Escherichia coli, Pseudomonas aeruginosa and Citrobacter freundii) and 2 Gram-positive bacteria , employing the response surface methodology and a Eubacteria probe. In general, it was observed that a high NaCl concentration is beneficial (from 2 M to 5 M), regardless of the denaturant used. Urea, formamide and ethylene carbonate are suitable denaturants for LNA/2’OMe-FISH applications; but urea provides higher fluorescence intensities among the different bacteria, especially for gram-positive bacteria and for P. aeruginosa. However, a unique optimal protocol was not found for all tested bacteria. Despite this, the results indicate that a hybridization solution with 2 M of urea and 4 M of NaCl would be a proper starting point. Furthermore, a hybridization temperature around 62°C, for 14 bp probes with LNA monomers at every third position of 2′OMe and 64% of GC content, should be use in initial optimization of new LNA/2’OMe-FISH protocols.Despite the successful application of LNA/2’OMe-FISH procedures for bacteria detection, there is a lack of knowledge on the properties that affect hybridization. Such information is crucial for the rational design of protocols. Hence, this work aimed to evaluate the effect of three essential factors on the LNA/2’OMe hybridization step—hybridization temperature, NaCl concentration and type and concentration of denaturant . This optimization was performed for 3 Gram-negative bacteria ( This. Thisin gy  have emerged for ea e.g. –13). In . In in vea e.g. , 6), to , to in vea  [The hybridization step in FISH procedure is influenced by several factors including temperature, ionic strength and denaturing conditions . MoreoveEUB388)  through (EUB388) , 38.8  througEscherichia coli CECT 515, Pseudomonas aeruginosa PAO1, Citrobacter freundii SGSC 5345, Staphylococcus epidermidis RP62A and Enterococcus faecalis CECT 184. All cultures were grown on tryptic soy agar  tryptic soy broth and 1.5% (w/v) agar) , overnight at 37 °C.The bacterial strains selected for this study were et al. [et al. [Eubacteria domain, was used during the FISH experiments. LNAs (represented by “l”) were placed at every third 2’OMe (represented by “m”) monomer. The LNA/2’OMe probe EUB338 also included a phosphorothioate backbone (PS linkage represented by “*”) instead of a phosphodiester. The choice of this type of probe design was based on previous works of our group  and NaCl concentration on the fluorescence intensity (a.u.) of LNA/2’OMe-FISH method, the hybridizations were performed in suspension based on Azevedo et al. , followe [et al. , specifiup e.g. , 7), whi, whiet a9 cells.ml-1. Then, for sample fixation/permeabilization, cell suspensions were centrifuged  and fixed in 400 μL of 4% (w/v) paraformaldehyde  for 1 hour at room temperature. After centrifugation, the fixed cells were resuspended in 500 μL of 50% (v/v) ethanol and incubated at -20°C for at least 30 min. For hybridization, 100 μL of the fixed cells were pelleted by centrifugation  and resuspended in 100 μL of hybridization solution containing 200 nM of LNA/2’OMe probe EUB338, 50 mM Tris-HCl (pH 7.5) , denaturant  and NaCl . Samples were incubated during 60 min at the hybridization temperatures under study. Afterwards, samples were centrifuged, resuspended in 500 μL of washing solution containing 5 mM Tris-base , 15 mM NaCl  and 0.1% (v/v) Triton X-100  and incubated for 30 min at the same temperature used in the hybridization step. Finally, the suspension was pelleted by centrifugation (10 000 g for 5 min) and resuspended in 500 μL of 0.9% (w/v) NaCl . Each experiment was performed in triplicate. Negative controls were resuspended in hybridization solution with no probe. The samples were stored at 4 °C in the dark before flow cytometry analysis.First, the bacterial cells were harvested from TSA plates and suspended in sterile water to a final concentration of ~10The flow cytometry analysis was performed to evaluate the fluorescence intensity of hybridized samples and negative controls. For this, a Sony Biotechnology EC800  equipped with a 488 nm argon ion laser was used. Forward angle light scatter (FS), side angle light scatter (SS), and green (FL1) fluorescence were detected at logarithmic scale. A minimum of 40 000 events falling into the bacterial gate defined on the FSSS plot were acquired per sample. The data were analyzed with the EC800 software version 1.3.6. , and the average fluorescence intensity was determined for each triplicate experiment.E. coli CECT 515, P. aeruginosa PAO1, C. freundii SGSC 5345, S. epidermidis RP61A and E. faecalis CECT 184, using the statistical software Design Expert 11  to estimate the coefficients of the model. For the designs, the hybridization temperature (variable x1), denaturant concentration (x2) and salt concentration (x3) were considered the independent variables and the fluorescence intensity was the response. Each of the three variables assumed five different experimental values, defined according our previous studies and the results obtained within this study , 6 axial points (coded as ± α) that represent extreme values used for the estimation of the model curvature, and a center point  repeated 6 times to take into account the experimental error [RSM, a mathematical and statistical tool based on the fit of empirical models to the experimental data obtained in relation to experimental design , was empis study  Tables. al error . The desTo find the optimum hybridization conditions of the five species, the obtained fluorescence values were fitted to a quadratic model. Each model was analyzed using analysis of variance (ANOVA) to test the significance and adequacy of the model. The Fisher variance ratio  was also evaluated. Model F-value is a measurement of variance of data about the mean based on the ratio of mean square of group variance due to errors; high Model F-value indicates that implies the model is significant.Finally, the optimum conditions within the experimental range that maximize the fluorescence intensity were estimated using the optimization function of the Design Expert software. The value estimated for the optimum conditions was, then verified for each bacterium on a confirmation experiment, in triplicate.E. coli-specific LNA/2’OMe probe (5’-FAM-lC*mA*mC*lG*mC*mC*lT*mC*mA*lG*mC*mC*lT*mU*mG*lA*-3’) was tested against all 5 bacteria. The hybridizations were performed at the optimal hybridization conditions for E. coli as described above. Afterwards, 20 μl of the suspension was spread on a microscope slide which was allowed to air dry before microscopy visualization. Negative controls were included.To evaluate whether each bacterium can be detected with accuracy under optimum conditions, an Images were acquired using a Leica DM LB2 epifluorescence microscope  connected with Leica DFC300 FX camera  and equipped with a sensitive filter to the FAM fluorochrome . The images were captured using a Leica IM50 Image Manager with a magnification of x1000, and fluorescence intensities were quantified by using an open source image-processing ImageJ software . Data waDespite the successful applications of LNA/2′OMe-FISH in bacterial identification e.g. , 44, 45), 4544, 4Due to the large number of variables involved in a FISH method , optimization is normally performed using a trial-and-error approach. We are currently using response surface methodology (RSM), a statistical approach that allows to obtain an optimum in a cost-effective way. Using the RSM the number of experiments that need to be performed is significantly reduced, allowing us to test a large number of variables and better understand the relationship between them.E. coli, C. freundii and P. aeruginosa) and 2 Gram-positive species . Different genera were selected to include bacteria with different characteristics, including different cell wall composition. Furthermore, three denaturant agents were included, because even though the formamide is more frequently used in FISH methodology, there are also studies using less hazardous compounds as denaturant agents, including urea  and  and E. cea (e.g. ).p-values (p<0.05)  , confirmHence, using the successful modelling of the hybridization temperature, NaCl and denaturant concentration, we have been able to obtain the optimum hybridization conditions that lead to the maximum intensity fluorescence for all bacteria . In geneS. epidermidis, Listeria innocua, Bacillus subtilis) and, thus, low fluorescence intensities in FISH procedure could be observed [et al. [Recent studies on PNA-FISH optimization, have noticed that Gram-negative bacteria show higher fluorescence intensity signals compared with Gram-positive bacteria , 38. Thiobserved , 38, 47. [et al. , explainIn order to better understand the effect of the studied variables, the next section will discuss and explore the results in more detail for variables separately. Moreover, optimum ranges presented at the surface response plots  Figs wilet al. [+ and Mg2+) increased the stability of DNA probes containing LNA monomers. For LNA/2’OMe-FISH there are no studies that analyze the effect of NaCl concentration. In fact, NaCl is highly important in the hybridization to stabilize the repulsive interactions of LNA/2’OMe-rRNA duplexes. Hence, to understand the effect of NaCl concentration on LNA/2’OMe-FISH and select an optimum range of concentration . It is also important to note that PNA-FISH protocols have not described the same effect  to the FISH hybridization buffer have an important effect on thermodynamic stability , 50. Adjet al.  showed tntration , we plotntration  Figs. Asct e.g. , 38); wh; whet alThe optimum range hybridization temperatures is quite large, varying from ~50 °C to ~80 °C  Figs, reWhile it is expected that LNA/2’OMe probes with similar size and % GC content can work on this temperature range, it is important to bear in mind that this parameter is highly dependent on the sequence. Using the same sequence of LNA/2’OMe probe for the identification of all bacteria, it is expected to obtain the same optimum hybridization temperature. Surprisingly, as we can see in et al. [et al. [In another study with PNA-FISH, Santos et al.  have dem [et al.  reportedversus denaturant concentration, with the NaCl concentration at the optimal value for each bacterium , 5346, 5P. aeruginosa   , which mE. coli-specific LNA/2’OMe probe was tested against the non-target species enrolled in this study. The probe was tested against all 5 bacteria using the optimal hybridization conditions obtained for E. coli. E. coli-specific LNA/2’OMe showed hybridization with E. coli CECT 434, and no cross-hybridization was observed to other species , 4238, 4In general, the results showed that an optimized and standardized efficient LNA/2’OMe-FISH procedure is possible for bacterial detection, even when bacteria with different properties are present.Urea and high salt concentrations seem to be an adequate choice to balance the fluorescence signal among species and to reach a universal hybridization solution. General recommendations for, at least, the starting point on optimization experiments would include approximately 2 M of urea and 4 M of NaCl in hybridization solutions . In addiS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 TableThe table also shows the Analysis of variance (ANOVA) for response surface quadratic models obtained for each bacterium and denaturant.(DOCX)Click here for additional data file.S1 FigThe formamide (a), ethylene carbonate (b) and urea (c) concentration was kept constant at the optimum value for each bacterium. Fluorescence values are presented in arbitrary units (a.u).(DOCX)Click here for additional data file.S2 FigThe formamide (a), ethylene carbonate (b) and urea (c) concentration was kept constant at the optimum value for each bacterium. Fluorescence values are presented in arbitrary units (a.u).(DOCX)Click here for additional data file.


