Survival of respiratory viral pathogens in expelled saliva microdroplets is central to their transmission, yet the factors that determine survival in such microdroplets are not well understood. Here we combine microscopy imaging with virus viability assays to study survival of three bacteriophages suggested as good models for respiratory pathogens: the enveloped Phi6 (a surrogate for SARS-CoV-2), and the non-enveloped PhiX174 and MS2. We measured virus viability in human saliva microdroplets, SM buffer, and water following deposition on glass surfaces at various relative humidities (RH). Saliva and water microdroplets dried out rapidly, within minutes, at all tested RH levels , while SM microdroplets remained hydrated at RH ≥ 57%. Generally, the survival of all three viruses in dry saliva microdroplets was significantly greater than those in SM buffer and water under all RH (except PhiX174 in water under 57% RH survived the best among 3 media). Thus, atmosphere RH and microdroplet hydration state are not sufficient to explain virus survival, indicating that the virus-suspended medium, and association with saliva components in particular, likely play a role in virus survival. Uncovering the exact properties and components that make saliva a favorable environment for the survival of viruses, in particular enveloped ones like Phi6, is thus of great importance for reducing transmission of viral respiratory pathogens including SARS-CoV-2. These microdroplets, ranging in size from a few micrometers up to millimeters5 travel in the air, and some of them—larger ones in particular—settle on surfaces2. Thus, inanimate surfaces are a potential route of virus transmission7. The factors that affect virus survival in droplets that settle on surfaces are complex and not well understood. Survival is believed to depend upon the physicochemical characteristics and hydration conditions of the immediate microscopic environment of the virion. These in turn are determined by several factors, including the composition of the fluid comprising the droplets, the surface properties, the ambient temperature, and the relative humidity (RH). A preeminent source for expelled droplets is human saliva, a complex solution that contains salts, a variety of proteins, and surfactants11. It has been suggested that micrometer-sized dry deposits of saliva droplets on surfaces protect virions12. Still, it is not clear how well viruses survive in saliva microdroplets following their deposition on surfaces, and what factors are at play therein. The impact of RH on the stability and viability of SARS-CoV-2 and that of other enveloped viruses has been studied mostly in airborne droplets or aerosols19 and less on surfaces22.Microdroplets expelled from the human respiratory tract into the air through coughing, talking, and breathing are considered a key source of transmission of respiratory viruses including SARS-CoV-223. The underlying mechanism of this U-shaped survival as a function of RH is not clear16. Only a few studies have attempted to gain mechanistic understanding of how RH affects virus stability in microdroplets. Prominent among these are the pioneering studies of the Marr group24, which point to the role of the solvent composition, evaporation dynamics, and RH on virus survival in aerosols and sessile droplets.While survival varies between virus species, increased survival at both low (< 40%) and very high (> 90%) RH is often observed, with decreased survival at intermediate RH levels27. Accordingly, although many surfaces in the indoor environment appear dry, they are in fact covered by thin liquid films and micrometer-sized droplets, invisible to the naked eye, known as microscopic surface wetness (MSW) 28. MSW can be considered the “envelope” that accommodates microorganisms on surfaces, and as such has profound impact on many aspects of microbial life. For example, it can protect microbes from complete desiccation. However, MSW is a harsh micro-environment that differs in its properties from those of bulk liquid. MSW inherently arises from drying liquids that evaporate on a surface. This drying process is accompanied by physicochemical changes such as solute concentrations, pH, reactive oxygenic species, surface tension, and others. At the microscale, gradients, local densities, and surface irregularity introduce heterogeneity to MSW environments31. Collectively, MSW imposes severe stresses on microbes therein—including viruses—and affects their survival33.A key factor determining the evaporation rate and equilibrium hydration level of drying droplets on surfaces (and in air as well) is the deliquescence property of solutes, or mostly highly hygroscopic salts39. While not necessarily viable, SARS-CoV-2 RNA has been detected on surfaces in contaminated sites such as hospitals43. Here, we aim to explore the link between microscopic surface wetness and virus survival therein. We focus on two variables that directly affect MSW—solution composition and RH levels—and seek to determine whether and how it is reflected by virus survival trends.The current study was motivated by the urgent call for the scientific community to address SARS-CoV-2 spread. Like many other viruses, SARS-CoV-2 has been shown to survive well and remain viable on various surfaces, e.g., metal, glass, and plastic, for up to several days46: the enveloped Phi6, and two other tailless non-enveloped model bacteriophages as a reference. Phi6 is a dsRNA phage of the Cystoviridae family that has been suggested as a good surrogate for studying enveloped RNA viruses including SARS coronaviruses49; similar to SARS-CoV-2, it is enveloped by a lipid membrane, has spike proteins, and is of similar size (~ 80–100 nm). The other virus strains that we used are the well-studied, non-enveloped MS2 52 and PhiX174 53. To better understand how RH and the solution composition of microdroplets affect virus survival on surfaces, we combine microscopy imaging to assess MSW state with plaque assays to determine virus survival. We compare survival in ‘sprayed’ microdroplets of three suspensions: human saliva, SM buffer, and ‘pure’ water under a range of RH (23–78%) relevant to most indoor environments. The link between the microscopic environment of viruses, including hydration conditions, and virus viability, is discussed.We use three well-studied bacteriophages previously suggested as good, safe, and easy to work with, as model surrogates for studying surface and air survival of pathogenic viruses6/mL), were added to the suspension. Sprayed microdroplet size ranged between tens to hundreds of microns in diameter . The sprayed well plates were placed under constant temperature and RH conditions  for 14 h, and subsequently microscopy images were taken and virus survival was estimated by the plaque assay using the corresponding bacterial host as a reporter for virus viability . Beads were dispersed fairly uniformly within these microdroplet deposits, as previously demonstrated for virions11. While the beads are at least an order of magnitude larger than individual virions, their visualization can help us grasp the concentration of virions in droplets, and possibly their spatial distribution  to a maximum of ≈ 2 × 104 PFU/mL (RH = 23%) after 14 h in evaporated saliva droplets, compared to ≈ 1 × 104 PFU/mL in the control , again with higher survival than in the suspended medium (4 × 103 PFU/mL). MS2 survival in saliva microdroplets increased with RH, between ≈ 2 × 104 PFU/mL at RH = 23% to ≈ 105 PFU/mL at RH = 78%, compared to 5 × 105 PFU/mL in bulk , MS2 showed positive correlation with weak linear correlation (R2 = 0.31). PhiX174 showed a non-monotonous U-shaped survival as a function of RH, with close to zero correlation and low R2 values (R2 = 0.008).Next, we estimated virus survival under all tested conditions. The content of each well was suspended, and viral viability in the suspensions were evaluated (PFU/mL) by the plaque assay Fig. ”. Virus ulk Fig. , S2. Whi4 PFU/mL in the control, Fig. 2 = 0.87, Supp. Figs. The fact that the water microdroplets were dry at all RH levels, appears to have a large impact on the enveloped virus Phi6, which showed more than 4 log reduction in viability . Combining surface microscopy imaging and the virus viability assays, we were able to explore the link between the tested variables as manifested with respect to microscopic hydration state and virus survival. In summary, we observed significantly higher survival of all three viruses in evaporated saliva microdroplets in comparison to SM buffer and water, 14 h after deposition on a glass surface. The only exceptions were for Phix174 at the higher range of tested RH levels, where survival in saliva was comparable to that of other media. Phage survival in saliva microdroplets as a function of RH showed different trends (at the studied range of RH levels): Phi6 survival decreased with RH, MS2 survival increased with RH, and PhiX174 showed a U-shaped survival-RH relation.2O respectively  can protect the virions for prolonged periods (even days) in such dried saliva droplets on surfaces. In addition, the lower content of inorganic salts in saliva55 than that in SM buffer may explain part of the large differences in virion survival between the two solutions.Remarkably, both enveloped and non-enveloped viruses survive well in evaporated human saliva microdroplets deposited on glass surfaces at a wide range of RH levels. The observation that saliva droplets were completely dry even at 78% RH was somewhat surprising, as we expected saliva to effloresce at 40–50% RH ons see “”. Thus, 11. The absence of protecting components such as salt crystals or saliva proteins may explain the low survival rate.A second remarkable result is the very low survival (> 4 log reduction) of Phi6 in the evaporated water microdroplets, regardless of RH. This result may indicate damage associated with the lipid membrane of the enveloped phage under these conditions. We note that in the control bulk water solution, survival of Phi6 was high and comparable to that in SM buffer and saliva. A possible explanation therefor may be attributed to pH changes that occur during evaporation and drying of microdroplets16. Thus, complete dryness could in fact protect the virions from those high concentrations of dissolved salts and low pH12.This indicates that the physicochemical properties, experienced by the virions in the SM droplets from time of deposition through evaporation until reaching equilibrium , and for the distribution of virions in drying droplets. Large virion aggregates (> tens of virions) might also affect microdroplets’ drying dynamics due to capillary pinning, as we have shown for bacteria 28.Another less-understood issue is viral aggregation39. Because SARS-CoV-2 transmission and disease severity appear to depend upon viral load59, it is likely that in indoor environments where infected individuals stay for long periods, viable viruses persist on fomites for days. Thus, as long as not proved otherwise, indirect transmission through inanimate surfaces—in particular those with prolonged and high contact like mobile phones and touch screens—is not unlikely, and must be considered.The results of this study provide important insights concerning COVID-19. Although performed with a surrogate for SARS-CoV-2, it indicates, as have other studies, that enveloped viruses like SARS-CoV-2 survive well in evaporated saliva droplets on inanimate surfacesThis study provides further evidence that viruses can survive well in evaporated saliva microdroplets for hours after their deposition on surfaces. What makes human saliva a favorable environment for virus survival is not well understood and thus calls for further research. Interdisciplinary research is required to uncover how the physicochemical microenvironment surrounding the virion affects its survival in saliva microdroplets, and may pinpoint specific components of saliva, such as proteins, polysaccharides, salts or surfactants, that are essential for virus survival.10 PFU/mL. Overnight bacterial host cultures were diluted at 1:50 into 1 mL fresh media and grown to OD600 0.3. A single plaque isolate, suspended in 100 µL SM buffer, was used to infect the fresh culture (first propagation cycle). The infected culture was shake incubated at the appropriate temperature for the host strain for 3 h, or until lysis was observed. Meanwhile, the overnight host cultures were diluted again in 9 mL fresh media and grown to OD600 0.3. 