Precise quantification of molecular targets in a biological sample across a wide dynamic range is a key requirement in many diagnostic procedures, such as monitoring response to therapy or detection of measurable residual disease. State of the art digital PCR assays provide for a dynamic range of four orders of magnitude. However digital assays are complex and require sophisticated microfluidic tools. Here we present an assay format that enables ultra-precise quantification of RNA targets in a single measurement across a dynamic range of more than six orders of magnitude. The approach is based on hydrogel beads that provide for microfluidic free compartmentalization of the sample as they are used as nanoreactors for reverse transcription, PCR amplification and combined real time and digital detection of gene transcripts. We have applied these nanoreactor beads for establishing an assay for the detection and quantification of BCR-ABL1 fusion transcripts. The assay has been characterized for its precision and linear dynamic range. A comparison of the new method against conventional real time RT-PCR analysis (reference method) with clinical samples from patients with chronic myeloid leukemia (CML) revealed excellent concordance with Pearsons correlation coefficient of 0.983 and slope of 1.08. Digital amplification techniques  such a dPreviously crosslinked polymer beads have been used for templating digital reaction compartments. This approach provided for a small reaction space at the interface between the hydrogel bead and the surrounding oil. In this work we are employing monodisperse non-crosslinked hydrogel beads comprised of agarose and gelatin. The composition has been found mechanically stable to sustain the handling during the assay procedure. Moreover, the matrix binds RNA template in RT-PCR buffer. Another important feature of the bead matrix is its thermal hysteresis with a gelling temperature of 40°C and a melting temperature of 80°C in the employed RT-PCR buffer. This allows for robust handling below the melting temperature and for minimized inhibitory effects at the temperatures used during thermocycling for PCR which would be otherwise observed due to steric hindrance by the gel matrix. Fluorescence detection has been found equally possible at any temperature in hydrogel or liquid state .The nanoreactor beads are provided in RT-PCR buffer and are exposed to extracted RNA mixed with RT-PCR reagents. The hydrogel composition is designed to allow the reagents to diffuse into the interior of the beads. Following an incubation step for reagent take-up and nucleic acid binding the beads are transferred to a fluorocarbon oil containing an emulsifier. The bead suspension is then transferred to a reaction and detection chamber (RDC)  designedThe set-up has been applied to establish a detection format that combines endpoint  and real time detection in each individual sample partition provided by the respective nanoreactor bead. We found that by analyzing only a modest number of 10,000 beads if compared to other digital assay methods an assay with an unprecedented dynamic range can be realized , 9–11. TA solution containing 1.5% Acetone-insoluble gelatin from bovine skin type A G1890 (Sigma) or porcine skin type B G9391 (Sigma) and 0.5% Low-gelling 2-Hydroxyethyl agarose  has been prepared in nuclease-free water (Carl Roth) and incubated at 50°C under gentle agitation (750rpm). The solution has been used to generate low dispersity microbeads on the μEncapsulator system (Dolomite microfluidics) using a fluorophobic droplet junction chip (100μm) with a 4-way linear connector to interface the fluidic connection between tubing and chip. Two Mitos P-Pumps were used to deliver the hydrogel solution and the carrier oil . The system was modified with an integrated heating rig which is placed on top of a hot plate allowing maintaining the gelatin/agarose hybrid solution in liquid state and heating up the driving fluid ensuring consistent temperature for all components and liquids. Picosurf-1 and the hybrid hydrogel solution were both pre- filtered with a 0.22μm filter before they were placed into their respective reservoirs. Temperature of the heating rig is set to 55°C to heat the gelatin-agarose hydrogel as well as the droplet junction chip. The nanoreactor beads were collected in a tube on ice and incubated for a minimum of 48h at 4–8°C before phase conversion. Thereafter, the excess of Picosurf-1 was removed from the nanoreactor bead emulsion by aspiration with a pipet and 0.5 volume 1H,1H,2H,2H-perfluorooctanol  and 3 volume H2O were added to the same volume. The tube was vortexed to break the emulsion and centrifuged for 5s at 2,500 x g. The aqueous phase with the beads was transferred to a fresh tube and the beads were washed 3x with 7 volume H2O. Excess PFO was removed by transferring again the aqueous phase with the beads into a new tube. The nanoreactor beads were washed 4x with an equal volume of nuclease-free water, aliquoted and stored at 4°-8°C for further use.™ IV VILO™ from ThermoFisher, followed by a PCR as described elsewhere , but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.2.2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.  http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and  there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.”  the recruitment date range (month and year), b) a description of any inclusion/exclusion criteria that were applied to participant recruitment, c) a table of relevant demographic details, d) a statement as to whether your sample can be considered representative of a larger population, e) a description of how participants were recruited, and f) descriptions of where participants were recruited and where the research took place.4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository  and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.5. We note you have included a table to which you do not refer in the text of your manuscript. Please ensure that you refer to Table 2 in your text; if accepted, production will need this reference to link the reader to the Table.Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: YesReviewer #3: I Don't Know**********3. Have the authors made all data underlying the findings in their manuscript fully available?Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********5. Review Comments to the AuthorReviewer #1: Overall, the manuscript is well written. The following are some suggestions for the authors to consider for a revised manuscript.Major:Since this is mainly an assay development manuscript, the data on assay quality assessment are somewhat insufficient. Only the linear dynamic range is provided. What about assay specificity? Intra-day vs. inter-day reproducibility? Is there any optimization data on beads: samples ration? How about the LLOQ of this assay?Minor:All the texts in every figures are fuzzy and cannot to be recognized.Reviewer #2: The authors present a way to compartmentalize biomolecules using hydrogel beads. They explain very well why this is important in the introduction to achieve true quantitative measurements of nucleic acids as current systems having the q in their name are not really quantitative. This is a very important issue as the COVID19 pandemia have demonstrated. The manuscript is technically sound and well structured. I must say I enjoyed reviewing this manuscript. Hence, I do recommend its publication with some minor revisions. Below, there are a number of recommendations and questions that I'd like the authors to address.I suggest authors to add a sentence in the introduction to describe the differences between their method and the ones in ref 7 and 8 of their manuscript as this is an important justification of their work.Regarding the cell based assays authors should refer how cells were cultured and also number of cells used. These number of cells could be used to translate to the number of transcript per cell detected. The number of a transcript per cell is always difficult to determine hence this technology could be useful to do so.One aspect which is not clear to me is the melting and reforming step of the process. In Fig. 1 (iv.) is said "bead melting, PCR and fluorescence detection". As the reading is done with beads, how long does it take to reform the beads? Are beads melted during the whole PCR process? So, I suggest to explain this step with more detail and add pictures of the 3 min denaturation step either in the main text or supplementary information.When a sample gives #dark beads <0.5%, it is then quantified by Ct and calibration curve. If that sample is diluted to have a #dark beads >0.5%, it is quantified by Poisson analysis. Are the quantifications equal, taking into account the dilution factor? Fig. 5 might answer it but it is not clear to me if that samples are serial dilutions or not.Do you need to do a calibration curve per run?Duration of each step in the workflow scheme could be useful for the reader to understand how long the sample-to-result takes.More details of the commercial suppliers of reagents are needed. It might be good to have a General section at the beginning of the Material and Methods section containing these details.TaqMan probes references are not provided.While authors give a reference for primers and probes, a Table describing them would be very helpful for readersFig. 