Opioid Use Disorder (OUD) is a substance use disorder with a chronic course associated with comorbid mental and somatic disorders, a high burden of psychosocial problems and opioid maintenance treatment (OMT) as a standard treatment. In the US, OUD imposes a significant economic burden on society, with annual societal costs estimated at over 55 billion dollars. Surprisingly, in Europe and especially in Germany, there is currently no detailed information on the healthcare costs of patients with OUD. The goal of the present research is to gather cost information about OUD patients in OMT with a focus on maintenance medication and relapses.We analysed health claims data of four million persons covered by statutory health insurance in Germany, applying a cost-of-illness approach and aimed at examining the direct costs of OMT patients in Germany. Patients with an ICD-10 code F11.2 and at least one claim of an OMT medication were stratified into the treatment groups buprenorphine, methadone or levomethadone, based on the first prescription in each of the follow-up years. Costs were stratified for years with and without relapses. Group comparisons were performed with ANOVA.p < 0.0001). In years with relapses, costs were higher than in years without relapses . In years with relapses, hospital costs were the major cost driver.We analysed 3165 patient years, the total annual sickness funds costs were on average 7470 € per year and patient. Comparing costs of levomethadone , methadone  and buprenorphine  revealed significant lower costs of buprenorphine compared to levomethadone (The present study shows the costs of OUD patients in OMT for the first time with a German dataset. Healthcare costs for patients with an OUD in OMT are associated with more than two times the cost of an average German patients. Preventing relapses might have significant impact on costs. Patients in different OMT were dissimilar which may have affected the cost differences. Opioid use disorder (OUD) is a substance use disorder characterized by compulsive opioid use, typically resulting in the development of tolerance and withdrawal after stopping the intake of the drug. Currently, there are several options available for the treatment of OUD, including behavioral therapies, opioid maintenance therapy using full opioid agonists (e.g. methadone), partial μ-opioid agonists (buprenorphine) or medication assisted treatment with opioid antagonists .OUD imposes a significant economic burden on society in the US, with annual societal costs estimated between 55 billion dollars in 2007  and 78.5In a previous study, based on pharmacy and medical claims of 16 self-insured employer health plans in the US in the period of 1998 to 2002, opioid users were compared to a control group of non-abusers. Among these insured individuals, opioid abusers had higher rates of medical and pharmacy utilization and an increased number of comorbidities including poisoning, hepatitis, psychiatric illnesses, and pancreatitis. Healthcare expenditure for these individuals may have been as much as 8 times the total as non-drug users .In Europe, and especially in Germany, there are currently no detailed information about healthcare costs of OUD patients. Available research with regards to Germany, the Netherlands and Switzerland, that investigated the costs of treatment with heroin and methadone, was based on calculations using various assumptions. Due to the lack of data availability, it mainly used administrative costs, such as diagnosis related groups (DRG) costs or listed pharmaceutical prices .Therefore, to analyze healthcare costs of OUD patients in OMT, we designed a retrospective cost analysis in the German healthcare system from the statutory health insurance’s (SHI) perspective. The objective of the present study was to examine the overall healthcare costs of OUD patients in OMT using four cost categories: inpatient costs, outpatient costs, pharmacy costs and other costs that included sick pay, aids and remedies, and other medical services.In Germany, there are currently 78,500 OUD patients in OMT . ConcernUp to now, there are mixed findings regarding healthcare costs of different maintenance treatments: A systematic review indicates that pure pharmaceutical costs for methadone are somewhat less costly than buprenorphine . HoweverTaken together, the present study addresses the research gap of assessing the overall healthcare costs of patients treated with common opioid maintenance treatments Germany and thus provides a picture of the current cost situation of OMT treated OUD patients in Germany.This longitudinal nested case-control cohort study was based on an anonymized German health claims database, including 4 million insureds from 8 German statutory health insurances, that provided a research database that could be used for this study. The dataset included a 5% sample of overall German population covered by statutory health insurances from January 1, 2010 to December 31, 2015. All patients in the database used for this analysis had complete data across all years of the analysis (2010–2015). Patients with incomplete yearly follow-up data were excluded unless they died within the study period and were in the dataset until death.The dataset contained information on patients’ medical in- and outpatient claims, prescribed and delivered pharmaceuticals and insurance eligibility information. The whole study design was predefined by a detailed analysis protocol following the recommendation of the German society for epidemiology .index year for each patient was defined as year when the first diagnosis of F11.2 was coded in 2010 or 2011. Although the ICD-10 code for OUD provides no information on the underlying opioid, recent data show that heroin use still accounts for the majority of new opioid-related treatment demands in Europe with around 80% of all patients with OUD [Patients with OUD were identified using the International Classification of Diseases, 10th Edition German Modification (ICD-10-GM) code F11.2  in outpatient and/or inpatient care data in any of the quarters in the identification period (Quarter 01/2010 through 04/2011). For inpatient data, primary OUD discharge diagnoses as well as secondary diagnoses were considered. Outpatient physicians are required to designate their diagnoses as one of the following categories: validated, suspected, symptomless condition and excluded. A diagnoses becomes “validated” when it is established beyond doubt, e.g. by a lab test, a specialists’ assessment or the physicians’ own conclusions . For thewith OUD . FurtherOther diagnoses than OUD were not included in the current analysis, because OMT is by law only available to patients with opioid dependence, not with other opioid abuse diagnoses .relapse in the 12-months prior (see section Relapses for details). This was done because this study aimed to examine the healthcare costs of patients within OMT. Costs of OUD patients not in OMT were supposed to mainly occur within sectors outside of the health insurances and therefore not included in the database .Patients who were prescribed dihydrocodeine, diamorphine, or morphine between 2010 and 2015 were excluded from analyses. Moreover, patients who were younger than 18 years at their first OUD diagnoses in the inclusion period were excluded as they receive abstinence-oriented treatment, not OMT , 17. PatThe dataset included ICD-10-GM codes for all diagnoses given by inpatient or outpatient physicians that were analyzed in each quarter of the inclusion period. Comorbidities were assessed when assigned by a general practitioner or a specialist physician if the ICD-10-GM code was coded as “validated”: depression , personality disorder (F6X), anxiety , sleeping disorder (F51X), chronic hepatitis C (B18.2), chronic obstructive pulmonary disease , HIV/AIDS (B2X) and cardiovascular diseases (I20X-I25X).The dataset included Anatomical Therapeutic Chemical Classification (ATC) and pharmaceutical central numbers (PZN) for outpatient drugs prescribed and delivered.Hilfstaxe) [OMT medications delivered to a physician’s office for supervised, office-based OMT were analyzed using the ATC codes for methadone, levomethadone, or buprenorphine respectively. OMT medication prescribed for take-home treatment was identified by pharmaceutical central numbers (PZN) from German auxiliary tax (lfstaxe) .Table Patients were stratified in OMT groups with buprenorphine, methadone or levomethadone, based on the first prescription in each follow-up year. Patients could switch treatment groups in each follow-up year, according to their first prescription in each year, assessed in 12-month periods starting with each patient’s earliest relevant drug claim.The definition of relapses was based on a previous longitudinal cohort study  and summFor attributing patients’ years with and without relapses, hospital stays, and treatment interruptions were counted in the year they started, i.e. hospital stays were counted in the year of the admission stay and treatment interruptions on the first day without OMT medication.Annual costs from the statutory health insurances database were assessed for each follow-up year and for four different resource use categories: inpatient care , outpatient care , prescribed pharmaceuticals (outpatient setting only), and other costs that included sick pay, medical aids (e.g. wheelchairs) and remedies . Total costs were calculated as sum of the four resource use categories. Furthermore, costs were analyzed stratified for years with and without relapses.p-value of p < 0.05 was used in all statistical tests for determining statistical significance. We used the software MS Excel 2016 and SAS 9.4, SAS Institute, Cary (North Carolina), USA for statistical analyses.Variables were analyzed descriptively using frequencies and percentages for categorical variables and means with standard deviations for continuous variables. Group comparisons at baseline were performed using Chi-Squared tests and for categorial and t-tests for continuous variables. Group comparisons between different treatment groups were performed with analysis of variance (ANOVA) and least significant difference (LSD) post hoc tests. A Overall, 996 patients fulfilled the inclusion criteria, 41 of the patients were excluded for being under 18 years old. The remaining 955 patients had a mean age of 37.6 years (standard deviation (SD) = 8.5). For these 955 patients, a total of 3165 patient years in the follow-up period could be observed. In the index year, 350 patients were treated with buprenorphine, 336 with methadone, and 269 patients with levomethadone  were treated with buprenorphine in the first year, 269 with levomethadone (28.16%) and 336 with methadone (35.18%). During follow-up, 23 patients (2.4%) died of any cause. From these 23 patients, one patient was treated with buprenorphine, 10 patients with levomethadone and 12 with methadone at the time of death.Seven hundred twenty-seven patients (76.13%) stayed in OMT treatment during the whole four follow-up years, i.e. had at least one OMT prescription in each year. 59 patients (16.8%) changed from buprenorphine to methadone during the follow-up, 74 from buprenorphine to levomethadone (21.1%). From methadone, 30 patients (8.9%) changed to buprenorphine and 89 (26.4%) to levomethadone. 