Examining how travel distance is associated with travel mode choice is essential for understanding traveler travel patterns and the potential mechanisms of behavioral changes. Although existing studies have explored the effect of travel distance on travel mode choice, most overlook their non-linear relationship and the heterogeneity between groups. In this study, the correlation between travel distance and travel mode choice is explored by applying the random forest model based on resident travel survey data in Guiyang, China. The results show that travel distance is far more important than other determinants for understanding the mechanism of travel mode choice. Travel distance contributes to 42.28% of explanation power for predicting travel mode choice and even 63.24% for walking. Significant nonlinear associations and threshold effects are found between travel distance and travel mode choice, and such nonlinear associations vary significantly across different socioeconomic groups. Policymakers are recommended to understand the group heterogeneity of travel mode choice behavior and to make targeted interventions for different groups with different travel distances. These results can provide beneficial guidance for optimizing the spatial layout of transportation infrastructure and improving the operational efficiency of low-carbon transportation systems. Advocating for the common efforts of humans to combat climate change and continuously promoting green urban development is the way to build a sustainable society and promote the global achievement of carbon neutrality. However, with the accelerated urbanization and expansion of urban space, increasing travel distance impels residents to choose motorized modes, especially private cars, for their trips. Car-oriented travel demand has made urban traffic congestion, energy consumption and environmental pollution problems increasingly serious . PromotiMany studies have explored the relationship between travel distance and travel mode choice and concluded that travel distance is a significant factor in residents’ activity participation and mode choice ,4. For eThese findings provide important insights into enriching the complex association between travel distance and mode choice. However, current research still has the following issues that must be solved. First, few researchers paid attention to the nonlinear relationship between travel distance and mode choice. It should be noted that pre-specified or parametric relationships may lead to erroneous statistical inferences and policy implications . FurtherUsing the resident travel survey and built environment data from Guiyang, this paper employs a random forest model to explore the nonlinear and threshold effects of travel distance on travel mode and the group differences of such effects. We mainly address the following issues: (1) How important is travel distance to mode choice? (2) How does travel distance affect travel mode nonlinearly? (3) How does the nonlinear and threshold effect of travel distance on travel mode vary across groups?This paper is organized as follows. In The relationship between travel distance and travel mode choice has long been an interesting research topic for transportation scholars. The early modeling of the relationship between travel distance and travel mode choice was mainly based on discrete choice models, especially the widely used multinomial logit (MNL) model . ResearcTo guide the layout planning of transportation infrastructure considering the distance and location of travel mode facilities, scholars have studied acceptable travel distances. The concept of acceptable travel distance was proposed by Seneviratne . Arasan The literature also shows significant differences in acceptable travel distances across gender, age, income and other factors ,31. WomeIn recent years, machine learning models have been proven to be a promising alternative in modeling travel mode choices ,37. ThroTraditional pre-specified or parametric relationship modeling may lead to limitations for urban planners and policymakers in understanding the impact of travel distance on travel mode choice, which has side effects on developing low-carbon transportation modes and building sustainable cities and communities. As far as we know, few studies have revealed nonlinear associations between travel distance and travel mode choice, and there is a lack of exploration of the possible behavioral heterogeneity of nonlinear associations across groups.There were three phases of data collection: in the first phase, the travel information and socio-demographic data of residents were collected. In the second phase, we designed a program to obtain the travel distance. In the third phase, the built environment data regarding the residential locations in the research sample were obtained.2, with a population of roughly 3.17 million, accounting for 52.8% of the total population of Guiyang (about 6 million). In the end, a total of 30,741 trips from 15,169 households in the urban area were used for analysis.Travel information and socio-demographic data were collected using a large-scale resident travel survey conducted in Guiyang in January 2021. The survey used the built-in survey program of tablet computers to collect residents’ information. The survey content included three parts: household information, individual characteristics and all personal travel information within 24 h of the previous day. We selected the urban area of Guiyang City as the study area. Travel modes included walking, cycling (bicycles and electric bicycles), public transportation  and car, accounting for 43.12%, 4.89%, 24.08% and 27.91%, respectively. Due to the poor cycling conditions in Guiyang, residents were relatively less likely to choose cycling in their trips compared to the other three modes of transportation. Therefore, we only focused on three major modes: namely, walking (45.34%), public transportation (25.31%) and car (29.36%).Since the ordinary Euclidean distance overlooks tortuous paths and road conditions, calculating the travel distance in this way will lead to serious potential errors. Therefore, we designed a program to obtain more precise travel distances using the “Batch Road Calculation API” function of Baidu Maps. The longitude and latitude coordinates of the residents’ travel origin–destination obtained from the resident travel survey data were input into Baidu Map. The “batch route calculation API” module located the location of the origin and destination through accurate longitude and latitude and output the travel distance of common routes  in three modes, i.e., walking, public transportation and car ) and the seventh National Census data. The road network density data were collected from OpenStreetMap (https://www.openstreetmap.org (accessed on 10 July 2022)). The built environment variables were obtained using ArcGIS software (version 10.6), including land use mix, road network density, population density, distance to CBD and the distance to the nearest bus stop. The road network density and population density were assessed within a 500 m buffer zone. The distance to the nearest bus stop was obtained using the “near” function in ArcGIS software (version 10.6). We adopted the travel distance of the most preferred routes with Batch Route Calculation API as the distance from the house to the CBD. The land use mix is calculated by reclassifying the 17 types of POI data crawled by Baidu Map. We reclassified the 17 types of POIs into five categories: residential, public management and services, commercial services, industry, parks and squares. Based on the 500 m buffer zone, the information entropy, The built environment data were collected from Baidu Map, OpenStreetMap, WorldPop and the seventh National Census data. Among them, the population density data were obtained by a combination of both WorldPop (Random forest (RF) was used in this paper to examine the nonlinear relationship and threshold effects between travel distance and travel mode choice. In recent studies, some scholars have confirmed that random forest has good performance in analyzing travel mode choice. Hagenauer and Helbich  comparedIn the RF algorithm, the relative importance of explanatory variables is assessed based on the reduction of the Gini index.Assuming that there are The importance of feature In addition to the relative importance of variables, random forest can characterize the marginal effects of feature variables on dependent variables through partial dependence plots (PDP). Different from traditional statistical models, this relationship does not require prior assumptions and can better reflect the real relationship between independent variables and dependent variables. Assuming that the dependent variable is The random forest algorithm was implemented using the “randomForest” toolkit in the R software 4.1.3. We first divided the sample set into a training set and test set according to 7:3. Then, we tested different combinations of the number of split variables  and the number of decision trees  to determine the optimal parameters with the smallest out-of-bag error (OOB). The testing results showed that when the number of split variables was 4 and the number of decision trees was 100, the model performed optimally with an out-of-bag error (OOB) of 17.04%. The random forest model established with the above parameters was used for further analysis.In order to illustrate the impact of the travel distance variable on mode choice and model performance, we used the same training data and test data to establish two random forest models and two multinomial logit (MNL) models. The four models are described as follows:RF-Mode1: The random forest model considering travel distance variable in travel mode choice modeling.RF-Mode2: The random forest model without considering travel distance variable in travel mode choice modeling.MNL-Mode1: The multinomial logit model considering travel distance variable in travel mode choice modeling.MNL-Mode2: The multinomial logit model without considering travel distance variable in travel mode choice modeling.In order to compare the performance differences between the random forest model and the multinomial logit (MNL) model in predicting the travel mode choice, we used four evaluation indicators: accuracy, precision, recall and F1 value (F1 score) . The fouThe results of the four evaluation indicators in the four models are shown in We found that the accuracy, precision, recall and F1 score all significantly decreased after the travel distance variable was not considered in the model. Overall, the random forest model with the travel distance variable (RF-Mode1) had the best prediction performance.We present the relative importance of the explanatory variables in the two random forest models (RF-Model1 and RF-Model2) in Furthermore, the relative importance of the characteristic variables in RF-Model1 among the three travel modes is shown in The nonlinear relationship between travel distance and travel mode choice is illustrated using partial dependence plots (PDP). In In this section, the nonlinear relationship between the built environment and travel mode choice is elaborated. We compared the nonlinear effects of the built environment on travel mode choice in the random forest model with travel distance variables (RF-Model1) and the random forest model without travel distance variables (RF-Model2) by partial dependence plots (PDP). The X-axes represent the explanatory variables, and the Y-axes represent the choice probability of mode. We aimed to compare the nonlinear changes in the built environment in the two models so as to highlight the importance of travel distance in travel mode choice.The PDPs of the five built environment variables are shown in 2, the choice probability of public transportation increased by approximately 1 percentage point, and walking increased by roughly 2 percentage points, while the choice probability of cars decreased. This is reasonable because lower road network density means less convenience and accessibility by car, and residents tend to choose public transportation and walking [When the population density increased from 0 to 12,500 people/km, the probability of walking