1 mL of the first propagation sample was used to infect 9 mL of fresh host cultures (second propagation cycle) that was shake incubated for 3 h or until lysis was observed. The second bacteriophage propagation was centrifuged  and filtered through a 0.22 µm filter (Millex GV). Phages were stored at 4 °C and their concentrations were determined by the drop plaque assay. Pseudomonas syringae (DSM-21482), a host strain for bacteriophage Phi6, was purchased from DSMZ and cultivated in TSB (Tryptic Soy Broth) at 28 °C. Escherichia coli (DSM-13127), a host strain for bacteriophage PhiX174, was purchased from DSMZ and cultivated in LB (Luria–Bertani broth) at 37 °C. Escherichia coli  (kindly provided by Adi Stern), a host strain for bacteriophage MS2, was cultivated in LB at 37 °C.Bacteriophages Phi6 (DSM-21518), PhiX174 (DSM-4497), and MS2 (DSM-13767) were purchased from the German Collection of Microorganisms and Cell Cultures DSMZ. Vacuum-dried phages were revitalized as per DSMZ instructions. Bacteriophage propagation: A two-cycle bacteriophage propagation protocol was used to obtain ~ 10 mL titers of > 108 PFU/mL for Phi6; and MS2 and 107 PFU/mL for PhiX174) were diluted tenfold with: (1) SM Buffer  (2) filter-sterilized H2O  and (3) natural human saliva (donated by one of the authors). Fluorescent beads  were added to the suspension with a 5 × 102 final dilution. We aimed to have an equivalent concentration of beads to those of the Phi6 and MS2 viruses (~ 106/mL). The beads were added for two purposes: (i) to mimic micrometer-sized particulates  that are spread on real-world surfaces and have been shown to affect the formation of microscopic surface wetness28; and to (ii) help interpret the virion distribution within droplets. Solutions were loaded into 5 mL refillable spray bottles  and a portion of the load was sprayed on a 12-well glass bottom plate . Each well was sprayed by pressing the spray nozzle twice . Spray was applied to the well through a 50 mL falcon tube from which we chopped 1.5 cm from its conical end. The plates were placed without the cover lid inside a sealed plastic box. As this study’s focus is virus survival in the indoor environment, we chose to work at a temperature of 25 °C and a range of RH (23% to 78%), which spans most indoor environments. A 100 mL saturated salt solution  was placed in the bottom of each box to maintain relative humidity of 23%, 43%, 57%, and 78%. The boxes were placed in an incubator set at 25 °C for 14 h. Control: spray bottles with the remaining (unsprayed) suspensions were left in incubation for 14 h and sprayed onto the 12-well plates at the end of the incubation period. All plates were imaged at the end of the 14-h incubation period and then suspended with 500 µL of SM buffer for the drop plaque assay.Phage stock solution . On the day of the experiment, overnight bacterial host cultures were diluted at 1:50 into 1 mL fresh media and shake incubated until they reached OD600 0.3. Meanwhile, the top agar  was melted and kept in a 55 °C water bath. The bacterial host culture was combined with the top agar at a ratio of 1:40, and poured on top of the bottom layer. The phages were serially diluted in SM buffer, and after the top agar solidified, either 10 µL, 100 µL, or 1000 µL were pipetted and spread out onto either one quarter, one half, or a full plate (10 × 10 cm petri dish) respectively. Plates were left open until dry, and incubated at the appropriate temperature for the host strain (as described above) overnight.Plates containing the bottom agar layer were poured in advance  during image acquisition. Images were collected using an Eclipse Ti-E inverted epi-fluorescence microscope (Nikon) equipped with a Plan Apo 20x/0.75 NA air objective and the Perfect Focus System for maintenance of focus. An LED light source  was used for fluorescence excitation. Rhodamine B-marked fluorescent beads were excited through a 560/40 filter, and emission was collected with a T585lpxr dichroic mirror and 630/75 filter . Images were acquired with a SCMOS camera  controlled with NIS Elements 5.02 software (Nikon Instruments Inc.). 10 × 10 adjacent fields of views  were monitored per each well. Multiple stage positions were collected using a motorized encoded scanning stage .2, F-tests, and P-values for statistical significance of the linear models  and the independent variable of RH, we fitted statistical models in MATALB using ordinary least-squares regression. We evaluated linear correlations based on RStatistical significance of the differences in viability reductions between saliva and the other two aqueous media, under equal RH levels, was assessed using Welch t-test (see Supp. Fig 6 per mL. The original volume of a droplet that left a 100-µm diameter deposition was estimated at ~ 0.01 nL. Assuming there is no significant virus aggregation and that viruses are uniformly distributed, we obtain ~ 106 per mL × 10–5 = 10 virions per such microdroplet.The initial concentration of viruses was ~ 10Supplementary Video 1.Supplementary Video 2.Supplementary Video 3.Supplementary Information 1.


The huge genetic diversity of circulating viruses is a challenge for diagnostic assays for emerging or rare viral diseases. High-throughput technology offers a new opportunity to explore the global virome of patients without preconception about the culpable pathogens. It requires a solid reference dataset to be accurate. Virosaurus has been designed to offer a non-biased, automatized and annotated database for clinical metagenomics studies and diagnosis. Raw viral sequences have been extracted from GenBank, and cleaned up to remove potentially erroneous sequences. Complete sequences have been identified for all genera infecting vertebrates, plants and other eukaryotes . To facilitate the analysis of clinically relevant viruses, we have annotated all sequences with official and common virus names, acronym, genotypes, and genomic features . Sequences have been clustered to remove redundancy at 90% or 98% identity. The analysis of clustering results reveals the state of the virus genetic landscape knowledge. Because herpes and poxviruses were under-represented in complete genomes considering their potential diversity in nature, we used genes instead of complete genomes for those in Virosaurus. At least 130 different viruses infecting humans have been recorded so far . These tHigh-Throughput Sequencing (HTS) technology makes possible the detection of all genetic entities present in a sample. Thus, this method offers promising perspectives for virus detection and diagnosis . HTS proTo fill these requirements, Virosaurus (for Virus Thesaurus) is an unbiased viral database built from eukaryotic virus genomes publicly available, cleaned and annotated. The focus of this database is not only to provide accurate references of virus diversity, but also to add a layer of annotation for better clinical analysis of results. Other virus reference databases have been published but with a different scope or annotations. VirusSITE is a virus reference database published in 2016 , mainly Here, we describe the Virosaurus reference database and the method used to build and annotate it. We focused on complete virus genomes to be compatible with existing clinical pipelines ,8,9,10, All virus data  were stored in a relational database (PostgreSQL) with a Java written interface. FASTA files were extracted from this database.https://viralzone.expasy.org/655) have been kept and divided in vertebrate, plant and other eukaryotes, the others have been discarded. The list of vertebrate host viruses comprised 330 virus genera. Viral species were assigned to each sequence using the hierarchy of NCBI taxonomy, and the official ICTV species names . Low quality sequences were then removed through a series of quality controls: removal of sequences with too many “N”s (more than 10%) or at least one gap annotation of unknown length. Some sequences classified under viral taxonomy contained non-viral elements, like vectors or recombined oncogenes. Since those are liable to produce false positives during the analysis, they were removed by the application of keywords filtering (see main text). All quality controls were refined after testing against at least 10 known clinical blood samples at each release to identify false positives.All viral sequences were downloaded from the INSDC database at GenBank in FASTA file format. Because we aimed at creating a eukaryotic virus database, these sequences were filtered using the NCBI taxonomy hierarchy. Virus sequences belonging to genera able to infect eukaryotes as per ViralZone . Papillomaviruses and torquetenoviruses were not considered relevant in a clinical report, so for those the usual name is “HPV” or “TTV” in order to pool all these viruses into one entity.Genotype or serotype data were identified by using GenBank taxonomy classifying Annotations are stored in FASTA header. The header contains 11 different topics annotated by a controlled vocabulary. Data come from GenBank, ICTV, ViralZone and manual curation.<GenbankID> Genbank accession number of the sequence displayed in the FASTA.<SequenceID> By default Genbank accession number of the sequence displayed in the FASTA. If the displayed sequence is a portion of a GenBank entry, for example in the case of genes, the Sequence ID is a unique identifier like GENE_583-3988.Usual name = Name of clinical level entity; if the scientific name is not commonly used, the common clinical name replaces it, for example parvovirus B19 is the usual name of the Primate erythroparvovirus 1 species. Clinical level = Gives the taxonomic level suggested to be relevant for usual clinical diagnostics. By default <species>, but can be at <genus> level like for TTVs or HPVs.Clinical typing = Unknown by default. Otherwise contains data clinically relevant below the species level. This can be genotypes (example:HCV) or qualifiers . In rare cases of mixed clusters, several types are listed separated by a comma, this notably happens for some HPVs “low risk” and “undetermined risk” that are very similar.https://talk.ictvonline.org/taxonomy/. In rare cases of mixed clusters, several species are listed separated by a comma, this notably happens for some segments of rotavirus A and C, which are very similar within different species.Species = indicates the current official species name, as reported by the International Committee on Taxonomy of viruses (ICTV): https://www.ncbi.nlm.nih.gov/taxonomy of the taxonomic entity at species level.Taxid = Taxonomy identifier from NCBI taxonomy database: https://viralzone.expasy.org/resources/Acronyms.xlsx Nucleic acid= Nature of viral genome, either RNA or DNA for most viruses, RNA/DNA for retro-transcribing viruses .Acronym = Abbreviation referring to virus name as reported in ViralZone acronym list: Circular = Y or N for yes or no. This is essential to efficiently map reads at both extremities of the FASTA sequence.https://viralzone.expasy.org/Segment = N/A for non-segmented viruses. For segmented genomes: official segment name as reported in the ViralZone database: https://github.com/sib-swiss/virusscan) on a set of sequencing data ) obtained from 20 human clinical specimens . These samples were previously reported positive by HTS for RNA and/or DNA viruses known to infect humans [During development of the database, beta versions have been tested using an unpublished pipeline . Non-vertebrate eukaryotic viruses might be useful for research or non-clinical studies. We have identified 330 virus genera in which at least one virus infects vertebrates . All sequences have been quality-controlled to exclude low-quality data with gaps or stretches of “N”. Particular care has been devoted to the elimination of non-viral sequences that happen to be classified under “virus” in GenBank: sequencing or assembly artifacts, viral expression vectors, patented sequences, artificial recombinant sequences, and so on. These non-viral sequences can generate false positives, for example by being similar to human genomic fragments present in most clinical samples. Such erroneous sequences are removed by two means: sequences entries containing “patent”, “Vector”, “Artif*”, “recombinant” were excluded; and testing of the database on different types of characterized clinical samples allowed removing sequences producing false positives. In the 2020_04 release, 3,002,470 virus sequences have been recovered from GenBank, and 2,936,380 sequences have been selected by the pipeline