1. A scale bar is missingLine 247 there is a reference errorReviewer #3: The authors report the development of a 'naoreactor bead' based assay for the quantification of mRNA targets. It appears that the use of nanoreactor beads can be used in quantification of mRNA targets spanning six order of magnitude.The authors build their manuscript on an argument by stating that digital assays are complex and require sophisticated microfluidic tools. Unless I am missing some important aspects/details the authors do generate 'nanoreactor beads' using a microfluidics chip, and the proposed method by all means not an unsophisticated one. Hence it is not clear to me why they prefer to 'sell' their method to be microfluidics-free and 'easy'.The manuscript text appears to be not submission ready given the overwhelming number of points I could comment on. The text could benefit from a thorough editing, especially paying attention to inconsistent terminology use that confused me on multiple occasions, especially the nanoreactor, digital nanoreactors, and alike…There is also no statistical analysis section provided within the Materials and Methods section. And finally the method section can benefit from a detail oriented focus.Given the current status of the manuscript, I would conclude resubmission after major revision; however, in this journal it corresponds to major revision hence my decision.Below I list my various comments based on the page and line numbers:Cover page: there are typos in the keywords!Page 2, Line 16: good agreement -> Can the authors use a quantitative measure. Also while the title reads 'ultra-precise' the closing statement of the abstract states 'good agreement'. It is ultra-precise or good? P3, L23: partitions -> It is not clear what is meant by partition. Can the authors clarify (the figure 1 legend reads beads. Can you please tune the term use for consistency/clarity).P3, L24: Poisson criteria -> Poisson distribution (criteria?). Can you please clarify what the criteria is/are?P3, L29: digital compartments -> for consistent terminology use can you please stick to one term whether it is the digital compartments/beads/nanoreactor compartment/nanoreactor beads/microbeads/sub-nanoliter droplets (whichever term the authors prefer so long as it is used consistently throughout the manuscript.)P3, L29: generating digital compartments -> Can you present how your droplet generation does not necessitate microfluidics as compared to the standard. Also lay out the details of the concrete differences between the two techniques for droplet generation.P3, L35: 'amplification reagents and the extracted nucleic acid.' -> 'RT-PCR amplification reagents and the extracted nucleic acids (GUS8 and BCR-ABL RNA).'Also the text reads BCR-ABL1 RNA. Please stick to one term.P3, L 40: Please state the size of the beads.Figure 1. In panel a please add sub-sections i., ii, … so that the figure complies with the figure legend.Figure 1b and c. Can you provide the scale.P4, L47: extensive shaking -> Can you provide reproducible quantitative information.Figure 2 legend:. Can you please use the same terms both in the figure and the legend, i.e. Fluorescence Micro-imager vs epi-fluorescence microscope, BLINK vs Blink…. Can you provide the technical details of the chamber i.e. material, dimensions, thickness, alike… in the legend.. Please state the company and also provide a link to the software/website.P5, L69: nanoreactor compartment -> see comment P3, L29P5, L70: modest number -> Please provide quantitative information wherever possible.P5, L71: highly precise -> Can the authors avoid the use of qualitative adverb and state the quantitative findings.P5, L89: fluidic connection between tubing and chip -> Please provide a link to the chip or provide the design in supplementary.P5, L99: was removed -> was removed by?P6, L112: e13a2 or e14a2 BCR-ABL1 -> Please describe what e13a2 and e14a2 are.P7, L113: BCR-ABL-1 -> BCR-ABL1P7, L119: HL60 and K562 -> Please describe what are HL60 and K562.P7, L124: as described elsewhere -> Please provide all the necessary details in the methods section.P8, L143: latter -> There is a single removal step in the previous sentence. Please re-phrase.P8, L148: parameters -> Please provide the full details.Figure 3 legend: Please describe the different beads in the legend. Preferably arrows can also be used to highlighting the specific beads.P11, L208: ERM-AD623 -> What is ERM-AD623? Please describe.P11, L209: (data not shown) -> Please provide the necessary data.P11, L211: high copy numbers -> high copy numbers (of what?)P11, L213: The concentration levels represent the expected measuring range for clin(ic)al samples -> Please provide data or a reference to support this statement.Figure 5: BCRLAB -> BCR-ABL1.Also please use different colours for dots and triangles.P13, L247: Table 1. Error! Reference source not found. -> Cross reference error?P14, L260; L267: The molecular response (MR) -> Acronym was already introduced in line 258.P14, L267: considered -> Please provide a reference for this statement.P14, L273: precision -> Please provide a reference for this statement.P15, L285: 53.000 -> 53,000?P15, L294: Blink test -> What is a Blink test?P16, L303: the reference method -> Please help the reader by providing a legend that provides all the relevant information.P16, L307: same data -> which data?P16, L318: microfluidics free digital molecular assays -> Unless I am missing the picture, the nanoreactor beads were generated using a Dolomite microfluidics chip. So how can the authors claim that the process is microfluidics free?P16, L320: bead-nanoreactors -> is this the same as nanoreactor beads?P16, L324: for the ratios -> for the ratios ofP16, L329: current laboratory standard -> which standard?Figure 6 and 7: BCRLAB -> BCR-ABL1.(applies to the entire text). real time - real-time. rtPCR - RT-PCR. have a white space before units, e.g. #µL -> # µL.. em dash between numerical values, e.g. 4-8°C -> 4–8°C. use a minus sign and not a dash sign.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  is said "bead melting, PCR and fluorescence detection". As the reading is done with beads, how long does it take to reform the beads? Are beads melted during the whole PCR process? So, I suggest to explain this step with more detail and add pictures of the 3 min denaturation step either in the main text or supplementary information. • This is now better explained in the text (line 42-71)When a sample gives #dark beads <0.5%, it is then quantified by Ct and calibration curve. If that sample is diluted to have a #dark beads >0.5%, it is quantified by Poisson analysis. Are the quantifications equal, taking into account the dilution factor? Fig. 5 might answer it but it is not clear to me if that samples are serial dilutions or not.Do you need to do a calibration curve per run? • A calibration curve is required for the real-time analysis part of the assay, this is if less than 0.5% present in the imaged area remain dark. If the number is >0.5% absolute quantification based on Poisson analysis is feasible. We have added a clarifying statement to the data analysis section.• Adjustment of the low, middle, and high level of BCR-ABL1/ratio is not done by serial dilution. We have mixed extracted RNA from different cell lines at different ratios to order to obtain three distinct levels of BCR-ABL1/GUSB ratios within 6 orders of magnitude.Duration of each step in the workflow scheme could be useful for the reader to understand how long the sample-to-result takes.• The duration of the test from sample to result is added (L188-L190)More details of the commercial suppliers of reagents are needed. It might be good to have a General section at the beginning of the Material and Methods section containing these details.TaqMan probes references are not provided.While authors give a reference for primers and probes, a Table describing them would be very helpful for readers• A table including all sequences for primers and probes has been added to the text. Wherever appropriate additional information on the source for the used materials has been provided.Fig. 1. A scale bar is missing • addedLine 247 there is a reference error • correctedResponse to Reviewer 3 The authors report the development of a 'nanoreactor bead' based assay for the quantification of mRNA targets. It appears that the use of nanoreactor beads can be used in quantification of mRNA targets spanning six order of magnitude.The authors build their manuscript on an argument by stating that digital assays are complex and require sophisticated microfluidic tools. Unless I am missing some important aspects/details the authors do generate 'nanoreactor beads' using a microfluidics chip, and the proposed method by all means not an unsophisticated one. Hence it is not clear to me why they prefer to 'sell' their method to be microfluidics-free and 'easy'. • The presented assay format is based on the use of hydrogel beads. These beads are made in bulk in large quantities and stored until they are needed. Contrary conventional droplet PCR requires microfluidic tools and specialized equipment to generate mono-disperse droplet nano-compartments to run the actual test, whereas in the presented format the beads in combination with a fluorocarbon oil and a suitable emulsifier are sufficient for partitioning of the sample into sub-nanoliter sized digital reaction compartments.We have now made this clear in the text.The manuscript text appears to be not submission ready given the overwhelming number of points I could comment on. The text could benefit from a thorough editing, especially paying attention to inconsistent terminology use that confused me on multiple occasions, especially the nanoreactor, digital nanoreactors, and alike… - • Thank you for this input: We have checked the text for consistency with regards to the terminology - - nanoreactor beads is now the only term used for the beads- Reaction and detection chamber us used throughout the textThere is also no statistical analysis section provided within the Materials and Methods section. • A statistical analysis section has been added to the Materials and MethodsAnd finally the method section can benefit from a detail oriented focus.Given the current status of the manuscript, I would conclude resubmission after major revision; however, in this journal it corresponds to major revision hence my decision. Below I list my various comments based on the page and line numbers:Cover page: there are typos in the keywords! • CorrectedPage 2, Line 16: good agreement -> Can the authors use a quantitative measure. Also while the title reads 'ultra-precise' the closing statement of the abstract states 'good agreement'. It is ultra-precise or good?  • Level of concordance has been quantified by Pearsons correlation coefficient of 0.983 and slope of 1.08.P3, L23: partitions -> It is not clear what is meant by partition. Can the authors clarify (the figure 1 legend reads beads. Can you please tune the term use for consistency/clarity). • We have added a description what partition means in line 23. In this manuscript we are referring to the basic concepts of digital PCR whereof sample partitioning is the fundamental approach. The sample is divided into a number of identical sub-volumes. A partition represents such a unit sub-volume. Since Fig 1 is referring to the newly developed process the figure capture refers to “nanoreactor beads”, whereas the introductory sentences are referring to digital analysis in general, hence our use of “partitions” there.P3, L24: Poisson criteria -> Poisson distribution (criteria?). Can you please clarify what the criteria is/are? • The unclear statement has been removed. The underlying mathematical model is explained in the quoted reference [5].P3, L29: digital compartments -> for consistent terminology use can you please stick to one term whether it is the digital compartments/beads/nanoreactor compartment/nanoreactor beads/microbeads/sub-nanoliter droplets (whichever term the authors prefer so long as it is used consistently throughout the manuscript.)