increased by approximately 15 percentage points, but the probability of cars and public transportation decreased rapidly. When the population density exceeded 12,500 people/km, the choice probability of cars started to increase, and walking gradually stabilized. This is consistent with the study of Eom and Cho . When ro walking . When thHowever, we found that once the travel distance variable was considered in RF-Model1, the nonlinear effect of the built environment on mode choice seemed to become relatively weak compared with RF-Model2. In particular, the nonlinear effects of land use mix, road network density and distance to the nearest bus stop were not obvious. The nonlinear effect of travel distance on the distance to CBD was also significant. We found that in RF-Model1, the nonlinear effect of distance to CBD on walking and public transport did not show a significant peak change. In addition, among the three travel modes, the nonlinear change of built environment on the probability of walking choice seemed to be most obviously influenced by the variable of travel distance. We found that after considering the variable of travel distance when modeling mode choice, the built environment variables had little nonlinear effects on walking. This finding may again demonstrate the importance of travel distance for predicting travel mode choice, and that travel distance may weaken the non-linear effects of the built environment on travel mode choice.In As shown in Differences in the nonlinear effect of travel distance on travel mode choice between household income groups are shown in Differences in the nonlinear effect of travel distance on travel mode choice between travel purpose groups are shown in The relationship between travel distance and mode choice has been the focus of researchers, but there is still a lack of research on the nonlinear association and group heterogeneity between both of them. Based on survey data from Guiyang City, China, this paper explores the nonlinear association and group differences between travel distance and travel mode choice using a random forest model.As expected, we found that travel distance is more important than built environment variables for understanding the mechanism of travel mode choice. Travel distance has the largest contribution of explanatory power for predicting travel mode choice, with a contribution of 42.28%, and even contributes 63.24% of explanation power for walking. The relative importance of car ownership ranks second in predicting travel mode choice, accounting for 10.43%. In addition, car ownership makes the largest contribution to predicting car choice at 29.22%, but its relative importance in predicting walking choice is only 3.44%. We also found that the relative importance of the built environment decreases significantly, from 58.96% to 24.80%, when the travel distance variable is taken into account in the model. This phenomenon confirms the importance of travel distance in the mode choice model, where travel distance may weaken the relative importance of the built environment. We call on researchers and planners to deeply explore the potential mechanism of travel behavior and make decisions with a cautious and sensible attitude when evaluating the importance and impact of the built environment [This study confirms that travel distance has nonlinear and threshold effects on travel mode choice. In this study, the choice probability of walking is negatively correlated with travel distance. The probability of choose walking within 1.4 km decreases slowly. From 1.4 km to 3.5 km, the probability of walking choice decreases sharply in the form of an almost linear change of approximately 90 percent. The probability of choosing a car increases nonlinearly with travel distance, and there is a significant threshold effect between travel distance and public transportation mode choice. The probability of choosing public transportation peaks at approximately 6 km. Understanding how travel distance affects travel mode-choosing behavior can help policymakers optimize public transportation infrastructure and non-motorized transportation environments. This study shows that before 2.5 km, the choice probability of walking is greater than the car and public transportation. We recommend planners optimize pedestrian infrastructure within 2.5 km of residential areas, especially planning for pedestrian-friendly environmental communities within 1.4 km. Within 2.5–12.5 km of residential areas, planners should focus on optimizing the layout of public transportation infrastructure, such as increasing public transportation routes, reducing the number of transfers and developing customized buses and community buses to encourage residents to use public transportation as much as possible in medium and long-distance trips and reduce the use of cars by urban residents.Further, the nonlinear effect of travel distance on travel mode choice was significantly different among different groups. The increase in travel distance may encourage travelers to give up walking and choose a faster travel mode, but we found that the increase in travel distance does not encourage all travelers to choose cars. There is also significant group heterogeneity in the choice behavior of travel patterns. In this paper, male travelers, travelers aged 31–45, commuters, people with regular jobs (including public institutions and civil servants and enterprise staff), people with higher education levels, travelers from high-income families and travelers with household car availability have a relatively lower probability of walking more than 3 km. When the travel distance exceeds 2.5 or 3 km, such travelers tend to choose cars. Most of these groups are relatively free in choosing their residence locations and show dependence on cars during their trips. As a result, they should be the target group guided to transfer to low-carbon transportation. A recent study indicates that travelers with higher affordability are more likely to frequently use customized buses (CB), and CB are more advantageous than public transportation for long-distance trips . The CB On the other hand, female travelers, the elderly (over 60 years old), unemployed or retired people, people with lower education levels, travelers from low-income families, non-commuter travel  and travelers without household car availability have a relatively high probability of walking 0–2 km. When the travel distance exceeds 2.5 or 3 km, such travelers tend to choose public transportation. These groups usually have poor mobility. As Simcock et al.  stated, This study has some limitations. These nonlinear effects are likely to vary at spatial scales . This st


Affiliation 1. Instead of “Department of Neurovascular Intervention and Neurosurgery, School of Medicine, Shanghai General Hospital, Shanghai Jiao Tong University, Shanghai, China”, it should be “Department of Neurovascular Intervention, Clinical Center of Neuroscience, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China”.In the published article, there was an error in The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Cyclin11 promoter affected the ESL among different lines. Cyclin11 showed the highest expression in the ear shank 15 days after silking among diverse tissues of maize, suggesting its role in modulating ESL. Our study contributes to the understanding of the genetic mechanism underlying maize ESL and genetic modification of maize dehydration rate and kernel yield.Ear shank length (ESL) has significant effects on grain yield and kernel dehydration rate in maize. Herein, linkage mapping and genome-wide association study were combined to reveal the genetic architecture of maize ESL. Sixteen quantitative trait loci (QTL) were identified in the segregation population, among which five were repeatedly detected across multiple environments. Meanwhile, 23 single nucleotide polymorphisms were associated with the ESL in the association panel, of which four were located in the QTL identified by linkage mapping and were designated as the population-common loci. A total of 42 genes residing in the linkage disequilibrium regions of these common variants and 12 of them were responsive to ear shank elongation. Of the 12 genes, five encode leucine-rich repeat receptor-like protein kinases, proline-rich proteins, and cyclin11, respectively, which were previously shown to regulate cell division, expansion, and elongation. Gene-based association analyses revealed that the variant located in  Zea mays L.) is an important crop, which serves as animal feed, human food, and industrial raw materials [Maize  evaluate the phenotypic variation of the ESL in the segregation population and association panel, (2) identify the significant genomic regions responsible for the ESL using QTL mapping and GWAS, (3) detect co-localized genetic loci across different populations, and (4) reveal the key genes controlling the ESL by referring to the published transcriptome data and using gene-based association analysis. The results will provide a foundation for understanding the genetic mechanism underlying the ESL and contribute to molecular marker-assisted selection (MAS) of breeding maize varieties with high yield and KDR. t-test showed that the ESL was significantly (p < 0.01) different between the parents (B73 and Mo17) of IBM Syn 10 DH population under these environments except in XSBN (p < 0.01) different among the genotypes (G), environments (E), and genotype × environment interactions (G × E), for both populations , maximum, minimum, and coefficient of variation (CV) for the ESL in the segregation population and association panel across three environments, Chongzhou (CZ), Yan’an (YA), and Xishuangbana (XSBN). A  in XSBN . In the  in XSBN . The SD  in XSBN . In the  in XSBN A,B. The  in XSBN C–H, confulations . AdditioTo identify the QTL controlling maize ESL, we conducted a linkage mapping using the ESL phenotypic values of the IBM Syn 10 DH population across three environments and 6618 bin markers of this population. Finally, a total of 28 QTL with the PVE ranging from 3.39 to 9.80% were identified on all maize chromosomes except chromosomes 6 and 9 across three environments . These Qp-value exclusively in the tail region , with the PVE of 5.59% , mixed linear model (MLM), and fixed and random model circulating probability unification (FarmCPU) model. The results showed that the quantile-quantile (Q-Q) plot from the FarmCPU model was highly consistent with the expected distribution, which abruptly deviated from the expected l region . Therefoocations . These Sof 5.59% A.A total of 30 elite lines that have been serving as the parents of commercial cultivars were included in the association panel , which ahttps://www.ncbi.nlm.nih.gov/sra/PRJNA738962, accessed on 1 August 2022). We then identified the DEGs with the absolute log2 fold change ≥ 2 between each comparison among the four stages. Finally, among the 42 genes, 12 were differentially expressed in at least one of the comparisons. According to the expression patterns  receptor-like protein kinases [Zm00001d026282 and Zm00001d026297 were annotated as proline-rich proteins (PRPs) [Zm00001d005293 encodes Cyclin11 [Zm00001d005293, Zm00001d026282, Zm00001d005298, Zm00001d026297, and Zm00001d026306, respectively (Zm00001d005293 was significantly (p ≤ 0.05) associated with the ESL under the environments CZ and YA and using the BLUP values, this variant was located in the promoter region  larger ESL than those with the T allele . Finally, Cyclin11 displayed the lowest expression in maize tassels. However, ear shanks had the highest expression of Cyclin11 among the seven tissues, followed by leaves and ears, implicating its crucial role in regulating ear shank development (Cyclin11 in ear shanks of a maize line (SCL212) with a large ESL at different developmental stages. As a result, the Cyclin11 expression was gradually upregulated from 5 days after silking (DAS) to 15 DAS, and was then downregulated from 15 to 30 DAS, suggesting that Cyclin11 played a significant role at the early stage of ear development  family, which contributes to cell division in plants [Cyclin11 was significantly associated with maize ESL across different environments. Cyclin11 