for sequence completeness.Virosaurus has been created using all GenBank genetic data (release 19-03-2020) . This meIdentifying complete genomes was more challenging for segmented viruses, composed of two to twelve genetic segments. The complexity lies in the fact that segment naming is not consistent in GenBank deposition, and isolate names happen to be different across all segments. For those viruses, we have decided to work with complete segments instead of complete genomes. To harmonize and correct segment naming, all segments have been identified by BLAST against Swiss-Prot, and named using official nomenclature. Then, minimum and maximum size range of complete segments has been manually defined as above. Although heuristic, this method gives optimal results with the current data. Moreover, it is automated, which is an asset for updating the database.Putting together complete genomes for non-segmented viruses, and complete segments for segmented viruses, creates the dataset we call the “complete virus sequence dataset”.The complete virus sequence dataset has been annotated by adding controlled vocabulary metadata in the FASTA files . All seqMetadata about the viral genome comprises GenBank accession number, nucleic acid nature, number of segments, circular/linear and sequence. Considering that some sample preparation protocols target either RNA or DNA, it is essential to know if the viral genome is DNA or RNA for virus detection. For example, RNA virus detection in DNA preparations should trigger further investigations to rule-out contaminants. Whether the genome is circular or linear matters if users want to assemble HTS reads. In the case of circular genomes, reads can overlap at the start and end of the sequence. All metadata are added to the FASTA files in the header section of each sequence .Each sequence in the INSDC database is linked to a virus isolate. Nonetheless, isolate data are not useful for clinicians in daily routine. To propose a relevant output, the Virosaurus database was designed to offer a broader classification of viruses at a higher level . We haveBecause virus genomes are small and thus relatively easy to sequence, thousands of genome sequences are available, and these datasets are highly redundant. Redundancy can result in bias, and increases computational time with no benefits. Clustering computer programs are designed to removing redundancy by alignment methods. This consists of calculating clusters of similar sequences at a given percentage of similarity, and selecting one sequence to represent the cluster. We used CD-HIT to cluster our complete virus sequence dataset at 90% and 98% similarity thereby creating Virosaurus90 and Virosaurus98 . ClusterDetection of viruses by HTS and bioinformatics analysis depends on the quality of the references. If the latter is suboptimal, this could lead to false negatives in the detection results for important viruses. Do we have enough knowledge of each viruses’ genetics to detect circulating viruses? The clustering method allows estimation of the genetic variability of a virus. A virus for which the genetic landscape has been thoroughly mapped will comprise many sequences in each cluster. On the other hand, we have only partial knowledge when clusters are isolated and contain few or a single sequence. This lack of redundancy suggests that herpesviruses and poxviruses are more diverse than we know, and we might be unable to detect all circulating viruses with our reference dataset. We decided to use a different method to gather sequences for these two virus families. Since there are too few complete genomes, we have used genes instead of genomes . Using gThe Virosaurus database 2018_11 has been tested on sequencing data from 20 human clinical samples previously characterized by HTS using the ezVIR pipeline  9,19,20,23,24,25https://viralzone.expasy.org/8676 and is licensed Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0). We plan to update the database every year with new sequences, and to apply all changes in the taxonomy. The pipeline to create the database has been automatized, thereby lowering the cost and time of such an exercise. Moreover, we plan to apply quick hotfixes whenever erroneous sequences are reported, or when a new virus emerges. For example, the SARS-Cov-2 isolate Wuhan-Hu-1 complete genome was first added by patch in January 2020, and then fully included in the April 2020 release.Virosaurus can be downloaded online at The creation of a eukaryotic virus reference database using taxonomic clusters requires a list of complete virus genomes and segments. There was no database of complete virus genomes available; therefore, we developed a method to identify those genomes. This dataset of complete virus sequences is the first to be created, to our knowledge, and may be useful for further references or epidemiologic studies. By using complete genomes or segments, Virosaurus allows seamless integration with many existing pipelines in hospitals and helps the final interpretation of results. The underrepresentation of herpes and poxviruses among complete sequences led to the use of genes instead of genomes to broaden the genetic diversity covered by the reference dataset without affecting the methods used and the final output. Indeed, all reads are mapped to reference sequences, which are in turn mapped to a virus species or genus. Whether the sequences are complete genomes or single genes does not matter. The only inconvenience is that, for these two virus families, we lost the possibility of tracking the genome coverage by HTS reads. This metric is invaluable to assess artifacts or contamination, but is not mandatory. Indeed, PCR methods rely on the specific detection of only a small part of viral genomes and are accurate.It is essential to minimize the risk of false negatives and false positives in diagnosis. False negatives could partially result from missing essential information. We estimated the quantity of references available for most human viruses and it revealed that herpes and poxviruses did not reach an adequate threshold. This highlights the need to sequence additional genomes for a better coverage of these viruses’ genetic landscape. Many false positives were easy to spot because of the similarity with the human genome and therefore were detected in most samples. We observed those false positives resulted mostly from sequences containing non-viral genes that were not filtered out by our method. False positives need to be evaluated at each release and manually excluded from the database. Another way to deal with sequences similar to human sequences would be to cross-reference the human genome with the virus database and exclude sequences from the latter, but this may remove too many sequences because of sequences of viral origin within the human genome.The dataset of virus infecting vertebrates should be valuable for clinical diagnosis. In the last 100 years all emerging viruses have jumped to humans from vertebrate host reservoirs . Nonetheless, it is not impossible that some viruses belonging to a genus only known to infect non-vertebrate hosts do indeed infect humans in unknown conditions . Moreove


Rhabdoviridae: Ephemerovirus) (BEFV) causes bovine ephemeral fever (BEF), an economically important disease of cattle and water buffalo. Outbreaks of BEF in Africa, Australia, Asia and the Middle East are characterized by high rates of morbidity and highly efficient transmission between cattle hosts. Despite this, the vectors of BEFV remain poorly defined.Bovine ephemeral fever virus (Culicoides sonorensis) and mosquitoes  were infected with a strain of BEFV originating from Israel by feeding on blood–virus suspensions and by intrathoracic inoculation. In addition, in vivo transmission of BEFV was also assessed by allowing C. sonorensis inoculated by the intrathoracic route to feed on male 6 month-old Holstein-Friesian calves.Colony lines of biting midges  in mosquitoes of three different species was found. Evidence was found for infection of  BEFV is the causative agent of bovine ephemeral fever (BEF), also known as ‘3 day sickness’, a non-contagious disease of acute febrile clinical signs in cattle and water buffalo. Clinical disease is usually mild in younger animals, often including bi-phasic fever, salivation, lameness and muscle stiffness  collected near clinical cases of BEFV led to the isolation of virus on the fourth mouse-brain passage, confirmed by a complement fixation reaction . Alternatively, using detection of BEFV RNA in the body at levels above that on day 0 (i.e. 3 of 170 midges), the estimated vector competence was estimated to be 1.8% (95% CI 1.0–2.5%).C. sonorensis IT inoculated with 0.2 µl BEFV, 19 (38%) survived the 5-day incubation period. The five C. sonorensis processed immediately following intrathoracic inoculation were tested for the presence of BEFV by RT-qPCR; median Cq was 31.6 (range 31.0–33.2). In comparison, the 19 C. sonorensis processed following incubation had a significantly  lower median Cq of 25.0 (range 23.7–27.3)  individuals survived the incubation period. The median Cq of the seven C. sonorensis processed immediately following intrathoracic inoculation was 29.8 (range 28.7–31.7). Of the 42 insects processed following incubation, 21 were processed whole and 21 heads and bodies were processed separately  and whole insects . BEFV RNA was also detected in 19 of the 21 tested heads . The median Cq values differed significantly  among these groups, with the highest Cq values (denoting low levels of detected BEFV RNA) obtained in the midges tested at day 0, followed by the heads of insects tested at day 6, then the bodies of insects tested at day 6, with the lowest Cq values detected in whole insects tested at day 6 , with Cq values that were significantly  lower (denoting more BEFV RNA) than those in midges tested at day 0  . No BEFV RNA was detectable by RT-qPCR in any insect on day of feeding  or after 8 days of incubation (study day 15).On study day 7, 7 days after the cattle were initially exposed to No clinical signs of viral infection in the calves were observed at any point during the study. Temperatures of all animals remained within ± 1 °C of baseline value across the study. BEFV RNA was not detected within any of the blood samples collected from any calf throughout the study, even after additional BEFV exposure by intradermal/subcutaneous inoculation on study day 11. No antibodies against BEFV were detected by ELISA in serum collected from any calf. Signs of ascites and pericardial fluid were observed at post-mortem; however, no BEFV RNA was detected by RT-qPCR in any of the fluids, and no antibodies were detected by ELISA.Based on the outcome of the transmission attempts , the maximum likelihood estimate for the probability of transmission from vector to host was zero (upper 95% confidence limit 0.016).Culicoides midges (C. sonorensis), inferred by comparing the level of viral RNA in incubated flies with that in those killed immediately following blood feeding. The vast majority of insects tested were capable of clearing ingested BEFV to the extent that no viral RNA was detectable, including all of the mosquito species tested . In C. sonorensis, a total of 170 individuals were tested, of which 13 returned a positive signal for BEFV RNA when their bodies were processed, including three individuals that demonstrated higher viral RNA levels  than those obtained in individuals processed immediately after blood-feeding (day 0 C. sonorensis), indicating viral replication. Studies of infection in C. sonorensis with other viruses (e.g. BTV) have previously demonstrated infections where there was potential both for retention of virus at low and sub-transmissible levels of infection [C. sonorensis and BTV has included salivary glands [C. sonorensis has the potential to act as a vector for BEFV, with a low vector competency, in the field despite no overlap in current geographic range of this species and the virus. To further investigate the risk of BEFV transmission within a European context, field-caught Culicoides species should be tested for susceptibility to infection through oral exposure to virus.The aim of this study was to provide evidence for potential vectors of BEFV, particularly those that might transmit the virus to cattle. Experiments that fed artificial blood meals containing BEFV to colony lines of potential dipteran vectors found evidence for replication in nfection . This canfection . The eviy glands . This reC. sonorensis that were IT inoculated with BEFV contained RNA at 5, 6 or 7 days post-infection. This technique bypasses the mesenteron infection and escape barriers as well as the hemocoel dissemination barrier and results in full infection, as demonstrated in C. sonorensis IT inoculated with BTV. In addition, while C. sonorensis infected via this route consistently infect ruminants with BTV with high efficacy [Culicoides fed on each calf would have been more than sufficient to establish infection and viremia in BTV/sheep infection studies [Culicoides-borne virus, but which could be further explored in future studies through the use of honey-impregnated filters or direct saliva collections [All efficacy , this wa studies . It is plections . It is min vitro cultivation of BEFV from initial isolation leading to the adaptation of BEFV to the cell line used. This has previously been reported for tissue culture-adapted foot and mouth disease virus type O pre-selecting for heparin-binding viruses, which were subsequently attenuated in the natural host [in vivo infection of virus isolates compared to non-tissue adapted virus. It was hoped that infection and replication within the potential Culicoides vector might select viral variants still capable of replicating in the ruminant host as an approximation of alternating viral replication between insect and mammalian hosts. Indeed, previous studies determined that the genetic diversity of BTV passaged on mammalian cells was significantly reduced when compared to BTV passaged on insect cell lines (KC cells derived from embryonic C. sonorensis) , with tral host . The funrensis) ( discusserensis) .The successful detection of BEFV genome by RT-qPCR, but the lack of successful virus titration from the original insect material were not surprising considering the potential for losses in infectivity during the freeze–thaw cycles between collection, processing for RT-qPCR and titration, enhanced by an additional freeze–thaw cycle due to the malfunction of the − 80 °C storage freezer between the PCR analyses and the titration experiments. The potential for a loss of infectivity was supported by an observed tenfold drop in infectious virus between titrations of the BEFV, and the impact of repeated freeze–thaw cycles has been previously noted as a reason for a lack of re-isolation of BEFV from original insect material .Culicoides, and mosquitoes, such as Culex annulirostris, found in Australia, parts of Southeast Asia and islands of the western Pacific. Oral infection studies with BEFV did not lead to infection of Culex pipiens, Cx. quinquefasciatus or Aedes aegypti mosquitoes with this virus, but evidence was found for infection in Culicoides biting midges. It should be noted that although only a relatively small number of mosquitoes were preliminarily investigated for BEFV infection compared to the number of Culicoides, this does reflect field densities of these vectors, where a low vector competence in Culicoides may be compensated by the very high local abundance and high biting rate. An estimated competence of < 10% for all the mosquito species tested and absence of evidence of dissemination suggest a low vectorial capacity for BEFV. This result implicates Culicoides—rather than mosquitoes—as a likely vector for the virus should it be introduced into Europe, and points to midge control measures as being the most effective to implement.Published data on the arthropod vectors of BEFV have identified a number of potential vectors, including biting midges of the genus C. sonorensis was demonstrated through both membrane feeding on blood–virus suspension and intrathoracic inoculation. Despite this, an in vivo study aimed at establishing a bovid: C. sonorensis: BEFV model failed, due to a lack of transmission from what were hypothesized to be fully infected insects into the calves. The reasons for this failure remain undetermined but may relate to previously encountered challenges in working with intensively cell culture-passaged BEFV strains. Future studies will examine dissemination of BEFV within this Culicoides species and why onwards transmission to calves did not result in virus replication in the ruminant host despite successful insect blood-feeding and dissemination of the virus in the insect vector.This study highlights the challenges of carrying out studies on the transmission of BEFV under laboratory conditions. Evidence of replication and dissemination of the bovine/Israel/2005-6 BEFV strain in C. sonorensis for the first time, both by feeding through membranes on blood–virus suspensions and through intrathoracic inoculation. No replication was observed in mosquitoes. Attempts to transmit BEFV via C. sonorensis feeding on cattle, however, failed, and thus the dissemination of the virus in the insect vector requires further investigation.To the best of our knowledge, the results of this study demonstrate the replication of BEFV in Additional file 1: Animal temperatures and clinical scoring notes.Additional file 2: RT-qPCR and titration results for Culicoides sonorensis orallyfed BEFV and intrathoracically inoculated with BEFV.


Molecular motors are microtubule-based proteins which contribute to many cell functions, such as intracellular transportation and cell division. The details of the nature of the mutual interactions between motors and microtubules still needs to be extensively explored. However, electrostatic interaction is known as one of the key factors making motor-microtubule association possible. The association rate of molecular motors to microtubules is a way to observe and evaluate the charge of the bio-motors in vivo. Growing evidence indicates that microtubules with distinct structural compositions in terms of beta tubulin isotypes carry different charges. Therefore, the electrostatic-driven association rate of motors–microtubules, which is a base for identifying the charge of motors, can be more likely influenced. Here, we present a novel method to experimentally confirm the charge of molecular motors in vitro. The offered nanotechnology-based approach can validate the charge of motors in the absence of any cellular components through the observation and analysis of the changes that biomolecular motors can cause on the dynamic of charged microspheres inside a uniform electric field produced by a microscope slide-based nanocapacitor. This new in vitro experimental method is significant as it minimizes the intracellular factors that may interfere the electric charge that molecular motors carry. Biomolecular motors are one of the cellular components carrying a significant role in cell functions ,2. SinglSome members of the kinesin family, such as kinesin-1, are known for their ability to move along microtubules ,5,6. TheNew evidence indicates that the functions of motors can be regulated by the structural specifications of microtubules that they employ as their track. The changes in biomechanical specifications of molecular motors, including processivity, velocity, and their binding time to microtubules, which has been reported in some studies, strengthen the possibility that the function of molecular motors can be cell specific ,10,11,12In the present work, we demonstrate a new method to confirm the charges of molecular motors in vitro. Through implementing this method, we have successfully identified and confirmed the electric charge of one of the molecular motors, kinesin-1.In this study, we confirmed the positive electric charge of one sub-group of kinesin motors, kinesin-1, in the absence of other cellular components in vitro, by utilizing an experimental nanotechnology-based approach. In this experimental design, we developed a method to monitor the behavior of kinesin-coated microspheres under the influence of a uniform electric field.It was particularly important to first monitor the behavior of plain microspheres inside the electric field. To conduct these control experiments, 1 to 1.2 µL of prepared beads, described in the Materials and Methods section, was transferred and confined in the space between the electrodes of the microscope slide-based nano-capacitors. The area between electrodes and consequently the beads in this area were visualized under darkfield microscopy. Upon the implementation of the DC voltage and establishing a uniform electric field between electrodes, suspended microspheres in the surrounding liquid media started to move toward the positive electrode of the capacitor. This was an indication that the beads were originally negatively charged and started to move due to the electrophoretic force.The behavior of these plain beads in a kinesin free environment was assessed under two different implemented voltages of 1 and 1.5 volts. The uniform electric fields of 66 V/cm and 100 V/cm that can be produced between electrode gaps in the absence of any dielectric was affected, due to the dielectric specifications of the beads and the surrounding liquid media.In our experimental design, we did not quantify the dielectric constant and the actual value of the produced electric field was not precisely achievable due to the random number of beads, as well as the random density of molecular motors in the capacitor gap. While the value of the dielectric constant was not a factor in our experiment, we know that the actual value of the strength of the electric field reduces due to the presence of the media between electrodes. In The observed displacement of each individual bead primarily resulted from interactions of three different forces: the electrophoretic force created by the electric field on the originally negatively charged beads; the drag force on the beads due to passing through the surrounding solution; a possible dielectrophoresis force associated with the differential polarization of the object and the environment around it ,23,24.The displacement of several prepared beads, as described in Materials and Methods, under the two different implemented voltages and, consequently, two distinct uniform electric fields, was observed, recorded, and analyzed by Image J. The data collected from several individual beads were then collectively used to express displacement in terms of mean ± SEM at several sequential time frames, as shown in t = 1 s were 33.67 ± 4.6 µm  and 25.7 ± 4.9 µm , with the two tailed p-value equal to 0.25. Additionally, a higher value of the velocity, which was observed in the stronger electric field was an assurance for the applicability and reliability of the designed experiment. Furthermore, the motion started with high velocity, which was reduced significantly under the effect of the drag force [Illustrated in To explore the existent kinesin-1′s electric charge, the behavior of kinesin-coated beads under the influence of the uniform electric field was evaluated in different sets of experiments and under the same experimental conditions.We first mixed the prepared beads with a concentration of 0.25 mg/mL kinesin-1. This mixture was then immediately used in making samples by transferring 1.2 µL of the mixture to the micro-capacitor’s gap. We refer to the beads in such samples as “un-incubated beads”. In the next set of experiments, the mixture of the kinesin-1 and the beads was incubated at room temperature for 30 min before the samples were prepared and the displacements of the beads were analyzed. Finally, we conducted the same experiment when prepared beads were incubated under the same conditions in a concentration of 0.5 mg/mL kinesin-1 protein.The drifted motion of the beads in all experiments showed the same pattern as those obtained from the beads tested in control experiments. As the data indicate, in samples of un-incubated beads, the average displacement of those beads was reduced, compared with that calculated from the measurements of the beads in the control experiments (in the kinesin-free environment). This reduction is more likely linked to the change in several factors—the presence of kinesin-1 protein in the solution elevated the viscosity of the solution and, consequently, increased the strength of the drag force acting on the beads. In addition, the dielectric constant of the liquid environment between the two electrodes of the nanocapacitor was affected, which may cause a reduction in the strength of the electric field between two electrodes.n = 5 measurements), which was significantly different from the displacement obtained from the beads in the control experiments (33.67 ± 4.6 µm) which