Aedes aegypti mosquito and causes non-specific symptoms such as fever, myalgia, and generalized weakness. In addition to these symptoms, it has been noted the ZIKV is capable of causing associated conditions in adults, particularly in pregnant women as well as in newborns via vertical transmission. These manifestations include microcephaly, lissencephaly, ventriculomegaly, optic neuropathy, and congenital glaucoma, arthralgia, maculopapular rash, and cardiovascular anomalies such as atrial fibrillation. It is important to understand the reason for this specific set of associated conditions that emerge with ZIKV. This paper aims to identify the manifestations of ZIKV in adults and neonates in detail and attempts to understand the pathophysiology behind each one.The Zika Virus (ZIKV) has been slowly becoming an epidemic in different parts of the world. Since its discovery in 1947, there have been numerous outbreaks affecting many different populations. Currently, there is an ongoing threat of ZIKV in Latin America and the United States of America. ZIKV is mainly spread by the  The Zika virus (ZIKV) is an arthropod-borne virus, of the genus, flavivirus. It is enveloped and spherical-shaped with an icosahedral capsid containing a non-segmented, positive-sense RNA genome. The entire structure is about 50 nm in diameter. The genome itself is made up of 10,794 bases and has two non-coding flanking regions on the 5’ and 3’ ends. There is a single open reading frame that codes for a polyprotein subsequently cleaved into capsid (C), precursor membrane (prM), envelope (E), and non-structural proteins (NS) . SequencAedes mosquito, Aedes aegypti. In addition, unborn babies can be affected through vertical transmission in the womb. It is related to other members of the flaviviruses namely; yellow fever virus, Japanese encephalitis viruses, West Nile virus and the dengue virus [ZIKV is transmitted to humans primarily through the bite of an ue virus . ZIKV haue virus . ZIKV haue virus  Currentlue virus , 6.History and epidemiologyThe ZIKV was first isolated in 1947 from a serum of a sentinel rhesus macaque monkey in the Zika forest of Uganda. After a course of 60 years, the virus emerged in the Yap Island, the Federated States of Micronesia in 2007 and affected approximately 75% of the population within four months . The outZIKV-associated symptoms, complications, and manifestations in neonates and adults are illustrated in Figure A. Life cycle of ZIKVAedes genus. The Aedes mosquitos lay eggs in areas of high moisture which undergo metamorphosis from larvae to pupae and eventually to the adult form. This cycle takes approximately one-and-a-half to three weeks to complete. Once the female adult mosquito takes a blood meal, it can produce on average 100 to 200 eggs per batch, yielding on average five batches of eggs during her lifetime [As mentioned earlier, ZIKV is transmitted primarily by the mosquitos of the lifetime .Aedes mosquitos, Ae. africanus, Ae. furcifer-taylori, Ae. dalzieli take blood meals from chimpanzees, monkeys, and baboons. Humans are affected when the Sylvatic cycle transitions into the Urban cycle. In this case, the subspecies of the Aedes mosquitos, Ae. aegypti and Ae. albopictustake a blood meal from humans. Lastly, it can be transmitted from human to human through sexual intercourse, blood transfusions, and in utero [The adult female form acquires the blood to produce eggs by biting humans and animals. The virus originated in non-human primates in tropical rainforests and infected them as described in the Sylvatic cycle. The subspecies of the in utero .Aedes mosquito saliva, contaminated with ZIKV and human blood, is exchanged, the E-proteins on the surface of ZIKV are primarily involved in the attachment to the host cell membrane. The virus is internalized by endocytosis. The capsid is broken apart in the cytoplasm to release to the viral RNA. The positive sense RNA genome is translated using host ribosomes attached to the endoplasmic reticulum resulting in a polyprotein processed by proteolytic activity to structural and non-structural proteins. Once the assembly of all parts is completed, the virion is transported out of the cell using endosomal sorting complexes to the Golgi apparatus. The mature virion exits the cell by exocytosis [When the ocytosis , 9. In tB. Vertical transmissionThe vertical mode of transmission provides passage to a disease-causing agent from mother to baby in utero or after birth. This can occur across the placenta, in the breast milk, or by direct contact during the delivery process . For thiThe best possibility to avoid the consequences of vertical transmission of ZIKV is to prevent the infection from occurring in pregnant women. A study conducted in Nature demonstrated how the live-attenuated ZIKV vaccine prevented viral transmission during pregnancy. This vaccine contained deletions in the 3’UTR (untranslated region) of the ZIKV genome. After a single dose of the vaccine, pregnant mice were challenged with ZIKV on embryonic day 6. An evaluation at embryonic day 13 showed markedly decreased levels of viral RNA in maternal, placental, and fetal tissues. The data collected from this study showed promising results of live-vaccines in preventing vertical transmission of ZIKV. However, the live-attenuated ZIKV vaccine is still in the phases of clinical trials .C. Neonatal manifestationVertically transmitted ZIKV manifestations in neonates are commonly noted to be congenital microcephaly, optic neuropathy, and congenital glaucoma, ventriculomegaly, and lissencephaly. The following is an identification and discussion of each presentation of ZIKV in neonates.Congenital MicrocephalyCongenital ZIKV can cause microcephaly and other brain abnormalities , 16-17. The pathogenesis of postnatal microcephaly from ZIKV is not known, however; the decrease in head circumference and brain growth might be the consequence of the in-utero destruction of neuroprogenitors, neural cells, and persistent inflammatory response-associated molecules -18. The Optic Neuropathy and Congenital GlaucomaOptic neuropathy is defined as damage to the optic nerve, clinically manifested by partial or complete loss of vision . The eyeGlaucoma the second leading cause of irreversible blindness in the world, affecting more than 70 million people worldwide with approximately 10% being bilaterally blind . GlaucomVentriculomegalyThe human brain contains a set of four ventricles connected to one another. There are a set of two lateral ventricles, which are the largest in size, connected to the interventricular foramen (foramen of Monro). This foramen connects the two lateral ventricles to the third ventricle, which is subsequently connected to the fourth ventricle via the cerebral aqueduct. These are the sites of cerebrospinal fluid (CSF) production. Ventriculomegaly is the dilation of the lateral cerebral ventricles usually greater than 10mm at the level of the atria . The atrVentriculomegaly can result in hydrocephalus, which is an increase in pressure of the CSF within the ventricles. The increased flexibility of the skull in children allows it to be susceptible to skull enlargement secondary to increased CSF pressure. This compresses and stretches the adjacent brain tissue . This reVentriculomegaly is treated after birth, usually by surgical means. The first step is to identify the etiology behind this manifestation. The next step is to take into consideration the age of the patient, rate of postoperative infection or malfunction, technical feasibility, and the great plasticity of a young brain . One surLissencephalyThe human brain consists of folds and grooves known as the sulci and gyri. The brain normally begins to fold during fetal developmental stages . This reD. Adult manifestationGeneralized symptoms of Zika virus in adults present as conjunctivitis, mild fever, headache, skin rash, and diarrhea. However, more specifically ZIKV can manifest in adults as arthralgia, Guillain Barré Syndrome, and cardiovascular anomalies.ArthralgiaArthralgia is characterized by joint pain which causes injuries that affect the ligaments, bursa, or tendons surrounding the joints . The paiGuillain-Barré Syndrome (GBS)In the recent researches conducted, it has been proven that the relationship between the ZIKV and the nervous system, known as the Guillain-Barré Syndrome, has risen in different countries especially in America. GBS is a demyelinating condition in which the body's immune system attacks the peripheral nervous system -41. ThisCardiovascular AnomaliesAtrial fibrillation, a cardiovascular condition, involves a rapid heartbeat leading to an increased risk of contracting stroke, heart failure, and other complications . The patThe association with atrial fibrillation and other cardiovascular (CVS) complications was made in a study comprised of adults with no prior history of CVS disease in Caracas, Venezuela, an epicenter of ZIKV outbreaks. The study resulted in all but one patient developing a dangerous heart rhythm condition and two-thirds of the subjects showed evidence of heart failure .E. Current treatment optionsZIKV has been making headlines in different countries due to its causal relationship to the microcephaly, severe brain defects along with diseases that severely affects the nervous system in unborn babies especially if the pregnant mothers who contract the virus . This isSymptomatic treatments of ZIKV associated conditions are also available, such as the VP shunt in microcephaly, drugs to treat heart rhythm problems, pain medication for arthralgia, glaucoma management, and other palliative treatment options. Conditions like microcephaly or lissencephaly are much more difficult to treat as they are congenital and require a more invasive approach.Aedes mosquito and its associated conditions can be fatal. Once the virus has infected an individual, it has no specific treatments, and only requires proper medical care. The diseases associated with the virus mainly infects the unborn fetus due to their weak immune system. This occurs after the virus carrying Aedes mosquito bites a pregnant woman. A majority of the people who are infected by this virus do not develop symptoms. If symptoms develop, they are initially generalized, presenting as fever, rash, and headaches among others. The virus has the ability to create various complications during pregnancy, but also in non-pregnant adults.ZIKV is spread by the ZIKV needs to be studied further to determine the exact mechanism through which these conditions arise. The virus is very likely to migrate beyond the Americas, and this research will attempt to provide the pathophysiology to eventually aid in the prevention of these associated conditions.


