The Northern Australia Quarantine Strategy (NAQS) is a biosecurity initiative operated by the Australian federal government’s Department of Agriculture, Water and the Environment (DAWE). It is unique worldwide because it deals specifically with the potential arrival via unregulated pathways of exotic threats from overseas in a vast and sparsely populated region. It aims to protect the nation’s animal- and plant-based production industries, as well as the environment, from incursions of organisms from countries that lie immediately to the north. These are diseases, pests, and weeds present in these countries that are currently either absent from, or under active containment in, Australia and may arrive by natural or human-assisted means. This review article focuses on the plant viruses and virus-like diseases that are most highly targeted by the NAQS program. It presents eight pathogen species/group entries in the NAQS A list of target pathogens, providing an overview of the historical and current situation, and collates some new data obtained from surveillance activities conducted in northern Australia and collaborative work overseas. NAQS was established in 1989 to specifically address unique plant and animal biosecurity challenges facing Australia’s northern coastline, from Broome in Western Australia to Cairns in Queensland. These arise directly from Australia’s close proximity to Indonesia, Timor-Leste and Papua New Guinea (PNG), which lie to the north and have a dramatically different plant and animal health status to that of Australia. At its closest point, the distance between Australia’s Saibai Island and the coast of PNG is about 4 km.The Australian federal government’s Department of Agriculture, Water and the Environment (DAWE) operates a multi-faceted program called the Northern Australia Quarantine Strategy (NAQS) (2) or silica gel at a temperature of 4 °C until fully desiccated, then stored at −20 °C thereafter. Samples collected overseas are subjected to gamma irradiation at 25 kGy upon return to Australia to fulfil import permit requirements.From a plant health perspective, movement south of insect pests, diseases and weeds can occur naturally, such as with winds created by wet-season monsoon-trough weather systems, or by human-mediated means, such as legal and illegal vessel arrivals. A cornerstone of the NAQS plant health program has been regular surveillance conducted by experienced plant scientists across Australia’s north aiming for early detection of new incursions. This would allow eradication before they reach plant-based industries or threatened populations of plants of environmental significance to the south. Another pillar of the plant health strategy has been collaborative surveys with government biosecurity scientists in these neighboring countries to obtain an ‘over the horizon’ view of current threats. This included several surveys in Indonesia in the 1990’s, including the Indonesian provinces of Papua and West Papua (then known as Irian Jaya), which occupy most of the western half of the island of New Guinea. Annual surveys have also been conducted in PNG and Timor-Leste from the early 1990’s to present. This collaboration continues today under the auspices of DAWE’s International Plant Health Surveillance Program with PNG’s National Agricultural Quarantine and Inspection Authority (NAQIA) and Timor-Leste’s National Directorate for Quarantine and Biosecurity (DNQB). This surveillance relies heavily on target lists of threats. Most are exotic to Australia, although some do occur within the country but are of limited distribution and subject to government biosecurity controls. The highest priority plant disease targets are placed on an A list, and the plant virus and virus-like  pathogens from the NAQS A list are presented in Bean common mosaic virus  causes peanut stripe disease. This virus, previously known as peanut stripe virus, assumed international biosecurity importance following introduction into the USA from China in 1979 [The peanut stripe strain of the virus species  in 1979 . Southea in 1979 . A threa in 1979 . Austral in 1979 . For sev in 1979 . It is a in 1979 . This vi in 1979 . These fRamu stunt virus (RmSV), a virus which has homology to the genus Tenuivirus, causes Ramu stunt disease of sugarcane, which is unique to PNG. The disease first appeared as a severe epidemic of stunting, yellowing and other symptoms at the Ramu Sugar Plantation in Morobe Province in 1986, with a 40% reduction in total sugarcane production [Eumetopina flavipes, was implicated as the vector of the disease before etiology was fully understood [E. flavipes is absent from commercial sugarcane fields in Australia but is common on the Torres Strait Islands and has also been recorded in Queensland’s Northern Peninsula Area [Saccharum officinarum rather than commercial Saccharum hybrids) is a common food plant in gardens throughout the Torres Strait Islands and northern Australia. This potentially provides an establishment and spread pathway to commercial production areas further south, valued at AUD 1.25 billion in 2019 [S. Officinarum samples by SRA using these diagnostic tests has so far returned no evidence for the presence of this disease in PNG’s Western Province or the Torres Strait Islands  is the causal agent of cotton blue disease. CLRDV is now considered to be synonymous to the previously reported chickpea stunt disease-associated virus (CpSDaV) [Aphis gossypii), which has a worldwide distribution, and it is not seed- or mechanically transmitted. Reported hosts of CLRDV include the malvaceous species Gossypium hirsutum, Gossypium barbardense, Hibiscus sabdariffa and Sida acuta [Cicer arietum), known symptomless hosts include several plants in the Amaranthaceae and Fabaceae families [Hibiscus rosa sinensis . Cotton blue disease was believed to be far from Australia until it was reported in Thailand [G. barbadense planting. As there is no commercial cotton production in the country, it now remains to be seen if CLRDV from Timor-Leste is capable of causing cotton blue disease in G. hirsutum. Simple pathogenicity tests are now needed in Timor-Leste to determine the risk to Australia’s cotton industry, which is currently expanding rapidly in northern regions. If pathogenic, a possible threat to Australia could be via direct introductions of viruliferous vector aphids via wind during the monsoon wet season. Similar insect movements have been postulated to have occurred from Timor-Leste to parts of the Northern Territory and northern Western Australia [G. barbadense, which is found sporadically in communities as a legacy of historical plantings for cotton production, and H. rosa sinensis, which is a very common garden and amenity ornamental. The Mitchell Plateau region in northern Western Australia is also a diversity hotspot for the genus Gossypium [The virus species (CpSDaV)  and both(CpSDaV) . This vi(CpSDaV) . CLRDV ida acuta . Althougfamilies . CLDRV hThailand  and was Thailand . The virustralia ,37. On aossypium , which mossypium . Althougossypium , diagnosBanana bunchy top virus  has been in Australia since the early 1900’s, where it has caused extensive losses [Pentalonia nigronervosa, a species which is already widespread throughout this region [Pentalonia caladii [The virus species e losses , but it e losses , the hige losses . As the e losses , and thes region . It is p caladii , were prFiji disease virus  causes Fiji leaf gall disease in sugarcane. Fiji leaf gall disease has caused devastating losses and has threatened the existence of the sugar industry in areas of Fiji and southern Queensland [Perkinsiella. P. saccharicida is the only known vector present in Australia and is widespread in commercial cropping areas, and it is also present in PNG [P. saccharicida is known to travel long distances, at least 30 km and perhaps up to 400 km [S. officinarum) is a common food plant in gardens throughout the Torres Strait Islands and northern Queensland. Whilst the vector is present in commercial cropping areas, there are no records from the Torres Strait or Cape York Peninsula (NAQS unpublished data). Although a high incidence of Fiji disease in PNG, ranging from the coastal swamps to the mountainous highlands, has been reported [Perkinsiella sp. from sugarcane have been made from this province in this series of surveys (NAQIA/NAQS unpublished data). The virus species eensland . Althougeensland . As the eensland . In neigeensland . The virt in PNG . P. sacco 400 km . If presreported , its disBegomovirus (family: Geminiviridae) is highly complex, fluid and constantly changing, with many unassigned isolates and numerous sequence variants within individual species [Bemisia tabaci and was regarded as part of a large species complex. Today, B. tabaci is not considered to be made up of biotypes and is instead a complex of 11 well-defined high-level groups containing at least 24 morphologically indistinguishable species in which this vector is known as B. tabaci Middle East-Asia Minor 1 (MEAM1) [B. tabaci populations [G. hirsutum), tomato (Solanum lycopersicum), chili (Capsicum annuum var. annuum) and sweetpotato (Ipomoea batatas). They also infect numerous weeds. In contrast to southeast Asia and India, Oceania has comparatively few records of begomovirus diseases. In important crops, this is tomato yellow leaf curl virus—Israel (TYLCV-IL), which is now considered a ‘severe’ strain of the species Tomato yellow leaf curl virus which is present in Queensland [Tomato leaf curl virus , which has been known in the Northern Territory since 1970 and reached parts of Queensland’s Cape York Peninsula in the late 1990s [Sweet potato leaf curl virus (SPLCV) in Queensland [The genus  species ; by 2020 species . Since t species ,53. This (MEAM1) . The keyulations . As wellulations . Since iulations , MEAM1 iulations . Begomoveensland , New Caleensland  and Freneensland . Also inte 1990s . A noveleensland , Westerneensland . Unpublieensland . Here, neensland  and in Peensland . It may eensland . Begomoveensland . Surveileensland  and SPG1eensland . DNA seqCandidatus Liberibacter asiaticus’. ‘Ca. L. asiaticus’ is vectored by the Asian citrus psyllid (ACP) Diaphorina citri, and long distance spread occurs via infected and infested planting material. Since the first detection of ACP in the northwest of Indonesia’s West Papua Province (then Irian Jaya) in 1990 [D. citri and highly limited disease focus expansion, only in and around the town of Vanimo. Since then, general surveillance has also prioritized this disease, and a body of additional data supports the claim that the distribution of this disease remains highly restricted. By 2021, 107 citrus trees had been sampled because they were showing possible HLB-like symptoms, which are similar to nutrient deficiencies and other stresses. All indexed negative in Australia for the disease in molecular testing , are used to assign phytoplasmas to known 16Sr groups and subgroups. In PNG, a serious wilt disease among cooking banana plants (ABB genomic group) associated with phytoplasmas was first described in 2012, and the phytoplasmas involved were named banana wilt-associated phytoplasmas (BWAP) [Cocos nucifera), known as Bogia coconut syndrome (BCS), was first associated with a phytoplasma in the Bogia district of PNG’s Madang Province [Candidatus Phytoplasma noviguineense’ [Ca. P. noviguineense’ from PNG’s northwest. It is possible that they represent another novel ‘Candidatus Phytoplasma’ taxon. When these phytoplasmas (from both east and west) were analyzed using the iPhyclassifier program [Ca. P. noviguineense’ have been studied extensively by an Australian Centre for International Agricultural Research project in the region in recent years [Zophiuma pupillata and Lophops saccharicida as species of particular importance [Areca catechu) and taro (Colocasia esculenta) as key alternative hosts using nested PCR and sequence analysis [Phytoplasmas are similar to ‘RNA gene . Restrics (BWAP) . This waProvince . Later, ineense’ ,79, it c program , they co program . The clont years . These sportance . Subsequportance . The BCSanalysis . Ca. P. noviguineense’ are likely to be widespread across the country [Losses of both cooking banana plants and coconut palms have been extensive, reaching thousands of plant deaths. To date, there have been no reports of a BCS-like disease epidemic from any areas outside of Madang Province. This has been backed up by opportunistic sampling and molecular testing of palms encountered on plant health surveys conducted by NAQIA/NAQS. This has targeted palms found showing any symptoms remotely similar to those of BCS since the outbreak started in Bogia District. A total of 65 palms have been indexed negative for phytoplasma . These c country . AnotherThe case histories outlined here are a testament to the great value of inter-agency and inter-governmental collaboration. Whilst conducting ‘over the horizon’ surveillance for Australia, mutually beneficial biosecurity outcomes are achieved for Australia and partner countries. Half of the virus/virus-like disease threats discussed here are new to the region, having arrived from southeast Asia, and two others probably originated on the island of New Guinea. It is valuable to raise awareness of these diseases which not only pose a potential biosecurity risk to Australia but also to other island nations further to the east in the Pacific.


