Background: Glycogen storage diseases (GSDs) are known as a group of disorders characterized by genetic errors leading to accumulation of glycogen in various tissues. Since different types of GSD can sometimes be clinically indistinguishable, next generation sequencing is becoming a powerful tool for clinical diagnosis.Methods: 12 patients with suspected GSDs and their parents were enrolled in this study. The clinical and laboratory data of the patients were reviewed. Causative gene variants were identified in the patients using whole exome sequencing (WES) and verified by Sanger sequencing.Results: Genetic testing and analysis showed that 7 patients were diagnosed with GSD II (Pompe disease), 2 patients with GSD III, 1 patient with GSD VI, and 2 patients with GSD IXα. A total number of 18 variants were identified in 12 patients including 11 variants in GAA gene, 3 variants in AGL gene, 2 variants in PYGL gene and 2 variants in PHKA2 gene, of which 9 variants were reported and 9 variants were novel. SIFT, Polyphen-2, Mutation Taster, and REVEL predicted the novel variants (except GAA c.1052_1075 + 47del) to be disease-causing. The 3D structures of wild/mutant type GAA protein were predicted indicating that variants p. Trp621Gly, p. Pro541Leu, p. Ser800Ile and p. Gly293Trp might affect the proteins function via destroying hydrogen bonds or conformational constraints. Neither liver size nor laboratory findings allow for a differentiation among GSD III, GSD VI and GSD IXα.Conclusion: Our study expanded the variation spectrum of genes associated with GSDs. WES, in combination with clinical, biochemical, and pathological hallmarks, could provide accurate results for diagnosing and sub-typing GSD and related diseases in clinical setting. GAA gene and in GSD III, AGL gene. In GSD VI, the clinical manifestations are associated with mutations in PYGL gene and in GSD IXα, PHKA1 and PHKA2 gene (Glycogen storage diseases (GSDs) as the innate defects of carbohydrate metabolism are caused by enzyme deficiency in the glycogenolysis or gluconeogenesis . There aKA2 gene .GSDs are a group of clinically and genetically heterogenous diseases. The clinical manifestations of GSDs range from almost no symptoms to severe cardiac dysfunction, respiratory insufficiency, and sudden death . GSDs arGAA, AGL, PHKA2 and PYGL gene related to GSD and the 12 patients were diagnosed with GSD types II, III, VI, and IXα respectively.In this study, by clinical and next-generation sequencing analysis of 12 patients with suspected GSDs, we aimed to give them molecular diagnosis of GSD and determine the spectrum of genetic analysis specific to our cohort. As a result, we found 18 variants in A total of 12 hospitalized children  in unrelated families with clinically suspected GSD were recruited for this study in Children’s Hospital Affiliated to Shandong University  from November 2018 to October 2021. All patients from the Han Chinese population in Shandong Province, China were examined and diagnosed by experienced pediatric specialists from the Neurology or Gastroenterology Department. The clinical records of 12 patients with GSD were retrospectively reviewed.http://www.1000genomes.org), ExAC (http://exac.broadinstitute.org/), Exome Variant Server  and in-house database to remove common variants . Frameshift variants, missense variants, premature stop-gain, initiation codon loss, and typical splicing site changes were prioritized. Effect on protein function of variations were predicted by SIFT, PolyPhen-2, MutationTaster, and REVEL. Descriptions of variants in Human Gene Mutation Database  and ClinVar (http://www.ncbi.nlm.nih.gov/clinvar) also were referenced. Swiss-Pdb Viewer 4.1 software was utilized to generate the 3D protein plots of wild-type and mutant type for novel missense variants. The variants identified in this study were classified according to the 2015 American College of Medical Genetics and Genomics (ACMG) guidelines . Mutation screening of the patients was applied by Whole exome sequencing (WES) with the Human Exome Probes P039Exome  on the Illumina NovaSeq5000 platform . The obtained mean exome coverage was more than 95% . Paired-end alignment was performed with version GRCh37/hg19 of the human genome on Burrows-Wheeler Aligner software (BWA Version: 0.7.10). Variant frequencies were determined in thousands of genomes .P < 0.05 (two-tailed) were considered statistically significant.Statistical analysis was performed on SPSS 24.0 software . The Median and Interquartile range (IQR) of onset age and diagnosis age was calculated. The Shapiro–Wilk test was used to test for normality of the data. For data with non-normal distribution, Kruskal-Wallis H test was used for multiple group comparisons and Mann-Whitney U test for two group. Clinical details were available for 12 patients. The age, sex, physical examination and laboratory findings at the time of diagnosis were recorded and showed in GAA gene (P1-P7), AGL gene , or PYGL gene (P10) were detected in our patients, respectively. Patients 11–12 were detected as heterozygotes for the PHKA2 gene variant. Totally 18 different variants in four GSD related genes were found , of which 9 variants were reported and 9 variants were novel. The variants in the patients were inherited from their carrier parents, except for 2 male patients with PHKA2 gene variant on the X chromosome were from their mother. GSD type, variation location, variation, amino acid variation, variant type, and mutation analysis results of the variations are given in Compound heterozygous/homozygous variants in The nine novel variants were determined after checked in all available databases and publications, including 1000 Genomes, ExAC, gnomAD, in-house databases and published articles. The scores of SIFT, Polyphen-2, Mutation Taster, and REVEL predicted that the novel variants (except c.1052_1075 + 47del) have a deleterious effect on the function of the protein . No predGAA and 1 of PHKA2 in the novel missense variants. The 3D protein structures of GAA with novel missense variants were predicted by Swiss-Pdb Viewer 4.1 indicating the variants might affect the proteins function via destroying hydrogen bonds, or conformational constraints which resulted in the proteins into disordered extended structures , followed by GSD III (n = 2) and IXα with 16.7% (n = 2).p > 0.05).Seven GSD II patients were divided into infantile-onset Pompe disease (IOPD)  group and late-onset Pompe disease (LOPD) (P3-P6) group according to appearance cardiomyopathy within/out 12 months and age of onset . The inip > 0.05). At diagnosis, both patients with GSD IX had a height shorter than -2 SDS, while growth retardation was not found in GSD III and GSD VI patients. Between GSD II group and the hepatic GSD group, significant differences in liver size and partial laboratory findings  were found.There were 5 patients with hepatic GSD in our study, including 2 patients with GSD III, 1 patient with GSD VI, and 2 patients with GSD IXα. Hepatomegaly, hypoglycemia and hypertransaminase were observed in all patients, hypertriglyceridemia in 80% (4/5) patients and hyperlactate in 60%  patients. The first and main symptom of patients  with GSD III was progressive abdominal distention (100%) due to enlarged liver. The main symptom observed in the patient (P10-P12) with GSD VI or IXα was hypertransaminasemia (100%) detected on laboratory examination accompanied by hepatomegaly. Neither liver size nor laboratory findings allow for a differentiation among GSD III, GSD VI and GSD IXα (®) injection, 4 of whom responded to treatment, 2 were discontinued due to death with severe myocardial hypertrophy and pulmonary hypertension and hypersensitivity, and 1 patient lost visit. Two patients with GSD III received oral raw cornstarch and increased dietary protein after diagnosis. During treatment, they occasionally experienced convulsions due to fasting hypoglycemia, and developed symptoms of short stature and delayed motor development. In patients with GSD VI and IXα, after receiving oral raw cornstarch, the clinical symptoms basically disappeared except for the hypertransaminases.Of seven GSD II patients, six received enzyme replacement therapy (ERT) , 2 carrying AGL mutations , 1 carrying PYGL mutation , and 2 carrying PHKA2 mutations . According to the ACMG guidelines , GSD IX (PHKB and PHKG2) was the major type of GSD  manifesting generalized myasthenia and hypertrophic cardiomyopathy within the first months of life were IOPD. Two of them also showed severe malnutrition. Four patients (P3-P6) who presented as progressive limb-girdle muscle weakness with hyperCKemia were LOPD. Noteworthily, although P4 developed dyspnea shortly after birth due to respiratory muscle involvement, but she had relatively mild myocardial damage and was classified as LOPD (GSD II (MIM# 232300), also known as Pompe disease, caused by deficiency of the enzyme acid α-glucosidase encoded by truction . GSD II truction . In our  as LOPD .GAA variants reported worldwide, c.-32-13T>G is the most common, especially in Caucasian populations. In Asia Pacific, p. Asp645Glu, p. Trp746Cys, p. Gly576Ser, p. Glu888X were most reported , VI  and IXα  belong to hepatic GSD with basic features of hepatomegaly and hypoglycemia , among which missense/nonsense mutations are the most common. So far, 17 Chinese patients with GSD VI have been reported, and no PYGL hotspot variant has been found in this group  being the most common, followed by small deletion variants. In this report, we detected two novel variants in PHKA2, c.3507_3520del (p.Gln1169Hisfs*29) and p. Arg295Gly. p. Gln1169Hisfs*29 lead to fourteen-nucleotide deletion within exon 32 of PHKA2, that seemed to result in a frameshift introducing a premature stop codon after 2602 bp of amino acid residue 1196 of 1235. The variant was predicted disease-causing by Mutation taster and evaluated as a pathogenic variant refer to ACMG guidelines. Notably, the PHKA2 gene had a total of 33 exons, and one-fifth (34/166) of the reported variants were located in or spanned exons 30–33, suggesting that the C-terminus of the PHKA2 was a variant-dense region. p. Arg295Gly is an unreported missense variant, but p. Arg295His  by whole-exome sequencing. Our study expanded the variation spectrum of gene associated with GSDs. Next-generation sequencing was considered the preferred diagnostic method for GSD because invasive liver biopsy can be avoided.This study reported 12 Chinese patients with Glycogen storage disease and identified 18 gene variants of