Caudovirales tailed virus morphotypes, a novel family of nontailed viruses, and the smallest  and largest  Vibrio virus genomes described. We provide general characterization and annotation of the viruses and describe read-mapping protocols to standardize genome presentation. The rich ecological and genomic contextualization of hosts and viruses make the Nahant Collection a unique platform for high-resolution studies of environmental virus-host infection networks.Viruses are highly discriminating in their interactions with host cells and are thought to play a major role in maintaining diversity of environmental microbes. However, large-scale ecological and genomic studies of co-occurring virus-host pairs, required to characterize the mechanistic and genomic foundations of virus-host interactions, are lacking. Here, we present the largest dataset of cultivated and sequenced co-occurring virus-host pairs that captures ecologically representative fine-scale diversity. Using the ubiquitous and ecologically diverse marine Vibrionaceae as a host platform, we isolate and sequence 251 dsDNA viruses and their hosts from three time points within a 93-day time-series study. The virus collection includes representatives of the three  However, understanding the structure of virus-host interaction networks in the wild still poses a major challenge as pair-wise interactions between specific viruses and their hosts cannot be predicted without isolate based studies. Thus, though viruses have been predicted to play a major role in maintaining the extensive fine-scale genomic diversity of environmental microbes, it has not been possible to systematically evaluate the mechanistic or genomic foundations of these interactions, nor their ecological and evolutionary consequences.Viruses influence the structure, function, ecology, and evolution of microbial communities. They represent the richest reservoir of nucleic acid diversity3–7 and time series studies8 available.Here, we present the annotated viral genomes of the Nahant Collection, a large-scale virus-host model system of cultivated and genome-sequenced bacterial and viral isolates, built on the extensively characterized environmental marine Vibrionaceae model system. By capturing large numbers of closely related host and virus strains, the Nahant Collection allows evaluation of the impact of ecologically relevant fine scale diversity on the interactions between bacteria and lytic viruses. This collection of 251 virus genomes and their associated pairs is a resource for interrogating the determinants of host range and the molecular bases of specific virus-host interactions within one of the most richly contextualized environmental microbial model systems8. All viruses were isolated on hosts collected on the same day, and hosts were nearly exclusively Vibrio. The Vibrio are a well-suited host group for the evaluation of the role of ecology and evolution in structuring virus-host interactions – they are ubiquitous in marine systems, ecologically diverse, and are among the most thoroughly characterized model systems for the study of bacterial populations in the wild10. The viruses were recovered using approaches designed to yield representation of diverse viruses, including: isolation from concentrates by plating in agar overlays to allow for more representative recovery of both fast- and slow-growing viruses; use of 2-week incubation times to allow for appearance of plaques by slow plaque-formers; and inclusion of additives to media to improve plaque visualization (glycerol11) and mimic environmental substrates (chitin) that might be necessary to induce expression of host receptors.Viruses and hosts were isolated from samples collected at three time points within a 93-consecutive-day study of littoral marine microbial communities, the Nahant Time SeriesTo standardize assemblies of purified viral isolate genomes we used an approach informed by predicted differences in packaging strategies among viruses, described in greater detail in the methods. This approach suggests that viruses of the Nahant Collection include members with diverse packaged genome types, including cohesive end overhangs, inverted terminal repeats, headful-packaging type terminal redundancy, and Mu-like host ends. To evaluate whether any of the viruses were prophages derived from the host of isolation, rather than environmentally-derived isolates, sequence-based searches between virus and isolation host genomes were performed; only one case of prophage purification was identified among the virus strains with sequenced host genomes .Vibrio virus genomes . Using the Virfam12Caudovirales classifier, we find that the tailed viruses include representatives of all Virfam Types and Clusters previously identified as associated with Proteobacteria, including: Type 1 Clusters 3, 5, 6, 7, 8, 9 (Siphoviridae and Myoviridae); Type 2 (Myoviridae); and Type 3 (Podoviridae), as well as 26 viruses not associated to any previously identified Virfam Types or Clusters. Analyzing portal protein phylogeny revealed groups of closely related viruses as well as extensive collection-wide portal protein diversity  and largest  described iversity . The non8. The integration of ecological context, sequence-information, and cultivation-based study available for this model system make the Nahant Collection a unique and robust foundation for the study of the role of viruses in the ecology and evolution of their bacterial hosts.The viruses and hosts of the Nahant Collection, the largest available dataset of sequenced co-occurring cultivated virus-host pairs, are embedded within the rich contextualization of the 93-consecutive-day Nahant Time Series study8 of coastal marine microbial communities at Canoe Cove, Nahant, MA, USA in 2010 : August 10 , September 18 , and October 13 . Bacteria were collected using a size-fractionation approach4 designed to partition co-occurring strains on the basis of differential associations in the water column. Here, as in previous studies of the Vibrio4, bacteria were isolated by dilution series plating of material resuspended from 63 μm, 5 μm, 1 μm, and 0.2 μm size-fractions onto vibrio-selective media (MTCBS) for colony growth and serial purification. We purified 3,456 bacterial isolates comprised of 1,152 strains from each of 3 days, evenly distributed over the size-fractions. Samples collected for isolation of viruses were 0.2 μm-filtered to remove bacteria, flocculated by addition of iron chloride, and the flocs collected on 0.2 μm filters and re-dissolved in oxalate for storage at 4˚C , and bottom agar containing glycerol and chitin . Glycerol was added to increase the visibility of plaques11, chitin was added to increase the probability of recovery of viruses dependent on chitin-induced receptors, and low density top agar was used to increase the probability of plaque formation by larger viruses16. Agar overlays were wrapped with plastic to reduce desiccation and held at room temperature for 14-16 days. Virus plaques were harvested at the end of the incubation period and archived by filtration of plaque eluates, as described in  were mixed with host cultures to form agar-overlays within which discrete plaques could form and from which viruses could be isolatedTo build a diverse and representative collection of virus-host pairs, at least one randomly selected virus was purified from each bacterial strain for which plaques appeared in the agar overlay plating of environmental concentrate. To achieve this, we serially purified viruses recovered from archived material, prepared small-scale lysates to boost viral titer, and then generated high titer stocks by confluent lysis in agar overlays. Purification resulted in genome sequencing of 283 viral strains (from 251 independent plaques) from 246 hosts, described below. Viral and host strain naming conventions are described in 17. Constructs were enriched by PCR using paired-end (PE) primers following qPCR-based normalization of template concentrations. Enrichment PCRs were prepared in eight replicate 25 μl volumes, with the recipe: 1 μl Illumina construct template, 5 μl 5x Phusion polymerase buffer, 0.5 μl 10 mM dNTPs, 0.25 μl 40 μm IGA-PCR-PE-F primer, 0.25 μl 40 μm IGA-PCR-PE-R primer, 0.25 μl Phusion polymerase, 17.75 μl PCR-grade water. PCR thermocycling conditions were as follows: initial denaturation at 98 °C for 20 sec; batch dependent number of cycles of 98 °C for 15 sec, 60 °C for 20 sec, 72 °C for 20 sec; final annealing at 72 °C for 5 min; hold at 10 °C. For each sample 8 replicate enrichment PCR reactions were pooled and purified by 0.8x SPRI bead clean-up. Each sample was then checked by Bioanalyzer (2100 expert High Sensitivity DNA Assay) to confirm the presence of a unimodal distribution of fragments with a peak between 350-500 bp. Sequencing of viral genomes was distributed over 4 paired-end sequencing runs as follows: 1 lane on the Illumina HiSeq2000 , 3 lanes on the Illumina MiSeq . Raw paired-end Illumina reads were imported and demultiplexed using CLC Genomics Workbench v.6.5.1 (https://www.qiagenbioinformatics.com/). Sequencing and assembly of genomes of bacterial hosts is described elsewhere13.Viral genomes were prepared from lysates of the host of isolation, as follows. Lysates were concentrated on centrifugal filtration devices , washed with 1:100 2216MB, and concentrates treated with nucleases to digest unencapsidated nucleic acids . Nuclease-treated samples were extracted by addition of 0.1 final volume of SDS mix , 30 min incubation at 65˚C, addition of 0.125 volumes 8 M potassium acetate, 60 min incubation on ice, addition of 0.5 volumes of phenol-chloroform, and recovery of nucleic acids from aqueous phase by isopropanol and ethanol precipitation. Illumina sequencing libraries of each extract were prepared as follows. Sample DNA (5 μg in 100 μl) was sheared by sonication  to enrich for fragment sizes of ~300 bp. Sequencing constructs were prepared by end repair of sheared DNA, 0.72x/0.21x dSPRI size selection to enrich for ~300 bp sized fragments, ligation of Illumina adapters and unique pairs of forward and reverse barcodes for each sample, SPRI bead clean-up, nick translation, and final SPRI bead clean-up18. Common examples of such strategies include production of virions with genomes that have: variable termini comprised of host DNA (Mu-like viruses); 5′ or 3′ single strand terminal overhangs (cos-viruses); or different start sequences and terminal redundancies ranging from 10 s to 10,000 s of bases (pac-viruses). To inform final curation of genome sequences, we first performed initial assemblies to group similar genomes and allow identification of the packaging-associated large subunit terminase gene (TerL) where possible. We then evaluated read mapping profiles within groups, considering terminase-predicted packaging strategy, to define final genome start sites. We next used an iterative approach, as described below, to standardize genome assemblies with conserved gene orders and genomic start positions for related viruses, and to place genomic termini at the contig ends.Differences in packaging strategies among viruses yield distinctive and characteristic distributions of packaged physical genomes in progeny virionsde novo assembly tool in CLC Genomics WorkBench