was conducted in the absence of any kinesin-1 proteins (p = 0.002). The reduction in the average displacement of the un-incubated beads inside the surrounding media containing kinesin proteins is an indication that the factors explained above  were affected. We conducted the next set of experiments to confirm the charge of kinesin. To do so, the behavior of microsphere beads incubated with kinesin proteins was evaluated under the same implemented voltage of 1.5 V.The average displacement for un-incubated beads after 1.0 s and under electric field produce by the voltage of 1.5 V was obtained to be 8.84 ± 1.8 µm . As compared with the displacement of un-incubated beads after the same time, the further reduction observed in the displacement of beads in these samples was noticeable (p = 0.08).The average displacement for beads incubated in 0.25 mg/mL kinesin-1 for 30 min was obtained to be 5.7 ± 0.7 µm . This value for the incubated beads in the same protein concentration was obtained to be 3.2 ± 0.5 µm . Once again, incubated beads showed a more significant reduction in their displacements (p = 0.09).The average displacement of un-incubated beads in the samples with the higher concentration of protein was 4.52 ± 0.43 µm (p = 0.01 for the average displacement obtained from incubated samples in two concentrations and p = 0.02 for the similar parameter obtained from unincubated samples).Additionally, beads in higher protein concentration showed an average displacement almost equal to 40% of that observed in samples with lower protein concentration. This difference is statistically significant . The thickness of the ITO was almost 5500 Å. The uncoated gap created in the center of the microscope slide was 150 µm . Upon connection to a DC power supply, a uniform electric field was created in the gap of the micron-sized capacitor built on each microscope slide.The implemented voltage in this study was 1.5 volts . However, in our controlled experiments the implemented voltages were 1 and 1.5 volts, as explained in the next section. The magnitudes of the electric fields in the absence of a dielectric material under these voltages were almost 66 V/cm and 100 V/cm accordingly.3. Due to the high concentration of beads, they were diluted 104 times by serial suspension in purified water. We conducted parallel experiments under the same experimental conditions. Therefore, no attempt was made to wash the beads and separate them from their liquid phase prior to the serial dilution of the beads. We refer to these beads diluted by water as “prepared beads”.The Silica microspheres employed in this experiment was purchased from Bangs Laboratories, Inc. . The Sio2 microsphere (beads) were available as an aqueous suspension. The diameter of individual beads was 0.5 µm with a density of 2 g/cmKinesin-1 was obtained from Cytoskeleton Inc. . The lyophilized kinesin-1  was first re-suspended in purified water to achieve the concentration of 1 mg/mL, divided to small allocations, and stored at −20 °C. To reach a lower concentration of kinesin at the time of the experiment, each allocation was mixed with prepared beads (explained in the previous section). The finalized concentration of molecular motors used was 0.25 mg/mL and 0.5 mg/mL. The mixture of molecular motors–beads was either immediately used or was incubated for 30 min at room temperature prior to being used. This incubation time created an environment for the molecular motors to attach to the beads. The 1–1.2 µL of these un-incubated kinesin-beads or incubated kinesin beads were then transferred to the micro-capacitor. The sample was then covered by a clean coverslip  and completely sealed with vacuum grease, which created a confined geometry with inner glass, surfaced around 5 µm. Through this procedure, beads were randomly distributed in the created enclosed area. Among them, some of the beads fell in the 150 µm area between two electrodes of the microscope slide-based capacitor. The beads that fell between two electrodes of the capacitor were visualized and the behavior of the beads in that area was monitored, as explained below.It should be emphasized that the goal of the experiment was to confirm the nature of the charge of kinesin proteins. To achieve this goal, we eliminated the interaction of kinesin proteins with other potentially charged proteins by preparing samples in the absence of any blocking solutions (such as casein solution). This choice of experiment had two effects: it increased (a) the possibility of un-wanted binding of protein coated beads with each other and consequently the formation of the clumping of the beads; (b) the immobilization of the beads due to un wanted adhesion to the surface of the microscope slide.The majority of the beads in the samples were clumped after 40–50 min of the incubation. This was an indication that the beads were covered by kinesin protein. To observe the behavior of single beads, with a reasonable level of certainty that the beads were incubated long enough to be coated by the protein, the mixture of protein–beads was ideally incubated for 30 min.In addition, the nano-capacitors could easily burnout by increasing the voltage. As we added the kinesin protein, no movement was observed in the initial voltage of 1 volt at the presence of 0.25 mg/mL or less of kinesin proteins. Our experimental conditions were adjusted by increasing the voltage to 1.5 volts. At this set voltage, we could repeatedly observe the change, which resulted in the displacement of the beads in the presence of kinesin in different samples. However, while we could potentially see the bead movements in the presence of lower protein concentration with increasing the voltage, this approach was not applicable, due to the limitation of the functionality of the capacitors under higher voltages. We did not observe the beads’ movements in the concentrations higher than 5 mg/mL, mainly due to the binding of the beads to the glass surface or one another. The choice of protein concentrations in this study provided us with the experimental frame that the beads’ movements and any changes in their movements were significant and were observed under our experimental limitations.https://imagej.nih.gov/ij/) to assess any possible changes in the size of the beads due to the coverage with the protein. In incubated samples with 0.5 mg/mL of protein, the average diameter obtained was 0.52 ± 0.006 µm, which is comparable with the original sizes of the prepared beads. Therefore, no evidence of significant changes in the sizes of the beads after the incubation was observed.Further, the average diameter of the beads after the incubation with the high concentration of kinesin protein was measured , equipped with a 100×/1.25 NA oil immersion objective lens, and a 1.43–1.20 oil dark-field condenser. The microscope was connected to a Lumenera- Infinity 1-3C camera with Infinity Analyze software . The movement of the individual beads was then recorded through video microscopy. The movies taken through this procedure were then analyzed by ImageJ .p value was then obtained.In our study, we measured the displacement of the different beads. However, as explained above, we experienced the unwanted binding of the beads with the microscope slide surface in the absence of the blocking solutions. Therefore, to obtain data, we relied on one-time recording of the movement of a single bead under the uniform electric field. Therefore, our data are obtained from several samples prepared under the same experimental conditions. After collecting the data from different samples, the mean ± STEM of the displacements was calculated. Consequently, the associated


Solanum tuberosum) is a major food source in the whole world including Bangladesh. Viral diseases are the key constraint for sustainable potato production by reducing both quality and quantity. To determine the present status of eight important potato viruses in Bangladesh, tuber samples were collected from three major potato growing regions  in January–February 2017 and February 2018. Reverse transcription polymerase chain reaction (RT-PCR) with coat protein (CP)-specific primers were used to amplify CP sequences of the respective viruses, and confirmed by sequencing, which were deposited in the GenBank. Results indicated that the tuber samples were subjected to Potato leafroll virus (PLRV), Potato virus X (PVX), Potato virus Y (PVY), Potato virus S (PVS), Potato virus H (PVH), Potato aucuba mosaic virus (PAMV) and Potato virus M (PVM) infection, whereas mixed infections were very common. Phylogenetic analysis revealed that the PLRV from this study was closely related to a Canadian and a Chinese isolate, respectively; PVX was closely related to a Canadian and a Chinese isolate, respectively; PVY was closely related to a Chinese isolate; PVS was closely related to a Chinese and an Iranian isolate, respectively; PAMV was closely related to a Canadian isolate; PVH was closely related to a Huhhot isolate of China; and PVM was closely related to an Indian and an Iranian isolate, respectively. As far as we know, PAMV in this study is the first report in Bangladesh. These findings will provide a great scope for appropriate virus control strategies to virus free potato production in Bangladesh.Potato ( Potato leafroll virus , Potato virus X , Potato virus Y , Potato virus S , Potato virus H , Potato virus M  and Potato virus A  have been reported so far [Potato aucuba mosaic virus  is an emerging potato virus worldwide, especially in Southeast Asia [2 to 105 times more sensitive in comparison to traditional ELISA technique for potato virus detection [Potato, the power house of energy, is the principal vegetable crop in Bangladesh . In 2018d so far ,10,11,12d so far ,14,15. Bast Asia ,16,17. Aast Asia . Accuracast Asia ,22. In aetection . Althougetection ,25,26. Netection ,28,29. Hetection .>In the present study, RT-PCR based detection followed by subsequent sequencing was performed to identify and characterize eight important potato viruses  in three key potato growing regions  of Bangladesh and also accomplish their relative incidence. Additionally, phylogenetic association with formerly reported virus isolates from the National Center for Biotechnology Information (NCBI) database was also analyzed. Findings from this study will provide an excellent scope for future research on the biological features of these viruses, including the effects on yield and quality, host range and distribution, and also for virus free potato production.Rapid and exact detection of viruses are pre-requisite for virus-free seed potato production, and planting of these virus-free potato seed is an important approach to control potato viruses in field to enhance the commercial potato production . RT-PCR Sequence analysis from this study with the BLASTn server of NCBI confirmed the presence of PLRV, PVX, PVY, PVS, PVH, PAMV and PVM . All theComparison of viral sequences found in Bangladesh showed that PLRV isolates shared a maximum 95.69% nucleotide (nt) identity with each other; PVX shared a maximum 96.36%, 97.06% and 99.30% nt identity with each other; PVS shared a maximum 93.79% nt identity with each other; PVM shared a maximum 80.59% nt identity with each other; and PAMV shared a maximum 96.93%, 97.47% and 97.60% nt identity with each other. Comparison of viral sequences from the present study with the respective viral sequences from the GenBank database further exhibited that PLRV from this study shared a maximum 95.52% and 100% nt identity with a Chinese isolate (GenBank accession no. MF062487) and a Canadian isolate (GenBank accession no. D13954), respectively; PVX shared a maximum 98.31%, 99.72% and 98.59% nt identity with a Scottish isolate (GenBank accession no. GU144353), a Shandong isolate (GenBank accession no. AF528555) and a Heilongjiang isolate of China (GenBank accession no. GU373815), respectively; PVY shared a maximum 99.38% nt identity with a Chinese isolate (GenBank accession no. KC296825); PVS shared a maximum 98.87% and 95.14% nt identity with a Chinese isolate (GenBank accession no. AY512653) and an Iranian isolate (GenBank accession no. MH159208), respectively; PVH shared a maximum 100% nt identity with a Huhhot isolate of China (GenBank accession no. HM584819); PAMV shared a maximum 97.07–97.60% nt identity with a Canadian isolate (GenBank accession no. S73580); and PVM shared a maximum 93.68% and 82.24% nt identity with an Indian isolate (GenBank accession no. KJ473993) and an