We found RB-UV was a very effective procedure for inactivating viruses of amnion and chorion tissues, which could be used as a complementary method to Co-60 irradiation. In addition, we also screened the washing solutions and drying methods for the retention of growth factors.Allogeneic amnion tissues have been widely used in tissue repair and regeneration, especially a remarkable trend of clinical uses in chronic wound repair. The virus inactivation procedures are necessary and required to be verified for the clinical use and approval of biological products. Cobalt-60 (Co-60) or electron-beam (e-beam) is the common procedure for virus and bacterial reduction, but the excessive dose of irradiation was reported to be harmful to biological products. Herein, we present a riboflavin (RB)-ultraviolet light (UV) method for virus inactivation of amnion and chorion tissues. We used the standard  Amnion is an avascular, aneural, and alymphatic tissue and has limited expression of human leukocyte antigen (HLA)-I antigens . The immAs a biocompatible and bioactive allogenic tissue-repairing material, amnion has been widely used in biomedical applications, including corneal ulcers, diabetic foot ulcers, venous ulcers, postoperative dehiscence, and burn wounds . Amnion Currently, solely in the United States, there are more than 100 amnion or chorion products in the market . Most ofThere are three popular ways of preserving amnion or chorion tissues, including air-drying, lyophilization, and cryopreservation . The dehAllogeneic and xenogeneic biological products usually require virus inactivation processes for clinical use and marketing approval. In the past decades, the blood-transmitted diseases such as human immunodeficiency virus (HIV) and hepatitis B virus (HBV) have been greatly reduced because more attention was paid to the risks and more sensitive detection methods were invented to screen the donor samples for blood transfusion or for making biological products . The winThe currently used amnion and chorion are donated placenta tissues from birth delivery. Both amnion and chorion membrane tissues are usually dissected and cut off from a placenta disk. Both layers are used either individually or in combination as a complex for tissue substitution . SteriliTissue procurement and ethics statements were provided by Qilu Hospital. Tissues were collected from eligible donors after obtaining written, informed consent.2O) depending on the experiment design. Amnion was washed twice, each for 1 h, rinsed in pure water, then kept in the fridge with normal saline overnight for the following virus inactivation procedures. Chorion was washed three times, each time for 1 h, and washed once again for another 2 h with 0.5% Triton X-100 in pure water, rinsed with pure water, then kept in the fridge with 0.1% EDTA-2Na in PBS overnight for the following virus inactivation procedures.Amnions and chorions from caesarean delivery were isolated as described elsewhere . In brie2 as required by the experiments, the samples were turned over after each cycle of 1 J exposure.RB is also known as vitamin B2 (VB2). 5 ml of 500 μmol/L RB stock solution  was diluted into 50 ml working solutions using normal saline. The washed samples described earlier were immersed within the aforementioned RB working solution for 2 h. Amnion or chorion was then spread on a tray and incubated in a UVB crosslinker  with an energy of 3–7 J/cmThe amnion and the chorion after virus inactivation were then rinsed in pure water and dried on a patterned drying board, which was designed locally, either separately or combined as a multilayer amnion/chorion complex . The airThe amnion, chorion, or the dual-layer complex was then cut and packed using a blister package and a Tyvek lid for the following Co-60 irradiation. Co-60 irradiation was performed by Zhongjin Irradiation Incorporated Company. The Co-60 irradiation doses were monitored using dosimeters (Harwell Red 4034 pmma).bFGF and HGF were detected using a Human bFGF ELISA kit  and a Human HGF ELISA kit . Experiments were performed using a standard curve method according to the user manuals. For all the tests, the standard curves reached an R-square > 0.99. Total protein was detected using a BCA kit . The testing samples were cut into minimal particles and homogenized using a homogenizer  in an IP and western cell lysis buffer  with PMSF  according to the user manual, followed by the full lysis using the lysis buffer within the kit at 4°C overnight and a centrifuge process, the lysate was collected and measured using the aforementioned ELISA assay kits. The bFGF and HGF results were normalized using the total protein concentration of each sample lysate. The high-throughput multicytokine-array screening of growth factors and cytokines was performed by Raybiotech using a Human Cytokine Array Q440 kit (QAH-CAA-440).in vitro limiting dilution assay , and the virus load was compared to the control to calculate the fold reduction of virus. For Co-60 irradiation, the amnion/chorion complex was challenged with 10 kGy, 15 kGy, or 17.5 kGy Co-60 irradiation to determine the reduction of the virus titer.Viruses were loaded on actual processed samples before the virus inactivation point designed to mimic the real circumstances. For the initial loading, amnions, chorions, or amnion/chorion complexes were immersed within viral solutions overnight at 2–8°C. Next, the concentrations of the viruses were detected and recorded as the initial loading. The virus-loaded samples were immersed within the 50 μmol/L RB working solution for 2 h. The virus loading was recorded and detected as the control. Then, samples were challenged with 0, 3, 5, or 7 J/cmTM  and the secondary wound dressing of Tegaderm, or Tegaderm alone as blank control. Animal weights and wound photos were collected weekly. The wound area was calculated using ImageJ. All animal procedures and animal care were performed in accordance with the Shandong Laboratory Animal Regulations. All animal procedures were approved by the Animal Welfare and Ethics Committee of Shandong Xinbo Pharm. Ltd.Animal experiments were performed by Shandong Xinbo Pharm. Ltd. A chronic wound model with an induced toxic level of reactive oxygen species (ROS) around the wound area was developed as previously described . In brieFor histology tests, the samples were fixed with 4% paraformaldehyde for at least 24 h, embedded with paraffin, sliced, and subjected to HE, Verhoeff–Van Gieson, and Masson trichrome staining procedures using standard protocols.The statistic calculation method was indicated at the end of the annotation of each figure or table.It was reported that amnion and chorion tissues could be preserved by dehydration and the following sterilization steps for long-term storage without losing their tissue-repairing potency . Before Not a surprising result showed that hypertonic saline in the washing step has a general tendency of reducing the growth factors of bFGF and HGF, compared with normal saline or pure water . SpecifiThe air-dry procedure maintained similar content of bFGF and HGF growth factors compared with the freeze-dry procedure . We did 6-fold reduction of virus  has been used for virus inactivation of whole blood or blood components to close the window period of virus screening. The method is known for its biosafety and nonharms for whole blood and blood components . But theThe commonly using virus inactivation technologies include the treatment with gamma irradiation, heat or UV-radiation; the treatment with photoactive compounds such as methylene blue, psoralens, or 1.5-iodonaphtyl azide; the treatment with ionic or non-ionic detergents such as Tween-20/Tween-80, Triton X-100 or sodium dodecyl sulfate and with solvents such as alcohol and acetone . The com–6. The dose was determined and verified according to a standard method  of 10d method . The bioWe also analyzed whether the growth factor content of amnion and chorion was affected by the washing reagents. Amnion has been reported to contain numerous growth factors and MMPs, including bFGF, HGF, KGF, EGF, and PDGF-BB. We found that, compared with normal saline, hypertonic saline substantially reduced the number of growth factors, using bFGF and HGF as the marker. We also compared the freeze-dry and air-dry procedures, amnion/chorion tissues derived from both drying methods reserved similar growth factor contents. The main contaminating maternal cells in chorion and amnion are usually blood cells. The vigorous washing steps were required to remove most of the blood cells within the tissue, although there were still rare blood cells sparsely residing within the chorion tissue with minimal risks. The washing process can be simply monitored and verified by urine occult blood test strips to minimize the risks. In addition, there were residues of degenerated maternity-origin decidual layer tissues attached to the chorion layer, although there is no report on the immunogenicity of chorion tissues despite their broad clinical applications.Using the washing, drying, and viral inactivation methods described earlier, we made an amnion/chorion complex graft and tested the tissue-repairing capacity of it. We compared the amnion/chorion complex, Mepitel, and the blank control on a diabetic wound model. The application of the amnion/chorion complex and Mepitel has a tendency of accelerated recovery in terms of wound area compared with control although data were not statistically significant. The data favored the amnion/chorion group compared with the blank control on day 7 and day 14 in terms of the number of fully recovered animals.We herein provide an RB-UV method for virus inactivation of amnion and chorion tissues. The present article also describes a way of washing and drying of amnion and chorion tissues, which greatly retained the growth factors.


Dysmorphic uterus or T-shaped uterus is an increasingly frequent diagnosis among the infertile population that has been associated to worse reproductive results. Hysteroscopic metroplasty is a safe and simple procedure that can improve the reproductive outcomes in this group of patients, although the benefits of this procedure remains controversial due to the lack of adequate scientific evidence.To analyze the hysteroscopic metroplasty using the SWOT  methodology.An electronic search from inception each database up to December 2021 including the following databases was conducted: PubMed-MEDLINE, EMBASE, Web of Science, The Cochrane Library, and Google Scholar.Studies reporting outcomes of patients undergoing hysteroscopic metroplasty were included.Not applicableClinical evidence from the included studies suggests an improvement in reproductive results after performing hysteroscopic metroplasty especially in women with recurrent pregnancy loss and previous infertility, but all of them have relevant methodological limitations. For this reason, benefits, risks and alternatives of this intervention should be considered with caution.Evidence from published data shows a probable association between dysmorphic uterus and poor reproductive outcomes. Hysteroscopic metroplasty in patients with dysmorphic uterus could improve pregnancy outcomes, but there is need of properly designed prospective controlled studies to determine the benefits of this technique. Since tn et al. , DU U1a n et al. . Althougn et al. –8. DU U1n et al. .The prevalence of dysmorphic uterus in the general population has not been determined  because Some controversial issues regarding dysmorphic uterus among the existing classification systems have generated conflict in its definition, resulting in added difficulty in establishing the prevalence of this malformation. T-shaped uterus was first included in 1988 American Association of Reproductive Medicine (ASRM) classification of uterine anomalies in class VII (DES-related) . Later, Several studies , 18 haveHowever, it is important to highlight that the evidence regarding the efficacy of this procedure must be considered of very low quality as the available studies are mainly retrospective, observational and lack a control group, as it has been highlighted in three recently published review papers by Garzon et al. , de FranFor the above mentioned reasons, we have conducted a SWOT  analysis to evaluate the available scientific evidence on the impact of hysteroscopic metroplasty in women not exposed to DES who are diagnosed with dysmorphic uterus and desire of future fertility .https://www.epigear.com/index_files/metaxl.html epigear.com.) The global pooled pregnancy rate after hysteroscopic metroplasty was 68.9%  and global pooled live birth rate was 56.2% . In patients with history of recurrent pregnancy loss, pooled pregnancy rate after metroplasty was 78.9%  and live birth rate was 62.8% . In patients with primary infertility, the pooled pregnancy rate was 65.6%  and live birth rate was 54.5% . In patients with recurrent implantation failure the pooled pregnancy rate was 71.5%  and live birth rate was 63.2%  and performed a pooled analysis of the obstetrical outcomes. After the search, eleven studies were selected Alonso et al. , AdriaenThe described results indicate a clear improvement in the obstetrical outcomes of patients with dysmorphic uterus after hysteroscopic metroplasty. All the included studies reported the live birth rate before and after the hysteroscopic metroplasty revealing a dramatic increase. The average live birth rate before the metroplasty was under 2%. After the procedure, the average live birth rate was over 55%, with some series showing live birth rates as high as 78% . RegardiIn all the included studies except one , the hysAll the included studies were performed by