v.6.5.1 with default parameters following trimming of reads . Open reading frames (ORFs) were identified using Prodigal19 with default parameters, and reciprocal best BLAST hits with ≥75% coverage of the longer sequence and e-value of ≤10−5 were clustered using OrthoMCL20. Viral genomes were clustered into genome groups on the basis of shared protein clusters using the FT algorithm of the ClustnSee21 plug-in in Cytoscape22. Preliminary curation of individual groups to assess synteny between closely related and replicate viruses  using LAST23 indicated that assemblies began at different locations, suggesting that virus genome characteristics were confounding consistency in contig start and end sites.Initial assembly and clustering of viral genomes identified groups of related viruses , but alsTo systematically address the inconsistency in contigs produced by assemblies of closely related viruses, assemblies were repeated and curated based on read mapping patterns and terminase similarities, as described below.https://www.qiagenbioinformatics.com/), using default assembly parameters and an insert size setting of 100 to 300 bp; 154 out of 285 assemblies resulted in one contiguous sequence (contig). For virus assemblies producing more than one contig, the highest coverage contig was extracted and considered the target viral genome contig; lower coverage contigs were considered contamination from host genome or prophages.Viral genomes were re-assembled using the command clc_assembler from CLC Assembly Cell  were identified using Prodigal version 2.6.1 with the -p meta flag to identify small ORFspatterns .To standardize final gene order presentations, start and stop positions for each genome were defined manually, considering three criteria: 1) terminase identity, as identified by UBLAST and OrthoMCL clustering; 2) read coverage; and 3) comparison of contigs between viruses within the genome groups identified in the initial assembly. All members of each group were assigned to a common inferred genome packaging strategy category , though best hits to short direct terminal repeat  and 5′-cohesive ends  viruses were also identified, along with cases of no similarity to reference virus terminases (20). Read coverage patterns among these viruses were dominated by either a pattern of gradual decreases/shifts (112) consistent with a ‘headful’ or packaging site (‘pac’) – based genome packaging strategy, or even coverage (46); though other patterns of coverage including short peaks  and multiple coverage peaks (2) were also observed. Examination of read coverage following TerL-based rearrangement of contigs , the majority of viruses from group 3, and a single virus from group 7.The majority of viral genomes (168/283) in the collection were standardized by circularizing the  contigs  often shThe second most commonly applied strategy for standardization (66/283) was circularization of contigs followed by re-linearization by cutting in the middle of a short region of either aberrantly low (36), or high (30), coverage . As a whIf viruses did not follow the patterns described above, but closely related members of the same group  did follow a particular pattern, viruses were assembled using closely related strains as a scaffold; this approach was used for genomes in group 5 (3).Singleton viruses with no similar members within the dataset were treated based on closest terminase identity and read coverage pattern, but if no distinct pattern was observed the original assemblies were maintained. This approach was used for 16/283 viruses, including viruses in groups 2 (8), 3 (1), 7 (5), 11 (1), and 12 (1).A subset of viruses (9/283) were found by BLAST comparison to possess Mu-like terminases, suggesting that they also used a Mu-like replicative transposition headful mechanism that incorporates host DNA upstream and downstream of the site of insertion. Read coverage patterns of initial assemblies of Mu-like viruses exhibited sharp drops in coverage at the termini followed by regions of low coverage , these r13, had distinctively short (~10 kb) genomes and did not contain predicted terminases. BLAST comparison of ORFs from these genomes showed similarity to the protein-primed DNA polymerase of viruses of the Tectiviridae, which have linear genomes and inverted terminal repeats (ITR), and thus these viruses were also evaluated for ITRs. Final assemblies for this group were performed iteratively, as follows. Following initial assembly, second and third assembly iterations were performed using an increased word size of 64, and the largest contig from the previous assembly was included as one of the “reads” for the successive round of assembly. The longest contigs from each of the three assemblies were then compared and the longest contig that also exhibited ITRs was used, when none of the contigs contained ITRs the longest assembled contig was determined to be the final assembly.A subset of the viruses (21/283), described elsewhere as a new familyViral genomes were annotated using multiple approaches and tools, as described below. Genomes are available through Genbank (Data Citation 1).12 classifier, which identifies multiple genes of the head-neck-tail modules of viral genomes and assigns viral genomes to morphotypes within Types and Clusters on the basis of previous characterization of diverse tailed viruses. Annotation was performed individually per genome by submission to the Virfam webserver (http://biodev.cea.fr/virfam/). Output reports for all Virfam annotation runs are available through figshare (Data Citation 2).Viral proteins were annotated using the Virfam28, COG29, eggnog30, Pfam31, ACLAME32, CAMERA Viral Proteins (CVP)33 and the OM-RGC collection of sequences34 via UBLAST24. Annotations were determined as the best hit (maximum bit score) to a non-hypothetical protein from EggNOG, KEGG, COG, ACLAME or Pfam . Best hits to remaining databases as well as CVP and OM-RGC are reported as notes within the final annotations. Annotations were combined with annotations identified using InterProScan version 5.17-56.0 using the iprlookup, goterms, and pathways options. InterProScan is a program from EMBL-EBI that uses the InterPro database for annotations. The InterPro database contains by default 13 databases, which are listed here: https://github.com/ebi-pf-team/interproscan/wiki/HowToRun#included-analyses. For these annotations, two optional databases were included: TMHMM for predicted transmembrane proteins and SignalP for predicted signal peptide cleavage sites. tRNA sequences were identified using tRNAscan-SE version 1.23 (ref. 36.Phage proteomes were compared to KEGGhttp://biodev.cea.fr/virfam/), and an HMM generated by performing 3 iterations of Jackhmmer37https://www.ebi.ac.uk/Tools/hmmer/search/jackhmmer. Searches of the Nahant Collection virus proteins with this collection of HMMs using the hmmsearch38 tool (hmmer version 3.1b2) identified putative portal proteins in 241 genomes, these 241 together with 6 portal proteins identified directly through the Virfam web page, were used to search the Nahant Collection with blastp39, identifying putative portal proteins in 262 of the 263 Caudovirales . In the 2 cases where the predicted portal proteins differed across the two methods (40 (https://toolkit.tuebingen.mpg.de/#/tools/hhpred) was used to evaluate both predictions and the protein with the longer sequence similarity to a portal protein was selected. The portal protein in virus 1.031.O could only be predicted using the Virfam approach and this protein was included, though HHpred and Phyre2 , specific accession numbers for each strain are provided in Given the known abundance of prophages in bacterial genomes we evaluated whether any viruses in the collection represented induced prophages from the host of isolation. Using megaBLAST in Geneious 6.1.8 we searched all virus genomes against all sequenced hosts, we identified only a single case of a high query cover and high identity match. The virus 1.202.O  shared a 30,051 bp 100% identity match with its host 10N.222.45.E8; this match region occurred within a larger host contig of 120,557 bp, suggesting that the failure to achieve a full match with the remaining 1,963 bp region of the virus genome contig was not due to incomplete assembly of the associated host region. Full genomes for the host of isolation were not available for 29 viruses and thus this prophage derivation could not be assessed for these strains, information on host sequence availability is provided in This dataset contained sets of virus pairs and triplets that served as biological replicates for assembly optimization. Such sets derive from instances of independent purification of viral sub-lineages from a parent plaque due to the occurrence of variable plaque morphology. Though they exhibited sites of polymorphism at the nucleotide level, ranging from 0 to 4 SNPs, and indels of up to 201 bp , membersHow to cite this article: Kauffman, K. M. et al. Viruses of the Nahant Collection, characterization of 251 marine Vibrionaceae viruses. Sci. Data 5:180114 doi: 10.1038/sdata.2018.114 (2018).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Fusarium is an important genus of plant pathogenic fungi, and is widely distributed in soil and associated with plants worldwide. The diversity of mycoviruses in Fusarium is increasing continuously due to the development and extensive use of state-of-the-art RNA deep sequencing techniques. To date, fully-sequenced mycoviruses have been reported in 13 Fusarium species: Fusarium asiaticum, F. boothii, F. circinatum, F. coeruleum, F. globosum, F. graminearum, F. incarnatum, F. langsethiae, F. oxysporum, F. poae, F. pseudograminearum, F. solani, and F. virguliforme. Most Fusarium mycoviruses establish latent infections, but some mycoviruses such as Fusarium graminearum virus 1 (FgV1), Fusarium graminearum virus-ch9 (FgV-ch9), Fusarium graminearum hypovirus 2 (FgHV2), and Fusarium oxysporum f. sp. dianthi mycovirus 1 (FodV1) cause hypovirulence. Rapid advances in various omics technologies used to elucidate genes or biological processes can facilitate an improved understanding of mycovirus-host interactions. The review aims to illuminate the recent advances in studies of mycoviruses in Fusarium, including those related to diversity, molecular mechanisms of virus-host interaction. We also discuss the induction and suppression of RNA silencing including the role of RNAi components as an antiviral defense response. Chrysoviridae, Endornaviridae, Megabirnaviridae, Quadriviridae, Partitiviridae, Reoviridae, Totiviridae, and Botybirnavirus, respectively), six linear positive-sense ssRNA virus families , one linear negative-sense ssRNA virus family (Mymonaviridae), two reverse transcribing linear ssRNA virus families (Metaviridae and Pseudoviridae), and one circular ssDNA virus family (Genomoviridae) , fungal viruses or mycoviruses have been classified into seven linear dsRNA virus families and one linear dsRNA virus genus , which can extracellularly infect fungi  and its derivatives  and one unassigned dsRNA group, 11 (+)ssRNA viruses from five established or provisionally designated families  and one unassigned (+)ssRNA group, and one (–)ssRNA virus from the family Mymonaviridae.Due to the advancement and widespread use of next