33 patients who were treated with levomethadone in the index prescription changed to buprenorphine (12.3%) and 78 changed to methadone (28.9%).Across all treatment groups, the most frequent comorbidities at index date were depression (42.1%), chronic hepatitis c 38.2%) and personality disorder (15.0%) . Post hoc LSD test showed a significant difference between buprenorphine and levomethadone . , methadone  and buprenorphine  revealed significant differences between the treatment groups  outpatient costs: 28.30% (1887 €); pharmacy costs: 32.41% (2162 €) and other costs: 4.28% (286 €). Costs of the methadone group consisted of inpatient costs accounting 38.83% (2753 €); outpatient costs: 25.02% (1774 €); pharmacy costs: 28.80% (2042 €) and other costs: 7.35% (521 €). And costs of the levomethadone group included 41.84% inpatient costs (3514 €), 24.39% outpatient costs (2049 €); 30.56% pharmacy costs (2567 €) and 3.21% other costs (269 €).We compared treatment costs in years with and without relapses. Results revealed that costs in years with relapses were about 10% higher than in years without relapses with hospital costs as cost driver   above years without relapses, indicating that preventing relapses might have positive effects on healthcare costs. This is in line with previous research that found associations between relapses and higher healthcare costs in other substance abuse patients: For alcohol addicted patients it was shown that patient groups with a lower risk of relapse also had lower healthcare costs .The strength of this study is that it used a large sample of the German statutory health insurances to assess healthcare costs directly from a database with health insurance claims data. To our best knowledge, this is the first study to examine costs of patients in OMT in a major European country.The limitations are as follows:a) The statutory health insurances database uses billing data which are designed for invoices and not for research activities. Therefore, typically not all necessary information is available, or it has limited quality.b) Persons entitled to other healthcare funds (e.g. members of the police), but also imprisoned patients are not covered by present data. About 10% of German population are members of private health insurance companies. There are differences between insureds of statutory and private health insurance companies in their socioeconomic status and health (care) behavior . For thic) We assume that total costs for patients without OMT are higher than indicated by present data because of social costs that are not reflected by statutory health insurances. Previous research  pointed d) There was no randomized assignment to the three drug groups. There is evidence that severely affected patients are more likely to receive methadone than buprenorphine , 32. We e) Since the cause of death was not included in the database, death cannot directly be attributed to OUD. The assumption, that every death is counted as a relapse therefore overestimated the number of relapses. Since only 2.4% of the study population died within follow-up, this should, however, only have little influence on the results.In this study we analysed the annual healthcare costs of patients with OUD in opioid maintenance therapy from the perspective of the statutory health insurance in Germany. We showed that costs attributed to patients with an OUD and OMT are more than double the cost of an average patient in Germany. The present study presented costs of OUD for the first time with a large German dataset, comparing patients with different OMT. In sum, we found overall costs for buprenorphine treatment were lower than those for methadone and levomethadone treatment, although the reasons for this need further investigation. Furthermore, preventing relapses might have significant impact on reduction of costs. Which risk groups should be assessed and which OMT and dosages should be used for such relapse prevention, requires further research beyond the scope of this paper.


Overexpression of URG11 promoted the expression of β-catenin, the growth, the migration, and invasion ability of prostate cancer cells. Taken together, this study reveals that URG11 is critical for the proliferation, migration, and invasion in prostate cancer cells, providing the evidence of URG11 to be a novel potential therapeutic target of prostate cancer.Upregulated gene 11 (URG11), a new gene upregulated by hepatitis B virus X protein, is involved in the development and progression of several tumors, including liver, stomach, lung, and colon cancers. However, the role of URG11 in prostate cancer remains yet to be elucidated. By determined expression in human prostate cancer tissues, URG11 was found significantly upregulated and positively correlated with the severity of prostate cancer, compared with that in benign prostatic hyperplasia tissues. Further, the mRNA and protein levels of URG11 were significantly upregulated in human prostate cancer cell lines , compared with human prostate epithelial cell line (RWPE-1). Moreover, by the application of siRNA against URG11, the proliferation, migration, and invasion of prostate cancer cells were markedly inhibited. Genetic knockdown of URG11 also induced cell cycle arrest at G1/S phase, induced apoptosis, and decreased the expression level of  Prostate cancer (PCa) is one of the most common cancers of urinary system and the second most common cause of cancer-related death in men in western world . MetastaUpregulated gene 11 (URG11) is a newly identified gene upregulated by hepatitis B virus X protein, located on the long arm of human chromosome 11, encoding a 70-kDa protein that consists of five chordin-like cysteine-rich repeats and a single C-type lectin domain . These dHowever, the role of URG11 in human prostate cancer remains to be determined. Therefore, the aim of the present study is to explore the role of URG11 in human prostate cancer, trying to reveal the expression of URG11 in human prostate cancer cells and in clinical human prostate cancer tissues and the relationship of URG11 with cell growth, migration, and invasion. The data demonstrates that URG11 may play a critical role in human prostate cancer progression.All human and animal experiments were approved by the Jinan University. All experiments using human specimens were performed in accordance with the Declaration of Helsinki. Informed consent was received from all participated subjects prior to the study. 68 cases of human tissue samples of prostate cancer and 74 cases of benign prostatic hyperplasia were obtained from prostate patients in the First Affiliated Hospital of Jinan University and Sun Yat-sen University Cancer Center, from Jun 2010 to Jun 2013. All animal procedures followed the humane care guidelines of the Chinese National Institute of Health, and the protocols were approved by the Committee on Animal Research of Jinan University.μg/ml) at 37°C in a humidified atmosphere with 5% CO2.Cell lines human prostate cancer DU145 and LNCaP were from the First Affiliated Hospital of Jinan University, human prostate cancer PC3 cell was a gift from NanFang Hospital of Southern Medical University, and nontumor human prostate epithelial cells (RWPE-1) were a gift from School of Pharmaceutical Sciences of Guangzhou Medical University. Cells were maintained in medium RPMI 1640  supplemented with 10% fetal bovine serum , penicillin , and streptomycin  according to the manufacturer's protocol. For real-time quantitative PCR, equal amounts of cDNA were added to SYBR premix EX Taq II (Toyobo) and run in Stepone real-time PCR system . The cycling program was 95°C for 5 s and 60°C for 30 s. The primers for real-time PCR are URG11 sense 5'-TGAATCAAGGAGTCGCTGGAC-3'; URG11 anti-sense 5'-GCATCTCACTGGAACACAAG-3'; GAPHD sense 5'-CAACGGATTTGGTCGTATTGGG-3'; GAPHD antisense 5'-CCTGGAAGATGGTGATGGGATT -3'. Each sample was assayed in triplicate, and GAPDH was used as an endogenous control. Results were normalization to the expression of GAPDH.siRNA duplexes (mixture of multiple siRNA fragments) targeting URG11 were synthesized by GenePharm . The siRNA sequences against URG11 are CAGACGGAUUGCUGUACUU and ACACAGACUUUACCUACAA . Optimalβ-catenin were obtained from Santa Cruz Biotechnology, Santa Cruz, CA, USA). GAPDH (Santa Cruz) was used as the loading control. Polyvinylidene fluoride (PVDF) membrane  transferred with proteins were washed with PBS containing 0.1% Tween-20 five times, the membrane was incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies  for 1 h, and bands were detected by enhanced chemiluminescence . Densitometric values were normalized to GAPDH levels.Cells were lysed in sample buffer and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described previously . Primary® AQueous One Solution Cell Proliferation Assay Kit  accordingly. In each group, cells were allowed to grow for 6, 24, 48, 72, and 96 h. At harvesting, 20 μl of CellTiter 96 AQueous One Solution reagent was added to each well in a total volume of 100 μl of medium for 3–4 h. Absorbance was measured at 490 nm using an ELISA plate reader. The growth rate was calculated from the absorbance, and the readings at 6 h time points in each group were set to 100%.MTS assay was applied to detect cancer cell growth as previously reported . Cell prμl binding buffer at 106 cells/ml, labeled with annexin V-FITC for 15 min and with PI for another 5 min; then cells were analysed by flow cytometry by a FACScan flow cytometer .Annexin V-FITC Apoptosis Detection Kit  was used to determine apoptotic cells and PI staining was used to reveal cell cycle stage. In brief, cells were resuspended with 400 2 incubator for 48 h at 37°C. Images of each well were acquired immediately and at 6, 12, and 24 h in four random fields using an inverted fluorescence microscope  at ×100 magnification. Wound closure was expressed as the average ± SD of the difference between the measurements at time zero and each time period.Cells were grown in six-well dishes until confluence; then cell culture was scraped with a pipette tip. Cells were washed twice with PBS and cultured in a 5% COμM), and 90% DMEM containing 10% FBS was added to the lower chamber and incubated for 24 h. The remaining cells in the upper chamber