• Thank you, this has been addressed P3, L29: generating digital compartments -> Can you present how your droplet generation does not necessitate microfluidics as compared to the standard. Also lay out the details of the concrete differences between the two techniques for droplet generation. • A clarifying sentence has been added P3, L35: 'amplification reagents and the extracted nucleic acid.' -> 'RT-PCR amplification reagents and the extracted nucleic acids (GUS8 and BCR-ABL RNA).'Also the text reads BCR-ABL1 RNA. Please stick to one term. • We have checked the text for consistency with regards to the terminology.P3, L 40: Please state the size of the beads.• Scale bar and information on size and volume of the beads has been added to the figure caption Figure 1. In panel a please add sub-sections i., ii, … so that the figure complies with the figure legend.• The changes have been made to Fig 1Figure 1b and c. Can you provide the scale.• Scale bars have been added to the imageP4, L47: extensive shaking -> Can you provide reproducible quantitative information.• The process is now described in more detail in line 167-171 in the materials and methods section. Figure 2 legend:Can you please use the same terms both in the figure and the legend, i.e. Fluorescence Micro-imager vs epi-fluorescence microscope, BLINK vs Blink… the text was checked for consistency. Can you provide the technical details of the chamber i.e. material, dimensions, thickness, alike… in the legend. Please state the company and also provide a link to the software/website.• Information has been added to figure legendP5, L69: nanoreactor compartment -> see comment P3, L29• The sentence has been modified for clarityP5, L70: modest number -> Please provide quantitative information wherever possible.• The quantity has been added.P5, L71: highly precise -> Can the authors avoid the use of qualitative adverb and state the quantitative findings.• We have modified the sentence.P5, L89: fluidic connection between tubing and chip -> Please provide a link to the chip or provide the design in supplementary. • the described set up used for generating nanoreactor beads represents a variant of the commercially available µEncapsulator system. The setup and components are described in detail in the product literature of Dolomite microfluidic. The manufacturer is referenced accordingly.P5, L99: was removed -> was removed by? • Sentence has been correctedP6, L112: e13a2 or e14a2 BCR-ABL1 -> Please describe what e13a2 and e14a2 are. • we have changed the names of the transcripts to the more common and known nomenclature (b2a2 and b3a2) and added a short description.These are transcript variants that are typical for the CML phenotype. A possible differential impact of the respective transcript variants e13a2 ("b2a2") and e14a2 ("b3a2") on disease phenotype and outcome is still a subject of debate. • We confined the analysis to samples with the common genotypes in order to focus on the technology rather than the disease. Thus, we also added a statement to the sample selection and sample exclusion strategy in section samples.P7, L113: BCR-ABL-1 -> BCR-ABL1 OK• Now consistent BCR-ABL1.P7, L119: HL60 and K562 -> Please describe what are HL60 and K562. • A description and references are added (L166-L167).P7, L124: as described elsewhere -> Please provide all the necessary details in the methods section. • A table is added including all sequences for primers and probes P8, L143: latter -> There is a single removal step in the previous sentence. Please re-phrase. • OKP8, L148: parameters -> Please provide the full details.• all necessary information is provided in the subsequent sentence.Figure 3 legend: Please describe the different beads in the legend. Preferably arrows can also be used to highlighting the specific beads. • Arrows have been added to the images for better understanding and the legend modified, accordingly P11, L208: ERM-AD623 -> What is ERM-AD623? Please describe. • ERM-AD623 is described in L172-175 P11, L209: (data not shown) -> Please provide the necessary data.• Removed, as not necessary in this contextP11, L211: high copy numbers -> high copy numbers (of what?)• Sentence has been correctedP11, L213: The concentration levels represent the expected measuring range for clin(ic)al samples -> Please provide data or a reference to support this statement. • References supporting the statement have been includedFigure 5: BCRLAB -> BCR-ABL1. Also please use different colours for dots and triangles.• Images have been modifiedP13, L247: Table 1. Error! Reference source not found. -> Cross reference error?• CorrectedP14, L260; L267: The molecular response (MR) -> Acronym was already introduced in line 258.• CorrectedP14, L267: considered -> Please provide a reference for this statement.• References supporting the statement have been includedP14, L273: precision -> Please provide a reference for this statement. • References supporting the statement have been includedP15, L285: 53.000 -> 53,000?• CorrectedP15, L294: Blink test -> What is a Blink test? • Replaced with “new test”P16, L303: the reference method -> Please help the reader by providing a legend that provides all the relevant information.• Reference method has been explained in line 139 - 140P16, L307: same data -> which data? Fig. 7 • Rephrased and information competed P16, L318: microfluidics free digital molecular assays -> Unless I am missing the picture, the nanoreactor beads were generated using a Dolomite microfluidics chip. So how can the authors claim that the process is microfluidics free? • Our statement is correct, as no specific microfluidic tools are required to split the sample into digital partitions during the test, instead we use pre-made hydrogel beads off the shelf for partitioning the targets into digital compartments.P16, L320: bead-nanoreactors -> is this the same as nanoreactor beads?• Corrected for consistencyP16, L324: for the ratios -> for the ratios of • correctedP16, L329: current laboratory standard -> which standard? • Rephrased for clarityAttachmentResponse to Riviewers.docxSubmitted filename: Click here for additional data file. 5 Feb 2021Ultra-precise quantification of mRNA targets across a broad dynamic range with nanoreactor beadsPONE-D-20-33329R1Dear Dr. Loncarevic,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Ruslan Kalendar, PhDAcademic EditorPLOS ONEReviewers' comments:Reviewer's Responses to QuestionsComments to the AuthorReviewer #1: All comments have been addressedReviewer #3: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?Reviewer #1: YesReviewer #3: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #3: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?Reviewer #1: YesReviewer #3: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?Reviewer #1: YesReviewer #3: Yes**********6. Review Comments to the AuthorReviewer #1: (No Response)Reviewer #3: I would like to thank authors for implementing the changes that improved the manuscript.There is still a (cross-reference) error in line 344.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article (Yes: Tai-Du LinReviewer #1: Yes: Rahmi LaleReviewer #3:  8 Mar 2021PONE-D-20-33329R1 Ultra-precise quantification of mRNA targets across a broad dynamic range with nanoreactor beads Dear Dr. Loncarevic:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofProf. Ruslan Kalendar Academic EditorPLOS ONE


The present study proposes a simple yet effective method of cellulose coating onto chitosan (CS) hydrogel beads and application thereof as drug carriers. The beads were coated with cellulose dissolved in 1-ethyl-3-methylimidazolium acetate, an ionic liquid (IL) via a one-pot one-step process. Water molecules present in the CS beads diffused outward upon contact with the cellulose–IL mixture and acted as an anti-solvent. This allowed the surface of the beads to be coated with the regenerated cellulose. The regenerated cellulose was characterized by FE-SEM, FT-IR, and XRD analyses. To test potential application of the cellulose-coated CS hydrogel beads as a drug carrier, verapamil hydrochloride (VRP), used as a model drug, was impregnated into the beads. When the VRP-impregnated beads were immersed in the simulated gastric fluid (pH 1.2), the VRP was released in an almost ideal linear pattern. This easily fabricated cellulose-coated CS beads showed the possibility for application as carriers for drug release control. Hydrogels manufactured from renewable natural resources like polysaccharides being extensively used in the medical and pharmaceutical fields6. Especially hydrogels made up of chitosan (CS), the cationic polysaccharide is of high priority owing to their impressive properties such as biodegradability, low toxicity, and biocompatibility9. However, the use of CS as drug carriers has been limited due to its weak physical properties and low acid resistance in gastric fluid when applied in oral delivery12. Therefore, in the present study, we used cellulose as a coating layer onto the surface of CS hydrogel beads to improve their stability in biomedical applications14.Hydrogels are natural or synthetic hydrophilic structures capable of holding significant amounts of water or biological fluids in their three-dimensional structures16. Cellulose is a crucial ingredient for fabricating various products such as paper, textiles, membranes, biofuel, and value-added chemical products. However, due to the strong inter- and intra-molecular hydrogen bonding, cellulose possesses high order crystalline domains18. Therefore, cellulose does not melt and is insoluble in conventional solvents such as water, dilute acids, alkali solutions, and the common organic solvents19, which limits the effective utilization of cellulose. Harsh conditions (high temperature and acid conditions) are usually required to disrupt the hydrogen bond network for the successful dissolution of the cellulose. These conventional methods require high energy consumption, and the use of toxic chemicals, thus, is not suitable for application in the biomedical field. As a result of thorough investigations, ionic liquids (ILs) have