Vitis vinifera L.), the most important indigenous red grapevine cultivar in Croatia, was tested for the presence of 16 grapevine viruses. Thirty-five samples from the collection vineyard were tested for the presence of grapevine leafroll-associated viruses-1, -2, and -3 , grapevine fanleaf virus (GFLV), arabis mosaic virus (ArMV), grapevine virus-A (GVA), -B (GVB), -G (GVG), -H (GVH), -I (GVI), -J (GVJ), grapevine fleck virus (GFkV), grapevine rupestris stem pitting associated virus (GRSPaV), and grapevine pinot gris virus (GPGV) by reverse transcription–polymerase chain reaction (RT-PCR). Furthermore, standard PCR was conducted for grapevine badnavirus 1 (GBV-1) and grapevine red blotch virus (GRBV). Mixed infections were most common and GLRaV-3, the most abundant virus found in 85.71% of the vines tested, was further molecularly characterised. Different genomic variants of the heat shock protein homologue (HSP70h) were separated by cloning, detected by single-strand conformation polymorphism (SSCP) analysis, sequenced, and phylogenetically analysed. The presence of phylogenetic groups I and II was only confirmed. This study demonstrates the high virus infection rate of Plavac Mali vines and the heterogeneity of GLRaV-3 present nowadays in a collection vineyard.The cultivar Plavac Mali ( Vitis vinifera L.) is the most important Croatian grapevine cultivar used for red wine production and the second rated cultivar in terms of acreages represented [Plavac Mali . Grapevine fleck virus (GFkV) from genus Maculavirus represents a latent threat particularly to rootstock material [Grapevine viability and performance can be affected by various agents. One of the crucial factors is the presence of viruses and virus-like diseases, which have a negative impact on every aspect of plant life and, potentially, wine quality. Grapevine could be affected by more than 80 viruses and virus-like agents . Those wGLRaV-1; ) and Clomaterial  and therVitivirus associated with this disease are emerging from different wine-growing regions around the world [Trichovirus, has recently come into focus for its harmful effects on grapevine plants [Other viral agents in some countries are considered less pathogenic to grapevine growing and are mandatory in certification programs of planting material depending on the country. These are, for example, grapevine rupestris stem pitting associated virus  and grahe world , and somhe world . Likewise plants . With thEven though grapevine virome is mostly consisted of RNA viruses, in recent years, by implementing high-throughput sequencing (HTS) in plant virome research, several DNA viruses were detected in grapevine . One of Identifying the individual variants which constitute virus inoculum and expanding the knowledge on their interaction with the host plant further contribute to efforts to control and monitor this virus. Since the first complete study was undertaken by Turturo et al.  on the GIn this study, using sensitive and reliable PCR-based detection methods, the presence of 16 economically important viruses was tested, and analysis of GLRaV-3 genetic variants was undertaken. This study provides valuable data on the sanitary status of Croatia’s most important red-berried cultivar and represents an important prerequisite for the production of healthy certified grapevine planting material.Molecular detection using primers specific for 16 grapevine viruses confirmed that neither one of 35 analysed plants of Croatian indigenous cultivar Plavac Mali was virus-free. Plants infected with four and six viruses represent the majority of the plant population, as four viruses were detected in 31.43% and six viruses in 20% of samples tested. Only 5.71% of analysed plants were infected with only one virus .The highest virus prevalence was reported for GLRaV-3, detected in 85.71% of samples. The GVA was detected in 77.14%, and GRSPaV was detected in 40% of tested samples. The lowest prevalence was observed for GVB and GVH, which were detected in 5.71% of the tested samples, while the prevalence of the other viruses detected in the sample ranged from 14.29% to 37.14% . The GVGRegardless of the high virus prevalence and frequent mixed infections, the symptoms of grapevine leafroll disease observed in the field plants were not unique, and they could not be associated with particular virus composition. In 2020 most of the symptoms observed on individual plants were ranked as moderate (34.29%), which presumes that more than 50% of leaves of the tested plants were diagnosed with interveinal reddening and downroll, the main characteristic of GLD . These cNine plants originating from nine scattered wine-growing areas were further selected based on the symptom severity observed in the first year of assessment for the molecular characterization of the HSP70h region of GLRaV-3. Clones 020 and 268 expressed severe symptoms, clones 214, 036, and 096 moderate, clones 041 and 225 mild, while clones 201 and 008 displayed no symptoms.The SSCP analysis was performed on approximately 20 randomly selected bacterial colonies per sample (nine selected plants). In total, 177 virus variants were analysed, and all variants of each sample displaying different SSCP patterns were sequenced. The phylogenetic analysis revealed that most of them (69.66%) belong to GLRav-3 phylogenetic group II, while 30.43% of variants belong to phylogenetic group I, all supported by high bootstrap values .. When aGrapevine viruses and viral diseases have a long history of scientific investigation in Croatia. Since the first report on the presence of the infection degeneration complex in Croatia by Šarić and Corte , continuAlthough most of the studies conducted so far are based on serological ELISA testing ,33,34, sCoccidae and Pseudococcidae. Zdunić et al. [We assume that the horizontal transmission of the viruses between clonal candidates has been occurring since the collection establishment in 2005, even though visual inspection gave negative results regarding ć et al. ,37. Accoć et al. . Furthermore, clones infected with numerous viruses, such as clone 020 infected with six different viruses, developed severe virus symptoms, resulting in the host plant decline and death. With no physiology analyses of those plants of cv. Plavac Mali, we can only assume that besides observed symptomatology, virus composition influenced plant metabolism and potentially wine quality. Hančević et al.  showed tAccording to the HSP70h, phylogenetic groups I and II were detected in this study, showing relatively low genetic diversity of GLRaV-3 based on SSCP method and sequence variants, similarly as in other native vines from the Mediterranean part of Croatia found in the same collection . MonophyVitivirus, GVH, was detected in 5.71% of samples originating from only one location  was slightly higher than previously suggested by other authors , and thelocation . Its worThe prevalence of nepoviruses (ArMV 22.86% and GFLV 17.14%) were lower than suggested by the previous study of cv. Plavac Mali  and highColomerus vitis (Pagenstecher), a mite described as a vector for GPGV [A possible reason for the high occurrence of GPGV (37.14%) is the presence of for GPGV , visuallfor GPGV  and is cfor GPGV , no specCaulimoviridae, is a nouvelle grapevine virus which, to our knowledge, has been found only in the Croatian autochthonous grapevine cultivars Ljutun and Vlaška, whose origin is also in the Mediterranean part of Croatia [Grapevine badnavirus 1, a member of the family  Croatia . TestingVitis riparia 183G × Vitis cinerea Arnold). Clones included in this study were selected based on the presence of visual symptoms of viral infections, and in total, thirty-five samples originating from 13 different wine-growing locations were inspected and assayed . Cortical scrapings were homogenized (250 mg of tissue) using liquid nitrogen, and RNA extraction was performed as described by MacKenzie et al.  using RNDetection of grapevine leafroll associated viruses-1, -2, and -3, , grapevine virus A (GVA), grapevine virus B (GVB), grapevine rupestris stem pitting associated virus (GRSPaV), grapevine fanleaf virus (GFLV), arabis mosaic virus (ArMV), and grapevine fleck virus (GFkV) was performed by multiplex RT-PCR reaction as described by Gambino and Gribaudo . PrimersGrapevine Pinot gris virus (GPGV) detection was performed separately using PCR conditions by Saldarelli et al.  and primDetection of GVG, GVH, GVI and GVJ was performed with the One-step RT-PCR kit  using primers described by Diaz Lara et al. . The masDetection of GRBV was performed with primers GVGF1/GVGR1 as described by Al Rwahnih et al. , while dAll GLRaV-3 positive samples were further analysed to determine their genetic variability. To obtain the longer product, primers (LC1 and LC2) amplifying the 545 bp long region of the HSP70h gene were used . The reaE. coli JM109-High-Efficiency Competence cells . The presence of the insert was confirmed by the white–blue selection, and at least 20 transformed colonies per sample were further characterised.To separate different genomic variants from each sample, amplicons were TA-cloned using pGEM-T Easy Vector Sistem I  and ligation products were used to transform To identify separated variants by SSCP, inserts originating from selected colonies were amplified using the same primer set (LC1/LC2) as in the initial reaction. 2 µL of amplicons were denatured and analysed after silver staining of polyacrylamide gels as described by Černi et al. . ExampleAfter identifying different sequence variants, at least two of them displaying the same SSCP pattern were sent for sequencing with Macrogen Europe Inc. . Representative sequences were deposited in GeneBank under the accession numbers: MZ450926-MZ450948. Sequences obtained were aligned in Clustal X 2.1  and analWe confirmed the deteriorating sanitary status of the most widespread Croatian indigenous red cultivar Plavac Mali. Mixed infections were common in the tested vines, and none of the tested clones proved to be virus-free. GLRaV-3 was the most common virus detected in 85.71% of the samples. Among its phylogenetic variants according to HSP70h, groups I and II were detected, with the monophyletic type of infections as predominant. Diagnostic tools improvement and knowledge about the sanitary status of grapevine plants with more detailed insight into genetic differences within the GLRaV-3 population should lead to better planning and management strategy for this valued wine cultivar in the Mediterranean Croatia viticulture. Promoting the production and usage of virus-free material should be taken more seriously in the future in order to realize the full potential of Plavac Mali.