experienced hysteroscopic surgeons in the field of reproductive surgery from different parts of the world, which supports the consistency of the results.An important source of heterogeneity is the absence of a consensus regarding the definition of dysmorphic uterus. Among the studies included in our systematic review, seven used the ESHRE-ESGE classification Adriaensen et al. , Di Spiein utero to dietilestilbestrol, date from 1977  and 3D transvaginal ultrasound (3D-US), as both provide a coronal view of the uterine cavity. Due to the simplicity and accessibility of the 3D US technology, it is becoming the diagnostic modality most commonly used for the diagnosis of Müllerian anomalies.In our review, five of the eleven included studies used 3D transvaginal ultrasound Alonso et al. , Boza etDespite the increasing number of publications Alonso et al. , AdriaenAnother important limitation is that the published studies reporting obstetrical outcomes after hysteroscopic metroplasty included a heterogeneous group of women, some of them with diagnosis of primary infertility Alonso et al. , AdriaenAnother source of bias is that the metroplasty was performed using different hysteroscopic tools. Currently, the use of hysteroscopic 5 French scissors and normal saline as the distention media represents the most common technique Alonso et al. , AdriaenAlonso et al.  have desAnother threat present in any surgical procedure is the risk of complications. In light of the data published in the literature we can consider that intraoperative complications during metroplasty for dysmorphic uterus are rare. Ducellier-Azzola et al.  in one oWith regard to safety, it is well known that expert high volume surgeons have better surgical outcomes and lower complication rates for any surgical procedure .The incidence of obstetrical complications such as late pregnancy loss or premature delivery was not augmented by the hysteroscopic metroplasty. An important aspect to consider is whether pregnant patients who have had hysteroscopic metroplasty before conception would benefit from an elective cesarean section as a delivery route. Although vaginal delivery is not contraindicated, a high rate of cesarean sections (33%–61%) has been reported in patients with history of metroplasty , 31, 35.There has been a considerable debate on the role of office hysteroscopy in the work up of infertile patients before Assisted Reproductive Techniques (ART) . A recenSeveral authors, as Di Spiezio et al.  and AlonOne of the main threats is the lack of a consensus for the diagnosis of dysmorphic uterus. This terminology was established by the ESHRE-ESGE classification, corresponding to the Class U1 anomaly  includinThe diagnosis proposed by ESHRE-ESGE and the former AFS classifications is based on subjective impression of the visual analysis of the coronal view of the uterus . This generates a high risk of biased interpretation and high inter-observer variability when establishing the diagnosis. In an attempt to overcome this problem, some authors have proposed objective criteria for establishing the diagnosis of dysmorphic uterus . HoweverAnother threat is related to the expertise of those who obtain and interpret the images of patients with dysmorphic uterus. To date, there is no evidence about the learning curve for achieving competency for this specific diagnosis.Several studies in the literature have tried to establish the optimal interval between hysteroscopic surgery and embryo transfer, but there are no studies that address this issue after metroplasty for dysmorphic uterus. It is clear that the endometrial healing time will depend on the procedure performed. After polypectomy, it has been seen that intervals greater than 120 days do not provide advantages and are even associated with worse biochemical and clinical pregnancy rates . AnotherIn patients undergoing hysteroscopic metroplasty, attempting a normal vaginal delivery is not contraindicated, even if the uterus could be considered scarred. Unfortunately, very high rates for cesarean section in patients with a history of hysteroscopic metroplasty who subsequently conceived are reported in the literature (33% to 57%). We have also confirmed this finding with a striking 61% cesarean section rate , 31, 35.Over the last decade, dysmorphic uterus has gained attention in the field of reproductive medicine, being increasingly referred as a potential cause of fertility impairment. From the perspective of the actual definition, this anomaly shows notorious differences with its precursor DES derived T-shaped uterus.Lack of consensus of a universally accepted diagnostic criteria of this anomaly limits the accuracy of its clinical characterization, and perhaps the validity of analytical studies assessing the potential effects on reproductive outcomes.Currently, there is no high-quality scientific evidence that supports performing hysteroscopic metroplasty in patients with DU U1a, although the case series included in this review suggest that in patients with previous adverse obstetric outcomes this minimally-invasive procedure can improve pregnancy and live birth rates.Although the efficacy of this technique needs to be confirmed with properly conducted prospective randomized controlled studies, due to its simplicity and low complication rates, hysteroscopic metroplasty could be offered to patients with a DU U1a and history of poor reproductive outcomes, provided a thorough discussion with the patient.Responsible for the document drafting and revision. All authors contributed to the article and approved the submitted version.


In response to the enormous threat to human survival and development caused by the large number of viruses, it is necessary to strengthen the defense against and elimination of viruses. Metallic materials have been used against viruses for thousands of years due to their broad-spectrum antiviral properties, wide sources and excellent physicochemical properties; in particular, metal nanoparticles have advanced biomedical research. However, researchers in different fields hold dissimilar views on the antiviral mechanisms, which has slowed down the antiviral application of metal nanoparticles. As such, this review begins with an exhaustive compilation of previously published work on the antiviral capacity of metal nanoparticles and other materials. Afterwards, the discussion is centered on the antiviral mechanisms of metal nanoparticles at the biological and physicochemical levels. Emphasis is placed on the fact that the strong reducibility of metal nanoparticles may be the main reason for their efficient inactivation of viruses. We hope that this review will benefit the promotion of metal nanoparticles in the antiviral field and expedite the construction of a barrier between humans and viruses. For decades, human survival and the global order have been severely threatened by viruses, such as novel coronavirus disease 2019 (COVID-19), Ebola virus (EBoV), severe acute respiratory syndrome coronavirus (SARS-CoV) and human immunodeficiency virus (HIV), etc. ,4. Even Over a long time, enormous contributions have been made to virus inactivation by many conventional disinfection techniques. For instance, physical techniques consist of ultraviolet (UV) radiation, heating, reverse osmosis, filtration and high pressure ,9,10. ChAs a novel technology that has flourished for decades, the unique properties of nanomaterials may provide an alternative solution for controlling the transmission of viruses ,18,19. AIn reality, the chemical nature of the metal elements may determine the antiviral properties rather than the morphological characteristics of the nanoparticles. To this end, this review begins with an exhaustive compilation of the antiviral performances of metal nanoparticles and other metallic materials. Afterwards, we comprehensively evaluate the antiviral heterogeneity of metallic materials according to the chemical nature of metal elements. Together, we hope that this review will help researchers in various fields to select suitable substrates for antiviral materials based on the chemical nature of metal elements, which will promote the development of novel powerful weapons for virus elimination.Thus far, metal mineral resources are still abundant in the crust of the earth, and humans have been exploiting metal ore for tens of thousands of years on earth . MeanwhiThe performances and mechanisms of metallic materials as antibacterial agents have been exhaustively summarized ,39,40,41To date, metallic materials with proven antiviral capability are mainly composed of noble metal elements  and transition metal elements  ,47,48,49In previous studies, a variety of antiviral efficiencies and mechanisms were shown to exist due to the different conditions comprising forms of metallic materials and test environments, which confounds the judgment on the critical mechanism of virus inactivation by metals. Hence, in order to assess whether the chemical nature of the metal element or the morphology of the material is the major contributor to the antiviral effect, we provide a synopsis of the antiviral performances and mechanisms of metal nanoparticles, metal ions, pure metals and alloys and metal compounds.Nanotechnology is an emerging field that has flourished in recent decades, and a large number of materials with nanostructures have been synthesized by this technique, such as nanoparticles (NPs), nanowires, nanorods and nanofilms ,18,19. A6 PFU/mL (plaque assay) decreased 3.5 log10 dramatically after 60 min of exposure to CuI NPs. The median particle diameter (D50) of CuI NPs was 160 nm, and the concentration for 50% of the maximal effect (EC50) was approximately 17 μg/mL.Copper iodide (CuI) is an active inorganic catalyst with excellent photodegradation ability , and it 2•−), which are generated by the reductant Cu+ released from CuI in water. The reaction is described in Equations (1)–(3)  = 0.2 mg/L) in one apiary as the test group. No Ag+ was added to the brown sugar syrup of another apiary as a control group. The results showed that the population density and activity of the bees in the test group remained well within 30 days of observation, and the bees survived longer. However, the population density of bees in the control group decreased rapidly after 8 days of observation, the worker bees stopped their daily work, and many bees even escaped from the hives. These results demonstrated that Ag+ is therapeutic but not curative for bees infected with SBV.In another study, the ability of Agstigated . In this2+) had no direct inactivation ability prior to TGEV infection and could not prevent the virus from binding to host cells. However, the titer of TGEV (106.7 TCID50/mL) significantly decreased by 1.4 log10 after the addition of Zn2+. Zn2+ mediated antiviral effects by inhibiting viral penetration or reducing the life cycle of the virus inside host cells, and researchers speculated that the intracellular target of Zn2+ might be viral RNA polymerase. In addition, the virus inactivation efficiency was positively correlated with the concentration of Zn2+, but Zn2+ could cause cytotoxic effects at high concentrations.Zinc is an essential trace element in organisms, that not only participates in cell division and energy metabolism ,92, but 2+ doped porphyrin complex had stronger virus suppression ability than the porphyrin complex not doped with Zn2+. Moreover, the nitroporphyrin-zinc complex was not specific for virus inhibition, that is, it could effectively inhibit both HIV-1 and SIVmac. Researchers found that porphyrins might enter the host cell membrane and inhibit fusion between the virus and the cell membrane, thereby hindering viral spread  was demonstrated in another investigation . In thisl spread . However5 TCID50/mL) decreased by 3.1 log10 after 20 min of exposure on the coating. The inactivation rate of other viruses on the coating was slower, for instance, the titers of influenza (3.63 × 105 TCID50/mL), dengue (3.98 × 106 TCID50/mL) and herpesviruses (1.00 × 106 TCID50/mL) decreased by 1.3 log10, 2.3 log10 and 5.0 log10, respectively, after 