showed a higher expression in ear shanks than the other maize tissues. Moreover, Cyclin11 was upregulated during ear development and its expression reached a peak on 15 DAS, which was consistent with the characteristics of ear shank elongation in maize. All these results supported that the five genes were potential casual genes for maize ESL, among which Cyclin11 can be used to develop functional markers for genetic modification of maize ESL.Cell division and expansion play decisive roles in cell number and size in a mature organ , and theelopment . Overexpelopment . The recelopment , consideelopment . Zm00001elopment . EXTENSIelopment . In addielopment . The cann plants . In Arabn plants . Furthern plants . Forzanin plants . RemarkaIn this study, an IBM Syn10 DH population that included 267 DH lines was used for linkage mapping of maize ESL . An assoFor each plant, the ear with its shank was collected after ear maturity. Three ears with consistent growth were selected to measure the ESL for each line in a replication. The ESL was measured using a soft ruler, which was equal to the distance between the stem attachment point to the ear bottom  Figure . The meahttps://www.ibm.com/analytics/spss-statistics-software, accessed on 21 July 2022) was used to analyze the phenotypic data, including mean, range, standard deviation (SD), coefficient of variation (CV), and variance. The broad-sense heritability (IBM SPSS Statistics 27 software (n+σ2/nb) . Herein,A high-density genetic map with 6618 bin markers was previously constructed for the IBM Syn 10 DH population. The average distance between adjacent markers was 0.48 cM . The Winp-value for association identification was set to p = 1 × 10−4 [https://www.maizegdb.org/, accessed on 25 July 2022).The association panel was previously genotyped using the Illumina Maize SNP50 BeadChip, which contained 56,110 SNPs . Genotyp2 fold change = 2. The DEG expression patterns are clustered by the OmicShare tool, an online platform for data analysis and visualization .These genes that were simultaneously located within the LD regions of GWAS and QTL intervals of linkage mapping were extracted for the analysis of differential expression based on the published data of transcriptomes regarding maize ear elongation . The sighttps://www.ncbi.nlm.nih.gov/tool/primer-blast, accessed on 27 July 2022). The sequence analysis software DNAMAN  was used to align and analyze the sequences of the amplified fragments. The results of DNAMAN alignment were output in the Phylip format and then edited and modified using the BioEdit software . The nucleotide polymorphisms among the 77 lines were analyzed using the DnaSP software . After removing the variants with MAF < 0.05 or heterozygosity rate > 20%, the remaining SNPs and insertion/deletions (InDels) of each gene were used to detect the associations with maize ESL by GLM in Tassel . The significance threshold of associated locus identification was set to p-value = 0.05. The haplotypes of the 77 lines were divided according to the significantly associated loci for each gene using the Haploview software [t-test was used to detect the significance of difference in the ESL between different haplotypes.According to the functional annotations, the DEGs involved in cell division, expansion, and elongation were subjected to gene-based association analyses using 77 lines randomly selected from the association panel. Genomic DNA was extracted from the leaves of the 77 lines using the CTAB method ,52. The software . A t-tesCyclin11, including roots (silking stage), stems (silking stage), leaf (silking stage), tassels (silking stage), ears (silking stage), kernels (6 DAP), and ear shank (15 DAS). Meanwhile, a line (SCL212) with long ear shank was subjected to analysis of Cyclin11 expression patterns in ear shanks during different developmental stages. The ear shanks were sampled on 5, 10, 15, 20, 25, and 30 DAS, respectively. The above 13 samples were individually used for total RNA extraction with TRIzol Reagent . The first-strand cDNA was synthesized from approximately 2 µg of the total RNA with a reverse transcription kit  according to the manufacturer’s instruction. The Cyclin11 expression levels were quantified by conducting RT-qPCR with the SYBR qPCR SuperMix Plus , the primers of RT-qPCR were listed in ZmActin served as a reference gene, and the 2−ΔΔCT method was employed to calculate the relative expressions of Cyclin11 [Seven tissues were collected from the line B73 and were subjected to expression analysis of Cyclin11 . Three bCyclin11, we cloned the full-length coding sequence of Cyclin11 without the stop codon into the reporter vector pCAMBIA2300-eGFP, generating the p35S: Cyclin11-eGFP fusion expression vector. The fusion vector was then introduced into the Nicotiana benthamiana leaves for transient expression using the Agrobacterium-mediated method [To understand the subcellular localization of d method . The p35Cyclin11 showed the highest expression in 15 DAS-ear shank among diverse tissues, and a variant located in the Cyclin11 promoter affected ESL among different lines. Our study contributes to the understanding of genetic mechanisms underlying maize ESL and the genetic modification of maize dehydration rate. In this study, QTL mapping and GWAS were combined to dissect the genetic basis and key genes controlling maize ESL. A total of 16 QTL were identified in the segregation population. Meanwhile, 23 SNPs were associated with the ESL in GWAS, of which four were located in the QTL identified by linkage mapping and were thus considered the population-common loci. In the LD regions of these common variants, 42 genes were identified and 12 of them were responsive to ear shank elongation. Of the 12 genes, five were involved in cell division, expansion, and elongation, which were designated as the key genes for maize ESL. Remarkably,


All animals developed clinical disease within two days and FMDV was reisolated from serum and saliva samples. In the second trial, another group of six cattle was exposed to FMDV RNA by instilling it on the tongue and spraying it into the nose. The animals were observed for 10 days after exposure. All animals remained clinically unremarkable and virus isolation as well as FMDV genome detection in serum and saliva were negative. No transfection reagent was used for any of the animal inoculations. In conclusion, cattle can be infected by injection with naked FMDV RNA, but not by non-invasive exposure to the RNA. Inactivated FMDV samples that contain full-length viral RNA carry only a negligible risk of infecting animals.Safe sample transport is of great importance for infectious diseases diagnostics. Various treatments and buffers are used to inactivate pathogens in diagnostic samples. At the same time, adequate sample preservation, particularly of nucleic acids, is essential to allow an accurate laboratory diagnosis. For viruses with single-stranded RNA genomes of positive polarity, such as foot-and-mouth disease virus (FMDV), however, naked full-length viral RNA can itself be infectious. In order to assess the risk of infection from inactivated FMDV samples, two animal experiments were performed. In the first trial, six cattle were injected with FMDV RNA (isolate A Diagnostic samples can contain large amounts of infectious disease agents and can be a source of contagion. Inactivating agents or treatments can be used at the point of care to mitigate the risks associated with these samples without compromising their diagnostic utility. For many infectious diseases, the target of first-line diagnostic tests is pathogen-specific nucleic acid. Therefore, inactivating agents such as alcohols or acids, which denature proteins and dissolve lipids, but do not destroy nucleic acids, are often preferred.Picornaviridae family. Like all picornaviruses, FMDV is a non-enveloped virus with a non-segmented single-stranded RNA genome of positive polarity [Acid treatment is particularly suitable for foot-and-mouth disease virus (FMDV) , a high-polarity . Unlike polarity . Therefopolarity .The FMDV capsid readily disassembles at low pH, leading to a rapid loss of infectivity . AccordiSeveral research groups have demonstrated that animals can be infected by naked single-stranded positive-sense genomic RNA of various viruses when transfection reagent is added to the RNA before inoculation ,8,9. ForTo investigate the risk of FMDV infection from inactivated diagnostic samples, in this study cattle were exposed to naked FMD RNA by injection and by non-invasive mucosal deposition.Two cell lines were used for the cultivation of FMDV: BHK-21 cells (clone “Tübingen”)  grown in minimal essential medium (MEM) with Hank’s and Earles’ salts and non-essential amino acids and LFBK-αVβ6 cells   in each well. The plates were then exposed to lengthwise temperature gradients  of 55–75 °C and 75–95 °C for 10 min in a thermocycler. The heat-treated samples were brought to room temperature, transferred to a tissue culture plate containing either BHK-21 or LFBK-αVβ6 cells, then incubated for 3 days at 37 °C and 5% CO2, and subsequently evaluated for cytopathic effect (CPE). A second experiment was completed in the same way, but half of the replicates at each temperature were treated with 5µL RNase A (200 µL/mL) after heating and before cell culture inoculation (BHK-21 cells only).To prevent false-positive results in the virus isolation, it is essential that infection of the cell culture is only caused by intact virus particles and there is no spontaneous uptake of free viral RNA by the cells. Accordingly, both cell lines were tested for their self-transfection ability. Two identical 96-well PCR plates were prepared with 200 µL FMDV suspension , and O/FRA/1/2001 (lineage O/ME-SA/PanAsia), were propagated on BHK-21 cells. The virus preparations were analyzed for infectivity by titration on LFBK-αVβ6 cells and for FMDV genome content by RT-qPCR. To test their transfectability, the virus preparations were serially diluted 10-fold in serum-free DMEM down to a final dilution of 10−5. Total RNA was extracted from the dilutions and the extracted RNA was analyzed by RT-qPCR and transfection as described below.Three isolates of FMDV, namely A/IRN/8/2015 (lineage A/ASIA/G-VII), A22/IRQ/24/64 were prepared in BHK-21 cells grown in MEM supplemented with 5% FBS. In a 75 cm2 cell culture flask with 16 mL culture medium, 50 µL of a previous preparation of A22/IRQ/24/64 were used to inoculate a 90% confluent cell monolayer. About 80% CPE was detected after one day of incubation at 37 °C. After freezing and thawing, the virus suspension was clarified by centrifugation at 1000× g for 5 min, aliquoted and stored at −80 °C. Virus and viral RNA content were quantified by end-point titration on LFBK-αVβ6 cells and RT-qPCR.Master stocks of FMDV A2.RNA was extracted from cell culture supernatants using TRIzol LS Reagent  as directed by the manufacturer. The transfections for the preliminary tests and the positive control were performed with Lipofectamine 3000 (Thermo Fisher Scientific). Per well, 1 µL of Lipofectamine 3000 reagent was diluted in 25 µL of DMEM and mixed gently without vortexing. In parallel, 3 µL of extracted RNA, 0.5 µL of P 3000 Reagent, and 25 µL of DMEM were mixed the same way. The two preparations were combined and incubated for 10 min at room temperature, before 50 µL per well were added dropwise to a 24-well plate with near-confluent LFBK-αVβ6 cells. Care was taken not to touch the cell monolayer. After 15 min of incubation at 37 °C, 500 µL of DMEM supplemented with 5% FBS were added per well. CPE was evaluated after 2 days at 37 °C with 5% COFMDV RNA was quantified with an RT-qPCR assay targeting the 3D coding region , using A22/IRQ/24/64 culture was serially diluted in nuclease-free water and transfected into LFBK-αVβ6 cells using two replicate wells per dilution. The other half was nebulized using a syringe-mounted atomization device . Nebulization was performed in a biological safety cabinet and the nebulized RNA was collected in a 50 mL conical tube for dilution and transfection. After 2 days of incubation at 37 °C with 5% CO2, the transfected cells were examined for CPE.Before the animal experiment, it was tested if nebulization of FMDV RNA