Poaceae community types that represent a gradient of grass species richness and management intensity, from highly managed crop monocultures to little-managed, species-rich grasslands. We performed a large-scale study on 950 wild and cultivated Poaceae over 2 years, combining untargeted virome analysis down to the virus species level with targeted detection of three plant viruses. Deep sequencing revealed (i) a diversified and largely unknown Poaceae virome (at least 51 virus species or taxa), with an abundance of so-called persistent viruses; (ii) an increase of virome richness with grass species richness within the community; (iii) stability of virome richness over time but a large viral intraspecific variability; and (iv) contrasting patterns of virus prevalence, coinfections, and spatial distribution among plant communities and species. Our findings highlight the complex structure of plant virus communities in nature and suggest the influence of anthropogenic management on viral distribution and prevalence.Modern agriculture has influenced plant virus emergence through ecosystem simplification, introduction of new host species, and reduction in crop genetic diversity. Therefore, it is crucial to better understand virus distributions across cultivated and uncultivated communities in agro-ecological interfaces, as well as virus exchange among them. Here, we advance fundamental understanding in this area by characterizing the virome of three co-occurring replicated IMPORTANCE Because viruses have been mostly studied in cultivated plants, little is known about virus diversity and ecology in less-managed vegetation or about the influence of human management and agriculture on virome composition. Poaceae (grass family)-dominated communities provide invaluable opportunities to examine these ecological issues, as they are distributed worldwide across agro-ecological gradients, are essential for food security and conservation, and can be infected by numerous viruses. Here, we used multiple levels of analysis that considered plant communities, individual plants, virus species, and haplotypes to broaden understanding of the Poaceae virome and to evaluate host-parasite richness relationships within agro-ecological landscapes in our study area. We emphasized the influence of grass diversity and land use on the composition of viral communities and their life history strategies, and we demonstrated the complexity of plant-virus interactions in less-managed grass communities, such as the higher virus prevalence and overrepresentation of mixed virus infection compared to theoretical predictions. Viruses are among the smallest but potentially most powerful biological entities, and they significantly influence the functioning of plant communities . While t9–Poaceae (grass family) community types that represent a gradient of grass species richness and management intensity, from highly managed crop monocultures to little-managed, species-rich grasslands. The Poaceae virome is one of the most important to understand globally, because collectively, humanity depends on Poaceae species, directly or indirectly, for two-thirds of its caloric consumption, making this plant family essential for world food security  where possible. Second, we aimed to evaluate the relationship between host plant richness and virus richness, as evident among these plant communities, and its temporal stability over 2 years. Third, we investigated whether the species composition of the plant community viromes was more strongly associated with plant community type  or site location. Finally, we examined three individual virus species in detail to determine their prevalence and spatial distributions within communities, and we investigated the genetic diversity of haplotypes of one latent virus within and between communities. We expected to find high virus prevalence and more severe symptoms in crop fields and lower virus prevalence with fewer symptoms and high rates of coinfection in the less-managed communities. Combining different analysis levels , we presented a broader view of plant-virus interactions at agro-ecological landscapes. This is of particular interest as most host-parasite richness relationships have so far been considered for a limited number of plant viruses and hosts (–High-throughput sequencing (HTS) is well-suited for characterizing plant viruses, both known and unknown . Whether viromes . Our firnd hosts , 28. In nd hosts  or used nd hosts –31.Poaceae-dominated communities that represented a gradient of plant species richness and management intensity . We studied these community types across 2 years (2017 and 2018) at three replicated sites 10 km apart  within the Belgian Natural Park Burdinale-Mehaigne  and used a viral metagenomic approach (virion-associated nucleic acids [VANA]) on pools of 50 samples per community (Table S2). Libraries contained on average 17.9% of viral reads (ranging from 0% to 78% [Table S3]). A low level of cross-sample contamination was observed, as the alien target sequences  detected in the Poaceae samples represented on average 0.01% of the total reads in the libraries. This number was used as the detection threshold for the present study, meaning that no viral detection below this level was considered. Fifty-one consensus plant virus genomes were assembled , covering for 47 of all known open reading frames (ORFs) described in the corresponding genera or families, thus permitting their assignment to species. We also identified four incomplete virus genomes  that represented putative novel species belonging to Closterovirus, Rymovirus, Varicosavirus, and Amalgavirus genera . These 51 RNA viruses were assigned to 16 virus families representing 21 virus genera  corresponding to putative novel virus species . Very few or no virus species were found in cereal fields, while a diversified virome was observed in more-diverse communities, with up to 22 virus species detected in grasslands . However, at the species level, grasslands were marginally more diverse than pastures . Linear regression models were used to show the relationship between plant species richness and virus family richness , virus genera richness , and virus species richness   .Poaceae communities were visualized using a network analysis of the virus species identified within each grass community  and some nonpersistent (or acute) viruses . In addition to this shared virome, virus species (mostly nonpersistent viruses) were detected in specific communities, for example, brome mosaic virus in wheat crop, ryegrass mosaic virus in pastures, and cocksfoot mottle virus in grasslands. Interestingly, the network analysis showed association between Poaceae communities and virus biology. Pasture viromes were dominated by nonpersistent viruses , while grasslands were more associated with persistent viruses and mycoviruses   . This paommunity .Poaceae virome composition in plant pools, we analyzed virus prevalence, coinfection, and distribution within the plots. For that, we performed virus detection in individual plants from the Antheit site in 2018 for three viruses detected by HTS: barley yellow dwarf virus-PAV  and two novel secovirids (Poaceae Liege nepovirus A [PoLNVA] and Poaceae Liege virus 1 [PoLV1]), which were recently characterized  were analyzed. Infirming our hypotheses, we observed high virus prevalence with an absence of visible symptoms in less-managed communities . The PoLNVA nepovirus was highly prevalent in both pastures and grasslands and very frequently detected within the host species L. perenne and P. trivialis (maximum of 88% found in L. perenne in pasture). In contrast, BYDV-PAV and PoLV1 had divergent prevalence patterns across plant community types and species  was distributed throughout the plots, with just a few clusters of noninfected plants , except for the P. trivialis pasture with a random distribution of noninfected spots . In contrast, virus infections transmitted by insects  were more clustered . Virus distributions were also compared to each other, and contrasted associations were shown between insect-transmitted viruses according to the plant species: mostly positive association for P. trivialis  and negative association for L. perenne  (Table S5).Virus spatial patterns were analyzed for lustered . SpatialLolavirus, family Alphaflexiviridae), which made it interesting to examine the virus genetic structure among communities and sites. This analysis was performed in L. perenne, as this species seemed to be the major host for LLV . Preliminary results of BLASTn searches revealed a high level of variability of LLV contigs that shared 70% to 99% nucleotide identity with LLV isolates in the NCBI GenBank database. Calculating the pairwise identity shared by the RdRp region of LLV isolates detected in this study further confirmed their genetic diversity. Hence, the LLV RdRp pairwise identity matrix (Table S6) presented a series of clusters and subclusters with 75 to 99% nucleotide identity between LLV RdRp sequences recovered from this study. Phylogenetic analyses performed on LLV RdRp sequences  and for PLNA, respectively, in Antheit pasture between both sampling years. However, more variability was found for Lolium latent virus   . Note thPoaceae communities, with at least 51 viruses belonging to 21 genera and 16 families . The overrepresentation of mixed infection compared to the predicted coinfection rate, as observed in Poa trivialis, suggested that the viruses are not circulating independently and some factors such as cotransmission, assistance, or attraction of vectors could have an effect. Results in individual plants, all asymptomatic, confirmed that virus infection is very frequent in nature and generally unapparent  may have also impacted virus distribution within the plot , as illuAlphachrysovirus, Partitivirus, Totivirus, and Victorivirus genera). Indeed, ICTV genome-based species demarcation criteria differ strongly among taxa: from 80 to 90% amino acid sequence identity for partitiviruses to 53 to 70% for alphachrysoviruses, 60% for victoriviruses, and even 50% for totiviruses. Biological criteria such as the host species are also considered (Saccharomyces cerevisiae virus L-A and Saccharomyces cerevisiae virus L-BC in the 717-amino-acid (aa) region of highest similarity) (Totivirus species identified here and in other metagenomics studies (Totiviridae family and present a demarcation threshold of 60% aa identity).Besides its implications from an ecological perspective, the identification of novel plant viruses down to species level also contributes to discussions on the species demarcation criteria proposed by ICTV, in particular for persistent viruses and mycoviruses (nsidered . In the ilarity) . However studies , 50 presPoaceae communities, illustrating the influence of plant diversity and land use on the composition of viral communities. The present study represents an avenue for future research in virus ecology, in particular, for virus vectors  in virus metagenomics studies to analyze spillover and spill-back between wild and agricultural reservoirs . This could provide a broader overview of the virus communities present in these mixed species communities and aid in understanding the ecology of plant viruses across agro-ecological interfaces, a domain still in its infancy.In summary, this large-scale study revealed a diversified and largely unknown virome in cultivated and noncultivated plant communities, with high viral prevalences and coinfection observed in less-managed communities and with the virome richness increasing with grass species richness in the plant communities. The virome network highlighted different virus lifestyles among wild servoirs . In addiPoaceae-dominated communities that represent a gradient of plant species richness and management intensity: (i) cereal crops ; (ii) pastures grazed throughout the year by cattle , and (iii) naturalistic grasslands managed