were removed. Cells migrating into the lower surface of the filter were fixed by PFA and stained with 2% crystal violet for quantification. For the invasion assay, the Transwell filter membrane was coated with Matrigel . Cell numbers were expressed as the mean ± SD.Migration and invasion assays were performed using a Transwell system , accordingly. Cells were seeded in the upper chamber of the Transwell insert and incubated with 0.5% DMSO or deguelin . Correlations between URG11 expression levels and clinic-pathological parameters are presented in p < 0.05). The URG11 levels in progressive TNM stages (III + IV stages) showed higher expressions than that in localized stages (I + II stages) (p < 0.05). The expression levels in metastatic prostate cancer showed higher staining signals than that in nonmetastatic cancer (p < 0.05). There was statistically significant correlation between URG11 expression level and histologic grade ( p < 0.05); also there was positive correlation between URG11 expression level and TNM stage ( p < 0.05). These data suggest that URG11 is highly expressed in prostate cancer samples and positively correlated with histologic grade and TNM stage. Further, we determined the expression levels in prostate cancer cells, including DU145, LNCa, and PC3 cell, and nontumor human prostate epithelial cells (RWPE-1). As shown in We first investigated the expression levels of URG11 in clinical prostate cancer specimens. Surgical specimens from 68 cases of prostate cancer and 74 cases of benign prostatic hyperplasia collected from the First Affiliated Hospital of Jinan University and Sun Yat-sen University Cancer Center were immunohistochemically stained for URG11. As shown in To further explore the role of URG11 in prostate cancer cells, we applied siRNA approach to silence URG11. Accordingly, we synthesized siRNA fragments against URG11 which had been proved functional . On the p < 0.05). Further, by annexin V and PI staining under flow cytometric analysis, URG11 silencing significantly induced cell apoptosis, compared with those in normal culture and NC group (Figures β-catenin. Together, these data suggest that URG11 is involved in cell cycle progression and inhibition of URG11 would induce cell cycle arrest and cell apoptosis, where the molecular signaling pathway via β-catenin may be involved.To better understand the role of URG11 in prostate cancer metastasis and the detailed mechanism, flow cytometric analysis was performed with PI- and/or annexin V staining for apoptosis and cell cycle analysis. URG11 knockdown showed significant cell cycle arrest, compared with that in normal culture and in NC control group Figures . URG11 s Figures . Moreoveβ-catenin along with URG11 overexpression (Above data showed that inhibition of URG11 markedly suppressed cell proliferation, migration, and invasion of prostate cells. Then we asked whether overexpression of URG11 would result in the opposite. We constructed an URG11 encoding plasmid and overexpressed the plasmid in LNCaP prostate cancer cells. The expression of URG11 was confirmed by western blot and the results revealed the upregulation of pression . The stapression  and by wpression . Furtherpression . These dβ-catenin in prostate cancer cells. Overexpression of URG11 significantly increased the growth, migration, and invasion of prostate cancer cells. Taken together, these data indicate that URG11 may function as an oncogene in prostate cancer. URG11 may be involved in the early formation and development of prostate cancer, of which the early stage is androgen dependent.URG11, as an effector of hepatitis B virus X protein, is upregulated in a variety of human cancers, including hepatocellular cancer, gastric cancer, colon cancer, lung cancer, esophageal cancer, and breast cancer . URG11 pAberrantly expression of URG11 is critical in carcinogenesis. According to the literature, silencing URG11 suppressed the proliferation of hepatocellular carcinoma cells via downregulation of several G1/S phase-related proteins and attenuates hepatocellular tumor growth in nude mice . By geneβ-catenin level was potently decreased upon URG11 knockdown and increased with URG11 overexpression. β-catenin is the main structure of adhesion between cells and is necessary for the creation and maintenance of epithelial cell layer. β-catenin is a critical end component in the Wnt signaling pathway, which is involved in promoting tumor progression, regulation of cell growth, proliferation, and invasion [β-catenin pathway controls the expression of many downstream target genes such as cyclin D1, matrix metalloprotease-7, and c-Myc, leading to uncontrolled cell growth and invasion [β-catenin pathway is implicated in prostate cancer tumorigenesis [β-catenin and the downstream targets including c-Myc, cyclin D1, and MMPs [β-catenin, suggesting the same inactivation of Wnt/β-catenin signaling pathway. Detailed exploring of URG11 regulated β-catenin would be performed in our further study, and role of URG11 would be determined in animal model in vivo.Interestingly, we found that invasion –19. Wnt/invasion –23. Accuigenesis –27. URG1and MMPs . Consistβ-catenin levels in prostate cancer cells. URG11 might be a potential novel clinical target for prostate cancer.Taken together, the study here provides evidence that URG11 is positively correlated to prostate cancer tumorigenesis and metastasis, inhibition of URG11 suppresses proliferation, migration/invasion, and


To investigate the function of ac111, an ac111-knockout AcMNPV (vAc111KO) was constructed through homologous recombination in Escherichia coli. Viral growth curve analysis and plaque assays showed that the deletion of ac111 had no effect on infectious budded virion production. Quantitative real-time polymerase chain reaction analysis confirmed that viral DNA replication was unaffected in the absence of ac111. Electron microscopy revealed that the ac111 deletion did not affect nucleocapsid assembly, occlusion-derived virion formation, or the embedding of occlusion-derived virions into the occlusion bodies. However, in vivo bioassays showed that although the deletion of ac111 did not affect the per os infectivity of AcMNPV in Spodoptera exigua larvae, it led to an approximately five-fold reduction in infectivity of AcMNPV in Trichoplusia ni larvae, and vAc111KO took approximately 21 h longer to kill Trichoplusia ni larvae than the wild-type viruses. Taken together, our results demonstrated that although ac111 is not essential for virus replication in vitro, it plays an important role in the per os infectivity of AcMNPV in a host-dependent manner.The Autographa californica multiple nucleopolyhedrovirus (AcMNPV)  Baculoviridae consists of Alphabaculovirus, Betabaculovirus, Gammabaculovirus, and Deltabaculovirus [polyhedrin (polh) gene, Alphabaculovirus can be further subdivided into Group I and Group II nucleopolyhedroviruses (NPVs) [Baculoviridae family and the first baculovirus to be completely genome-sequenced [Baculoviruses are a family of rod-shaped, enveloped, double-stranded DNA viruses which are specifically pathogenic to invertebrate species, mainly belonging to the orders Lepidoptera, Hymenoptera, and Diptera . Baculovulovirus . Based os (NPVs) . Autograequenced . The AcMequenced . To dateequenced ,5,6,7.During the baculovirus infection cycle, two morphologically distinct but genetically identical progeny virion phenotypes, budded virions (BVs), and occlusion-derived virions (ODVs), are produced. Although BVs and ODVs are similar in their nucleocapsid structure, they differ in their origin and the composition of their envelopes, which reflects their distinct roles in the baculovirus life cycle ,9. In thper os infectivity factors (PIFs), including P74, and PIF-1 to 8 [Spodoptera frugiperda NPV, Sf58, which is only conserved in lepidopteran baculoviruses, has been shown to be a PIF of lepidopteran-infecting baculoviruses [ac18 is a highly conserved gene in lepidopteran NPVs, though ac18 deletion did not affect viral propagation in vitro or AcMNPV infectivity for Trichoplusia ni larvae; however, the LT50 was 24 h longer for the ac18 knockout virus in T. ni larvae compared to the wild-type virus [ac114 and ac124 are nonessential for AcMNPV replication in vitro, ac114 and ac124 selectively affect viral infectivity and viral killing speed, respectively, of AcMNPV in S. exigua larvae [per os infection is a complex process involving numerous virus proteins coordinating the establishment of the primary infection.The site of baculovirus primary infection is the midgut of host insects. Upon oral ingestion, the OBs are dissolved under alkaline conditions in the insect midgut, and they release ODVs, which then penetrate the peritrophic matrix and infect epithelial cells. A number of ODV envelope-associated proteins that play important roles in oral infectivity are designated as F-1 to 8 ,21,22,23oviruses . Moreoveoviruses ,29,30,31pe virus . Previoua larvae ,29. Therorf111 (ac111) is located between AcMNPV nucleotides (nt) 101,624 and 101,827 [ac111 is transcribed as an early gene [Bombyx mori NPV, Bm93, has been found not to be essential for virus replication [ac111 in the AcMNPV life cycle is still unknown.The AcMNPV rly gene . Recentllication . Howeverac111 knockout AcMNPV (vAc111KO) was constructed to investigate the role of ac111 in the AcMNPV life cycle. We found that the ac111 deletion had no significant effect on infectious BV production, viral DNA replication, or viral morphogenesis. However, the oral infectivity bioassays showed that, in T. ni larvae, the infectivity of vAc111KO was reduced by about five times relative to the wild-type virus, and the mutant virus took approximately 21 h longer to kill these larvae. Interestingly, the deletion of ac111 did not affect the infectivity of AcMNPV in another lepidopteran host, S. exigua larvae. Therefore, our results suggest that Ac111 plays an important role in virus per os infectivity in a host-dependent manner.In this study, an HHpred analysis , NCBI’s Spodoptera frugiperd and S. exigua, respectively, were cultured at 27 °C in TNM-FH medium  supplemented with 10% fetal bovine serum, penicillin (100 μg/mL), and streptomycin (30 μg/mL). The bMON14272 bacmid , which contains the AcMNPV genome, was propagated in DH10B cells as previously described [T. ni and S. exigua larvae were reared on an artificial diet at 28 °C [50) endpoint dilution assay in Sf9 cells or Se301 cells, as previously described [The Sf9 cell line and Se301 cell line , which aescribed . T. ni aescribed .ac111-knockout AcMNPV bacmid (bAc111KO) was generated through ET homologous recombination in Escherichia coli, as previously described [ac111 ORF  was PCR-amplified from bMON14272 using the primers ac111PF1/ac111PR1  was PCR-amplified from bMON14272 