leads to significant fragmentation, as had been observed for large DNA molecules . Half ofTo prevent the viral RNA from being damaged by freezing and thawing and obtain as many intact full-length viral genomes as possible, it was extracted immediately before inoculation of the animals in each experiment. For the first experiment, the pooled RNA of 18 replicate extractions was diluted 1:5 in nuclease-free water. For the second experiment, the pooled RNA was also diluted 1:5, but half of the 1:5 dilution was then further diluted to provide an adequate volume for intranasal administration by atomizer. For this purpose, 1.2 mL of the diluted pooled RNA were added to 11.4 mL of nuclease-free water. The freshly extracted and diluted RNA was transported to the animal room on ice. No transfection reagent was added to the viral RNA for any of the animal inoculations.The leftover RNA inoculum was brought back to the laboratory on wet ice for confirmatory tests.To confirm that it did not contain any intact viral particles, 50 µL of the RNA inoculum without transfection reagent were added to an intact monolayer of LFBK-αVβ6 cells and incubated at 37 °C for 3 days. The test was carried out in three replicates. After 3 days, the cultures were frozen and thawed and the cell lysate was passaged onto fresh LFBK-αVβ6 cells. After another 3 days of incubation, the cultures were examined for CPE.To confirm that intact full-length FMDV genomes were present in the inoculum, it was serially diluted in nuclease-free water and transfected into LFBK-αVβ6 cells using lipofectamine as described above. The viral RNA content of the inoculum was quantified with the FMDV 3D RT-qPCR assay. A serial dilution of an in vitro transcribed FMDV RNA standard was included to construct a calibration curve and calculate genome copy numbers.Six Holstein-Friesian cattle were used for each trial. The animals were brought to the BSL4vet facility of the FLI Riems one week before the start of the trial to ensure proper acclimation. In the first experiment, the animals were between 5 and 8 months of age. In the second experiment, they were between 3 and 4 months.For the inoculations and clinical exams, the animals were kept off feed overnight and sedated by intramuscular injection of 0.3 mg xylazine per kg body mass into the hind quarters. The sedation was reversed by intramuscular injection of 0.03 mg atipamezole per kg body mass.All work with animals occurred after ethical review and in compliance with local, state, and national animal welfare regulations. The experimental protocol was filed with the State Office for Agriculture, Food and Fisheries of Mecklenburg-Vorpommern (LALLF M-V), the competent authority for animal experiments conducted at FLI Riems (file no. 7221.3-2-82-026/17). The animals were handled in accordance with all applicable European and German guidelines for the use of experimental animals.The first animal experiment was designed to clarify whether it is at all possible to cause an FMDV infection in cattle with naked RNA. Accordingly, the most reliable route of inoculation was used: injection into the epithelium of the tongue . The tonIn the second experiment, less invasive routes of inoculation were used. Half of the 1:5 diluted RNA  was dripped onto the tongue of the sedated animals. The tongues were inspected before application of the RNA to make sure that there were no visible breaks in the epithelium. For each animal, 2.1 mL of the further diluted RNA preparation was applied to the nasal mucosa by spraying into a nostril using a syringe-mounted atomizer . The stepwise application of the total volume of RNA was timed to coincide with inspiration.Rectal body temperature was documented daily during the acclimation and experimental period. After inoculation, the animals were examined each day for signs of FMD. A clinical score was calculated by evaluation of the general attitude, feed intake, body temperature, and gait . Saliva was collected by swabbing the oral cavity with a human vaginal tampon  to quickly obtain a large quantity of fluid. Samples were transferred back to the laboratory on wet ice, centrifuged immediately for 10 min at 2000× At the end of the trial or when a humane endpoint was reached, the animals were deeply sedated as described above and then euthanized by intravenous injection of an overdose of sodium pentobarbital . Samples of the lesions on the tongues and in the interdigital spaces were taken at necropsy and placed in 10% formalin for histopathological examination.For determination of the viral RNA content, small pieces of vesicular epithelium (2 mm × 2 mm) from the same sites were collected in a screw-cap tube without fixative.2 tissue culture flasks with 90% confluent cell monolayers were inoculated with 50 µL of sample material. The assay was performed in duplicate. After incubation for 3 days at 37 °C, the flasks were evaluated for CPE and frozen at −80 °C to disrupt the cells. After thawing, 1 mL of the lysate was passaged onto fresh LFBK-αVβ6 monolayers, incubated, and again evaluated for CPE.The collected serum and saliva samples were checked for the presence of infectious virus by isolation on LFBK-αVβ6 cells. For this purpose, 12.5 cmg at 4 °C, the supernatant was collected and used for RNA extraction with the QIAamp Viral RNA Kit (Qiagen).The tissue samples collected for RT-qPCR were homogenized in 500 µL serum-free DMEM with a 4 mm stainless steel bead at 30 shakes/s for 3 min in a Tissue Lyser II . The homogenate was centrifuged for 2 min at 20,000× The viral RNA content of the serum and saliva samples, as well as of vesicular fluid collected from lesions and homogenized vesicular epithelium, was quantified with the 3D RT-qPCR assay.The serum samples of all animals from day 2 of the first experiment were used for FMDV sequence analysis. RNA was extracted using the QIAamp Viral RNA Kit (Qiagen). The VP1 coding region was amplified using the previously published primers FMD-3161-F and FMD-4303-R [Histological sections of all tissue samples taken during the first animal experiment were prepared. These were each stained with hematoxylin and eosin (H.E.) as well as with an in-house polyclonal rabbit anti-FMDV antiserum (1:2000). Adjacent tissue sections treated with pre-immune serum served as a negative control.Since there were no FMDV-positive ex vivo samples and no conspicuous lesions in the second animal experiment, no histological examination was performed.https://epitools.ausvet.com.au/ciproportion) (accessed on 21 July 2022).For the statistical analysis, the animal inoculations were considered Bernoulli trials with a binary outcome (infection/no infection). Accordingly, each animal experiment comprised six independent observations, from which 95% confidence intervals for the proportion of infection were calculated using Jeffreys’ method as implemented in the Epitools suite (Without RNase treatment of the heated virus suspension, there was a clear difference between LFBK-αVβ6 and BHK-21 cells. After heating to about 60 °C for 10 min, the virus suspension no longer caused infection in LFBK-αVβ6 cells. In BHK-21 cells, on the other hand, CPE did still occur up to a temperature of about 80 °C . When RN22/IRQ/24/64, and O/FRA/1/2001 had virus titers of 107.3, 108.4, and 105.3 TCID50/mL with Ct values of 12.3, 12.5, and 14.4, respectively. The transfection was successful for O/FRA/1/2001 down to a dilution of 10−2, for A/IRN/8/2015 to 10−3, and for A22/IRQ/24/64 to a dilution of 10−4, which corresponded to a Ct value of 26.5. Based on these results, A22/IRQ/24/64 was selected for the animal experiments, because it provided the largest margin of safety for successful transfection.The preparations of FMDV A/IRN/8/2015, A22/IRQ/24/64 used in the second animal trial had a virus titer of 107.8 TCID50/mL with a Ct value of 14.4.The master stock of A−4.The comparison of untreated and previously nebulized RNA showed no difference in transfectability. For both preparations, transfection was successful in at least one of two replicates down to a dilution of 10t values of 13.7 in the first experiment and 13.3 in the second, corresponding to about 1011 copies of FMDV genome in each animal dose.The RNA preparations used to inoculate the animals had FMDV CCultures of LFBK-αVβ6 cells inoculated with the RNA preparations without transfection reagent did not exhibit any CPE in either the first or second animal experiment.−3. For the second experiment, in vitro transfection of the inoculum was successful down to a dilution of 10−4.With lipofectamine, the RNA inoculum from the first experiment was successfully transfected into LFBK-αVβ6 down to a dilution of 10In the first animal experiment, excessive salivation was observed in all animals 24 h after inoculation. In addition, two animals exhibited an increased body temperature of more than 39.5 °C see . CursoryIn contrast, no clinical signs of FMD were seen in any of the 6 cattle in the second animal experiment.All samples taken on days 1 and 2 after inoculation in the first animal experiment were positive in the virus isolation. No positive samples were found in the second experiment.q value of 16.6 in the FMDV RT-qPCR.No FMDV genome was detected in the serum and saliva samples collected before infection in the first animal experiment. Beginning on the first day after inoculation, however, a steady increase in the viral genome load was observed in serum and saliva see . The homIn the second animal experiment, no FMDV genome was detected in any sample.22/IRQ/24/64 to at least 99.7%.The viral sequence recovered from the samples of the first animal experiment matched the database sequence of FMDV AThe clinical findings of the first animal trial were confirmed during necropsy see . The tonNo lesions of this kind were found in the second animal experiment.Vesiculoulcerative tissue damage can be seen in the H.E. staining in all samples of the first animal experiment . This coIn the first experiment, the proportion of animals in which the injection of FMDV RNA into the tongue led to infection was 100%, with a lower bound of the 95% confidence interval of 67%.In the second animal experiment, none of the animals developed FMD, corresponding to an observed proportion of infection after non-invasive exposure to FMDV RNA of 0%. The upper bound of the 95% confidence interval was 33%.2 pressure, and ambient pH prevail, and the cellular membrane of BHK-21 cells may have become increasingly permeable over the course of many passages. This can be beneficial in certain applications, e.g., for the rescue of infectious virus in reverse genetics.The critical temperature for FMDV inactivation is about 60 °C . Above tHowever, the potential for “self-transfection” must be kept in mind when setting up virological assays. When BHK-21 cells are used for virus titrations, CPE is not only induced by intact virions but also by naked RNA and it is conceivable that slightly higher titer readings are obtained compared to other cells. Self-transfection can also lead to divergent results in inactivation tests of FMDV, which are carried out to assess the degree of stability of the virus particle when exposed to chemicals, heat, or radiation. The treated virus preparation is inoculated into cell culture and the cultures are evaluated for the development of CPE ,18,19,20We did not see any indication of self-transfection with LFBK-αVβ6 cells. Based on this observation and on their high sensitivity for FMDV infection, we used these cells exclusively for all tests of the RNA inoculum and the samples from the animal experiments.11 FMDV genomes is an upper bound, because the RT-qPCR assay does not distinguish between whole genomes and viral RNA fragments, as long as they contain the part of the 3D-coding region targeted by the PCR.The first experiment clearly demonstrated that cattle can be infected with FMDV by a superficial injection of naked viral RNA into the tongue. The occurrence of lesions in the interdigital space indicates that the infection became generalized within less than two days. The calculated inoculation dose of 10Certainly, the large amount of viral genome from a preparation selected for high transfectability created optimal conditions for successful infection. The rapid course of disease—no slower than after inoculation of infectious FMDV —suggests2 Spain, A5 Eystrup, or C1 Tölz was infectious for cattle and guinea pigs.When conceiving of this study, we were not aware of any prior work where FMDV RNA was injected into animals without a transfection reagent. It has since been brought to our attention that there was in fact such a study in the 1950s , which aThe non-invasive application of FMDV RNA in the second experiment, i.e., instillation on the tongue and intranasal nebulization, did not result in infection despite the very high amount of viral RNA. This is probably because an intact cellular membrane of epithelial cells efficiently prevents the entry of naked RNA under natural conditions. Saliva also contains a large number of different ribonucleases ,24. ThesHowever, as soon as the barrier of at least one cell is breached, e.g., if it is nicked by a hypodermic needle, a single intact full genome that is introduced to the cytoplasm can cause a productive FMDV infection. For this to occur, it is important that the damage is just large enough for the RNA to gain entry to the cell, but not so large that the cell dies. While a similar injury can be caused by chewing on a jagged piece of plastic from a damaged LFD, it must be emphasized that this scenario is highly contrived and seems very unlikely.The main risk of spreading FMDV over longer distances certainly lies elsewhere, such as in trade with infected animals or contaminated materials . Based o