Helicobacter pylori, herpes simplex virus 1, influenza virus subtype H1N1, influenza virus subtype H3N2, measles virus, Saccharomyces cerevisiae, Theiler’s virus, Toxoplasma gondii, and SAG1 protein domain, a surface antigen of Toxoplasma gondii measured for greater precision. We performed genome-wide association analyses of antibody levels against these 14 infections (N = 357 – 5010) and identified three genome-wide signals (P < 5×10-8), two associated with measles virus antibodies and one with Toxoplasma gondii antibodies. In an association analysis focused on the human leukocyte antigen (HLA) region of the genome, we further detected 15 HLA alleles at a two-digit resolution and 23 HLA alleles at a four-digit resolution associated with five antibodies, with eight HLA alleles associated with Epstein-Barr virus antibodies showing strong evidence of replication in UK Biobank. We discuss how our findings from antibody levels complement other studies using self-reported phenotypes in understanding the architecture of host genetics related to infections.The burden of infections on an individual and public health is profound. Many observational studies have shown a link between infections and the pathogenesis of disease; however a greater understanding of the role of host genetics is essential. Children from the longitudinal birth cohort, the Avon Longitudinal Study of Parents and Children, had 14 antibodies measured in plasma at age 7: Alpha-casein protein, beta-casein protein, cytomegalovirus, Epstein-Barr virus, feline herpes virus,  Helicobacter pylori has been linked to myocardial infarction  cohort by: (1) Investigating the options for analysing antibody titers to model antibody levels in terms of whether using measures as a continuous variable or thresholding to define infection status is most appropriate; (2) Identifying genome-wide single nucleotide polymorphisms (SNPs) strongly associated with the antibodies; (3) Identifying HLA alleles strongly associated with the antibodies; (4) Assessing consistency of genetic signals associated with the antibodies at different time points in ALSPAC and in an independent cohort (UK Biobank). (5) Evaluating whether any of the genetic signals identified overlap with SARS-CoV-2 infection (COVID-19 Host Genetics Initiative). This study extends the scope of understanding infection susceptibility and host genetics by increasing the range of infections examined and investigates infections in children.Pregnant women resident in Avon, UK with expected dates of delivery 1st April 1991 to 31st December 1992 were invited to take part in the ALSPAC study , 16. TheWhen the oldest children were approximately seven years of age, an attempt was made to bolster the initial sample with eligible cases who had failed to join the study originally. As a result, when considering variables collected from the age of seven onwards  there are data available for more than the 14,541 pregnancies mentioned above. The number of new pregnancies not in the initial sample (known as Phase I enrolment) that are currently represented on the built files and reflecting enrolment status at the age of 24 is 913 , resulting in an additional 913 children being enrolled. The phases of enrolment are described in more detail in the cohort profile paper and its update , 16. TheA 10% sample of the ALSPAC cohort, known as the Children in Focus (CiF) group, attended clinics at the University of Bristol at various time intervals between 4 to 61 months of age. The CiF group were chosen at random from the last 6 months of ALSPAC births (1432 families attended at least one clinic). Excluded were those mothers who had moved out of the area or were lost to follow-up, and those partaking in another study of infant development in Avon.http://www.bristol.ac.uk/alspac/researchers/our-data/). Ethical approval for the study was obtained from the ALSPAC Ethics and Law Committee and the Local Research Ethics Committees. Consent for biological samples has been collected in accordance with the Human Tissue Act (2004).The study website contains details of all the data that is available through a fully searchable data dictionary and variable search tool  between September 1998 and October 2000 . AttendeSacchraromyces cerevisiae.Whole blood samples were collected and processed by centrifugation at 3500rpm, for 10 minutes at 4-5°C . SubsequAll target antigens and source of target antigens for each infection of interest have been detailed elsewhere . Serum aToxoplasma gondii (T. gondii), surface antigen 1 (SAG1) protein domain of T.gondii, cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus type 1 (HSV1), influenza virus subtypes H1N1 and H3N2, measles virus, Saccharomyces cerevisiae (S. cerevisiae), Helicobacter pylori (H. pylori), feline herpes virus (FHV), Theiler’s virus (TV), bovine casein alpha protein  and beta protein (beta-casein).The antibodies examined in this analysis are against the following antigens: For each antibody, measurements of optical density were read directly from the ELISA plate. The ratio to standards were then derived from the standards measured on each plate. These ratios were then standardised to produce z-scores with a mean of two and a standard deviation of one per plate  . In addiNot all participants were measured for all 14 antibodies at every clinical time point. The antibody levels for each antigen at the seven-year time point were analysed in the primary analysis as they represented the largest sample size, and the distribution of z-scores for each infection did not largely differ across time points.-7. Cryptic relatedness was assessed as the proportion of identity by descent (IBD >0.1) described previously  subcontracting from the Wellcome Trust Sanger Institute, Cambridge, UK and the Laboratory Corporation of America, Burlington, NC, US. Genotypes were called with Illumina GenomeStudio and PLINK(v1.07)  was usedeviously .After quality control steps, a total of 9,115 individuals and 500,527 SNPs passed these filters. ALSPAC children were phased using ShapeIt v2  to phaseTwo software packages, HLA*IMP:03  and SNP2SNP2HLA_package_v1.0.3  was utilFor HLA*IMP:03 (v.0.1.0) , HLA impT, to determine concordance (-0.5 ≤ T ≤ 0.5) or discordance (-0.5 ≥ T ≥ 0.5) between the two approaches. The proportion of concordant and discordant HLA alleles at each given HLA loci were then tabulated. In addition, allele frequencies for each HLA loci were calculated for each HLA imputation approach, and values were compared to the T1DGC reference panel.Concordance of HLA imputation using the two packages were compared by assessing the difference in dosages by providing a call threshold, -6) from an analysis where the antibody measures were treated as a continuous variable. .It would be desirable to be able to stratify individuals into seropositive and seronegative for each antibody analysed. However, determining where to call the threshold for continuous antibody data is complex due to the unavailability of reliable assays on reliably determined controls to establish uninfected population baseline distributions ; (2) A swww.cog-genomics.org/plink/2.0/) (www.cog-genomics.org/plink/2.0/)  (nk/2.0/)  for eachnk/2.0/)  on all fwww.cog-genomics.org/plink/1.9/) (http://neurogenetics.qimrberghofer.edu.au/matSpDlite/)   and adjupDlite/)  was usedpDlite/)  was emplHelicobacter pylori, and Toxoplasma gondii. Analyses were performed using the initial assessment time point for all antigens . Antibody measurements were rank-based inverse normal transformed for all GWAS and HLA association analyses and beta estimates presented are on this scale. To give an idea of the magnitude of these effects, the main results are also presented using the z-scores of the ratio-to-standard measures, where beta estimates are a change in standard deviation per allele.Comprehensive description of UK Biobank’s cohort profile, serological measures and analyses performed are available in P-values were compared.To investigate genetic overlap, ALSPAC antibody association results and the SARS-CoV-2 GWAS meta-analysis findings (C2_ALL_eur) N = 1,683,784) Helicobacter pylori, Toxoplasma gondii, SAG1 protein domain), and eight had <700 individuals  (Distribution of the ratio to standards of the 14 antibodies are shown in evisiae) .We explored the utility of analysing the 14 antibodies as continuous or thresholded variables in ALSPAC children. Four threshold approaches (as described in the methods) were used: (1) Ratio to standards greater than or equal to 1; (2) A subjective threshold based visually identifying the trough in distributions that were clearly bimodal; (3) The threshold which demonstrated the most signal in a GWAS analysis; (4) The threshold that best recapitulated continuous GWAS results. These were also compared to proportions of seroprevalence measures reported in the literature  (versus 15-25%)  from the continuous GWASs across the thresholded GWASs for each specific antibody, to see if any thresholds gave similar association results on an individual locus basis. The thresholds which gave the most consistent results (to continuous) are shown in We compared the top SNP associations (P < 5 × 10-8) to two antibodies (Measles virus or T. gondii) and 26 suggestive SNPs (P < 1 × 10-6) associated with 12 antibodies. These SNPs are presented in T. gondii antibody, was retained after adjusting for the 14 antibodies tested (P < 3.6×10-9).We performed genome-wide association analyses of all continuous antibody measures at the seven-year clinic in ALSPAC and identified a total of three SNPs with strong associations , an intergenic SNP located between LOC105370271 and SCEL on chromosome 13q22.3. Followed by rs28617484 , also an intergenic SNP, between LINC01920 and LINC02612 on chromosome 2q23.3. In addition, stratified analysis by measles, mumps and rubella (MMR) vaccination status was performed on the genome-wide and suggestive SNPs. Findings in P > 0.001), with environmental exposure of the measles virus showing greater genetic signal (P > 2.6×10-6).The strongest association for measles virus was rs506576  located in GRAM Domain Containing 1B (GRAMD1B) on 11q24.1 . No evidence of an association was identified, with effect size shown in the opposite direction.For  11q24.1 –37. A loP < 4.3 ×10-4, adjusted for the three additional timepoints and the 29 associations tested), with all estimates showing consistency in the direction of effects  and antibodies against T.gondii, with effect sizes similar to that observed at the 7-year clinic (β = 0.20) also seen at the 11-year clinic  and the 15-year clinic , but this association was not seen at the earlier five-year clinic (P = 0.45).The strongest associations at different time points were demonstrated between rs36020612 (P = 7.1 × 10-7); rs117760947 . These effect sizes were similar to the effect sizes exhibited at the seven-year clinic: rs3104369 (β = -0.19); rs117760947 (β = 0.48), however these associations were not observed at the five-year and 15-year clinic (P < 0.83).For antibodies against feline herpes virus, two SNPs replicated at the 11-year clinic time point: rs3104369  and the eleven-year clinic , with comparable effect sizes shown at the two time points. These associations however were not able to be replicated at the five-year and 15-year clinic.Furthermore, rs35030589 was shown to be associated with antibodies against P = 5.6×10-5) and the 15-year clinic , although this association was not observed at the five-year clinic (P < 0.07).For antibodies against herpes simplex virus, rs10961934 showed the strongest associations at different timepoints. The effect size at the seven-year clinic (β = 0.53) were similarly demonstrated at the 11-year clinic , and the effect size was similar to the seven-year clinic (β = 0.33). Neither of the SNPs that associated strongly with measles showed good evidence for association at other timepoints, with the effect appearing to attenuate with age beyond seven-years.Lastly, replication