Slaked lime  is a commonly used disinfectant for fecal sludge. Although viruses are inactivated by lime treatment, whether RNA viruses adapt to lime treatment has not yet been determined. Here, we show that murine norovirus developed higher tolerance during serial passages with lime treatment. We compared synonymous and non-synonymous nucleotide diversities of the three open reading frames of viral genome and revealed that virus populations were subjected to enhanced purifying selection over the course of serial passages with lime treatment. Virus adaptation to lime treatment was coincident with amino acid substitution of lysine to arginine at position 345 (K345R) on the major capsid protein VP1, which accounted for more than 90% of the population. The infectious clones with the K345R produced using a plasmid-based reverse genetics system exhibited greater tolerance in a lime solution, which indicated that the specific amino acid substitution was solely involved in the viral tolerance in lime treatment. Caliciviridae, is known as a major cause of acute gastroenteritis. According to a systematic review of 175 studies, the pooled prevalence of norovirus in 187,336 patients with acute gastroenteritis was 18% (95% CI:17–20%) worldwide  are dominant types of the lime disinfectant, because they are highly reactive and generate hydroxide ions, which are strong bases  are determined on the basis of the tolerances of reference viruses in a disinfection study. Meanwhile, previous studies have reported the heterogeneous susceptibilities of reference viruses and indicators for waterborne enteric viruses within and among genetically relative strains. For example, disinfection studies of the virus B5 . These svirus B5 . Similarvirus B5 .The varied sensitivities in RNA viruses and some DNA viruses are explained by their distinctive features, high mutation rates, and continuous production of variant genomes that provide a rich substrate for selection and random drift to act on . A viralNorovirus, is used as a model virus for human norovirus in disinfection studies on inactivation efficiency and mechanisms 3, 2 mM L-glutamine, 10 mM non-essential amino acids, penicillin (100 mg/ml), and streptomycin (100 U/ml).Prof. Yukinobu Tohya, Nihon University, Japan, provided the MNV S7 PP3 strain. MNV S7 PP3 was propagated, enumerated, and purified according to the published protocols. RAW 264.7 cells [American Type Culture Collection (ATCC) TIB-71] were used as host cells and were cultured in Dulbecco’s Modified Eagle Medium (Nissui) containing 10% (v/v) fetal bovine serum (Gibco), 0.1% (w/v) NaHCOnth passage without lime treatment (−lime) and with lime treatment (+lime) were designated as pn + lime and pn−lime virus, respectively .A virus population was taken from a laboratory strain of MNV S7 PP3 as an ancestor of serial passages with and without lime treatment . MNV popn + lime MNV suspension in the culture medium [106–107 plaque forming unit (PFU)/ml] was exposed to 900 μl of lime solution at room temperature (22°C) and inactivated to 103 PFU/ml. The treatment time for less sensitive populations was prolonged so that the infectious titer would be 103 PFU/ml after treatment. A 100-μl pn−lime MNV suspension in the culture medium was diluted with the culture medium to create 103 PFU/ml, in parallel. Each of the virus suspension was inoculated onto the confluent RAW cells cultured in T75 flask containing 25 ml of the culture medium [the multiplicity of infection (MOI) was approximately 10–4]. The MNV was incubated at 37°C with 5% CO2 for 3 days. The cell suspension was subjected to three rounds of freeze-thawing and then transferred to a 50-ml falcon tube. The falcon tube was centrifuged at 10,000 g for 10 min at 4°C, and the supernatant was passed through a PolyVinylidene DiFluoride (PVDF) membrane filter (0.2-μm pore). The collected MNV suspension, pn+1+lime and pn+1−lime, was exposed to the lime solution or diluted with the culture medium and then inoculated onto the confluent RAW cells (°C until the disinfection experiment and next generation sequencing (NGS) library preparation.The lime solution was prepared by dissolving 15 mg of calcium hydroxide  in 30 ml of sterilized pure water so that the pH value increased to approximately 12. A 100-μl pAW cells . These pThe lime sensitivity of viruses was evaluated on the basis of the reduction of infectious titers by exposure to lime solutions. The lime sensitivity assay was conducted in triplicate for each sample. MNV suspension (100 μl) in the cultured medium was added to a 1.5-ml tube containing 900 μl of the lime solution. Experiments were carried out at 22°C. In the sensitivity assay for the populations in −lime and +lime lineages, samples were periodically taken during the 5-min exposure to the lime solution and immediately diluted with the culture medium. In the sensitivity assay for the plaque-purified isolates and the infectious clones, samples were taken at 4 min because MNV was not always detected after the longer exposure time.2 for 90 min with periodical swaying. The virus suspension was removed after the 90-min incubation, and 2 ml of the agar medium (1.25% agar) were added. The six-well plates were incubated for 2 days at 37°C with 5% CO2 and then dyed with 0.03% neutral red solution and incubated for 2–3 h. The neutral red solution was removed and the six-well plates were incubated for 1 h, and then, the plaques were counted.Murine norovirus infectivity was determined by plaque assays in the sensitivity assay for the populations in −lime and +lime lineages. One milliliter of the sample was inoculated onto the confluent RAW cell cultured in the six-well plate and incubated at 37°C with 5% CO50) assays in the sensitivity assay for the plaque-purified isolates and the infectious clones produced using the plasmid-based reverse genetics system. We performed the TCID50 assays because these clones and isolates did not form clear plaques with unknown reasons. We did not compare the data obtained using the two different methods for titration, and we evaluated the lime sensitivity within the data obtained from identical method. Three-fold serial dilutions of virus were prepared in 96-well plates containing culture medium. Fifty microliters of the RAW cell suspension were inoculated in each well. The 96-well plates were incubated at 37°C with 5% CO2. After 2 days, a cytopathic effect was observed with light microscopy. TCID50 values were calculated according to the Spearman and Kärber algorithm (Murine norovirus infectivity was determined by Median Tissue Culture Infectious Dose (TCIDlgorithm .–4). Plaques were formed using the method described above. Well-separated plaque from any other plaques was taken using 1,000 μl of barrier tips cut by a sterilized cutter, placed into a 1.5-ml tube containing 500 μl of culture medium, and vortexed. The isolate suspensions were incubated overnight at 4°C and then passed through a membrane filter (0.2-μm pore). The first plaque-purified virus suspension was subjected to a second round of plaque purification. After the third round of plaque purification, virus suspensions were stored at −80°C until the subsequent analysis. Finally, we obtained six isolates originated from the p5 + lime populations, six isolates originated from the p5−lime populations, and three isolates originated from the ancestor population in the experimental adaptation . Supernatants from transfected cultures were collected at 48 h after transfection and inoculated onto Huh7.5.1 CD300lf cells cultured in six-well plates. The virus was passaged three times in Huh7.5.1 CD300lf cells to obtain a sufficient titer for disinfection experiments. The whole genome sequence of propagated virus was confirmed using next-generation sequencing.Infectious clones (referred to as clone MNV) were produced using a plasmid-based reverse genetics system as described previously . HEK293TViral RNA was extracted from the MNV suspension using the QIAamp Viral RNA Mini kit  according to the manufacturer’s instructions. Ribosomal RNA (rRNA) in RNA suspension was removed using the NEBNext rRNA Depletion Kit (Human/Mouse/Rat) . A 300-bp fragment library was constructed for each sample using the NEBNext Ultra II RNA Library Prep Kit for Illumina according to the manufacturer’s instructions. Samples were added bar codes for multiplexing using the NEBNext Multiplex Oligos for Illumina . Library purification was done using Agencourt AMPure XP magnetic beads  according to the NEBNext Protocol. The quality of the purified libraries was assessed on an Agilent 2100 Bioanalyzer using a High Sensitivity DNA Kit . The concentration of the purified library was determined on a Qubit 2.0 fluorometer using the Qubit HS DNA Assay  and the NEBNext Library Quant Kit for Illumina. Libraries generated from the populations obtained in serial passages, plaque purified isolates, and infectious MNV were sequenced with paired-end 150-bp reads on an Illumina NovaSeq 6000, paired-end 250-bp reads on an Illumina MiSeq, and paired-end 150-bp reads on an Illumina iSeq , respectively.Trim Sequences function trimmed index primer sequences used for library amplification and sequencing. The MNV S7 PP3 genome sequence obtained from National Center for Biotechnology Information (NCBI) (GenBank: AB435515.1) was used as the reference sequence. The trimmed sequence reads were aligned to the reference sequence using the Map Reads to Reference function, and then realigned using the Local Realignment function. Sequence coverages were obtained by the locally realigned read files to SAMtools . The SAMtools . The conLow Frequency Variant Detection function. The threshold value of the error rate was set to 1% to avoid the false positive of variant detection. SNPs with a coverage depth of at least 10 were considered according to the default setting of the CLC Genomics Workbench. Genome-wide and site-specific nucleotide diversities (πS and πN) were calculated with SNPGenie (For detection of the single-nucleotide polymorphisms (SNPs) in the MNV isolates, the MNV S7 PP3 genome sequence (GenBank: AB435515.1) was used as a reference sequence. The frequency of SNPs was determined using the SNPGenie .We performed a regularized regression modeling to evaluate the significance of SNPs for the viral lime sensitivity using the NGS data and sensitivity data of the MNV isolates. We considered the following multiple linear regression model:y is the logarithm of the inactivation ratio [the log reduction values (LRVs)] of viruses at 4 min after exposure to lime solutions, β is a regression