Transmission is a crucial part of a viral life cycle and transmission mode can have an important impact on virus biology. It was demonstrated that transmission mode can influence the virulence and evolution of a virus; however, few empirical data are available to describe the direct underlying changes in virus population structure dynamics within the host. Potato virus Y (PVY) is an RNA virus and one of the most damaging pathogens of potato. It comprises several genetically variable strains that are transmitted between plants via different transmission modes. To investigate how transmission modes affect the within-plant viral population structure, we have used a deep sequencing approach to examine the changes in the genetic structure of populations (in leaves and tubers) of three PVY strains after successive passages by horizontal  and vertical (via tubers) transmission modes. Nucleotide diversities of viral populations were significantly influenced by transmission modes; lineages transmitted by aphids were the least diverse, whereas lineages transmitted by tubers were the most diverse. Differences in nucleotide diversities of viral populations between leaves and tubers were transmission mode-dependent, with higher diversities in tubers than in leaves for aphid and mechanically transmitted lineages. Furthermore, aphid and tuber transmissions were shown to impose stronger genetic bottlenecks than mechanical transmission. To better understand the structure of virus populations within the host, transmission mode, movement of the virus within the host, and the number of replication cycles after transmission event need to be considered. Collectively, our results suggest a significant impact of virus transmission modes on the within-plant diversity of virus populations and provide quantitative fundamental data for understanding how transmission can shape virus diversity in the natural ecosystems, where different transmission modes are expected to affect virus population structure and consequently its evolution. Viruses can be transmitted between hosts via different modes, that can differently shape the population of transmitted viruses and thus have important implications for their biology and evolution. Potato virus Y, a damaging potato pathogen, can be transmitted between potato plants by aphid feeding, by wounding, or via tubers (mother to daughter plants). In this study, we have investigated how these three transmission modes directly affect the structure of the virus populations within the potato plants. We discovered that the mode of virus transmission significantly affected the population of the virus transmitted to the new plant. For example, virus populations transmitted by aphids were the least diverse while the ones transmitted vertically through tubers were the most diverse. To fully understand the effect of transmission modes on virus populations we needed to consider the events following the transmission, such as viral movement within a plant. Understanding the role that transmission modes play on the diversity and evolution of virus populations helps us to elucidate the mechanisms shaping the diversity of the virus in the field, and provides valuable information for future plant virus epidemiological studies that could promote the development of novel management strategies. It can define highly relevant characteristics of virus-host interactions such as virulence, virus fitness ,2, and ton modes . Experimon modes . Moreovetlenecks ,9 and sttlenecks , narrow tlenecks  and strotlenecks . Howeveri.e., mutant swarms or mutant clouds, within an infected host [quasispecies [e.g., vector transmission and within-host movement) have been linked to both evolutionary processes [Most of the known viruses infecting plants are RNA viruses. The high replication rate of RNA viruses coupled with a lack of proofreading ability by their RNA-dependent RNA-polymerases can lead to genetically diverse virus populations, ted host . RNA virispecies , which aispecies , rather ispecies . Two evoispecies . Selectiispecies . It is drocesses . Neverthrocesses . Combinerocesses –20.Potyvirus, family Potyviridae) is a major virus pathogen of potatoes (Solanum tuberosum L. subsp. tuberosum) [O and PVYN being parents to numerous emerging recombinant strains, several of which have become prevalent in potato fields worldwide [Potato virus Y  . The virorldwide . PVY canorldwide , and anoorldwide . Numerouorldwide . The DAGorldwide .vs tubers) for different transmission modes, (3) if the strength of the transmission bottlenecks occurring in each transmission mode can be inferred from population genetics data, and (4) if differences existed between different strains of PVY in regard to the effect of transmission mode on the virus population structure. We used Illumina HTS technology and state of-the-art population genetics algorithms to address these objectives.In summary, PVY can be transmitted vertically and horizontally with known specific interactions between the virion and the aphid vector, and exhibits, as an RNA virus, the potential for rapid evolution. All these features make PVY an ideal system to test important research questions regarding the effect of different transmission modes on the structure of virus populations within its host. Here we describe experiments to characterize the changes in within-plant population structures of different PVY strains prior to and following passage of the virus by three different transmission modes  to shed light on the role of transmission modes in the evolution and diversification of the PVY populations. We aimed to investigate: (1) if the structure of the PVY within-plant populations changed according to the transmission mode, (2) if the diversity of the viral populations differed among different organs  at 0.161 mL/m2) once a week to avoid aphid infestation and any cross-contamination among treatments and PVY strains. The three PVY isolates used in this study were collected from infected potato plants grown in Wisconsin , Minnesota , and Montana . The isolates were maintained long term in lyophilized tobacco tissue stored at −40°C and their identities were confirmed using a strain-specific RT-PCR multiplex diagnostic assay[Potato plants, cv Goldrush, vegetatively propagated from cuttings , were grtic assay.Lyophilized PVY-infected tobacco tissue (for each of three strains) was homogenized in 10 volumes of phosphate-buffered saline  and used to mechanically inoculate a founding potato plant . Three wMyzus persicae Sulzer), reared on turnip (Brassica rapa L.), were collected, starved for two hours, then allowed a five min acquisition access period on the terminal leaflet. Ten aphids were moved to a clip cage enclosing the terminal leaflet of the youngest fully mature leaf on each of the recipient plants and allowed a 24 h inoculation access period. Subsequently, aphids were killed by fogging plants with a mixture of pymetrozine (Endeavor 50WG) at 17.20 mg/m2 and bifenthrin  at 0.161 mL/m2, and plants moved to a greenhouse.The following procedures