only by annual mowing . We studied these community types (Hordeum vulgare subsp. vulgare L. and Hordeum vulgare subsp. hexastichum (L.) Celak, respectively] and winter wheat (Triticum aestivum L.). The crop fields were cultivated conventionally  at Héron and Latinne and with organic practices (organic fertilization and no pesticides) in Antheit. In contrast, the grazed pastures were sown a century ago and contain four dominant grass species, all of which are cool-season C3 perennials native to Eurasia: Agrostis capillaris L., Dactylis glomerata L., Lolium perenne L., and Poa trivialis L. Lastly, the grasslands were communities with high conservation value, containing up to 14 Poaceae species and additional forbs. These grasslands had been mowed once a year in mid-July for the past 15 to 30 years (see Table S1), except in Héron, which was mowed in mid-May in 2017.We characterized the virome of three co-occurring ty types  across 2Poaceae communities, we surveyed them twice: from mid-May to mid-June of 2017 and 2018 . In 2018, we sampled an additional six-row barley field (AF-3) adjacent to the pasture plot in Antheit, because it presented interesting symptoms of fungal infection. Thus, there were 19 surveys in total. For each survey, we randomly placed 50 quadrats (1 m by 1 m) in the target area (1.1 to 2.7 ha). Within each quadrat, we inventoried all Poaceae species and noted any viral or fungal symptoms. We harvested one randomly chosen individual (one stem with leaves) of each species identified within that specific quadrat. Harvested plant material was kept cool on ice freeze packs in the field and then stored at −80°C later that day.To capture the virome of these cool-season Poaceae species encountered in that survey. For instance, 20% of the grasses within the 2017 Antheit Grassland survey were L. perenne (47/232 plants sampled) (Table S1), so this species was represented in the 50-sample pool by 10 plants. Non-Poaceae species were not surveyed.To compare the virome captured in each survey, we prepared 19 corresponding pools of 50 individual plants each, based on botanical inventory of the 50 quadrats visited in each survey (Table S2). Each pool reflected the relative abundance of the 2HPO4, 0.07 g glucose, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4, 4.2 mM NaHCO3), using a tissue homogenizer. The homogenized plant extracts were centrifuged at 3,200 × g for 5 min. Supernatants were further centrifuged at 8,200 × g for 3 min. Resulting supernatants were then filtered through a 0.45-μm sterile syringe filter, and 25 mL of supernatant was put into an ultracentrifuge tube. Then, a sucrose cushion, made of 3 mL of 30% sucrose in 0.2 M potassium phosphate (pH 7.0), was added. Extracts were then centrifuged at 148,000 × g for 2.5 h at 4°C using an SW 28 Ti rotor (Beckman). The resulting pellet was resuspended overnight at 4°C in 1.5 mL of HBSS. Unencapsidated nucleic acids were eliminated by adding 15 U of bovine pancreas DNase I (Euromedex) and 1.9 U of bovine pancreas RNase A  to 200 μL of resuspension. Samples were incubated at 37°C for 90 min. VANA were then extracted from 200 μL of resuspended virions by using a PureLink viral RNA/DNA minikit  following the manufacturer’s protocol.Both DNA and RNA viruses were targeted in this study. The 19 pools of plants were prepared by virus-like particle (VLP) enrichment, followed by the extraction and sequencing of VANA, as described previously . To obtaReverse transcription, Klenow fragment treatment, and amplification of the viral DNA and RNA using barcoded PCR primers were performed according to a protocol described elsewhere . AmplifiPolerovirus, RNA virus) and banana streak virus  were selected. The presence of reads from alien viruses in the samples correspond to cross-contamination between samples and can support the establishment of a threshold below which presence of a virus is considered background cross-contamination.To control proper extraction of RNA and DNA viruses , a series of blank (water) and alien controls were used in each batch of sequenced samples. An alien control, which has been recommended in recent guidelines , correspde novo assembled into contigs  (−20 cutoff) were isolated for further analyses using Geneious Prime 2019.2.1. In order to obtain complete or nearly complete virus genomes, we assembled the virus contigs and then mapped the reads on these contigs . Novel virus species were further tentatively identified following host and genomic ICTV species demarcation criteria for each virus family (host range and nucleotide or protein identity percentage on RdRp and coat protein). We putatively attributed the host (plants or fungi) of the novel viruses according to the known host range of the related virus family or genus and the closest virus hit after the BLAST analysis. In order to obtain robust results, we performed the analyses  on complete (or nearly complete) virus genomes, excluding the small contigs (less than 500 nt) that did not allow formation of complete virus sequences.After quality check, raw reads were demultiplexed and cleaned according to their MID linkers with Cutadapt . Then, rf 35 nt)  on the Df 35 nt)  and NCBILolium latent virus (LLV) over years, communities, and sites. The abundance of this virus in almost all the pools, including Lolium samples  allowed us to analyze the LLV intraspecific genetic structure, phylogeny, and variants among and within plant communities. As the complete genome of LLV was not obtained for all samples, the analysis focused on the RdRp gene, which was better covered . After obtaining de novo LLV contigs for each sample, a multiple nucleotide alignment of the 31 contigs was performed with MUSCLE . The aim of this analysis was to visualize how virus species were distributed among the communities and to determine any association between virus taxa and plants, e.g., comparing persistent and nonpersistent (acute) virus lifestyles, according to definitions reported elsewhere (https://software.broadinstitute.org/morpheus), recursively merging objects  based on their pairwise distance.Viromes at the family, genus, and species levels were inventoried from the 19 pools. Statistical analyses were performed using Minitab19 to determine whether virome richness levels  were significantly different between plant communities and anthropogenic management methods. Population structure was analyzed through the distribution normality and variance homogeneity. As the two hypotheses were not fulfilled for all the conditions, we used the Kruskal-Wallis test to analyze all communities and the Mann-Whitney test to compare two community categories . Both tests were performed with a 95% confidence level. In addition, network analyses of the virome composition  in the different lsewhere  (see TabTaq enzyme kit (Bioline) in a 25-μL reaction mix composed of 2.5 μL of cDNA, 12.5 μL of RNase-free water, 5 μL of 5× PCR buffer, 0.5 μL of dNTP mix (10 mM), 1 μL of each primer (20 μM), and 0.5 μL of Mango Taq enzyme (5 U/μL). Primers (listed in Table S8) were previously designed for each targeted virus  from the HTS data using Geneious Prime 2019.2.1 software. Thermal cycling corresponded to 94°C for 4 min, 35 cycles at 94°C for 45 s, Ta for 1 min, and 72°C for 30 s, and a final at 72°C extension for 10 min. PCR products were analyzed by electrophoresis on a 1% agarose gel in Tris-acetate-EDTA buffer, stained with GelRed nucleic acid gel stain (Biotium), and visualized under UV light.Total RNA was extracted according to methods described previously . ReverseL. perenne and P. trivialis were analyzed with the SADIE technique using SADIEShell version 2.0 and N_AShell version 1.0 . This approach is used to study spatial patterns in count-based data where location is known and to test the statistical significance  that the counts are arranged randomly with respect to one another    . Clusterctively) . For BYDPRJNA882095. All data related to virus genome assembly and identification, and their NCBI GenBank accession numbers (OL330737 to OL330775 and ON137708 to ON137726) are listed in Table S4. All other data are provided in the supplemental material.Raw sequencing reads were submitted in the NCBI Sequence Read Archive, with the BioProject accession number


Begomovirus, family Geminiviridae) in association with a disease-specific betasatellite. However, another satellite-like molecule, alphasatellite, is occasionally found associated with this disease complex. A quantitative real-time PCR assay for the virus/satellite components causing CLCuD was used to investigate the performance of selected cotton varieties in the 2014–2015 National Coordinated Varietal Trials (NCVT) in Pakistan. The DNA levels of virus and satellites in cotton plants were determined for five cotton varieties across three geographic locations and compared with seed cotton yield (SCY) as a measure of the plant performance. The highest virus titer was detected in B-10 (0.972 ng·µg−1) from Vehari and the lowest in B-3 (0.006 ng·µg−1) from Faisalabad. Likewise, the highest alphasatellite titer was found in B-1 (0.055 ng·µg−1) from Vehari and the lowest in B-1 and B-2 (0.001 ng·µg−1) from Faisalabad. The highest betasatellite titer was found in B-23 (1.156 ng·µg−1) from Faisalabad and the lowest in B-12 (0.072 ng·µg−1) from Multan. Virus/satellite DNA levels, symptoms, and SCY were found to be highly variable between the varieties and between the locations. Nevertheless, statistical analysis of the results suggested that betasatellite DNA levels, rather than virus or alphasatellite DNA levels, were the important variable in plant performance, having an inverse relationship with SCY (−0.447). This quantitative assay will be useful in breeding programs for development of virus resistant plants and varietal trials, such as the NCVT, to select suitable varieties of cotton with mild (preferably no) symptoms and low (preferably no) virus/satellite. At present, no such molecular techniques are used in resistance breeding programs or varietal trials in Pakistan.Cotton leaf curl disease (CLCuD) is a significant constraint to the economies of Pakistan and India. The disease is caused by different begomoviruses (genus  Gossypium spp.) is the world’s most important fiber-producing plant, providing fiber for the textile industry as well as a significant portion of animal feed and edible oil. In terms of production, Pakistan ranks among the top ten cotton-producing countries. Cotton is grown on 2.96 million hectares in Pakistan, with an average annual lint yield of 670 kg ha−1 [Begomovirus (family Geminiviridae) and is transmitted by an insect vector, Bemisia tabaci (whitefly). It is frequently associated with a disease-specific DNA-satellite molecule, cotton leaf curl Multan betasatellite (CLCuMuB) [cotton leaf curl Multan alphasatellite (CLCuMuA) is occasionally found associated with CLCuD [Cotton (CLCuMuB) . Nonetheth CLCuD . The preCotton leaf curl Kokhran virus (CLCuKoV) and Cotton leaf curl Multan virus (CLCuMuV) associated with CLCuMuB [Cotton leaf curl Kokhran virus Burewala strain (CLCuKoV-Bur), previously known as Cotton leaf curl Burewala virus (CLCuBuV), and was associated with a recombinant betasatellite, termed as Cotton leaf curl Multan Betasatellite Burewala strain (CLCuMuBBur) [Bur) complex infects all the previously resistance and tolerant cotton varieties in Pakistan and India [Since the first discovery of CLCuD-associated begomovirus in 1960, southern Asia has experienced four stages of CLCuD progression: pre-epidemic, epidemic, resistance breaking, and post-resistance breaking. The pre-epidemic phase of CLCuD was first experienced in 1967 near the city of Multan, Pakistan, but remained a sporadic problem affecting only a few plants . In 1988 CLCuMuB . HoweveruMuBBur) . This rend India ,12,13. Cnd India  and Indind India . The onsnd India , and connd India . Tolecusatellitidae (genus Betasatellite), and it has a small genome