of rs1634083 associated with antibodies against measles virus was shown at the 11-year clinic , an intronic variant located in genes SUGCT and LOC105375245. However, the effect size was in the opposite direction.Furthermore, we attempted to replicate the associations identified in the ALSPAC seven-year clinic in an independent cohort, UK Biobank , and findings are shown in P < 5×10-8) with Epstein-Barr virus (10 associations), herpes simplex virus 1 (one association) and T. gondii (one association) in UK Biobank (P < 0.001): rs9264759, rs1043620, rs2523502, rs3117139 and rs3096695, with effects in the same direction compared to UK Biobank, with the exception of rs1043620 in which the effect was in the opposite direction (P > 0.485). In addition, three EBV antibody associations were not able to be replicated as no suitable LD proxy SNP was identified (R2 < 0.6) in ALSPAC, and similarly none of the associations with antibodies against herpes simplex virus 1 or T. gondii were able to be replicated.We also conducted the analysis in the opposite direction, with a GWAS in UK Biobank as a discovery cohort and ALSPAC seven-year clinic as replication. Five antibodies in UK Biobank overlapped with ALSPAC  . We found very high concordance between posterior probabilities of imputed genotypes between the two methods at two- and four-digit resolutions (P < 6.2 × 10-4) and at a 4-digit resolution (P < 5.0 × 10-4) are shown in We performed HLA association analyses for 14 antibodies at two- and four-digit resolutions using both HLA imputation methods in ALSPAC. The associations that exceeded the HLA-wide corrected -value thresholds at a 2-digit resolution (P < 4.4×10-5) remained associated with antibodies against beta-casein protein, feline herpes virus and H. pylori. At a 4-digit resolution (P < 3.5×10-5), six HLA alleles remained associated with antibodies against beta-casein protein, Epstein-Barr virus, feline herpes virus, and H. pylori.When further correcting for the 14 antibodies tested, seven HLA alleles at a 2-digit resolution (H. pylori antibodies (other infections were not available). All eight Epstein Barr virus antibodies associations showed strong replication signals with the same direction of effect, with P <1×10-9. However, the four associations with H. pylori did not replicate , and 17 HLA associations were identified with five antibodies at a four-digit resolution (P < 2.8×10-5) in UK Biobank (P < 0.003) and five HLA alleles at a four-digit resolution (P < 0.003) associated with Epstein-Barr virus antibodies replicated in ALSPAC, with all effect estimates consistently showing the same direction of effects  with two antibodies, measles virus and T. gondii, at the ALSPAC seven-year clinical time point. Associations linking these loci with their respective antibodies have not been previously reported. Assessing the 26 associations that met a suggestive threshold (P < 1 × 10-6), the association between rs186721582 and Epstein-Barr virus antibodies replicated in UK Biobank. The associations between rs36020612 and T. gondii did not replicate (P > 5.9 × 10-4) and antibody data on the measles virus was not available. Stratifying individuals by MMR vaccine status for SNPs associated with antibodies against measles virus at the seven-year clinic showed stronger signal for individuals with an unknown status . For FHV antibodies, H. pylori and measles virus, the observed associations disappeared at the 15-year time point, which could be attributable to the reduction in sample size, lack of phenotypic variance, and possible false-positive associations identified at the seven-year time point.In genome-wide association analysis, we observed three genetic signals strongly associated (GRAMD1B with antibodies against T. gondii (P = 0.78), an antigen of T. gondii¸ with the effect size in the opposite direction. This lack of association could be due to the difference in statistical power contributing to the sample size of SAG1 protein domain (N = 1228) compared to T. gondii (N = 5010).Of particular interest in the main ALSPAC GWAS, was the association between rs36020612 located on . gondii –40, lymp. gondii , mean co. gondii , 43, ery. gondii , 44, blo. gondii , and imm. gondii , 46. HowMHC) class II region in ALSPAC and identified 20 associations of interest with antibodies against beta-casein protein, Epstein-Barr virus, feline herpes virus, H. pylori, and S. cerevisiae  which could possibly be a result of false-positive associations in ALSPAC at the seven-year clinic, and lack of phenotypic variance. To date, no studies have investigated the association of these HLA alleles and H. pylori in individuals of European ancestry. Studies using other ancestral populations have shown that HLA-DQB1*05:01 strongly increased the risk of gastric cancer in individuals of Mexican descent  associated with antibodies against measles virus, however we were unable to test for replication in UK Biobank due to data unavailability. Furthermore, we observed strong evidence of replication of the suggestive SNP (rs186721582) associated with Epstein-Barr virus antibodies in UK Biobank, suggesting the genetic effect is stronger in adults. Our study provides a useful resource for future studies looking to use ALSPAC antibody measurements as well as HLA alleles. It indicates that for future use of the measured antibodies in ALSPAC the most appropriate approach is to use the antibody level measures as a continuous variable. This study has highlighted the potential for identification of host genetic risk factors for several common infections, and demonstrates that if similar data is collected in other cohorts, future meta-analysis GWAS is likely to be a fruitful endeavour in uncovering the genetic and biological mechanisms of infection susceptibility.In summary, our study has confirmed known HLA allele associations with Epstein-Barr virus in a younger age group (seven-years) and replicated results in the adult population, UK Biobank. In addition, in the discovery phase we have identified four potentially novel genetic associations, with rs36020612 showing strong evidence of association with http://www.bristol.ac.uk/alspac/researchers/our-data/). Access to ALSPAC research data must be requested using the formal procedures and is subject to eligibility, the ALSPAC funder’s terms and conditions and University of Bristol policies and procedures. Requests to access these datasets should be directed to alspac-exec@bristol.ac.uk.The ALSPAC study website contains details of all the data that is available through a fully searchable data dictionary and variable search tool . This research was specifically funded by Wellcome Trust and MRC, 076467/Z/05/Z. AC is funded by the Jonathan and Georgina de Pass studentship. This work was supported by the Integrative Epidemiology Unit, which receives funding from the UK Medical Research Council and the University of Bristol .The UK Medical Research Council and Wellcome (Grant ref: 217065/Z/19/Z) and the University of Bristol provide core support for ALSPAC. A comprehensive list of grants funding is available on the ALSPAC website (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cryptococcus spp. represents a potential risk for immunosuppressed and healthy persons. In Egypt, epidemiological data on cryptococcal infection in horses are limited. The current study was carried out to investigate the occurrence of Cryptococcus spp. in horses and its possible role in the epidemiology of such disease in Egypt.Cryptococcosis is an opportunistic mycozoonosis of global significance in a wide variety of host species. In equines, cryptococcosis is uncommon, and sporadic cases have been reported with rhinitis, sinusitis, pneumonia, and meningitis. Cryptococcus spp. were performed. Molecular serotyping of Cryptococcus spp. was determined by multiplex PCR using CNa-70S/A-CNb-49S/A. The virulence genes  of the identified isolates were detected by PCR. Moreover, sequencing and phylogenetic analysis of the C. gattii gene from horses, humans, and soil isolates found nearby were performed.A total of 223 samples was collected from different localities in Egypt included 183 nasal swabs from horses, 28 nasal swabs from humans, and 12 soil samples. Bacteriological examination and the identification of Cryptococcus spp. in horses were 9.3, 25, and 10.7% in horses, the soil, and humans, respectively. Molecular serotyping of the Cryptococcus spp. isolates recovered from the nasal passages of horses proved that C. gattii (B), C. neoformans, and two hybrids between C. neoformans (A) and C. gattii (B) were identified. Meanwhile, in case of soil samples, the isolates were identified as C. gattii (B). The human isolates were serotyped as C. gattii in two isolates and C. neoformans in only one isolate. Molecular detection of some virulence genes (LAC1), (CAP59), and (PLB1) were identified in both C. gattii and C. neoformans isolates. The C. gattii gene amplicons of the isolates from horses, humans, and the soil were closely related.The overall occurrence of Cryptococcus species and highlights the role of horses as asymptomatic carriers in disseminating the potentially pathogenic Cryptococcus spp. It also presents the possible risk of cryptococcosis infection in humans.This study provides the first insights into the Egyptian horse ecology of  Cryptococcus neoformans species complex and the Cryptococcus gattii species complex and is usually associated with pulmonary and systemic infections in humans and animals [Cryptococcosis is a life-threatening systemic mycosis of global significance that affects a wide variety of host species. It is caused by the  animals , 2.C. neoformans species complex includes two separate species, C. neoformans (serotype A) represented by genotypes VNI, VNII, and VNB and C. deneoformans (serotype D) represented by genotype VNIV. The C. gattii species complex has five cryptic species with serotypes B and C and genotype from VGI to V. Moreover, interspecies hybrids between C. neoformans and C. gattii have been detected [C. neoformans (A) is found worldwide, whereas C. gattii (B) has been restricted geographically to tropical and sub-tropical regions [C. gattii has recently been identified in temperate climates, which suggests an ecological shift that is possibly associated with global temperature and moisture changes [The detected –5. C. ne regions . C. gatt changes , 7.C. neoformans) or soil debris and decaying trees like eucalyptus trees (C. gattii) [Cryptococcosis has been described in a wide variety of host species, including cats, dogs, ferrets, llamas, porpoises, birds, and horses, affecting many, and often multiple, organ systems . Further gattii) .Cryptococcus is able to rapidly and effectively adjust to variable conditions, favoring its survival in the environment and in the infected host [C. neoformans that enables it to reside both in the environment and in mammalian hosts.There is evidence to prove that humans and animals usually get the infection from environmental sources by inhaling fungal cells . The humted host . The surted host . These vC. neoformans is the most common fungal pathogen that infects the human central nervous system [C. neoformans causes life-threatening infections as meningoencephalitis primarily in immunocompromised hosts  [C. neoformans, C. gattii causes meningoencephalitis and pulmonary infections mainly in immunocompetent hosts.s system . C. neofth AIDS) . Unlike C. gattii infection was identified in an Arabian horse imported from South Africa into the United Arab Emirates and identified as C. gattii VGII, which was closely related to another C. gattii VGII isolate from the Middle East [In horses, the distribution of cryptococcosis is worldwide, whereas the majority of clinical infections are reported from Western Australia and Vancouver Island, British Columbia, Canada , 15. Mordle East .C. neoformans spp. complex isolated from either environmental (Plant or Bird origin) or clinical samples  is C. neoformans (serotype A) [C. neoformans and C. gattii from pigeon dropping. Furthermore, plants from Qutur and Tanta area carried C. gattii [Cryptococcus serotypes A and A/D in clinical specimens . But to date, little epidemiological data are available among cryptococcal infection in horses and humans in contact in Egypt.In Egypt, most studies at the beginning of the year 2003 reported that the major subtype of the otype A) , 18. Besotype A)  revealed. gattii  reportedCryptococcus spp. among horses and the role played by horses in the distribution of such pathogens in the surrounding environment and humans occupationally in contact with such animals.Therefore, the current study was carried out to determine the prevalence of A total of 223 samples were collected from different localities in Egypt  over a period of 1 year from December 2018 to December 2019. These samples included 183 nasal swabs from horses and 28 nasal swabs from humans. In addition, 12 soil samples were collected from the houses of horses. Data collected from each human and animal included age, gender, and underlying health problems.The nasal swabs were taken using sterile bacteriological swabs under complete antisepsis conditions and inserted into both nasal vestibules and rotated vigorously. The collected samples were immediately transported in ice boxes to the laboratory and then inoculated into sterile Sabouraud dextrose broth (Oxoid) supplemented with chloramphenicol (0.1 g/L) (HiMedia).Soil samples were collected in sterile plastic bags. The samples were prepared by mixing about 3–5 g of each sample into sterile test tubes containing 10 ml of Sabouraud dextrose broth supplemented with chloramphenicol. The tubes were shaken vigorously by vortex and allowed to stand for about 15 min .Cryptococcus colonies with a mucoid appearance were selected and identified by the microscopic morphology of yeast cells.The inoculated swab samples of humans and horses in addition to the prepared soil samples were incubated at 37 °C for 24 h according to Horta et al. . The supCryptococcus isolates were confirmed based on melanin synthesis (brown color effect) by streaking on the tobacco agar plates [Cryptococcus isolates was done using the Rapid yeast plus system  [r plates . The ideel, USA) .Cryptococcus isolates was extracted using the boiling method according to Mohammadi et al. [Genomic DNA from the pure i et al. . The extC. neoformans serotype A and C. gattii serotype B as previously described by Aoki et al. [Multiplex PCR was carried out to detect i et al.  using spLAC1, CAP59, and PLB1 genes was performed according to the method of Meyer et al. [Conventional PCR to amplify the r et al. . BrieflyC. gattii isolates from horses, humans, and the soil were selected and purified using the QIAquick gel extraction kit  according to the manufacturer’s instructions and sequenced at the Animal Health Research Institute  using the forward and reverse primers of C. gattii (CNb-49 s and CNb-94A).The amplification products of three The nucleotide sequences have been deposited in the National Center for Biotechnology Information (NCBI) GenBank database under the accession numbers MT520167-MT520169.C. gattii isolates were compared with the sequences available in the public domain using the NCBI BLAST server. Publicly available gene sequences were retrieved from the NCBI GenBank and aligned using CLUSTALW in BioEdit version 7.0.1.4. Phylogenetic analysis was performed with MEGA version X using the neighbor-joining approach. The bootstrap consensus tree was estimated from 1000 replicates.The nucleotide sequences of Cryptococcus spp. collected from 183 nasal swabs of horses at different localities in Egypt was 17/183 (9.3%). Molecular serotyping of the Cryptococcus spp. isolates recovered from the nasal passage of horses proved that C. gattii B was identified in 12 isolates and C. neoformans A in 3 isolates. Two hybrids between C. neoformans A and C. gattii B were also identified. In the case of soil samples, C. gattii B was detected in 3 out of 12 samples (25%) collected from horse houses. The human isolates were serotyped as C. gattii in two isolates (7.1%) and C. neoformans in only one isolate (3.6%).The overall occurrence of Cryptococcus spp. in healthy horses showed that C. gattii was identified in 5 out of 44 samples (11.4%). In the case of diseased horses, the detection rate was 12/139 (8.6%), and the identified Cryptococcus spp. in each case mentioned in , and C. neoformans serotype (A) was isolated from the nasal passage of an immune-compromised male suffering from chest allergy. In addition, C. gattii was isolated from two asymptomatic individuals. The positive samples belonged to humans working in stud farms where the horses’ positive samples were collected. Our results indicated that Cryptococcus spp. was more prevalent in old male individuals. Moreover, we found that Cryptococcus spp. more frequently colonized the nasal passages of smokers than those of non-smokers as shown in Table Regarding the human samples, LAC1, CAP59, and PLB1 was carried out in both C. gattii and C. neoformans isolates  in healthy examined horses, which probably indicates that apparently healthy horses could be asymptomatic carriers of Cryptococcus spp. These findings are supported by those of Malik et al. [Cryptococcus spp. from the nasal passages of dogs, cats, koalas, and donkeys without any evidence of disease, suggesting the asymptomatic colonization of the nasal mucosa following environmental exposure.In the current study, the overall recorded prevalence of k et al. , Connollk et al. , Duncan k et al. , and Mohk et al. , who isoCryptococcus appeared to be higher with C. gattii (12 isolates) in examined horses than with C. neoformans (3 isolates). This result was similar to that of Mcgill et al. [Cryptococcus spp. from 20 out of 155 affected horses in Western Australia with a high frequency of C. gattii, and Duncan et al. [C. gattii in the nasal passage of horses in Canada.The species identification of l et al. , who detn et al. , who ideC. neoformans (A) and C. gattii (B) were also identified in two isolates, which is in line with the findings of Bovers et al. [C. gattii is capable of forming hybrids with C. neoformans. Interspecific cryptococcal hybrids have been reported earlier in Germany [Cryptococcus are more virulent and cause mammalian diseases [C. neoformans and is able to infect immunocompetent humans like C. gattii, combining the malicious characteristics of both species [Hybrids between s et al.  and Amins et al. , who rep Germany , Denmark Germany , and the Germany . Inter-vdiseases . This hy species .C. neoformans and C. gattii infect immune-suppressed and immune-competent individuals, respectively [Cryptococcus spp. from human nasal passages are very rare. In this study, we isolated Cryptococcus spp. from people without cryptococcosis, Morera et al. [C. gattii in humans sharing the same house with domestic ferret having cryptococcosis.The pathogenic species ectively , 40. Howa et al.  supporteCryptococcus spp. in the nasal passage of humans who have occupational contact with infected horses was 10.7% (3 out 28); two of them were reported in aged individuals who were smokers, which is in line with the findings of Forthal et al. [The occurrence of l et al.  and Pourl et al. , who repC. neoformans serotype A was identified from the nasal passage of an immune-compromised male suffering from chest allergy. In addition, the C. gattii isolate was isolated from two asymptomatic individuals in contact with horses. In this context, a clinical isolate was obtained from an immigrant worker from Kuwait that is closely related to a case of C. gattii in an Arabian horse in the United Arab Emirates [In this study, the Emirates .C. gattii. Besides, the pigeon is a natural reservoir of C. neoformans, and it usually eats the remains of equine food  from the feed troughs and defecates in the feed and water trough.The epidemiological significance of this research lies in its proving that both horses and humans are at risk of environmental contamination in stud farms. In these farms, eucalyptus trees are used for horse shelter and shadowing , and theCryptococcus spp. and the result revealed 25% from which all isolates were C. gattii. This may be attributed to the fact that that soil contaminated with plant debris and decaying wood is a major environmental source of C. gattii [To support our epidemiological view, the soil around horses included in this research was examined for . gattii .C. gattii from the nasal passage of horses, humans, and soil samples in close vicinity to the examined horses point to the presence of a common issue between these isolates, which was predicted to be the excessive presence of eucalyptus trees in the collection sites. This result is in line with that of Mcgill et al. [C. gattii than with C. neoformans, and this was attributed to horse population’s close association with eucalyptus trees, a regional environmental reservoir of C. gattii.The isolation of l et al. , who repCryptococcus spp. This is enhanced by some virulent determinants, which are enzyme secretion, the addition of melanin to the fungal cell wall, and the abundant secretion of polysaccharides that may surround the cell to form a capsular structure [Human cryptococcosis develops following environmental exposure and inhalation of the infectious spores of tructure .LAC1), capsular-associated gene (CAP59), and phospholipase (PLB1) in the identified Cryptococcus spp. isolates. The International Society of Human and Animal Mycology (ISHAM) group selected the Multilocus Sequence Type (MLST) analysis as the method of choice for global molecular epidemiological typing of Cryptococcus species using seven housekeeping loci  [LAC1) gene, which is the main protein involved in the production of the melanin pigment, in all C. neoformans and C. gattii isolates [CAP59 gene in the identified isolates. This capsular gene has a vital role in Cryptococcus virulence. Robertson et al. [C. neoformans during the course of the infection) in all isolates [In this study, we investigated the presence of some virulence genes such as laccase (nd IGS1) . The stuisolates , 47. Then et al.  demonstrisolates .The studied virulence genes have been generally found to be recovered from the horses, horses’ environments, and humans in contact with the horses, which is in line with the findings of Firacative et al. , who supCryptococcus. C. gattii sequenced from different sources  confirmed that it can be isolated from those three sources with the same sequence type and indicated that C. gattii was shared between humans, animals, and the environment. The phylogenetic tree showed that the three sequences were found on the same clade, which explains the probability of the transmission of the infection between these sources. The presence of eucalyptus trees in equine farms could be one of the environmental sources of infection of horses with C. gattii, which would explain how the people in the surroundings get infected. The same theory was put forward by Farrer et al. [C. gattii VGV in samples from Hyrax-associated environments, which is suggestive of an association with these mammals that can spill over into humans.In the present study, a phylogenetic relationship was required to predict the epidemiological infection cycle of r et al. , who disC. neoformans and C. gattii from clinical samples  and C. gattii from environmental samples (soil) with clarifications of the possible epidemiological relationship between environmental and clinical isolates. Further studies need to be carried out on virulence by measuring capsule size and enzyme production. The research results provide insights into the potential exposure risk of humans in contact with cryptococcal infection in the investigated locations.The present study confirmed the presence of