were used for all subsequent passages. At harvest (14 weeks after planting), the terminal leaflet of the youngest fully expanded leaf of each source plant was used as virus source tissue for the aphid transmission passage while the leaflet was attached to the plant. Adult apterous green peach aphids  and used to mechanically inoculate the terminal leaflet of the youngest fully mature leaf of a potato seedling . Then, the terminal leaflet from the youngest fully expanded leaf was collected for total RNA extraction, and tubers were harvested. The largest infected tuber from each plant was sampled and used for tuber transmission assay as described above . Five paN strain and 5 for PVYO and PVYN-Wi strains, 1122 to 3775 bp long) along the entire genome with at least a 76 base pair overlap between amplicons—each primer set amplifies only its specific PVY strain , following manufacturer’s directions. Total RNA concentration and quality was checked on a NanoDrop 200 Spectrophotometer  and by agarose gel electrophoresis prior to RT-PCR amplification. We designed a set of PVY-specific primers for each strain that allowed amplification of fragments  with a target peak size ranging from 150 to 500 bp. Libraries were constructed from 1–2 μg of DNA following a published protocol [Amplicons were sheared by sonication using a Covaris S2 Focused-ultrasonicator , 130 μL microTUBE AFA Fiber Pre-Slit Snap-Cap 6×16 mm (cat# 520045), and the microTUBE Holder (cat# 500114). A 50 μL sample was sheared using the following parameters: Intensity of 5, duty cycle of 10%, 200 cycles per burst, temperature of 7 protocol , with mohttps://www.qiagenbioinformatics.com/), where most of the following analyses were conducted. We first trimmed adaptor and amplification primer sequences from the reads and then performed quality-based trimming of the reads ends . Next, using the FASTX-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) we discarded all of the reads containing quality values lower than 20. In CLC Genomics Workbench, we then constructed consensus sequences of the three viral strains  present in the founding populations by mapping the reads  to respective most similar reference sequences available in NCBI GenBank . Consensus sequences of the three founding populations were used as references in all the subsequent analyses.Raw Illumina reads were first demultiplexed using Illumina’s CASAVA pipeline v1.8.2, and then imported into CLC Genomics Workbench v11.0  and CLC Genomics Low Frequency Variant Detection v2.02 tool was used to call the SNPs in each of the samples (Required significance (%) = 1.0, Minimum frequency (%) = 0.1, Ignore non-specific matches = Reads, Minimum coverage = 10, and Minimum count = 2).Trimmed and quality filtered reads of each of the samples from the experiment were first mapped to consensus reference sequences of all three PVY strains used in the experiment . To avoid possible contaminant reads of other strains, resulting from, To remove possible reverse-transcription, amplification, and sequencing artifacts we further filtered SNPs; only the SNPs that appeared in at least one time point (within a single lineage) at a frequency higher than 1% were retained for further analyses. Moreover, SNPs with the forward/reverse balance < 0.1% in majority of sequenced time points, SNPs positioned at the known polymerase slippage site in the PVY genome and SNPs at the sites with sequencing coverage < 100×, were removed from further analysis. Such cleaned SNP tables were then used as an input for subsequent analyses.π), for synonymous sites (πS), and for nonsynonymous sites (πN). SNPGenie also calculated the mean number of nonsynonymous differences from the reference per nonsynonymous site (dN) and the mean number of synonymous differences from the reference per synonymous site (dS). The indices were averaged over a complete viral genome or over separate PVY cistrons. Nucleotide diversities (π) for leaf samples were plotted through time as line plots for each lineage and the average was calculated and plotted through time for all of the lineages corresponding to the respective transmission modes. Likewise, dN and dS were plotted through time. Generalized linear mixed model (GLMM) analysis in SPSS Statistics version 26  was used to test for factors contributing to differences in nucleotide diversities for the samples of passages 1–5. The three orthogonal factors included in the model were transmission mode (T), viral strain (S), and evolutionary passage (P) and all their interactions, using the following model equation: πijkl = Π + Tk + Si + Pj + (T×S)ik + (T×P)jk + (S×P)ij + (T×S×P)ijk + εijkl, where Π represents the grand mean nucleotide diversity and εijkl the error term assumed to be normally distributed. Nucleotide diversities were compared between leaf and tuber samples of the same plant by plotting the values for π in leaves against the value of π in tubers. We calculated the ratios of π in tubers to π in leaves and plotted their decimal logarithmic values for separate transmission modes as histograms. One-sample Wilcoxon signed rank tests were used to test if the median of the distributions of the ratios for each transmission mode is higher than one (greater diversity in tubers) or lower than one (greater diversity in leaves).Filtered SNP tables were used as an input for SNPGenie  to calcuπS and πN) were calculated separately for each of the PVY cistrons in each of the samples. The results were plotted for each cistron in time as line plots. To test for possible difference between the values for different cistrons, the cumulative distributions were calculated  and plotted for each cistron for both πS and πN. Pairwise Kolmogorov-Smirnov tests were used to compare the cumulative distributions for all of the pairs of PVY cistrons for πS and πN values.Nucleotide diversities  and locus-specific selection coefficients, s, of the observed SNPs, we employed the time-series information about frequency of observed SNPs. Filtered SNP tables of each lineage were converted to input files for ApproxWF analysis [Ne and s were estimated using task = estimate setting with h = 0.5 , a log-uniform prior for Ne, logNe ~ U, and normal prior for s, s ~ N, and run with 51 states for 100,000 MCMC iterations. Results were further analyzed in R v3.6.1 [Ne of each lineage were plotted as kernel density plots and their modes were calculated. Modes for each of the lineages were plotted and used to compare the Ne between different transmission modes using paired two sample Wilcoxon signed-rank test. For s, mean, median, and 95% highest posterior density intervals were calculated and plotted for each analyzed SNP of all analyzed lineages.To estimate effective population sizes, analysis , includiR v3.6.1 . First 1n-dimensional vector, dimensions representing frequency of the SNP at each sequenced time point. Heatmap.2 library [To better understand the dynamic changes in viral population structures we plotted the changes in frequency of each SNP through the experiment for each of the lineages using R