coefficient, and ε is the residuals. The vectors y and ε consist of n elements that correspond to the number of samples with a set of the LRVs and SNPs. The vector β consists of p + 1 elements that are allocated to p regression coefficients and one intercept. X is a n × (p + 1) design matrix that includes the frequency of SNPs at p polymorphic sites in n samples. We assumed that the value of βi indicated the contribution of the SNPs at site i in the viral tolerance to lime treatment. The Elastic Net (EN) method (β in Eq. 1 as follows:where ) method  was emplk-fold cross-validation to minimize the mean square value plus standard error (minMSE+1SE).where λ(λ > 0) is a regularizing parameter and α(0 < α < 1) is a penalty weight for regularization. The EN regression was performed by the glmnet package  on the Rp = 0.55 and p = 0.15 in the first round and the second round, by linear regression non-zero slope T-test; p = 0.0048 and p = 0.031 in the first and second round, by linear regression non-zero slope T-test; p = 2.5 × 10–5 and p = 5.1 × 10–6 in the first and second rounds, respectively, using the Mann–Whitney U test).Inactivation of the +lime and −lime populations is shown in S) and non-synonymous nucleotide diversity (πN) for the three protein coding genes  separately  accounted for over the 90% of the virus populations , C6289G (ORF2), T6605C (ORF2), and T6751C (ORF3). The consensus sequence of the ancestor population was used as the reference sequence for the SNPs detection from populations obtained in the serial passages.T561C and T7279C accounted for over 35% of the −lime populations . T561C aIn the +lime lineages, one synonymous SNP (A2519G) and one non-synonymous SNP (A6089G) arose in the populations with high frequency . In the A2519G and A6089G did not demonstrate parallel trajectories indicative of genetic hitchhiking. In the first round, the frequency of A2519G increased to 66.4% by passage 3, and, similarly, in the second round, the frequency of A2519G increased to 54.7% by passage 4 . The traThe frequency of A6089G and A2519G was associated with the greater tolerance of virus populations to the lime treatment, and thus, we hypothesized that those two SNPs contributed to the lime sensitivity of MNV. To verify this hypothesis, we increased the sample size by plaque isolation from the sensitive and less sensitive MNV populations and evaluated the association between the SNPs frequency and the LRVs of the isolates.p = 0.027 by Mann–Whitney U test; The LRVs were smaller in the isolate originated from the less sensitive populations  compared to the other isolates . The lesp = 0.004 by Mann–Whitney U test; The sole effect of A6089G on the lime sensitivity was verified by comparing the LRVs of infectious clones produced using a plasmid-based reverse genetics system (clone MNV) and ancestor virus used for the serial passage with lime treatment. Those two populations shared non-synonymous mutations on ORF2 (C6289G and T6605C) and ORF3 (T6751C), whereas clone MNV had a specific mutation on ORF2 (A6089G). The LRVs were significantly smaller in clone MNV than ancestor virus . Data obtained in the disinfection experiment suggested that viral populations evolved by different mechanisms in addition to bottleneck constraints, which led to different sensitivities to the lime treatment. The LRVs of the +lime populations decreased with the increase in frequency of A6089G and A2519G , 5. A seS and πN were compared between the +lime and −lime populations . If A6089G is maintained due to the presence of lime, then it may disappear from populations after additional passages without lime treatment although it needs to be confirmd in future studies. Future studies also need to assess cross-resistance with other disinfectants which has been demonstrated previously . Kadoya This study used MNV as a surrogate of human noroviruses which is of concern in fecal sludge treatment. Although we identify the mutation in the VP1, that is responsible for the decrease in lime sensitivity of an MNV population, it remains unclear whether human noroviruses acquire the lower sensitivity by the same mechanism . Because genetically diverse populations of human norovirus are observed in specimens and wastewater , we can DRA013422.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: DDBJ database with accession WO and DS designed the study and wrote the manuscript. WO performed the experiments, bioinformatics, and statistical analyses. RT-T and RI performed the experiments. KaK coordinated the research activity planning and execution. KH, KeK, and MS supervised the use of NGS and bioinformatic analysis. All authors critically revised the manuscript and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Garlic lost its ability to produce true seeds millennia ago, and today non-fertile commercial cultivars are propagated only vegetatively. Garlic viruses are commonly carried over from one generation of vegetative propagules to the other, while nematodes and arthropods further transmit the pathogens from infected to healthy plants. A recent breakthrough in the production of true  garlic seeds resulted in rapid scientific progress, but the question of whether viruses are transmitted via seeds remains open and is important for the further development of commercial seed production. We combined morpho-physiological analysis, fluorescence in situ hybridization (FISH), and PCR analysis to follow potyvirus localization and translocation within garlic fertile plants and seeds. Spatial distribution was recorded in both vegetative and reproductive organs. We conclude that garlic potyviruses are translocated to the seeds from the infected mother plant during flower development and post-fertilization, while pollen remains virus-free and does not contribute to seed infection. Therefore, the main practical goal for virus-clean seed production in garlic is the careful maintenance of virus-free mother plants. Although garlic pollen is free of potyviral infection, the male parents’ plants also need to be protected from contamination, since viral infection weakens plants, reducing flowering ability and pollen production. Allium sativum L., is one of the oldest vegetables in the human diet. Originating in East–Central Asia, this important crop and nutraceutical condiment is currently farmed worldwide, in many climatic zones, for its bulbs, green leaves, and young floral stalks [Garlic, l stalks . China, l stalks , providel stalks  due to ol stalks .Similar to other vegetatively propagated crops, most commercial garlic clones harbor numerous viruses, thus suffering 25 to 50% yield losses and reduced quality. The viral infection is further promoted by international trade and germplasm exchange . SimilarGarlic plants are commonly infected by the members of three virus families: Potyviridae (genus Potyvirus), Betaflexiviridae (genus Carlavirus), and Alphaflexiviridae (genus Allexivirus) . The mosIn the absence of seeds, only vegetative cloves and aerial bulbils (topsets) are used for garlic propagation. This vegetative propagation, however, suffers many disadvantages, such as a low multiplication rate and voluminous storage space. In storage, heavy losses may occur due to deterioration and loss of viability, sprouting, and pathogens infection. Moreover, vegetative propagation leads to the perpetuation of diseases, severe sanitary issues, and heavy losses in yield and quality ,13.In an attempt to reduce infection load, garlic propagation by aerial bulbils and biotechnological methods are becoming popular ,15,16. SIn the last decades, the breakthrough in fertility restoration and production of true garlic seeds resulted in rapid scientific progress on one hand and opened the way for advanced research, as well as breeding of modern and improved garlic varieties on the other . The queIn general, seed-borne diseases and the quality of seeds have a significant impact on the development and trade of crops . About 1The ability of viruses to infect male and female gametophytes is crucial in determining the virus transmission potential through the embryo . AdditioAllium species, OYDV is considered the most economically devastating pathogen [In the pathogen ,30. Althpathogen , its trapathogen ,33. Morepathogen ,35.If garlic true seeds transmit viruses, it is important to clarify their localization and what the sources of contamination are: is it the mother’s reproductive systems, the pollen, or both? In the case of surface localization, certain disinfection procedures can readily inactivate such external virus associations and their translocation to the germinating seedlings. However, where viruses infect inner tissues  , the eraWe hereby report on the presence of potyviruses both in the vegetative and reproductive plant tissues and in true garlic seeds. This is the first report on virus transmission in sexually propagated garlic.True garlic seeds harvested from various garlic accessions are kept at the Israeli Gene Bank. Selfed seed populations of the fertile accession #87 from the seasons of 2015–2017  were sow2 in 200 L containers filled with a mixture of 50% ground coconut shell: 20% volcanic tuff particles: 20 peat: 10% compost .A collection of fertile garlic clones is maintained in ARO, The Volcani Center, Rishon Lezion, Israel. In July 2021, freshly harvested bulbs of 12 of the fertile seed-producing accessions were harvested and stored under ambient conditions in a roofed open shed. Cloves were planted in November 2021 at 80 plants/mIn February 2022, the plants of 12 fertile accessions were screened for the presence of potyviruses. Sections of the fourth youngest leaf were collected from two plants of each accession, and both OYDV and LYSV were detected by using PCR. Two accessions, 3376 and 3379, were confirmed as infected by potyviruses and used for future analyses. Samples from three plants of these lines were collected at the stages of bulbil sprouting, mature leaves, young inflorescences, and individual young and mature flowers and seeds. In addition, at least 20 flowers, seeds, or seedling were pooled for the RNA extraction .Single-stranded DNA oligos of 24 nucleotides labeled with the fluorophore cyanine-Cy3 were used as a probe for RNA hybridization. The labeled probe (5′-TGC TGT GTG CCT CTC CGT GTC CTC-3′) was designed as a specific universal primer RS1 for the detection of conserved potyviruses’ genomic sequences, including LYSV and OYDV.Our initial attempts to perform virus analysis using published fluorescence in situ hybridization (FISH) protocols ,38 faile2O2 solution in EtOH for 1 h. Then