of 1350 nt with a conserved region and adenine-rich region. The indispensability of CLCuMuB highlights its importance and pivotal role. CLCuMuB encodes a multifunctional βC1 protein that mediates all the functions of a betasatellite. The βC1 protein has a major role in virulence and symptoms determination as well as manipulate the host cellular functions such as autophagy, ubiquitination, and suppression of gene silencing to support the CLCuD infection [Alphasatellitidae [AlphaRep); Although, alphasatellites are self-replicating molecules and helper begomovirus mediates their encapsidation and movement. Alphasatellites have not yet been assigned a specific function, and neither the development of symptoms of infection nor the manifestation of disease is dependent on them. Alphasatellite, on the contrary, reduces symptoms in plants infected with begomovirus-betasatellite complex by regulating the virus and/or betasatellite DNA titers [AlphaRep of some alphasatellites can, however, suppress the host defense at the post-transcriptional gene silencing level [It is worth noting that, aside from the sporadic identification of other CLCuD-associated begomoviruses and a mastrevirus from cotton throughout all epidemics, only a single species of betasatellite, CLCuMuB, was found associated with all the CLCuD-associated begomoviruses. The CLCuMuB belongs to a newly established family, nfection ,19. On tnfection  that belllitidae . AlphasaA titers ,22. The ng level ,24.Bur complex in a large collection of available germplasm have turned susceptible [Bur complex. A more concerted effort is being made to look for resistant cotton because local cotton varieties in Pakistan are extremely susceptible to CLCuD. However, a few promising cotton lines are under investigation [Several lines that were initially identified as resistant to the CLCuKoV-Bur/CLCuMuBceptible . Currenttigation . In PakiCotton yield is quantified by seed cotton yield (SCY), lint yield, lint percentage, boll number per plant, boll weight, lint index, and seed index. The most important of these are SCY and lint yield; the former is the total weight of seed and fiber, which reflects the potential productivity contributing to cotton fiber, whereas the latter is only the weight of fiber that is directly related to the textile industry . SCY wasThis study was aimed to envision the potential of qPCR to assess the titers of CLCuD-associated begomovirus and their effect on SCY. The results demonstrated that qPCR is a useful technique for assessing the virus/satellite DNA levels and revealed a significant negative correlation between betasatellite titer and SCY. From the ten NCVT sites, three were chosen: the Central Cotton Research Institute (CCRI) in Multan, the Nuclear Institute for Agriculture and Biology (NIAB) in Faisalabad, and the Cotton Research Station (CRS) in Vehari . Across all three sampling sites, the five selected varieties showed symptoms characteristic of CLCuD, indicating that no variety was immune . A wide The standard curves for virus and satellites were obtained through linear regression analysis of threshold cycles (Ct) values plotted against the input DNA amount. The results showed a linear relationship between the Ct and the amount of input DNA over six log units . In addi−1) from Vehari, followed by B-2 (0.876 ng·µg−1) and B-1 (0.528 ng·µg−1) from Multan. The least virus titer was found in B-3 (0.006 ng·µg−1) from Faisalabad (−1), followed by B-10 (0.052 ng·µg−1), and B-2 (0.045 ng·µg−1), and all were from Vehari. The samples collected from Faisalabad had the lowest alphasatellite titer, with the least alphasatellite titer (0.001 ng·µg−1) in B-1 and B-2. The betasatellite titer was overall low in Multan relative to Faisalabad and Vehari (−1) from Faisalabad, while the betasatellite titer in B-1 and B-2 from Faisalabad and Vehari was the same (0.959 ng·µg−1). B-12 from Multan had the lowest betasatellite titer (0.072 ng·µg−1)  . p ≤ 0.05) correlation with betasatellite titer, alphasatellite titer, and SCY. Alphasatellite titer showed a non-significant, negative correlation with SCY (p ≤ 0.05), whereas begomovirus and betasatellite titers showed a non-significant positive correlation. Notably, betasatellite titer showed a significant and negative correlation with SCY. Correlation between the DNA levels of the three components, virus, beta-, and alphasatellite, of the CLCuD complex and SCY showed a highly varying pattern at a significance level of 5% . The begPath coefficient analysis revealed that virus titer had a positive effect on SCY and a negative effect on symptom severity, but in conjunction with betasatellite titer, it had a negative effect on both SCY and symptom severity . BegomovThe NCVT is a yearly series of trials designed to identify the best cotton varieties for cultivation in various agro-ecological zones of Pakistan. In this program, a dozen cotton varieties developed by federal, provincial, and private organizations are tested across at least twenty sites. Due to the scarcity of consumables, it was impossible to conduct a comprehensive analysis of all varieties across all sites. Instead, three sites and ten cotton varieties were selected to assess the possible use of qPCR to provide a quantitative measure of the susceptibility of a cotton variety to the virus, beta- and alphasatellite causing CLCuD.Here, SCY was used to assess varietal performance. Clearly, cotton lint yield would have been a better indicator of varietal performance. Cotton lint yield and quality are the most important indicators of crop price. Unfortunately, the NCVT does not measure the lint yield for each variety at each location. Rather, lint yield for each variety is measured across the entire trial. SCY was the only available measure for each variety at each location, so this had to be used here as the measure of plant performance. It is unclear whether this choice has any effect on the results, but it will be interesting, in the future, to decipher the relationship between virus/satellite DNA levels and lint yield.Considerable variability was found in both SCY and virus/satellite titer in the study shown here. The variability of the SCY between the varieties in a specific location may be due to their different genetic makeup, so they have differing levels of resistance/tolerance to the CLCuD complex. The variability of the SCY for one variety at different locations may be due to environmental conditions. It may also be the case that different locations have different “strains/variants” of the begomovirus/betasatellite/alphasatellite, yielding a different intensity of infection. There may also be some abiotic stresses, such as water stress which can also cause variability in SCY as well as other biotic stresses which may differ between the locations. It is precisely for this reason that the NCVT are conducted at multiple locations.The results obtained here suggest that it is the betasatellite level that has the greatest effect on cotton performance rather than virus or alphasatellite titer, or even virus and betasatellite titer. This may not be surprising, even though the betasatellite depends entirely on the helper begomovirus for its encapsidation, replication, and movement . BetasatAlphasatellite titer, either alone or in combination with begomovirus titer, has a negative effect on both symptom severity and SCY. Although alphasatellites are not required for geminivirus infection, the advantage of having one is subtle. Despite this, the presence of alphasatellites suggests that these molecules confer a significant selective advantage. The selective advantage of having an alphasatellite may be the ability to modulate symptoms to appeal insect vector behavior and performance, allowing swift viral propagation ,45. FurtIt is evident that the performance of cotton under varying environmental conditions and CLCuD pressure is complex, hence the need for trials such as those conducted under the NCVT. Nevertheless, the qPCR method outlined here would provide an additional character, other than symptoms, to determine/quantify cotton response to the viruses causing the disease. This can be useful in the decision-making process on which cotton varieties to carry forward in the development pipeline for commercially acceptable, high yielding varieties. Put simply, in the absence of immunity it is desirable for a variety to show mild symptoms (preferably no symptoms) and to harbor a low virus titer (preferably no virus), noting that the results obtained here suggest that it is betasatellite titer that is important rather than virus. So, the present NCVT trials monitor disease severity and yield parameters. qPCR would provide an additional, important parameter: virus/satellite titer. The authorities conducting the NCVT, and similar trials of cotton varieties, should provide serious consideration to including a qPCR element in the evidence for deciding which varieties to recommend farmers to grow. The qPCR assay demonstrated here has successfully been implemented to quantify the titers of different begomoviruses and their associated DNA satellites in pumpkin plants , sunflowIn this study, only one attribute, SCY, was investigated to measure cotton yield. Nonetheless, other attributes such as lint yield, total biomass, lint percentage, and boll number per plant, can formulate a broader perspective and can be included in futuristic studies. Betasatellite performs several key functions and βC1 induces virus-like symptoms , which uTen leaf samples from ten different plants of the same variety were collected from three of the ten sites of the 2014–2015 NCVT trials; Nuclear Institute for Agriculture and Biology (NIAB) Faisalabad , the Central Cotton Research Institute (CCRI) Multan , and the Cotton Research Station (CRS) Vehari . The inclusion of the five cotton varieties  in the study was based on their susceptibility to CLCuD. SCY data from 100 plants of each variety were collected from three of ten sites of the 2014–2015 NCVT trials. Plants were photographed prior to the leaf collection , and colDNA was extracted from the collected leaves and the control healthy cotton plants (grown in greenhouse conditions) by the CTAB method . The resSad1  values Standards for the absolute quantification and condition for qPCR for the identification of CLCuD associated with begomoviruses and their associated satellites were described previously .A total of 25 µL were used for each reaction, of which half was SYBER Green Super Mix . Thermocycler  was used, and the final settings were demonstrated earlier . In qPCRThen qPCR was performed with begomoviruses and their cognate satellites under the same conditions used in . Each tehttp://www.spss.com.hk/statistics/; accessed on 20 January 2017) was used at a significance level of 5%. Path coefficient analysis was performed at a significance level of 5% to assess the correlation of combinations of components of the CLCuD complex SCY by using the software IBM SPSS Amos graphics .To assess the correlation structure between the titers of CLCuD components and SCY, PASW Statistics 18 (formerly SPSS Statistics; SPSS Inc., Chicago, IL, USA (Conclusively, the qPCR assay was found to be a useful tool in examining the response of cotton plants to the virus complex that causes CLCuD, identifying resistant sources against CLCuD in breeding programs, and monitoring the spread and population of CLCuD-associated viruses in epidemiological surveys. As the titers of begomovirus and alphasatellite varied greatly, no relationship was found between the titers of begomovirus and alphasatellite to SCY was concluded. Nonetheless, a statistically significant and inverse relationship between betasatellite titer and SCY was noted.Differences in yield at different locations could be accredited to differences in environmental conditions and, in the case of viruses and satellites, differences in virus/satellite “strains” between locations.