Recently, new orthopoxviruses, including some able to infect humans, have been found and their complete genomes have been sequenced. Questions about the orthopoxvirus mutation rate and the emergence of new threats to humankind as a result of the evolution of circulating orthopoxviruses remain open. Based on contemporary data on ancient VARV DNA and DNA of new orthopoxvirus species, an analysis of the molecular evolution of orthopoxviruses was carried out and the timescale of their emergence was estimated. It was calculated that the orthopoxviruses of the Old and New Worlds separated approximately 40,000 years ago; the recently discovered Akhmeta virus and Alaskapox virus separated from other orthopoxviruses approximately 10,000–20,000 years ago; the rest of modern orthopoxvirus species originated from 1700 to 6000 years ago, with the exception of VARV, which emerged in approximately 300 AD. Later, there was a separation of genetic variants of some orthopoxvirus species, so the monkeypox virus West African subtype originated approximately 600 years ago, and the VARV minor alastrim subtype emerged approximately 300 years ago.Although variola virus (VARV) has been eradicated through widespread vaccination, other  Orthopoxvirus is the most notorious genus of the Poxviridae family, which comprises complex DNA viruses. Several viruses of the genus represent a potential threat to humans. The most dangerous member of this genus has been variola virus (VARV), which can cause smallpox. According to various estimations, smallpox killed 300–500 million people in the 20th century [orthopoxvirus infections. Besides VARV and MPXV, cowpox virus (CPXV) and VACV can infect humans; however, generalized infection can appear in individuals with impaired immunity. CPXV can infect many mammals, and various genetic variants of CPXV circulate in nature. Researchers believe that the ancestor of CPXV, with a wide range of susceptible hosts, is the progenitor of most of the currently studied specialized Old World orthopoxviruses. The main vectors of CPXV are numerous rodent species of a variety of habitats. This is a condition for the molecular evolution and diversity of CPXV and creates the possibility of the emergence of new orthopoxviruses pathogenic for humans and other mammals [ century ,2. By 19 century . Monkeyp century , and the mammals ,8.orthopoxviruses is expanding constantly. In addition to the aforementioned human pathogenic orthopoxviruses, Abatino virus, Akhmeta virus, and Alaskapox virus have recently been discovered in Italy, Georgia, and the United States, respectively [orthopoxviruses on the species phylogram of this genus, whereas Abatino clusters with ectromelia virus (ECTV). Progress in molecular methods has made it possible to study the genomic sequences of ancient VARV [orthopoxviruses and more reliably determine their time of origin and the rate of genetic variation.The range of studied ectively ,10,11. Tent VARV ,14,15,16orthopoxviruses and VARV in particular. In the previous studies, the rate of evolution has been calculated based on the central conservative region of the genome. The rate of mutation accumulation in the genomes of orthopoxviruses has been estimated to be approximately (4–6) × 10−6 substitutions/site/year [−6 substitutions/site/year and the time of VARV occurrence was calculated as 3000–4000 years ago [−5 substitutions/site/year. However, all analyzed VARV strains were isolated in the 20th century.A number of studies have been devoted to assessing the evolutionary rate of ite/year . The firears ago ,19,20. Wears ago  estimate−6 substitutions/site/year [−6 [−6 substitutions/site/year [Recently, ancient VARV DNA has been isolated from samples of a Lithuanian child mummy dating between 1643 and 1665 , from twite/year  and . The orthopoxvirus strains analyzed in this work are listed in orthopoxvirus strains included in the analysis had reliable collection dates.Nucleotide sequences of F4L and A24R (according to the nomenclature of the VACV strain Copenhagen) were isolated from genome sequences of orthopoxviruses  xviruses  and alighttp://iqtree.cibiv.univie.ac.at/, accessed on 10 November 2021) [A maximum likelihood phylogenetic tree was constructed by using the IQ-TREE 2.0 software (er 2021)  in assocer 2021)  and bootMolecular dating was performed by using the Bayesian Markov chain Monte Carlo (MCMC) inference method with BEAST 2  software . BEAST 2orthopoxviruses, various species and strains of orthopoxviruses were selected, including currently known strains of different genotypes of orthopoxviruses and all unique VARV strains (orthopoxvirus genome (approximately 102 kb).To study the molecular evolution of  strains . Notablyorthopoxviruses  years ago according to the results of the analysis. The ancestor of raccoonpox virus separated from skunkpox virus and volepox virus of the New World about 22,000  years ago. The last viruses, in turn, split about 13,000 years ago.From the analysis of the chronogram , it follorthopoxviruses, separated approximately 19,000  and 11,000  years ago, respectively (orthopoxviruses began their independent evolution approximately 5000 (95% HPD 4000–9000) years ago; the separation of MPXV took place about 3500 (95% HPD 2200–5400) years ago within this clade. The further appearance of the MPXV West African subtype occurred about 600 (95% HPD 300–1000) years ago.It should be noted that Alaskapox virus and Akhmeta virus, belonging to the Old World ectively . The braorthopoxviruses that are dangerous for humans, such as MPXV, remain in nature. Modern CPXV can be considered the closest relative of the progenitor of VARV, MPXV, VACV, CMLV, TATV, and ECTV  substitutions/site/year. The range of values in different branches varied from 1.6 × 10−6 to 8.2 × 10−6 substitutions/site/year. These values are consistent with estimates of the rate of variation of the VARV genomes obtained by Mühlemann et al. [Considering new VARV sequences from ancient burials, we re-calculated the rate of molecular evolution of  viruses . The gen is high ,23, a fa is high  and compn et al. .A group of ancient northern European strains—VK388, VK382, VK281, and VK470  from othAs was first established by Babkina et al. , all VARVARV strains of the P2 clade cause smallpox, characterized by a significantly lower mortality rate < 1%) than strains of the P1 clade causing variola major with a mortality rate of 8%–12% . It can % than stWith the updated data on ancient DNA, it became possible to recalculate the time of VARV emergence. In 2012, Biagini et al.  had estiAnalysis of the P1 VARV group shows the trends of grouping these 20th century strains by geography, with VARV strains from India present in most of these groups . This inorthopoxviruses  AD (Gerbilliscus kempi), respectively. Sole gerbils live in the savannas and dry forests of Africa. The overlap of the habitats of these animals indicates the probable origin of these viruses in the Middle East approximately in the 3rd century AD. The significance of such a biogeographic calibration was assessed by Ho et al. [Our updated reconstruction of the evolutionary history of –494) AD , Table 2 95% HPD 3–494 AD o et al. . In a re 95% HPD 3–494 AD orthopoxviruses in this study is a relative value, based on the smallpox virus, and may be inaccurate. The solution to this problem is possible with further discovery and extensive studies of ancient orthopoxviral DNA. New paleontological data on the expansion of the habitat of the orthopoxviruses hosts and consideration of biogeographic calibrations can also improve the dating. Further increasing knowledge will allow the evolutionary history of orthopoxviruses to be reconstructed with much greater accuracy.The dating of orthopoxviruses than previously thought. Circulation of various genovariants of CPXV and the recently discovered Alaskapox virus and Akhmeta virus in nature can lead to the emergence of new variants of orthopoxviruses with unknown properties. Further study of ancient fossil viral pathogens and the search for new orthopoxviruses circulating in nature will deepen our understanding of the evolutionary history of these viruses.In conclusion, the emergence of new genetic data has pushed the date of VARV origin from 3000–4000 years ago to about 1700 years ago. This indicates a higher rate of variability and evolutionary potential of pathogenic


Growing studies are revealing the critical manifestations of influenza, dengue virus (DENV) infection, Zika virus (ZIKV) disease, and Ebola virus disease (EVD) as emerging infectious diseases. However, their corresponding mechanisms of major complications headed for neuronal dysfunction are not entirely understood. From the mechanistic point of view, inflammatory/oxidative mediators are activated during emerging infectious diseases towards less cell migration, neurogenesis impairment, and neuronal death. Accordingly, the virus life cycle and associated enzymes, as well as host receptors, cytokine storm, and multiple signaling mediators, are the leading players of emerging infectious diseases. Consequently, chemokines, interleukins, interferons, carbohydrate molecules, toll-like receptors (TLRs), and tyrosine kinases are leading orchestrates of peripheral and central complications which are in near interconnections. Some of the resulting neuronal manifestations have attracted much attention, including inflammatory polyneuropathy, encephalopathy, meningitis, myelitis, stroke, Guillain-Barré syndrome (GBS), radiculomyelitis, meningoencephalitis, memory loss, headaches, cranial nerve abnormalities, tremor, and seizure. The complex pathophysiological mechanism behind the aforementioned complications urges the need for finding multi-target agents with higher efficacy and lower side effects. In recent decades, the natural kingdom has been highlighted as promising neuroprotective natural products in modulating several dysregulated signaling pathways/mediators. The present study provides neuronal manifestations of some emerging infectious diseases and underlying pathophysiological mechanisms. Besides, a mechanistic-based strategy is developed to introduce candidate natural products as promising multi-target agents in combating major dysregulated pathways towards neuroprotection in influenza, DENV infection, ZIKV disease, and EVD. As emerging infectious diseases, influenza, dengue virus (DENV) infection, Zika virus (ZIKV) disease, and Ebola virus disease (EVD) demonstrate various peripheral and central complications. Studies have shown that neurological manifestations have been a critical part of the aforementioned emerging infections , throughDespite advances in revealing the pathophysiology of emerging infectious diseases, underlying neuronal mechanisms require further investigation . On the Besides, limited studies reported natural secondary metabolites and candidate phytochemicals as promising agents for the prevention/treatment of emerging infections. This is the first comprehensive review on neurological manifestations of emerging infectious diseases and associated dysregulated pathways as well as the modulatory effects of candidate phytochemicals on the associated signaling pathways.Scopus, Medline, PubMed, Cochrane, and Web of Science were employed as electronic databases to conduct the comprehensive review. Besides, related articles in other sources were included. Accordingly, keywords (“Influenza” OR “Dengue” OR “Zika” OR “Ebola”) AND  (title/abstract/keywords) were used. All the phytochemicals possessing both the antiviral and neuroprotective activities within the classes  were also searched in the whole text. Overall, the entire plant-derived secondary metabolites with neuroprotective and antiviral effects, modulating neurological complications of emerging infectious diseases, were included. Data were collected without date and language restrictions until March 2021. The screening procedure of retrieved articles was also performed on the reference citation/lists. Regarding completing the search on electronic databases, hand searching also was provided relying on the authors’ expertise on the neuronal