the sampled tissues were pre-hybridized in hybridization buffer , 30% formamide) for 1 h, followed by overnight hybridization in an incubator equipped with an orbital shaker (25 rpm) at 25, 37, or 50 °C in a hybridization buffer containing the fluorescent probe (3 or 5 pmol/mL).All tissue samples were fixed overnight in Carnoy’s fixative , followed by two decolorization steps in 6% HThe hybridized samples were washed twice in sodium chlorides/sodium citrate (SSC) mixed with 0.1% SDS solution for 10 min, and once with a mixture of 0.5 × SSC and 0.1% SDS for 10 min at room temperature. Samples were stored in SSC at 4 °C, until studied under a Leica SP8 laser scanning microscope  that was equipped with solid-state lasers with 405 and 552 nm light, HCX PL APO CS 10 × /0.40 or HC PL APO CS2 20 × /0.75 objective , and Leica Application Suite X software . Then 4′,6-Diamidino-2-phenylindole (DAPI) and Cy3 emission signals were detected with PMT and HyD (hybrid) detectors in ranges of 415–490 and 560–660 nm, respectively.Control tissues were similarly treated, but the fluorescent probe was replaced by water.DAPI staining that indicates membrane viability is used extensively in fluorescence microscopy to stain both live and fixed cells. In this study, staining with 0.1 mM DAPI solution  was employed following RNA hybridization with Cy3 (see above) and examined under a confocal microscope.For plastic embedding, flowers fixed in FAA were dehydrated in a graded series of ethanol concentrations , followed by immersion in acetone and gradual replacement by LR-White resin . Following polymerization at 60 °C for 48–72 h, 2 µm–thick slices were obtained by using a rotary microtome (LeicaRM2245) and stained with 0.05% toluidine blue.For paraffin embedding, samples were fixed in FAA, dehydrated in alcohol series as described above, cleared with xylene, and embedded in paraffin, using Paraplast Plus tissue-embedding medium. Xylene was gradually replaced with paraffin (melting point of 58–60 °C) at 60 °C for 4 four days. Cross-sections of 12 μm–thick slices were cut, using a Leica RM2245 microtome , and stained with Safranin-Fast Green. The tissue slices were studied under a light microscope .Viral RNA was extracted from 100 mg of fresh tissues, using the AccuPreo Viral RNA Extraction Kit , according to the manufacturer’s instructions. The RNA concentrations were determined spectrophotometrically . The remaining RNAs served as templates for cDNA synthesis, using 1 µg total RNA, 0.1 µL random primer, and 0.9 µL oligo dT primer . The PCR reactions were performed in 20 μL, containing 1 μL of cDNA as template, 10 pmol of each primer, 0.2 mM of each dNTP, 2 mM MgCl2, 0.5 U of Taq DNA polymerase, and 1× PCR buffer, using a T-personal thermal cycler . PCRs consisted of an initial incubation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and 72 °C for 1 min. Final elongation at 72 °C was carried out for 10 min. PCR amplification products were visualized by electrophoresis in 1.0% agarose gels stained with ethidium bromide. The specific primers for LYSV and OYDV were previously reported , while tTarget amplification was confirmed by cloning and Sanger sequencing  of the PCR reaction products.To optimize the FISH protocol for garlic tissues, we hybridized Cy-3 probe at 3 or 5 pmol/mL overnight at 25, 37, or 50 °C. The fluorescent signals were highly visible when 3 pmol/mL Cy-3 was hybridized at 50 °C . Hence, In January 2022, we screened twelve selected garlic fertile clones and three seed populations for the presence of potyviruses. Plants of two vegetatively propagated accessions, 3376 and 3379, were identified as infected.Allium-specific Allexiviruses, which are considered harmless to the crops despite the induced mosaic and ringspot symptoms [Allium species and the transmission of Allexiviruses in vegetative and reproductive organs.In the next PCR analysis, we tested specific tissues  of thosesymptoms ,40. Allesymptoms , thus suCarlaviruses were detected only in one young inflorescence and in one seed population, while the most harmful and dangerous potyviruses, LYSV and OYDV, were detected in all vegetative and reproductive organs of the selected clonally propagated genotypes 3376 and 3379. Seeds and seedlings also contained potyviruses. Interestingly, seeds from the 2016 harvest did not contain potyviruses, but their presence was detected in two other seed populations .The results of PCR analysis of LYSDV, OYDV, GarV-D, and GCLV were cross-validated with Sanger sequencing.Individual garlic flowers comprise perianth, androecium, and gynoecium. The androecium consists of six stamens composed of a filament and a bi-lobed anther; each lobe contains two pollen sacs A. When tThe gynoecium develops in the center of the flower and consists of ovary and style. The ovary includes the carpel, divided into three locules, containing two ovules in each locule C,D.The FISH analyses during the vegetative and reproductive development of garlic revealed potyviruses in foliage leaves, floral stem, floral meristem, and developing flowers . In seedWith flower development and maturation, the signals of viral RNA became stronger in the female reproductive tissues—ovary, style, and stigma A,B. In tSeed infection, including virus presence inside and/or on the seed coat, may result from either the maternal or paternal parent, or both . Since aStudies on bulb onion  suggesteAllium crystallinum, A. filidens, and A. fritschii, which, like garlic, belong to the subgenus Allium, the epidermis of the mature anther consists of large bladder-shaped cells with strongly thickened outer walls covered by a toothed cuticle [In many plant species, viruses can be transmitted by infected pollen both vertically and horizontally . This is cuticle . It is nAnother explanation for virus-free pollen in the infected plant can be a possible protection mechanism(s) during pollen differentiation. In general, the systemic viruses’ movement within the plants through vascular bundles or through plasmodesmata is protected by the capsid proteins. Since viruses are too large to move through unmodified plasmodesmata, they employ movement proteins dedicated to the enlarging of pores for the active transport of viral RNA ,50,51. UVirus translocation and localization within the seed varies with plant species. For instance, in infected cucumber and melon plants, the fluorescence signal of Tobamovirus cucumber green mottle mosaic virus (CGMMV) was recorded in flowers, both before and following anthesis. Later, the virus was detected in the perisperm, endosperm envelope, which underlies the seed coat and surrounds the embryo . SimilarPlant viruses and other pathogens are transmitted both horizontally and vertically. Similar to other vegetatively propagated crops  , garlic Allium species . While Allexivirus and Carlaviruses are not reported as devastating pathogens, their interaction still can weaken garlic plants and reduce crop productivity [Further research is required to assess the transmission of the other viral groups via the sexual reproductive system of garlic and elucidate the reason for the significant difference between various uctivity .Our observations suggest that the sexual progeny remains vigorous and highly productive for at least four or five years, despite the present findings on virus transmission via seed. Possibly, since seeds are free from other pathogens, such as fungi and some bacteria, sexually propagated plants are able to withstand viral infection.In conclusion, garlic potyviruses are translocated from the infected mother plant during flower development or post-fertilization, while pollen remains virus-free and does not contribute to seed infection. Therefore, the main practical goal of virus-clean seed production requires careful maintenance of virus-free mother plants. Although garlic pollen is free of potyviral infection, the male parents’ plants also need to be protected from contamination, since viral infection weakens plants, reducing flowering ability and pollen production.


Potato is a major food crop that has the potential to feed the increasing global population. Potato is the fourth most important crop and a staple food for many people worldwide. The traditional breeding of potato poses many challenges because of its autotetraploid nature and its tendency toward inbreeding depression. Moreover, potato crops suffer considerable production losses because of infections caused by plant viruses. In this context, RNA silencing technology has been successfully applied in model and crop species. In this review, we describe the RNA interference (RNAi) mechanisms, including small-interfering RNA, microRNA, and artificial microRNA, which may be used to engineer resistance against potato viruses. We also explore the latest advances in the development of antiviral strategies to enhance resistance against potato virus X, potato virus Y, potato virus A, potato leafroll virus, and potato spindle tuber viroid. Furthermore, the challenges in RNAi that need to be overcome are described in this review. Altogether, this report would be insightful for the researchers attempting to understand the RNAi-mediated resistance against viruses in potato. Solanum tuberosum L.) is ranked fourth worldwide among all food crops in terms of its total production, next to rice, wheat, and corn. Potato continues to be recommended as an important component of diversified cropping systems, especially in the face of the current population expansion, to overcome problems in food supply, nutrition, and food security as well as global climate change challenges [Potato  were first described by Hamilton and Baulcombe in 1999, who confirmed that naturally occurring siRNAs in plants can cause post-transcriptional gene silencing . Plant vRNA silencing  provides an antiviral immune system that can be applied to plants and has attracted increasing attention in plant–virus interaction studies ,8,9. RNAIn the RNA silencing pathway, dsRNA is cleaved into fragments of 18–27 nucleotides and non-coding interfering RNAs, such as siRNAs and microRNAs (miRNAs). Both siRNAs and miRNAs may be associated with various plant proteins to form the RNA-induced silencing complex (RISC) ,15,16.In RNA-containing plant viruses, the dsRNA structures of viral RNAs are processed by Dicer-like proteins into long (24–26 nt) and short 21–23 nt) siRNA fragments ,18. Doub–23 nt siMIR genes by RNA polymerase II (Pol II), have a hairpin-like structure consisting of a terminal loop, an upper stem, the miRNA/miRNA* region (miRNA* refers to the passenger strand), a