using the primers ac111PF2/ac111PR2. The 5′- and 3′-PCR products were digested with PstI/HindIII and SacI/BamHI, respectively, and ligated into pUC18-Cm [Cm) gene cassette to generate pUC18-111US-Cm-111DS. The plasmid was then digested with SacI and HindIII to obtain a linear 1778-bp fragment in which the Cm gene cassette was flanked by ac111 5′- and 3′-regions. Homologous recombination between the deletion region of ac111  and the Cm cassette in the linear fragment was performed to obtain an ac111-knockout bacmid, bAc111KO. The replacement in bAc111KO was confirmed by PCR and sequencing.Employing the bacmid bMON14272, an escribed . BrieflypUC18-Cm , which cpolh gene and the enhanced green fluorescence protein  gene, was transformed into electrocompetent DH10B cells harboring the bAc111KO bacmid and the pMON7124 helper plasmid as previously described [ac111-knockout virus, vAc111KO. To generate the ac111-repaired virus, vAc111:HA, the donor plasmid pFB1-Ac111:HA-PH-GFP was constructed using the following steps. The 531-bp DNA fragment containing the promoter and the ORF of ac111 tagged with an HA epitope prior to the stop codon was PCR amplified from bMON14272 using the primers ac111PF3/ac111PR3, and it was cloned into the SacI/BamHI-digested pUC18-SV40 plasmid [SacI/XbaI, and subcloned into SacI/XbaI-digested pFB1-PH-GFP to generate the donor plasmid pFB1-Ac111:HA-PH-GFP. The pFB1-Ac111:HA-PH-GFP was then transformed into electrocompetent DH10B cells harboring the bAc111KO bacmid and the helper plasmid pMON7124 to generate the ac111-repaired virus, vAc111:HA. Similarly, vAcWT, a wild type virus, was also constructed by inserting polh and gfp genes into bMON14272.To facilitate the detection of viral infection, the pFB1-PH-GFP donor plasmid, containing the AcMNPV escribed , to gene plasmid  to const6) were transfected with 1.0 μg bacmid DNA using the Cellfectin liposome reagent . For infection, Sf9 cells (1.0 × 106) or Se301 cells (1.0 × 106) were infected with BVs at a multiplicity of infection (MOI) of 5 TCID50/cell. At the designated time points post transfection (p.t.) or post infection (p.i.), the supernatants containing BVs were harvested and titered. To further evaluate the effect of the ac111 deletion on BV production, a plaque assay was performed as described previously [Sf9 cells , 1 μL water (control) or 1 μL OB suspensions of each virus was administered to a small piece of diet of newly molted third-instar T. ni or S. exigua larvae, respectively. For each virus in T. ni, the doses were 9, 30, 90, 3 × 102, and 9 × 102 OBs per larva. For each virus in S. exigua, the doses were 9 × 102, 3 × 103, 9 × 103, 3 × 104, and 9 × 104 OBs per larva. Larvae were reared individually in 24-well plates until all of the diet was consumed, then fresh diet was added. Only the larvae that completely ingested the diet with OBs within 12 h were further reared and monitored daily, until all larvae had either pupated or died. The median lethal time (LT50) was determined using the droplet feeding method [T. ni or fourth-instar S. exigua larvae were starved for 6 h, and then they were fed with 1 μL OB suspensions of each virus with concentrations of 1 × 107 or 1 × 108 OBs/mL, respectively. For each virus, the final mortalities were about 99% or 86% in T. ni or S. exigua, respectively. The larvae that have ingested virus were reared individually and the mortality was recorded every 12 h until all larvae had either pupated or died. The experiments with each dose in both oral and injection treatments were performed three times (30 larvae per dose). The LD50 value and the LT50 value were determined by probit analysis and the Kaplan-Meier estimator, respectively.In order to determine whether the eviously . Brieflyeviously . Differeg method . The newac111 are present in lepidopteran-specific baculoviruses. Sequence-based searches have shown that Ac111 belongs to the Baculo-8kDa superfamily with no homology to other identified functional domains. The alignment of Ac111 orthologs from several selected lepidopteran-specific baculovirus groups, including AcMNPV, Hyphantria cunea NPV (HcNPV), Orgyia pseudotsugata MNPV (OpMNPV), Rachiplusia ou MNPV (RoMNPV), Euproctis pseudoconspersa NPV (EupsNPV), Chrysodeixis chalcites NPV (ChchNPV), Helicoverpa armigera NPV (HearNPV), Lymantria dispar MNPV (LdMNPV), Xestia c-nigrum GV (XecnGV), and Spodoptera litura GV (SpliGV), revealed that a T-T/S-L-H/Y-X2-N motif (where X was any other amino acid) was conserved in all Ac111 homologues  or vAcWT (63%)    was approximately five times more than that of vAcWT  was comparable to that of vAcWT (85 h), but the LT50 value of vAc111KO (106 h) was 21 h longer than that of vAcWT  (ac111 did not impact per os infectivity of AcMNPV in S. exigua larvae.To determine the effect of p> 0.05) . Intrahep> 0.05) . Howeverof vAcWT . The LT5of vAcWT . Therefonterval) . This suac111 plays a role in primary oral infection of AcMNPV and the effect is host dependent.Taken together, the bioassays demonstrated that ac111 is one of the highly conserved lepidopteran-specific baculovirus genes, but its role in the AcMNPV replication remains unknown. In this study, we demonstrated that ac111 is important for AcMNPV to efficiently establish per os infection in T. ni larvae specifically, while is dispensable for per os infectivity of AcMNPV in S. exigua larvae, thus providing evidence for its important role as a conserved baculovirus gene.ac111 in the AcMNPV life cycle, an ac111-knockout virus was generated and characterized. We found that ac111 was dispensable for virus replication in vitro as evidenced by indistinguishable characteristics such as a normal viral growth curve, normal plaque, steady viral DNA replication and normal virus morphogenesis, compared to those of vAcWT. These results were consistent with the study on Bm93 [50 and LT50 of vAc111KO OBs and those of vAcWT in S. exigua larvae. However, the LD50 of vAc111KO OBs was approximate five times higher than that of vAcWT OBs in T. ni larvae, and the LT50 of vAc111KO was 21 h longer than that of vAcWT in T. ni larvae. Furthermore, the deletion of ac111 did not affect BV infectivity through intrahemocoelic injection in T. ni and S. exigua larvae. Taken together, these results indicated that ac111 affects the per os infectivity of AcMNPV in a host-dependent manner.To investigate the function of  on Bm93 . Bioassapifs and several virus virulence-related genes, such as ac111, ac145, and ac150. Baculoviruses initiate infection orally when their insect hosts consume and digest OBs in the alkaline pH of the midgut, releasing ODVs to infect the midgut epithelial cells. A number of viral membrane proteins, including the PIF proteins, have been identified to be essential for this process. Considering the pivotal role of PIFs, these proteins are encoded by baculovirus core genes [per os infection of all baculoviruses and thus have been conserved during baculovirus evolution. Other virus virulence-related genes are only conserved in certain baculoviruses, implying that they might function distinctly compared to PIFs in a host dependent manner and facilitate the efficient establishment of baculovirus infection in specific hosts.Of the 154 putative ORFs encoded in the AcMNPV genome, the function of more than 70% of these ORFs has been determined. While some genes were found to be nonessential and could be deleted without affecting virus replication, some genes are essential for virus replication in vitro and participate in critical steps in the virus life cycle, such as DNA replication, early/late gene transcription, or nucleocapsid assembly. There are some other genes that are dispensable for virus replication in vitro but function during oral infection of insect larvae. Genes belonging to this category include the nine re genes , indicatac111, previous studies have demonstrated that two AcMNPV genes, ac145 and ac150 affect oral infectivity in a host dependent manner [ac145 and ac150 encode proteins related to the 11K proteins with a characteristic C6 motif of conserved cysteine residues in a well-defined spacing pattern. Previous studies have found that the deletion of ac145 led to a six-fold drop in infectivity of AcMNPV in T. ni larvae but not in Heliothis virescens larvae [ac150 had no effect on infectivity of AcMNPV in either host [ac145 and ac150 significantly reduced the infectivity of AcMNPV (39-fold) in H. virescens larvae [50 for ac150 deletion AcMNPVcompared with wild-type virus in H. virescens, S. exigua, and T. ni larvae , suggesting that Ac150 indeed had an enhancing effect on per os infection and that the effect was host-specific [ac111 specifically affected viral infectivity and killing speed of AcMNPV in T. ni larvae but is dispensable in S. exigua larvae. Therefore, although AcMNPV could establish infection in T. ni, S. exigua, and H. virescens larvae, differences might exist that subtly affect the fitness of AcMNPV in these different insect hosts. It is intriguing to speculate that a group of proteins in AcMNPV, such as Ac111, Ac145, and Ac150, might specifically affect the infectivity of a virus in different hosts. We also cannot rule out the possibility that these viral proteins might interact with each other or with specific host proteins, and these interactions may eventually determine the host tropism of AcMNPV. Further studies are needed to verify this assumption.Similar to t manner . ac145 as larvae . On the her host . Howevers larvae . In anotspecific . In thisac111 was not required for AcMNPV replication in vitro, but it played a pivotal role in the viral infectivity in vivo in T. ni but not in S. exigua larvae. Further experiments are needed to unravel the specific role of ac111 during AcMNPV infection in T. ni larvae. These studies will likely shed light on our understanding of the mechanism of baculovirus–insect interactions, to ascertain the fitness of baculoviruses in different hosts.Taken together, this study has shown that