pathophysiological mechanisms of emerging infectious diseases and candidate phytochemicals.As provided, influenza, DENV infection, ZIKV, and EVD show neurological manifestations passing through multiple signaling pathways.Influenza virus, belonging to the RNA viruses in the Orthomyxoviridae family, is categorized into four virus types based on antigenically distinct of A, B, C, and D. However, influenza virus types C and D are not considered as health threats . In a re1N1 and associated pandemic in 2009, displayed a meaningful impact on both the central nervous system (CNS) and peripheral nervous system (PNS)  . Accordiem (PNS) , febrileem (PNS) .In a retrospective study, neurologic complications of the influenza virus were also evaluated. The findings showed that 4% of patients displayed associated neurological manifestations. In this line, the most reiterated complication was influenza-associated encephalitis (IAE) in 65% of patients, and 13% were categorized as having neurological residuals. In addition, 16% showed epileptic seizures, 5% demonstrated acute inflammatory demyelinating polyneuropathy (AIDP), and 14% were classified as having an infection-associated stroke . One of . study, the neurological involvement of DENV, in the form of atypical cases, was discussed and based on pathogenic mechanisms was categorized into metabolic disturbance , viral invasion , and immune-mediated reactions, inclusive of neuromyelitis optica, myelitis, ADEM, optic neuritis, encephalopathy, neuroophthalmic complications, and GBS  from the junction of white and grey matter , an RNA virus from the Filoviridae family, consists of five species, including olavirus . Experimolavirus  associatolavirus .The CNS neurological manifestations of EVD include seizures, meningoencephalitis , encephaIn a study by Bowen and coworkers, a certain degree of objective abnormality was observed on neurologic examination of EVD, such as impairments of either saccades or pursuits, tremors, abnormal sensory manifestations or abnormal reflexes, and frontal signs. Consistent focal deficits along with stroke have been also presented in several survivors of EVD, such as those with homonymous hemiparesis, hemianopia, and cranial nerve palsies .., 7 months of monitoring the physical and neurological manifestation in EVD showed that individuals presented decreased executive function and chronic fatigue  molecule is efficiently cleaved and producing the viral particles is the epithelium of the respiratory system. Influenza transmission eventuates from respiratory fomite or aerosols of a susceptible individual that comes into contact with other ones. Investigations in ferrets have shown that the soft palate is the primary source of the influenza virus that could be transmitted between people. Significantly, the soft palate is a rich source of α-2,6-linked sialic acids selected by the HA proteins recently detected in human influenza viruses . The patA case report study observed an increase in the levels of CSF neopterin in viral infection-related acute encephalopathy and encephalitis , which iReactive astrocytes, microglia, and glial cells have been detected as a pathologic sign of immune-mediated illness of the CNS. CD14 molecules are expressed on the surface of microglia , could bFrom another mechanistic point of view, following the entry of influenza virus, related M2 proton channels are activated by low pH of the associated endosome, thereby acidify the viral interior, and weaken the electrostatic interaction, which allows viral uncoating. In this line, inhibiting the function of M2 ion channel prevents the uncoating of the influenza virus. On the other hand, the HA of the influenza virus binds to receptors with neuraminic acid. The enzymatic activity of neuraminidase (NA) releases viruses by removing neuraminic acids from oligosaccharide chains of receptors. So, NA inhibitors (NAIs) are another class of anti-influenza drugs .in vitro infection of BV2 microglia cell line with DENV resulted in increased expression of proinflammatory cytokines, including IFN-γ, TNF-α, IL-6, IL-1β, IL-10, and monocyte chemoattractant protein-1 (MCP-1), as well as matrix metalloproteinase- (MMP-) 2 and MMP-9 . ZIKV encodes three structural proteins, that is, called envelope (E), capsid (C), and precursor membrane (prM), and seven nonstructural proteins, including NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. The structural proteins, E, and prM are used by the virus to attach to the neural cell membrane of the host. The nonstructural proteins and C protein localize to different organelles of the neuron such as Golgi apparatus, nucleus and its nucleoli, and cytoplasm lipid droplet, leading to apoptosis, cell cycle arrest, and cell death .via the nonstructural proteins-mediated interferon antagonism or by reducing the IFN production that is organizing the inhibition of ZIKV replication in human cells  intracellular cholesterol transporter 1 receptor  and tissue factor, reduced secretion of proinflammatory cytokines, and enhanced production of chemokines, for example, IL-8, C-C motif chemokine 2 (CCL2), CCL3, and CCL4  class. The rapid growth of viral resistance has appeared as the prevailing liability of DAAs, in particular when used against RNA viruses with error-prone polymerases such as the influenza viruses  or the rle Golgi .In spite of the fact that there are no approved biologic drugs for influenza treatment hitherto, neutralizing antibodies (nAbs) have been examined. Therapeutics-based antibodies, in most cases, are well-tolerated and demonstrate desired pharmacokinetic profiles . InfluenImmunization based on RNA has appeared as hopeful strategies in comparison with conventional approaches like vaccines . RecentlFor brain complications of influenza virus, best treatment practice includes initiate antiviral treatment during 48 h of onset , administration of a high dose of gamma globulin, and at the same time hormone shock therapy, which decrease the brain metabolism. Plasma exchange therapy is offered when disseminated intravascular coagulation (DIC) and/or MOF exist . PlasmapCurrently, a chimeric, attenuated vaccine against DENV   was provCurrently, there is no approved antiviral agent or vaccine against ZIKV disease. The major strategy for controlling ZIKV is suppressing mosquito breeding . The comin vitro models  can bind to the HA molecule in the influenza virus, thereby inhibit virus adsorption to the host cell, and lead to the inhibition of influenza infection . It has via antioxidant potential as well as inhibition of TLR signaling pathway and inhibiting caspase-3 activity  has inhibitory activity against various viruses and also can inhibit influenza virus replication affecting inducing IFN-γ secretion and also reduced neurological signs  suppresses the expression of RIG-1 and also leads to the reduction of IFNs and cytokines in influenza virus infection/replication and associated apoptosis . Resveraoduction . Based oectively . Consideectively . Evidencectively .As a triterpene saponin, glycyrrhizin has shown anti-inflammatory and antiviral activities . It decl. observed that berberine inhibited NLR family pyrin domain containing 3 (NLRP3) inflammasome activation in the influenza virus as well as a decline in ROS generation  significantly inhibited the level of TNF-α, IFN-γ, and IL-6 . Consistneration . In thisneration .Li et al. indicated that aloe emodin (an anthraquinone) binds to virus envelope and leads to the inhibition of influenza A replication and regulated the level of IFN-γ, IFN-β, and double-stranded-RNA-activated protein kinase in influenza A virus . Dai et As another phytochemical, carvacrol (a phenol) has shown anti-inflammatory, antiviral, and antioxidant effects, with inhibitory effects on influenza virus infection. Carvacrol acts through the regulation of the IFN-γ pathway, modulation of IL-6, IL-17, TGF-β, IL-4, and IL-10, and reduction of TLR7, myeloid differentiation primary response 88 (MyD88), interleukin-1 receptor-associated kinase 4 (IRAK4), TNF receptor-associated factor 6 (TRAF6), and NF-κB . In thisvia interacting with the HA type 2 subunit of the influenza HA protein. In this line, the inhibitory effect of quercetin against both H1N1 and H3N2 virus leads to the inhibition of virus replication towards the reduction of neurological manifestations , have anti-inflammatory, antioxidant, and antiviral effects of inhibiting TLR7, RIG-1, and AQP5 in the alveolar epithelial cells. It also inhibited the influenza virus and neurological symptoms  has anti-DENV activity, as it interferes with the late phase of the DENV lifecycle , which leads to the suppressed synthesis of viral proteins and declined level of NF-κB, towards suppressing viral replication during the early phase of DENV infection  suppressed the entry phase of the viral replication cycle and also can affect viral RNA replication and viral protein synthesis, as it has antiviral effects against other RNA viruses. One of them is ZIKV, a flavivirus related to DENV   . Similarr (EGFR) .in silico evidence has shown curcumin, rutin, sanggenon, delphinidin, isoquercetin, naringenin, and EGCG as antiviral activity against the ZIKV NS2B-NS3 protease activity and can inhibit replication of the virus to reduce neurological sign  Benn. can suppress ZIKV and DENV infection via inhibiting the virus entry phase (Silymarin and pinocembrin, as other natural compounds, also inhibit the production of ZIKV in the early phase . Evidencry phase .Overall, by targeting ZIKV proteases, phytochemicals seem to be promising candidates in combating emerging infectious diseases. Additionally, modulation of TAM may be a critical way for phytochemicals to combat ZIKV.Potential plant-derived secondary metabolites are shown to combat EVD. In this regard, curcumin blocks cytokine storm by suppressing IL-1, IL-6, and TNF-α, which correlates with clinical manifestations of Ebola . AdditioO-D-glucoside protected against Ebola in vivo. Moreover, it was shown that this quercetin derivative inhibited the early steps of viral entry (Recently, quercetin and other derivatives have been identified as affecting the entry process of EBOV . Anotheral entry . The mecal entry .in vitro antiviral effect of oleandrin acts as a novel cardiac glycoside against the EBOV (Other studies indicated that genistein has antiviral effects and inhibited EBOV replication . Ellagicthe EBOV . Some prthe EBOV . AltogetPhytochemicals have been excellent sources of alternative therapeutic agents and lead compounds in combating viral diseases. As a top global priority, novel plant-derived antiviral agents are promising candidates in combating neuronal manifestations of emerging infectious diseases. Consequently, inflammatory/oxidative pathways are critical targets, including ILs, TLR, NF-κB, MAPK, iNOS, and AQP, as well as several enzymes involved in the virus life cycle . The potin vitro and in vivo experimentation to highlight the significant pathophysiological mechanisms of viral diseases. Introducing other effective and novel plant-derived antiviral lead compounds through modulating such dysregulated pathways could pave the road in combating viral infections. It should be followed by well-controlled clinical trials to assess phytochemicals as multi-target alternative agents. Such reports will help reveal more applications of phytochemicals in the prevention, management, and treatment of emerging viral diseases.In the present review, a mechanistic approach has been employed on plant-derived antiviral compounds with related pharmacological mechanisms, as alternative therapies against neuronal signs of emerging infections . Future