Culex pipiens (Linnaeus) (87%) and Aedes albopictus (Skuse) (13%). We decided to further analyze the Culex pipiens mosquitoes, due to the more extensive number of samples collected. Our approach was to utilize an unbiased total RNA-seq for pathogen discovery in order to explore the mosquito virome. The viral reads from this analysis were mostly aligned to Insect-specific viruses from two main families, the Iflaviridae; a positive-stranded RNA virus and the Rhabdoviridae; a negative- and single-stranded RNA virus. Our pathogen discovery analysis revealed viral reads aligning to the Merida-like virus Turkey (MERDLVT) strain among the Rhabdoviridae. To further validate this result, we conducted a BLAST sequence comparison analysis of our samples with the MERDLVT strain. Our positive samples aligned to the MERDLVT strain with 96–100% sequence identity and 99.7–100% sequence coverage. A bootstrapped maximum-likelihood phylogenetic tree was used to evaluate the evolutionary relationships among these positive pooled specimens with the (MERDLVT) strain. The Georgia samples clustered most closely with two strains from Turkey, the Merida-like virus KE-2017a isolate 139-1-21 and the Merida-like virus Turkey isolate P431. Collectively, these results show the presence of the MERDLVT strain in Georgia.Arbovirus surveillance is fundamental for the discovery of novel viruses and prevention of febrile vector-borne illnesses. Vector-borne pathogens can rapidly expand and adapt in new geographic and environmental conditions. In this study, metagenomic surveillance was conducted to identify novel viruses in the Country of Georgia. A total of 521 mosquitoes were captured near a military training facility and pooled from species  Flaviviridae family. Flaviviruses are positive-stranded RNA viruses, that are recognized to have the capacity to globally infect and cause a spectrum of several diseases  are one of the main causes of human infections worldwide . The geodiseases . This fadiseases . Anothertebrates . Two weltebrates . This coIflaviridae family; a positive-stranded RNA virus . ISVs can naturally infect and replicate in arthropod hosts. An example of ISVs is the Culex Iflavi-like virus 4 which belongs to the NA virus . In receNA virus . In addiNA virus . ISVs isNA virus .Culex pipiens mosquitoes collected in various sites of a Georgia military facility. Metagenomic analysis was executed using the Chan Zuckerberg ID (CZ ID) cloud-based pipeline; results were further evaluated using phlyogenetics to determine the likely evolutionary origin of our samples. The Merida virus is classified as a rhabdovirus, a single-stranded, negative RNA virus that was originally discovered in the Yucatan Peninsula of Mexico , CDC light trap , Stealth Trap , and Fay-Prince Trap . Collection traps were placed on a tree that was either next to a pond or a pile of tires. Larval dipping collection focused on small water filled containers and larger water pools in order to collect immature mosquitoes. The larval mosquitoes were then allowed to mature in sealed containers then collected as adults. Mosquito collections were conducted between August 2018 and June 2019, in the town of Senaki, located in the Samegrelo-Zemo Svaneti region of Georgia. Mosquitoes were morphologically identified using a stereomicroscope  and the ECDC MosKey Tool.Mosquitos were homogenized by bead-beating with the Bio Spec Mini-Bead Beater 16 . The mosquitos’ homogenates were centrifuged, and the supernatant was treated with DNase I, Benzonase nuclease and RNase A as described in ™ Pico Green™ dsDNA Assay (Thermo Fisher Scientific). Library prep was made using the DNA Prep library prep kit  (Product # 20060059). Libraries were quantitated using Tape Station, D5000 Screen Tape , pooled at equal molar concentrations, and sequenced using Illumina MiSeq system and Sequencing Reagent Kit v3 (600-cycle) (Product # MS-102-3003).Random reverse transcription and PCR amplification (RT-PCR) was conducted on the purified nucleic acid as described by CZID.org  was used as a reference to map sequence reads on Geneious prime. The resulting sequences were then aligned to remove sequencing artifacts followed by a BLAST to show sequence similarity and identity. We then identified the individual ORFs using BLASTp for our annotation.RASTtk  with theDrosophila melanogaster sigmavirus AP30, Wuhan Louse Fly Virus 9 strain, Iriri virus nucleoprotein, European bat 1 lyssavirus, Australian bat lyssavirus, Rabies virus, Bole Tick Virus 2 strain, Culex tritaeniorhynchus rhabdovirus, Culex rhabdovirus strain CRV , Merida virus isolate XY14959 nucleoprotein, Merida virus isolate CC_H, Culex rhabdovirus strain CRV , Merida virus isolate MERDV-2020, Merida virus isolate CMS002, Merida virus isolate MERD-Mex07, Merida virus OTU4, Merida-like virus KE-2017a isolate, and Merida-like virus isolate P431. The sequences were aligned using MAFFT version v7.475 using the high-speed setting (We compared 10 whole genome Merida-like virus (Georgia) sequences with 30 publicly available Merida-like viruses sequences, including Hirame rhabdovirus, Infectious hematopoietic necrosis virus, Zahedan rhabdovirus, Long Island tick rhabdovirus, Moussa virus, Arborteum virus, Puerto Almendras virus, Bovine ephemeral fever virus, Kimberley virus, Coastal Plain virus, Tibrogargan virus, Shayang Fly Virus 2 strain,  setting . This al setting . The resCulex pipiens (87%) and Aedes albopictus (13%)  . A totalus (13%) . Due to To explore the viral diversity of the town of Senaki, we conducted an unbiased total RNA-seq approach to identify viruses in this specific area. This methodology was previously described and published by us . A totalIflaviridae; a positive-stranded RNA virus and the Rhabdoviridae; a single-stranded, negative RNA virus. We found the Merida-like virus KE-2017a in most of the collected pooled samples from 2018  can be found at: JP-B: Formal analysis, Methodology, Writing – original draft, Writing – review & editing, Conceptualization, Investigation, Visualization. AP: Formal analysis, Methodology, Writing – original draft, Writing – review & editing, Conceptualization, Investigation. BK: Methodology, Project administration, Writing – review & editing, Investigation, Formal analysis. TC: Methodology, Writing – review & editing, Investigation. CF: Formal analysis, Methodology, Software, Visualization, Writing – review & editing, Validation. MC: Formal analysis, Methodology, Writing – review & editing, Software, Visualization, Validation. DR-W: Formal analysis, Funding acquisition, Methodology, Project administration, Supervision, Writing – review & editing, Investigation, Resources. JH: Formal analysis, Funding acquisition, Methodology, Project administration, Supervision, Writing – review & editing, Investigation, Resources, Validation, Visualization.