been considered a promising solvent that dissolves cellulose without any formation of derivatives22.Cellulose consists of β-(1 → 4)-linked glucose units and is one of the naturally abundant vital biomass components in the earth24. In recent years, ILs have been widely used to replace conventional organic solvents in various industrial applications. In 2002, Rogers et al.25 reported a method to dissolve cellulose by using ILs under mild conditions without derivatization. The dissolved cellulose after that could be solidified by using anti-solvent such as water and ethanol and regenerated in various forms such as fiber26 film27 or membrane28, according to the manufacturing conditions.By definition, ILs are room temperature molten salts that consist of cations and anions with melting points of less than 100 °CIn this study, CS beads were coated with cellulose dissolved in IL using only water molecules contained in hydrogels , the cellulose coating formed was uneven, rough and tend to clump up. Moreover, when the bead was dried or had no water inside, it was impossible to form the cellulose coating due to the lack of water molecules to act as anti-solvents. Thus, the best condition for this idea to be realized was to control the moisture content of the bead by effectively wiping off surface water. For this purpose, the wet bead was placed on gauze to remove adhering water from the surface of the bead before use.The conceptual strategy for the cellulose coating on hydrogel is shown in Fig. To understand the texture, the cellulose-coated CS bead was cut in half, and the morphology was analyzed using FE-SEM, as shown in Fig. −1 was due to the intermolecular O–H stretching vibration of the cellulose molecule. The peak at 2,898 cm−1 observed for native cellulose was due to the C–H stretching vibration of CH2 and CH3 groups. This band was not affected by changes of crystallinity; thus, there was no significant change of this band in the regenerated cellulose spectral data. The peak at 1,428 cm−1 in the original cellulose was assigned to the crystallized cellulose I and amorphous cellulose, whereas in the case of the regenerated cellulose, this band shifted to 1,419 cm−1, representing cellulose II and amorphous cellulose30. The peak at 1,428 cm−1 in the cellulose spectra was assigned to symmetric CH2 bending vibration. The peak at 1,103 cm−1, which appeared in the spectrum of the native cellulose was not observed for the regenerated cellulose. This further confirmed the prevalence of crystalline cellulose II7. In the regenerated cellulose spectra, the new peak at 1,750 cm−1 was attributed to the C=O in the acetate31. This is good evidence that cellulose dissolved in [Emim] [Ac] had been regenerated. The band observed at around 895 cm−1 was due to the stretching vibration of C–O bond in the amorphous regions of cellulose and regenerated cellulose.Prior to FT-IR analysis, the cellulose-coated CS bead was separated into the shell (regenerated cellulose) and core (CS) part to confirm the functional groups of each section. FT-IR spectroscopic analysis of the original cellulose and regenerated cellulose on CS bead are shown in Fig. 33. Due to the pronounced quantity of amorphous cellulose existing in the regenerated cellulose, it showed a lower crystallinity than the original cellulose. It could be explained by the fact that [Emim][Ac] broke the inter- and intramolecular hydrogen bonds and reduced the crystallinity of the original cellulose during the dissolution process.The typical XRD profiles of the original cellulose and regenerated cellulose on CS bead are shown in Fig. Since cellulose coatings may improve the physical and chemical stability of the hydrogels, cellulose-coated CS beads may have a wide range of applications. One of the attractive applications of the cellulose-coated hydrogels could be in the biomedical area because both cellulose and CS are biocompatible and biodegradable.34. The release experiments were studied in different release fluids, including simulated gastric fluid  and simulated intestinal fluid . The release profiles corresponding to a formulation consisting of CS beads and cellulose-coated CS beads are displayed in Figs. Hence we tested the cellulose-coated CS beads for in vitro verapamil hydrochloride (VRP) release applications. VRP is a calcium channel blocker used for the treatment of hypertension, angina and myocardial infarction SIF, pH .8. The r SIF, pH .8. The rn) for CS beads are smaller than 0.45 in SGF (0.0752) and SIF (0.3217), indicating that the release of VRP from the CS beads are a Quasi-Fickian diffusion mechanism  and verapamil hydrochloride (VRP) were purchased from Fluka and Sigma-Aldrich, respectively. CS  was purchased from YBBio Co., Ltd. (Korea), at which CS was obtained on an industrial scale by the deacetylation of chitin from the red crab. The ILs 1-Ethyl-3-methylimidazolium acetate  was purchased from Ionic Liquids Technologies (Germany), and acetic acid (above 99.7%) was obtained from Junsei Chemical Co., Ltd. All of the other reagents used in this study were also of analytical grade. In addition, the plastic hub needle used to fabricate the CS beads were purchased from Teaha Co., Korea.Cellulose/[Emim][Ac] solution was first prepared by dissolving 6 g of cellulose powder in 100 ml of [Emim][Ac] at 120 °C, and the obtained 6% (w/v) cellulose/[Emim][Ac] solution was used to manufacture the regenerated cellulose-coated CS beads.For the manufacture of the CS beads, CS powder (2 g) was dispersed in 100 ml of distilled water containing 2 ml of acetic acid solution at room temperature. The mixture was left overnight with continuous mechanical stirring to prepare completely dissolved CS solution. The as-obtained mixture was dropped through a plastic hub needle with a diameter of 310 µm into 1 M NaOH solution under gentle magnetic stirring at room temperature to obtain CS beads. After 2 h, the hydrogel beads were separated from the NaOH solution and washed with distilled water.To fabricate the VRP-loaded CS beads, 1,000 mg of VRP was added to 10 ml of 2% CS solution and stirred for 30 min to obtain a homogeneous dispersion. The VRP-loaded CS beads were prepared in the same dropwise method, as described in the previous section.The wet CS beads were dropped into the 6% (w/v) cellulose dissolved in [Emim][Ac] solution and kept for 5 min, resulting in the formation of cellulose coat on the surface of CS beads. Then, the cellulose-coated CS beads were separated from the solution using gauze mesh and washed with deionized water three or more times.The VRP-loaded cellulose-coated CS beads were fabricated by first manufacturing the VRP-loaded CS beads and then coating it with cellulose. The verapamil encapsulation efficiency (EE) and loading capacity (LC) of cellulose-coated CS beads used in drug release experiments were 100 ± 0.0% and 8.62 ± 0.07%, respectively. Encapsulation efficiency (EE) and loading capacity (LC) were calculated as follows:The in vitro release studies were performed in simulated gastric fluid  and simulated intestinal fluid . Ten beads were placed in plastic bottles containing 50 ml of the release medium. After the cellulose coating, the weight per bead was increased by cellulose coating, so the number of beads was used instead of the weight of beads. The drug release experiments were carried out at 100 rpm and 37 ± 0.5 °C in a shaker. At predetermined time intervals, samples of 0.5 ml were collected from the release medium and replaced with fresh SGF and SIF solution, respectively. The experiments were duplicated.To estimate the initial concentration of VRP entrapped into the beads, the VRP concentration was measured after crushing and dissolving 10 VRP-loaded CS beads in 50 ml of medium, which is the same condition as the release experiment.In the dried cellulose-coated CS bead, the core and the shell part are easily separated. In this study, a cross-section was cut using a stationery knife and tweezers and then FE-SEM analysis was performed, and FT-IR and XRD analyses were performed by separating the core and shell. The morphology of the core (CS) and shell (cellulose) parts of the cellulose-coated CS beads was examined using an FE-SEM  equipment. FT-IR  was used to obtain the functional groups present in the fabricated materials. Multipurpose high-performance X-ray diffractometer  was used to analyze the crystallographic nature of the samples. Furthermore, the concentration of drug in the solution was assayed by using high-performance liquid chromatography  at 278 nm. The mobile phase condition was 75% methanol and 25% buffer solution of 25 mM ammonium formate. The used column was Zorbax Eclipse XDB-C18 .Supplementary Information.


Hurst exponent and fractal dimension are found to be optimal at a critical pressure (Pm), whereas, for P > Pm and P < Pm, we found rich genome diversity relating to complicated genome organisation and virulence of the virus. The values of these complexity measurement parameters are found to be increased linearly with health index values.One of the main concerns about the fast spreading coronavirus disease 2019 (Covid-19) pandemic is how to intervene. We analysed severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) isolates data using the multifractal approach and found a rich in viral genome diversity, which could be one of the root causes of the fast Covid-19 pandemic and is strongly affected by pressure and health index of the hosts inhabited regions. The calculated mutation rate ( SARS-CoV-2), which is a member of the β-CoV genus ) can be related to Λ by using (dP[Λ]/dΛ) =   − dg at hydrostatic equilibrium, and ideal gas equation, P =  (d/N)RT where d is the air density, g is the acceleration due to gravity, N is the air molar mass, R is the universal gas constant and T is the standard temperature , β = (Mg/2r2LRT) and γ = (Mg/r2LRT)[2r −  (T0/L)]. Equation (P[Λ] decreases quite fast as Λ increases. The mutation rate of the isolates (rm) is found to be relatively high (rm ∈ (10−4−10−5)), and increases as Λ increases (decrease in pressure P[Λ]) upto a maximum value (at around Λ ~ 1.7 × 102m), and then decreases as Λ increases  from seven countries . We thenxtracted . The heiperature , and is rocesses  and coulrm with Γ can be obtained by fitting the data and is found to obey, rm ~ baeΓ, where a = 1.3713 and b = 0.3681 are constants. The Pearson correlation coefficient value of the fitted function to the data points is r2 = 0.8313.The health index of a place characterises the overall health of the population