Increases in bin ranges for these characteristics are simultaneously accompanied by a decrease in hybridization specificity—the ratio between specific and cross-hybridization signals. However, both averaged hybridization signals exhibit growing trends along with an increase of probes’ binding energy, where the hybridization specific signal increases significantly faster in comparison to the cross-hybridization. The same trend is evident for the S function, which serves as a combined evaluation of probe binding energy and occurrence of probe’s k-mers in the genome. Application of S allows extracting a larger number of specific probes, as compared to using only binding energy. Thus, we showed that high values of specific and cross-hybridization signals are not mutually exclusive for probes with high values of binding energy and S. In this study, the application of a new set of sequence characteristics allows detection of probes that are highly specific to their targets for array design and other bio-techniques that require selection of specific probes.Off-target oligoprobe’s interaction with partially complementary nucleotide sequences represents a problem for many bio-techniques. The goal of the study was to identify oligoprobe sequence characteristics that control the ratio between on-target and off-target hybridization. To understand the complex interplay between specific and genome-wide off-target (cross-hybridization) signals, we analyzed a database derived from genomic comparison hybridization experiments performed with an Affymetrix tiling array. The database included two types of probes with signals derived from (i) a combination of specific signal and cross-hybridization and (ii) genomic cross-hybridization only. All probes from the database were grouped into bins according to their sequence characteristics, where both hybridization signals were averaged separately. For selection of specific probes, we analyzed the following sequence characteristics: vulnerability to self-folding, nucleotide composition bias, numbers of G nucleotides and GGG-blocks, and occurrence of probe’s  Many biotechnology applications involve oligoprobe hybridization with complementary targets in DNA or RNA as a basic procedural step. One such application is microarray technology. High throughput sequencing is gradually replacing microarrays as the preferred method for studying cellular transcript expression levels. However, microarrays are still dominating certain applications, such as identification of transcription binding sites , and genDesirable reactions between an oligoprobe and its complementary target are frequently complicated by other undesirable interactions of the probe. Particularly problematic is off-target probe binding with partially complementary DNA or RNA sequences, which are almost always present in a hybridization mixture. These interactions happen in parallel with on-target interactions. Microarray hybridization is an excellent technology for characterizing an oligoprobe’s on-target and off-target interactions. A single microarray experiment, especially with comparative genomic hybridization (CGH) –6, allowOne microarray application is the evaluation of gene copy number variation, and can be performed using CGH experiments  with AffHybridization signals derived from one microarray experiment even with the same target concentration are variable. Uneven hybridization conditions , target A number of studies have analyzed factors that influence genome-wide cross-hybridization levels of microarray probes. Duplexes of 10–16 nucleotides that are complementary to targets may be sufficient to generate a cross-hybridization signal –28. For To standardize terminology definition, we recommend that the scientific community discriminate between absolute cross-hybridization signals and relative cross-hybridization values. The term “absolute” cross-hybridization is used to identify signals that derive from probes that interact with partially complemented (off target) sequences only, e.g. without fully complemented targets. The term “relative” cross-hybridization represents the proportion of absolute cross-hybridization in an overall hybridization signal. Finally, the overall hybridization signal is represented by a sum of target specific and absolute cross-hybridization signals.Why would such terminology and discrimination be useful for microarray hybridization studies? Two probes might have similar absolute, but different relative, cross-hybridization values. The latter is more important for probe design than the former. Relative cross-hybridization in an optimal probe should be low, whereas the same is not necessary for absolute cross-hybridization. Moreover, probes with low absolute cross-hybridization might have a low specific signal component and might be unsuitable for sensitive target detection and, consequently, for array design. A limited number of studies have analyzed relationships between probes’ sequence characteristics and target specific and/or cross-hybridization signals: two publications describe such analysis for 50-nt  and 25-nThis study describes the analysis of relationships between sequence characteristics and hybridization signals of probes. The focus of this study is not limited to hybridization specificity or cross-hybridization signals. We have concentrated on the difference between absolute and relative cross-hybridizations and their divergent behavior according to changes in various sequence characteristics. These findings could be used for further optimization of recent advanced probe-target hybridization models  as well In normal human somatic chromosomes, each gene is represented by two copies . In the 4 data points from the same hybridization experiment, performed with the same chip  is represented by signals from probes with targets. Overall hybridization combines target specific and cross-hybridization signals.Absolute cross-hybridization (A) is represented by signals that derive from interactions of probes with partially complemented (off target) sequences only, e.g. without fully complemented targets.Relative cross-hybridization (R) is the proportion of absolute cross-hybridization in the overall hybridization signal: R = A/OTarget Specific hybridization (Sh) is the difference between overall hybridization and absolute cross-hybridization: Sh = O − AHybridization specificity (HS) is the ratio between target-specific and absolute cross-hybridizations: HS = Sh/A = (O-A)/A = 1/R-1ftp://ncbi.nlm.nih.gov/genomes/H_sapiens/ and created a table of occurrences of k-mers (where 7 ≤ k ≤ 11) for the human chromosomes. The frequencies of k-mers (where 7 ≤ k ≤ 11) we define as a number of occurrences of the k-mer normalized by total number of all k-mers occurrences in the human chromosomes. For each oligonucleotide probe in our set of 20K oligonucleotides, we calculated the minimum, maximum and total number of occurrence of all k-mers: fifteen 11-mers, sixteen 10-mers, seventeen 9-mers, eighteen 8-mers and nineteen 7-mers, presented in the 25-nt oligonucleotide passenger strand. Genomic occurrence of each k-mer in an oligoprobe was calculated using in-house scripts [We downloaded publicly available human genome sequences for the GRCh38.p7 version of the genome assembly  scripts . In thisS function using the earlier described model and approach for calculation of predicted probes’ specificity [S is based on the following formulas:S is the ratio between predicted specific signal (numerator) and predicted accumulative cross-hybridization signal (denominator); ΔGspec is the free energy change related to the reaction of fully paired duplex formation between an oligoprobe and target sequence; ΔGcross is the free energy change related to the reaction of duplex formation between an oligoprobe and partially complementary sequence in genomic DNA; Xolig is the theoretical estimation of the accumulative cross-hybridization component of the oligonucleotide. Assuming every target has a core of 7-11nt of exact complementarity, we can estimate Xolig by the following expression:i is a position of n-mer in the oligonucleotide, and Nn,i − the total number of occurrences of n-mer (started from i position of n-mer in the oligonucleotide) in the human genome.We estimate theoretical values of cificity . CalculaWe have recently evaluated the sequence complexity and sequence asymmetry (SC and SA) scores  based onSAS = (fA − fT)2 + (fG − fC)2, where SAS is the sequence asymmetry and simplicity score of a probe, and fN is a frequency of N nucleotide  in a probe.The sum of the squared differences between frequencies of A and T nucleotides and of G and C nucleotides represents the SAS score of a probe: Theoretically, the SAS score’s range varies from 0 to 1, where 0 corresponds to probes with equal proportions of each nucleotide and 1 corresponds to a probe that consists of only a single type of nucleotide. So, for example a score of 1 would represent a probe comprised entirely of As. In this database, the SAS score values range from 0.0015 to 0.4.ΔGfolding and ΔGbinding respectively. ΔGfolding was calculated by the A-Fold software [ΔGbinding was evaluated using in-house scripts and previously published nearest neighbor parameters [The probe’s self-folding and binding energies were evaluated by calculating software –42, whilrameters –45.ΔG) and theoretically estimated function S, where S is calculated based on the combined evaluations of probe binding energy and genomic occurrence of k-mers (see above).Averaging of hybridization signals from consecutively positioned tiled probes targeting the same gene is a routine procedure for CGH data analysis. Such averaging diminishes problems related to signal variability from individual probes, and in turn improves detection of deleted gene regions versus those with variable copy numbers. We applied a signal averaging procedure to analyzed data, where we sorted probes into bins according to their sequence characteristics and then averaged signals from each bin. The list of probe characteristics that was used as categorization criteria for binning and averaging included G-count, GGG-block count, nucleotide composition bias measured as SAS score (see below), self-folding, genomic occurrence of 11-mers, binding energy  a combination of specific signal and cross-hybridization or (ii) genomic cross-hybridization only. Absolute cross-hybridization applies to cross-hybridization signal that derives from contributions of all off-target interactions of each probe. The relative cross-hybridization term applies to a proportion of absolute cross-hybridization in an overall probe’s signal, which includes two components: the target specific signal and cross-hybridization. Therefore, while the absolute cross-hybridization represents a signal, the relative one represents a calculated signals’ ratio.All probes from the database were categorized into bins according to their sequence characteristics and hybridization signals in each bin were averaged. The difference between bins with averaged signals of probes with and without a target represents averaged specific hybridization for all probes characterized by similar sequence characteristic. The calculation of the difference was used for evaluation of hybridization specificity, which is a ratio between specific- and absolute cross-hybridizations. The binning and averaging approach allowed finding and analyzing trends in relationships between probes’ hybridization and sequence characteristics without knowledge of all hybridization characteristics of each individual probe.Hybridization specificity is negatively affected by high G-count and/or by G-block presence in a probe . We collMultiple studies suggest that array probes with four G nucleotides in a row (GGGG-block) are responsible for hybridization artifacts because of their involvement in non-WC interactions –18. We fWe measured each probe’s nucleotide bias by calculating the probe’s SAS score see , which vΔG values of probe’s self-folding varies from -14 to 0 kcal/mol. The hybridization specificity of comparatively open probes  is at least twice as high versus the specificity of those with high self-folding vulnerability .Open probes with low self-folding potential are more specific with low absolute . ΔG valuWe found that measurement of genomic occurrence of 11-mers in oligoprobes can be used for further increasing hybridization specificity. The minimum values of genomic occurrence of all 11-mers in a probe are particularly suitable for this purpose. Filtering out all probes with minimum values above 250 caused a decrease of cross-hybridization and an increase of hybridization specificity .