lower stem, and two arms [miRNAs are a class of short non-coding RNAs that contain approximately 20–24 nucleotides and are derived from endogenous precursor transcripts containing double-stranded  regions in plants ,24. Primtwo arms . Short ptwo arms . Pre-miRtwo arms . The guitwo arms .Artificial microRNAs (amiRNAs) are a class of artificial small RNAs that have been engineered to silence specific transcripts in plants. amiRNAs consist of two components: a pri-miRNA scaffold and an siRNA insert. This pre-amiRNA is processed via a series of DCL1 slicing events to generate mature amiRNA/amiRNA* duplexes . The matIn this review, we have focused on siRNA- and miRNA-based strategies for the engineering of virus resistance in potatoes, and we present an overview of the RNA-silencing-mediated control of plant viral disease in potato, e.g., potato virus X (PVX), potato virus Y (PVY), potato virus A (PVA), potato leafroll virus (PLRV), and potato spindle tuber viroid (PSTVd). Because of the large number of publications on RNA silencing, a summary of this topic is provided in Potexvirus and a positive-strand RNA virus that infects potatoes [Nicotiana benthamiana plants transgenic for P25 are less susceptible to PVX [PVX is a type member of the genus potatoes ,43. Althpotatoes ,45. PVX potatoes ,47. Moree to PVX .AS) constructed by Faivre et al. [Solanum species. This silencing state can be transmitted and detected for several generations via vegetative propagation.The development of PVX-based virus-induced gene silencing (VIGS) vectors in transgenic plants was central to the suppression of silencing; furthermore, VIGS triggers an RNA-mediated defense mechanism that directly targets the integrity of the invading viral genome . The PVXe et al.  was demoAgrobacterium infiltration bind directly to the endogenous miRNA and block its function [Another study has reported that virus X-based miRNA silencing (VbMS) vectors silence endogenous miRNAs in potato. The researchers designed the target mimic molecules of miRNA and demonstrated that the VbMS vectors expressed in potato (cv. Katahdin and Russet Burbank) by function .Potyvirus belonging to the family Potyviridae and is one of the most important plant viruses affecting potato production [N, PVYO, and PVYNTN).PVY is an aphid-borne virus of the genus oduction . PVY sproduction . Controloduction ,53. Petroduction  proposedoduction  expresseThe combination of transgene and transcription strength is functional for constructing efficient resistance to viruses. The data generated from high-throughput sequencing (HTS) can be analyzed to identify the regulatory micro RNAs (miRNA) that are involved in gene regulation as well as the small inhibitory RNAs (siRNA) that confer virus resistance. Potato cv. Atlantic and Ranger Russet were transformed with an inverted hairpin RNA (ihRNA)-generating construct, with the tail-to-tail repeat of the PVY coat protein (PVY-CP)-coding region set apart, and HTS data indicated high levels of siRNA production from the transgenic hairpin construct .Potyvirus within the picorna-like supergroup of single-stranded, positive-sense RNA viruses [PVA is a member of the genus  viruses ,55. Alth viruses . The vir viruses ,55. VPg  viruses .The PVA-CP has an intrinsic capacity to self-assemble into filamentous virus-like particles. However, the mechanism responsible for the initiation of viral RNA encapsidation in vivo remains unclear. In addition to virion assembly, PVA-CP is also involved in the inhibition of viral RNA translation .Chuang et al.  documentPolerovirus within the Luteoviridae family, causes a yield loss of >20 million tons of infected potatoes every year [PLRV, which is a member of the genus ery year ,59,60. Iery year . Kumari ery year  designedCP gene expression in potato. Potato plants that were agroinfiltrated with siRNA constructs against a CP containing an ATPase domain exhibited no rolling symptoms upon PLRV infection. This finding further confirmed that the silencing of CP gene expression is an efficient method for generating PLRV-resistant potato plants [CP and MP genes, ORF2b of PLRV could also confer resistance to PLRV via a PTGS mechanism, and grafting infection experiments showed that resistant transgenic plants could be obtained in this way [Recently, Kumar et al. used the RNAi approach to prevent PLRV genome packaging and assembly via the suppression of o plants . In addithis way .Orbegozo et al.  reportedPospiviroidae. PSTVd alone or in combination with other viruses can decrease the yield of susceptible potato varieties from 40% to 70% [PSTVd is a member of the viroid family % to 70% . Symptom% to 70% .StTCP23, which encodes a potato transcription factor derived from an siRNA of PSTVd, could cause PSTVd disease. The authors demonstrated that the expression of StTCP23 is repressed and accompanied by StTCP23 transcript cleavage within the identified region of complementarity in PSTVd-infected potato. The authors also claimed that the expression of virulence-modulating-region sequences as 21-nt amiRNAs of StTCP23 in potato resulted in reduced StTCP23 transcript abundance and PSTVd-like disease symptoms [RNAi-based strategies have been successfully applied for the development of plants that are resistant to PSTVd. Bao et al. reported that the silencing of symptoms .The use of RNAi may be an effective approach for engineering crop resistance to a broad range of viroids. Schwind et al. reported that transgenic plants expressing an hpRNA construct derived from PSTVd sequences exhibited resistance to PSTVd infection and that hpRNA-derived siRNAs (hp-siRNAs) could target the mature viroid RNA and prevent its replication . FurtherCarbonell et al.  suggesteORF2 gene of PVX, the HC-Pro gene of PVY, and the CP gene of PLRV, was constructed to generate transgenic potatoes of cultivars Kuroda and Desiree, with 20% of the transgenic plants being immune to all three viruses because of simultaneous RNA silencing. Other studies have reported a single chimeric hairpin that was expressed in a cassette of fused viral coat protein-coding sequences from PVX, PVY, and PVS as a 600-bp inverted repeat to generate dsRNAs having a hairpin loop configuration. Compared with the severe viral disease symptoms observed in the untransformed controls, the transgenic potato lines (cv. Desiree) showed nearly 100% resistance against PVX, PVY, and PVS infections simultaneously [In potato, the simultaneous coinfection of multiple viruses may result in pronounced disease phenotypes and lead to crop failure because of synergistic or antagonistic interactions among the viruses. For example, the yield loss can reach 55% under PVX mixed infection with PVY, whereas a single infection with PVX only causes a 15–30% yield loss . An RNA aneously .Moreover, the endogenous tRNA-processing system has been used to generate multiple amiRNAs from tRNA-pre-amiRNA tandem repeats for multiplex gene silencing . An amiRArabidopsisthaliana was modified to express an amiRNA construct targeting P25, HC-Pro, and Virp1 of PVX, PVY, and PSTVd, respectively. The amiRNA construct was stably transformed in potatoes. Viral challenge assays revealed that these transgenic plants exhibited resistance against PVX, PVY, and PSTVd coinfection simultaneously, whereas the untransformed controls developed severe viral disease symptoms. These results indicated a strategy to engineer resistance to several viruses simultaneously via the c-expression of multiple amiRNAs (unpublished).In our recent study, the miRNA159 backbone precursor from RNAi is a direct target of RNA silencing mechanisms that is used to regulate gene expression, control developmental processes, and maintain genome integrity. Moreover, hpRNA-based constructs targeting viral RNAs have proven to be superior to previous transgenic approaches for generating resistance in plants against viruses ,73. The CP gene, which is an important part of the viral structure [Viral assembly may be prevented by interfering with the expression of the tructure . As is ktructure . TherefoSelective gene silencing, albeit without absolute specificity, is offered by the RNAi-mediated strategy, and transgene-derived siRNA-based silencing of host genes based on sufficient nucleotide sequence complementarity may represent an off-site targeting risk . TherefoamiRNA-mediated gene silencing has a high degree of specificity and effectiveness and enables genome stability and hereditability of phenotypes. Moreover, it has become one of the most important tools in genetic engineering currently. However, different resistance levels were observed when the amiRNAs targeted different viral functional genes or were allocated at different positions in the same functional gene.When devising strategies for expressing amiRNAs in plants, it may be difficult to stably suppress a target virus gene because of the rapid mutation rate of viruses. Hence, it is essential to target highly conserved regions of the viral genome and express more than one amiRNA sequence in one vector to avoid viral escape resulting from the emergence of evading mutations. It has been proven that the coexpression of multiple antiviral amiRNAs is effective in several viral pathosystems. Despite the successful generation of multiple-virus-resistant plants, immense time would be necessary to generate such amiRNA constructs.Modern genome-editing techniques, such as the clustered regularly interspaced short palindromic repeats/CRISPR associated 9 (Cas9) (CRISPR/Cas9) and Cas13a/sgRNA  system, have extended the possibilities of engineering virus resistance in plants ,81. CompAlthough the CRISPR technology may represent new technologies to engineer antiviral resistance and in some cases rivals or exceeds the RNAi technologies, it has several disadvantages. Firstly, CRISPR-Cas components can only be carried through transgenic expression, whereas RNAi-based technology is transgene-free; secondly, genome-editing techniques are only feasible within a limited range of potato varieties and it needs a longer time select the edited plants (months or years); thirdly, CRISPR technologies induce permanent gene edits in potato, while RNAi induces transient knockdown of gene expression. Therefore, RNAi approaches remain the main tool for targeting viral pathogens in potato ,84.Finally, RNAi will likely continue to be used in potato crop research and development for assessing the function of specific genes because of its high specificity. Providing long-term protection against the targeted viral pathogen to plant cells is a critical future research goal in this field.


