest virus titres, respectively, and were used as resistant and susceptible models for comparative analyses with other genotypes. In the greenhouse, the best results were obtained with the genotypes ‘OP-8143 DH’ (2.94 × 105 copies), OP-BN-72 (3.29 × 105 copies), ‘Navajo’ (3.58 × 105 copies) and ‘SG-C 21215’ (4.09 × 105 copies), which reached virus titres about 2 times higher than the minimum virus concentration measured in ‘DK Temptation’ (1.80 × 105 copies). In the field trials, the genotypes ‘Navajo’ (3.39 × 105 copies), ‘OP-8148 DH’ (4.44 × 105 copies), ‘SG-C 21215’ (6.80 × 105 copies) and OP-8480 (7.19 × 105 copies) had the lowest virus titres and reached about 3 times the virus titre of DK Temptation (2.54 × 105 copies). Both trials showed that at least two commercial varieties  and three advanced breeding lines  had low titres of the virus after TuYV infection. This indicates a high level of resistance to TuYV in ‘Navajo’ or the newly developed breeding lines and the basis of resistance is probably different from R54 (as in ‘DK Temptation’). Furthermore, the greenhouse trials together with RT -qPCR-based virus titre analysis could be a cost-effective and efficient method to assess the level of resistance of a given genotype to TuYV infection compared to the field trials. However, further research is needed to identify the underlying mechanisms causing this difference in susceptibility.Turnip yellows virus (TuYV), is one of the most important pathogens of oilseed rape, which has caused enormous yield losses in all growing regions of the world in recent years. Therefore, there is a need for resistant varieties for sustainable crop protection. We have investigated the resistance of known varieties and newly developed advanced-breeding lines of oilseed rape to TuYV in greenhouse and field trials. We have analysed the TuYV titre of individual genotypes inoculated with the virus using viruliferous aphids  Brassica napus L.) is a highly valuable crop grown in many countries and is considered the most important source of vegetable oil in Europe [Polerovirus in the family Solemoviridae [Oilseed rape , which is known to be the main vector of TuYV with a transmission efficiency of over 90%, and the cabbage aphid Brevicoryne brassicae , which has been reported to have a lower transmission efficiency [TuYV is a phloem-limited virus that is transmitted by many different aphid species in a persistent, circulative, and non-propagative manner. The spread of the virus depends on the abundance and movement of the main natural vectors, namely the green peach aphid ficiency ,19,20,21ficiency ,22. For ficiency . TherefoB. napus  and the Korean spring variety B. napus var. oleifera ‘Yudal’ [B. napus variety with high erucic acid content and is so far the only line that has shown genetically characterised resistance to TuYV [In the past, chemical insecticides were widely used to control outbreaks of the virus by targeting the aphid vector. However, studies have shown that reducing aphid numbers does not necessarily reduce the proportion of plants infected with TuYV. Even low numbers of aphids and low numbers of infested plants can lead to high rates of infection with the virus . In addi ‘Yudal’ ,26. A pr to TuYV ,28. Both to TuYV ,29. In r to TuYV ,31). In  to TuYV . The impIn this study, we analysed virus titre in relation to the resistance of oilseed rape genotypes to TuYV. Seed treatments with neonicotinoids have traditionally been used against the aphids’ vector of the virus. However, almost all oilseed production areas in the world have restricted the use of insecticides for environmental protection. We studied genotypes of different origins, including varieties grown in our regions and advanced-breeding lines developed in our earlier research. We found several genotypes in both the varieties and the breeding lines that had low virus titres both in the greenhouse and in the field trails. Our results provide information on the level of resistance of commercially available varieties, as well as of newly developed breeding lines of oilseed rape, and therefore may contribute to reducing the yield losses caused by the virus infection and finding new sources of resistance for crop improvement.p < 0.0001) in the mean TuYV titre levels between the genotypes. Tukey’s multiple comparisons test revealed a statistically significant difference in the mean TuYV titre between the control and inoculated plants for each genotype (6 copies), while ‘DK Temptation’ had the lowest virus titre (1.80 × 105 copies)  determined 4 weeks after infection using RT-qPCR . The onegenotype . The tit copies) . Based o copies) . Overall copies) .M. persicae to inoculate TuYV in the experimental plots proved successful in all three years of the study. The one-way ANOVA