In sports psychology, the issue of finding resources to overcome stress remains relevant at present. Currently, the priority is the search for personal resources that can help overcome difficult life situations. Currently, the priority is the search for personal resources that can help overcome difficult life situations. Research by many psychologists  is devoted to this topic. Among the coping resources, the authors single out motivation, locus of control, resilience, self-control, purposefulness, outlook, intelligence, etc.The purpose of the study is the analysis of literary sources regarding the study of the locus of control as a personal coping resource of an athlete.To realize the goal of the work, the following were used: theoretical analysis and generalization of literary sources and Internet data.According to the results of research by domestic scientists, the locus of control and responsibility can act as a coping resource. It was found that the internal locus of control is associated with a low level of anxiety, and the external one with a high one; in addition, an inverse relationship between the locus of control and the level of neuroticism was found . This indicates that respondents with an internal locus of control are more stable in their behavior in situations that provoke anxiety and actualize personal anxiety. Internality allows a person to maintain a sense of control over the situation and his condition. It has been proven that the relationship between the locus of control and the state of anxiety depends on volitional self-control. However, along with this, the phenomenon of “heaviness of responsibility” is known in the scientific literature, which manifests itself in a sharp increase in anxiety with an internal locus of control. This may indicate that internality “loses” its resourcefulness under certain conditions: relationships between internality and anxiety are evident only at a low level of self-control. In the case when a person has developed self-control, such a connection is broken.The generally accepted structure of personal resources, which determine the effective behavior of an individual under conditions of stress, has not yet been formed. Locus of control can act as a coping resource in the structure of self-regulation of an individual.None Declared


During the last 70 years, the bull semen industry has been trying to maximize reproduction efficiency to meet demands. Changes in public attitudes towards the conditions under which domestic animals are kept have led to questions being raised about animal husbandry and its impact on animal welfare. Protocols for bull welfare assessment in artificial insemination centers and how welfare disturbances can reflect on bull productivity have not previously been taken into consideration. Welfare is important for the bull industry because, apart from the known consequences of stress on reproductive parameters and performance, stress can also influence the onset of puberty and cause other health problems. Therefore, it would be useful to have an early indicator of an incipient welfare problem so that countermeasures could be taken in time to prevent such long-term effects on the animals. Different protocols have been developed for specific animal species and production groups/systems based on their biology, husbandry, management, and breeding, and care guidelines formulated. Different housing conditions, poor feeding during rearing and production, as well as poor health status have all been shown to affect bulls negatively and are reflected in sperm quality and animal fertility. ® project. Unfortunately, there is a lack of such summarized information about bull welfare assessment in artificial insemination stations or about how disturbed welfare can be reflected in their productivity. Animal reproduction is the basis for the production of meat and milk; therefore, factors contributing to reduced fertility in bulls are not only indicators of animal welfare but also have implications for human health and the environment. Optimizing the reproductive efficiency of bulls at an early age can help to reduce greenhouse gas emissions. In this review, welfare quality assessment will be evaluated for these production animals using reproduction efficiency as a key area, focusing on stress as a main effect of poor animal welfare and, thereby, reduced fertility. We will address various welfare aspects and possible changes in resources or management to improve outcomes. Animal welfare is a complex subject; as such, it requires a multidimensional approach with the main aim of providing the animals with the “five freedoms”. The violations of any one of these freedoms could have an influence on animal wellbeing on different levels. Over the years, many welfare quality protocols were developed in the EU thanks to the Welfare Quality Modern animal husbandry relies on artificial insemination (AI) as a substitute for natural mating in some species, such as dairy cattle. One of the reasons for the introduction of AI was to control the transmission of diseases between animals. Historically, AI has offered several advantages over natural mating. The most recently advocated one is an increased rate of genetic improvement in intensive animal husbandry . Other hOne aim of holding bulls in AICs (AI bulls) is sperm production. Housing bulls in AICs varies between and within countries, but their objectives are to minimize the spreading of sexually transmitted diseases and to maximize the reproductive efficiency of the animals. Thus, a large number of males of different ages are kept in relatively static conditions at an AIC, without access to females. Compromised welfare can manifest in several ways, depending on the severity of the stressor and its duration of action. For example, long-term impairment of animal welfare can have profound effects on the animal, such as sub-fertility, reduced life expectancy, impaired growth, body damage, disease, immunosuppression, adrenal activity, behavior anomalies, and self-narcotization . One of The concept of AICs is based on keeping male animals isolated from animals of the opposite sex. Due to the fact that breeding bulls in AICs is different than in natural conditions and that the process of semen collection is not normal reproductive behavior, requiring close human–animal interaction, there are many critical points in the semen production process that can cause animals to express inappropriate behavior. On the other hand, inadequate space and insufficient nutrition can cause health problems that further decrease animal quality of life. Therefore, the structure of this review paper is based on the main principles and requirements for welfare standards. The welfare assessment recommendations are based on indicators in the Welfare Quality Assessment Protocol for Cattle , which iThe articles considered in this review were identified using the main search engines. The abstracts of potentially relevant articles were evaluated, and, finally, 80 articles were included and reviewed. The relevant keywords, such as animal welfare, bull welfare, poor welfare, bull reproduction, etc., were first used in the search. In the case of a lack of such literature for this specific production group, we linked it with other production groups, with animals of the opposite sex from the same species, and, eventually, with other species in a similar production group.The application of animal welfare science occurs at three levels of research: (i) fundamental research that provides a basic understanding of animal welfare, (ii) research that develops ways of assessing animal welfare, and (iii) practice-oriented research, the actions that are operationalized in welfare standards or criteria . ApplicaThe requirements in animal welfare standards can be viewed as serving four broad objectives ,10, refl® (WQ) assessment protocols [These objectives were the basis for the welfare principles of the Welfare Qualityrotocols  (WAPs), Since the beginning, the Five Domains Model  was deveSince there is little research conducted on bull welfare, specifically on AI bulls, our work in this review will be mostly based on the Five Freedoms Concept through the WQ principles and criteria. We indicate husbandry conditions for AI bulls and summarize basic knowledge of potential negative effects, mostly on animal-based requirements. However, we found the human–animal interaction in the Five Domains Model  to be anStress appears when environmental demands exceed the regulatory capacity of an organism, particularly when an animal perceives a given situation as unpredictable and uncontrollable . The twoThe physiological response to stress can be described by measures of HPA axis activity. Earlier in stress research, it was assumed that increased HPA activity was a common response to all stressors, both internal and external . HoweverBased on the duration, stress can be acute, which lasts for minutes or days, or chronic, which lasts for weeks or months (and even years) . Blood sAnimals subject to chronic stress generally suffer from metabolic disturbances associated with reduced feed intake, a negative energy balance, an increased metabolic rate, and, subsequently, loss of body weight or reduced growth ,29. On tLiestner and Menke  reviewedWe will now consider the three main types of requirements for animal welfare standards, according to the classification by Blokhius et al. , in the For resource-based requirements, research often tries to identify critical levels or thresholds beyond which animal welfare indicators are affected. Such work has typically found that each production system has its specific welfare challenges and that outcomes differ considerably within the same type of system ,34.There are studies showing that space allowance and type of bedding have an influence on animal production ,36. To o2. For bulls weighing over one ton, the sleeping area should be at least 1 m2 for every 60 kg of live weight. If a bull is not regularly and routinely exercised outside the bull pen, the pen should include an exercise area at least twice as large as the sleeping area. In some countries, tethered housing was previously widely used. However, this system was shown to induce abnormal behavior in young bulls, such as abnormal licking of equipment or leaning against equipment, compared with loose-housed bulls. Sufficient bedding and/or rubber matting for stall flooring helps to prevent and/or minimize foot, leg, back, and spinal column discomfort [There are different housing systems for bulls in AICs, but currently, bulls are frequently kept in individual pens with access to outdoor space. The UK codes of recommendations for the welfare of cattle livestock , give spscomfort .Since the WQ protocol for dairy cattle has no developed measure of thermal comfort, we will introduce sperm quality evaluation as a potential method based on extensive research conducted on thermal discomfort and sperm quality results. Bos taurus and Bos indicus) were noted. Bos taurus bulls are more sensitive to high ambient temperatures than Bos indicus bulls. Even the semen quality parameters of crossbred bulls (Bos taurus × Bos indicus) recover more rapidly after heat stress than for Bos taurus bulls. Morrell [A high testicular temperature has an effect on sperm quality; an increased testicular temperature leads to increased production of defective spermatozoa. Physiological mechanisms to avoid an increase in testis temperature include heat loss from the scrotum via scrotal sweating, heat exchange in the testicular vascular cone, relaxation of scrotal muscles, and whole-body responses. For the production of fertile spermatozoa, testicular temperatures should be between two and six degrees Celsius lower than the bull body temperature . When th Morrell  indicate Morrell . On the other hand, few studies have been conducted to explore the influence of air pollution on fertility in farm animals. There are systematic reviews that link the impact of air pollution with reproduction in women  and laboAccess to clean drinking water and a diet that is appropriate to a species and can keep them healthy is one of the five freedoms and therefore needs to be assessed to meet animal welfare demands. It is known that stress influences feed intake in animals, but there are few reports on how feeding time and frequency influence animal welfare by causing a stress response. Calamari et al.  conducteRecent studies on the nutritional control of puberty