v3.6.1. Each SNP was treated as a  library  was usedO, 58 samples for PVYN, and 57 samples for PVYN-Wi. Detailed information about each of the analyzed samples is given in We collected and sequenced 183 samples from potato leaves and tubers during the course of the experiment , passage number (P), and their interaction on the diversity of viral populations (S) on the diversity of viral populations , for each transmission mode, supported significantly higher diversity of the viral populations in tubers than in leaves for MI and AT transmission modes. The median of the πtuber/πleaf for IT was < 1, suggesting a lower diversity of the viral populations in tubers in this transmission mode; however, the difference was not statistically significant  by using the information on how SNP frequencies are changing through time (passages). Ne represents the number of individuals from one passage that contribute to the establishment of the population in the next passage. This number is influenced by the sizes of genetic bottlenecks and can, in these experiments, be understood as a number of viral genome units that were transmitted from one to the other passage. We compared the viral populations in systemically infected leaves of source and inoculated plants, thus estimating Ne values for the whole transmission process . We observed that PVY was subjected to narrower population bottlenecks in the AT and IT modes, compared to MI mode. Estimations for most of the lineages of the AT and IT modes were very low, i.e., mostly 10 or less viral genome units , pointing to very strong genetic bottlenecks occurring when PVY was transmitted by aphids or through tubers  for all three strains. For the AT mode, higher estimates of Ne were calculated for two out of the three experimental lineages of the PVYN strain (18 and 25)  . This imNe in the IT lineages compared to the MI lineages .To gain further insight into the dynamics of the viral populations transmitted by different transmission modes, we visualized the changes in SNPs' frequencies in each of the experimental lineages as heatmaps . This kis values, which indicate if specific SNPs in the population are subjected to positive or negative selection. All of the estimations had wide intervals and we were not able to reliably detect any sites under selection  during the course of the experiment in two of the nine MI lineages (2 consensus level SNPs per lineage), seven of nine IT lineages (1–4 consensus level SNPs per lineage) and seven of nine AT lineages (1–5 consensus SNPs per lineage). Their dynamics can be observed in detail on πS and πN for each specific cistron changed through the experiment  comparable to the empirically determined number of PVY viral particles transmitted by aphids (0.5 to 3.2 per single aphid) [Ne values for PVYO and PVYN-Wi strains, however, the Ne values for PVYN strains were higher for two of three experimental lineages. We can speculate this may imply a larger number of viral particles transmitted by aphids for this strain (either larger number of particles transmitted by a single aphid or a larger chance of virus acquisition and transfer by aphids). Differences in the efficiency of transmission of different PVY strains by aphids could have important implications in the field, where they could lead to changes in prevalence of PVY strains infecting the potato crop. However, further experiments would be needed to confirm this observation, since the transmission efficiency of different PVY strains by green peach aphid can be influenced by many different factors. Transmission efficiency for PVYN and PVYN-Wi was shown not to differ in a previous study [Ne values estimated for this two strains in our study. Even though some studies demonstrated different efficiency of transmission for different PVY strains [e.g., potato cultivar, viral titer in the source leaf [We observed a significant impact of PVY transmission modes on the within-plant diversity of the PVY populations. Viral diversities were consistently low in all of the samples collected from AT lineages, as expected due to the strong population bottlenecks demonstrated for aphid transmission of PVY . The obse aphid) . Furtherus study , which i strains , conclus strains  and can rce leaf ,38, viruNe values for MI lineages (from 17 to 32). Nevertheless, these estimates are still relatively low, considering high number of viral particles expected to be present in an infected leaf. Previous research on contact transmission of another plant virus demonstrated that, even though the source material for mechanical transmission of viruses might contain very high number of viral particles, e.g., up to 109 tobacco mosaic virus particles in a tobacco leaf [Ne, likely also in our experiment. We demonstrated in this experiment that the Ne for the MI mode is indeed relatively low, but still significantly higher than for the AT or IT modes. This observation has wide implications in plant virology research. The studies performed in laboratory are predominately performed using mechanical transmission, which is expected to be less frequent in the field. Quantitative estimation of the difference in a population bottlenecks expected from mechanical transmission in comparison to aphid transmission or transmission via vegetative propagation suggests that mechanical-transmission based experiments would be subjected to smaller effects of genetic drift, compared to the processes in the field.The diversities of viral populations were higher in the MI mode than in the AT mode, and they increased during the experiment. This observation agreed with the estimation of consistently higher cco leaf ; only a cco leaf , suggestcco leaf , which hNe values for IT lineages were very low . Even though this result seems counter intuitive, it makes sense, when inspecting the dynamics of separate SNPs in the population. Most of the variability in the IT lineages was generated de novo after each passage  (Ne) was indeed very low.For the IT mode, we observed increases in PVY population diversity throughout the experiment. Viral diversities for IT lineages were higher than for the AT or MI lineages. However, the estimated ppeared)  inferrinπ for leaves and tubers of the same plants revealed significantly higher population diversities in tubers for AT and MI transmission modes and slightly lower  diversity in tubers for IT transmission modes  is accompanied by significant genetic bottlenecks [Comparison of on modes . Observetlenecks –43, whictlenecks ,42.i.e., as a sink, tubers collect variants from many leaves).In our experiments, in the AT and MI modes, virus enters a plant through the inoculated leaf and then moves to other leaves  via sourOn the other hand, in the IT mode, the virus is present already in the sprouted plantlet and thus has more time to diversify (more replication cycles) in each of the leaves, compared to the MI and AT modes. This might contribute to higher observed diversity of the viral populations in leaves in the IT mode, compared to the MI and AT modes . In the Ne do not always