at that particular place. The calculated mutation rate is strongly dependent on the health index (H), and generalised fractal dimension (D) [P and Γ is as follows. First, we converted the symbolic genome sequence to time series like DNA walk by taking purine (A or G) as step-up (+1) and pyrimidine as step down (−1) [DNA walk is a map of the genome's cumulative sum, which carries the complex information of the genome. Then DNA walks of 42 SARS-CoV-2 isolates collected from the patients' data are calculated as a function of Λ and Γ. Now consider a DNA walk of length N of a particular isolate. From this DNA walk, we explain the procedure briefly for calculating various multifractal parameters discussed as follows [λj's from the DNA walk of length N, with j = 1, 2, 3, …, N, such that, λj = 0 is considered to be insignificant. Then, this function Y(i) is divided into Nx = int(N/x) equal non-overlapping segments of length x. To incorporate the end effects of Y(i), 2Nx segments of length x are considered by taking into account opposite end repetition in the simulation. The local trend of fluctuation of each 2Nx was estimated from the variance, which was calculated by the least-squares fitting procedure for each segment series. Averaging over all the calculated local fluctuations of all 2Nx segments, the q (order parameter) dependent qfluctuation function F(x) for the considered virus isolate, and found to follow fractal nature qF(x) ~ Hqx, where, qH is the q order Hurst exponent [qF(x) is both q-dependent , each genome exhibits multifractal property [H of each genome is obtained by H = <qH>. The calculated values of H of all SARS-CoV-2 isolates are found to be quite sensitive to q, indicating rich heterogeneous structures in the genomes. Further, it is also found that 1 > H>0.5  < 1, one can approximate the factor s = (1/β)(r + α(L/T0)). Further, for positive Λ > 0, it can be shown that P0 > P. Now, putting the expression for Λ to the equation of H, and after simplification, we arrive atNext, we study the impact of Λ and Γ on the genome complexity of the SARS-CoV-2 isolates by calculating the complexity measurement parameters, Hurst exponent (sion (D) , 18. Thesion (D) . These vsion (D) –22, affesion (D) . From thsion (D) , 24, andsion (D) . Moreovesion (D) , 27. Henown (−1) . By consexponent , 29. Sinependent ) and x -property , 18, 30.P > mP and P < mP (mP → Λ = 887m) exhibit complicated divergence indicating more virulence attempting to establish long-range correlation within the genome during the virus−host interaction. However, the virus may likely cause minimal harm to the host adapted to the regions at and around P → mP. The pressure variation driven by the change in altitude Λ can perturb gene expression and physiological changes of the organisms and micro-organisms [m i.e. as pressure decreases till Pm  ~ νz [ν is Holder exponent [D can be obtained by D = <D>. Similar to H, D is also quite sensitive to q, indicating rich heterogeneous structure in each virus isolate [D is found to be maximum at Λ = 887m . The degree of viral complexity measured by H and D is dependent on Hhealth index I  because of multifractality of the SARS-CoV-2 viral genomes. However, here, H and D are calculated average values of each genome.Now, rganisms –22. It mrganisms . Our dat(z) ~ zν , where νexponent . The fra isolate , where lIn conclusion, it is quite evident that the mutation rate and rich diversity in SARS-CoV-2 genome complexity are strongly driven by pressure levels of the hosts' inhabited regions and health index. Our results show clear exponential dependence of health index with mutation rate, which may trigger the SARS-CoV-2 virus's virulence, which may increase the infection rate. From our analysis, since higher health index people have high mutation rates causing higher infection rates, proper precautions like WHO guidelines should be strictly followed, and the immune system has to be kept strong to fight the virus infection. Our analysis of the viral isolates data show a critical pressure at which the virus causes minimal harm to the host and beyond which the viral evolution preserves rich diversity relating to virulence. The virus's complexity increases as the hosts' health index, probably for its survival and then attacks the hosts. The variation in atmospheric pressure triggers significant changes in gene expressions leading to indicative biological and physiological changes in the macro and micro-organisms –22. In t


In Sudan, many Butana cattle farmers practice indiscriminate crossbreeding to improve the milk yield performance of cows, as organized breeding programs are lacking. Objectives of this study were to identify the current production conditions of Butana cattle and to determine farmers’ production objectives and trait preferences using a field survey. The overall aim was to explore the possibility of establishing a community-based breeding program for the genetic improvement of the breed. A semi-structured questionnaire and field visits were used to collect data from 202 Butana cattle owners. Data were analyzed using chi-squared test, multiple response analysis, and binary logistic regression. Our results showed that Butana cattle farmers mainly raised their animals for milk production. On a five-point scale (5 = most important), milk yield (4.6 ± 0.05), growth performance (4.0 ± 0.07), and lactation length (3.9 ± 0.08) were highly preferred for future development of the breed. One-third of the farmers kept crossbred cattle with on average 4 crossbred animals per herd. About two-thirds of respondents were willing to adopt crossbreeding using exotic breeds to increase milk performance and about the same proportion were willing to exchange breeding bulls and establish farmers’ associations. None of the respondents kept written performance records. However, educated farmers were more likely to adopt record keeping. Farmers’ willingness to engage in associations could be useful for the establishment of a community-based breeding program. Based on the current farmers’ production objectives, the future breeding program should emphasize increasing milk production of the Butana cattle by using improved Butana bulls in village herds. Indigenous livestock contribute to milk and meat supply, and represent an essential source of employment, income creation, and export earnings of many communities in rural areas in African countries FAO . In SudaAs early as 1945, the Sudan government began a within-breed selection improvement program for Butana cattle by establishing the Atbara Livestock Research Station  socio-economic characteristics; (2) herd sizes and composition; (3) production objectives; (4) feeding management; (5) animal health management; (6) breeding management; (7) farm income and milk marketing; (8) farmers’ evaluation of adaptation and production traits; (9) production challenges; and (10) future development aspects. Based on a five-point scale (from 1 = very poor to 5 = very good), respondents were asked to evaluate the performance of Butana cattle for milk production, lactation length, growth rate, disease tolerance, and grazing ability . The respondents were also asked to score their preferences for the traits’ improvement in the future on a scale of five points (from 1 = not important to 5 = most important). On the same basis, respondents were asked to score five major challenges of cattle production including high costs of concentrate feeds, lack of financial supports, lack of cattle improvement services, scarcity of rangeland, and competition from crossbred dairy cattle in terms of milk yield and selling price. The selection of these challenges was motivated by reports of previous studies, e.g., Musa et al.  and AhmeThe collected data were analyzed using the statistical package for social sciences (SPSS) version 20 IBM SPSS  and the All household heads interviewed were men. The majority were above 49 years (43.5%) or between 30 and 39 years (32.7%) old, while 11.9% were between 40 and 49 or below 30 years old, respectively. Primary education was predominant among older (> 49 years) and middle age (30–39 years) farmers and the former had a high proportion of individuals who had only informal education . There was no significant association between herd size and farmer’s age . On average, cattle herds were composed of 8.2 ± 0.70 cows and 1.2 ± 0.04 breeding bulls (Table n = 61) had on average 3.8 ± 0.65 animals per herd. Farmers were not asked to provide information about the level of exotic blood of their crossbred cattle.The majority of farmers (65.3%) owned between 1 and 11 cattle heads, and the proportions were 18.4% and 16.3% for farmers who owned 12 to 20 heads, and above 20 heads, respectively. There was a significant relationship between herd size and the employment of laborers on the farm  kept cattle for meat production, meaning that they sold live cattle for slaughter. Only 9% and 8% of the farmers used cattle for draught power  and as an insurance against financial difficulties, respectively. We observed that keeping cattle for draught power was more important for farmers found in areas close to riverbanks, representing about 7% of the respondents.Clitoria (Clitoria ternatea), Abu-70 (Sorghum bicolor), or alfalfa (Medicago sativa) fed as green or dry. In addition, 65.8% of the farmers offered their cattle concentrate feeds, which were primarily composed of oilseed cakes of sesame (Sesame indicum) or groundnuts (Arachis hypogaea L.), wheat (Triticum L.), bran, sorghum grain, sugar cane (Saccharum officinarum), molasses, and cotton (Gossypium hirsutum) seed cake. According to farmers, the concentrate feeds were usually mixed on the farm from all or some of these ingredients, but farmers also purchased concentrate feeds from the markets. The concentrate feeds were usually provided to lactating cows during milking period and to crossbred animals. The provision of concentrate feeds was significantly associated  with the type of roughages offered to cattle . Only 24.8% of the respondents fed their cattle crop residues as supplement mainly in the dry season. There was no significant association between the types of roughages offered to cattle and farmer’s age . The crop residues comprised stubble of sugar cane and sorghum or groundnut hulls. The majority (62.4%) of the farmers reported moving their cattle out of the permanent homes to communal grazing areas during the wet season that spans between July and October in order to access green pastures freely. During the dry season (from November up to June), cattle are returned back to the permanent homes, where they graze on rangelands close to riverbanks. In this season, cattle are supplied with roughage fodder (irrigated or crop residues) and concentrates.The majority of the respondents (75.2%) fed their cattle irrigated fodders. Irrigated fodders comprised le Table . FarmersAccording to farmers, private veterinarians provided veterinary services. The majority of respondents (79%) reported that they had limited access to appropriate animal health services, whereas only 21% were satisfied with the animal health services. The latter were specifically those farmers who lived in urban areas. During the survey, we observed that in case of disease occurrence, farmers brought a veterinarian to the farm to diagnose the disease, and to provide treatment for the animal. The most common diseases that farmers experienced on their farms included foot and mouth disease (31%), contagious bovine pleuropneumonia (14%), jaundice disease (14%), mastitis (11%), cowpox (10%), heart water (9%), bovine ephemeral fever (6%), and trypanosomiasis (5%). Farmers mentioned the local names of the diseases, which were then translated by a local veterinarian.