Iranian isolate (GenBank accession no. KC129092), respectively. As PVH-CP sequence exhibited 100% nt similarity with a Huhhot isolate of China, we executed an additional RT-PCR with a primer pair encoding the triplegene block-1 region, whereas the sequence (GenBank accession no. MH932394) shared a maximum 99.86% nt identity with the same isolate.To assess the evolutionary relationship, nucleotide sequences from this study and existing sequences in NCBI database of respective viruses were aligned by ClustalW . Here, iViruses are the principal threat for potato production all over the world, including Bangladesh ,10,34. APhylogenetic analysis denotes various important information including the degree of relationship among genera, species and strains, diversity of geographical isolates, and the origin and evolution of plant viruses ,43. An aGlobally, a number of researchers used identical potato virus identification techniques to our study ,47,48,49Potato tuber samples  from the three major potato growing regions of Bangladesh, Munshiganj , Jessore  and Bogra  districts were collected at harvesting stage in January–February 2017 and February 2018. Altogether, a total of 220 tuber samples  were picked randomly from 44 small fields  taking five tubers from each field for further RT-PCR detection.The occurrence of eight economically important potato viruses in three major potato growing regions  of Bangladesh were determined by RT-PCR and confirmed by successive sequencing. Total RNAs from all the five tubers of 44 sample lots were extracted using the method described formerly by Han et al. . BrieflyThe cDNAs were synthesized using Moloney murine leukemia virus (M-MLV) reverse transcriptase  as described by Khine et al. . These cEscherichia coli strain MC1022  as described earlier by Sambrook et al. [AxyPrepTM DNA gel extraction kit  was used to purify amplified products from the agarose gel. The purified PCR harvests were ligated to pMD19-T (Simple) vector  following the manufactures instruction and subsequently transformed into the competent cell of k et al. . RecombiSequences from this study were aligned with related available sequences from the NCBI database by ClustalW . PhylogeViruses are major constraint for global potato production, including Bangladesh. Along with the expanding global trade, chance to migrate potato viruses is mounting day-by-day. Consequently, rapid and reliable potato virus detection techniques are mandatory to stop these migration as well as for virus free seed potato production. This study confirmed the presence of PLRV, PVX, PVY, PVS, PVH, PAMV and PVM in the three major potato growing regions  of Bangladesh. In addition, virus incidence and their evolutionary relationship were also demonstrated here. As far as we know, this is the first report of PAMV in Bangladesh. Our findings will provide a base for further research on the biological features of the above-mentioned viruses, including the effects on yield and quality, host range, distribution, and also to develop appropriate management strategies for virus free potato production.


Understanding motorcyclists’ on-the-road behaviour is critical for developing and evaluating interventions specifically targeting them. Risky on-the-road behaviours have historically been subdivided into errors and violations of the rules of road use. Police records could be used to record cyclists’ behaviours, however these documents do not necessarily capture all errors and violations. None use of protective wears have been documented in many studies conducted on Boda-boda drivers in Uganda. The relationship between drivers’ economic stress and road safety have been studied for many years but the effects of global and economic stress, its joint effects on behaviours of drivers in relations to accidents have received very little attention. This study aimed at assessing the relationship between injuries and the Boda-boda drivers’ behaviours in Northern Uganda.Two hundred Boda-boda drivers from Gulu Municipality had face-to-face interviews to assess views and underlying factors on the issue of high prevalence of motorcycle road traffic accidents and injuries. Descriptive statistics were used to describe factors on road accidents including personal information, knowledge, skills and attitudes towards road use and safety. The study was approved by a local Institutional Review Board (IRB).Collectively, the results showed that economic stress is an important factor that affects drivers’ behaviour and increases their risks to motorcycle accidents.These authors suggest that more studies should be conducted to determine the relationship between economic stress, anger and dangerous behaviours among Boda-boda drivers and accidents in Gulu Municipality using a Motorcycle Rider Behaviour Questionnaire (MRBQ). Psychometric research within road traffic safety has for a long time produced various questionnaires and scales for measurement of risky drivers’ behaviours and similar constructs have been made with the intention of predicting individual differences in safety . UnderstIt has been noted in some studies that, drivers’ stress mostly occur when an individual perceives that his driving ability is insufficient, to manage the demands, and to handle the dangers of driving, and thus induce a dislike for driving and hazard monitoring , 18. In Study design: a cross-sectional study was conducted in four divisions of Gulu Municipality, Uganda in 2015.Study settings: the study was conducted at the Boda-boda road-side stages in  of Gulu Municipality, Northern Uganda.Study participants: Boda-boda drivers who were registered and were already operating at Gulu Municipality road-side stages three months prior to this study. Written informed consent was a requirement for enrolment.Study instrument: we used questionnaires to conduct face-to-face interviews with two hundred out of six hundred registered Boda-boda drivers in Gulu Municipality. All 200 Boda-boda drivers completed the interviews (100% completion rate). The questionnaire was constructed in two major parts: Structured and unstructured questions. The first part contained; socio-demographic characteristics of the drivers and questions on road traffic accidents, knowledge on road safety measures, attitudes and skills in Boda-boda driving. The second part contained questions which were open handed and gave the drivers the opportunity to express their views, thoughts and opinions freely with minimal limitations. All observations were summarized into descriptive statistics with numbers and percentages. The questions in the questionnaire had an internal validity Cronbach’s alpha coefficient of more than 0.73 . Each participant gave a written informed consent and their personal information was kept confidential. We ensured Privacy of Boda-boda drivers at a distance in, the quiet of their road-side stages. Records of the interviews were maintained and kept anonymous, ensuring that confidentiality of personal information was carefully and properly maintained.Data analysis: data was analyzed in two parts: We summarized their qualitative answers into themes with numbers, and percentages and where required, STATA software version 14.1 was used for further statistical analysis.In summary, the socio-demographic characteristics of Boda-boda drivers were, sex, 198(99.0%) males and 2(1.0%) females, mean age 29(SD+13.6) years, and age ranged from 16-48 years. The majority were; Catholics (58.5%), Acholi (87.5%), Boda-boda, the main occupation (89.5%), certificate, the highest level of education (46.0%), mainly from Bardege division (28.5%), Single (not married) (50.0%), owned their motorcycles (50.5%); and had work experience of 5-9 years (52%). A total of 180(90%) drivers reported the years of work experience ranging from 1 to 14 years. A total of 104(53.6%) drivers reported to have been involved in road traffic accidents in the last one year; majority (77.8%) earned less than $5 a day; (54.7%) had a child and (79.5%) of those had more than two children; (71.5%) had no house and (70%) could not afford to pay rent and lived with friends or relatives; of those renting, most (98.3%) could afford a monthly house rent less than $12.5; (68.3%) of the houses had no running water and (67.8%) no electricity. Where there was electricity (32.2%), the majority (53.3%) could only afford less than UGX20,000/= [equivalent of $5.3] per month. Similarly, for running water (31.7%), the majority (81.3%) could only afford less than UGX20,000/= [equivalent of $5.3] per month . The majHowever, only (38.9%) believed that the police arrests those drivers without driving permit. In addition, (72%) agreed that drivers who cause accidents should be arrested by the police, and (74.6%) agreed that training of drivers would significantly help in reducing the rampant road traffic accidents in the Municipality due to poor motorcycle driving behaviours . Most drInternal validity of the instrument: the internal validity and consistency of the questions indexed by the Cronbach’s alpha coefficient [fficient  were as From Road traffic injuries are the eighth leading cause of death globally and the leading cause of death among young people aged 15-29 years and nearly 3,400 people die on the world's roads every day . A disprThe African region has the highest road traffic fatality rate  and it has been observed that motorcyclists are among the most vulnerable road users  with neaThere is a high prevalence of road traffic accidents in Uganda due to Boda-boda drivers and particularly in Gulu, Northern Uganda. This study showed that a number of issues were associated with Boda-boda accidents and among the many were, poor daily and monthly incomes of the drivers , Table 3In addition, Boda-boda drivers work for far too long with an average of 14 hours a day, driving with very limited time for rest and no regulation on their activities . This loFunding: this study was partly funded by the University of Oxford under the COOL project in addition to the support from individual authors of this paper.Driving safety is influenced by human factors in various ways and actions. In one part the knowledge, attitudes and behaviours of the drivers and on the other the pedestrians. These two issues together with the environmental factors markedly influence the outcome of driving especially, accidents. In addition, the everyday life stresses of drivers which are specific and require drivers’ attention are considered factors in determining the daily outcome of motorcycle driving. The effects of poor daily and monthly incomes where drivers were unable to meet their life demands, perhaps contributed to the bnormal behaviours of Boda-boda drivers in actions such as; risky over-taking, over-speeding, driving for very long hours, driving under the influence of alcohol and drugs, not respecting other road users and not following road signs. These observations highlight the need for continuous training of Boda-boda drivers and pedestrians in Gulu Municipality on rules and regulations of road safety in light of the new developments in Gulu town. In some of these trainings, the use of protective wears, road safety regulations, use of alcohol and drugs should be included in the curriculum of the training. Furthermore, Boda-boda drivers need to start other new economic activities since this motorcycle business alone cannot meet their economic demands in Gulu Municipality. It is also suggested that future studies on Boda-boda drivers in Gulu Municipality should be conducted using Motorcycle Rider Behaviour Questionnaire (MRBQ) to assess in details the relationships between drivers’ stress, anger, behaviours and accidents.It’s one of the commonest cause of road traffic accidents in Uganda;It’s one of the cheapest, agile, affordable and convenient method of transportation;It is a source of livelihood for many unemployed youth.Poor daily income contributes to the increased cases of accidents in Gulu, Northern Uganda; in addition, economic stress due to fast urbanization of Gulu town with increasing population, improved social amenities, high prizes of goods and services makes life in Gulu Municipality very competitive for the Boda-boda drivers;Long hours of driving unregulated contribute to fatigue, lack of concentration and accidents;Uninformed pedestrians from the rural areas who do not know how to cross roads in the town contribute to the increased incidences of accidents with the drivers.The authors declare no competing interests.