240 min of exposure to the coating. In particular, the titer of the unenveloped coxsackievirus B3 did not change significantly.Besides the study of single ions, other research also demonstrated the antiviral properties of hybrid coatings synthesized from silver, copper and zinc cations against HIV-1, influenza virus H1N1, human coxsackievirus B3, human herpes virus 1 and dengue virus . The stu2+ and Ag+ killed the virus by directly cleaving the envelope or binding to the thiol group of the protein, while the exact mechanism of virus inactivation by Zn2+ remains unknown. Furthermore, the research team also found that the activity of mammalian cells did not change significantly even after four hours of exposure. In the future, metal ion hybrid coatings can be applied in various devices in the medical field to block the spread of pathogenic microorganisms on solid surfaces. The antiviral performances of metal ions are summarized in In reality, a major contribution to the inactivation of these viruses was the release of metal ions from the hybrid coating. CuPure metals and alloys are widely distributed and abundantly stored in the crust of the earth. They can be industrialized and commercialized without processing or light processing ,98. FurtPrevious studies have shown that the virus could not only spread through water and air, but could also adhere to the surface of solid materials and survive for a period of time . When thThe application of copper in disease treatment can be traced back to the 19th century or even earlier. In the past two decades, some studies have proven that different types of viruses, bacteria and fungi can be quickly killed on the surface of copper . For ins6 virus particles/mL) only decreased by 0.3 log10 after 6 h of incubation on the stainless steel surface, and decreased by 0.6 log10 after 24 h of incubation. However, after 30 min of incubation on the copper surface, the number of viruses dropped by 0.6 log10, and the number of viruses dropped by 4 log10 after incubation for 6 h. Otherwise, the researchers speculated that copper might prevent virus replication by damaging viral RNA.Moreover, another research team compared the effects of copper and stainless steel surfaces on the number of influenza A viruses . In this4 PFU/mL of HCoV-229E was stained with copper, copper-nickel alloys (containing 90% copper) and brass (containing 70% copper) in the range of 1 cm2, respectively. The results showed that the virus inactivation rate was directly proportional to the percentage of copper in the material. Almost all coronaviruses can be inactivated (3 log10 reduction) within 30 min of exposure to 90% copper alloys. Even if exposed to alloys with low copper content (70%), the coronavirus can be completely inactivated (3 log10 reduction) within 60 min. In particular, copper caused the fragmentation of the viral genome during inactivation, thus ensuring that inactivation was irreversible. Moreover, the researchers found that the Cu2+ and Cu+ dissolved from the copper alloy played a major role in virus inactivation, and O2•− enhanced the inactivation effect. However, they did not clarify which component of the virus was destroyed during the inactivation process by copper ions.More recently, the efficacy of a series of copper alloys to inactivate human coronavirus (HCoV-229E) was determined through experiments . In this5 PFU/mL) within 9–10 min of contact time, and the removal efficiency was 4.5–6 log10. In addition, the researchers also found that the virus removal efficiency of ZVI was positively correlated with the virus concentration in the water. The antiviral performances of pure metals and alloys are summarized in Zero-valent iron (ZVI) is an active metal with strong reducibility that has been widely used in wastewater treatment, drinking water purification and groundwater remediation . In addiMetal compounds  are also widely distributed in nature, and these metal compounds have thermal stability, chemical stability, photocatalysis and other characteristics ,108. Alt2O) and cuprous oxide (CuO) are the two oxide forms of copper, and the inactivation effects of these two compounds on influenza A virus and bacteriophage Qβ have been reported by researchers . M. M2) is 2-modified hydroxyapatite composites (HA/TiO2) was reported in an earlier study  is the concentration of Mn+ in the solution, (mM); t is the incubation time of Mn+ and virus, h.When metal nanoparticles are mixed with viruses, a portion of the Mdatabase  134]. T. Tn+ reln+  cannot form stable complexes with EDTA, while alkaline earth Mn+ and transition state Mn+ can form stable complexes with EDTA. These results indirectly prove that many Mn+ have the potential to combine with viral biomacromolecules through chelation reactions. However, there is no significant correlation between the equilibrium constant of the chelation reaction and the antiviral rate of Mn+, and few studies have shown that Mn+ such as Cd2+ (lg K0 = 18.24) and Ca2+ (lg K0 = 12.39) have significant antiviral properties  on the non-enveloped virus protein coat results in the accumulation of a net charge on the viral surface . On the ractions .The ionic potential (Φ) is a measure of charge density, which is used to evaluate the ability of ions to electrostatically attract ions with opposite charges and repel with the same charge . The ionn+ with a larger ionic potential means that it is more electrostatically attractive to the virus. As shown in 3+ and Fe3+ have larger ionic potential and stronger electrostatic attraction with viruses, while Ag+ has smaller ionic potential and weaker electrostatic attraction with viruses. In fact, Ag+ has excellent antiviral ability, while Cr3+ and Fe3+ can hardly inactivate viruses effectively. The strong electrostatic interaction force does not represent excellent virus inactivation ability; thus, we conjecture that the ionic potential is also not a determining factor affecting the antiviral ability of metallic materials. For Mn+ with larger ionic potential, stronger electrostatic attraction may contribute to limiting virus spread and blocking virus invasion into host cells.Generally, in the case of a net negative charge on the viral surface, Mn+ or metal nanoparticles, which may weaken the antiviral ability of metallic materials. Certainly, increasing the dose of metallic materials can overcome these factors, which affect the antiviral capacity, but this does not make sense as high doses can also cause damage to normal surviving host cells.In addition, the electrostatic interactions between viruses and metallic materials are also affected by ion concentration, solution pH and coat protein functional groups. For example, viruses exhibit significant aggregation between particles due to weakened electrostatic repulsive interactions at lower pH . Signifin+ in a low valence state with stronger reducibility. Mn+ with stronger reducibility is more likely to react with oxygen in the environment to generate ROS that is lethal to the virus, and the reaction equation is described in Equation (16) (the products are exemplified by O2•−). In order to verify this conjecture, we queried the previous manuals and literature to find standard electrode potentials for each metal element, which is an important indicator to measure their redox ability  is superior to copper (Ⅱ), ferrous (Ⅱ) is superior to ferric (Ⅲ)) ,111. We  ability  145]. infect and kill cancer cells without harming normal cells . Thus, vUp to now, there have been a large number of studies that have reported on the antiviral performances of metal nanoparticles, including the virus inactivation efficiency, the defense and inactivation mechanisms, and the factors affecting antiviral activity. From the analysis of the chemical nature of metal elements, we conclude that redox ability may be a key factor affecting the antiviral ability, and the chelation reaction equilibrium constant, hydrate ion radius and ionic potential are the secondary factors. We hope that this conclusion will help researchers in various fields to select suitable substrates for antiviral materials based on the chemical nature of metal elements, and promote the development of novel powerful weapons for virus elimination.Metal nanoparticles not only have antiviral activity against viruses transmitted from person to person, but also have promising therapeutic effects on virus-infected animal and plant groups. In addition, considering the high cost and time-consuming process of new drugs discovery and the excellent physicochemical properties of metal nanoparticles, the proposal to use metal nanoparticles to inhibit the spread of viruses is valuable. Together, it is necessary to accelerate the research and development of metal nanoparticles, and more efforts need to be made to drive the translation of research results into actual industrialized products and devices. This will be beneficial to control the spread and infection of the virus, reduce the use of pesticides, and ultimately reduce the threat of the virus to human survival and environmental health.


Rubella is a common inherited infection resulting in congenital cataracts and a significant cause of permanent vision loss in developing countries. In 2016, Indonesia had the highest number of congenital rubella syndrome (CRS) cases globally. Here, we report the first genotype of the rubella virus extracted from the eye lens from a child with congenital cataracts due to CRS.A female neonate was delivered by an elective caesarean delivery with normal birth weight at term from a 24-year-old mother in the rural setting. The baby presented with bilateral congenital cataracts, small-moderate secundum atrial septal defect, severe supravalvular pulmonary stenosis, and profound bilateral hearing loss. She also had microcephaly and splenomegaly. The patient's serology showed persistent positive IgG for rubella virus at the age of four years and four months. Following extraction during cataract surgery, viral detection of the lenses identified the presence of rubella. Phylogenetic analysis confirmed that the virus was grouped into genotype 1E.Our study reports the first phylogenetic analysis of the rubella virus extracted from the eye lens of a child with CRS in Indonesia. The detection of the rubella virus from eye lenses is remarkably promising. Our findings also emphasize the importance of molecular epidemiology in tracking the origin of rubella infection toward achieving virus eradication.The online version contains supplementary material available at 10.1186/s12887-022-03775-4. Rubella or German measles is caused by the rubella virus that belongs to the family of Matonaviridae and is the only member of the genus Rubivirus . Its genRubella typically manifests as a mild disease with symptoms such as fever and skin rash. However, rubella infection during pregnancy, especially in the first trimester, results in congenital anomalies, prematurity, and fetal death. Infected newborns along with the anatomic defects constitute the congenital rubella syndrome (CRS) . In 2010Genotype analyses of the rubella virus are essential for achieving virus eradication. These data can be used to track virus control and elimination progress, help case classification, and trace transmission pathways . AnalyseA female neonate was born by an elective cesarean section due to cephalopelvic disproportion, with a normal birth weight  at term, to a 24-year-old secundigravida mother in the rural setting. Serological testing for TORCH infection for the mother was not done previously. Routine ultrasound screening was within the normal limit. The patient's mother was unaware of having any rubella symptoms nor did she have contact with any patient with rubella during pregnancy. Her mother also had never received the rubella vaccine. There was no history of consanguinity, no known inherited conditions, nor congenital abnormalities and no other ocular malformations occurring in the family.On physical examination, the neonate had microcephaly and a non-dysmorphic face. Complete cardiac examinations showed a small-moderate secundum atrial septal defect and