ΔG values vary from -16 to -33 kcal/mol. Probes’ overall hybridization and their absolute cross-hybridization values increase with probe binding energy  is at least three times higher versus the specificity of probes with low binding energy . Moreover, hybridization specificity of probes with the lowest binding energy  is close to zero  more than doubled after filtration steps described above were applied , and high self-folding potential. Each step of probe removal improved specificity of remaining probes, independent from the order of the applied filtration parameters. The effect that increased specificity was more pronounced among the probes with high binding energy. The specificity of probes with maximal binding energy  and could be used for the prediction of model experiments. We also estimated the function S, related to the predicted probe’s specificity, using the earlier described approach [Xolig, the theoretical estimation of accumulative cross-hybridization, was used as the denominator  SAS score above 0.05; (iii) G-count above 7 nt; (iv) occurrence of two GGG-blocks anywhere in a probe sequence; (v) one GGG block being present at any of the first four nucleotide positions from probe’s 5’ end; (vi) genomic occurrence of 11-mers above 250 : (i) -Each negative characteristic with the indicated cut-off point diminishes hybridization specificity by a factor of at least two. Thus, the hybridization specificity of remaining probes is two times higher in comparison with the hybridization specificity of filtered probes (0.25 versus 0.12). Approximately 50% of probes analyzed in this work have at least one of these negative sequence characteristics. The “specific probes” or “optimal probes” are those that do not have any negative characteristics, or by definition, these probes contain the following characteristics:ΔGbinding above 23.5 kcal/mol;-ΔGfolding below 1.5 kcal/mol;- SAS score below 0.05;G-count below 8 nt;no GGG-blocks.genomic occurrence of 11-mers below 250.The hybridization specificity of these selected probes is approximately two times higher in comparison with the hybridization specificity of remaining probes (0.5 versus 0.25). Approximately 4% of probes considered in this work belong to the “specific” category (hybridization specificity ~0.5).S  allows the user to extract a larger number of specific probes (8%). The default parameters for the specific probe design are following:There is an option to classify the oligoprobes based on the estimated values of S , insteadS above 12.5;ΔGfolding below 1.5 kcal/mol;- SAS score below 0.05;G-count below 8 nt;no GGG-blocks.In this study, we demonstrated that elevated levels of hybridization specificity and absolute cross-hybridization are not mutually exclusive and may be attributed to the same probe sets. Moreover, trends in which hybridization specificity and absolute cross-hybridization are changing along with a sequence characteristic can differ significantly. They might be either both positive, either both negative, or opposite of each other, depending on the sequence probe characteristic.Low cross-hybridization is not necessarily indicative of a probe’s high specificity. The low value of cross-hybridization is frequently a result of the poor ability of the probe to interact in general; in such cases, both specific and cross-hybridizations are low. Conversely, the probes that generate high specific hybridization could also generate high cross-hybridization because of their better interaction ability, both on- and off-target. It is important to differentiate between absolute and relative cross-hybridization values that also account for the specific hybridization. Some published studies suggest avoiding probes with high binding energy because of their high vulnerability to cross-hybridization . Our stuk-mers in human genomes, binding energy, and partially through SAS scores and self-folding evaluation of probes, while their off-target interactions based on non-WC pairing are partially evaluated through assessment of G-effects. These evaluations are helpful for detection of probes that are involved in off-target hybridization reactions and for removal of such probes from the pool of all probes. Such filtering significantly improves hybridization specificity of the remaining probes on average, and could be used during the array design process. The improvement is more pronounced in the category of the probes with high binding energy because of the proportion of the probes involved with parallel hybridization reactions is larger in this category compared to ones with lower binding energy. These probes have high nucleotide bias: they are GC-rich and capable of stable self-folding, specifically, enriched with G and GGG blocks.Specific probe-target interactions occur in parallel with non-specific interactions of two types, WC and non-WC. Potential off-target interactions of probes based on WC pairing are mainly evaluated through estimation of target This study demonstrates that bins of probes with high binding energy are enriched with more specific oligos and show significantly greater averaged hybridization specificity. However, an increase of target concentration (gene copy numbers for CGH) may also affect the behavior of the probes with high binding energy, where the trend may be different, or hybridization specificity may reach a plateau. Hadiwikarta and co-authors  stated bMany bio-techniques, beyond microarrays technology, rely on specific interactions of the oligoprobe with complementary targets. Even though the results of the study presented here are derived from microarray experiments, the physicochemical principals underlying our findings are “microarray-free” and most of the steps in the design procedure may be extrapolated universally.Hybridization specificity and absolute cross-hybridization values of oligoprobes both increase with increasing binding energy of probes in the analyzed bins. We showed that high specific hybridization and high cross-hybridization are not mutually exclusive, and may be attributed to the same probe sets. In other words, the level of non-specific interactions for some molecules may be high, but the ratio between off-target and total hybridization signals may be low. This also means that the specific signal is sufficient in magnitude for a high on-target/off-target ratio, which defines interaction specificity.k-mers in an oligoprobe decreases the probe’s hybridization specificity.Low hybridization specificity of a probe is related to its high self-folding vulnerability, nucleotide composition bias, G-richness and GGG-block presence. Probes with these characteristics have high absolute and high relative cross-hybridization values. High genomic occurrence of S as the combination of probe binding energy and occurrence of probe’s k-mers (7 ≤ k ≤ 11) in the genome for efficient oligoprobe design. Along with an increase of S function of probes, both averaged hybridization signals (specific and cross-hybridization) exhibit growing trends, where the hybridization specific signal increases significantly faster in comparison to the cross-hybridization. The application of S allows extracting a larger number of specific probes, as compared to using only binding energy. Thus, the S function together with other described sequence characteristics are promising features for further improvement of oligoprobe design for bio-techniques that require selection of most specific probes.In this study, we suggested applying the function S1 Figst positions of the probes; C) the numbers of GGG-blocks located in 2nd to 4th positions of the probes; D) the numbers of GGG-blocks located in in any position from the 5th to 23rd; E) probes’ SAS score; F) self-folding vulnerabilities in the probes; G) probes’ binding energies.Oligoprobes were categorized into bins according to their variable sequence characteristics. The relative cross-hybridization value was calculated for each bin as an average ratio of absolute cross-hybridization versus overall-combined hybridization value. Relationship between probes’ relative cross-hybridization values and A) the numbers of G in the probes; B) the numbers of GGG-blocks in the1(EPS)Click here for additional data file.S2 FigΔGfolding above 1.5 kcal/mol, SAS score above 0.05, or G-count above 7 nucleotides, and GGG-block(s) presence.Oligoprobes were categorized into bins according to variable probes characteristics. Averaged absolute or relative cross-hybridization signals were calculated for each bin and their values are shown as columns. Absolute cross-hybridization values are shown on the primary Y axis and relative cross-hybridization is shown on the secondary Y axis. Categorization according to A) G-count ; B) SAS score ; C) self-folding vulnerability ; D) probes categorization according to binding energy. Probes with characteristics that negatively affect their hybridization specificity were excluded from the categorization procedure. The exclusion criteria were -(EPS)Click here for additional data file.S3 FigOligoprobes were categorized into bins according to variable probes’ sequence characteristics. Averaged absolute cross-hybridization signals or hybridization specificity were calculated for each bin. Absolute cross-hybridization values are shown according to primary Y axis and hybridization specificity is shown on the secondary Y axis. All probes were categorized according to A) G-count; B) SAS score; C) probes’ self-folding vulnerability; D) probes’ binding energy. Probes with characteristics that negatively affect their hybridization specificity were excluded from the categorization procedure. The exclusion criteria were the same as described in (EPS)Click here for additional data file.