correlate with observed π, highlight the fact that transmission event itself cannot be the only factor explaining the underlying differences in population structures of PVY within plants in different transmission modes. Differences in the number of replication cycles of the virus, movement of the virus within the plant, admixture etc., need to be considered to better understand how the structure of within-plant virus population is shaped by different transmission modes.The differences in population diversities between leaves and tubers, and the observation that Ne values, if these changes could be attributed to selection and/or genetic drift. It is possible that both, strong genetic drift and selection, contribute to the observed patterns; however, different experimental approaches would be needed to test such hypotheses. When investigating the position of fixed nonsynonymous mutations, we did not observe SNPs causing changes directly in the motifs known to be involved in the PVY-aphid interactions in any lineage. Nevertheless, we observed a fixed change in lineage AT3 that might be particularly interesting; it is located in HC-Pro (conservative substitution D334E), in the relatively close proximity (15 amino acids upstream) to the PTK motif, which is known to interact with CP during the aphid transmission. Moreover, observed increases in nucleotide diversities for the 6K2 and P3N-PIPO cistrons hint that these may be under different selection pressures than other PVY cistrons.Although we detected several SNPs rising in frequency or even being fixed in the populations , it was not possible to distinguish, due to the small estimated dS and dN) for lineages of both transmission modes are very similar , which is the main experimental approach for virus transmission, is less likely to occur in the field [Ne  of PVY populations in plants might slow down virus adaptation and consequently, the emergence of resistant-breaking virus isolates [Ne of the populations simultaneously and subsequently. Based on our results, we can hypothesize that tuber transmission represents a potent generator of diverse viral populations, providing new alleles to the PVY metapopulation (field-wise) and the diversity can then be (randomly) subsampled, when PVY is spreading in field through aphid transmission. We cannot directly link these processes to predict, e.g., the severity of disease in the field, however, the quantitative data presented in this paper enable the extension of epidemiology models of plant virus diseases by integrating the population genetics parameters underlying different transmission modes. For example, under the scenarios described above, we can predict reduced within-plant and increased within-field diversity for PVY, when aphid transmission is not decreased by control measures. At the same time, one could expect increased diversity of PVY populations within infected plants generated through vegetative propagation (tubers) or via mechanical transmission. Carefully planned experimental work and/or in-silico simulations would be needed to determine how the described differences in population genetic parameters for different transmission modes influence the adaptation of the virus to different environmental conditions .Transmission of potyviruses by aphids have an important epidemiological role for the spread of the virus mostly on relatively short distances , e.g., whe field . It willhe field , will inisolates . HoweverS1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Tablepost-hoc pairwise comparisons of estimated marginal means for groups defined by factors STRAIN, TRANSMISSION MODE, PASSAGE and TRANSMISSION MODE×PASSAGE.Results of (XLSX)Click here for additional data file.S1 FigO samples in the experiment. Samples are color coded according to the legend below the plot. The mapped read depth values are log10-transformed.Each line represents mapped read depth across the genome of the virus for each of the PVY(PDF)Click here for additional data file.S2 FigN samples in the experiment. Samples are color coded according to the legend below the plot. The mapped read depth values are log10-transformed.Each line represents mapped read depth across the genome of the virus for each of the PVY(PDF)Click here for additional data file.S3 FigN-Wi samples in the experiment. Samples are color coded according to the legend below the plot. The mapped read depth values are log10-transformed.Each line represents mapped read depth across the genome of the virus for each of the PVY(PDF)Click here for additional data file.S4 Figs value estimations, blue dots represent their median and green dots their mean. AT–aphid transmission, MI–mechanical transmission, IT–tuber transmission.Each plot represents results of the s values estimations for a separate evolutionary lineage of the experiment. Horizontal lines represent 95% highest posterior density intervals for SNPs (PDF)Click here for additional data file.S5 Figs values estimations for a separate evolutionary lineage of the experiment. Horizontal lines represent 95% highest posterior density intervals for SNPs s value estimations, blue dots represent their median and green dots their mean. AT–aphid transmission, MI–mechanical transmission, IT–tuber transmission.Each plot represents results of the (PDF)Click here for additional data file.S6 Figs values estimations for a separate evolutionary lineage of the experiment. Horizontal lines represent 95% highest posterior density intervals for SNPs s value estimations, blue dots represent their median and green dots their mean. AT–aphid transmission, MI–mechanical transmission, IT–tuber transmission.Each plot represents results of the (PDF)Click here for additional data file.S7 FigThe heatmap within each rectangle represent dynamics of SNPs frequencies in particular lineage during the experiment. Lines represent specific SNPs, rows represent passages (S–source); SNPs frequencies are represented by color according to the color key at the bottom of the figure.(PDF)Click here for additional data file.S8 FigπS data for all the samples from the experiment separated by cistron (color coded). Lower panel represents cumulative distribution functions for πN data for all the samples from the experiment separated by cistron (color coded). Within the panels the networks summarize the results of Kolmogorov-Smirnov tests for the same data (edges connect the cistron pairs for which Kolmogorov-Smirnov test did not show a significant difference).Upper panel represents cumulative distribution functions for (PDF)Click here for additional data file.S9 FigdS (upper panel) or dN (lower panel) values for separate lineages over the course of the experiment. Colors represent three different transmission modes , line types represent three different PVY strains used in the experiment. Thick translucent lines connect average values for all of the lineages within the same transmission mode over the course of the experiment.Thin lines connect (PDF)Click here for additional data file.S1 File(ZIP)Click here for additional data file.


Betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has rapidly evolved into a pandemic. Though its origin has been linked to the Wuhan City of China’s Hubei Province in December 2019, recent reports claim that the original animal-to-human transmission of the virus probably happened sometime between September and October 2019 in Guangdong Province, rather than Hubei. As of July 3, 2020, India has reported a case positivity rate of 6.5% and a fatality rate of 2.8%, which are among the lowest in the world. Also, the severity of the disease is much less among Indians as evidenced by the low rate of ICU admission (15.3%) and the need for mechanical ventilation (4.16%). As per the World Health Organization (WHO) situation report 165 on July 3, 2020, India has one of the lowest deaths per 100,000 population . Several factors related to the pathogen, host and environment might have some role in reducing the susceptibility of Indians to COVID-19. These include some ongoing mutations that can alter the virulence of the circulating SARS-CoV-2 strains, host factors like innate immunity, genetic diversity in immune responses, epigenetic factors, genetic polymorphisms of ACE2 receptors, micro RNAs and universal BCG vaccination, and environmental factors like high temperature and humidity which may alter the viability and transmissibility of the strain. This perspective -highlights the potential factors that might be responsible for the observed low COVID-19 fatality rate in Indian population. It puts forward several hypotheses which can be a ground for future studies determining individual and population susceptibility to COVID-19 and thus, may offer a new dimension to our current understanding of the disease.Coronavirus disease 2019 (COVID-19), an acute onset pneumonia caused by a novel  Betacoronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was reported from the Wuhan City of China’s Hubei Province. The outbreak was declared as a Public Health Emergency of International Concern by the World Health Organization (WHO) on January 30, 2020 and a global pandemic on March 11, 2020.1,2 Though its origin has been linked to Hubei Province, recent phylogenetic analysis claims that the original animal-to-human transmission of the virus probably happened some 3 to 4 months before December 2019 in Guangdong Province, rather than Hubei , caused by a novel an Hubei . As of Jan Hubei . As of JThe traditional model for any infectious disease consists of a triad of the causative agent, the host, and an environment in which the agent and host are brought together, causing the disease to occur in the host. Little is known regarding the origin of SARS-CoV-2. There have been speculations that the virus got transmitted to humans from bats or pangolins, but conclusive evidence regarding the same is lacking. Genomic sequence data indicate that SARS-CoV-2 has 96.2% sequence homology with a bat coronavirus RaTG13 , and 91%Earlier, six genera of coronaviruses were known to cause human disease, of which four  are responsible for mild respiratory infections, mostly in pediatric age group while two (SARS-CoV and Middle East Respiratory Syndrome coronavirus [MERS CoV]) are associated with severe outbreaks . SARS-Co5,6 These observations indicate that strain difference has some impact on virulence and pathogenesis. Scientists from the National Institute of Biomedical Genomics, India, performed phylodynamic analyses and examined the temporal and spatial evolution of the virus in 3636 SAR-CoV-2 sequences deposited in Global Initiative on Sharing All Influenza Data (GISAID) from 55 countries and observed that at least 10 distinct clades  of the virus have originated from the ancestral clade O , indicating that the virus might have originated in China and eventually spread worldwide . The Ind clade O . Though  clade O . Phyloge clade O . An anal clade O . Mutatio clade O . Researc clade O . A tripl clade O . Besides clade O . The RNA clade O . A study clade O . A compa clade O . Figure 3 India has reported only 1.32 deaths per 100,000 population against a global average of 6.04, which is among the lowest in the world.7 The potential host factors that might alter the susceptibility of the Indian population to SARS-CoV-2 are discussed below.The host-pathogen interaction in COVID-19 has shown striking variations in fatality rates across different geographical regions, depending on age, smoking habits and pre-existing comorbidities . China, 8 Given the immunomodulatory and antiviral properties of these drugs, their widespread usage might have contributed to defense against SARS-CoV-2.First, the Indians are exposed to an enormous microbial load early in life which confers a broad-based immunity to the population. The wide variety of pathogens, including bacteria, viruses, fungi, and parasites to which the Indians are exposed since birth, leads to induction of immune responses and also adaptive changes in cell frequencies and functions. A comparative analysis of the immune phenotypes in Indian and American newborns revealed that Indian infants had a higher proportion of dendritic cells, monocytes, natural killer (NK) cells, memory CD4 + T cells, and naïve B cells, compared to American infants . Such vaCurcuma longa, is a rhizomatous herbaceous perennial plant belonging to the ginger family that is routinely used in Indian households as a culinary spice and as a component in religious ceremonies. It has also been used as a traditional medicine over centuries. Curcumin, the bioactive compound in turmeric has been recognized for its potent antioxidant, anti-inflammatory, antimutagenic, antimicrobial, and anticancer properties. It has demonstrated antiviral activity against a wide range of viruses including Human Immunodeficiency Virus 1 and 2, Influenza Virus , Herpes Simplex Virus (HSV) 1 and 2, Coxsackievirus, Hepatitis B and C viruses, Human Papillomavirus (HPV) types 16 and 18, Japanese Encephalitis virus, and Human T cell Lymphotropic Virus type 1 (Allium sativum), a popular condiment used in Indian foods, has been shown to possess antiviral activity against HSV-1 and -2, Parainfluenza Virus type 3, and Human Rhinovirus type 2  and unique haplotypes (HLA-A2-B50-DR3) have been observed in the Indian population, which are not known to occur in other ethnic groups  complex, much more extensive than the Caucasian population and this is attributed to the high microbial load to which the Indians are exposed during life . The HLAc groups . WhetherFourth, allelic variation in angiotensin converting enzyme 2 (ACE2) receptor can be an important factor determining the susceptibility to SARS-CoV-2 infection. Cell culture assays and molecular superimposition studies indicate that SARS-CoV-2 spike (S) protein has got high affinity for human ACE2 receptors and utilizes the same for gaining entry into the target cells. ACE2 receptors are widely distributed in body tissues, the highest expression being observed in type II alveolar epithelial cells . Recent Fifth, the microRNAs (miRNAs) might play a crucial role in defense against SARS-CoV-2. miRNAs are small non-coding RNAs that are thought to regulate gene expression and cell differentiation, and have the ability to inhibit viral replication in a sequence-specific manner. Considering their unique mechanism of