severe supravalvular pulmonary stenosis which were confirmed using echocardiography. Assessment for hearing impairment using the Brainstem Evoked Response Audiometry showed bilateral profound hearing loss. The abdominal examination identified splenomegaly. Unfortunately, the serology testing for diagnosis of congenital infection, including rubella, was not conducted in the first year of life due to the late case presentation and findings. Based on these clinical findings, she was categorized as clinically compatible CRS .At four years and three months old, due to visual impairment, the child was brought to the ophthalmologist for an eye examination Fig. . The patViral RNA was extracted from lens samples using the QiaAmp Viral RNA Mini Kit . Reverse transcription-polymerase chain reaction (RT-PCR) assay was used to detect the existence of rubella virus RNA in lens samples using the SensiFAST™ Probe Lo-ROX One-Step Kit (Bioline). The assay amplified a 185 nucleotides fragment of the E1 coding region. The nested RT-PCR assays were designed to amplify the RV E1 coding area segments, including the WHO-recommended 739nt region for rubella genotyping using the Center of Disease Control (CDC) protocol   program , 15. PhySubsequently, we performed a phylogenetic analysis using the maximum-likelihood method to classify genotype 1E into five distinct lineages L0-L4). Based on the classification, our sample was grouped into L2 . Before the campaign, Genotype 1E was the most prominent rubella genotype that circulated throughout Indonesia . HoweverOur study reported one genotype of rubella which is 1E. In 2011, the same genotype was registered in GenBank as the first sequence from an Indonesian patient who returned to the United States . In 2017More than a million children in Asia are blind due to pediatric cataracts . RubellaOur findings also prove that the rubella virus can still be found in cataract lenses of patients with CRS even though rubella IgM was negative for the patient in the serology examination. It can also be found when the patient's age has already passed twelve months, even until almost five years. This finding indicates that the rubella virus can still be found within a few years after birth in lens material , 25. In The diagnosis of CRS in this study was made relatively late. Studies showed that most laboratory-confirmed CRS cases were diagnosed among cases who were 0- < 6 months of age which contradicts the late presentation of our patient –28. In IIn all of the cases reported in this study, the mothers of the patients were unaware of the symptoms of rubella infection during pregnancy due to the mild symptoms of a rubella infection such as fever, rash, cough, and adenopathy, which are usually considered as common ailments. Therefore, the diagnosis of rubella infection in pregnancy is very difficult . For devThe challenge of eliminating congenital rubella syndrome is also more significant than before the COVID-19 pandemic. Several immunization programs have declined due to parents' fear of coming to health service providers, including the hospital. The decline in the measles-rubella vaccination coverage is likely to increase their mortality and morbidity. The pandemic also slowed the implementation of measles and rubella mass vaccination campaigns in many regions and delayed various monitoring and evaluation activities. According to UNICEF and the WHO, many children in remote areas do not receive the first dose of a measles-containing vaccine as part of the routine immunization program each year . As the Rubella is a common inherited infection that causes congenital cataracts and is a major cause of lifelong vision loss and poor quality of life in children and their families. Children born with congenital cataracts can still achieve the best visual acuity if they follow a well-defined treatment protocol that includes the timing of surgery, postoperative visual rehabilitation, with proper and prompt management of complications. Unfortunately, the cataract case in our patient was discovered very late after she presented to the hospital with light perception in visual acuity. Early detection and intervention can improve the prognosis for children's vision.Our study presented the first genotype of the rubella virus extracted from the eye lens, which was discovered in a child with congenital cataracts caused by congenital rubella infection. Additionally, our findings demonstrate that the rubella virus can be detected in the cataract lenses of patients with CRS even when the patient tested negative for rubella IgM in the serology examination. It can also be detected when the patient is older than twelve months, and up to five years old. The persistence of the rubella virus in lenses of CRS patients with ocular abnormalities is due to the virus infecting the lens before the lens capsule develops, which normally serves as a barrier to the virus.Based on the genotype 1E lineage classification, our discovery is classified as L2, which are indigenous strains that circulate throughout Indonesia. This case suggests that 1E is still circulating, albeit in a limited geographical region. The detection of rubella virus RNA using lens material is remarkably promising. This approach can help establish the diagnosis of CRS in older children. This information can be critical for ophthalmologists and pediatricians, especially in patients with unusual clinical signs.Our findings also contribute to establishing baseline data of RV genotypes in Indonesia. These results emphasize the relevance of molecular epidemiology in tracking the origin of rubella outbreaks, such as in Indonesia, and the importance of generating enough sequencing data to be made available in GenBank. Furthermore, our data emphasize the importance of strengthening laboratory and epidemiologic surveillance of rubella, particularly in Indonesia, which is mandatory for rubella control and elimination.Additional file 1: Supplementary Figure 1. Representative electrophoresis result of nested-PCR electrophoresis.


Rhipicephalus (97.3%) and Amblyomma (2.7%)] collected from 15 vertebrate taxa were sequenced in 46 pools. Fifty-six pathogenic bacterial species were detected in 35 pools analyzed for pathogens and relative abundances of major phyla. The most frequently observed species was Escherichia coli (62.8%), followed by Proteus mirabilis (48.5%) and Coxiella burnetii (45.7%). Francisella tularemia and Jingmen tick virus (JMTV) were detected in 14.2 and 13% of the pools, respectively, in ticks collected from wild animals and cattle. This is one of the first reports of JMTV in Kenya, and phylogenetic reconstruction revealed significant divergence from previously known isolates and related viruses. Eight fungal species with human pathogenicity were detected in 5 pools (10.8%). The vector-borne filarial pathogens , protozoa , and environmental and water-/food-borne pathogens  were detected. Documented viruses included human mastadenovirus C, Epstein-Barr virus and bovine herpesvirus 5, Trinbago virus, and Guarapuava tymovirus-like virus 1. Our findings confirmed that host-feeding ticks are an efficient sentinel for xenosurveillance and demonstrate clear potential for wildlife-livestock-human pathogen transfer in the Kenyan landscape.Focusing on the utility of ticks as xenosurveillance sentinels to expose circulating pathogens in Kenyan drylands, host-feeding ticks collected from wild ungulates , carnivores , as well as regular domestic and Boran cattle were screened for pathogens using metagenomics. A total of 75 host-feeding ticks [ Tick-borne infections account for a major portion of all vector-borne diseases in many developed and underdeveloped countries, with significant health, economic, and food security consequences . The incTicks can transmit a diverse array of microbial pathogens including viruses, bacteria, and protozoans to humans and susceptible animals . Tick-boThe Republic of Kenya is a large sub-Saharan country in eastern Africa, with an area of 580,367 square kilometers and a population of over 47 million . In KenyDiceros bicornis), buffalo (Syncerus caffer), elephant (Loxodonta africana), giraffe , Grévy’s zebra (Equus grevyi), hartebeest (Alcelaphus buselaphus), impala (Aepyceros melampus), leopard (Panthera pardus), lion (Panthera leo), plains zebra (Equus quagga), spotted hyena (Crocuta crocuta), white rhinoceros (Ceratotherium simum), and wild dog (Lycaon pictus), as well as farmed Boran (Bos indicus) and cattle (Bos taurus)  from animals in wildlife conservancy sites in Isiolo county, Kalama, Lewa, Ol Pejeta, Samburu, Sangare gardens, Sarara, Solio ranch and Oljogi, between October 2013 and April 2019 . The hos taurus) . The Bortraction .Tubes containing tick pools (1–8 ticks/pool) were dipped in liquid nitrogen and homogenized using beads. Nucleic acid purification was conducted using the RNeasy Mini Kit , and complementary DNA (cDNA) synthesis with random hexamers was performed using the RevertAid First Strand cDNA Synthesis Kit , as per the manufacturer’s recommendations. Tick nucleic acid extraction and cDNA conversion were carried out at IPR laboratories in Kenya, and cDNAs were sent to WRBU (United States) for further testing.oC, ligation step of 90 min, and 20 PCR cycles. Library quantification and quality control were performed using the TapeStation 4,200 Automated Electrophoresis instrument . Excess adapter dimer and small fragments were removed using KAPA pure beads . Unbiased metagenomic sequencing was performed on the NovaSeq platform  (PE 2 × 150) at the Walter Reed Army Institute of Research (WRAIR). Samples were run on one lane of an S4 flow cell with XP workflow designed to maximize the overall raw reads output. Read quality was assessed using fastqc  on a Fluoroskan FL instrument . Libraries were prepared using KAPA HyperPlus Kits , as recommended by the manufacturer, with fragmentation step of 20 min at 35g fastqc , and adag fastqc .Jingmen tick virus (JMTV) screening was performed by a specific nested PCR assay, targeting the viral NS5-like protein located on the genome segment 1, as described previously . Amplifi2 and expressed as taxon read count/total bacteria read count. Reads and contigs from viruses, parasites, and tick-borne bacteria were manually reviewed with the taxon identity confirmed using BLAST (Raw data from the Illumina NovaSeq sequencing runs were uploaded to the CZ-ID platform (formerly ID-Seq), a publicly accessible cloud-based storage and analysis platform for metagenomic pathogen detection , and werng BLAST .Rhipicephalus sanguineus, ASM1333969v1). Data were then clustered using vsearch  to give “virus-like” sequence matches. These were then passed to a second step alignment to the complete NCBI NT and NR databases  for final classification. We used CZ-ID for the initial workup and VirIdAl on samples with detectable virus signal. Outputs from both pipelines were combined for producing final contigs, following duplicate removal.Viral reads were also recovered using the VirIdAl pipeline . This pi vsearch  to recov1. Nucleotide and deduced amino acid sequence alignments and pairwise comparisons were generated using CLUSTAL W  biosample database with the ID: SUB11147880.The raw reads from tick pools tested in this study are freely available in Rhipicephalus spp.  and Amblyomma spp. . Host sequence data were utilized to confirm the genus-level identification in the tick pools. The average raw reads count of 9,313,080 was recorded per sample, whereas it was noted as 190,915 following the low-quality read and host removal.A total of 75 adult ticks collected from 15 animal species were sequenced in 46 pools. Most of the pools (84.8%) comprised single ticks, whereas multiple ticks  were screened in 7 of the 46 pools (15.2%) . Majorit2). Members of the phylum Proteobacteria were abundant in the bacterial composition of the tick pools, comprising 51.6–97.5% of the total bacterial reads in 17 (48.6%) pools, regardless of the host species  and 2 (5.7%) pools, respectively. Predominance of a single bacterial species (with reads constituting over 50%) within a phylum