in female cattle demonstrate that maternal nutrition during gestation can induce morphological and functional changes in the hypothalamus system, which can persist long after the birth of female offspring, influencing reproductive performance even in adulthood . FurtherFor many animal-based measures, such as the proportion of lame or injured animals, there are no non-zero values where welfare is not affected . AssessiAbsence of pain is one of the main principles of good animal welfare, as well as absence of illness. There are two groups of markers of farm animal welfare that can be measured and that are health-related. The first group comprises physical indicators including cut injury, body damage, abscess formation, swelling of the joints, and loss of hair or wool. The other group consists of physiological indicators, which include cortisol level, reduced feed intake, immunosuppression, adrenal activity, and altered physiological responses, e.g. depressed reproductive parameters. Health indicators included in the welfare assessment of bulls in AICs have not been described. Physical indicators modified from the protocol for dairy cows  are presThe Office International des Epizooties (OIE) Terrestrial Animal Health Code, which implements improvement standards for animal and public health and animal welfare from a veterinary viewpoint, summarized general considerations and conditions applicable to AICs and to breeding bulls. Instructions for general hygiene in AICs are provided in Chapter 4.5 of the Health Code, although the requirements that bulls should fulfill before entering AICs are based only on potentially pathogenic infectious agents. Furthermore, recommendations are given that would reduce semen contamination with common microorganisms. However, additional details are needed, which could include an assessment of welfare biomarkers, if sufficiently sensitive indicators could be identified. The study by Cojkic et al.  indicateExcellent hoof condition is one of the most important physical aspects of pain-free semen collection. Lameness, whether from sole abscesses, foot rot, interdigital dermatitis, hoof cracks, laminitis, improper hoof trimming, etc., will often result in bulls being reluctant to mount. Furthermore, lame bulls often overcompensate for the foot pain and exert abnormal pressure on other bone and muscle groups, placing the bull at risk for greater injury .Puberty and social and sexual development in bulls are under strong genetic regulation. There are breed differences, as well as age differences, in the beginning of the manifestation of, and changes in, sexual behavior . Young cRearing calves to become bulls in Artificial Insemination Centers (AICs) is quite unlike a situation in the wild or under extensive farm husbandry conditions. In the wild, or under extensive farm husbandry conditions, the social ranking of bulls is based on size and age within the group . In a heThere are certain behaviors that indicate a problem with animal welfare. Some of these include an increased respiratory rate or panting, decreased feed intake, and demonstration of aggressive, depressive, stereotypic, and/or other abdominal behaviors . PhysiolIn the study by Lange et al. , a good The frequency of semen collection is positively correlated with bull reaction time and novelty . ManagemFor management-based requirements, science has been used in two main ways. One is simply to assess how animal welfare is affected by management practices, and the second application is to develop and test practices that improve animal welfare. For example, in the “Welfare Quality” project, a large cooperative research program that developed standards for cattle, pigs, and chickens, the scoring systems rely heavily on animal-based measures such as body condition, lameness, lesions, and agonistic behavior . Such results contribute to a growing recognition that good animal welfare is a complex outcome of different factors including animal genetics, management, and environment. During the last 70 years, efforts in the bull semen industry have been made to maximize reproduction efficiencies to meet demands. For this to be achieved, there is a need for management schemes to be efficient to exploit bulls’ reproductive potential and, at the same time, to minimize the risk of injury associated with it. At present, bulls younger than 15 months old are commonly included in intensive semen collection . HoweverManagement requirements should be evaluated based on welfare principles and criteria. The welfare quality assessment protocol for cattle  summarizAntimicrobial use is one of the management requirements in the process of semen preservation since the antibiotics are used without previous bacterial identification and testing of antimicrobial susceptibility. The sources of bacterial contamination in bull semen are the animals themselves, their environment, and the process of semen collection and preservation. The European Union provides government directives on the use of antibiotics in semen for international trade , aiming The main aim of this review was to summarize the critical points in bull semen production where animal welfare could be compromised. Furthermore, we explained how insufficiently welfare-oriented production criteria lead to stress and, subsequently, decreased reproduction efficiency. Assessment of animal welfare can be challenging due to its complexity and multifactorial nature. Poor animal welfare can be easily missed due to a lack of knowledge of the species and insufficient evaluation. This can be avoided by using appropriate methods for animal welfare assessment. Two primary methods which can be used are (1) objective evaluation by scoring specific criteria and (2) use of subjective judgments and evaluation. There are differences in the scoring systems between different countries ,75, whicThe development of systems to evaluate animal welfare was based on scientific interest as well as public concern about the raising conditions and treatment of animals. The first versions were developed for animals used in research, teaching, and testing (RTT). In subsequent years, questions were raised about other animals and their keeping conditions. Thereafter, hundreds of welfare assessment protocols were developed around the world; all differ from one another based not only on the animal species and production group but also on the place where the animals are kept at the time of evaluation. ® Assessment Protocol dairy cattle (WQ), the U.S. National Dairy Farmers Assuring Responsible Management Program (FARM), and the New Zealand Code of Welfare: Dairy Cattle (The Code). The fourth, upcoming program, the Integrated Diagnostic System Welfare (IDSW), was also discussed. Some of them are voluntary , and some of them are required to be followed by law (The Code). Some are more animal-oriented (WQ), and others include environmental and management-based requirements . Looking at all of them from the bull perspective, just two of them include bull breeding (FARM and The Code), and only one protocol includes reproduction data (IDSW) for the welfare evaluation but is not complete. The FARM program includes bull breeding, based on the fact that all bulls being raised as dairy steers should be castrated, and how the procedure should be performed, with the remainder being on pain management. On the other hand, The Code gives more recommendations about natural service bulls, regarding the number of cows that needs to be served, nutrition, and environmental requirements. Some of them can be implemented for AI bulls, such as the size of the sleeping and exercise area requirement, but the rest cannot, based on the environmental conditions that animals are kept in and the level of social interaction between humans and other animals. Animals experience the same environment in different ways, based on their genetics, early experiences, and temperament [Since AI technology is mainly used in the dairy industry, dairy bulls are usually the cattle kept in AICs. Krueger A et al.  summarizperament . Bulls kept in AICs are unique in many ways; they are mostly dairy breeds with specific metabolic requirements due to the intensive selection for milk production. Furthermore, husbandry conditions for AI bulls differ slightly between countries at present but have huge differences compared with other animals from the same species and between production groups (beef and dairy). In addition, semen collection is not a natural way of expressing reproduction behavior. All of these differences and specifics require detailed knowledge of AI bull physiology, behavior, and husbandry before defining criteria for welfare assessment. It might be possible to create a unique WAP for the specific conditions of AI bulls by analyzing different existing welfare assessment protocols and approaches to welfare assessment. ® designed to determine the welfare status of cattle, pigs, and poultry in animal units. However, creating such an assessment system for other animal types, in our case for bulls in AICs, has its difficulties due to the multidimensional and multidisciplinary approach of animal welfare [Subjective evaluation is difficult to eliminate in total because of the observers´ projection of their own impression. This can be minimized by the personnel responsible for these assessments attending training programs and by establishing criteria that define good welfare customized to the species, sex, age of animals, and production purpose. The whole goal of assessment programs should focus on minimizing the subjective component. One objective assessment system, which has been developed during the past few decades, was Welfare Quality welfare . The focus of all further assessment protocols should be the same, i.e., reducing stress in animals from the beginning but also promoting positive stimulants. The reason why this demand is important for the bull industry is that, apart from the known consequences of stress on reproductive parameters and performance, stress can also influence the onset of puberty . DelayedThere is a lack of information on bull welfare assessment in AICs or on how disturbed welfare can be reflected in bull productivity. Production systems for breeding bulls should be described and evaluated to meet the standards of good animal welfare. More research is needed on welfare throughout an animal´s life, i.e., from when calves are selected to become breeding bulls to the end of the bulls’ productive lives. Furthermore, specific protocols for welfare quality evaluation for this specific production group of animals should be created, focusing on their physiological and behavioral needs, with the aim of achieving optimal productivity.