χ2 = 25.47, p = 0.001) ; rather, they depended on their neighbors’ bulls . Conversely, farmers who had larger herd sizes (> 20 heads of cattle) tended to possess their own breeding bulls  and were less likely to depend on neighbors’ bulls .All farmers interviewed reported that natural mating was the only breeding method practiced since artificial insemination service using local bulls’ semen was not developed. However, the Sudani Center for Artificial Insemination and a Sudanese-Turkish Center for semen production provide services for cattle insemination in Sudan using semen from exotic bulls. The breeding bulls were allowed to run with the cows all year round. The majority of farmers 62.9%) selected the breeding bulls from their own herd, while 31.7% relied on bulls from their neighbors. Only 5.4% of the farmers relied on communal bulls, which were available at grazing sites. The source of breeding bulls was significantly associated with herd size . The dark red coat color was preferred by the respondents, because dark red cattle were considered better milk producers, having heavier body weight. A similar proportion of farmers (48%) selected their bulls based on the observed milk yield of the bull’s dam while only a few of them (3.3%) based their selection on observed phenotypes  of the bull’s sire. The average age of a breeding bull at selection was 25.6 ± 1.56 months, and the length of time for using bulls for mating was 5.9 ± 0.23 years.Without exception, none of the respondents reported keeping written records of production, reproduction, or animal pedigree. The main reason was that farmers claimed they had abilities to memorize all relevant information (81.7%). About 13.3% of the respondents had difficulties to keep written records. Accordingly, all information related to cattle production and reproduction characteristics, pedigree, and bulls’ selection criteria was based on farmers’ memories.Nearly three-quarters (74.3%) of respondents indicated that they generated income from the sale of milk, followed by selling of live animals for slaughter (43.6%). Only 11.9% of farmers indicated that they derived their income from the sale of crops. About 48% of the farmers who sold milk indicated that they delivered the milk to final consumers using donkeys, followed by 27.3% who reported the use of cars or bicycles for milk delivery. The remaining 24.7% of the farmers sold their milk at the farm gate.Evaluating trait performances of the Butana cattle breed, the highest scores were given to grazing ability (4.3 ± 0.86) and disease tolerance (4.3 ± 0.78), indicating that Butana cattle were able to walk long distances in search of pastures and watering points, and were disease tolerant Fig. . ModeratBased on mean score values, respondents considered high costs of concentrate feeds (3.7 ± 0.10), lack of financial supports (3.7 ± 0.10), competition from crossbred dairy cattle in term of milk yield and selling price (3.7 ± 0.12), and lack of cattle improvement services (3.6 ± 0.08) as the major challenges associated with Butana cattle production Table . Scarcitp < 0.001) (Table p < 0.05). On the contrary, the willingness to exchange information, establish farmers’ associations, keep written records, and exchange breeding bulls were not influenced by the farmer’s educational level.Table 1) Table . Highly The development and successful implementation of a breeding program for local breeds require the definition of a comprehensive breeding objective, a holistic description of the production system, and the involvement of producers at every stage in the planning and implementation process  who considered the sale of milk as the main source of farm income. This result is consistent with the findings of Musa et al.  who repoSelection criteria of breeding bulls reported in the present study are in accordance with previous findings. For instance, the milk yield of a bull’s dam  reported by 89.6% Butana cattle farmers in the current study is a common practice found in many parts of Africa. For example, Ankole cattle in Burundi, Tanzania, Rwanda, and Uganda  have also been reported in previous studies (Musa et al. Animal records are strongly required to establish genetic improvement programs and to support selection decisions in the long term. Our results revealed a complete absence of an official system for animal identification and performance recording. Instead, farmers purely relied on recalled memory. The absence of performance recording has also been reported in previous studies for other indigenous cattle breeds (Opoola et al. The present study highlighted the need for establishing community-based breeding programs for smallholder dairy cattle producers in Central Sudan. Our results showed that Butana cattle farmers mainly raised their animals for milk production. This was reinforced by farmers’ reliance on the sale of milk as the predominant source of income and the prioritization of milk yield as the most important trait for improvement. The willingness of farmers to engage in associations will be useful for the establishment of a community-based breeding program.


Due to the pandemic caused by the novel coronavirus disease, researchers internationally have started to utilize their efforts in understanding its pathophysiology and method of action . OngoingIn addition, the form of the SARS-Cov-2 infection that has been established in the literature, and its mechanism of action, are similar to that of the SARS-Cov virus . Using tTaking into account all this information, several issues around the Coronavirus to semen and male fertility relationships are being raised, including:Can SARS-Cov-2 be found in semen?Is the seminal sample a means of transmission for this virus?Can this viral infection lead to loss of gonadal function or changes in male fertility potential?If this loss or change is proven, is it a reversible or permanent?In relation to assisted reproduction techniques (ART), will future conduct in this area need to be reprogrammed?Some studies have attempted to answer these questions; however, they contain biases that can alter their results, such as a low number of sample patients or due to the moment when the analyses were performed  -18.The importance of obtaining these answers lies in the fact that there is a need for a change of conduct in the face of the new reality imposed by the novel coronavirus. If the presence of SARS-Cov-2 in semen is confirmed, the methods of assisted human reproduction conduct should be modified, ensuring the timely safety of couples; however, current information about the virus raises other issues, such as: if seminal transmission exists, should a couple avoid sexual intercourse or use a barrier method if the male partner is known to be positive for the coronavirus disease 2019 (COVID-19)? As for ART, should we test all men for COVID-19 just as we do for HIV, hepatitis B and C, syphilis, HTLV I/II, and Zika before the procedure? If there is SARS-Cov-2 in an infected male’s semen, is double sperm washing effective in isolating the virus as it is for HIV and hepatitis C? Should we take special care when cryopreserving semen from COVID-19 positive men? These questions’ answers will have a direct impact on the daily routine of Andrologists and Reproductive Endocrinologists.In addition, even with the absence of the virus in the seminal sample, a study has reported the presence of orchialgia in men diagnosed with COVID-19 , which iIn conclusion, the COVID-19 pandemic has brought many uncertainties to the future of ART and male infertility management. Additional studies are necessary to answer all these questions; however, while we do not currently have all the answers, the most effective conduct is to be cautious and evaluate each reproduction treatment step individually when dealing with a couple in which the man has been diagnosed with COVID-19.De Carvalho RC wrote the manuscript and conceived this research. Groner MF, Camillo J, Ferreira PRA analyzed the topic in the literature. Fraietta R conceived the study and coordinated this research. All authors revised and accepted the final version of the manuscript.