Solanum tuberosum through the stimulation of RNA interference (RNAi)-based antiviral defenses. Expression cassettes carrying inverted repeats of potato virus S  movement or coat protein sequences were used for the transformation of potato cultivars naturally infected with PVS and/or a related carlavirus potato virus M (PVM), without or with potato virus Y . A high proportion of transformants PCR-positive for transgenes were cured from both carlaviruses and PVY. After 3-year field trials, 22 transgenic lines representing seven cultivars remained free of any virus or became infected only with PVY. Vegetative progenies of the transgenic lines of cultivar Zeren , sampled after in vitro propagation or field trials, and other field cultivars accumulated transgene-derived 21, 22, and 24 nt small interfering (si)RNAs almost exclusively from the PVS inverted repeats. Additionally, some field progenies accumulated 21–22 nt siRNAs from the entire PVY genome, confirming PVY infection. Taken together, transgenic RNAi is effective for virus elimination from naturally infected potato cultivars and their sequence-specific immunization against new infections.Tissue culture methods enable virus elimination from vegetatively propagated crop plants but cannot prevent new infections. Here we used a tissue culture transgenic approach for curing field cultivars of  Solanum tuberosum) is the world’s third most important food crop after wheat and rice. A large proportion of produced potato tubers are also used as livestock feed and as seed material [Potyvirus, family Potyviridae), potato leaf roll virus , potato virus X , and potato viruses S and M  [Cultivated potato  ,4.In Kazakhstan, the carlaviruses PVS and PVM are the most widespread and economically important potato viruses, followed by PVY and PVX . PVS is PVS has a ~8.5 Kb positive-sense single-stranded RNA genome that contains six conserved open reading frames (ORFs) encoding a 223 KDa (K) RNA-dependent RNA polymerase (RdRP) mediating viral genomic RNA replication; triple gene-block (TGB) proteins TGBp1 (25K), TGBp1 (12K), and TGBp3 7K) mediating cell-to-cell movement; a 34K coat protein (CP); and an 11K terminal cysteine-rich nucleic acid binding protein (NABP) implicated in suppression of RNA interference (RNAi)-based antiviral defenses [K mediatiMeristem culture, thermotherapy, electrotherapy, cryotherapy, and chemotherapy and their combinations are the main methods to eliminate viruses from potato plants. These methods are sufficiently effective, but none of them protects the cured plants from new infections . IdentifRNAi is a conserved mechanism that regulates gene expression and defends against invasive nucleic acids such as transposons, transgenes, and viruses in most eukaryotes ,13,14,15Boosting natural RNAi-based resistance to viral infection can be achieved by transgene expression of viral genome fragments in sense or antisense orientation, or as inverted repeats separated by a spacer or an intron. In the latter case, a so-called intron-hairpin RNA transcribed by RNA polymerase II (Pol II), after splicing and formation of dsRNA structure, is directly processed by DCL4, DCL2, and DCL3 into 21, 22, and 24 nt viral siRNAs, resulting in complete (or near-complete) immunity to cognate/target virus infection . NotablSolanum tuberosum from natural infections with PVS and/or PVM, or their mixed infections with PVY, and thereby obtained transgenic plants with durable resistance to carlavirus infection.In this study, we tested an intron-hairpin RNA-based stable transformation method to cure field cultivars of Ricinus communis catalase gene cat1 intron I. In the resulting constructs, the inverted repeats of CP or 25K sequences, separated by the intron, were inserted between the cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthase (NOS) terminator  with the parameters 25 mF, 1.8 kV, and 200 Ohm. Transformed cells were selected on standard Luria-Bertani medium with kanamycin (50 mg/L) and then were used for stable transformation of potato plants.Total RNA was extracted from PVS (Kazakh isolate MK442089)-infected potato leaves, using TRI Reagent , according to the manufacturer’s protocol. Reverse transcription (RT) was performed by using 5 μg total RNA, oligo(dT)18 primer, and Maxima Reverse Transcriptase  according to the manufacturer’s protocol. Then 884 and 732 nt coding sequences of the PVS CP and 25K (TGBp1) proteins, respectively, were amplified by PCR , using the PVS sequence-specific primers  containirminator . The bin405nm and A492nm) were recorded using EL311 Microplate Autoreader  after sample incubation with substrate for 1 h at room temperature (22 °C), with 3 repeats for each sample.Double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) analysis of leaf samples of potato plants cultivated under field or greenhouse conditions was carried out for detection of PVS, PVM, and PVY, using BIOREBA kits (Switzerland) according to the manufacturer’s protocol. Absorbance values  in the standard Murashige and Skoog (MS) salts containing liquid medium supplemented with 2% sucrose for 30 min and then placed on the solid callus medium  for 2 days for callus formation. The explants with calli were placed on the solid callus medium supplemented with 0.5 g/L cefotaxime for two weeks to eliminate agrobacteria. Then the explants were placed on the solid morphogenesis medium containing standard MS salts, 1.6% glucose, 40 mg/L adenine, 5 mg/L calcium pantothenate, 1 mg/L biotin, 1.5 mg/L trans-zeatin, 0.5 mg/L gibberellic acid, 0.7% plant agar, 0.5 g/L cefotaxime, and 25 mg/L kanamycin and cultivated for shoot regeneration. The regenerated shoots of 0.7–1.0 cm in size were cut and placed in vitro on the standard MS medium supplemented with 2.6% sucrose, 1 g/L glycine, 0.8% plant agar, and 0.25 g/L cefotaxime for rooting and further growth under sterile conditions.Stem internode explants of virus-infected potato plants were transformed by co-cultivation with cat1 intron-specific primers   .www.fasteris.com), using TruSeq small RNA protocol for library preparation and multiplexing 28 libraries (including 5 potato libraries ALYU-75-79 and 23 non-potato libraries) in one SP flowcell of NovaSeq 6000, which yielded for the potato libraries 31,627,721 to 35,869,294 reads with Q30 = 94.42 to 94.66. The libraries were de-multiplexed, followed by adapter trimming. The resulting reads, most of which were within a 20–25 nt size range, containing functional plant miRNAs and siRNAs, as well as viral siRNAs , using Burrows–Wheeler Aligner (BWA) 0.7.12 and scaffolding non-plant contigs with SeqMan module of Lasergene DNASTAR software suite 7.1.0. The SeqMan contigs were manually corrected (if needed) and verified by mapping back redundant 20–25 nt reads and analyzing the mapping results with MISIS-2 [RNA integrity was verified by capillary electrophoresis on LabChip GX (Perkin Elmer). Illumina sequencing was performed at Fasteris AG  and PVY , and the intron-hairpin RNA construct expression cassette S-25K-AS  reads from each library were mapped onto reference sequences of the S-25K-AS .Eleven potato cultivars naturally infected with up to three different viruses, including at least one carlavirus (PVS or PVM), as evaluated by ELISA , were trA total of fifty PCR-positive potato transformants (lines) grown up in vitro were placed in pots and cultivated under greenhouse conditions for six months . Every tRemarkably, in the case of the cultivar Zeren, which was initially infected with PVS, PVM, and PVY, two of the five PCR-positive lines carrying 25K transgenes were free of any virus. Likewise, PVY infection was eliminated from three of the nine lines of Picasso and from all three lines of Sante, but it remained to be detectable in all eight lines of Albinka .Tubers obtained for PCR-positive transformant lines of the cultivars Albinka (n = 8), Dunyasha (n = 11), Fortuna (n = 3), Kormilitsa (n = 1), Picasso (n = 11), Sante (n = 3), and Zeren (n = 5), as well as progeny tubers of the respective non-transformed parental plants and three additional cultivars, were taken for 3-year field trials in 2017, 2018, and 2019 . Each liIn 2018, tubers from 30 transgenic lines (including 13 virus-free lines) from the first season were again planted in the field . By the In 2019, field trials performed for 29 PCR-positive lines from the second season revealed that most of them were free of PVS (n = 23) and PVM (n = 27), but they were infected with PVY (n = 19) . No viruIn conclusion, the tissue culture transgenic RNAi approach was found to be effective for elimination of both target (PVS) and non-target (PVM) carlaviruses from naturally infected potato cultivars with subsequent resistance to those two carlaviruses under the field conditions over 3 years of trials.Tubers of three transgenic lines carrying the S-25K-AS construct and representing the cultivars Dunyasha (#61) and Kormilitsa (#103), both being ELISA-negative for virus infection after the third year of trials, and the cultivar Zeren (line #119), which scored ELISA-positive for PVY alone after the third year of trials , were coThe small RNA blot hybridization analysis using a 22 nt DNA probe complementary to the PVS 25K antisense strand revealed that all the transgenic lines produce transgene-derived siRNAs of ca. 21–22 and 24 nts in size, with the in vitro line #119 accumulating lower levels of siRNAs than the field lines #119, #61, and #103, all carrying the same transgene , Lane 1.To reconstruct virome components (if any) present in each leaf sample, we used a previously developed pipeline for de novo assembly of viral genomes from siRNAs ,29 see . As a reTo understand the biogenesis and function of intron-hairpin RNA transgene-derived siRNAs, we performed a bioinformatic analysis of their size, polarity, 5′-nucleotide identity. and hotspot profiles, in comparison with the respective profiles of virus (PVS and PVY)-derived siRNAs and plant sRNAs .Our analysis of single-nucleotide resolution maps and counts of 15–34 nt sRNAs revealed that, in each of the four transgenic plant samples, transgene-derived sRNAs are produced almost exclusively from the inverted repeats of the PVS 25K coding sequence and belong predominantly to the 21 nt class, followed by less abundant 22 and 24 nt classes . For eacOur analysis of the 5′-terminal nucleotide identity of transgene-derived siRNAs revealed that 21 and 22 nt classes are enriched in 5′U (39 to 76%), followed by 5′A (14 to 41%) and 5′C (4 to 17%) , suggestBased on these findings that are generally consistent with those previously reported for intron-hairpin RNA transgenes ,31,32,33Begomovirus) [Negligible amounts of sRNAs derived from the transgene expression cassette outside of the PVS 25K inverted repeats  indicate limited involvement (if any) of RDR-dependent production of secondary siRNAs and transitive  silencing, thus explaining the robust production of transgene-derived siRNAs over three years of field trials and their durable protective effect. This is consistent with the results of field trials of intron-hairpin RNA transgenic tomato plants that exhibited immunity to tomato yellow leaf curl virus (genus movirus) .Our analysis of 15–34 nt sRNAs from the non-transformed Zeren plant (ALYU-75) coinfected with PVS and PVY revealed that viral siRNAs are derived from both strands of the entire PVS and PVY genomes and belong predominantly to the 21 nt and less abundant 22 nt classes, likely produced by DCL4 and DCL2, respectively, while other size classes accumulate at lower levels and are derived mostly from the sense strand , suggestMapping of sRNAs from the transgenic plants  onto the PVS genome reconstructed only from the non-transgenic plant (ALYU-75) revealed that they match exclusively to the PVS 25K coding sequence , thus coNotably, counts of the PVS 25K transgene-derived siRNAs in the Zeren line #119 from the field (ALYU-76) and the whole PVS genome-derived viral siRNAs in the non-transformed Zeren plant from the field (ALYU-75) were comparable: 6617 and 6066 reads per million (RPM) of total 20–25 nt sRNAs, respectively  . Given tConsistent with sRNA blot hybridization results, the in vitro propagated transgenic Zeren line #119 accumulated lower amounts of transgene-derived siRNAs  than the field progeny of this line, whereas the field progenies of transgenic Dunyasha and Kormilitsa lines #61 and #103 accumulated higher amounts of transgene-derived siRNAs . Since the Dunyasha line #61 is free of any virus, while the Kormilitsa line #103 is infected with PVY, PVY infection does not appear to interfere with production of transgene-derived siRNAs. This is despite the fact that the accumulation levels of PVY-derived viral siRNAs are much higher than those of PVS- and transgene-derived siRNAs . A high Our finding that intron-hairpin RNA transgenes had curing and protective effects not only against the target carlavirus PVS but also a non-target carlavirus (PVM) is consistent with the results of a previous study in which one of the cucumber lines expressing the highest amounts of intron-hairpin RNA transgene-derived 21, 22, and 24 nt siRNAs exhibited resistance to infections with both a target potyvirus and two non-target potyviruses that share 67% and 63% identity with the transgene sequence . Our ana