Clostridium autoethanogenum obtained using three commercial rRNA removal and strand-specific library construction products of NuGEN Technologies, Qiagen, and Zymo Research and assesses their performance relative to published data. While all three vendors advertise their products as suitable for prokaryotes, we found significant differences in their performance regarding rRNA removal, strand specificity, and most importantly, transcript abundance distribution profiles. Notably, RNA-seq data obtained with Qiagen products were most similar to published data and delivered the best results in terms of library strandedness and transcript abundance distribution range. Our results highlight the importance of finding appropriate organism-specific workflows and library preparation products for RNA-seq studies.Transcriptome analysis via RNA sequencing (RNA-seq) has become a standard technique employed across various biological fields of study. The rapid adoption of the RNA-seq approach has been mediated, in part, by the development of different commercial RNA-seq library preparation kits compatible with standard next-generation sequencing (NGS) platforms. Generally, the essential steps of library preparation, such as rRNA depletion and first-strand cDNA synthesis, are tailored to a specific group of organisms  or genomic GC content. Therefore, the selection of appropriate commercial products is of crucial importance to capture the transcriptome of interest as closely to the native state as possible without introduction of technical bias. However, researchers rarely have the resources and time to test various commercial RNA-seq kits for their samples. This work reports a side-by-side comparison of RNA-seq data from IMPORTANCE RNA-seq is a powerful technique for transcriptome profiling while involving elaborate sample processing before library sequencing. We show that RNA-seq library preparation kits can strongly affect the outcome of an RNA-seq experiment. Although library preparation benefits from the availability of various commercial kits, choosing appropriate products for the specific samples can be challenging for new users or for users working with unconventional organisms. Evaluating the performance of different commercial products requires significant financial and time investments infeasible for most researchers. Therefore, users are often guided in their choice of kits by published data involving similar input samples. We conclude that important consideration should be given to selecting sample processing workflows for any given organism. Gene transcription is a fundamental process that mediates a vast number of intracellular and environmental responses in every cell. Therefore, understanding transcriptional states of any organism of choice can shed light on basic biological processes as well as ways to direct and control cellular behavior. Insights into cellular transcriptional profiles or transcriptomes  have vastly expanded in the last decade due to rapid development and high accessibility of next-generation sequencing (NGS) platforms . MeanwhiWhile RNA-seq has become widely used across all fields of biological sciences, obtaining high-quality data of the transcriptome under investigation nevertheless requires careful planning, extensive sample processing, and considerable resources. The availability of commercial RNA-seq library preparation kits tailored to a variety of organisms, experimental approaches, and sequencing platforms has made RNA-seq accessible even to nonexpert users. When planning to do an RNA-seq experiment for the first time, researchers often consult existing literature to see which sample preparation protocols and products have been previously used with their organism of interest. However, working with unconventional microorganisms that have not yet been extensively studied via RNA-seq can make it difficult to decide which commercial kits might be most suitable.Clostridium autoethanogenum  and dilution rates . Each sample represented a single biological replicate. Both feed gas composition  for RNA-seq analysis of cultures . To asseposition  and specposition  of the canogenum . Next, tanogenum , 13.C. autoethanogenumNC_022592.1  . Again, stark differences between the kits were observed, confirming that rRNA depletion from bacterial samples is nontrivial . The ZymC. autoethanogenum culture conditions (GSE90792). Notably, Qiagen data were grouped the closest to this reference data set, with NuGEN transcriptome profiles separating most distinctively  . Within C. autoethanogenum (2 RPKM) (2 RPKM), while Zymo data were positioned between Qiagen and NuGEN. According to Zymo , such condensed distribution profiles could be caused by higher sensitivity of the workflow toward the presence of genomic DNA in the input sample, which can artificially inflate mRNA reads and with a more prominent effect on low-abundance transcripts, thereby pushing the left tail of the distribution to the right. This would also be consistent with the poorer strandedness of the Zymo and NuGEN data . Transcript levels in bacteria generally span over 4 orders of magnitude –17, incl–anogenum  and otheanogenum , 19. Aga(2 RPKM) . StrikinGEN data  that aroanogenum  efficienC. autoethanogenum RNA-seq studies, the Illumina and Qiagen kits are most suitable, by providing high sensitivity across a wide range of transcript levels, superior strand specificity, and sufficient rRNA removal, ensuring high coverage of transcriptome-wide mRNA detection and quantification.Our work is important, as researchers rarely have the resources and time to test various commercial RNA-seq kits, which are advertised as suitable for multiple organisms with different genomic GC content, for their samples. The ability to capture the spectrum of transcript abundances as closely to the true cellular state as possible is crucial to accurately address research questions investigated via RNA-seq. Our work shows that rRNA removal and library construction kits can strongly affect RNA-seq outcomes. This is highly relevant for anyone establishing an RNA-seq pipeline for an organism or for researchers puzzled by unexpected RNA-seq results. We conclude that, at least for Clostridium autoethanogenum DSM 10061 strain, namely DSM 23693, deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ), was used in all experiments and stored as a glycerol stock at −80°C. Full details of the cultivation conditions are reported in previous work . Four independent experiments were conducted with cultures grown at dilution rates (D) of ~1.0 and ~2.0 day−1 on both feed gas mixes. Chemostat cultures were performed in 1.4-liter Multifors bioreactors (Infors AG) at a working volume of 750 mL and connected to a Hiden HPR-20-QIC mass spectrometer  for online high-resolution off-gas analysis. The system was equipped with peristaltic pumps, mass flow controllers, and pH, oxidation-reduction potential, and temperature sensors. Cultures were sampled for RNA extraction and subsequent transcriptome analysis using RNA-seq after the optical density, gas uptake, and production rates had been stable for at least one working volume. Each sample represented a single biological replicate.A derivate of ous work . Shortlyg for 10 min at 4°C) and treated with RNAlater  overnight at 4°C, and pellets were stored at −80°C until RNA extraction. Thawed cell pellets were disrupted with glass beads in 800 μL of RLT buffer  containing 10 μL of β-mercaptoethanol by using the Precellys 24 instrument with liquid nitrogen cooling (Bertin Technologies) before extracting total RNA using the RNeasy minikit . Next, RNA extracts were depleted of DNA using off-column TURBO DNase  followed by purification using the RNA Clean and Concentrator kit . We used the NanoDrop 1000 instrument (Thermo Fisher Scientific) for verifying efficiency of RNA purification. The high quality and integrity of the total RNA extracts were confirmed by RNA integrity numbers (RIN) above 8.2 with TapeStation 2200 equipment (Agilent Technologies). Total RNA and residual DNA concentrations were determined using the Qubit 2.0 instrument (Invitrogen).Full details of culture sampling, RNA extraction, and purification are reported in previous work . BrieflyTotal RNA extracts for each sample were split between the NuGEN, Qiagen, and Zymo kits for rRNA removal and strand-specific RNA-seq library construction according to vendor instructions. Samples referred to as “NuGEN” were processed with Universal Prokaryotic RNA-seq, prokaryotic AnyDeplete ; “Qiagen” samples were processed with a QIAseq FastSelect 5S/16S/23S kit   and a QIAseq stranded RNA Lib kit  (for library construction); “Zymo” samples were processed with a Zymo-Seq RiboFree total RNA library kit .GSE90792) (C. autoethanogenumNC_022592.1 (RNA sequencing of the 12 mRNA libraries (four cultures times three kits) was performed on a MiSeq instrument (Illumina) using the MiSeq v3 150 cycle sequencing kit  with paired-end 2 × 75 bp reads. Raw RNA-seq data of the reference data set   were anaSE90792) . ShortlySE90792)  and presSE90792) . High-qu022592.1  and geno022592.1 . Finally022592.1  (see Tab022592.1  was perfGSE200959.The RNA-seq data have been deposited in the NCBI Gene Expression Omnibus repository under accession number