action, miRNAs have become attractive candidates as diagnostic biomarkers and therapeutic targets . SeveralSixth, the Indians have a higher percentage of nature killer (NK) cells, compared to other ethnic groups . It has 9 Two randomized controlled trials, BCG-CORONA (NCT04328441) and BRACE (NCT04327206) trials are underway, evaluating the efficacy of BCG vaccine in reducing the viral load and/or sequelae associated with COVID-19. One limitation of BCG vaccination is seroconversion, which is the basis for diagnosis of TB in several countries. Therefore, MMR, another live attenuated vaccine, might be a potential option for inducing beneficial non-specific effects in human populations and thus provide protection against the catastrophic sequelae of COVID-19. A strong correlation has been observed between individuals in geographical locations who received MMR vaccine and reduced COVID-19 death rates  and Mumps, Measles, Rubella (MMR) commonly administered during childhood, have been found to confer non-specific protection against unrelated lethal infections by inducing “trained” non-specific innate immune cells for more efficient host response against wide range of pathogens . The BCGth rates . Interesth rates . Though 0) of the virus from 224 Chinese cities revealed that high temperature, humidity and UV radiation has no significant impact on cumulative incidence rate and transmission of SARS-CoV-2. Infact, countries like Iran and Australia continued to have rapid disease transmission and exponential rise in the number of cases despite high temperature and humidity, indicating that the inverse relationship between ambient temperature and COVID-19 transmission lacks rationality. Studies have shown that an interplay between macrodomain (MAC) and papain-like protease 2 (PLP2) domain of coronavirus NSP3 impacts viral replication, host innate immune antagonism and virulence. Strains harboring mutations in these two domains are more rapidly degraded at non-permissive temperatures (37°C and 40°C) than the wild-type strains  radiation indices. Based on these facts, it was assumed that COVID-19 transmission might decrease or even disappear with the rise in ambient temperature and UV radiation during the summer months. However, a study by  strains , indicat3,4, 10 Strict lockdown measures implemented nationwide for more than two months might have kept the transmission and deaths in check. This must have contributed significantly to the much desired flattening of the epidemic curve. There are speculations that India’s predominantly young population as compared to the western world (with a higher proportion of elderly people), and the presence of a less virulent strain of the virus in circulation are helping keep fatalities low in India. However, there are no evidences in support of these claims. In fact, phylogenetic analysis has revealed that four clades of the virus: O , B (from China), A3 (from Iran) and A2a  are in circulation in India with A2a being the dominant type  and cell-mediated immunity  . Also, the disease has been observed to be less severe among Indians with a significantly higher recovery rate  and doubling time (20.3 days) as compared to the populations elsewhere.ant type . There ad cells) . There ad cells) . A stronThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.AS and RG: conceptualization, data curation, and methodology. AS, RG, and VN: supervision. AS, RG, VN, and SM: validation, and visualization. AS and RG: writing – original draft. AS, RG, VN, and SM: writing – review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Shrink-swell soils are predominant in various parts of the parts of the world. Lime has been extensively used to reduce the shrink-swell mechanism as it chemically reacts with soil minerals forming pozzolanic products such as calcite and calcium-silicate-hydrate (C-S-H). Conventionally, whether chemical treatment of soils results in effective pozzolanic stabilization reactions is determined anecdotally through engineering tests including unconfined compressive strength, plasticity index (PI), and pH tests. This study builds on existing literature regarding how more direct quantification of pozzolanic products can be obtained through tests that directly identify and quantify pozzolanic products, specifically in lime-treated clay soils. Specifically, x-ray diffraction (XRD) and differential thermogravimetric analysis (DTA) are used for this testing. Expansive soils with plasticity indices above 25% were selected for this study. Engineering tests on these lime-treated soils indicated significant improvement in strength and reduction in PI. In XRD analysis, pozzolanic products are assessed by the location and intensity of x-ray peak(s). The XRD data show a decrease in the intensity of alumio-silicate minerals such as kaolinite and smectite; silica and alumina are dissolved at a high pH and converted to pozzolanic products such as calcium-silicate-hydrate (C-S-H). DTA indicates the presence of C-S-H with the characteristic weight loss from 140°C to 250°C.The methodology describes the following:● Sample preparation steps for XRD and DTA analysis.● Analysis of XRD results and DTA analysis.  Specifications TableExpansive clay minerals are prevalent in many parts of the western and southwestern U.S. Soils containing such expansive minerals are referred to as shrink-swell soils due to their swelling characteristics in the presence of moisture and shrinking characteristics during moisture loss. The amount of actual swell is dependent on the moisture condition, climatic conditions, and the type of expansive clay mineral present. In the U.S. alone the annual cost due to damage caused by expansive soils is estimated to be $2.3 billion Several researchers have studied the change in physical and engineering properties for lime-treated soils 1.Describe detailed sample preparation steps for analytical tests such as XRD and DTA.2.Detail steps to analyse XRD and DTA results.3.Compare XRD and DTA results with conventional engineering test results such as unconfined compressive strength (UCS) and Plasticity Index (PI).There is a deficit of literature that provides step-by-step instructions on how to prepare and test samples in order to perform x-ray diffraction (XRD) and differential thermogravimteric analysis (DTA). For example, there is a lack of information on the recommended instrumentation parameters and analysis steps for XRD and DTA. This study addresses those shortcomings by building on existing literature and providing a detailed step-by-step approach that can be readily followed by practicing engineers on field collected soil samples. This study emphasizes sample preparation and analysis methodology for XRD and DTA. The objectives of the study are:2) improves the compressive strength of the stabilized material 2+ and OH− ions. The release of OH− ions increases the pH to 12.4, which causes silica and alumina from clay minerals to dissolve, and in combination with Ca2+ ions form calcium-silicate-hydrates (C-S-H) and calcium-aluminate-hydrates (C-A-H). The increase in strength is largely attributed to the formation of these C-S-H and C-A-H products, an amorphous gel that binds the soil matrix. The quantity of C-S-H and C-A H that can form at chemical equilibrium is dependent on the soil mineralogy, pH