Aedes mosquito bite. The infection is not severe in most cases; however, there is evidence that infection during pregnancy may be associated with fetal genetic abnormalities . In addition to microcephaly and other malformations, some specific lesions in the central nervous system have been reported. The aim of this systematic review was to determine the risk of developing microcephaly in infants whose mothers were infected with Zika virus in pregnancy. Epidemiological studies and case reports were incorporated in our review, finally including 15 articles from an initial pool of 355 related papers. Most studies have linked maternal infection during pregnancy to the development of neonatal microcephaly. The period considered most dangerous is the first trimester and the beginning or the whole of the second trimester. In order to understand the relationship between Zika virus and microcephaly in infants, a cohort study will be able to estimate the time from the onset of Zika infection and the full spectrum of adverse pregnancy outcomes.The global epidemic of Zika virus has been a major public health problem affecting pregnant women and their infants. Zika virus causes a viral disease transmitted to humans mainly by the infected  The purpose of the present study was to analyze all publicly available data to promote an informed discussion of trends in microcephaly cases in infants after mothers’ infection with Zika virus during pregnancy, in Brazil mainly and elsewhere. The study approached the issue after the end of the pandemic.After Zika virus was first identified in 1947, from the blood of rhesus monkeys in the Zika forest in Uganda, there were few cases reported in humans. In 1952, the first human cases of Zika were detected and since then, outbreaks of Zika have been reported in tropical Africa, Southeast Asia, the Pacific Islands, and Southern America. Before 2007, at least 14 cases of Zika had been documented [Aedes aegypti mosquitoes [The Centers for Disease Control and Prevention (CDC) defines the Zika virus as a flavivirus transmitted in humans by squitoes  and infesquitoes . Microcesquitoes . A higheSince 2015, various studies have been conducted, such as reviews and case reports with contradictory results on important points. According to Ellington et al. in 2016, the probability of microcephaly in infants was about 1% to 13%, with limited data for the second and third trimesters . Other iAedes aegypti and Aedes albopictus mosquitoes, which are the species more commonly found at 2000 m above sea level. Zika virus is an arthropod-borne virus that is a member of Flavivirus, Pegivirus, and Pestivirus. Flaviviruses include the Zika virus, yellow fever virus, West Nile virus, and dengue virus, while Hepacivirus comprises the hepatitis C virus [Zika virus is transmitted from  C virus .Aedes mosquito’s bite is the most common one. Transmission from the infected mother to the fetus through the placenta has also been reported. Additionally, Zika virus has the ability to be transmitted through sexual contact; in 2016 the first male-to-male transmission was reported in Texas, United States of America USA). However, the mosquito bite is the most serious threat of transmission [Zika virus is transmitted in various ways. The smission . According to the CDC, primate mammals are the main “tanks” of the virus, while human-to-human (mosquito-borne) transmission occurs during the viral outbreak. This means that an infected mosquito, can infect a second mosquito not only directly but also through the blood of a human: the first infected mosquito bites the human, transmits the virus, and then a second healthy mosquito bites the same human and gets the virus through the blood. The cycle starts when a mosquito bites an infected person. After a 10 day incubation period, the mosquito’s saliva becomes infected and from that moment a mosquito becomes a vector able to infect a human .Zika virus infection in acute stage is believed to be asymptomatic in up to 80% of the infected people and it is classically characterized by low fever, arthralgia, maculopapular rash accompanied by pruritis, and conjunctivitis. After a 12 day incubation period, symptoms usually last only a few days. Apart from the consequences in pregnancy, infections in healthy adults were associated with Guillain–Barre syndrome as well . HoweverThe diagnostic value of Zika virus disease differs between countries and specific population groups. Travelers in high-risk areas or people in contact with an infected person must be screened with a serological blood test. Furthermore, pregnant women with possible Zika virus exposure, with or without symptoms and women who were diagnosed with fetal microcephaly must be tested for Zika virus infection. Exposed neonates should be evaluated with special Zika Outcomes and Development in Infants and Children (ZODIAC) tools. The current protocol of Zika infection management involves only symptomatic care. Due to the serious problems faced by children exposed during pregnancy and their adverse effects on the economy and society, the early diagnosis of exposure is very important ,16.Aedes albopictus was reported in Europe [Aedes aegypti mosquito has not been recorded for many decades (since the early 1950s); however, in 2003–2004 the Aedes albopictus mosquito was recorded in the country and from then on it was recorded in many parts of the country. In Greece, there is also the risk of the introduction of Zika virus mainly by infected travelers from countries with active virus transmission  [According to the World Health Organization (WHO), there is a Zika virus transmission risk in Europe and this varies from country to country. Thus, on 2 June 2016, the ongoing transmission of Zika virus by mosquitoes was reported from 60 countries/regions worldwide . In Euron Europe . In GreeThe objective was to systematically review the available evidence that causally links Zika virus and neonatal microcephaly. We conducted a review of different outbreaks of Zika virus disease, including investigations and case reports studies. The research was carried out based on PubMed, Crossref, and Google Scholar. The keywords used were: “Zika virus and microcephaly”, “Zika virus and fetal toxicity”, “Zika virus and craniostenosis”, “Zika virus and congenital malformations”, “Zika virus and teratogenesis”, “Zika virus and children’s mental health”. The timeline was set from February 2016 to December 2019 and out of 355 studies, only 15 were included in the review. More specifically, the articles identified through the initial search strategy were first screened by abstract and title. The full texts of appropriate studies were examined against the inclusion and exclusion criteria and from a total of 338 studies, a total of 150 reviews, systematic reviews, and meta-analyses were rejected. After further screening, 173 articles including other genetic disorders related to Zika, i.e., hearing problems, eye problems, cerebellum problems, hydrocephalus, intrauterine growth restriction, or even the Guillain–Barre syndrome, were rejected too . The stuThe 15 articles included to this systematic review have been carried out in various countries in the territory of Latin America, USA and Europe. Specifically, eight studies were conducted in Brazil, one in Colombia, five in USA, and one in Finland. Of the total studies, six were cross-sectional studies, one case control, two clinical trials with experimental animals, five case studies, and one cohort study . AccordiMore detailed, in the study of Magalhães et al., 2016 , which wp = 0.12).The case–control research of de Araújo et al., 2016 [There were three case studies in our review. In the case report of Driggers et al., 2016 , a womanToxoplasma, rubella, herpes simplex virus, HIV, Treponema pallidum, and B19 parvovirus were all negative.In the cross-sectional study of Pacheco et al., 2016 , data onDuring the period October 2015–February 2016, several cases of fetuses with microcephaly were reported to the Paraiba Institute of Embryonic Medicine, Brazil. Eleven of the 150 pregnant women were selected for this particular study of Melo et al., 2016 , due to In addition, a study by Rice et al., 2018 , performFinally, Miner J et al., 2016 , in an eThe present study provides strong evidence for a probable link between Zika virus infection and congenital malformations resulting in serious malformations in children. We see a clear lead in studies from Brazil and Latin America in general that the virus is in the form of an epidemic and the lack of prospective studies so far are due to the short time of the virus and microcephaly worldwide.The results, according to the articles, show that the prevalence of microcephaly cases in Southern America (confirmed by laboratory tests), has been shown to be related to maternal infection with Zika virus in pregnancy, especially in the first and second trimesters . ParticuAedes mosquitoes. Inoculation attempts in experimental animals before pregnancy, early, or mid-pregnancy, indicate fetal death or genetic abnormalities with microcephaly [Certainly, the presence of these brain lesions is not necessary for the diagnosis of microcephaly. Some infants with the outcome did not show any brain damage during the imaging procedures presented. This suggests that congenital Zika syndrome may occur in infants with microcephaly and without the presence of imaging brain abnormalities. According to these findings, public health surveillance authorities should not disregard patients with microcephaly without findings from imaging methods . Concernocephaly .Regarding preventive measures, international organizations (CDC/MEDICHEM/WHO) have made recommendations on Zika virus and microcephaly in infants. The CDC recommends measures to prevent disease and transmission. The best way to prevent is a protection with special mosquito repellent measures and the avoidance of direct contact of people with Zika virus, due to patient’s body fluids as the infection is transmitted in this way . PreventThe World Health Organization (WHO), in response to the outbreak of Zika virus, has set out a response plan, outlining four key goals with emphasis on the prevention and management of neonatal microcephaly caused by Zika infection. These goals include detection, prevention, care support, and research. Given the current situation, the spread of Zika virus is expected to have long-term effects on health, for families, communities, and countries whose health systems will be called upon to care for children born with these complications for years to come . FinallyIn the present study, an attempt was made to approximate the frequency and conditions under which Zika may cause microcephaly as well as factors increasing the risk of vertical transmission in pregnancy.Most studies have linked maternal infection during pregnancy to the appearance of brain damage in newborns. Consequently, congenital microcephaly is the hallmark of Zika’s intrauterine infection. The period considered most dangerous for the vertical transmission of the infection is the first trimester and the beginning of the second or the entire second trimester, as with the Toxoplasmosis, Rubella, Cytomegalovirus and Herpes TORCH and all pathogens.Therefore, for countries with outbreaks of Zika, it is advisable to prevent unintended pregnancies and take measures to combat transmission of Zika virus from infected mosquitoes, especially during the first and second trimesters of the nervous system development.In order to better understand the relationship between Zika infection and neonatal microcephaly, further research will be needed, because the prevalence of Zika infection and risk of vertical transmission remains high. A cohort study of pregnant women will be able to estimate the time from onset of Zika infection and associate it with the full range of adverse pregnancy outcomes.Zika virus epidemics have significant short and long-term impacts in the socioeconomic spheres in the Americas. In addition to the huge financial losses due to a heavy dependence on tourism and increased stress on the healthcare system, affected individuals with microcephaly may be unable to join the labor force in the future. The Zika epidemic has disproportionately affected the poorest countries, as well as the vulnerable groups creating unequal social and health service and contributing to widening inequalities in the region. As recently observed, epidemics spread by mosquitoes  can expand rapidly, including to other parts, which highlights the need for effective control of the vectors.