Sexual and reproductive health (SRH) is at the base of young people's living and wellbeing. A significant number of young peoples are affected by avoidable SRH problems due to a lack of appropriate knowledge regarding SRH. Parent-young communication on SRH is critical in informing them about risk and protective behaviors which in turn decrease the likelihood of involvement in risky sexual behaviors. Therefore, the purpose of this study was to evaluate the parent-young communication on SRH issues and associated factors among secondary and preparatory school students at Agaro town, Southwestern Ethiopia.School based cross-sectional study was conducted from April 13 to April 20, 2019 using stratified random sampling technique. A total of 315 students were included to the study. Data were collected using pretested interviewer-administered structured questionnaire entered into Epi data version 3.1; and analyzed using SPSS version 20. A variable having a p-value of < 0.25 in the bivariable logistic regression model was subjected to multivariable logistic regression analysis to avoid the confounding variable’s effect. Adjusted odds ratios were calculated at the 95% confidence interval and considered significant with a p-value of < 0.05.The mean age of the respondents was 20.2 ± 2.6 years. The study finding showed that 61.3% of the participants were discussed on SRH issues with their parents. According to multivariate analysis; educational status of mother ,educational status of father[primary education ],having family size of < 5  and having boy/girlfriend were significantly associated with parent-young people communication.About two third of the participants communicate with their parents about SRH issues and parents’ educational status, family size of < 5 and having boy/girlfriend were significantly associated with it. The main reasons for not communicated was cultural taboos, shame and parents lack of knowledge. Therefore, it is necessary to educate and equip students and parents to address the identified problems.The online version contains supplementary material available at 10.1186/s12978-022-01553-0. Sexual and reproductive health (SRH) is at the base of young people's living and wellbeing. A significant number of young peoples are affected by avoidable SRH problems. Parent-young communication on SRH is critical in informing them about risk and protective behaviors which in turn decrease the likelihood of involvement in risky sexual behaviors. This study was intended to assess this issue on students learning at Agaro town, Ethiopia during April 13 to April 20, 2019 using pretested interviewer-administered structured questionnaire with sample size of 315.The study finding showed that 61.3% of the participants were discussed on SRH issues with their parents. According to multivariable logistic regression analysis educational status of parents, having family size of < 5 and boy/girlfriend were significantly associated with parent adolescent communication. Cultural taboos, shame and parents lack of knowledge were the major barriers for communication. Thus, it is necessary to educate students and parents to address the identified problems thereby prevent young population from risky sexual behaviors.The online version contains supplementary material available at 10.1186/s12978-022-01553-0. Globally, there are 1.8 billion young people age 10–24 years) and 90% of them live in developing countries 4 years a. Unsafe Each year around the globe sixteen million late adolescent girls give birth, in which 95 percent of them occur in developing countries –8. GlobaOne of the priorities of the Sustainable Development Goals is improving the SRH of the young population . To thisParent-young population communication on SRH issues is vital in reducing risky sexual behaviors and negative its consequences . It is f. As a result, most of the young people attempts regarding sexual matters is generally misguided by their peer group of the same sex and many teenagers do not have access to reliable information regarding their SRH needs. A significant number of young peoples are affected by avoidable SRH problems due to a lack of appropriate knowledge regarding SRH. Parent-young communication on SRH is critical in informing them about risk and protective behaviors were significantly associated with parent-young communication after controlling for confounders Table .Table 4BThis study has attempted to assess the proportion of parent-young communication on SRH issues and its associated factors in Agaro secondary and preparatory school students, Southwest Ethiopia. The magnitude of parental communication on SRH issues was reported to be only 61.3% (193) . This finding was in line with two independent studies done in Ethiopia, 59.1% in Yergalem, South Ethiopia  and 57.6In contrast, the finding of the current study was lower than that of a study conducted in Hayik and Wolaita Sodo, Ethiopia were 83%  and 85%,The major topics discussed among participants were premarital sex, puberty and STIs/HIV/AIDS, while topics like contraception was discussed less among participants. The possible justification might be as majority of the participants were Muslim followers and their religion did not encourage them to use contraception including condoms which might affect their discussion. Thus, we recommend future study supported with qualitative findings to explore on this issue.Regarding challenges for not discussing SRH issues shame, cultural taboos, embarrassments and parents' lack of knowledge were mentioned by the majority of participants. For instance, almost three quarters of participants were not discussed regarding unwanted pregnancy due to shame and parents’ lack of knowledge 46% and 24.8%, respectively. This was consistent with previous studies , 27. ThiIn agreement with earlier studies, in this study educational status of father and mother showed statistical significance with parent adolescent communication about SRH issues , 36, 37.In the same manner, the study showed that those students whose family size was less than five were more likely to communicate which agreed with previous study . This rend this study was done to assess parent young-people communication were based on young population perceptions, which may not reflect what parents were actually doing. 3rd, since it was based on self-reporting, it might be affected by social desirability bias because of sensitive nature and cultural barrier for open discussion and finally additional qualitative study should be done to explore more on young and parents communication.1st due to cross-sectional nature of the study, it was difficult to imply cause–effect relationship. 2This study showed about two third of young population had discussion on SRH issues with their parents on at least two SRH issues. Educational level of parents, having family size of < 5 and having boy/girlfriend were significantly associated with communication. The main reasons for not communicated was cultural taboos, shame and parents lack of knowledge. Thus, it is necessary to educate and equip students and parents to address the identified problems.Students and their parents should receive health education on the significance of talking about SRH issues and related consequences of risky sexual behaviors. To promote and support school sex education, teachers should receive SRH training. Moreover, more research is required to identify barriers especially with regard to parents.In order to build a supportive atmosphere for parent-young people communication regarding SRH, policy makers will need to design tailored action to create supportive environment for parent young people communication concerning SRH.Additional file 1: Figure S1. Conceptual framework showing the possiblefactors that could contribute to the Parent young communication [nication , 35–37.


The lips are a common location for minor salivary gland tumors, including adenoid cystic carcinoma (ACC).50) CIRT delivered in 16 fractions, along with the inclusion of teeth within the planning target volume (PTV), are significant independent risk factors for ORN.50 is required, and intraoral devices are one modality by which the lip and jaw are positioned to reduce the dose. However, no devices to prevent ORN from lip cancer have been reported for CIRT.Osteoradionecrosis (ORN) is a serious adverse event in patients with head and neck cancer who receive CIRT. Grade ≥2 maxillary ORN after CIRT was reported at 19.0% in 2014.50 in patients with ACC of the lip(s) treated with CIRT.In this technical report, we introduce and evaluate the efficacy of a custom-made mouthpiece with a lip bumper for the reduction of maxilla VAn 85-year-old male patient presented to our hospital with a primary ACC located on the right side of the upper lip without invasion of the maxilla. The patient found the esthetic impact and articulatory dysfunction after surgery to be unacceptable, and was subsequently referred for CIRT. The patient had no other known risk factors associated with maxillary ORN, including tobacco or alcohol use or a surgical history. The patient had 28 teeth, and maintained adequate oral hygiene.,,A custom-made mouthpiece is created at our institution for all patients with head and neck cancer, as described previously.The CIRT technique has been described previously.After CIRT, maxillary ORN was evaluated based on clinical symptoms, macroscopic examination, and the findings of the CT and magnetic resonance imaging conducted every 2 to 3 months. The National Cancer Institute's Common Terminology Criteria for Adverse Events, version 3.0, was used to define toxicity.50 with and without the spacer was calculated. A dose-volume histogram (DVH) analysis was performed using MIM software  to compare the irradiation dose with the maxilla volume with and without the mouthpiece. Pretreatment diagnostic CT images in the same case were used to simulate treatment without a mouthpiece  compared with nonusage .50 (≥3.0 mL) and the presence of teeth in the PTV were identified as independent risk factors.50 from 4.11 mL to 2.21 mL with a mouthpiece. The lip bumper appears effective in reducing maxillary dose and, by extension, the risk of maxillary ORN for lip ACC treated with CIRT.A previous study reported on the independent risk factors to develop maxillary ORN after CIRT. On multivariate analysis, VSimilarly, dosimetric parameters have been reported as risk factors for ORN in the mandible. Musha et al reported that doses of 30 Gy (RBE) to the mandible and teeth caused ORN at 29.5 ± 6.7 cc and 3.9 ± 1.8 cc, respectively, with cutoff values of 16.5 cc and 1.8 cc, respectively.,Lip cancer is a common cancer of the head and neck, and is generally SCC.In non-SCC cases requiring escalated treatment doses, charged particle therapy  may be considered, offering improvement in RBE and dose distribution in comparison with conventional RT. Charged particle therapy, in particular, should be considered for radioresistant diseases.At our hospital, we use floor-mounted kV x-ray image guided RT systemsA custom-made mouthpiece with a lip bumper was employed in CIRT to reduce the maxillary dose. The patient tolerated treatment well with acceptable treatment toxicity, and the DVH simulation demonstrated a 46% reduction in maxillary dose with expected concomitant reduced ORN risk. Further evaluation in a dedicated cohort is warranted. In addition, evaluation of the dental sequelae of CIRT is also a future concern.The authors thank Minami Ikawa for the preparation of the medical illustrations.


Gammacoronavirus genus and Igacovirus subgenus. The phylogenetic tree plotted using the ML method revealed that those sequences formed a group most similar to pigeon coronavirus strains from China, Finland, and Poland, and to a single strain from a common starling from Poland, which suggests wide geographical distribution of the virus and its possible transmission between various species.While disease control in racing pigeons and the potential role of pigeons as vectors transmitting viruses to poultry are of importance, there is still a paucity of data concerning the occurrence of coronaviruses in pigeons. In this study, 215 domestic pigeons were tested for the presence of coronaviral genetic material using the nested PCR method, which revealed 57 positive samples (26.51%). The difference in coronavirus prevalence between young and adult pigeons  has been found statistically significant. In contrast, no statistically significant difference has been demonstrated between the prevalence in symptomatic and asymptomatic birds, leaving the influence of coronavirus presence on pigeon health uncertain. Phylogenetic analysis of the RdRp gene fragment allowed us to assign all the obtained strains to the  Orthocoronavirinae subfamily, i.e., those representing the Deltacoronavirus and Gammacoronavirus genera, are relatively widespread across bird species [Gammacoronavirus genus are responsible for infections of poultry and wild birds, although the latter are known to also be infected by viruses from the Deltacoronavirus genus [Columba livia species [Coronaviruses from the  species . Virusesus genus ,3. Virus species ,5. Coron species ,7. Racin species . Keeping species . As stat species . Pigeons species ,13,14,15To date, coronaviruses genetically similar to infectious bronchitis virus (IBV) have been confirmed to occur in pigeons. Detection of viral strains with a nucleotide sequence of the S1 fragment nearly identical to that of Massachusetts and Connecticut vaccine serotypes in asymptomatic feral pigeons suggests the possible transfer of IBV between pigeons and chickens . While IAvian coronaviral infections have been a focus of interest of researchers for the past few years; however, most