was observed in certain pools, namely, Proteus mirabilis , Clostridium botulinum , Coxiella burnetii (pool #21), and Enterobacter cloacae (pool #78). Other than these instances, no particular tendency in relative bacterial abundances or host species was noted.Of the 46 pools sequenced, 35 tick pools were analyzed for pathogenic bacterial species and relative abundances of major phyla. Eleven pools were omitted from the analysis due to the relatively low number of total bacteria reads , followed by P. mirabilis  and C. burnetii . C. burnetii is the etiologic agent of Q fever, characterized by febrile and respiratory symptoms. Infections are zoonotic and are initiated by inhalation of particles contaminated by infected animals, most commonly sheep, goats, and cattle, with ticks implicated in transmission  collected from cattle, lions, an elephant, and a Grevy’s zebra, whereas B. anthracis was identified in a single pool  collected from an elephant. A Gram-negative facultative intracellular bacterium, F. tularensis, causes tularemia, an anthropozoonosis that occurs via contact with wild animals, arthropod bites, or environment through respiratory, oral, or cutaneous routes  identified in ticks collected from a hartebeest and a lion  or diagnosed as opportunistic pathogens in immunocompromised hosts, with the exception of Crypotococcus spp., and are mainly disseminated by inhalation of the infectious particles from environmental sources  with low number of reads (range: 2-7). d a lion . It is ad a lion . Not bei sources .Trichinella spiralis, detected in 6 pools (13%) collected from various hosts. It is a nematodal parasite of many carnivorous and omnivorous animals, where infections occur by ingestion of muscle tissue with encysted larvae  and pool #47 from a lion ] strongly suggests an environmental origin for most of the identified pathogens. Moreover, human Plasmodium falciparum sequences detected most likely indicate prior feeding of the ticks on infected humans, evidencing the potential for cross-species zoonotic pathogen transfer from wildlife to humans via tick bites in African dryland ecosystems , mostly in low to moderate number of reads (range: 2–736) . The mosd larvae . Human i6.5–8.7% . Howeverosystems .We recovered 4 viruses with significant human or veterinary health impact . Human mVaricellovirus genus of the Alphaherpesvirinae subfamily. Similar to other alphaherpesviruses, BHV-5 establishes latency in the central nervous system of the exposed animals and is excreted in ocular, nasal and genital secretions upon reactivation  was detected in 4 tick pools (8.7%), including one collected from cattle pool #3, . It is ativation . It caustivation . It is ativation .Flaviviridae family (OM913596). The sequence displayed up to 94.62% nucleotide and 99.23% deduced amino acid identities to previously characterized JMTVs. Maximum likelihood analysis showed that the sequence is phylogenetically distinct from all previously described JMTV clades from various regions, as well as from related viruses such as Mogiana tick virus (Piliocolobus rufomitratus) from Uganda.Jingmen tick virus is the only virus where tick-borne transmission appears as the main mode of spread in the study. JMTV sequences were detected in a total of 6 pools (13%) . Metagene family . JMTV poe family . It is re family . We recock virus . The cloPestivirus genus of the family Flaviviridae (R. sanguineus ticks from Trinidad and Tobago) and closely related Bole tick virus 4 isolate Thailand tick flavivirus  of the pools, sequences with the highest nucleotide and deduced amino acid identities to Trinbago virus (TBOV) were detected. TBOV is a recently described virus, distantly related to iviridae . The TBOiviridae . The lonavivirus .Tymovirales (We further identified Guarapuava tymovirus-like virus (GTLV) sequences in 4 (8.7%) pools and pairwise comparisons revealed the highest identities to GTLV-1 . GTLVs hovirales . PartialSimilar to GTLVs, two open reading frames (ORFs) are recognized in the GTLV-1 isolate Kenya-P43 genome, encoding for the ORF1 polyprotein (nucleotides 1–5031) and viral capsid  . AnalysiF. tularemia and JMTV in 14.2 and 13% of the tick pools, respectively, with both agents observed in ticks collected from wild animals and cattle. Currently, tularemia is considered a global re-emerging zoonotic disease and F. tularemia as a potential agent of biological warfare, due to its ease of aerosol dissemination, low infectious dose, and potential fatal outcomes , a critically endangered primate  collected feeding on an elephant, demonstrating ongoing activity in wildlife. Botulinum toxin produced by the anaerobic rod C. botulinum causes severe poisoning, frequently occurring due to oral intake. C. botulinum is ubiquitously found in soil and aquatic sediments that serve as an environment for sporulation  collected from cattle, buffalo, and endangered Grevy’s zebra. In this study, the source is likely to be environmental in origin. However, the presence of C. botulinum as the single bacterial species in 3 pools suggests that it can readily predominate and pose a threat to human and animal health without particular precautions.Other bacterial agents with high pathogenicity identified in the study include in Kenya . Infectiin Kenya . In thisrulation . Botulinin Kenya . We idenE. coli and P. mirabilis constitute the most frequent species, identified in 62.8% and 48.5% of the pools, respectively. Furthermore, several other species of Enterobacteriaceae, Staphylococci, Streptococci, and Clostridia spp. other than C. botulinum were present in tick pools . Similarly, we identified several fungi including Aspergillus, Candida, Cladophialophora, Cryptococcus, Pneumocystis, and Rhizopus species, found widespread in nature, that mostly cause opportunistic infections. However, Cryptococcus neoformans and Cryptococcus gattii, detected in the study in 4.2% of the pools, are documented to produce severe infections in immunocompetent individuals as well  and protozoa (Plasmodium spp. and T. cruzi) detected in ticks probably represent blood meals from infected hosts. The environmental and water-/food-borne pathogens Entamoeba histolytica, Encephalitozoon intestinalis, Naegleria fowleri, Schistosoma spp., Toxoplasma gondii, and Trichinella spiralis were detected, which indicate, along with the diversity of parasites detected in single ticks, contamination from environmental sources. These findings demonstrate a marked potential for wildlife-livestock-human pathogen transfer. This is also supported by the frequent detection of adenovirus and herpesvirus that cause human or cattle infections  Biosample and Sequence Read Archive (SRA) under accession numbers 28626882–28626931.The raw reads from tick pools tested in this study are available in KE: laboratory analysis, data analysis and interpretation, and manuscript drafting. MM: wildlife specimen collection, transport, and initial processing. BB: laboratory analysis and data analysis and interpretation. SJ: laboratory analysis and data analysis. LC-Q: laboratory analysis and data analysis. JK: wildlife specimen collection, transport, and initial processing. SaM: wildlife specimen collection, transport, and initial processing. IA: wildlife specimen collection, transport, and initial processing. EC: wildlife specimen collection, transport, and initial processing. FG: wildlife specimen collection, transport, and initial processing. PO: wildlife specimen collection, transport, and initial processing. SuM: study design. DZ: study design and data interpretation. Y-ML: study design and conception, data interpretation, and manuscript drafting. All authors contributed, read and approved the submitted manuscript.MM was employed by Kenya Wildlife Services Corporation. The remaining 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.


Culex tritaeniorhynchuses–specific virome, 6400 C. tritaeniorhynchuses were collected in Honghe autonomous prefecture, China. Abundant virus sequences were obtained from 28 viral families using metavirome sequencing. Herein, several viruses in C. tritaeniorhynchuses virome were verified using the PCR technique, which covers Japanese encephalitis virus (JEV), Getah virus, and even Chikungunya virus (CHIKV). Seven JEV gene sequences were amplified successfully, of which JEV-China/CT2016E-1 shared the highest homology with the known JEV sequence isolated in Korea, 1946, with at least 96.1% nucleotide (nt) identity, which belonged to genotype III. Nine CHIKV gene sequences were amplified, which shared the highest with at least 93.0% nt identity with CHIKV from Thailand isolated in 2007, which was assigned to genotype Asian. Remarkably, CHIKV was isolated from C. tritaeniorhynchus in China for the first time. It was initially confirmed that the isolated virus CHIKV-China/CT2016-1 may increase infectivity after passaging in Vero cells from BHK-21 cells. Collectively, our study reveals the diversity, properties, and potential virus susceptibility dynamics of the C. tritaeniorhynchus virome and sheds new perspectives on the viral ecology in other important biological vectors.To explore the  Culex tritaeniorhynchus is the dominant mosquito species in the region  and WB (Western blotting), can accurately identify several viruses , Chikungunya virus (CHIKV) was isolated in C. tritaeniorhynchus from China for the first time. Our preliminary study on the metavirome of C. tritaeniorhynchus provides valuable information for the discovery, diversity, and monitoring of the virome in other mosquito species.The purpose in our study was to explore the C. tritaeniorhynchus into 2-ml U-bottom microcentrifuge tubes with a maximum of 100 mosquitoes per tube and then stored at −80°C.Female mosquitoes were collected daily from barns in Honghe autonomous prefecture , Figure 1https://blast.ncbi.nlm.nih.gov/Blast.cgi) was employed for contig comparison to the GenBank, in which the E value ≤10e−5 was incorporated into the adoption. To further validate the metaviral sequencing results, RT-PCR was applied for confirming viral sequences using specific primers  with 8% fetal bovine serum added. In a nutshell, after centrifugation, 20 μl of the supernatant of the mosquito-grinding solution was added to the monolayer of BHK-21 cells, on which 80 μl of DMEM was contained and incubated at 37°C for 6 days until the cells were observed to have a cytopathic effect (CPE) induced by the virus. Blind passage was performed for 3 times when no CPE appeared at the first time. CHIKV was searched in supernatants after virus isolation using CHIKV-specific primers (Table 2) targeting 326 nt of the E1 gene. Furthermore, the expression of E1 protein was tested through Western blotting with the anti-E1 monoclonal antibody  and an HRP-conjugated rabbit anti-mouse antibody . Additionally, negative-stained electron microscopy was employed for the observation of CHIKV particles, which were prepared using supernatant of infected BHK-21 cells mingled 1:1 with 6.1% paraformaldehyde, mounted on copper grids, with the treatment of 3% phosphotungstic acid.50 of the 5th and 10th passages from BHK and the 15th and 20th from Vero cells was tested according to the Reed−Muench method  19.0, in which a comparison between groups was performed using the T-test. Each experiment was validated iteratively by setting up three replicates and repeating it three times. Differences were considered significant when the P-value was less than 0.05.The data produced in metagenomic sequencing have been deposited in the GenBank Sequence Reads Archive with the accession number of SRR15291873. The amplified viral sequences have been deposited in the GenBank, in which the accession numbers were covered: JEV-China/CT2016E-1/2/3(OM799545–OM799547), JEV-China/CT2016NS1-1/2/3/4 (OM799554–OM799557), GETV-China/CT2016E1-1/2/3 (OM799565–OM799567), CHIKV-China/CT2016E1-1/2/3/4 (OM799570–OM799573), and CHIKV-China/CT2016C-1/2/3/4/5 , respectively.C. tritaeniorhynchus were collected for metaviral sequencing. Sequences were sorted as viruses with 7.02 × 106 reads, including known and unclassified viruses. Many mosquito viruses