generation sequencing (NGS) techniques, knowledge of the diversity of known mycoviruses has rapidly increased in the past few years. To date, complete genome sequences are available for 29 mycoviruses, which have been identified from different species of Fusarium , includiCrysoviridae have been isolated from Fusarium, Fusarium graminearum virus-ch9 (FgV-ch9) from F. graminearum strain China 9  from F. oxysporum f. sp. dianthi strain 116   from Fusarium solani f. sp. rohiniae strain SUF704 , and dsRNA2 (encoding the CP). However, the attempt to clone dsRNA2 of FsPV2 has failed   from F. graminearum strain AH11 , and Fusarium poae dsRNA virus 3 (FpV3) isolated from the same Fusarium poae strain SX63  has been isolated from Deltaflexiviridae has been isolated from F. graminearum strain BJ59 and is designated as Fusarium graminearum deltaflexivirus 1 (FgDFV1)  from Fusarium graminearum strain HN10  have been isolated from Fusarium, Fusarium circinatum mitovirus 1 (FcMV1) from Fusarium circinatum strain FcCa070  and Fusarium globosum (MAFF No. 237511), respectively   has been isolated from Mymonaviridae has been isolated from Fusarium graminearum strain HN1 and is designated Fusarium graminearum negative-stranded RNA virus 1 (FgNSRV-1)  and genes required for transcription and signal transduction , were significantly up-regulated in fungal host cells, which seems to be related to virus replication. In contrast, significant down-regulation of genes required for metabolism  and transporting systems in a fungal host containing the virus appears to be related to the host defense mechanism and fungal virulence  and two non hypovirulent mycoviruses (FgV3 and FgV4), respectively. Interestingly, changes in the host transcriptome caused by different mycoviruses are not always correlated with observed host phenotypes. The fungal host transcriptome was more affected by FgV1 and FgV4 infections than by FgV2 and FgV3 infections. In addition, 12 differentially expressed genes were identified in response to all four mycovirus infections, but functions of most of these genes are still unknown  was conducted with recombinant FgHex1 protein and RNA sequences derived from various regions of FgV1 genomic RNA. These analyses demonstrated that the FgHex1 functions in the synthesis of both strands of FgV1 RNA and therefore in FgV1 replication probably by specifically binding the FgV1 genomic RNA. This is the first report about the regulation of viral RNA replication by a fungal cellular protein that can directly bind to viral genomic RNA  of FgHal2 was investigated in F. graminearum using gene deletion and gene over-expression mutants. It was found that deletion of FgHal2 reduced conidiation, mycelial growth, and the production of secondary metabolites. Moreover, deletion of FgHal2 decreased viral RNA accumulation and the vertical transmission of FgV1 via conidia. Together, these results indicate that F. graminearum can down-regulate one of its major multifunctional genes, FgHal2, in response to FgV1 infection  was identified to be involved in symptom expression in F. graminearum. Downregulation of vr1 in the virus FgV-ch9 infected fungus and vr1 deletion evoke virus-infection like symptoms while constitutive expression overrules the cytopathic effects of the virus infection. Intriguingly, the presence of a specific viral structural protein P3 is sufficient to trigger the fungal response, i.e., vr1 downregulation, and symptom development similar to virus infection. Hence, vr1 represents a fundamental host factor for the expression of virus-related symptoms and helps to understand the underlying mechanism of hypovirulence. The advancements in understanding fungal infection and response may aid biological pest control approaches using mycoviruses or viral proteins to prevent future Fusarium epidemics  functions as an antiviral defense mechanism in eukaryotic organisms. The core elements of the cross-kingdom RNA silencing defense response consists of conserved ribonucleases: members of the Dicer-like (DCL) and Argonaute-like (AGO) protein families. Dicer nucleases recognize viral double-stranded and structured RNAs and use the associated RNase III-type activity to process these RNAs into small RNAs of 21–24 nts in length, termed virus-derived small (vs.) RNAs. The vsRNAs are then incorporated into an effector complex termed the RNA-induced silencing complex (RISC) with the aid of an Argonaute family protein. One strand of the vsRNA is degraded and the remaining guide strand targets the effector complex to the cognate viral RNA, which is then cleaved by the Argonaute-associated RNAse H-like activity. Subsequently, the antiviral RNA silencing response is further amplified by host RNA-dependent RNA polymerases (RdRPs) Ding, . To combem Ding, . More enF. graminearum contains genes encoding two dicer proteins (FgDicer1 and FgDicer2), two Argonaute proteins (FgAgo1 and FgAgo2), and five RdRp proteins (FgRdRp1 to 5). Chen et al. showed that RNAi machinery was not involved in growth, abiotic stress and pathogenesis in F. graminearum. In addition, it was demonstrated that the hairpin RNA (hpRNA) can efficiently silence the expression level of target gene and the argonaute protein FgAgo1 and dicer protein FgDicer2 played a critical role in the hpRNA mediated gene silencing process  and reduction of SW, LZ, and IA performed the literature search and prepared the manuscript table. LG drafted and revised the manuscript. PL and PB contributed equally to writing the manuscript. All authors revised and agree with the final manuscript 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.


Chikungunya virus (CHIKV) infection is a long known mosquito-borne disease that is associated with severe morbidity, characterized by fever, headache, rashes, joint pain, and myalgia. It is believed that virus load has relation with severity of clinical features.We performed this study to assess the relationship between virus load and clinical features in children during the acute phase of CHIKV infection, in order to draw insights for better-informed treatment.Between June 1, 2009, and May 31, 2010, 338 patients with fever and susceptive to CHIKV during first 4 days of illness were prospectively enrolled from Karnataka Institute of Medical Sciences, Hubli in our hospital based cross sectional observational study. Sybr green quantitative reverse transcription polymerase chain reaction was performed to estimate the virus load.8 copies/ml (1.7x105-9.9x109 copies/ml). Among the observed clinical features, a statistically significant difference in log mean virus load was found between patients with and without myalgia .Quantitative RT-PCR was positive for CHIKV in 54 patients. The median copy number of CHIKV was 1.3x 10Patients with myalgia had lower virus load and those without myalgia had a higher virus load. Aedes genus  in the endemic regions and it has a short incubation period. Transmission of the disease is favoured by damp climatic conditions and more cases are usually registered during monsoon season. Increased global travel by humans also contributed to the wide spread of the disease [Though Chikungunya virus (CHIKV) was known since 1952, effective and timely management options are at various stages of development [ disease . Quantit disease . Studies disease .. The variation in the amount of these individual components during different days of illness reflects in the form of varied symptoms at different time points.[In virus detection laboratories, it is an established fact that antigen based tests (PCR) give positive results during viremic phase (1–4 days of infection) and antibody based tests give positive results seroconversion phase (after 5 days of infection) . The syme points.. The tare points.. Hence we points.. In a ree points.. This prBetween June 1, 2009, and May 31, 2010, we enrolled 338 patients (2 months to 18 years old) with suspected CHIKV symptoms during the acute phase of infection (day 1–4) at the outpatient department of Karnataka Institute of Medical Sciences (KIMS), Hubli. CHIKV symptoms were defined as the presence of fever (≥ 38°C) plus any symptoms like joint pain, rash, and myalgia and/or retro-orbital pain. Splenomegaly and hepatomegaly were detected and recorded after physical examination by the clinician following standard clinical methods. Patients who are already on any prescribed mode of treatment  for present episodes, were excluded from this study.Blood samples (3-5ml) were collected in sodium citrate vials. Plasma was separated by centrifugation, three aliquots were made for each sample and sent in dry ice to the department of pediatrics, All India Institute of Medical Sciences (AIIMS), New Delhi for testing. Once the samples were received (2009–2010), one aliquot was used for IgM testing and RNA extraction and the rest two aliquots were stored at -80°C for future use.RNA was extracted using QIAamp Viral RNA Mini Kit as per manufacturer’s instructions. The extracted RNA was kept at -20°C and used for PCR testing within a week. Standard PCR with three sets of primers targeting E1 gene  was used to confirm the CHIKV positivity before their inclusion for quantification. Dengue (DENV) PCR test for all four serotypes was performed to exclude dengue infected and co-infected samples (CHIKV & DENV) from this analysis.5’CAGCAAGAAAGGCAAGTGTGCG 3’,  (21b) and reverse primer 5’TGACTATGTGGTCCTTCGGAGGG3’,  (22b) were used. Reverse transcription was performed at 25°C for 10min, 37°C for 120 min and 85°C for 5 sec followed by 40 cycles of PCR . Copy numbers in test samples were determined using a standard plot (lower limit of detection is 450 copies/ml) that was generated using in-house CHIKV RNA standards [5’-3’ CAGCAAGAAAGGCAAGTGTGCG,  and reverse primer 5’-3’ TGACTATGTGGTCCTTCGGAGGG,  and CHIKV RNA standards were prepared in house. Briefly, primers specific to 200bp region of E1 were designed to amplify the African CHIKV culture RNA. The forward primer used here was with T7 promoter sequence elongation (Forward TAATACGACTCACTATAGGGCAGCAAGAAAGGCAAGTGTGCG). The resulted PCR product was with T7 elongation and was gel purified. This purified product was used as the template for generating RNA using invitro transcription kit  following manufacturer’s instructions. The resulting RNA (sub genomic transcript) was quantified by nanodrop and concentration in nano grams was converted into copy numbers by using the formula and used as a standard after making serial dilutions. The whole experiment was performed on ABI7500 real-time machine. Briefly, RNA was extracted from 140ul of plasma samples using the QiAMp viral RNA mini kit . RNA was converted to cDNA by using high capacity cDNA reverse transcription kit  according to manufacturer’s instructions and the cDNA was subjected to amplification of 200bp region of E1 gene in Sybr green real time PCR assay setup (ABI7000) using target specific primer set. Cycle threshold (ct) values obtained were plotted against the log dilutions of the RNA generated by Invitro transcription as described earlier [For quantitative RT-PCR, RNA extraction from plasma samples was performed using viral RNA extraction kit in accordance with the manufacturer’s instructions . Sybr green quantitative RT-PCR was used to detect CHIKV by targeting 200bp region of E1, envelope