studies addressing this issue concern poultry kept in the intensive production system . Most reFifty-seven out of the two hundred fifteen studied samples (26.51%) tested positive for coronaviral genetic material. Twenty-three out of one hundred sixteen samples (19.83%) and thirty-four out of ninety-nine samples (34.34%) derived respectively from adult and young birds were found to be positive. Seventeen out of eighty-seven samples (19.54%) acquired from asymptomatic pigeons and forty out of one hundred twenty-eight samples (31.25%) acquired from symptomatic pigeons turned out to be positive . Additio2 = 5.78, p = 0.0162). Differences between asymptomatic birds and symptomatic birds, asymptomatic adult birds and symptomatic adult birds, asymptomatic young birds and symptomatic young birds, asymptomatic adult birds and asymptomatic young birds, and between symptomatic adult birds and symptomatic young birds have proven to be statistically insignificant . There was also a significant difference  in coronavirus prevalence between live and dead birds.Despite the very distinct difference in the prevalence of coronaviral genetic material between symptomatic and asymptomatic pigeons, the only statistically significant difference has been found in coronavirus prevalence between adult and young birds  and feral pigeons from Finland and Poland   A,B. Two Our study revealed that over a quarter of the samples tested contained coronaviral RNA. However, quite high coronavirus prevalence values have also been found in other avian species to date  ,24,25. TThe majority of positive samples were acquired from young pigeons, which has been proved to be statistically significant. The higher prevalence of coronaviruses in the group of young pigeons might be caused by their not yet fully developed immune system. It could also be due to the stress related to leaving the parental nest and, in the case of racing pigeons, to racing training and transport. This phenomenon has been confirmed for pigeon circovirus, pigeon herpesvirus, and pigeon rotavirus, and could be likely also in the present study ,32.Despite the lack of statistical differences between the prevalence of coronaviruses in asymptomatic pigeons and pigeons with selected clinical symptoms, a certain trend was noticeable also in young birds, because the prevalence of the genetic material of coronaviruses was about 70% higher in young pigeons showing selected disease symptoms than in asymptomatic young pigeons. Moreover, higher prevalence was determined in the samples collected from live than dead birds, and this finding has been proven statistically significant. Therefore, one may assume that the coronavirus infection either may not be related to the birds’ death, might have overlapped with another health problem, or was a part of a complex infection with other pathogens. However, this finding might not be entirely reliable due to the small number of samples collected from dead birds. We have not investigated the samples for the presence of genetic material of other known viruses pathogenic to pigeons. Due to some contradictions in the results obtained, it cannot be clearly stated whether pigeon coronaviruses are pathogenic to this bird species and whether they can induce enteric problems. The above analysis is also hindered by the very common asymptomatic infections in pigeons, which was also confirmed in our current research. The ideal solution to confirm the potential role of pigeon coronaviruses as triggers of enteropathies would be an experimental infection with laboratory-propagated isolates of these viruses, which would allow for meeting Koch’s postulates. Unfortunately, these assumptions cannot be addressed because other research indicates great difficulties with the cultivation of these viruses in laboratory conditions, which makes it impossible to obtain a pure isolate for experimental inoculation ,34.Gammacoronavirus genus. Only one case of discovery of a virus from the Deltacoronavirus genus in pigeon was documented among all surveillances to date [Analysis of the RdRp gene fragment, which is a conserved region of a coronaviral genome used for classification, allowed us to assign all strains obtained in the current research to the  to date . FurtherAnalysis of the phylogenetic tree revealed that pigeons were hosts of the coronavirus typical of this bird species, which is confirmed by the close position of the Polish strains and those originating from pigeons from other European countries and China on the phylogenetic tree. The above finding was consistent with the results reported by Zhuang et al., who investigated 170 pigeon coronavirus sequences obtained in China in the years 2013, 2014, and 2018, constituting one large group of strains, acquired mainly from pigeons, called pigeon-dominant coronavirus . The simZhuang et al. also recognized several smaller groups, associated with sequences derived mainly from wild birds and domestic waterfowl, closely related to pigeon strains. According to our phylogenetic tree, two strains acquired from ducks from China were closely related to Polish pigeon strains. These two strains were identified by the researchers as pigeon-dominant, suggesting that such strains are most commonly isolated from pigeons and have a very high resemblance to pigeon strains ,33.Interestingly, the pigeon coronavirus strains from Poland are positioned closely to the coronavirus obtained from the common starling on the phylogenetic tree. Although we are unable to provide a clear explanation for their close relationship, the possible contact of racing pigeons with wild birds during free flights in the areas surrounding lofts might be a trigger of cross-infection. Moreover, the very low genetic identity of the strains studied with IBV and turkey coronavirus sequences indicates that those viruses definitely belong to different species.Coronaviridae family generally have very low mutation rates compared to other RNA viruses because of the presence of exonuclease in nsp14 protein, which remarkably increases replication fidelity by the removal of misincorporated nucleotides [Recently, coronaviral infections have been receiving a significant amount of attention from both the public and researchers. Despite the very large genome, viruses belonging to the leotides ,37,38. Hleotides ,40. Whilleotides ,42. Consleotides ,44,45,46No ethical approval was required for this study, as the samples consisted of cloacal swabs collected from birds being patients of the Department of Poultry Diseases, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Poland, during standard diagnostic procedures, for which the consent of their owners was obtained. No other manipulations were performed on live animals. Tissue samples were collected during necropsy of dead birds delivered to the Department of Poultry Diseases.Experimental material was collected from 215 domestic pigeons originating from 67 breeding facilities located in multiple regions of Poland between January 2016 and August 2020 . The pigThe total RNA was extracted from the samples suspended in Fenozol solution using a Total RNA Mini Plus kit  in accordance with the manufacturers’ protocol, and eluted in 50 μL of RNase-free water. Purity and concentration of the obtained RNA were measured with a NanoDrop 2000 Microvolume Spectrophotometer. cDNA was generated through reverse-transcribing 2 μL of eluted RNA using a High-Capacity cDNA Reverse Transcription Kit . 2 µL of the acquired cDNA were then used for nested PCR for the RNA-dependent RNA polymerase—gene (RdRp gene) fragment in the final volume of 20 µL. A HotStarTaq Plus Master Mix Kit  was used to prepare the reaction mixture. Two rounds of PCR were performed according to the method used by Chu et al., in a MasterCycler Pro thermocycler  . The secThe sequences obtained were assembled into the contigs in the SeqMan Pro software version 14.1  and manually checked for sequencing errors using the same software. These sequences, compiled with RdRp gene sequences of other avian coronaviruses retrieved from the GenBank database (NCBI) using the BLAST tool, were aligned with the MUSCLE method using Molecular Evolutionary Genetics Analysis software, version 11 (MEGA 11) . All aliViral sequences obtained in this study and used in this paper have been deposited in GenBank under accession numbers OM366000 to OM366006, OM366008 to OM366013, OM366015, OM366016, and OM366018 to OM366034.2), chi-square test with Yates correction, and V-square test (V2) have been used to determine the significance of differences in selected parameters, depending on the expected values in the compared groups. Differences were considered significant with p < 0.05.The data obtained has been subjected to a statistical analysis using Statistica 13.3 software . The chi-square test (χ


In this study, we developed a novel, multiplex qPCR assay for simultaneous detection of RIG-1, MDA5, and IFIT-1 at the mRNA level. The assay was validated in A549 cells transfected with in vitro transcribed RNAs. Both exogenous RNA-GFP and self-amplifying (saRNA-GFP) induced significant expression of RIG-1, MDA5, IFIT-1, as well as type I and III interferons. In contrast, native RNA from intact A549 cells did not upregulate expression of these genes. Next, we evaluated RIG-1, MDA5, and IFIT-1 mRNA levels in the white blood cells of patients with influenza A virus (H3N2) or SARS-CoV-2. In acute phase (about 4 days after disease onset) both viruses induced these genes expression. Clinical observations of SARS-CoV-2 typically describe a two-step disease progression, starting with a mild-to-moderate presentation followed by a secondary respiratory worsening 9 to 12 days after the first onset of symptoms. It revealed that the expression of RIG-1, MDA5, and MxA was not increased after 2 and 3 weeks from the onset the disease, while for IFIT-1 it was observed the second peak at 21 day post infection. It is well known that RIG-1, MDA5, and IFIT-1 expression is induced by the action of interferons. Due to the ability of SOCS-1 to inhibit interferon-dependent signaling, and the distinct antagonism of SARS-CoV-2 in relation to interferon-stimulated genes expression, we assessed SOCS-1 mRNA levels in white blood cells. SARS-CoV-2 patients had increased SOCS-1 expression, while the influenza-infected group did not differ from heathy donors. Moreover, SOCS-1 mRNA expression remained stably elevated during the course of the disease. It can be assumed that augmented SOCS-1 expression is one of multiple mechanisms that allow SARS-CoV-2 to escape from the interferon-mediated immune response. Our results implicate SOCS-1 involvement in the pathogenesis of SARS-CoV-2. Under homeostatic conditions, cells produce interferons (IFNs) at extremely low, often undetectable, levels. Induction of IFN synthesis is mediated by the activation of pattern recognition receptors (PRRs). Cytosolic sensors that belong to the category of RIG-1 like receptors (RLR) play a key role in IFN production in response to the penetration of foreign RNAs . All RLRIFNs do not have a direct antiviral effect . They acThe aim of the study was to develop a multiplex qPCR system for simultaneous detection of MDA5, RIG-1, and IFIT-1 mRNA levels and to evaluate these genes’ expression in the white blood cells (WBC) of influenza and SARS-CoV-2 patients.2 with humidification). The A549 (CCL-185) cell line was obtained from the American Type Culture Collection . A549 cells  were cultured in F12K medium  supplemented with 10% fetal bovine serum . Treated cells and intact (mock) cells were incubated at 37 °C  storage medium containing 10% DMSO, 50% FBS). Blood samples for WBC isolation from IAV patients were collected on the 3–4 days after onset of clinical symptoms. In total, the study involved 14 healthy donors, 13 patients with influenza A/H3N2 (2018–2019 epidemic season), and 17 SARS-CoV-2 pneumonia patients being treated at clinics in St. Petersburg (Russia) in 2019–2020. The following symptoms were observed as the most striking clinical manifestations in patients: fever; intoxication syndrome ; and/or catarrhal syndrome . Inclusion of patients in the A/H3N2 and SARS-CoV-2 groups was carried out based on positive results from laboratory diagnostics of the