Real‐time polymerase chain reaction was used to detect 22 distinct viruses from collected samples. The overall prevalence of herpesviruses was 89% and respiratory viruses 94%. Human herpesviruses 6 (HHV6), 7 (HHV7), and Epstein–Barr virus (EBV) were found, respectively, in adenoids , tonsils , and NPA . Copy numbers of the HHV6 and HHV7 genome were significantly higher in tonsils than in adenoids. Patients with intra‐adenoid HHV6 were younger than those without. Detection rates of EBV and HHV7 showed agreement between corresponding sample types. This study shows that adenoid and tonsil tissues commonly harbor human herpes‐ and respiratory viruses, and it shows the differences in virus findings between sample types.Adenoids and tonsils have gained interest as a new in vivo model to study local immune functions and virus reservoirs. Especially herpesviruses are interesting because their prevalence and persistence in local lymphoid tissue are incompletely known. Our aim was to study herpesvirus and common respiratory virus infections in nonacutely ill adenotonsillar surgery patients. Adenoid and/or palatine tonsil tissue and nasopharyngeal aspirate (NPA) samples were collected from elective adenoidectomy ( The primary infections are generally asymptomatic or induce mild symptoms. Herpesviruses escape the host's immune system and persist for life. HHVs can cause severe infections and predispose immunocompromised hosts to end‐organ disease and lymphomas.There are several studies concerning respiratory viruses of tonsil and adenoid tissue or sole herpesviruses of tonsil and adenoid tissue. However, there are limited data of persistent herpesvirus and respiratory virus infections in autologous adenoid, tonsil, and NPA samples.22.12.1.1NPA samples, adenoid, and/or tonsil tissue samples were collected from 89 elective adenoidectomy or adenotonsillectomy patients at Satakunta Central Hospital in Pori, Finland, between April 2008 and March 2009. Inclusion criteria were adenoidectomy or adenotonsillectomy according to clinical indication and written informed consent from the study subject or his/her guardian.2.2The internal part of the tonsil and/or adenoid tissue removed from the patient was instantly cut into 3–4 mm cubes and stored in RNAlater (Qiagen), first at +2–8°C until the next working day, and then at −80°C.2.3The primary outcome of this study was to analyze the presence of different human herpesviruses and respiratory viruses in adenoid tissues. The secondary outcome was to compare common herpes‐ and respiratory virus findings between adenoid, tonsil, and NPA samples.2.4Viruses in adenoid tissue, tonsil tissue, and NPA were detected by polymerase chain reactions  on nucleic acid extracts.For respiratory virus detection in all sample types, laboratory design real‐time PCRs were used for HBoV1, EV, RV, and RSV, as described earlier.2.5U test due to skewed distribution. Cohen's Kappa was used to determine agreement between most common intratonsillar, intra‐adenoid, and NPA viruses. Agreement statistics between results from PCR tests from intra‐adenoid, intratonsillar, and NPA virus diagnostics were calculated with kappa coefficients and could be interpreted as follows: <0.40 = poor, ≥0.40 to <0.75 = fair to good, and ≥0.75 = excellent.p < 0.05.Data were analyzed using SPSS software Statistics for Windows, version 27.0 (IBM SPSS Statistics for Macintosh). Categorical variables are expressed as frequencies and percentages and were analyzed by using a Chi‐square test or Fisher's exact test (when counts <5). Continuous variables are described as medians and were analyzed using the Mann–Whitney 33.1Initially, 200 adeno‐ and/or tonsillectomy patients were consecutively recruited year‐round. Of these study patients, 45 received adenoidectomies and 44 had adenotonsillectomies, and they were included. Sole tonsillectomy patients (111) were excluded from this study since their tonsil samples have been utilized and studied earlier.3.2n = 89) was 5 years (range: 1–20); 62% were male. The most common indication for the operation was adenoid and/or tonsillar hypertrophy in 43 (48%). There were mixed indications  in 33 (37%) cases. Mild upper respiratory tract symptoms such as rhinitis, cough, throat pain, otalgia, and upper respiratory tract obstruction during the operation day were reported by 24/77 (31%) of the study patients (Supporting Information: Table The median age of the study patients (3.3Ninety‐seven percent (86/89) of the adenoid tissue samples were positive for ≥1 viruses. At least one intratonsillar virus was detected in 95% (42/44) of adenotonsillectomy patients. Moreover, one or more viruses were detected in 94% (84/89) of the NPA samples (Table 3.43) of HHV6 and HHV7 were significantly higher in tonsil tissue compared to adenoid tissue . EBV genome levels were not significantly different in adenoid and tonsil tissues. In one case, the EBV virus DNA load was 210 000 copies/mm3 in tonsil tissue, which could be considered as a marker for acute infectious mononucleosis or reactivation of the virus (Table p = 0.017)  of the adenoid samples, followed by HHV7 26%) and EBV (25%). Only one adenoid sample was positive for HSV1. VZV and HSV2 were not detected (Table 6% and EB3% of the3.5p = 0.001 and p = <0.001). Additionally, patients with a positive adenovirus finding in intra‐adenoid tissue were younger than those having a negative PCR test . An HBoV1‐negative result was more often associated with male gender than female gender (p = 0.019). No other associations between age or gender and virus findings in adenoid or tonsil tissue were found.RV (60%) was the most prevalent respiratory virus in adenoid tissue samples followed by HBoV1 (58%). Other respiratory viruses detected in adenoid tissue were parainfluenza viruses 1–4 13%), adenovirus (12%), and others (<10% each). In tonsils, HBoV1 (23%) and adenovirus (18%) were the two most prevalent respiratory viruses. Moreover, in NPA samples RV (63%) was the most common respiratory virus . Similarly, detection of EBV in adenoids agreed strongly with detection of the virus in the corresponding tonsils . Furthermore, EBV detection showed agreement between adenoid and NPA samples  and between NPA and tonsil samples . Of the two most commonly detected respiratory viruses, HBoV1 and RV, HBoV1 was more frequently detected in adenoids than in tonsils  or NPAs . RV was more frequently detected in adenoids and NPA than in tonsils  and respiratory viruses in adenoid, tonsil, and NPA samples. Our study shows a few essential findings of prevalent virus infections in adenoid and tonsils of nonacutely ill adenotonsillar surgery patients. First, overall herpesvirus prevalence was high. HHV6, HHV7, and EBV were commonly found in adenoids and tonsils. Additionally, there were observed agreements in the detection of HHV7 in adenoids and corresponding tonsils and EBV in all sample types. Second, higher herpesvirus genome loads were found in tonsils compared to adenoids. Only detection of HHV6 was associated with age. Third, RV and HBoV1 were the most frequently detected respiratory viruses overall. For neither virus were the detections in corresponding samples from different sites connected.At least one virus was found in 97% of the adenoid samples, 95% of the tonsil samples, and 94% of the NPA samples. In an earlier study concerning only respiratory viruses, the detection rates in the same sample types were 86%, 69%, and 79%, respectively.HHV6 was the most prevalent (33%) intra‐adenoid herpesvirus, and the prevalence was similar compared to earlier studies.HHV7 was the most common virus detected in tonsils. HHV6 and HHV7 virus DNA levels were significantly higher in tonsil tissues compared to adenoid tissues. Our study agrees with earlier results concerning HHV7, but contradicts those of HHV6.In the present study, the prevalence of EBV was similar in all corresponding samples compared. Similar results have been seen in an earlier study comparing EBV DNA load between adenoids and tonsils.It is known that 90% of people are infected by EBV. In Finland and Western countries, 50%–60% of primary EBV infections occur in childhood without symptoms or with mild symptoms. In adolescents and young adults EBV can cause acute mononucleosis. Typical symptoms and findings include fever, sore throat, and swollen and exudative tonsils.In our study, CMV was detected in NPA but not in tonsils and only in one adenoid sample. Our results are in line with the SPLIT study, but another study has detected CMV in adenoids and tonsils.The prevalence of common respiratory viruses in adenoid, tonsils, and NPA is well known.HBoV1 was the most prevalent respiratory virus in tonsil tissue (23%), and the frequency of HBoV1 was also high in adenoid tissue and NPA samples. Detection of HBoV1 or RV did not show agreement between sample types, which is in line with earlier results.The strength of this study is the characterization of common herpesviruses and respiratory viruses in three different sample types obtained from the same patient. The present study shows that many different viruses are prevalent in secondary lymphoid tissue in chronic adenotonsillar patients. Adenoid and tonsil tissues serve as reservoirs for multiple herpes‐ and respiratory viruses. Since certain viruses have the potential to modulate immune functions and associate with carcinogenesis, our results offer interesting knowledge of virus prevalence in active secondary lymphoid tissues.The limitations of the study include that we did not study healthy control patients for ethical reasons. Additionally, the respiratory virus tests from tonsils and NPA were done earlier than those from adenoids using a test kit version, which was later replaced with a new test kit by the manufacturer. Virus infections were not studied at the cellular level.5This study shows high overall virus prevalence and characterizes the wide spectrum of different herpesviruses and common respiratory viruses in adenoid and tonsil tissues of nonacutely ill adenotonsillar surgery patients. Furthermore, it shows the differences in herpesvirus prevalence between adenoid, tonsils, and NPA sample types. Adenoid and tonsil tissues may serve as virus reservoirs in nonacutely ill patients and warrant future studies.The study protocol and manuscript were written by the investigators. Data were collected by Tuomo Puhakka, Lotta E. Ivaska, and Emilia Mikola and analyzed by Lotta E. Ivaska, Antti Silvoniemi, and Tuomas Jartti. Viral analyses were carried out by Matti Waris and Tytti Vuorinen. The study was supervised by Tuomas Jartti. The first draft was written by Lotta E. Ivaska. All authors contributed to the revision of the final manuscript.The authors declare no conflicts of interest.Supporting information.Click here for additional data file.