The rapid spread of influenza viruses (IV) from person to person during seasonal epidemics causes acute respiratory infections that can lead to hospitalizations and life-threatening illness. Atmospheric conditions such as relative humidity (RH) can impact the viability of IV released into the air. To understand how different IV are affected by their environment, we compared the levels of stability of human-pathogenic seasonal and avian IV under a range of RH conditions and found that highly transmissible seasonal IV were less sensitive to decay under midrange RH conditions in droplets. We observed that certain RH conditions can support the persistence of infectious viruses on surfaces and in the air for extended periods of time. Together, our findings will facilitate understanding of factors affecting the persistence and spread of IV in our environment. Highly transmissible influenza viruses (IV) must remain stable and infectious under a wide range of environmental conditions following release from the respiratory tract into the air. Understanding how expelled IV persist in the environment is critical to limiting the spread of these viruses. Little is known about how the stability of different IV in expelled aerosols is impacted by exposure to environmental stressors, such as relative humidity (RH). Given that not all IV are equally capable of efficient airborne transmission in people, we anticipated that not all IV would respond uniformly to ambient RH. Therefore, we have examined the stability of human-pathogenic seasonal and avian IV in suspended aerosols and stationary droplets under a range of RH conditions. H3N2 and influenza B virus (IBV) isolates are resistant to RH-dependent decay in aerosols in the presence of human airway surface liquid, but we observed strain-dependent variations in the longevities of H1N1, H3N2, and IBV in droplets. Surprisingly, low-pathogenicity avian influenza H6N1 and H9N2 viruses, which cause sporadic infections in humans but are unable to transmit person to person, demonstrated a trend toward increased sensitivity at midrange to high-range RH. Taken together, our observations suggest that the levels of vulnerability to decay at midrange RH differ with virus type and host origin.IMPORTANCE The rapid spread of influenza viruses (IV) from person to person during seasonal epidemics causes acute respiratory infections that can lead to hospitalizations and life-threatening illness. Atmospheric conditions such as relative humidity (RH) can impact the viability of IV released into the air. To understand how different IV are affected by their environment, we compared the levels of stability of human-pathogenic seasonal and avian IV under a range of RH conditions and found that highly transmissible seasonal IV were less sensitive to decay under midrange RH conditions in droplets. We observed that certain RH conditions can support the persistence of infectious viruses on surfaces and in the air for extended periods of time. Together, our findings will facilitate understanding of factors affecting the persistence and spread of IV in our environment. Seasonal influenza viruses (IV) cause yearly infection cycles that tend to peak in the winter in temperate regions and during rainy periods in tropical climates . Seasona3–Variations in ambient RH have previously been shown to directly affect transmission of IV in animal models , 8. Hist9–https://www.cdc.gov/flu/weekly/index.htm)  and influenza B viruses (IBV), which persist in the population despite significant surveillance and intervention efforts . The epidex.htm) –21, whicex.htm) –. The impex.htm) ––26 but fx.htm) –––30. Prev.htm) –––, 32. TheIn this study, we examined the environmental persistence of six IV, including human-pathogenic seasonal and LPAI viruses , but thi10.1128/mSphere.00552-19.9TABLE S1Table S1, DOCX file, 0.01 MB.Influenza virus passage history and references. Download Copyright © 2019 Kormuth et al.2019Kormuth et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Recently, we demonstrated that the presence of airway surface liquid (ASL), collected from HBE cells grown at an air-liquid interface, confers a protective effect against RH-dependent decay to H1N1pdm virus grown in MDCK (Madin-Darby canine kidney) cells in aerosols and droplets . H1N1pdm50 (50% tissue culture infectious dose) assay results between unaged aerosols and aerosols aged for 1 h in the drum determined on MDCK cells. Log decay was corrected for both physical loss and dilution in the drum at each RH  and dilution (dk) coefficients under each set of RH conditions in the rotating drum and confirmation of virus conservation in the drum. Physical loss and dilution coefficients were determined following measurement of aerosol volume concentration using scanning mobility and aerodynamic particle sizers. Coefficients were determined by fitting the data to a first-order decay model. (A) pk and dk determined under each set of RH conditions using betapropiolactone (BPL)-inactivated Perth H3N2 plus ASL from HBE cells. (B) pk and dk determined under each set of RH conditions for BPL-inactivated IBV plus ASL from HBE cells. (C) To ensure that similar amounts of viral material were collected before and after aging during the temporal experiment performed with Perth H3N2, we performed qPCR on samples before and after aging for 15 min, 1 h, and 2 h. Data are representative of results from three replicate experiments (denoted by symbols), where viral aerosols were sampled independently three times from the RH drum. Bars represent means ± standard deviations. Download FIG S1, EPS file, 2.2 MB.Physical loss  of virus incubated in a sealed vial outside the chamber. The levels of viral stability determined under each set of RH conditions are presented as the raw viral titer and log decay, which is the change in infectivity between experimental and control samples. Consistent with our previous findings  strain . Similar10.1128/mSphere.00552-19.3FIG S3P values are reported using Bonferroni’s correction for multiple comparisons . Download FIG S3, EPS file, 2 MB.Pre-2009 A/Brisbane/59/2007, Bris H1N1, is protected from RH following growth in HBE cells. Bris H1N1 viruses grown in MDCK and HBE cells were exposed to seven different sets of RH conditions for 2 h in droplets. The infectious titer of the MDCK cell-grown virus showed that it was more sensitive to midrange RH than the HBE cell-grown stock, with significantly less decay of the HBE virus at 43%, 55%, and 75% RH. Data are presented as mean values ± standard deviations of the results from all of the experiments, each of which was conducted in triplicate, using virus from a single HBE cell line. Asterisks indicate significantly different mean levels of log decay over time, as determined by two-way ANOVA, and adjusted Copyright © 2019 Kormuth et al.2019Kormuth et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00552-19.4FIG S4FIG S4, EPS file, 1.9 MB.Sensitivity of HBE cell-grown Perth H3N2 in stationary droplets. Perth H3N2 was grown in primary HBE cell lines derived from 3 different patient samples. Droplets containing virus were incubated under a range of RH conditions for 2 h. Levels of decay of the virus were compared among three HBE cell lines and were not significantly different among the three HBE cell lines, as determined by one-way ANOVA with Tukey’s multiple-comparison test. Data are presented as mean values ± standard deviations of the results from all of the experiments, each of which was conducted in triplicate. Download Copyright © 2019 Kormuth et al.2019Kormuth et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10) occurring at 75% RH (10 at a midrange RH (55%). No significant difference in log decay was observed between the two propagation methods, suggesting that HBE ASL does not protect IBV from sensitivity to RH. This phenotype was reproduced in two additional HBE cultures derived from distinct patient samples  IBV was grown in primary HBE cell lines derived from 3 different patient samples. Droplets containing virus were incubated under a range of RH conditions for 2 h. Levels of decay of the virus were not significantly different among the three HBE cell lines, as determined by one-way ANOVA with Tukey’s multiple-comparison test. Data are presented as mean values ± standard deviations of the results from all of the experiments, each of which was conducted in triplicate. Download FIG S5, EPS file, 1.9 MB.Sensitivity of seasonal IBV in stationary droplets under conditions of virus growth in HBE cells. (A) Replication kinetics of IBV in HBE cells demonstrates efficient replication of the virus in a primary cell line. Data are shown as means ± standard deviations of values from three transwells representing TCIDCopyright © 2019 Kormuth et al.2019Kormuth et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The risk of infection by indirect contact, or by fomite transmission, of a seasonal IV