relevant pathogens in oropharyngeal swabs. Laboratory diagnosis of pathogens in selected swabs was performed by RT-PCR using certified kits. Blood for white blood cell (WBC) isolation was collected in vacuum tubes with sodium heparin. Eight milliliters of blood, diluted with DPBS to a volume of 12 mL, was introduced (avoiding mixing) into a tube containing 9 mL of Lymphosep . Tubes were then centrifuged at 400× For SARS-CoV-2 patients, the specific IgG antibody levels were measured. According to the results of serological examination, SARS-Cov-2 patients were divided into three groups: 3–4 days (IgG < 1); second (1 < IgG < 10) and third (IgG > 10) weeks after the onset of illness.This clinical investigation was approved by the Institutional Ethics Committee, and the corresponding protocol numbers are 118, dated 16 October 2017 and 164, dated 12 February 2021. Informed consent was obtained from all donors and patients who provided research materials. All biological experiments were performed in accordance with the relevant guidelines and regulations.Synthetic GFP-coding RNAs (IVT-RNA-GFP) were obtained by in vitro transcription using the mMESSAGE mMACHINE kit . The pJET1.2-GFP  and T7-VEE-GFP  plasmids, containing the T7 promoter and the GFP gene, were used as templates for RNA synthesis during in vitro transcription. One microgram of linearized plasmid template was used for each reaction. Anti-Reverse Cap Analog (ARCA)  was used for efficient translation of the RNA. To maximize RNA yield and the fraction of capped transcripts, we used the following cap analog/GTP ratios: 4:1 for RNA-GFP; and 10:1 for self-amplifying GFP-coding RNA (IVT-saRNA-GFP). Reactions were performed according to the manufacturer’s protocol. Following RNA synthesis, the DNA template was removed by subsequent digestion with DNase Turbo . The Poly(A) Tailing Kit  was used to polyadenylate the 3′-termini of transcribed RNA. Transcripts were purified by lithium chloride precipitation according to the recommended protocol . RNA samples were analyzed using electrophoretic separation under denaturing conditions. Five hundred nanograms of each RNA sample was mixed with an equal volume of Gel Loading Buffer II  and heated for 5 min at 80 °C. Samples were subsequently loaded into wells of 1% agarose gel (containing 0.5 µg/mL ethidium bromide) and run in 1x MOPS buffer at room temperature. 11 RNA molecules per well. For IVT RNA-GFP, total RNAs, mRNA, viral genomic RNA, LMW, and HMW, this was approximately 80, 160, 16, 40, 120, and 380 ng, respectively. The immunogenicity of the studied RNAs was evaluated 24 h after introduction of the complexes.High-molecular weight  and low-molecular weight  poly(I:C) dsRNAs, produced by InvivoGen , were used as PRR agonists. Total RNA was isolated from either uninfected A549 cells or cells infected with IAV A/California/07/09 (H1N1pdm09) (24 h post infection). Total RNA was extracted from cells with TRIzol reagent  in full accordance with manufacturer’s instructions. Viral genomic RNA was isolated from influenza virus A/California/07/09 (H1N1pdm09) using the RNeasy Mini Kit . RNA transfections were performed using Lipofectamine MessengerMAX Reagent  according to manufacturer instructions. There were approximately 0.6 µL of Lipofectamine Messenger Max Reagent and 1.40 × 10The target mRNA sequences of RIG-1 (DDX58), MDA5 (IFIH-1), IFIT-1, SOCS-1, AnxA-1, IFNA, IFNB, IFNL2/3, and MxA were downloaded from the GenBank database. Primers and fluorescent oligonucleotide probes, containing fluorescent reporter dyes at the 5′-end and a quencher at the 3′-end, were commercially synthesized and HPLC-purified .Total RNA was isolated from A549 cells and WBCs using TRIZol reagent  according to the manufacturer’s instructions. RNA concentrations were analyzed using a NanoDrop ND-1000 spectrophotometer .16 primers and RNAscribe RT . Complementary DNA synthesis was carried out at 50 °C for 50 min. Enzyme was inactivated at 80 °C for 5 min. Products were diluted (1:2) and stored at −20 °C until use.One microgram of total RNA was treated by DNase  and then directly reverse transcribed using oligo-dTReal-time PCR assays were performed using the CFX96 Real-Time PCR System . Multiplex qPCR was performed in 25 μL final reaction volumes containing 12.5 μL BioMaster HS-qPCR mix (2×)  and 2 μL cDNA (obtained from 1 μg of RNA). The reaction mixtures additionally contained 200 nM of each primer and probe for RIG-1 and IFIT-1 as well as 750 nM of each primer and 250 nM of probe for MDA5. Conditions of two-step PCR were 95 °C for 5 min, followed by 40 amplification cycles .Expression of other genes was assessed by monoplex two-step qPCR (500 nM of each primer and 200 nM of probe). IFNA expression was measured using HS-qPCR SYBR Blue (2×) . Human IL-8 concentrations in cell culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA) using the IL-8/CXCL-8 DuoSet ELISA commercial kit .Data processing was carried out in Microsoft Excel. GraphPad Prism was used to evaluate the statistical significance of differences.Multiplex qPCR is one of the most accurate and sensitive methods for simultaneous evaluation of gene expression . HoweverHome—Nucleotide—NCBI (nih.gov)). The designed sequences  should detect almost all of the transcriptional variants of the target genes both in monoplex and multiplex assay  to identify their possible range of nonspecific products. Sequences highly homologous to any other human genes were not found  features seven; the IFIT-1 gene features five; and the IFIH-1 gene (MDA5) features one (ex assay . Sequencot found . SpecifiE) of the target and reference genes must be almost equal. The E value for each amplified gene was determined by plotting a graph with the x-axis as log(quantity) and the y-axis as Ct obtained in monoplex and multiplex qPCR (E = 10(–1/slope) − 1. To achieve highly efficient amplification of specific fragments for all three genes in multiplex qPCR, primer and probe concentrations ranged from 50 to 1000 nM. The optimal primer and probe concentrations, determined for each gene, are presented in the Materials and Methods section. The corresponding multiplex PCR amplification E values were: RIG-1 (98%); MDA5 (98%); and IFIT-1 (97%).RNAs isolated from intact cells and infected cells (influenza A virus) were used to make cDNA matrix for qPCR optimization. Primer concentrations and thermal conditions were optimized to amplify all of the specific PCR products with high efficiency. The amplification efficiencies  RNAs); IVT-RNA-GFP; IVT-saRNA-GFP; or native RNAs . Transfection of A549 cells with IVT-RNA-GFP and IVT-saRNA-GFP resulted in stable translation of green fluorescent protein after 24 h .Both IVT-RNA-GFP and IVT-saRNA-GFP led to an increase in IFN mRNA synthesis. In addition, the magnitude of changes in IFNA mRNA levels was much less than IFNB or IFNL2/3 . It was Next, using the developed multiplex qPCR, we evaluated RIG-1, MDA5, and IFIT-1 levels in response to transfection. Stimulation with synthetic RNAs led to a strong increase in the expression of these genes in A549 cells . It is nIt was shown that both total RNA from infected cells and viral RNA are able to dramatically induce RIG-1, MDA5, and IFIT-1 expression. Total RNA from intact A549 cells did not influence these ISGs or IFNs at the mRNA level . Taking For practical evaluation of the developed multiplex qPCR, we carried out an experiment with human WBCs. Cells were isolated from the whole blood of healthy volunteers or respiratory illness patients (influenza A/H3N2 or SARS-CoV-2). ISGs analysis was carried out in the acute phase of illness . We used Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Actin beta (ACTB) as reference genes . In acutWe investigated possible correlation between IFIT-1 and RLR expression in WBCs in both infected groups and in healthy volunteers . There wA canonical antiviral ISG, MxA, which is considered a reliable marker of viral infection , was eleDue to the ability of SOCS-1 to inhibit IFN-dependent signaling ,16,17, aNext, to assess the formation of a systemic immune response in patients with SARS-CoV-2, the ISGs expression study was also performed in dynamics at different phases of the disease .Interestingly, expression of RIG-1, MDA5, and MxA was remarkably increased compared to the control in WBC of SARS-CoV-2 patients only at early stage of the disease a,b,d. InThe RNA helicases RIG-1 and MDA5 are key cytosolic sensors that induce type I and type III IFN expression. Simultaneous analysis of their mRNA levels enables assessment of IFN-mediated immune response activation. The third target gene for the developed multiplex is IFIT-1, a canonical ISG with distinct antiviral activity. It should be noted that IFN mRNA levels in WBCs can be extremely low or even undetectable. In our work, it was shown that the developed multiplex qPCR is suitable for RIG-1, MDA5, and IFIT-1 mRNA measurement in A549 cells and WBCs.A promising technology is vaccine development based on synthetic RNA molecules . The immSynthetic poly(I:C) dsRNA and long viral dsRNA are considered to be MDA5 agonists; uncapped ssRNA with a 5′-triphosphate (5′-PPP) or 5′-diphosphate (5′-PP) group activates RIG-1 . Thus, IInhibition of translation is associated with the recognition of foreign RNA by IFIT-1. IFIT-1 attenuates translation either at the level of 43S complex stabilization, or at the level of RNA binding, interacting through a 2′-O-unmethylated 5′ cap structure or 5′-PPP group ,10,21. TTherefore, the developed multiplex qPCR assay is suitable for assessing IFN-dependent response activation when cells are transfected with exogenous RNAs. This is especially useful for evaluating the immunogenicity of mRNA-based drugs. Estimation of IFIT-1 mRNA level is not only an indicator of ISG expression, but also indicates the degree of protein synthesis inhibition. Increases in the mRNA levels of RIG-1, MDA5, and IFIT-1 were correlated with augmented expression of IFNB, IFNL2/3, as well as SOCS-1. Interestingly, the IVT RNA, despite 5′-capping and 3′-polyadenylation, turned out to be an even more potent inducer of IFNs than control poly(I:C) dsRNA. These data once again emphasize the need to use modified bases and thorough purification of mRNA .Interferons exhibit distinct antiviral activity against SARS-CoV-2 ,23,24. SActivation of cytosolic RNA-sensors leads to IFN induction, while IFIT-1 can be considered an antiviral ISG that blocks the primary translation of (+) RNA viruses. At the early stage of the disease we have discovered that RIG-1, MDA5, IFIT-1, MxA, and SOCS-1 expression was elevated. High ISG expression at the acute phase of disease has been accompanied by low SARS-CoV-2 antibody levels (IgG < 1). These data can be considered as a common innate antiviral response. For instance, influenza infected patients had augmented mRNA level of the genes, with the exception of SOCS-1.Considering the complex dynamics of RNA sensing with the consequent dysregulation of IFN production, we compared ISG expression in WBCs of SARS-CoV-2 patients at approximately 14 and 21 days after disease onset. Our experiments demonstrated that only SOCS1 expression was significantly increased during 4, 14 and 21 days from onset of initial symptoms. At the same time, expression of other ISGs  at 14 and 21 days did not differ from the control group. In another research it was also demonstrated that at about ten days after disease manifestation, severe SARS-CoV-2 patients were characterized by downregulation of key antiviral ISGs . Our resSARS-CoV-2 utilizes many mechanisms to escape the immune response . It simuIt is well known that SOCS proteins negatively regulate IFN signaling by inhibiting the JAK-STAT signaling pathway . It has We developed a multiplex qPCR assay for simultaneous evaluation of RIG-1, MDA5, and IFIT-1 expression. Using the assay, we discovered that RIG-1, MDA5 and IFIT-1 mRNA levels were augmented in the WBCs of influenza infected patients, but not in those from SARS-CoV-2 patients after 2 weeks from the disease onset. This motivated us to assess SOCS-1 expression. It was revealed that SARS-CoV-2 patients had increased SOCS-1 expression during the course of the disease, while the influenza-infected group did not differ from healthy donors.