INK4A, and p21Waf1/Cip1 and performed cell nuclei measurements. The obtained results may contribute to an increase in the knowledge of the cellular effects of cobalamin deficiency in the context of astrocytes. In addition, the presented data suggest a potential causative agent of astrosenescence that has not been proven to date.Cobalamin (vitamin B12) deficiency is one of the major factors causing degenerative changes in the nervous system and, thus, various neurological and psychiatric symptoms. The underlying cellular mechanism of this phenomenon is not yet fully understood. An accumulation of senescent astrocytes has been shown to contribute to a wide range of pathologies of the nervous system, including neurodegenerative disorders. This study aimed to investigate whether cobalamin deficiency triggers astrosenescence. After inducing cobalamin deficiency in normal human astrocytes in vitro, we examined biomarkers of cellular senescence: SA-β-gal, p16 Cobalamin, also known as vitamin B12, is crucial for cell functioning, particularly DNA synthesis, methylation, and mitochondrial metabolism. A subclinical deficiency of this vitamin could affect approx. 26% of the general population. Risk factors of cobalamin deficiency include old age, gastric or small intestine resections, inflammatory bowel disease, and veganism, as well as the use of metformin, proton pump inhibitors or histamine H2 blockers. Vitamin B12 deficiency can present with non-specific clinical features and, in severe cases, with hematological or neurological abnormalities . The mosINK4A and p21Waf1/Cip1 protein expression . After inducing cobalamin deficiency in NHA, we examined biomarkers of cellular senescence: SA-β-gal, p16INK4A, and p21Waf1/Cip1 and measured cell nuclei. In addition, we examined whether the cobalamin deficiency state affects the sensitivity of cultured astrocytes to apoptosis. We believe that the obtained results will contribute to an increase in knowledge about the cellular effects of cobalamin deficiency in the context of astrocytes. In addition, the presented data suggest a potential causative agent of astrosenescence that has not been proven to date.The aim of the current study was to investigate whether cobalamin deficiency is an astrosenescence-inducer. For this purpose, we used a previously developed hypocobalaminemia model based on the culture of normal human astrocytes (NHA) in the presence of the cobalamin antagonist, hydroxycobalamin  (HCCL), which is a cobalamin antagonist. HCCL was synthesized by Prof. Dorota Gryko from Institute of Organic Chemistry, Polish Academy of Science. according to the previously described method [In the current study, we applied a previously developed  experimed method .The CellEvent Senescence Green Detection Kit  was used. The fluorescein-based probe contains two galactoside moieties, making it a target for cellular β-galactosidase. The enzymatically cleaved product emits fluorescence (absorption/emission maxima = 490/514 nm). In brief, the cells were grown on Nunc Lab-Tek 4-well Chambered Coverglass . After washing with PBS, the cells were fixed with 2% paraformaldehyde and then washed with 1% bovine serum albumin (BSA). Next, the cells were incubated with the diluted CellEvent Senescence Green Probe for 2 h and then washed 3 times with PBS. The samples were observed using a Nikon A1R Si confocal system . Nikon NIS Elements AR 4.51 software was applied for image analysis.INK4A rabbit Ab (1:1000 dilution); anti-p21Waf1/Cip1 rabbit Ab (1:1000 dilution); anti-GAPDH rabbit Ab (1:1000 dilution), all purchased from Cell Signaling .The analysis was performed according to the protocol presented in our previous article . In thisINK4A rabbit Ab (1:800 dilution), anti-p21Waf1/Cip1 rabbit antibody (1:400 dilution).The method used was described earlier . PrimaryThe JC-1 staining probe and image cytometer NucleoCounter NC-3000  was applied to determine the mitochondrial membrane potential (Δψm). The procedure was previously decribed in our paper .The annexin V assay and image cytometer NucleoCounter NC-3000 were applied to analyze the process of cell apoptosis. The protocol was described previously .t-test  or the Mann–Whitney U test , as indicated in the figure captions. A p-value < 0.05 was used as the cutoff for statistical significance.Statistical analysis obtained data was performed using GraphPad Prism 8.0.1 software . In all experiments mean values ± standard deviation (SD) of at least three separate experiments. Differences between groups were assessed by the unpaired SA-β-gal is an enzyme that accumulates in the lysosomes of senescent cells, where it hydrolyses β-galactoside . We analINK4A and p21Waf1/Cip1 may indicate senescent cells [INK4A protein compared to the control  analysis and annexin V assay were performed. Four populations of astrocytes were analyzed: (i) NHA cultured in a growth medium; (ii) NHA cultured in a growth medium with the addition of HCCL (20 µg/mL) for 27 days ; (iii) NHA cultured in a growth medium and then for 48 h with 300 µM etoposide; (iv) NHA cultured in a growth medium with the addition of HCCL (20 µg/mL) for 27 days, and then for 48 h with 300 µM etoposide. The treatment of astrocytes with etoposide resulted in the depolarization of mitochondria with a decrease in Δψm a,b. A slNeurological disorders are the main cause of disability and rank among the predominant causes of death worldwide . The eluCobalamin deficiency is one of the major factors causing degenerative changes in the nervous system, and thus neurological symptoms such as symmetric dysesthesia, disturbance of position sense, spastic paraparesis or tetraparesis and dementia ,25. ExisPreviously we developed an experimental model of cobalamin deficiency in normal human astrocytes . The modINK4A-positive) astrocytes in the frontal cortex of AD patients compared with non-AD adult control subjects of similar ages. Vazquez-Villaseñor et al. [INK4A-positive and p21Waf1/Cip1-positive cells in the frontal cortex of patients with motor neuron disease, which suggest the involvement of senescence in the progression of this disorder. Recently, it has been proposed that targeting senescent cells by senolytics can ameliorate the effect of brain aging, thus preserving cognitive function into old age [Recent studies suggest that astrosenescence has a severe impact on neurodegenerative disorders and brain aging ,32. Bhatr et al.  observed old age .The aim of the current study was to determine whether a senescence activation occurs in astrocytes under cobalamin deficiency. We induced cobalamin deficiency in the culture of normal human astrocytes and then examined key markers of cellular senescence.INK4A and p21Waf1/Cip1. The disturbance of DNA synthesis due to cobalamin deficiency may probably induce p21 expression, as demonstrated by Kwan et al. [INK4A and p21Waf1/Cip1, in cellular senescence has been widely investigated. p21Waf1/Cip1 blocks cyclin-dependent kinase 2 (CDK2) activity, leading to cell-cycle arrest and the initiation of senescence. Persistent cellular stress leads to the upregulation of p16INK4A, triggering the p16INK4A/RB pathway and irreversible cellular arrest [INK4A protein is highly dynamic, being largely undetectable in healthy young tissues, but rising in many tissues with aging or after injury [Waf1/Cip1 protein level in senescent astrocytes is accompanied by the overexpression of p16INK4A. Interestingly, according to the study by Bitto et al. [Our analysis indicated that vitamin B12-deficient astrocytes show increased SA-β-gal, a well-recognized biomarker of senescence. Moreover, we found that cobalamin deficiency in the studied cells may resulted in an overexpression of p16n et al.  for clotr arrest . The expr injury . Bitto er injury  indicateo et al. , astrocyRecently, Heckenbach et al.  examinedThe state of senescence may affect the susceptibility of cells to apoptosis; however, this interplay has been shown to be cell-type-specific . In the To the best of our knowledge, this is the first study to investigate whether cobalamin deficiency induces astrosenescence. Our research sheds light on the cellular mechanism of nervous system disorders associated with hypocobalaminemia, pointing to the potential role of senescent astrocytes. We believe that the findings could form the basis for further research into the potential efficacy of senolytics in the treatment of clinical implications of cobalamin deficiency.


Scientific Reportshttps://doi.org/10.1038/s41598-018-31148-4, published online 10 September 2018Correction to: The original version of this Article contained errors in the Results section under subheading ‘Virus discovery’ where,Penaeus chinensis hepandenovirus , Wenzhou shrimp virus 2 , Wenzhou shrimp virus 8 , Beihai picorna-like virus 2 , Wenzhou picorna-like virus 23 (551 nt) and Moloney murine leukaemia virus Pr180 sequence .”“The remaining 21 contigs had Top Hit E-value scores identifying them to be related most closely to strains of Gill-associated virus , now reads:Penaeus chinensis hepandenovirus , Whenzhou shrimp virus 2 , Whenzhou shrimp virus 8 , Beihai picorna-like virus 2 , Wenzhou picorna-like virus 23 (551 nt) and Moloney murine leukaemia virus Pr180 sequence .”“The remaining 21 contigs had Top Hit E-value scores identifying them to be related most closely to strains of Gill-associated virus , In the Discussion section,“The presence of near full-length ssRNA genome sequences for viruses such as gill-associated virus  and white spot virus 2  provided additional validation of the methods used to synthesize and assemble the transcriptome, and to its completeness as demonstrated by various metrics measuring the nature and number of endogenous gene transcripts.”now reads:“The presence of near full-length ssRNA genome sequences for viruses such as gill-associated virus  and two sequences  with high similarity to Whenzhou shrimp virus-2 L and M segments  provided additional validation of the methods used to synthesize and assemble the transcriptome, and to its completeness as demonstrated by various metrics measuring the nature and number of endogenous gene transcripts.”50,51 unknown until now to occur in Australian P. monodon.”“In addition to known endemic viruses, the transcriptome contained full-length or near full-length RNA transcripts related closely to the recently-described shrimp viruses WSV2 and WSV8now reads:50,51 unknown until now to occur in Australian P. monodon.”“In addition to known endemic viruses, the transcriptome contained full-length or near full-length RNA transcripts related closely to the recently-described shrimp viruses When-2 and When-8The original Article has been corrected.