depends on the ability of the viruses to remain infectious for a long period of time following deposition onto a surface. To understand the persistence of seasonal IV in droplets, we tested the stability of HBE cell-propagated H1N1pdm, Perth H3N2, and IBV after 2, 8, and 16 h under four different sets of RH conditions: 23%, 43%, 75%, and 98%. We confirmed that the RH chamber was capable of maintaining constant temperature and humidity for the duration of our studies . The rat10.1128/mSphere.00552-19.6FIG S6FIG S6, EPS file, 2.3 MB.RH chambers maintain stable RH and temperature over long incubation times. Longitudinal virus stability assays require extended incubations in the RH chamber. (A) Stable RH was maintained beyond 16 h for each set of RH conditions tested. Brief deviations in RH resulted from the chamber being opened to allow placement of the samples. (B) Stable temperature within the chambers was maintained beyond 16 h for each set of RH conditions tested. Download Copyright © 2019 Kormuth et al.2019Kormuth et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P < 0.01) and IBV (P < 0.05) at 43% RH as well as from that of IBV (P < 0.01) at 75% RH. Δ log decay of Perth H3N2 was significantly different from that of IBV (P < 0.05) at 75% by the same statistical analysis.Surprisingly, the Δ log decay of H1N1pdm in droplets was highly variable based on RH condition . InitialTogether, these data indicate that the levels of persistence of seasonal IV in droplets differ among virus types and subtypes but also that IV can remain stable and highly infectious for long periods of time under certain RH conditions.7 TCID50/ml  decayed significantly less than virus grown in HBE 0195 or HBE 0204 cells  Decay of LPAI viruses grown in HBE cells. LPAI viruses were propagated in three distinct HBE cell cultures, and virus stability was determined following a 2-h exposure to 7 different sets of RH conditions. For both LPAI virus experiments, the level of decay of viruses grown in HBE 0206 was significantly different from that seen with viruses grown in the other two HBE cell lines (determined by two-way ANOVA using Bonferroni’s correction for multiple comparisons). Data are presented as mean values ± standard deviations of the results from all of the experiments, each of which was conducted in triplicate. Asterisks represent RH conditions where decay of virus grown in HBE 0206 was significantly different from that seen with either one (*) or both (**) of the other two HBE cell lines. Download FIG S7, EPS file, 2.2 MB.LPAI viruses propagated in HBE cells are sensitive to RH-dependent decay in droplets. (A) Replication kinetics of avH6N1 (left) and avH9N2 (right) in HBE cells demonstrating that the avian viruses grow well in the human cell line. Data are shown as means ± standard deviations of values from three transwells representing the TCIDCopyright © 2019 Kormuth et al.2019Kormuth et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10 at 55% RH . Symbols represent individual decay values, and error bars represent means ± standard deviations. Download FIG S8, EPS file, 2 MB.A mutant H1N1pdm virus with a preference for binding avian cell surface receptors is less stable than wild-type H1N1pdm. α2,3 H1N1pdm was grown in three primary HBE cell lines. Combined droplet decay levels of wild-type H1N1pdm grown in three HBE cell lines were compared to the level of decay seen with the mutant virus grown in three HBE cell lines. Decay of virus from each individual cell line was determined from experiments conducted in triplicate. Significantly more decay of α2,3 H1N1pdm was detected under 43% RH conditions by two-way ANOVA, and adjusted Copyright © 2019 Kormuth et al.2019Kormuth et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the With this work, we have produced a comprehensive investigation of the relationship between RH and the stability of seasonal IV and LPAI in the environment. We found that stability of airborne IV in aerosols and droplets is not unique to H1N1pdm as previously reported  but is a9–Among seasonal IV, we identified differences between isolates of IBV and H3N2. Both viruses were stable in fine aerosols , but IBVAmong the IV exposed to a range of RH conditions in stationary droplets, all viruses responded to RH with generally more stability at low and high RH and the most decay under midrange conditions. However, the degrees of decay at midrange RH differed among the strains. Previous studies exploring the levels of stability of IAV and IBV smeared onto plastic  and bankWe have shown that, similarly to the RH sensitivity results, the persistence of infectious IV over time varied with virus strain background and with RH. For example, the rate of decay of H1N1pdm in droplets did not increase for up to 16 h after deposition at midrange RH, while both Perth H3N2 and IBV continued to decay over time under all RH conditions tested . PreviouIn contrast to seasonal IV, LPAI viruses, including avH6N1 and avH9N2, have not caused pandemic outbreaks in people . As with41–Here, we have clarified the relationship between RH and the stability of seasonal IV and LPAI strains resembling those that would be released into the environment from the airway of an infected person. This report provides a distinction between IV strains and virus stability in response to propagation method and specific environmental parameters. Aerosolized seasonal IV are highly resistant to RH-dependent decay, which suggests that removing them via increased air exchange rates, filtration, or UV germicidal irradiation may be critical to reducing the transmission of these viruses indoors. We found that RH is important for the stability of IV on surfaces but also that infectious viruses have the potential to persist on surfaces for hours in physiological droplets, reinforcing the need for surface decontamination in high-risk environments.l-glutamine, and penicillin-streptomycin. Influenza A viruses A/California/07/2009 (H1N1) pdm09 antiviral (AVR) reference virus M2—S31N NA—wild type (wt) (item number FR-458) and A/Brisbane/59/2007 (H1N1) FR-1 and influenza B virus B/Texas/02/2013  were obtained through the International Reagent Resource, Influenza Division, World Health Organization Collaborating Center for Surveillance, Epidemiology, and Control of Influenza, Centers for Disease Control and Prevention . Influenza A virus A/Perth/16/2009 (H3N2) was a generous gift from Zhiping Ye, Center for Biologics Evaluation and Research, Food and Drug Administration . LPAI influenza A viruses A/shorebird/DE Bay/230/2009 (H6N1) NR-45155 and A/shorebird/DE/127/2003 (H9N2) NR-45169 were obtained from BEI Resources, National Institute of Allergy and Infectious Diseases, National Institutes of Health . Virus stocks were propagated in MDCK cells, harvested following detection of cytopathic effect (CPE), and clarified by low-speed centrifugation. HBE virus stocks were prepared by inoculation of each Transwell with 103 TCID50/ml virus as previously described  in chambers conditioned for each RH using aqueous saturated salt solutions as previously described , 15. BriP values reported using Bonferroni’s multiple-comparison test. Significance was determined using a 95% confidence interval where P values of <0.05 are denoted by a single asterisk (*) and P values of <0.01 are denoted by double asterisks (**), unless otherwise indicated in the text. One-way ANOVA was used with a Tukey’s multiple-comparison test to compare levels of decay among three HBE patient cell lines for H1N1pdm, Perth H3N2, and IBV strains. Two-way ANOVA was used with Bonferroni’s multiple comparison’s test to compare levels of decay in three HBE patient cell lines for avH9N2 and avH6N1. Specific details regarding statistical tests are also provided in the figure legends. Statistical analyses were completed using GraphPad Prism version 8.0.0 software.Comparisons between levels of decay in MDCK cells versus HBE cells under all RH conditions were performed with a two-way ANOVA, with adjusted 2. Target RH was achieved by adjusting the flow rates of the aerosol, dry air, and saturated air into the drum. Once RH reached equilibrium, an aerosol sample representing time zero was collected onto a gelatin filter at a flow rate of 2 liters/min for 15 min. The drum was then sealed, and aerosolized virus was incubated for 1 h, after which another aerosol sample was collected. The filters were dissolved into 3 ml of prewarmed L-15 medium, and IV titers were determined by TCID50 assay. Decay rates were corrected by RH-specific aerosol physical loss rates (TC 1-step kit (Thermo Fisher)  and a StepOnePlus real-time PCR system (Applied Biosystems). Viral RNA was detected using a probe against the IAV M gene segment (5′-FAM-TCAGGCCCCCTCAAAGCCGA-BHQ1-3′) .Aerosolized viruses were exposed to selected RH conditions in a rotating, 27-liter aluminum drum housed within a biosafety cabinet, as previously described . Briefly