gene. SYBR Green is relatively cost effective and easy to use, technically based on binding the fluorescent dye to double-stranded deoxyribonucleic acid (dsDNA) and by optimization of SYBR Green method, its performance and quality could be comparable to other TaqMan method . Forwardtandards . ConfirmCategorical variables were analyzed by chi-square/Fischer's exact test. P- Values less than 0.05 and near were considered to be statistically significant. Stata 12.0 was used for statistical analysis.Clinical features: Clinical features of patients with confirmed CHIKV infection (n = 54) were recorded. Among them, 20 were females and 34 were males. The age of the subjects ranged from 8 months to 13 years. All the patients presented with fever and other clinical symptoms. The symptoms that were frequently observed among the confirmed CHIKV cases include joint pain (94.44%), myalgia (70.37%), headache (55.56%), vomiting (51.85%) and abdominal pain (51.85%) (51.85%) . All the.85% . Al8 copies/ml (1.7x105-9.9x109copies/ml).Considering the day of fever onset as day 1, samples were collected during first 4 days of illness (day 1–4). The median copy number of CHIKV in 54 qRT-PCR positive patients was 1.3 X 10Observed clinical features were compared with estimated virus load. A significant difference in virus load was found between patients with and without myalgia (p = 0.01). The log mean virus load of the patients without myalgia is higher than in the patients with myalgia (8.34 vs 7.50). CHIKV load did not differ significantly in children with or without other clinical symptoms .CHIKV infection is now a global problem, as its spread is not limited to certain boundaries. Increased transport activities supported the transmission of the virus and number of cases are getting registered due to infected travellers all over the world ,16. MultAs observed in other studies involving other alphaviruses like Ross River virus (RRV), Sindbis virus (SINV), Mayaro virus (MAYV), O'nyong-nyong virus (ONNV), and Barmah Forest virus (BFV), virus load might have a direct influence on the expression levels of various cytokines which in turn might influence the presentation of various clinical symptoms –23. StudJoint pain was found to be the prominent feature among most of the study subjects (94.44%). Analysis of difference in the presentation of clinical symptoms between two age groups (3–7 yrs and 8–13 yrs.) showed that headache was present more in older children (8-13yrs) compared to the younger children (3–7 yrs.), (P = 0.003) . SimilarAmong all the recorded clinical features, only the presentation of myalgia varied with the difference in virus load (P = 0.0105). Interestingly, the subjects who don’t have myalgia had more virus load in them compared to subjects with myalgia. The translocation of virus particles towards myocytes coupled with host’s protective immune responses at that point of time might have some association with the reduction in virus load and manifestation of myalgia. Usually, viremia disappears after 4–5 days of infection, that’s one reason why antibody-based tests are used for detection after 5 days of infection . Any feaLimitations of the study were, 1) the association between virus load and days of illness was not clearly identified in our study because we did not have serial samples from the same patient. 2) Not estimating the role of cytokines and antibody levels in manifestation of clinical symptoms.In our study we estimated virus load in 54 positive CHIKV patients by qRT-PCR and analysed it with presented clinical features . Virus lS1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Further, we focus on recent genetic association studies and investigations utilizing genetically modified mouse models that identified SLC26A9 as a disease modifier and supported an important role of this alternative chloride channel in the pathophysiology of several organ manifestations in CF, as well as other chronic lung diseases such as asthma and non-CF bronchiectasis. Collectively, these findings and the overlapping endogenous expression with CFTR suggest SLC26A9 an attractive novel therapeutic target that may be exploited to restore epithelial chloride secretion in patients with CF irrespective of their CFTR genotype. In addition, pharmacological activation of SLC26A9 may help to augment the effect of CFTR modulator therapies in patients with CF carrying responsive mutations such as the most common disease-causing mutation F508del-CFTR. However, future research and development including the identification of compounds that activate SLC26A9-mediated chloride transport are needed to explore this alternative chloride channel as a therapeutic target in CF and potentially other muco-obstructive lung diseases.The solute carrier family 26, member 9 (SLC26A9) is an epithelial chloride channel that is expressed in several organs affected in patients with cystic fibrosis (CF) including the lungs, the pancreas, and the intestine. Emerging evidence suggests SLC26A9 as a modulator of wild-type and mutant CFTR function, and as a potential alternative target to circumvent the basic ion transport defect caused by deficient CFTR-mediated chloride transport in CF. In this review, we summarize  CFTR variants have been identified in CF patients, with F508del being the most common accounting for 70% of all CF mutations (CFTR genotype (Cystic fibrosis (CF) is a severe life-shortening multiorgan disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) chloride channel, which plays a fundamental role in salt and fluid transport across the surfaces and ducts of many epithelial organs including the lungs, pancreas, and gastro-intestinal tract. In patients with CF, CFTR dysfunction results in impaired epithelial ion and water transport that leads to a characteristic multi-organ pathology . More, tutations . Diseaseutations . Drug deutations . These iutations . The larutations . Emergingenotype . In thisSLC26A9 is a member of the solute-linked carrier 26 (SLC26) anion transporter family that functions uniquely as a chloride channel with minimal conductance to bicarbonate . SLC26A92+ benzoic acid (NPPB), while anion-exchanger inhibitor 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) only partially inhibited SLC26A9 . On the The constitutive channel activity suggests that SLC26A9-mediated chloride secretion is controlled by the insertion and stability of SLC26A9 channels in the plasma membrane. Previous studies demonstrated that SLC26A9 cell surface expression is influenced by WNK (with no lysine [K]) kinases that have been implicated in the regulation of cell volume homeostasis and ion transport processes in epithelial cells . WNK kinNotably, there is a large overlap in the expression pattern of SLC26A9 and CFTR in epithelial tissues and emerging evidence suggests a complex, multifaceted interaction between the two channels, both under physiological and pathophysiological conditions. First, there is a functional interaction between the two channels mediated by the STAS domain of SLC26A9 and the R domain of CFTR. Although an inhibitory relationship has also been suggested , the natBiochemical and functional studies of two other SLC26 family members, SLC26A6 and SLC26A3, showed that following cAMP-dependent stimulation, the phosphorylated R domain can bind to STAS domain, which leads to mutual activation of both CFTR and SLC26 transporters . Coding +/H+ exchanger-3 regulatory factor 1 (NHERF1) and CFTR-associated ligand (CAL), which display antagonistic effects in CFTR trafficking .Third, numerous genetic association studies and investigations utilizing genetically modified mouse models supported an important role of SLC26A9 in the pathophysiology of several organ manifestations in CF, as well as other chronic lung diseases such as asthma and non-CF bronchiectasis . The resin vitro modulatory effect, the lack of SLC26A9 association with lung function in F508del homozygous patients is difficult to reconcile. One explanation may entail the F508del dependent trafficking defect of SLC26A9 and the necessity of CFTR plasma membrane targeting for SLC26A9 function discussed above  is a chronic lung disease involving airway mucus obstruction, recurrent infections, airway inflammation, and remodeling that resembles CF lung disease in many aspects . Interes− secretion, which prevented airway mucus plugging in presence of mucus hypersecretion (Slc26a9-deficient (Slc26a9−/−) mice displayed normal lung morphology and no differences in epithelial ion transport when compared to wild-type mice. However, in the presence of allergic inflammation Slc26a9−/− mice lacked upregulation of chloride secretion and mucus hypersecretion led to airway mucus plugging. Furthermore, three SNPs at 3′ UTR of SLC26A9 were identified that were associated with a risk for childhood asthma, with an odds ratio of 1.48. Functional investigations revealed that one of these non-coding variants altered a putative micro-RNA response element in the 3′ UTR of SLC26A9 and lead to micro-RNA mediated translational repression in vitro, indicating that decreased SLC26A9 protein expression may be the underlying mechanism for disease association  mice show a severe meconium ileus-like intestinal phenotype characterized by severe intestinal plugging. When −/−Cftr mice were crossbred with −/−Slc26a9 mice, mortality highly increased, indicating that SLC26A9-mediated anion secretion ameliorates meconium ileus  in CF is present from early life in nearly all patients with severe CFTR genotypes. CFTR is expressed in the pancreatic ductal epithelium and regulates the secretion of an alkaline, bicarbonate rich pancreatic fluid that washes out digestive enzymes produced by acinar cells. CFTR dysfunction leads to an acidic, low volume, protein rich secretion that can slow down or even block the outflow of zymogens from the ductal tree . Early hPancreatic insufficiency confers a major risk for CFRD. CFRD is an age-dependent complication that affects 2% of children and 50% of adults with high heritability . A GWAS CFTR genotype, including patients with non-sense or splicing mutations, where no CFTR protein is made, or other rare CFTR mutations that do not respond to emerging CFTR modulator therapies , pharmacological activation of SLC26A9 may also augment functional rescue of mutant CFTR achieved by CFTR modulator therapies . In addition to compounds that facilitate SLC26A9 trafficking to increase the number of channels, SLC26A9-mediated chloride secretion may be augmented by compounds that enhance the open probability of the channel or its stability in the plasma membrane. Single channel recordings demonstrated SLC26A9 channels with open and closed states and a conductance of ∼3.2 pS (In summary, SLC26A9 constitutes an alternative chloride channel that is implicated in coordinated ion and fluid secretion in various epithelial tissues affected in CF including the airways, the pancreas and the gastro-intestinal tract . Demonstherapies . Given therapies . However ∼3.2 pS  suggesti ∼3.2 pS . In addiAB and MM conceived and designed the review, contributed to analysis and interpretation of published literature, and drafted the article and revised it critically for important intellectual content.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.