multiple comparisons test revealed a statistically significant difference  = 7.546) (p < 0.0001) in the mean TuYV titre levels of the genotypes, including both autumn and spring sampling overall during the three-year field trials. The Tukey test for multiple comparisons revealed a statistically significant difference in the mean TuYV titre levels between the control and inoculated plants for each genotype, except for ‘Navajo’ and ‘OP-8480 DH’ (6 copies), while ‘DK Temptation’ had the lowest virus titre (2.54 × 105 copies)  . Similar copies) . Remarka copies) .p < 0.05) only for the genotypes ‘DK Temptation’, ‘OP-8480 DH’, ‘OP-BN-72’ and ‘Corida’, yet it remained significantly lower (p < 0.05) than the mean titre for the most susceptible genotype in the study  comparing mean TuYV titres (copy number) for each oilseed rape genotype between the autumn (year 2021 + year 2022) and spring (year 2022 + year 2023) samples are shown in he study . The greThe comparison of identification results from the greenhouse and field trials conducted over a three-year period (2020–2023) showed a remarkable similarity between the results of the field trials and those of the greenhouse trials in terms of resistant genotypes. Our results revealed that three of the five resistant genotypes  were observed together under both conditions. Furthermore, comparison of the results under both conditions showed no significant difference in the mean virus titre of all genotypes tested, except that four genotypes  had significantly higher virus concentrations under greenhouse conditions and two genotypes  under field conditions .M. persicae. A low virus titre indicates reduced virus replication, suggesting the high resistance [B. napus line ‘R54’, which confers incomplete resistance to TuYV [This study provides new information on the resistance or susceptibility of oilseed rape genotypes to turnip yellows virus (TuYV), including both cultivated varieties and newly developed breeding lines, including doubled haploids (DH). Twenty-three genotypes were tested for resistance in the greenhouse and field trials. Both trials showed that at least two commercial varieties  and three advanced breeding lines  had low titres of the virus and mild or no symptoms after inoculation of plants with viruliferous aphids sistance  of the gsistance , has the to TuYV ,29. Two  to TuYV . Our res to TuYV . TherefoAccurate quantification methods for measuring virus titres are incredibly important for the resistance screening of plant hosts, as mild or no symptoms may have a higher virus titre or vice-versa ,37,38. TWe found slight differences in the virus titres of the genotypes between the greenhouse and field trials. In all cases, the TuYV titre is much higher in the greenhouse trials than in the field, which may be due to the more efficient transmission by aphids and virus replication under control conditions. However, the performance of the individual genotypes and their resistance/susceptibility characteristics were more or less maintained in both types of trials see . In addiB. napus genotypes to TuYV. These included 11 commercial varieties  and 12 advanced breeding lines from the Czech oilseed rape breeding programme  (The present study was conducted over the last three years (2020–2023) under greenhouse and field conditions to investigate the resistance of 23  48916’) . The breM. persicae, in both greenhouse and field trials. The virus-free aphids, M. persicae, were reared on Chinese cabbage (Brassica rapa) in insect-proof cages in the greenhouse. Aphids were collected and allowed, then placed on TuYV-infected B. napus plants for three days of the acquisition-access-feeding period (AAFP) (under a 16 h photoperiod at 20 ± 2 °C and 60% humidity). Then, the viruliferous aphids were transferred into the greenhouse and the field trial plants  at the four-leaf stage for three days’ access to the inoculation feeding period (IAFP). For both the greenhouse and field trials, we used the same group of 23 genotypes as non-inoculated control plants as for the TuYV inoculation. In the greenhouse trials, the control plants were kept in insect-proof cages. In the field trials, they were covered with non-woven fabric to isolate them during artificial inoculation. Aphid feeding activity was then inactivated with an insecticide (0.25 mL/L H2O acetamiprid). The trials were conducted over three years from 2020 to 2023 for both conditions. The resistance assessment workflow explains the experimental setup in the greenhouse and field trials, which allows a comprehensive evaluation of the resistance of genotypes to TuYV infection under the given conditions  by the use of viruliferous aphids, nditions .In the greenhouse, the seeds of the genotypes were planted in 10 × 10 cm plastic pots filled with