Paramyxoviridae family with a high fatality rate. It causes various diseases such as respiratory ailments and encephalitis and poses a great threat to humans and livestock. Despite various efforts, there is no approved antiviral treatment available. Therefore, to expedite and assist the research, we have developed an integrative resource NipahVR (http://bioinfo.imtech.res.in/manojk/nipahvr/) for the multi-targeted putative therapeutics and epitopes for NiV. It is structured into different sections, i.e. genomes, codon usage, phylogenomics, molecular diagnostic primers, therapeutics  and vaccine epitopes . Most decisively, potentially efficient therapeutic regimens targeting different NiV proteins and genes were anticipated and projected. We hope this computational resource would be helpful in developing combating strategies against this deadly pathogen.Nipah virus (NiV) is an emerging and priority pathogen from the http://bioinfo.imtech.res.in/manojk/nipahvr/Database URL:  Henipavirus of the family Paramyxoviridae. NiV is an enveloped single-stranded RNA (ssRNA), a negative-sense virus of size ~18 250 nucleotides. It encodes nine proteins namely nucleoprotein (N), four proteins encoded by P gene (phosphoprotein (P), W, V, C protein), matrix (M) protein, fusion (F) glycoprotein, attachment glycoproteins (G) and a large polymerase (L) protein (Nipah virus (NiV) is a highly pathogenic virus closely related to the Hendra virus (HeV) from the genus  protein .Different NiV proteins play a cardinal role in viral infection and disease manifestation. Viral G protein (attachment protein) first binds to the Ephrin-B2 or B3 cellular receptors found on the neuron, smooth muscles, capillaries and arterial endothelial cells. G protein provides attachment to the host cell surface, which triggers the fusion by the F protein ,5. SubsePteropus are known as a natural reservoir and host of the NiV  mainly distributed in south Asian regions like Bangladesh, India and Pakistan  agent that signify a rolling threat to humans and animals worldwide ,8. It wa the NiV . Among aPakistan ,15. OtheCambodia ,17.Pteropus giganteus in different Indian states signifying it as a natural reservoir in India  were tried to eradicate the infection . Distincanti-Nipah’ for the identification of effective inhibitors against the NiV  through small interfering RNAs (siRNAs) are also used previously to inhibit N and L genes . Further the NiV . The alg the NiV .However, despite determinations, the Nipah Virus study is generally neglected. Currently, there is no Food and Drug Administration (FDA)-approved therapeutics or prophylactic vaccine available to treat NiV diseases in humans, and treatment is only supportive ,28,31. SComplete genome sequences of the NiV were searched and collected from the NCBI database. In total, 18 complete or near-complete sequences were obtained utilizing the length-filtering criteria of size more than 10 kb and provided on the resource . A facilCodon bias analysis of the complete genome sequence is performed to explore relative synonymous codon usage (RSCU) and codon frequency. Further, codon preference and context are analyzed employing the Anaconda program .We have performed phylogenomic analysis to understand the phylogenetic reconstruction of NiV genomes. In the current study, we have employed complete genomes of 15 NiV that cause outbreaks in various Asian countries like India, Bangladesh, Malaysia and Singapore from 1998–2018. The genomic information was extracted from various sources like NCBI, ViPR, Viral zone and research articles ,49. FurtIn order to provide diagnostic primers, two strategies were utilized. First published literature was searched for extracting the experimentally used primer pairs for the diagnosis of the Nipah virus along with relevant information. Secondly, putative primer pairs were also designed using the PrimerDesign-M tool , keepingFor the potential epitope identification, 9-mer overlapping peptides were generated for each NiV-encoded proteins, i.e. N, P, W, V, C, M F, G and L. Further, the analyses were performed in quest to find promiscuous immune response, inducing peptides against the virus as also described previously . In ordeHomo sapiens (human) genome assembly GRCh37 (hg19). Correspondingly, potential siRNAs using the threshold of 0.80 were also deduced applying the DesiRm algorithm. Moreover, the imRNA program with the ‘siRNA immunotoxicity’ option along with the ‘screen siRNA library’ module is used to explore the immunomodulatory or non-immunomodulatory potential of siRNAs. SiRNAs with a score of 4.5 and above are considered as immunomodulatory, and less than the threshold is non-immunomodulatory. Moreover, we have also predicted NiV miRNAs. First, the VMir algorithm  system-based genome editing employing sgRNAs also shown to have an application to target a particular genomic region  or viralThe eventual goal is to provide the web resource of the putative therapeutic regimens and solutions from the study to assist in fighting with the deadly Nipah virus and support scientific society in therapeutic development. This platform, ‘NipahVR’, is hosted on the Linux environment using a LAMP  open-source web development platform. The front-end of the web interface is built using the PHP, HTML, CSS and JavaScript as also accomplished earlier , and theNipahVR is an integrative and systematic resource mainly dedicated towards the putative therapeutics and vaccinome against the Nipah virus. A well-structured and dynamic web interface is developed for navigation. It is classified into different divisions like genomes, phylogenomics, molecular diagnostic primers and most importantly vaccine epitopes  and therapeutics . The complete architecture of the NipahVR compendium is shown in Genomic information of available 18 Nipah virus sequences was compiled and provided on the resource . It is eWe have calculated the codon frequency and pattern in the genomes, which vary due to the nucleotide composition, GC percentage, expression level, etc. Additionally, codon preference is represented through histogram, where rare codons are shown in blue and black color signify preferred codons . The mosThe reconstructed phylogenetic tree showed the sum of branch length of 0.11. Out of 15, 07 NiV genomes from Malaysia outbreaks (1998–99) were clustered together with the bootstrap of 100. Further, from the 04 NiV genomes from Bangladesh outbreak, two genomes from 2004 and 2010 outbreaks were clustered together. Interestingly, the 02 NiV genomes from the 2008 epidemic of Bangladesh were grouped with the NiV of 2007, West Bengal, India. Moreover, the recent outbreak of the NiV virus in Kerala, India, was found in the Indian and Bangladesh NiV genomes but at a distant branch. The phylogenetic reconstruction of NiV genomes is shown in We have collected primer pairs utilized for the detection of the NiV. Detailed information about primers like the respective primer name, a sequence of primer, its orientation (forward and reverse), genomic region and study reference is provided . OverallIn this study, efforts were made for the identification of potential vaccine candidates for the Nipah virus. Epitopes encompassing promising MHC I and II binders, CTL epitopes and B-cell epitopes is cataloged. 9-mer peptides were generated from the Nipah proteins. Overall, 979 MHC-I and 1628 MHC-II binding peptides were deduced and presented on the server. For both MHC-I and II binders, peptide sequence, respective protein region, MHC-alleles and counts were provided. The protein-wise number of peptides for both the MHC classes is shown in Additionally, we have also analyzed putative epitopes from all the arms of the immune system, i.e. B-cell epitopes, MHC-I and II binders and CTL epitopes to find the common epitopes, which could be promising and can be recommended as the potential vaccine candidates . We haveRNA-based therapeutic interventions provide another approach to counter lethal viruses through silencing the genes. In this study, we have also tried to provide a compendium of potent siRNAs against the individual NiV genes. Overall, 118 putative siRNAs with very few off-targets using the VIRsiRNApred algorithm, which is developed using the experimentally proven viral siRNAs and 441 siRNAs employing DesiRm tool, were cataloged along with inferred inhibition efficiency in percentage. Additionally, the immunomodulatory potential of these siRNAs is also predicted, which could be helpful and crucial in the development of vaccine adjuvant or RNA-based immunotherapy and therapeutics. Furthermore, the number of siRNA off-targets to the human genome is also presented. The resource provides a complete picture of these efficient siRNAs with a small number of off-targets and detail information such as sense–antisense sequence, gene region, start–end, efficacy scores and immunomodulatory potential. The set of efficacious siRNAs, i.e. 18 using the VIRsiRNApred algorithm  and 43 uFurthermore, we have also designed and identified the Nipah precursor and mature miRNAs. Totally, 22 precursor miRNAs (pre-miRNAs) were identified along with 44 mature Nipah-miRNAs (22 5p and 22 3p). Among these, 3 pre-miRNAs are from N, 3 from P/V/C, 1 from M, 2 from F, 3 from G and 10 belong to the L gene. Detailed information related to miRNAs, i.e. mature miRNA sequences (5p and 3p), genomic region, the precursor (hairpin) and mature miRNA location on Nipah genome, precursor (hairpin) length, GC content, score and rank is extracted and provided.On the basis of our analysis, overall, 1412 sgRNAs from the NiV were obtained. Out of these, we have found and listed 126 sgRNAs that can act as putative targets against the virus. Apart from this, a list of the 21 most efficient sgRNAs is given in Paramyxoviridae family and a BSL-4 agent, which causes various diseases such as encephalitis, respiratory illness and fever. Moreover, a high case fatality rate during epidemic, broad host range and lack of therapeutics or prophylactic vaccines critically demand efforts and a multidisciplinary approach to develop combat strategies against this virus. Up to now, very few computational studies focusing on the NiV are performed, and there is no such kind of therapeutic web resource available for it. In the current work, we have developed all-inclusive resource ‘NipahVR’ for the putative therapeutic solutions targeting individual Nipah genes and proteins. It provides a compendium of various components that includes genomics, diagnostic primers, vaccine epitopes  and therapeutics . Here, based on our analysis, we are also endorsing and providing a catalog of potential vaccine epitopes and efficient siRNAs, miRNAs and sgRNAs. However, medicinal plant-based antivirals and other chemical compounds are not in the scope of current work. We anticipate that NipahVR will be useful and assist the wider scientific community in determining efficient antiviral candidates to fight against the Nipah and exterminate the infectivity. We will periodically update and maintain the stable functioning of the NipahVR web resource.Nipah virus is a priority pathogen from the This study is conceived, designed and supervised by M.K. genomic data collection and curation; A.K.G., web server development; A.K.G., codon analysis; A.K.G., vaccine epitope analysis; A.K., K.M., A.K.G., phylogenetic analysis; A.R., sgRNAs and diagnostic primers; K.K., siRNA analysis; S.D., miRNA analysis; A.T., data interpretation; A.K.G., A.K., A.R., M.K.. Manuscript writing, A.K.G., A.R., K.K., M.K.The authors declare that they have no competing interests.Manuscript_color_change_baz159Click here for additional data file.Supplementary_Information_baz159Click here for additional data file.