A proportion of patients with Lyme borreliosis (LB) report long-term persisting signs and symptoms, even after recommended antibiotic treatment, which is termed post-treatment Lyme disease syndrome (PTLDS). Consensus on guidance regarding diagnosis and treatment is currently lacking. Consequently, patients suffer and are left searching for answers, negatively impacting their quality of life and healthcare expenditure. Yet, health economic data on PTLDS remain scarce. The aim of this article is therefore to assess the cost-of-illness related to PTLDS, including the patient perspective.N = 187) with confirmed diagnosis of LB were recruited by a patient organization. Patients completed a self-reported questionnaire on LB-related healthcare utilization, absence from work and unemployment. Unit costs (reference year 2018) were obtained from national databases and published literature. Mean costs and uncertainty intervals were calculated via bootstrapping. Data were extrapolated to the Belgian population. Generalized linear models were used to determine associated covariates with total direct costs and out-of-pocket expenditures.PTLDS patients , of which 49.5% were out-of-pocket expenditures. Mean annual indirect costs amounted to €36 081 (€31 312–40 923). Direct and indirect costs at the population level were estimated at €19.4 and 151.5 million, respectively. A sickness or disability benefit as source of income was associated with higher direct and out-of-pocket costs.The economic burden associated with PTLDS on patients and society is substantial, with patients consuming large amounts of non-reimbursed healthcare resources. Guidance on adequate diagnosis and treatment of PTLDS is needed. Borrelia burgdorferi (Bb) genospecies complex sensu lato, and is transmitted by ticks.,,Lyme borreliosis (LB) is caused by spirochetes of the Bb infection. Clinical manifestations of LB may be both cutaneous and systemic, and can have cardiovascular, neurological and musculoskeletal involvement.,,,Erythema migrans (EM), which manifests as expanding gyrate erythema at the site of a tick bite, is the most common objective manifestation of Few studies have analyzed the economic burden of Lyme disease in Europe.The same Belgian study included few patients with PTLSD and called for more research in this patient population.The study was performed within a cost-of-illness framework.Convenience sampling was applied via the digital newsletter and Facebook page of a non-profit Lyme disease patient association. The digital survey was available in Dutch and French. The study population included individuals of all ages living in Belgium with PTLDS, defined as LB-related persistent signs, with symptoms for more than 1 year after initial symptoms. To be included, individuals with PTLDS had to have a confirmation of (previous) Lyme disease, based on a physician and/or laboratory blood sample testing.Cross-sectional data collection was assimilated using an online self-reporting questionnaire. Self-reporting was chosen to capture all healthcare utilization, even reimbursed care, because of the limited use of administrative data in not well-defined patient populations, such as the PTLDS population at hand. Moreover, we aimed to identify LB-related healthcare utilization data only, which would have been difficult, if not impossible, using administrative claims data.Questions were related to: (i) sociodemographic information , (ii) disease characteristics , (iii) healthcare resource utilization  and (iv) impact on work situation (i.e. employment status and absenteeism). To limit recall bias, data collection on healthcare resource utilization covered a retrospective period of 6 months before the survey was completed, except for hospitalization, which covered a 1-year period. Employment-related questions also covered a 1-year period. The questionnaire was launched mid-September 2018 via Time for Lyme (a patient organization), and remained open until the end of October 2018. A reminder to participate was sent after 5 weeks. At the time of the survey, the patient organization had 260 members, 2510 followers on Facebook and 835 newsletter subscribers.Direct medical costs were calculated by applying unit costs to different types of healthcare resource utilization. Unit costs were obtained from the publicly available Nomenclature of Medical Performances database of the National Institute for Health and Disability Insurance (NIHDI). These costs were related to GPs, medical specialists, paramedics (other than psychologists), imaging and home-based nursing care. This database contains unit costs of all (partly) reimbursable performance that is included in compulsory health insurance.Hospitalization costs were based on the average costs for hospital stays at the all-patient diagnosis related groups (APR-DRG) level. These costs were obtained from the publicly available National Database of Medical Diagnosis, Care and Cost.Costs related to a day hospitalization (without an overnight stay) were obtained from a report on the financing of day hospitalization in Belgium.All unit costs were reported separately by both the patient and a third party . NIHDI and the Drug Repository database stratify between out-of-pocket and reimbursed costs, but hospitalization and day hospitalization costs were not stratified. For the latter, we assumed an out-of-pocket share based on a report of the largest Belgian health insurer.Indirect non-medical costs included productivity losses related to: (i) temporary absence from work during the 1-year reference period before the questionnaire was completed; and (ii) unemployment related to post-treatment Lyme Disease symptoms. The Human Capital Approach was applied to reflect a calculus expressing the full quantification of lost economic productivity, including the consequences of losing employment as a result of the condition. For temporary absence from work, costs were estimated by multiplying the reported number of days of absence from work by an average unit cost of €284/dayAll costs were converted to and reported in Euros based on 2018 values.P-value of ≤0.05 was considered statistically significant. Standard summary statistics were used to describe participant characteristics. Categorical variables were presented as frequencies and percentages. Continuous variables were presented as means with standard deviations or medians with interquartile ranges (IQRs).Data were analyzed with SPSS 27.0 software. A Mean direct medical costs were calculated for each category of healthcare utilization. 95% bias-corrected and accelerated confidence intervals were estimated using a bootstrap procedure with 1000 iterations. Missing data on healthcare utilization costs were interpolated using the mean value for a given category.Generalized linear models with log-link function and gamma distribution (Tweedie distribution for indirect costs), reflecting the positively skewed distribution with high kurtosis and a heavy tail, were applied to investigate potential predictors of four dependent variables: (i) total direct costs, (ii) out-of-pocket costs of patients, (iii) total costs and (iv) indirect costs. Six covariates were included in the four separate models; gender, age, region, education, income type and time to diagnosis (defined as the time between first signs/onset of symptoms and the moment of diagnosis by testing and/or by a physician). The former five covariates were chosen because of the general likelihood that they would have an association with healthcare utilization and related costs. Time to diagnosis was chosen as a covariate because of its importance in patients with PTLDS. Multicollinearity diagnostics were performed, but none was detected. Participants with missing data on one of the covariates were excluded from regression analyses, which did not undermine model fit. Akaike Information Criterion appeared to be better when all six covariates were retained in the model, despite increasing the number of case-wise deletions, when compared to the scenario in which the covariate ‘time to diagnosis’ was excluded. Time to diagnosis accounted for the larger part of missing data.Descriptive statistics were used to show the proportion of reimbursed versus out-of-pocket costs, as well as indirect non-medical costs.,,n = 905) to become an estimate of the number of persistent signs and symptoms cases in Belgium, being 447 per year. This incidence value was multiplied by a mean disease duration of 112.7 months (i.e. time between the first signs and symptoms of occurrence and Lyme diagnosis reported by participants at the time of filling out the questionnaire) resulting in a prevalence of 4199 patients .Numbers on the incidence of PTLDS are not available for Belgium. To extrapolate numbers for the Belgian population as a whole, we used data from the Netherlands,Characteristics of the 187 PTLDS patients who filled out the survey are provided in Total mean annual direct costs per patient amounted to €4618 , of which 49.5% were out-of-pocket costs . Out-of-Mean and median indirect costs amounted to €36 081 (€31 312–40 923) and €34 080 (IQR: €0–68 160), respectively. These costs were driven by the high number of patients  who reported to be unemployed or absent from work for more than 1 year due to Lyme disease.Extrapolation of the obtained results to the Belgian population, with an estimated PTLDS prevalence of 4199 patients, resulted in an estimated total annual disease burden of €19 389 848 for direct costs and €151 503 111 for indirect costs.The regression model with total direct costs as the dependent variable yielded significant results for the covariates age category and income type . PatientThe regression model with out-of-pocket costs as the dependent variable only yielded significant results for income type. Patients receiving sickness/disability benefit incurred 63.0% (15.7–129.6%) higher costs compared to patients receiving an income from work . SimilarThis study examined the direct and indirect costs of PTLDS-confirmed patients in Belgium. Based on an estimated population prevalence of 4199 patients, and direct and indirect costs of €4617 and 36 081 per patient, respectively, total annual societal costs were estimated as €170 892 959. Direct costs were high for out-of-pocket expenditures, largely due to the substantial contribution of both complementary therapies and non-prescribed medication.Our estimation is considerably higher than the recently calculated societal cost of €5.6M in the first year after diagnosis in Belgium.The cost of complementary therapies was previously assessed by van den Wijngaard et al.Another noticeable result was the high mean period of 112.7 months (±9.4 years) from first symptoms to receiving a Lyme diagnosis in our study population. Physicians may not always think of Lyme disease when patients present with mild to more severe symptoms including fatigue, cognitive difficulties and musculoskeletal pain. Such a scenario may result in non-ideal treatment initiation and consequently persistent symptoms. Although the majority of patients return to their pre-morbid health, a subset of patients continue to report so-called subjective but long-term symptoms. It is not easy to diagnose and treat PTLDS at that moment, as universal guidelines for diagnosis and treatment are lacking.Importantly, patients under 40 were found to incur more direct costs compared to patients over 40 and 60. Our data show that this difference could be assigned to differences in reimbursed care-related costs. This might be explained by a shorter time since diagnosis in these younger adults, and hence still more healthcare seeking behaviour in the regular care circuit.,Caution is required when comparing cost-of-illness studies across countries, due to differences in scientific methodology, healthcare structure and social security systems. Our results in Belgium were compared with results in neighbouring countries by calculating the cost per capita (i.e. costs divided by the country’s population in a given year). We found that estimated direct costs were substantially higher in our study (direct cost per capita of 1.69) compared to Germany (0.31),This study had several limitations inherent to retrospective self-reporting over long periods. First, information on the number of PTLDS patients in Belgium was lacking, which was required to extrapolate the results from the individual patient level to population level. Thus, we used data from a study in the NetherlandsFuture research should, among other elements, focus on possibly relevant covariates, such as ethnicity or belief systems to predict Lyme-related direct medical costs and the use of complementary therapies in particular. It is of major importance to gain insight into which patients have a greater likelihood to experience difficulties in navigating the health care system, and to develop tailored answers on their call for medical help and support. In that regard it is also beneficial to analyze patterns of patients’ healthcare seeking behaviour.This study presented the first cost-of-illness analysis on PTLDS patients in Belgium. The results indicate high patient out-of-pocket expenses, especially with respect to unprescribed medication and complementary therapies. This phenomenon was particularly noticeable for patients with an income stemming from sickness or disability benefits. In conclusion, patients, especially those with high disease severity, encounter difficulties in finding appropriate care via conventional medical circuits.Based on our study findings, the main implications are that more awareness should be created among policy makers about the health and economic consequences of PTLDS. There is also an urgent need for guidance on diagnosis and appropriate treatment.The study was performed in accordance with the Declaration of Helsinki and approval to perform the study was obtained from the Ethics Committee of the University Hospital in Ghent (Belgium) [reference 2018/0933]. Informed consent was obtained from all participants. They were informed about the purpose of the study that participation in the study was voluntary, the data analysis was anonymous and they could withdraw from the study at any time.ckad045_Supplementary_DataClick here for additional data file.