a premixed sterilised substrate, with one plant per pot. The pots were placed in a separate insect-proof cage and were regularly watered and fertilised. The greenhouses had temperatures of 16 °C to 22 °C, a photoperiod of 16/8 h (light/dark) and a humidity of 60%, which is optimal for both plant growth and virus infection. Each experiment consisted of two treatment groups: healthy control plants and inoculated plants. Each genotype was tested in three biological replicates for the control and inoculated plants in each trial, resulting in a total of nine replicates in three trials. Samples from each plant were collected four weeks after inoculation and frozen in liquid nitrogen, grinded and 100 mg aliquots were stored at 80 °C until use for the quantitative PCR analysis.2 per plot) in the experimental fields  during 3 growing seasons . Each genotype was tested in three biological replicates for the control variant (3 non-inoculated plots for each genotype) and the inoculated variant (3 TuYV-inoculated plots for each genotype). Leaf samples of at least fifteen plants were taken at random from each plot (bulk groupings of leaf of plants throughout the plot) at two time points, in autumn and early spring. Samples were frozen in liquid nitrogen, grinded and 100 mg aliquots were stored at 80 °C until use for quantitative PCR analysis.Three experiments were conducted in the field, under typical field conditions in small plots representing the control and inoculated plants, with an area of , according to the manufacturer’s protocol. The concentration and purity of the isolated RNA were quantified using the spectrophotometry method . Complementary DNA was synthesised using a Reverse Transcription System . A reaction mixture composed of 1 µg of total RNA, 0.5 µg random hexamer primer, and RevertAid reverse transcriptase 200 U/µL  was incubated at 25 °C for 10 min, 42 °C for 1 h, and then the reaction was stopped by heating to 70 °C for 10 min and subsequent chilling on ice following the manufacturer’s protocol.® 480  with SYBR Green I as described in [® 480 Instrument II  was used to measure the exact quantity of viral RNA copies present in the cDNA samples using the 384-well plate containing 12 µL of reaction solution per well. The qPCR reaction mixture was composed of 0.5 µL of the TuYVF-K1 and TuYVR-K1 primer pair mix at 10 µM [® 480 SYBR Green I Master mix with 2× concentrated , 4.5 µL of sterile nuclease-free water, and 1 µL of cDNA, resulting in a total volume of 12 µL. The amplification protocol was as follows: denaturation at 95 °C for 10 min, followed by 40 cycles at 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 20 s. Finally, a melting analysis of the PCR products was performed to confirm that only the specific products were amplified. To calculate the TuYVtitre in the tested samples, a ten-fold standard dilution curve was created using the plasmid DNA of a positive TuYV sample as described by Singh and Kundu [6 pg µg −1/660 pmol pg−1/Nb by using Avogadro’s constant (6.023 × 1023 molecules mol−1) [Quantitative analysis of TuYV was performed in each oilseed rape sample using RT-qPCR. RT-qPCR was performed using a LightCyclerribed in ,62. The at 10 µM , 6 µL ofnd Kundu . The fols mol−1) . Three tB. napus genotypes according to the procedure of Edwards et al. [Genomic DNA was extracted from leaf tissue of 23 s et al. . To detes et al. . The fraB. napus, causing enormous yield losses worldwide. The virus itself or its Asian strain, brassica yellows virus, is widespread in all growing regions of the world. Seed treatment with neonicotinoids is traditionally used against the aphids’ vector. However, almost all oilseed growing regions in the world have restricted the use of this insecticide for environmental protection. Together with the challenges of global warming, there is a need for sustainable solutions that contribute to the introduction of resistant oilseed varieties. Our results have shown that virus titre analysed by qPCR can effectively define the resistance of genotypes  to TuYV. However, additional data on yield reduction in individual genotypes due to TuYV infection could better explain their tolerance to the virus. The greenhouse trial together with RT-qPCR-based virus titre analysis could replace the expensive and laborious field trials to assess the level of resistance of a given genotype to the virus. Nevertheless, a sufficient number of biological and technical replications should be carried out to better assess the genetic resistance of oilseed rape. The development of DH breeding lines in combination with the described methods for resistance evaluation allow effective breeding of B. napus for resistance to viruses in general and to TuYV in particular.TuYV is one of the most important viral pathogens of