The emergence of coronavirus disease 2019 (COVID-19), a novel unknown virus that is challenging whole countries all over the world, has prompted different strategies from various governments. Iran, as one of the first countries to experience the onset of the virus outbreak, made and implemented some policies that should be assessed, so that lessons may be learned for the future. Although some negative actions and policies, such as delays in cancellation of international flights especially from China, not taking the disease seriously and comparing it with seasonal influenza, and the like, are hard to ignore, some impressive actions are also vividly clear. Policies, such as social distancing, dramatically increasing social awareness about preventive actions in terms of public health, and using masks and hand washing, were cost-effective policies that resulted in successful control of the virus in the first onset. While some quite clearly ineffective decisions were made by Iranian authorities, the huge catastrophic effect of sanctions cannot be forgotten. Possibly in level situations with similar countries, Iran will have far better results regarding preparedness for future pandemics like COVID-19. Taking the wrong steps in combination with inadequate policies has the power to endanger the lives of many. Considering the level of contagiousness, as well as its international reach, this virus has the potential to affect lives not just in 1 country but the population of the entire world.5Emerging from Wuhan, China, in early December of 2019, the virus has spread all over the world. Iran was one of the first countries affected by the virus, and although the authorities’ initial reaction at the beginning of the epidemic was to “keep calm, and ignore the seriousness of the situation by comparing it to seasonal influenza,” they subsequently had to change their position and consider tougher controls in the cities, as the course of the virus outbreak seemed to be out of control.6 In this regard, we are going to assess Iran’s steps and actions to assess these proceedings as well as outline and evaluate its impact.Iran’s health system, under overwhelming pressure because of COVID-19, is experiencing a unique situation in which very novel and yet valuable lessons can be learned for the years to come.7 This resulted in an increased risk of spreading the disease by infected people entering the country. Authorities are speculating that it was the students from Wuhan who were the main reason for spreading the virus into Guilan Province, a northern province of Iran. Clearly, given the high contagious characteristic of the virus in combination with communications and travel (air transport in particular), this had a potential to easily accelerate the spread of such diseases.8On a time-based approach, the first and foremost event was a delay in canceling all flights from China, while other countries simultaneously shut down all air travel from that region.9 Due to the political importance of this event and the lack of awareness of the disease within the country at the time, allowing such ceremonies to be held on a regular basis and with no distancing protocol could be another reason for a drastic increase of the spread of the disease in the country.The next event that increased the rate of disease prevalence is the demonstration on Independence Day, February 11th - 22 Bahman in the Persian calendar, while China had officially announced infection by the virus and were taking the measures of disallowing all travel from the region; this might have flattened the curve of the disease brought by the travelers who had not yet shown any symptoms. Numerous studies have shown that close contact, as well as disregarding the importance of social distancing, could be one of the factors causing the virus to spread rapidly.Moreover, despite the official announcement of the presence of the first infected patients with COVID-19, the decision to hold the parliament election turned out to be the third major factor in increasing the spread of the virus. Large gatherings of people in voting centers, despite all cautions about the COVID-19, took place, and this led to a further spread of the virus.5The fourth, and some would say the most important factor, was deciding not to quarantine Qom, the first city in which the virus was officially announced. The presence of a large number of religious school students from diverse ranges of countries, especially from China, made Qom the first city with a widespread prevalence of COVID-19. Given that the majority of students from different cities in Iran are present in Qom as well, it was not so hard to understand the virus would have travelled widely, with the city not being quarantined. While quarantine prevents the spread of contamination, it is one of the most primitive ways of controlling such diseases, inasmuch as in Wuhan, China, this strategy turned out to be very effective.Despite authorities’ primary denial of the COVID-19 incidence, the disease gradually was spreading to other cities. Consequently, with increasing concerns about the virus, schools and universities sent their students home. Delay in shutting the universities resulted in sending potentially contaminated students from big cities back to their homes, some of them small towns.Continuous disregard of the seriousness of pandemic presence within the society was partially, however, due to the primitive approaches of social media. National TV called it less fatal than influenza, and having announced the decision to impose several days off work to the public, some people took the opportunity to go on trips, mostly to northern parts of the country, which had already been red-marked in terms of the virus epidemic. After that, in the very last hours of the Friday, the government changed the plan by suspending the days-off work policy, but it was clearly too late.Concurrent with questionable actions of the public authorities, some opportunistic jobbers who claimed traditional medicine, they called it Islamic medicine, prescribed various alternative solutions that were in contrast with the practice of modern scientific medicine. This, alongside a bewilderment of public authorities, resulted in a confused population, which added to the ill handling of controlling the pandemic.One thing about lack of collaboration in various sections was obvious. For example, while the Ministry of Health authorities did their best to inform the public about the disease by advising people to stay at home on national TV, just after the speech, the TV played an advertisement promoting a sale event for one of the biggest chain stores in Iran, which clearly contradicted the messages from health-related authorities, like they were living in a whole another country.6 Lack of basic supplies, such as masks and general personal protective equipment (PPE), as well as inadequate numbers of beds per capita compared with other developed countries, caused a shock to the health system, particularly in hospital care and more specifically in intensive care unit (ICU) beds. Lack of medical equipment, such as computed tomography scanners and ventilators, forced healthcare managers to resort toward inevitable rationing. Not being able to screen everybody with symptoms due to limited availability of diagnostic kits was one of the main reasons for the World Health Organization (WHO) statement declaring that the official statistics in Iran are probably far less than the real figures.However, the effect of international sanctions on the economy of the country cannot be ignored.On the bright side, participation and voluntary collaboration of people’s efforts to decontaminate public spaces, spreading information about the disease, and several tele-medical approaches for screening people, were positive steps in controlling the disease.What is more, although during the New Year holiday (Nowruz) there were concerns about travelling to various provinces such as Isfahan, Yazd, and Fars, and at first no serious regulations were imposed as authorities resorted to advising the public, rather than enforcing the law, on the last days of the holidays, however, the government decided to put serious limitations in place, and although rather late, this might just be better late than never.10 and also improving their practices resulted in a somehow successful management of the virus in the first onset.Another positive aspect of Iran’s authorities’ actions was focusing on primary healthcare policies that are proven to be more cost-effective than just clinical tertiary care. Increasing public knowledge, promoting their attitude,Last but not least was the dedication and commitment of the personnel of the health-related centers, including primary, secondary, and tertiary levels of encountering the virus and infected people. Given the horrifying situations the virus can cause, these people proved their value, and it was one of the most memorable moments in the history of health care in Iran.To summarize the above, it seems that, although some good actions are hard to ignore, in aggregate, Iran’s crisis management procedures, in terms of handling epidemics, are far from perfect. There is a lot to be learned from this event to better prepare for any similar events in the future. After all, the cruel heavy sanctions imposed on the government hurt the common people; its role should be considered when judging Iran’s policy-making decisions about managing COVID-19.