Data on the economic burden of RSV-associated illness will inform decisions on the programmatic implementation of maternal vaccines and monoclonal antibodies. We estimated the cost of RSV-associated illness in fine age bands to allow more accurate cost-effectiveness models to account for a limited duration of protection conferred by short- or long-acting interventions.We conducted a costing study at sentinel sites across South Africa to estimate out-of-pocket and indirect costs for RSV-associated mild and severe illness. We collected facility-specific costs for staffing, equipment, services, diagnostic tests, and treatment. Using case-based data we calculated a patient day equivalent (PDE) for RSV-associated hospitalizations or clinic visits; the PDE was multiplied by the number of days of care to provide a case cost to the healthcare system. We estimated the costs in 3-month age intervals in children aged < 1 year and as a single group for children aged 1–4 years. We then applied our data to a modified version of the World Health Organization tool for estimating the mean annual national cost burden, including medically and non-medically attended RSV-associated illness.The estimated mean annual cost of RSV-associated illness in children aged < 5 years was US dollars ($)137,204,393, of which 76%  were healthcare system incurred, 6%  were out-of-pocket expenses and 13%  were indirect costs. Thirty-three percent  of the total cost in children aged < 5 years was in the < 3-month age group, of which 52%  were healthcare system incurred. The costs of non-medically attended cases increased with age from $3,307,218 in the < 3-month age group to $8,603,377 in the 9–11-month age group.Among children < 5 years of age with RSV in South Africa, the highest cost burden was in the youngest infants; therefore, interventions against RSV targeting this age group are important to reduce the health and cost burden of RSV-associated illness. Globally, an estimated 33 million RSV-associated lower respiratory tract infections (LRTI) and 118,000  94,600–149,400) deaths occur annually in children under 5 years of age .Global cost-effectiveness models based on estimates of burden in low- and middle-income countries suggest up to 1.2 million cases, 104 million hospital admissions, and 3000 deaths could be averted annually through maternal RSV vaccination, including a cost saving of 186 million US dollars ($) in healthcare costs [In South Africa the burden of RSV-associated mild and severe illness is substantial. From 2012 to 2016, the mean annual number of RSV-associated severe cases in children aged < 5 years was 96,220  66,470–132,844) with 78,571  severe cases in children aged < 1 year. In the same period the mean number of RSV-associated deaths in children aged < 5 years was 650 (95% CI 475–947) [We aimed to estimate the direct cost of services , direct and indirect costs to the caregiver (out-of-pocket costs and loss of income), and cost burden  associated with RSV-associated mild and severe illness in children aged < 5 years (in 3-month age groups from 0 to 11 months and then in those aged 1–4 years) in South Africa from 2011 through 2016. We also estimate the cost of illness in children who do not access healthcare for the episode .We collected all costs in South Africa Rands (ZAR) and converted to US dollars ($) using the average monthly ZAR to $ exchange in 2014 based on the South African all-items consumer price index . We usedWe conducted a costing study at sentinel surveillance sites  that are part of a national surveillance program for pneumonia to estimate out-of-pocket and indirect costs incurred by caregivers of children with RSV-associated mild (influenza-like illness (ILI)) and severe illness (severe acute respiratory illness (SARI)) from January through December 2014 providing a baseline of costing estimates (and adjusted for consumer price index for other years). The national surveillance program for pneumonia was started in 2009 and runs at sentinel sites across the country. In 2012, we added primary healthcare clinics in the sentinel hospital catchment area. Dedicated surveillance officers enroll patients Monday through Friday and collect demographic, clinical, and treatment data. Nasopharyngeal swabs are collected from patients and tested at the National Institute for Communicable Diseases (NICD) for a number of respiratory viruses, including RSV.We selected the children for the costing study from children enrolled in the surveillance program  based on target numbers of 2 children aged < 1 year and 2 children aged 1–4 years every week for 1 year irrespective of RSV result. These children were sampled systematically; we included the first two identified children meeting the study enrolment criteria each week. Children who were enrolled in the outpatient surveillance program and then subsequently admitted to the hospital within 14 days were excluded from enrolment in the costing study to avoid overlap between mild and severe cases.The case definition for ILI included fever and cough (duration of symptoms < 10 days). We refer to this as mild illness for ease of reading. This definition was used for children attending outpatient clinics. In addition, we used a physician diagnosis of LRTI for severe illness . We used the term severe illness to refer to this group of children. Children and caregivers were enrolled by dedicated surveillance nurses in the pediatrics ward or the outpatient clinic each week, based on the child’s symptoms fitting the case definition for the illness (mild or severe) and the parent’s willingness to consent. Following enrolment, a nasopharyngeal swab was collected for testing at the NICD by real-time polymerase chain reaction (rt-PCR) for RSV. Only data from children who tested positive for RSV were included in the costing analysis .Data for the costing study were collected by structured interview including caregiver out-of-pocket costs for the visit or admission at the time of hospitalization or clinic visit. These included the direct costs. such as transport, food, over-the-counter medication used, and cost of any private practitioner or traditional healer visits prior to study clinic visits or hospitalization. Indirect costs including loss of income were also collected from the caregiver. We applied the national minimum wage estimates to the days of lost income for the caregiver and to days of hospitalization for the unemployed caregiver. Minimum wage during the study period was $1.3 per day , 7.To calculate healthcare provider costs (inpatient and outpatient) we used case-based data to calculate a patient day equivalent (PDE), which was multiplied by the number of days of care (number of days of admission for severe cases and 1 day for mild cases) to provide a case-cost to the healthcare system. All hospitalization and clinic costs were assigned to the healthcare costs as healthcare is free of charge to children aged < 5 years in South Africa. Itemized hospital (and clinic) costs and procedures  were obtained from the National Department of Health (NDoH) and the state price list of the National Health Laboratory Service (NHLS) , 9. A siFor non-medically attended cases (mild and severe) costs outside of public facilities including over-the-counter (OTC) medications, consultation with traditional healers, direct non-medical costs (like transportation to collect medication), and indirect costs (loss of income) were obtained from the HUS (2013) in 4 of 9 provinces and adjusted for consumer price index .To estimate the national economic burden of RSV-associated illness, 2013 to 2015 data were entered into a modified version of the World Health Organization tool for estimating the mean annual national cost burden, this included the healthcare system costs, caregiver costs, and non-medically illness) , 10. We We adjusted the total cost by the gross domestic product (GDP) deflator for South Africa from 2014 to 2022, and then by the annual average ZAR to USD exchange rate to compare costs collected to present costs .Population data were obtained from Statistics South Africa as a mean annual population for 2011–2016 and case fatality ratios were obtained from surveillance data during the same time period . We estiWe calculated years of life lost by applying the life expectancy estimates for South Africa in 2016 to the years lost for deaths in each age group .We enrolled 527 children with mild illness and 675 children with severe illness in the costing study. The detection rate of RSV was 10% (57) in those with mild illness and 26% (178) in those with severe illness. Between 2% (children aged 9–11 months) and 4% (children aged 12–59 months) of children admitted with RSV-associated illness required intensive care admission  and the median was $475 (2.5–97.5% range $191–$1156) . ChildreHealthcare system costs were highest for the youngest infants (aged 0–2 months) at $921.66 per episode as compared with $623.59 for infants aged 3–5 months, $583.71 for infants aged 6–8 months, $716.68 for infants aged 9–11 months, and $591.44 for children aged 1–4 years. This cost is driven by a mean duration of hospitalization 6.2 days in infants aged 0–2 months as compared to 4.7 days, 4.4 days, 5.6 days, and 4.1 days in children aged 3–5 months, 6–8 months, 9–11 months, or 12–59 months, respectively. Healthcare system costs for mild illness were more uniform, ranging from $24.98 among infants aged 0–2 months to $25.66 in children aged 12–59 months per episode  at $77.55 as compared to $62.21 for infants 3–5 months, $51.27 for infants 6–8 months, $72.19 for infants 9–11 months, and $66.77 for children. For children admitted with severe illness, the cost to the caregiver was between 110% ($51.27/$46.58) and 166% ($77.55/$46.58) of the mean monthly household income. Caregiver costs were similar across the age groups for children with mild illness (range $11.28–$11.99)  are healthcare system incurred, 7%  are out-of-pocket expenses and 17%  are indirect costs. Thirty-three percent  of the total costs were in the 0–2-month age group (Table The cost of RSV-associated severe illness was highest in the youngest infants (0–2 months), with 43%  of the healthcare system costs in this age group. Similarly, the highest proportions of the total out-of-pocket and indirect costs were in this age group; 43%  and 43% , respectively. Healthcare system costs for severe illness were relatively low in the 12–59 age group with 22%  of the healthcare costs in this age group  of the healthcare system costs in this age group. Similarly, the cost burden in this age group for out-of-pocket costs and indirect costs are highest in this age group   of costs in the 1–4-year age group. Non-medically attended illness accounted for 11%  of the total costs of RSV-associated illness.We estimated years of life lost for deaths for children < 5 years with RSV-associated illness to be 39,625 years of which 45% 17,812) occurred in the 0–2-month age group  in the youngest infants (aged 0–2 months), largely driven by high healthcare system costs in this age group. Costs are high throughout the first 6 months of life, the age groups targeted for new interventions. In contrast, for milder and non-medically attended illnesses, the majority of costs were in children aged 1–4 years, with significant costs of non-medically attended illnesses. This is the first analysis of the cost and cost burden of RSV-associated illness using pathogen-specific cost data derived from South Africa and provides important data to support cost-effectiveness models for interventions such as maternal vaccination and long-acting monoclonal antibodies aimed at youngest infants.There are few data to compare to our analysis as few studies include finer age bands (most studies provide costs for children aged < 5 years) and most studies document the costs of pneumonia rather than pathogen-specific costs , 16. Of In a review article that included data from low and middle-income countries (LMIC) compared with high-income countries (HIC), the cost per episode of pneumonia ranged from $10–$1648 for inpatient care in LMIC (including data from South Africa), within the bounds of our estimate of $591.44–$921.66 for RSV-associated inpatient care. Similar to our study the costs for pneumonia were driven by the duration of hospitalization . This reStenberg et al. updated the WHO-CHOICE models in 2018 with data from 30 countries to develop a robust model to estimate country-level costs . Our aveHealthcare is free to all children aged < 5 years in South Africa, which means that the South African government budget carries most of the burden of the costs of RSV-associated illness. The South African healthcare budget in 2016, was 38,563 billion Rand ($33.53 billion) . AlthougAlthough out-of-pocket and indirect costs are smaller proportions of the total costs of RSV-associated illness, the percentage of household income for these costs is high, specifically in a setting with high unemployment. In addition, the non-medically attended illness costs, specifically in the 1–4-year age group adds additional pressure on cash-strapped households, with these cost amounting to between 100 and 170% of the mean monthly household income.Our study has several limitations. Although we enrolled children for a full year including the RSV season, there may be differences in severity and cost between years dependent on the subtype of RSV circulating. Our study included urban and rural sites but may not have been representative of all settings in the country. We were not able to follow cases enrolled into the HUS studies and so it is possible that children accessed care after the interview date leading to possible misclassification of non-medically attended cases. This is likely to be small as cases were asked about retrospective health-seeking behavior, in the past 14 days, i.e., most cases were not active at the time of the interview. The costs of long-term sequelae such as recurrent wheezing and loss of lung function following RSV infection are not captured in our study. In the South African Drakenstein cohort study a significant proportion of children experienced repeat admission and numerous follow-up visits to clinics for these complications . The incThe highest costs to the healthcare system for RSV-associated pediatric illness in South Africa were in very young infants resulting in the highest cost burden in this age group; therefore, use of these higher per-illness cost estimates for interventions targeting young infants with either maternal vaccine or longer-acting mAb may result in higher quality cost-effectiveness analyses. The substantial cost of RSV-associated mild illness and non-medically attended illness in older children aged 1–4 years suggests early childhood vaccination against RSV may also be a cost-effective intervention.