and percentage of Ca(OH)2 added. A simplified qualitative view of typical soil-lime reactions Chemical stabilization of expansive soils with additives such as lime, portland cement (cement) and fly ash has been successfully achieved as evinced by strength increase and reduction in shrink-swell behavior upon treatment 2 reacts with atmospheric CO2. This reaction is called carbonation. Calcite is crystalline and can also contribute to an increase in strength Calcite also forms when Ca(OH)2+ ions (from hydrated lime) are adsorbed to the clay surface, lowering plasticity and flocculating the clay colloids Engineering tests such as unconfined compressive strength (UCS) test and plasticity index (PI) test are good indicators by which to assess the effect of pozzolanic reactivity in lime-treated soils. Addition of hydrated lime to a clayey soil facilitates modification and stabilization Analytical methods including using X-Ray diffraction (XRD) and differential thermogravimetry (DTA) are used extensively to detect C-S-H in hydrated cement pastes. The accuracy of these methods depends on sample preparation methods and the analysis of results The physical properties of the untreated soil samples S1 and S2 are shown in D2166D4872D4318A design hydrated lime dosage of 6% was determined on the basis of a threshold pH of 12.45 at 25°C for both soils evaluated in this study following ASTMD 6726-19 1.Recover a 5 g representative sample from the both the treated and untreated specimen.2.Air-dry the sample for 72 hours.3.Grind the representative treated and untreated sample with a mortar and pestle until the ground material passes the No. 325 (0.44 mm) sieve. If the samples stick to the sides of the mortar and pestle during the grinding process, making it difficult to grind, extend the air-dry period by 24 hours.Addition of lime agglomerates clay sized fractions present in the soil and affects the XRD analysis. Therefore, the grinding process is important as it breaks down the agglomerated soil particles.1.Back load onto the XRD sample holder. Back loading reduces the chances of preferred orientation due to stacking of clay minerals.2.0 to 700 2θ with the suggested parameters in Run the XRD from 5The sample preparation method for untreated and lime-treated samples is as follows:1.Follow steps 1 to 4 for XRD sample preparation as previously described.2.4 as the desiccant for 14 days.Moisture equilibrate both treated and untreated samples in a desiccator with CaSO3.Test moisture equilibrated samples immediately at the following settings.The sample preparation steps used for the DTA analysis are as follows:Table 32 (am), equation 1) that can further react with Ca2+ (from hydrated lime) in the presence of H2O to form the pozzolanic product C-S-H. Furthermore, atmospheric CO2 can react with lime to produce calcite. The XRD diffractogram also shows the presence of calcite in the treated samples S1 and S2  is a widely applied technique to measure hydration and carbonation by cement scientists. The DTA analysis results of the treated and untreated samples are shown in The maximum dry density and optimum moisture content (OMC) of the treated and untreated samples are shown in 1.It is of key importance to be able to determine the degree of pozzolanic reaction developed in a lime-treated soil in order to assess the durability of the reactions responsible for changing the properties of the treated soil. The DTA and XRD analysis methods described herein are effective and efficient tools for assessing the degree of pozzolanic product.2.DTA can qualitatively evaluate C-S-H precipitation.3.The XRD diffractogram of the treated samples can quantify the level of reactivity and dissolution of quartz in the treated samples.4.DTA and XRD in combination with traditional pH, PI and UCS tests combine to provide an effective and efficient protocol for assessing the quality and durability of lime-treated soils.


Survivors of Ebola virus infection may present late with a spectrum of ocular manifestations, ranging from mild retinal scarring to complete bilateral blindness resulting from tractional retinal detachment, cataract and glaucoma secondary to intraocular inflammation.An infectious disease specialist's brush with death during the 2014 Ebola crisis in West Africa proved instructive to ophthalmologists.Ebolavirus (EBOV) genus, four infect humans, causing an acute haemorrhagic fever that is associated with very high case fatality. The finding of replicating Ebola virus in aqueous is not surprising: Marburg virus, a member of the same Filoviridae family , had been isolated from the intraocular fluid of infected persons several years before.Flaviviridae such as Zika, Dengue and West Nile viruses, and Togaviridae such as Chikungunya also cause intraocular inflammation.Of the six species in the “Ocular complications form part of post-Ebola virus disease syndrome, which also includes symptoms of joint and muscle pain and sometimes neurological problems.”Ocular complications form part of post-Ebola virus disease syndrome, which also includes symptoms of joint and muscle pain and sometimes neurological problems.A longitudinal study in Liberia established that EVD survivors have a significantly higher incidence of uveitis than a control group of close contacts.A range of signs are possible, including non-granulomatous anterior uveitis, posterior synechiae, iris atrophy, vitreous inflammation, chorioretinal scarring, panuveitis and optic neuropathy. These findings reflect direct viral invasion of the normally immune-privileged intraocular space. Inflammation can range from mild to severe, causing raised intraocular pressure, cataract , tractioA variety of chorioretinal lesions may be found in survivors, such as pigmented retinal scars with a hypopigmented halo .6 In a sTreatment options include topical or systemic ocular antihypertensives, cycloplegic and mydriatic agents for analgesia and to prevent synechiae, and topical corticosteroids.The role of antiviral drugs in eliminating intraocular infection with Ebola virus is currently being evaluated.In those recovering from Ebola infection, the finding of treatable eye disease which can lead to visual impairment has two implications for organisations providing health care to Ebola survivors.Health care workers should be prepared to assess the eye, which should include:Assessing visionExamining the fundusMeasuring intraocular pressure.Health workers can use a smartphone app to measure visual acuity, with the addition of a camera adaptor clip to visualise the fundus.Adequate treatment and referral pathways should be developed, potentially using retinal images for remote consultation with an ophthalmologist.Happily, the infectious disease specialist who developed Ebola uveitis partially recovered his vision, but others are not so lucky.Recent Ebola outbreaks have had the unexpected effect of exposing the striking shortage of ophthalmic services available in affected communities, as well as the lack of preparedness to deal with its ophthalmic consequences. For instance, some clinicians have been reluctant to treat survivors with cataract due to unfounded fears over persisting infection. This adds to the already overwhelming burden on health services in countries struggling with the aftermath of an Ebola epidemic.