In our study, we could break the long-standing assumption that the Lmeq isoform is an indicator for virus attenuation. Using recombinant viruses that express the different CVI-meq isoforms, we could demonstrate that both isoforms drastically differ in their abilities to promote pathogenesis and tumor formation in infected chickens.Marek’s disease virus (MDV) is one of several oncogenic herpesviruses and causes fatal lymphomas in chickens. The current “gold standard” vaccine is the live-attenuated MDV strain CVI988/Rispens (CVI), which is widely used and efficiently prevents tumor formation. Intriguingly, CVI expresses two predominant isoforms of the major MDV oncogene  meq. The most frequently used Marek’s disease vaccine is the live-attenuated CVI988/Rispens (CVI) strain, which efficiently protects chickens and prevents tumorigenesis. Intriguingly, CVI expresses at least two isoforms of meq; however, it remains unknown to what extent these isoforms contribute to virus attenuation. In this study, we individually examined the contribution of the two CVI-meq isoforms to the attenuation of the vaccine. We inserted the respective isoforms into a very virulent MDV (strain RB-1B), thereby replacing its original meq gene. Surprisingly, we could demonstrate that the longer isoform of meq strongly enhanced virus-induced pathogenesis and tumorigenesis, indicating that other mutations in the CVI genome contribute to virus attenuation. On the contrary, the shorter isoform completely abrogated pathogenesis, demonstrating that changes in the meq gene can indeed play a key role in virus attenuation. Taken together, our study provides important evidence on attenuation of one of the most frequently used veterinary vaccines worldwide.Vaccines play a crucial role in the protection of animals and humans from deadly pathogens. The first vaccine that also protected against cancer was developed against the highly oncogenic herpesvirus Marek’s disease virus (MDV). MDV infects chickens and causes severe immunosuppression, neurological signs, and fatal lymphomas, a process that requires the viral oncogene, IMPORTANCE Marek’s disease virus (MDV) is one of several oncogenic herpesviruses and causes fatal lymphomas in chickens. The current “gold standard” vaccine is the live-attenuated MDV strain CVI988/Rispens (CVI), which is widely used and efficiently prevents tumor formation. Intriguingly, CVI expresses two predominant isoforms of the major MDV oncogene meq: one variant with a regular size of meq (Smeq) and one long isoform (Lmeq) harboring an insertion of 180 bp in the transactivation domain. In our study, we could break the long-standing assumption that the Lmeq isoform is an indicator for virus attenuation. Using recombinant viruses that express the different CVI-meq isoforms, we could demonstrate that both isoforms drastically differ in their abilities to promote pathogenesis and tumor formation in infected chickens. Intriguingly, we found that viruses with these vaccine-derived meq isoforms strikingly differ in pathogenesis and oncogenesis in infected chickens.Marek’s disease virus (MDV) is a lymphotropic alphaherpesvirus that infects chickens and causes 1 to 2 billion dollar losses worldwide annually , 4. The meq . The Meqmeq –10. One NA (vTR) , a noncoNA (vTR) . One of s (Smeq)  13). Th. Thmeq  virus . We could demonstrate that the meq gene expression of vSmeq or vLmeq was comparable to the meq expression in the wild-type virus (To determine if insertion of the e) virus  and C, ipe virus .meq isoforms affect virus replication, pathogenesis, and/or tumor formation in vivo, we infected 1-day-old Valo specific-pathogen-free (SPF) chickens subcutaneously with 4,000 PFU of wild-type virus, vSmeq, or vLmeq. The viral load in the blood was assessed by qPCR and revealed that the recombinant viruses replicated efficiently in infected animals , while a lower incidence was observed for wild-type virus (84%). Consistently, contact animals infected via the natural route with vLmeq had a significantly higher disease incidence (45%) compared to the wild-type control (9%). No pathogenicity was observed in the vSmeq contact animals  compared to wild-type virus (76%)  , while nus (76%) . In lineus (76%) . In the us (76%) . This inus (76%) . Intriguus (76%) , highligLmeq isoform, we examined the expression of vTR in cells infected with the wild type, vSmeq, and vLmeq using RT-qPCR. Remarkably, the Lmeq isoform significantly upregulated vTR by 12-fold compared to the already highly expressed RB-1B-carried meq and Smeq, suggesting that the 180-bp insertion in the transactivation domain strongly influences vTR expression and in turn transformation efficiency in chickens  compared to the Smeq. Both isoforms are only expressed in the CVI vaccine and have not been detected in other MDV strains, such as RB-1B and MD5  plays a role in transformation and (ii) is regulated by the Meq protein. It has previously been shown that the Meq protein modulates the expression levels of vTR, which plays a crucial role in MDV-induced lymphomagenesis and tumor dissemination via cellular c-myc  were prepared from 11-day-old specific-pathogen-free (SPF) chicken embryos  as described previously . Cells wSmeq or Lmeq (GenBank accession no. AY243333 and AY243338) derived from the commercial CVI988/Rispens vaccine strain  of the very virulent MDV strain RB-1B, which lacks most of the internal repeat long region , which is rapidly restored upon virus reconstitution (meq region had to be manipulated by two-step Red-mediated mutagenesis as described previously (meq substitution in both loci as confirmed by PCR (meq gene and then introduced either Smeq (vSmeq) or Lmeq (vLmeq). We confirmed the BAC clones by RFLP, PCR, and Sanger and Illumina MiSeq sequencing (Illumina’s v3 chemistry for 600-bp paired‐end sequencing) to verify the integrity and the sequence of the entire virus genome. The primers used for mutagenesis and sequencing are listed in We generated recombinant viruses each harboring either e strain . Smeq antitution . Therefoeviously , 27, whid by PCR . First, n = 50) were taken, and plaque areas were determined using Image J software (NIH).The spread and replication of the recombinant viruses were first analyzed by plaque size assays as described previously . BrieflyPlaque size data were confirmed by qPCR-based multistep growth kinetics as described previously . Brieflymeq isoforms are expressed comparable to its counterpart in wild-type RB-1B, we quantified the expression levels of the different meqs using RT-qPCR as previously described . We also used this approach to examine the expression of vTR in cells infected with the wild type and vSmeq and vLmeq strains by RT-qPCR. The vTR expression levels were normalized to the expression levels of cellular GAPDH (To ensure that the CVI-escribed . Brieflyar GAPDH . The prin = 25), vSmeq (n = 25), and vLmeq (n = 25). Each group was cohoused with 11 noninfected contact animals to assess the natural transmission of the respective virus from experimentally infected birds. The animal experiment was approved by the Landesamt für Gesundheit und Soziales in Berlin, Germany  and was conducted according to relevant national and international guidelines for humane use of animals. Animals were monitored daily for clinical symptoms throughout the 86-day experiment.The replication properties, pathogenesis, and tumorigenesis of the recombinant viruses were assessed in specific-pathogen free (SPF) chickens as described previously . In the n = 16) or vLmeq (n = 17). This animal experiment was approved by the Agricultural Animal Care and Use Committee . Animals were monitored for clinical symptoms throughout the 43-day experiment.The phenotype of the vLmeq was confirmed in a second, independent animal experiment. White leghorn chickens  were inoculated with 1,000 PFU of either the wild type . Analysis for plaque size assays and growth kinetics included one-way analysis of variance (ANOVA). Fisher’s exact test and Kaplan-Meier survival analysis along with the log-rank test (Mantel-Cox test) were used for analyses of the animal experiment data with Bonferroni correction on multiple comparisons. Differences were considered significant if


Free from the limitations posed by exogenous scaffolds or extracellular matrix-based materials, scaffold-free engineered tissues have immense clinical potential. Biomaterials may produce adverse responses, interfere with cell–cell interaction, or affect the extracellular matrix integrity of cells. The scaffold-free Kenzan method can generate complex tissues using spheroids on an array of needles but could be inefficient in terms of time, as it moves and places only a single spheroid at a time. We aimed to design and construct a novel scaffold-free bioprinter that can print an entire layer of spheroids at once, effectively reducing the printing time. The bioprinter was designed using computer-aided design software and constructed from machined, 3D printed, and commercially available parts. The printing efficiency and the operating precision were examined using Zirconia and alginate beads, which mimic spheroids. In less than a minute, the printer could efficiently pick and transfer the beads to the printing surface and assemble them onto the 4 × 4 needles. The average overlap coefficient between layers was measured and found to be 0.997. As a proof of concept using human induced pluripotent stem cell-derived spheroids, we confirmed the ability of the bioprinter to place cellular spheroids onto the needles efficiently to print an entire layer of tissue. This novel layer-by-layer, scaffold-free bioprinter is efficient and precise in operation and can be easily scaled to print large tissues. With the development of cutting-edge strategies and techniques for generating complex, functional tissues, bioprinting, or three-dimensional 3D) printing of biological