SNHG14) is a host gene for small non-coding RNAs, including the SNORD116 small nucleolar C/D box RNA encoding locus. Large deletions of the SNHG14 locus, as well as microdeletions of the SNORD116 locus, lead to the neurodevelopmental genetic disorder Prader–Willi syndrome. This review will focus on the SNHG14 gene, its expression patterns, its role in human cancer, and the possibility that single nucleotide variants within the locus contribute to human phenotypes in the general population. This review will also include new in silico data analyses of the SNHG14 locus and new in situ RNA expression patterns of the Snhg14 RNA in mouse midbrain and hindbrain regions.The Small Nucleolar Host Gene 14 ( In the newest release of GRCh38p14 in the e!Ensembl database (July 2022), the number of non-coding genes exceeds the number of protein-coding genes by 5530  [Xist [SNHG14, is the subject of this review. In addition to relevant background materials, this review will include new in silico data analyses of the SNHG14 locus, focusing on a cluster of small nucleolar genes it hosts as part of the SNORD116 locus. The SNORD116 locus  encodes 30 small nucleolar C/D box RNAs that are processed from the host gene, SNHG14 [LncRNAs are classified as those ncRNAs > 200 nucleotides that are not translated and can exceed 17,000 nucleotides, i.e., Xist . Papers SNHG14 locus will be reviewed, and the expression patterns of Snhg14/SNHG14 in whole mouse brain and human RNA seq datasets will be examined. Single nucleotide variants (SNVs), as well as deletions contained within the SNHG14 locus, and their possible association with human disease in the general population, will be discussed.The UBE3A locus [UBE3A, maintaining the expression of the UBE3A mRNA only from the maternal chromosome in tissues, such as the brain. In that same year, a yeast artificial chromosome library was able to complete the gaps in the locus, mapping the rest of the region [SNHG14 RNA was first identified using reverse transcription PCR experiments, which had been seeking to amplify the e region .SNHG14 locus spans 965,992 base pairs (966 kb) on chromosome 15q11.2, with 148 exons (NCBI NR_14677) and more than 21 different transcripts (SNRPN (Gene ID 6638) and SNURF (Gene ID 8926) exist in a bicistronic transcript originating from different open reading frames on the transcripts. According to the latest updates of these entries in October and November 2022 from both the NCBI and UCSC databases, the entire lncRNA transcript has multiple start and termination sites and undergoes extensive alternative splicing, as described in a 2001 article [In humans, the nscripts . The regSNHG14 locus, which they called sno-lnc RNAs, as the RNAs were between 1200 and 2900 nucleotides with the SNORD116 snoRNAs at their 5′ and 3′ ends [SNHG14 locus was shown to sequester the Rbfox2 splicing factor, allowing for normal developmental processing of the mRNAs [SNORD116 region on the SNHG14 locus.In addition to the major exons of the locus, more than ten years ago, Yin and colleagues identified a new class of non-coding RNAs that are derived from the he mRNAs . SPA RNAhe mRNAs . Accordihe mRNAs . Both thSnhg14 is on chromosome 7B5, 34.04 cM (Gene ID 52480), and is longer than the human locus at 1,177,441 nucleotides (1177 KB), with 172 exons  . Interestingly, the mouse locus is missing some of the non-coding RNAs (ncRNAs) found in humans, including SNORD109a and SPA2 lncRNAs. SPA1 lncRNA is retained in approximately the same position as in humans. Copy numbers of the snoRNA clusters also differ [The mouse locus for 72 exons . Like th_146211) , and theSNHG14/Snhg14 gene is only expressed from the paternally inherited chromosome. This becomes important for the human conditions that result from the deletion or alteration of the locus. Nearly all known cases of PWS (OMIM 176270) result from the inheritance of a paternal deletion or other genetic situations, such as maternal uniparental disomy, an imprinting defect, or, rarely, chromosome translations. As previously shown, brain tissue from patients with PWS lacks an expression of the SNORD116 locus by Northern analysis [SNORD116 locus is caused by large or small deletions within the locus, with maternal uniparental disomy and epigenetic changes in the DNA’s methylation or loss of the imprinting control region, making up the other 40% of the cases [SNORD116 cluster of sno-RNAs  and, thSNHG14 is located between breakpoints 2 and 3. One PWS patient (patient #5) was recently described and had an atypical 126 kb deletion spanning from SNRPN through to SNORD109a but excluded the SNORD116 locus in the deletion (chr15: 25153293-25279455 in hg19) [SNORD116 locus. Chromosome 15q’s rearrangements and breaks are the most common chromosomal changes seen in the human population [SNHG14 can disrupt the overall mRNA stability and/or splicing of host RNAs. Several studies have addressed this question, finding that gene expression is generally repressed. For example, in a patient carrying a translocation between chromosome 15 and 19 46, XY, t , the lymphocyte expression of the paternally-expressed genes, such as SNORD116 and IPW, were completely absent [There are six common breakpoints within the proximal region of the human chromosome 15q . SNHG14 in hg19) . To datepulation . Of inteSNHG14 locus is large and serves as a host gene for more than 15 coding and non-coding genes. It is complex in producing multiple RNAs from the region, some of which may not yet be annotated. Finally, the deletion of the regions within the SNHG14 locus, or translocations affecting the expression of the transcripts from the host gene, or uniparental disomy leading to the normal expression of only some genes in the locus, lead to PWS.To summarize, the SNRPN [SNRPN locus  by at least one day during in vitro differentiation [Our laboratory recently showed that one of the Snord116 snoRNAs in mouse Snord116-3 could post-transcriptionally regulate the expression levels of Nhlh2, a basic helix-loop-helix transcription factor . We and ntiation . Howeverntiation . This pantiation ) and by ntiation ).SNHG14 , as well as in the cortical cells, the cerebellum, and the hindbrain . These aClose examination of the Snhg14 expression in the dentate gyrus of the hippocampus shows dense labeling of the granular cell layer, with fewer positive cells in the polymorphic and molecular layers B. This iSnord116 cassette [Snhg14, is retained in the nucleus near the site of the transcription [At increased magnification, the probe location shows what could be nucleolar localization C. Previocassette . Howevercription . The cencription . This wacription ,42. WhetThe high expression of the Snhg14 transcript is also present in the lateral habenula D. The haIn summary, the expression of the SNHG14 lncRNA is ubiquitous, with high expression found in the nervous system. Our work on mouse brain expression analysis for Snhg14 can be used to fine-tune the RNA-seq analysis to specific structures within the brain but should be considered with some caution with regards to what transcript is being detected. First, the probe set used was developed commercially by ACD/Biotechne  to detect both unprocessed and processed transcripts from the locus, but it may be able to detect some of the processed transcripts, as indicated by the nucleolar-like expression pattern. The expression of Snord116 snoRNA or other ncRNAs from the region will require the development of additional probes for this purpose. The nucleolar-specific co-expression of the Snhg14-processed transcripts with possible RNA targets may also be invaluable in clarifying the functional information of when and where the ncRNAs are working to regulate or modify target RNAs.SNHG14 host gene [SNHG14 locus. As shown in SNRPN locus, and the others are all 5′ of the region. While the upstream SNVs could be linked to the SNHG14 host gene through a commonly inherited haplotype, for the purpose of this review, we will focus just on those SNVs which are within the SNHG14 locus. With more non-coding annotated transcripts in e!Ensembl than protein-coding transcripts, genome-wide association studies (GWAS) generally identify many variants within ncRNA transcripts. To be classified as a variant, the change from the reference genome must be found in at least 1 percent of a population. It is estimated that variants occur once every ~1000 nucleotides, with ~4–5 million single nucleotide variants (SNVs) per person . In the ost gene ), there SNHG14 region: rs8033133 within the SNORD116 locus and rs67659323 within the SNORD115 locus. Using GWAS Central, an association was found for rs8033133 and blood osmolarity with a modest effect size of 0.09 for carriers of the risk allele A  with immune biomarker levels in bipolar disease. Worldwide, the variant allele G has a general population frequency of 4%, mainly in those of European ancestry. The SNV is found 51 nucleotides downstream of the encoding exons of SNORD115-41 but again intronic for the SNHG14 locus. Several studies have shown a higher frequency of bipolar disease in individuals with PWS due to maternal uniparental disomy , and thSNHG14 gene are intronic-relative to the known hosted genes. However, since GWAS identify areas that are linked to a phenotype and not always the causative allele, it is relevant to examine these further. Rs4906947 is identified as an associated allele for two studies on neuroticism. In a large study focusing on socioeconomic status and educational attainment, the SNV was associated with neuroticism conditioned on educational attainment (4 × 108 and 2 × 108) [7) [SNORD116 and SNORD115 loci, close to PWAR1. Co-morbid psychiatric illness is found in patients with PWS, so this marker may also provide insight into the role of the SNHG14 host gene and its functions. The same study on educational attainment found a significant association with rs2714720 and neuroticism, but this time in association with self-rated math ability (p = 3 × 108) and highest math class taken (p = 3 × 108), as well as the Townsend deprivation index (p = 3 × 108), a measure of socioeconomic status [PWAR1 and SNORD115. Finally, rs186045664 was found to be associated with myopia in an ophthalmologic GWAS study of over 53,000 individuals of both European and Asian descent (p = 5 × 106) [SNHG14 host gene can be of use to understanding both its functional role in general in individuals without PWS, as well as to pointing to specific parts of the transcript that could play a causative role in some of the phenotypes associated with PWS. Finally, and of the direct relevance to the phenotypes of hyperphagia, circadian rhythm imbalance, and sleep disturbances seen in patients with PWS [The three remaining SNVs in the 2 × 108) . In othe2 × 108) . A secon108) [7) . The C a108) [7) . This SNc status . This SN5 × 106) , using t5 × 106) . This SNwith PWS , sleep dSNHG14 locus, some of which, described here, are associated with human traits. It is likely that the associated variants are not the causative ones but rather linked to causative variants that have yet to be identified or were not directly tested in the GWAS. Either way, these associations are another way to help to clarify the roles of the lncRNA transcript and its host genes in human phenotypes. To date, 237,982 variants have been identified within the SNHG14 gene locus encodes a non-coding RNA, which is a member of the long non-coding RNA host gene family. Multiple small non-coding RNAs, as well as mRNAs, are also encoded or hosted within this locus. This locus is imprinted with only the paternal allele being expressed, providing some insight into its role in imprinted disease. The high expression occurs throughout the brain, but the expression is also found in many tissues of the body. While the function of an active SNHG14 lncRNA is still being characterized, paternal deletion of the locus, particularly those deletions involving transcripts from the SNORD116 locus, is associated with the genetic disorder, PWS. A GWAS’ analysis has linked SNVs’ gene locus with bi-polar disorder, blood osmolarity, neuroticism, and educational attainment phenotypes, opening up the possibility that this gene and its non-coding RNA transcript may control many facets of normal brain and body functioning, with the caveat that the paternal inheritance pattern of the locus will make the genetic analysis of any genotype–phenotype relationship more complicated. More basic research into the role of this lncRNA host gene is warranted, with the hopes that we can elucidate its function and how loss through large deletions or individual SNVs leads to neurological and other phenotypes.The