together with unknown or unclassified viruses were discovered, with potential infectious risks to human and animal. The abundance of viral reads varied largely by the virus taxonomy. A total of 28 viral families were detected, , 25× (239–916 nt), and 37× , respectively. Moreover, the JEV-like contigs, GETV-like contigs, and CHIKV-like contigs possessed 83.2%–95.7%, 82.6%–96.4%, and 81.9%–97.3% nt homology with the known JEV, GETV, and CHIKV sequences separately. Subsequently, virus-specific primers , followed by sequencing. Three 1,500-bp fragments (JEV-China/CT2016E-1/2/3) and four 1,056-bp fragments (JEV-China/CT2016NS1-1/2/3/4) were obtained. After alignment, JEV-China/CT2016E-1/2/3 shared ~96.5%–96.7% nt identity and ~90.6%–92.6% amino acid (aa) identity with one another. Additionally, JEV-China/CT2016NS1-1/2/3/4 possessed ~95.3%–96.6% nt identity and ~88.1%–91.5% aa identity between each other. Viral sequence analysis using blastn showed that the amplified JEV-like fragments shared the highest (nt identity at least 96.1% and aa identity at least 94.2%) identity with JEV from Korea isolated in 1946, which was assigned to genotype III JEV Figure 3Equus caballus, 1978 , followed by sequenced. Three 1,314-bp fragments (GETV-China/CT2016E1-1/2/3) were acquired. After alignment, GETV-China/CT2016E1-1/2/3 shared ~97.0%–97.4% nt identity and ~92.0%–93.8% aa identity with one another. Viral sequence analysis using blastn showed that the amplified GETV-like fragments shared the highest (nt identity at least 98.2% and aa identity at least 94.3%) identity with the E1 gene of GETV from Japan isolated in us, 1978 Figure 4The amplified CHIKV-like fragments were cloned into the pMD19-T vector , followed by sequencing. Four 1,317-bp fragments (CHIKV-China/CT2016E1-1/2/3/4) and five 783-bp fragments (CHIKV-China/CT2016C-1/2/3/4/5) were obtained. After alignment, CHIKV-China/CT2016E1-1/2/3/4 shared ~96.5%–97.3% nt identity and ~90.9%–93.8% aa identity with one another. Additionally, CHIKV-China/CT2016C-1/2/3/4/5 possessed ~92.0%–97.2% nt identity and ~91.2%–94.6% aa identity between each other. Viral sequences analysis using blastn showed that the amplified CHIKV-like fragments shared the highest (nt identity at least 93.0% and aa identity at least 94.3%) identity with CHIKV from Thailand isolated in 2007, which was assigned to genotype Asian Figure 5All validated viruses were attempted to be isolated, and only CHIKV was successfully isolated. The newly isolated CHIKV was verified by CPE  compared to the 5th and 10th generations.The infectious titer value of CHIKV-China/CT2016-1 at the 5th generation in BHK cells was assessed as 6.35 × 10C. tritaeniorhynchus was the dominant mosquito species in Yunnan province, China . Multiple studies found that C. tritaeniorhynchus carried a rich variety of viruses, covering Zika virus  could be used in future experiments. In addition, some viruses were not amplified, probably due to insufficient sample size or low viral load.Validation based on metaviral sequencing results, JEV and GETV were successfully amplified from rhynchus , suggestate host . The detAe. albopictus  compared to the 5th and 10th generations. As reported, E1-A226V mutation will significantly enhance the adaptability of CHIKV in bopictus . In our C. tritaeniorhynchus in this region. However, the specific viral profile of C. tritaeniorhynchus is incomplete. Unknown viruses of specific mosquito species need to be revealed through further research. In conclusion, our study provides valuable insights into the specific mosquito-borne virus spectrum and the excavation of its new susceptible viruses. This will provide an important reference value for the molecular epidemiological studies of arboviruses.The findings in this study demonstrated the potential viral reservoir in the The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.PX and NL conceived and designed the experiments. DZ, CP, CL, and HZ performed the experiments. PX and YL analyzed the data. CL contributed reagents/materials/analysis tools. PX and NL wrote the paper. All authors read and approved the final version of the manuscript.This work was supported by the National Natural Science Foundation of China [grant number 32002312], the Natural Science Foundation of Zhejiang Province [grant number LQ21H160001], Science and Technology Project of Wenzhou, Zhejiang, China [grant number Y20210080 and Y2020103].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.


Although wheat streak mosaic virus (WSMV) is a well-known pathogen inducing significant crop losses and endangering wheat production worldwide, the recent discovery of High Plains wheat mosaic virus (HPWMoV) in Ukraine raises questions on the co-existence of these two viruses having a similar host range and the same mite vector. Here we report on the screening of winter wheat industrial plantings in several important regions of Ukraine for WSMV and HPWMoV. WSMV was identified in an extremely high number of symptomatic plants (>85%) as compared to HPWMoV detected in 40% of wheat samples. Importantly, the preferred mode of HPWMoV circulation in Ukraine was mixed infection with WSMV (>30%) as opposed to WSMV, which was typically found in monoinfection (60%). Screening wheat varieties for possible virus resistance indicated that all but one were susceptible to WSMV, whereas over 50% of the same varieties were not naturally infected with HPWMoV. Overall, phylogenetic analysis of the collected WSMV and HPWMoV isolates indicated their high identity and similarity to other known isolates of the respective viruses. Here we first characterize WSMV isolates found in winter wheat plants in mono- or mixed infection with HPWMoV, which was recently reported as a typical wheat pathogen in Ukraine. Triticum aestivum L., and especially winter wheat, remains the second-most common grain cereal crop cultivated both in Ukraine and worldwide for centuries and is outcompeted only by corn.Thanks to its high yields on organic-rich chernozem soils, Depending on the year, winter wheat crops in Ukraine reach 6.4 to 7.3 million hectares and are of strategic importance for ensuring food safety and export trade. Viral diseases are major factors causing a significant reduction in wheat yields. Available evidence indicates that, nowadays, wheat streak mosaic virus (WSMV) is one of the most common and harmful wheat pathogens common in Europe, the United States, Canada, Australia, the Middle East, North Africa, Asia, and other wheat-growing regions of the world ,2,3,4.Tritimovirus genus of Potyviridae family, and has filamentous particles of ~700 nm in length and ~13 nm in diameter [WSMV is a type species of diameter . WSMV gediameter ,7.Poaceae family, which are routinely cultivated in Ukraine, including all wheat cultivars/varieties (T. aestivum L.). Most WSMV isolates can also infect oat (Avena sativa L.), barley (Hordeum vulgare L.), rye , some cultivars/varieties of corn (Zea mays L.), millet (Panicum miliaceum L.), and various grasses both among cultivated plants and wild flora acting as reservoir plants and ensuring overwintering of the virus [WSMV can naturally infect many species of plants of the he virus ,8,9,10.On grain cereals, WSMV typically causes symptoms of streak mosaic, but may also induce necrosis of shoots and overall plant stunting. Visual symptoms of the disease depend on the virus strain, species, and cultivar/variety of the infected plant, the period of infection, temperature, and other environmental conditions ,12. ThisPoacevirus genus of the Potyviridae family with filamentous particles of 680–750 nm in length and 12–15 nm in diameter. In turn, HPWMoV belongs to the Emaravirus genus of the Fimoviridae family and has double-membrane spherical particles 80–200 nm in diameter [In this complex, TriMV is another representative of the diameter .Aceria tosichella Keifer, also known as Aceria tritici, Acarina: Eriophyidae), the single known vector for these viruses [WSMV, HPWMoV, and TriMV are all efficiently transmitted from infected to healthy plants by the wheat curl mite , B , C (Iran and Middle Asia), and D  .Wsm1, Wsm2, and Wsm3 have been identified insofar. Recently, the most promising candidate gene, Wsm2, was successfully introduced into the germline of several wheat varieties [The resistance genes arieties . HoweverOutbreaks of the disease caused by WSMV in wheat crops have occasionally occurred in Ukraine ,15,16,32Winter wheat plant samples with visual symptoms typical of WSMV, BSMV, HPWMoV, and TriMV  were collected in May–June of 2017–2019. Sampling locations included crop-producing areas in Kyiv, Cherkassy, Vinnytsia, Dnipropetrovsk, Donetsk, Luhansk, Zaporizhia, Kharkiv, and Odessa regions.m/v), with the addition of 2% polyvinyl pyrrolidon, 0.05% Tween 20, and 0.2% dried milk. Sap from healthy plants was used as negative controls. Positive controls were commercial protein preparations from respective manufacturers. MICROCOLON™ microtitre plates  were coated with virus-specific polyclonal antibodies in carbonate buffer, pH 9.6. Leaf extracts were then added to the plates in wells and incubated overnight at 4 °C. Virus presence in the samples was detected in 200 μL homogenate by virus-specific antibodies conjugated to alkaline phosphatase using a p-nitrophenyl phosphate substrate . Absorbance values at 405 nm were measured using a Termo Labsystems Opsis MR microtiter plate reader  with Dynex Revelation Quicklink software v.4.2. Absorbance values measured 60 min after adding the substrate, greater than three times those of the negative controls, were considered positive.Samples collected from symptomatic plants were tested for WSMV and HPWMoV infection by a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), as described by Clark and Adams , using sTransmission electron microscopy for direct virus indication and studying virus morphology was conducted employing standard techniques of negative contrasting for clarified virus preparations. Briefly, copper grids  were coated with chloroform-dissolved 0.2% polyvinyl formaldehyde  and dried overnight on filter paper at room temperature. Then the coated grids were deposited in the clarified homogenate of symptomatic plant sap, incubated for 2–15 min at room temperature, and dried on filter paper for 1 min. The samples deposited onto grids were stained with 2% uranyl acetate  for 10 min, and examined using a JEM 1400  transmission electron microscope ,38. The Total RNA was extracted from fresh leaves of virus-infected wheat plants using a Gene JET RNA Purification kit  and following the manufacturer’s instructions.One-step RT-PCR was performed using a Qiagen one-Step RTPCR kit + Q-solution  according to the manufacturer’s instructions. Amplification was performed using Genetic Research Instrumentation Ltd. thermocycler . WSMV-specific oligonucleotide primers for amplifying a part of the coat protein gene , as wellThe amplification reactions were set up as follows: Initial denaturation for 3 min at 94 °C, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C/53 °C/45 °C (WSMV/HPWMoV/TriMV) for 30 s, and extension at 72 °C for 1 min. The final extension was at 72 °C for 7 min. PCR products with GeneRuler 100 bp Plus DNA Ladder  or PCRBIO Ladder III 50 bp DNA Marker  were separated in a 1.5% agarose gel stained with ethidium bromide, and visualized under UV light ,43.Fragments of the expected size were excised from the gels and cleaned using a QIAquick Gel Extraction Kit  following the manufacturer’s instructions. Purified amplicons were sequenced using the cycle sequencing technology on Applied Biosystems ABI 3730X1 DNA Analyzer  using the Big Dye Terminator v3.1 Cycle Sequencing kit .http://www.ncbi.nlm.nih.gov, accessed on 1 April 2022) using BLAST ). For phylogenetic analysis, the Neighbor-Joining (NJ) method was employed with the Jukes–Cantor model [The resulting sequences were aligned using Clustal W v.2.0 and further compared with the nucleotide sequences of respective viruses publicly available from the GenBank database . For phylogenetic analysis, in-built algorithms of MEGA 7 software were used.A. tosichella (A. tritici) mites were often spotted on such and/or neighboring plants during sampling in most locations, suggesting a means for possible vectored virus transmission.In May-June of 2017–2019, several regions of Ukraine representing ~60% of the total area of winter wheat production were visually screened for signs of viral diseases. Wheat plants exhibiting typical virus-like symptoms, in particular leaf blade mosaic and stunting , were coJudging by the symptoms it was presumed that such plants could have been infected by WSMV or BSMV in the form of either mono- or mixed infection. Selected samples were further serologically tested using DAS-ELISA.In 2017, WSMV was found to be widespread and circulating in 6/7 screened regions of Ukraine, with an overall incidence of 71.4% in symptomatic plants (50/70). Furthermore, BSMV was not detected .However, in 2017, there was a remarkable portion of WSMV/BSMV-negative samples characterized by severe stripe mosaic symptoms often accompanied by notable stunting, which indicated the possible presence of (an)other virus(es) inducing a similar pathology in wheat. Hence, wheat samples collected subsequently in 2018 and 2019 were also tested for HPWMoV. Because of the absence of ELISA-based diagnostic kits for TriMV detection, this pathogen was omitted at the stage of serological testing.In 2018, serological testing of symptomatic wheat samples showed the occurrence of WSMV in every region probed (6/6) with an incidence of 88.9% in symptomatic plants (80/90). HPWMoV, however, was identified exclusively in central-eastern regions of Ukraine  in 50% of the collected wheat samples (45/90). Similar to 2017, BSMV was never found . ImportaFurther screening in 2019 confirmed WSMV circulation in every region probed (6/6), whereas HPWMoV was detected only in central-eastern parts of the country. The incidence of WSMV infection for symptomatic plants was 94.1% (80/85). In contrast to WSMV, HPWMoV was found in 29.4% of collected wheat samples (25/85). Similar to previous years, BSMV was not found. In 2019, 70.6% of wheat plants (60/85) were monoinfected by WSMV, 5.9% of wheat plants (5/85) were monoinfected by HPWMoV, while mixed infection (WSMV + HPWMoV) was registered for 23.5% (20/85) of collected plants .Overall, a total of 245 plant samples showing virus-like symptoms  were collected in sampling sites in 2017–2019. On average, WSMV was detected by DAS-ELISA in 210/245 plant samples (85.7%), whereas HPWMoV was found in 70/175 plant samples (40%). BSMV was not detected in this work . WSMV waIn 2018–2019, the average incidence of WSMV monoinfection was 60% (105/175) of symptomatic wheat plants, HPWMoV monoinfection was registered for 8.6% (15/175) of collected samples, whereas mixed infection (WSMV + HPWMoV) was found in 31.4% of the samples (55/175).Furthermore, the ELISA results obtained are in agreement that mixed infection of winter wheat plants by WSMV and HPWMoV may correlate with more severe visual symptoms exhibited with pronounced yellow stripe mosaic, shorter spikes, and plant stunting, as shown in Further, we analyzed winter wheat varieties of Ukrainian and German selections widely cultivated in Ukraine for their natural infection by WSMV and/or HPWMoV .Our results showed that 12/13 of the varieties tested were definitely susceptible to WSMV, but WSMV was not found in the Ukrainian variety Novosmuhlyanka in the field. Surprisingly, a similar study for HPWMoV showed that 53.8% (7/13) of these varieties were not naturally infected by this pathogen in Ukrainian agroecosystems. This list included five varieties of Ukrainian selection  and two varieties of German selection (Emil and Cubus).Virus presence and morphology in the partly clarified sap of symptomatic wheat plants was determined using transmission electron microscopy .According to TEM data, filamentous viral particles ~700 nm in length and ~12–13 nm in diameter have been registered, in some cases accompanied by spherical double-membrane bodies ~90–110 nm in diameter. In all these cases, accurate morphological identification allowed us to attribute filamentous virions to potyviruses, whereas spherical particles were typical for emaraviruses.Winter wheat samples previously shown to be positive for WSMV and/or HPWMoV in serological screening see  were furPCR products of the expected size for WSMV and HPWMoV were successfully amplified. However, TriMV was not identified in PCR-tested samples.Amplified subgenomic sequences of WSMV and HPWMoV were determined and deposited in DDBJ/EMBL/GenBank databases as indicated in The results indicated that these sequences corresponded to the respective parts of the genomes of WSMV (coat protein gene) and HPWMoV (nucleoprotein gene).Pairwise comparison of nucleotide and amino acid sequences of the coat protein of five WSMV isolates used in this research showed that they were characterized by mutually high levels of identity (>97% at the nucleotide level). Moreover, these Ukrainian WSMV isolates demonstrated a high level of identity (>96.5% at the nucleotide level) with previously published wheat isolates from Ukraine , Poland , Slovakia , Serbia , Russia (AF454459.1), Czech Republic , Italy (FJ606885.1), Germany (HG810954.1), France (HG810953.1), Lithuania (KJ720819.1), Hungary (MT780557.1), and Turkey (FJ606886.1), recovered mostly from wheat but also from barley and millet. WSMV isolates from other groups, A, C, and D, as well as those recovered from wild-growing grasses, and surprisingly, two other Ukrainian wheat WSMV isolates reported previously (MH523357.1 and MH523356.1), demonstrated a much lower degree of identity (~60–93% at the nucleotide level) to the five Ukrainian WSMV isolates used in this research, as shown in For WSMV, NJ phylogenetic trees were initially calculated from the sequences of all isolates available from the GenBank. However, there were inconsistencies in, and poor bootstrap support for, some lineages in the resulting trees, as found previously for other potyviruses ,51,52. TThe tree partitioned most of the sequences into the four consistent phylogenetic groups: A, B, C, and D. All the phylogenetic groupings were supported by high bootstrap values. As expected, all Ukrainian isolates  to other wheat isolates originating from the USA . Surprisingly, despite the maize HPWMoV isolate from Ukraine being less related to other published sequences, it was also more similar to wheat isolates from the USA listed above, with MT563400.1 being the best hit, showing only ~83% of identity at the nucleotide level.For HPWMoV, an NJ phylogenetic tree was calculated using sequences of all 39 isolates available from the GenBank .Most of the HPWMoV sequences were partitioned into two phylogenetic groups, A and B, as supported by high bootstrap values. All three Ukrainian isolates described here and listed above in In the present study, WSMV was confirmed to be a predominant virus in every screened region in the eastern, southern, and central parts of Ukraine  , Table 2Complementing results of serological screening, direct indication, and molecular detection  proved tA. tosichella was mostly found in the eastern part of the country where the climatic conditions  may have favored its spread and overwintering, similar to other parts of the world [The significant spread of WSMV and HPWMoV and their dependence on a unique vector, wheat curl mite, indicates the importance of vector monitoring and control for the successful management of these viral diseases in Ukraine. For decades, he world ,54 The cInterestingly, as much as 60% of symptomatic plants were monoinfected with WSMV, whereas wheat monoinfection by HPWMoV was registered for only ~9% of plants. In line with the overall virus spread, this suggests prolonged circulation of WSMV in Ukrainian agroecosystems but the presumably recent introduction of HPWMoV. In contrast to the relatively small number of HPWMoV-moninfected plants, over 31% of wheat plants were mixed infected with both viruses, which correlated with more severe visual symptoms b. The wiThe screening of 13 commonly cultivated wheat varieties of Ukrainian and German selection confirmed that all but one variety were naturally infected with WSMV in field conditions. Symptomatic samples of the Novosmuhlyanka variety were not WSMV-infected . This piece of data indicates that all such varieties are not only susceptible to WSMV but also confer the survival of its vector , underpiOn the contrary, seven winter wheat varieties  were not HPWMoV-infected in the field . These vThe simplest interpretation of the phylogenetic analyses is that five WSMV isolates identified and studied in this work expectedly belonged to group B and were characterized by a high degree of similarity among themselves and to many other known isolates of this virus found in Ukraine and in neighboring or even more distant European countries . The line of evidence suggests high overall homogeneity of WSMV population(s) circulating in screened regions of Ukraine and Europe. However, two WSMV isolates (MH523357.1 and MH523356.1) found in the Kharkiv region (east of Ukraine) some time ago  were far more distant from the five WSMV isolates described in this study. This is indicative of the tentative occurrence of several lineages of WSMV circulating in Ukraine. Wide-scale populational research with full genomic sequencing of WSMV isolates will aid in establishing their possible origins.As for HPWMoV, the obtained data were in agreement with our previous results showing high homogeneity of two wheat isolates with a somewhat smaller degree of identity typical for the only maize HPWMoV isolate found until now. All these isolates, however, were most similar to the known sequences of this virus originating from the USA, suggesting the possible introduction of HPWMoV into Ukraine with seed/plant material, which may have occurred a number of times. Despite the currently limited geographical spread of HPWMoV in Ukraine, its occurrence in two different and widely cultivated cereal crops may pose a risk of consequential virus distribution into ‘new’ niches, following the footsteps of WSMV and their common vector.Interestingly, we were not able to trace any notable phylogenetic differences for WSMV and HPWMoV found in either mono- or mixed infection in winter wheat collected from various regions at different years. As shown in In summary, although an added understanding of WSMV and HPWMoV biology, epidemiology, and phylogeny was brought about with this study, the wide survey of these pathogens and their vector in different (mostly southern and western) regions of Ukraine is necessary to confirm our conclusions. However, to our knowledge, the present study shows the occurrence of wheat streak mosaic virus in mono- and mixed infection with High Plains wheat mosaic virus in winter wheat in Ukraine and describes the evolutionary relationships between Ukrainian isolates and the worldwide isolates of these pathogens previously reported.Complementing results of visual and serological screening, direct indication, and molecular detection prove the wide spread of WSMV (>85% incidence rate) and HPWMoV (40% incidence rate) in the central-eastern part of Ukraine  where these two pathogens may successfully survive in the form of mono- or mixed infections.In Ukraine, WSMV more frequently induces monoinfection (60%), whereas HPWMoV is typically found in mixed infection with WSMV (>31%) but not in monoinfection (<9%).Nearly all winter wheat varieties commonly cultivated in Ukraine and screened for natural virus infection are susceptible to WSMV. In contrast, 53% of these varieties (7/13) were not naturally infected with HPWMoV, possibly representing candidates for sources of resistance.Five WSMV isolates found in this research are characterized by high mutual identity (>97% at the nucleotide level) and high identity (>96.5% at the nucleotide level) to previously published wheat isolates from Ukraine and many other European countries. In turn, the three HPWMoV isolates described here are most similar (>85–98% at the nucleotide level) to the USA isolates of this virus found earlier.