Respirovirus. The present investigation contributes to the knowledge of respiratory infections in wild ruminants and raises questions on the epidemiological link between chamois and other animal species.The Alpine chamois is a free-living wild ruminant distributed across the mountainous areas of France, Italy, Switzerland, Liechtenstein, Germany, Austria, Slovenia and Croatia. This wide distribution favours interactions with other wild ruminants and livestock, implying the risk of cross-transmission of pathogens. Due to the impact of lung diseases on chamois populations, the investigation of respiratory pathogens is important for wildlife conservation and for the understanding of infection transmission at the livestock–wildlife interface. In the present study, a novel respirovirus was isolated from a chamois with lung lesions and characterized by full-genome sequence and phylogeny. The genome characterization of this novel virus revealed similarities to domestic ruminant respiroviruses, mainly of caprine and ovine origin. Overall, phylogenetic analyses indicated that the chamois virus is distinct from already defined species and suggested that it is a putative novel member of the genus Respirovirus genus, family Paramamixoviridae, includes respiratory viral pathogens. Here we report the identification and genetic characterization of a respirovirus in an Alpine chamois showing interstitial pneumonia associated with catarrhal bronchopneumonia. The full-genome characterization of this respirovirus, named ChamoisRV/IT2014, revealed low similarities to caprine respirovirus (77.1%), bovine respirovirus (74.5%) and human respirovirus (72.0%). The phylogenetic analyses based on the full-length genome sequence of the novel isolate and reference respirovirus strains showed that ChamoisRV/IT2014 clustered with caprine respirovirus but formed a separate branch. The phylogenetic tree topology of complete large protein amino acid sequences, representing the current species demarcation criterion for Respirovirus genus, showed a 0.05 branch length of ChamoisRV/IT2014 sequence between the nearest node and the tip of the branch, suggesting that this virus belongs to a novel species. This new isolate in a new host species raises several questions to be addressed on the epidemiological role of chamois and the risks of cross-transmission between wild ruminants and livestock.The  Respirovirus genus in the family Paramyxoviridae includes six species: human respirovirus 1 and 3 , porcine respirovirus 1 , bovine respirovirus 3 , caprine respirovirus 3  and murine respirovirus , as recently revised [In recent years, novel respiroviruses have been identified in goats suffering respiratory syndrome in China ,2 and in revised .CRV3 (formerly CPIV3) was first identified in goats  and receBRV3 (formerly BPIV3) is a worldwide cattle respiratory pathogen involved in bovine respiratory disease (BRD) , a multiRupicapra spp. with nearly 500,000 individuals distributed among France, Italy, Switzerland, Liechtenstein, Germany, Austria, Slovenia and Croatia [The Alpine chamois is the most abundant subspecies of  Croatia . This ex Croatia .Considering the need to widen the knowledge on respiratory infection of chamois, in the present study we report a novel respirovirus isolated from chamois with lung lesions that was characterized by the full-genome sequence and phylogeny.Capreolus capreolus), red deer (Cervus elaphus) and ibex (Capra ibex) are present. Furthermore, cattle and small ruminant herds share alpine pastures during summer season. Chamois, roe deer and red deer are legally selectively hunted in accordance with Italian law (N. 157 of 11/02/1992) and inspected by veterinarians at the wildlife control centres of the area. In autumn 2014, coughing chamois were reported by hunters. In September–October 2014, 191 chamois were legally selectively hunted . To collect data on respiratory infections, lungs were available only for 14 yearlings, both with and without macroscopic lesions, and for 7 adults with macroscopic lesions. Lung samples were collected and stored at −20 °C for 1–2 weeks in the control centres facility and subsequently transferred to the laboratory at −80 °C until further processing. Blood samples, collected from major blood vessels or heart clots, were also available for 1 kid, 3 yearlings and 7 adults. Samples were centrifuged and the serum was stored at −20 °C until further processing.The study area was located in the north-western Italian Alps  where chamois, roe deer (w/v) of lungs were inoculated in 24-well plates in sub-confluent monolayers of Madin–Darby bovine kidney cells (MDBK ATCC CCL-22). MDBK cells were maintained in minimal essential medium (MEM) with 1% L-glutamine 200 mM, 100 U/mL penicillin, 100 µg/mL streptomycin, 2.5 µg/mL fungizone and 10% of fetal bovine serum (FBS), free of antibodies to bovine herpes virus-1, bovine respiratory syncytial virus, bovine parainfluenza virus 3 and free of both virus and antibodies to bovine viral diarrhea virus. The inoculated plates were incubated at 37 °C in 5% CO2 and after a 1–2 h adsorption period the cell cultures were rinsed and maintenance medium was added. The cell cultures were observed daily for cytopathic effect (CPE) for 6 days. Two blind passages were made if no CPE was observed; the cell cultures were scraped and vigorously mixed with culture medium and used for inoculation of fresh monolayers.Tissue homogenates , according to the manufacturer’s instructions. The RNA was retrotranscribed using a QIAamp One-For-All Nucleic Acid kit  and cDNA was subject to PCR according to previous published protocols for bovine and ovine respiratory syncytial virus [Viral RNA was extracted from lysates of cell cultures showing CPE using TRIZOLal virus ,15, pan-al virus , pestivial virus ,18 and mal virus .Bacteriological examination of tissue samples was performed on blood–agar plates under aerobic and microaerophilic conditions. Plates were incubated at 37 ± 2 °C for 24 and 48 h.g using a rotor holding six 175-μL test tubes in which specific adapters for 3 mm carbon-coated Formvar copper grids were placed. Grids were stained with 2% NaPT, pH 6.8 for 1.5 min and examined at 13,500–43,000x by using a Tecnai G2 Spirit Biotwin transmission electron microscope  operating at 85 kV and at 20,500–43,000× for at least 15 min before being considered negative. Attempts to identify the observed viral particles were based on their morphological characteristics.Due to negative results from specific PCR panels, the supernatant fluid from cell culture showing CPE was submitted to negative staining electron microscopy (nsEM) using the Airfuge method . SamplesThe whole genome was amplified by using the sequence-independent single-primer amplification (SISPA method) and sequenced by Next Generation Sequencing (NGS) method . In detaFollowing this step, 5 µL of double-stranded DNA was added to a PCR master mix containing 5 µL of 10× AccuPrime PCR buffer I; 0.2 µL of AccuPrime Taq DNA Polymerase, high fidelity; 4 µL of 10 µM FR20RV (5′GCCGGAGCTCTGCAGATATC3′) and 35.8 µL of water. The incubation was performed under the following thermal conditions: 94 °C for two min, followed by 40 cycles of 94 °C for 30 s, 55 °C for one min and 68 °C for three min. The PCR product was purified and quantified using a TECAN plate reader. The sample was diluted to an initial concentration of 0.2 ng/µL in accordance with the Illumina protocol, and 1 ng was used for the library preparation .Genomic libraries were sequenced on the Illumina MiSeq platform  with 2 × 151 base pairs paired-end runs. Finally, the obtained reads were evaluated for sequence quality and read-pair length using FastQC ver. 0.11.5. The reads were assembled using Geneious software v. 11.1.5  and re-sequencing analysis was performed with the complete genomes retrieved from Genbank. The full-length genome sequence of the chamois isolate was deposited into GenBank (accession number MT180123).Respirovirus using Clustal W. The phylogeny of the complete genome was estimated by the neighbor-joining algorithm (NJ) and the maximum likelihood (ML) method (MEGA version X), with 1000 replicates. Sequence identity of the nucleotide and deduced amino acid sequences of genome segments were calculated against reference sequences of genus Respirovirus using Bioedit software version 7.0 [The novel complete genome and nucleotide sequences of genome segments were compared for similarity against all sequences available from GenBank using Basic Local Alignment Search Tool (BLAST) analysis. Multiple sequence alignments of the novel complete genome were performed with reference sequences of genus sion 7.0 . Analysision 7.0 . Estimatsion 7.0 . The non2 for 96 h for Chamois strain and BVDV, and for 5 days for BRSV. VN titer was defined as the highest serum dilution able to inhibit virus replication. A titer > 4 was defined as positive.Sera were tested by virus neutralization tests (VNT) against the novel chamois strain, bovine respiratory syncytial virus (BRSV Strain 375) and bovine viral diarrhea virus (BVDV strain NADL). Two-fold serial dilutions of heat-inactivated serum in duplicate were mixed with equal volumes of virus containing 100 TCID50. The VNT was performed on MDBK cells, maintained in MEM supplemented as previously described and with 10% of FBS. The plates were incubated at 37 °C with 5% of COAt the control center, macroscopic lesions of interstitial pneumonia in association with catarrhal bronchopneumonia were observed in four adult chamois  and interstitial pneumonia was observed in five yearlings  and three adult females. Relevant bacterial pathogens known to cause respiratory disease were not identified by bacteriological examination of the lungs. Evidence of virus growth was detected only from the lung of a 13-year-old hunted male chamois with interstitial pneumonia associated with catarrhal bronchopneumonia. The CPE was observed at the first passage of the cell lines from 72 h post infection .The CPE–positive sample was negative to specific PCR panels. The supernatant of the infected cell cultures preliminarily observed by the nsEM revealed the presence of virus particles with morphological features indicative of the paramyxovirus family. In particular, in addition to some intact virions, several disintegrating particles with free nucleocapsid chains were observed . SubsequBLAST analysis of the whole genome of the chamois virus, named ChamoisRV/IT2014, revealed CRV3 as the most similar sequence (accession number MK091103 80.1%). The whole genome of ChamoisRV/IT2014 showed 77.1% nucleotide identity with CRV3 reference strain JS2013, 74.5% with BRV3 genotype A (BRV3a) strain TVMDL60, 74.2% with BRV3 genotype B strain Q5592, 73.9% with BRV3 genotype C strain SD0835 and 72.0% with HRV3 strain AUS/5/2007.The phylogenetic tree with other respirovirus strains, including publicly available sequences of BRV3, CRV3, HRV1–3, PRV1 and MRV, showed that ChamoisRV/IT2014 clustered with RV3  and formParamyxoviridae showed a 0.05 branch length of ChamoisRV/IT2014 sequence between the nearest node and the tip of the branch  and by an analytical protocol focused on viral isolation. The phylogenetic analyses based on the full-length genome sequence of the novel isolate and reference respirovirus strains showed that ChamoisRV/IT2014 significantly clustered with CRV3 but formed a separated branch. Despite this, we generated a comprehensive dataset of animal RV3 publicly available from GenBank; there were relatively few whole genome sequences available, and no other RV3 sequence clustered with ChamoisRV/IT2014. The nucleotide identity for the ChamoisRV/IT2014 complete genome with respirovirus reference strains, including CRV3, was 80% or below, revealing nucleotide differences higher than the ones observed among genotypes and members of 0 °C and us genus . The comus genus , shows tus genus , confirmRespirovirus genus, indicate that ChamoisRV/IT2014 is distinct from already defined species and suggest that it is a putative novel member of the genus Respirovirus.Overall, the results of the full genome phylogenetic tree, sequence identities with other respirovirus and species demarcation criterion for the This new isolate in a new host species raises several questions to be addressed. Studies on the epidemiological role of chamois, the risks of cross-transmission between wild ruminant and livestock and the evolution mechanisms, including genomic changes driven by the adaptation of viruses to new hosts, are suggested. The new powerful methods for genomic characterization will contribute to increase the availability of whole genome sequences to deepen the understanding of the origin and evolution of respiroviruses.