materials, has become a critical tool in tissue engineering D printin,5,6,7. BCells, however, possess the innate ability to produce their scaffold material in the form of extracellular matrix (ECM) as they grow and interact with one another ,17,18,19The Kenzan method utilizes an array of stainless steel microneedles to provide a structure that the spheroids can be punctured onto to facilitate the fusion process without additional materials . This meWe hypothesize that a device which can print spheroids with a layer-by-layer method will not only reduce the manufacturing time of clinically relevant-sized tissues but also assists in advancing the field of tissue engineering by introducing an affordable alternative for printing scaffold-free, spheroid tissues. The research described here is focused on the design, construction, and testing of such a bioprinter. We also show that we were able to build a working and affordable prototype which efficiently and accurately transfers cellular spheroids to a needle array, one 3 mm × 3 mm × 1 mm layer at a time.The printing method used by the utilized bioprinter relies on the accurate and precise placement of needles for the proper fusion of spheroids into a single tissue. Needle arrays Aii,Fi weThe bioprinter A was desThis scaffold-free bioprinter was controlled using an Arduino  and a custom-written script. It controlled the stepper motor  for moving the print head, the servomotor  for moving the spheroid stage, the vacuum pump for aspirating the spheroids, and the end stop  mechanism for calibrating the stepper motor prior to beginning the printing process. The bioprinter was designed to first automatically calibrate to ensure that the print head is in the proper position before beginning. Once it has calibrated, the Arduino program prompts the user to load the spheroids into the spheroid stage in the round bottom plate B. Next, Glass beads were used to determine the efficiency of the vacuum and print head system. Glass beads approximately 700 µm in diameter  were used in place of cultured spheroids because of the number of tests that needed to be completed and the non-sterile conditions of the testing area. This diameter was chosen because it is similar to the 800 µm diameter of the spheroids that our lab has previously produced . These bAlginate beads were chosen as a reasonable spheroid surrogate to test the ability of the bioprinter to place spheroids on the needle array effectively. As mentioned previously, spheroids that would be used with this system are approximately 800 µm in diameter, so the alginate beads needed to have a similar diameter. These beads were generated by adding a solution of 1.5% sodium alginate  in deionized water (DI) water to a 100 mM solution of calcium chloride  in DI water . The alg2 with mTeSR . Fresh medium was added daily until the cells reached 90% confluency. The cells were seeded and grown in suspension according to the manufacturer’s protocol [5 cells per mL in 30 mL of mTeSR 3D Seed Media . This solution was then cultured in a 125 mL shaker flask at 70 rpm with daily media additions of 3.4 mL. Prior to their use with the bioprinter, the spheroids were filtered through a 500 µm reversible strainer  and collected in 5 mL of mTeSR.Human induced pluripotent stem cells (hiPSCs) were cultured on Matrigel-coated dishes at 37 °C under 5% COprotocol . BrieflyData are shown in the form mean ± standard error of the mean (SEM). Significance was chosen as p < 0.05. This was determined using both one- and two-way analysis of variance (ANOVA) along with the Student’s t-test. These analyses were performed utilizing Microsoft Excel’s data analysis software package.For image analysis, ImageJ  was used to estimate the overlap coefficient between each layer of beads and to generate representative pictures. Also, it was used in the diameter measurements of the alginate beads that were synthesized. Images and videos in this experiment were taken with a stereomicroscope , a phase-contrast microscope , and a Canon camera .To test the ability of the vacuum pump system and print head to pick up spheroids, glass beads similar in diameter to the average spheroids that would be used with this printing setup were used. These beads, according to the supplier’s specifications, had a diameter of 700 ± 70 µm on average A, which To further optimize the printing process, the time during which the print head remained in the bead container was varied  to determine the optimal time needed to pick up most of the beads. For each time, the corresponding efficiencies (n = 13 for each time point) were calculated E. From tIn addition, due to the fact that as spheroids are removed, more empty space in the spheroid container would be present, a test was done to determine if the number of beads within the container affected the capture efficiency. Beads were added to the dish at amounts of 100, 500, 1000, 2500, and 5000, estimated on a mass basis. The print head was used to capture beads a number of times (n = 10 for each group), and the subsequent efficiencies were determined (average efficiency: 97.6 ± 0.2%) F. There The repeatability of the bioprinter ability to place the beads in the same area with each subsequent layer was tested to ensure that the spheroids could accurately be placed on the needles when generating tissues. This was determined by placing a full layer of beads into a pre-determined area on a layer of silicone multiple times. After every single layer was placed, an image was taken of the beads, with each placed layer given a different color A. A compSmall beads made from alginate were chosen as a surrogate for cellular spheroids for testing the bioprinter’s ability to print onto the needle arrays correctly. Alginate beads were synthesized using a dropwise technique  and sepaAdditionally, to determine how effectively the bioprinter could print multiple layers of spheroids, two layers were printed onto the needles . From thWe confirmed the potential of our bioprinter to place cellular spheroids onto the needles in a layer-by-layer fashion. For this, hiPSC spheroids were grown to a similar diameter of, on average, 718 ± 77 µm  and addeIn this study, we were able to construct a novel and affordable bioprinting device with the ability to produce scaffold-free tissues through the utilization of layer-by-layer printing of cellular spheroids. This device could allow the enhanced production of viable tissues to be used in drug testing, translational research, and various clinics. With the design and development of the layer-by-layer print head, efficiently picking up the spheroids from a reservoir was paramount for the overall success of the bioprinter. Through testing with beads of size similar to that of the spheroids, we were able to achieve nearly 98% efficiency when picking up the beads in a single 4 × 4 layer. Although the print head designed in this project could only transfer a maximum of 16 beads or spheroids, changes can easily be made to the number and arrangement of holes in the bottom of the print head. This would allow for the construction of both larger tissues as well as custom arrangements to be used in different applications where 3D models of the native tissue microenvironment are needed ,31In addition, we were able to show the reliability of the bioprinter to print in a localized position repeatedly. The lowest overlap coefficient calculated between any two layers was 0.997. The closer to 1.0 this value is, the more colocalized the beads of different layers are. This corresponded to the precise placement of the spheroids on the needles when testing with hiPSC spheroids. This precision will allow for the proper alignment of layers in the construction of native tissue microenvironments as well as any cellular gradients found within tissues. The reproducibility and speed of printing viable tissues are critical for the success of bioprinting in the clinical environment ,33. The Compared to other methods which utilize single-spheroid printing, a process requiring a multitude of precise positioning controls and visual analysis, this layer-by-layer method allows to straightforwardly pick up and place the spheroids . This noDespite the advancements in scaffold-free spheroid printing that have been shown here, there remain a few limitations in this presented design. One in particular is the inability to customize the size and shape of the tissues. Future iterations will need to have the ability to print different tissue shapes  to expand the capabilities to other fields of tissue engineering. Also, the current design is limited to holding only one type of spheroid for printing. Having the choice to print layers of different types of spheroids would increase the customization potential. Further design changes could allow for the placement of multiple spheroid reservoirs on the spheroid bath stage of the bioprinter. In conclusion, we present here a custom device for printing spheroids using a novel layer-by-layer method. This bioprinter can efficiently pick up and transfer spheroids to the printing stage in a single, complete layer, decreasing the printing time of large tissue constructs. With this precise and accurate transfer of spheroid-like beads to the printing surface, custom tissues can easily be constructed on the needles in future experiments. Also, we were able to prove, using hiPSC spheroids, that we can successfully print a single layer of spheroids onto the needle array printing surface. In addition, we were able to build and manufacture this device at an affordable price, making it easier for other researchers to use the technology if brought to the market. Although there are additional design considerations that can be incorporated into future iterations, this device helps to advance and improve the growing area of scaffold-free bioprinting and could one day become a go-to method in tissue engineering.