KF] and extended [HFKE], extension [HE], abduction [HA], external [HER], and internal [HIR] rotation), knee (flexion [KF]) and ankle (dorsiflexion with knee flexed [ADFKF] and extended [ADFKE]) ROMs. Bayesian inferences exploring differences between player position, competitive level, sex and limb were made. A Bayesian correlation analysis was conducted to explore the influence of playing years on joints ROMs. The results showed no significant player position or competitive level related differences in any average ROM score. However, statistically significant sex-related differences were documented whereby female players reported higher hip and knee joints ROM average values than their male counterparts. Especially relevant were the proportions of males (72%) and players from teams engaged in the second division (61%) displaying limited HFKE ROMs. Likewise, around 35% of all players showed restricted ADFKF ROMs. In addition, approximately 21, 18, 22, and 25% of the futsal players were identified as having bilateral asymmetries (≥8°) for HA, HIR, HER, and KF ROMs, respectively. Finally, Bayesian correlation analysis did not report any significant association between years of playing futsal and ROM measures . The implications that these restricted HFKE and ADFKF ROMs and bilateral asymmetries in hip  and knee (flexion) ROMs caused by the practice of futsal may have on physical performance and injury risk warrant future research.The purposes of this study were to describe the lower extremities joints range of motion (ROM) profile using a comprehensive approach in futsal players and to examine potential player position , competitive level (first [top] division vs. second division), number of playing years, sex , and bilateral (dominant limb vs. non-dominant limb) differences. A total of 72 male and 67 female elite futsal players from 11 clubs were measured of passive hip (flexion with knee flexed [HF Futsal  is played worldwide with more than one million registered players all over the world . During It has been traditionally suggested that studies aimed at describing the effects of long-standing participation within a single sport on major joints ROMs are of interest because restricted values and bilateral asymmetries are not only detrimental to physical performance but also increase the risk of soft tissue injury . HoweverOnly Therefore, the purposes of this study were to describe the lower extremities joints ROM profile using a comprehensive approach in a large cohort of futsal players and to examine potential player position , competitive level (first [top] division vs. second division), number of playing years, sex , and bilateral (dominant limb vs. non-dominant limb) differences.A convenience sample of 139  elite futsal players from 12 different teams  completed this study. Before data collection, participants filled out a questionnaire containing questions about their sport-related background , anthropometric characteristics  and training regimen . Descriptive statistics for males and females are displayed in KF) and extended (HFKE), extension (HE), abduction (HA), external (HER) and internal (HIR) rotation; knee flexion (KF); and ankle dorsiflexion with knee flexed (ADFKF) and extended (ADFKE) ROMs of the dominant and non-dominant limbs were assessed following the methodology previously described . Prior to the testing session, all participants performed the dynamic warm-up designed by p > 0.05). Descriptive statistics (including means and standard deviations [SD]) were calculated for hip, knee and ankle ROM measures for all futsal players combined and also separately by player position , competitive level (first division vs. second division) and sex .Statistical analyses were performed using JASP software version 0.13.01 . Prior to the statistical analysis, the distribution of raw data sets was checked using the Shapiro–Wilk expanded test and demonstrated that all data had a normal distribution , from 0, from 0, from 0, from 0, from 1 to 3 = anecdotical evidence for alternative hypothesis (H1), from >3–10 = moderate evidence for H1, from >10 to 30 = strong evidence for H1, from >30–100 = very strong evidence for H1, > 100 extreme evidence for H1. Only those models that showed at least strong evidence for supporting H1 (BF10 > 10) with a percental error < 10 were considered robust enough to describe the main effects.Separate Bayesian paired samples uggested : < 1100 KF ROM (KE ROM (KE ROM (KF ROM (x2) test was used to examine potential player position , competitive level (first [top] division vs. second division) and sex  -related differences in the proportion of players showing limited scores and bilateral differences.Furthermore, and similar to what was carried out in previous studies on football players , in eachKF ROM , <80° , knee (KF), and ankle (ADFKF and ADFKE) joints for all players combined and separately by player position, competitive level and sex, respectively.t-tests did show no significant differences (BF10 < 10) between dominant and non-dominant limbs for each of the nine ROMs  and/or ADFKF (35%) ROMs  and 30% (HER ROM) of the outfield players. The Bayesian x2 tests did not show significant differences between the proportions of goalkeepers and outfield players with limited HFKE and ADFKF ROMs nor between those goalkeepers and outfield players who displayed bilateral differences ≥ 8° in these two ROMs. Approximately 26, 23, and 39% of players from teams engaged in the first division reported limited HFKE, KF and ADFKF ROM values, respectively. Furthermore, percentages of first division players ranged from 23 to 29% presented significant bilateral differences in their HFKE, HA, HIR, and HER ROMs. For its part, a percentage larger than 20% of the players from teams engaged in the second division showed limited HFKE (77%) and ADFKF (32%) ROMs as well as bilateral differences ≥ 8° in their HIR (30%), HER (31%), and KF (29%) ROMs. Significant differences in the proportion of first and second division players with limited ROM values were only found for the HFKE ROM (23% [first division] vs. 77% [second division]) (BFPoisson10  > 10). However, no significant differences were found between the proportions of first division and second division players who displayed bilateral differences ≥ 8° in their ROMs. Approximately 72, 24, and 35% of the male players displayed restrictions in their HFKE, KF and ADFKF ROM values, respectively. For females, 21 and 36% of them reported limited HFKE and ADFKF ROM values. The proportion of female players with limited HFKE ROM values was significantly lower than that of males (BFPoisson10  > 10). Likewise, 24% of the male players showed significant bilateral differences in their HER ROM whereas 22, 21, 24, 39, and 32% of the females presented inter-limb differences ≥ 8° in their HFKF, HFKE, HA, HIR and KF ROMs. The proportion of female players with bilateral differences ≥ 8° in the HFKE and HIR was significantly higher than that of males .The comprehensive analysis conducted in this study found that a significant number of the futsal players  demonstrate limited HF5%) ROMs . In addir values < 0.34).Finally, Bayesian correlation analysis did not report any significant correlation between years of playing futsal and ROM measures  and/or ADFKF (≈35%) ROMs, irrespective of their position, competitive level and sex. Previous studies using the same comprehensive analysis employed in the current study also identified a large number of youth (KE (≈45%) and/or ADFKF (30%) ROMs, despite the fact that their pooled average scores for these two ROMs were categorized as normal or non-limited [HFKE > 80° (KF > 34° (KE (≈47%) and/or ADFKF (35%) ROMs may be based on the demands of the game of futsal that requires players to perform several repeated high intensity movements such as sudden acceleration and deceleration, rapid changes of directions, kicking and tackling  for the factor sex on the ROM profile of the futsal players tested whereby females presented higher hip and knee ROM average values than male players. However, it should be noted that for both males and females, all ROM average values  were categorized as normal or non-limited. On the other hand, the comprehensive analysis conducted in this study also revealed that more than 20% of male and female players displayed restrictions in their HFKE and ADFKF ROM values. When potential sex-related differences in the proportion of players displaying limited HFKE and ADFKF ROM values were explored, only statistically significant differences were observed in favor of the males for the HFKE ROM . In practical terms, approximately 3 out of 4 diagnosed cases of limited HFKE ROM found in futsal players may be expected to be males. The fact that females presented lower volume of futsal practice per week than males (8.4 h vs. 10.8 h) alongside the larger number of high intensity movements that males perform during matches in comparison with females  may help elucidate the reasons why they show a high predisposition to suffer hip flexor muscles strains. For this specific HFKE ROM, the results also report that futsal teams engaged in the first division presented a lower proportion of players with limited values than their counterparts playing in the second division (27% vs. 61%). It is likely that the differences in terms of professionalism , match physical demands and training status of players between futsal’s first and second divisions may justify why the proportion of players showing limited HFKE ROM was lower in teams engaged in the first division than in the second division.The findings of this study also show that, and unlike the between-subject factors player position and competitive level, there was a significant main effect  ROMs over the course of a competitive season for professional male football (p < 0.05) decreases in ADFKF and HER ROMs from pre-season to post-season. Although these documented changes in ADFKF and HER ROMs were defined as statistically significant, their magnitudes  may be considered as not relevant from a clinical standpoint as they did not exceed the value of 1.5 times (80–90% of certainty) the magnitudes of the standard error of the measurement (SEM) reported in previously published reliability studies  makes comparisons difficult. Likewise, another limitation of this study relies on the fact that only ROM assessments were carried out and hence, it is not possible to determine whether the same bilateral differences could be found in other physical performance tests . The fact that the all the pre-season ROM assessments were not conducted in the same year  but in four different ones may be also considered a limitation as it cannot be assumed that all pre-seasons presented similar characteristics , which might have had an effect on players neuromuscular properties at the testing timepoint. For females, the phase of their menstrual cycle during testing was not recorded and this may have potentially influenced their ROM scores as fluctuating concentrations of estrogen throughout the menstrual cycle affect musculotendinous stiffness  and joinfootball  and basefootball  players, studies .KE ROMs. Moreover, around 35% of all players showed restricted ADFKF ROMs. The results of this study also indicate no significant differences in the ROM for the hip, knee and ankle joints between outfield players and goalkeepers. Likewise, it has been identified a large total number of players with bilateral differences ≥ 8° in HA, HIR, HER and KF ROMs. The number of females with bilateral asymmetries in HIR  and KF  is two time greater than in male players. The potential implications that these restricted HFKE and ADFKF ROMs and bilateral asymmetries in HA, HIR, HER, and KF ROMs caused by the practice of futsal may have on the physical performance and injury risk of the players warrant future research.The findings of this study reveal the existence of large proportions of males (72%) and players from teams engaged in the second division (61%) displaying limited HFThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Órgano Evaluador de Proyectos, Universidad Miguel Hernández de Elche approved the study (DPS.FAR.02.14). The patients/participants provided their written informed consent to participate in this study.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.