An evolutionary arms race has been ongoing between retroviruses and their primate hosts for millions of years. Within the last century, a zoonotic transmission introduced the Human Immunodeficiency Virus (HIV-1), a retrovirus, to the human population that has claimed the lives of millions of individuals and is still infecting over a million people every year. To counteract retroviruses such as this, primates including humans have evolved an innate immune sensor for the retroviral capsid lattice known as TRIM5α. Although the molecular basis for its ability to restrict retroviruses is debated, it is currently accepted that TRIM5α forms higher-order assemblies around the incoming retroviral capsid that are not only disruptive for the virus lifecycle, but also trigger the activation of an antiviral state. More recently, it was discovered that TRIM5α restriction is broader than previously thought because it restricts not only the human retroelement LINE-1, but also the tick-borne flaviviruses, an emergent group of RNA viruses that have vastly different strategies for replication compared to retroviruses. This review focuses on the underlying mechanisms of TRIM5α-mediated restriction of retroelements and flaviviruses and how they differ from the more widely known ability of TRIM5α to restrict retroviruses. The innate immune system evolved as an intracellular response to a variety of pathogens including viruses, bacteria, and fungi . Well stRhTRIM5α  was identified as the predominant block to retroviral infection by Simian Immunodeficiency Virus (SIV) in rhesus macaques as evidenced by its ability to limit cross-species transmission and drive the evolution of the retroviral capsid protein ,16. The The restriction and sensing of RNA viruses by TRIM5α, as well as other TRIM proteins, is intimately linked to ubiquitin signaling due to the conservation of an E3 ubiquitin ligase RING domain present in all of these family members . During In stark contrast to the role of ubiquitin in retroviral restriction, a clear role for K48 ubiquitin conjugation by the RING domain of TRIM5α has been described for the select restriction of several tick-borne flaviviruses like tick-borne encephalitis virus (TBEV) and Langat virus (LGTV), but not mosquito-borne flaviviruses like Dengue Virus (DENV) . During Renilla luciferase and dual luciferase assays were performed according to manufacturer’s protocol (Promega). As shown previously [IFNβ, CXCL10 or IL8 cytokines in LGTV-infected A549 cells was not affected by depletion of TRIM5 mRNA  NS3 protease-helicase . TRAF6 iHow TRIM5α has evolved to patrol two separate cellular compartments and overcome virus-induced barriers for the restriction of distinct viral and retroelement nucleoproteins remains unclear. Further still, molecular details regarding its protein targets remain obscure. For instance, the ability of mosquito-borne flaviviruses to evade immune detection by TRIM5α despite near complete structural conservation and 50% sequence identity across NS3 proteins, remains unanswered . Additio


Francisella tularensis, human immunodeficiency virus-1 (HIV-1),Toxoplasma gondii, Variola virus, andYersinia pestis. Results confirm that heptapeptide overlap is high between pathogens andHomo sapiens, but not between pathogens and primates. Data are discussed in light of the possible genetic bases that differently model primate phenomes, thus possibly underlying the zero/low level of molecular mimicry between infectious agents and primates. Notably, this study might help address preclinical vaccine tests that currently utilize primates as animal models, since autoimmune cross-reactions and the consequent adverse events cannot occurin absentiaof shared sequences.Recently, it was found that proteomes from poliovirus, measles virus, dengue virus, and severe acute respiratory syndrome-related Coronavirus 2 (SARS-CoV-2) have high molecular mimicry at the heptapeptide level with the human proteome, while heptapeptide commonality is minimal or absent with proteomes from nonhuman primates, that is, gorilla, chimpanzee, and rhesus macaque. To acquire more data on the issue, analyses here have been expanded to Ebola virus, Beginning in 2000, a high, unexpected level of molecular mimicry between microbial and human proteins has been repeatedly documented; accordingly, the consequent potential cross-reactivity following infections or active immunizations has been highlighted.As a matter of fact, pathogen-derived immunoreactive epitopes are mostly composed of peptide sequences present in human proteins,par excellence,namely, rhesus macaque (Macaca mulatta), which is a nonhuman primate phylogenetically close to humans.However, as recently underlined,primum movensof autoimmune pathologies via cross-reactivity, then different levels of peptide sharing with pathogens should characterize the proteomes of humans and primates.De facto, comparative amino acid (aa) sequence analyses documented that poliovirus, measles virus, dengue virus, and SARS-CoV-2 share peptide sequences almost exclusively with the human and murine proteomes, but not with primate proteomes.in absentiaof molecular mimicry.Therefore, it was hypothesized that if the peptide sharing between pathogens and humans is theYersinia pestisIn this research frame, the present study expands comparative sequence analyses to additional pathogens that, although thoroughly investigated, still remain without safe and efficacious therapies, for example,http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi): membrane protein/O-antigen protein fromFrancisella tularensis, 409 aa (177416); apical membrane antigen 1-like protein fromToxoplasma gondii, 651 aa (432359); surface antigen S from variola virus, 354 aa (587200); virulence-associated V antigen fromYersinia pestis, 326 aa (632); proteome from Ebola virus, 5494 aa (128952); proteome from HIV-1, 3134 aa (11676).Molecular mimicry analyses were conducted by using heptapeptides as immunobiological units. The analyzed pathogen proteins/proteomes are as follows (with NCBI TaxId in parentheses and further details atwww.uniprot.org/proteomes).Homo sapiens(9606); gorilla,Gorilla gorilla gorilla(9595); chimpanzee,Pan troglodytes(9598); and rhesus macaque,Macaca mulatta(9544). In addition, proteomes from the following mammalian organisms were analyzed as controls: cow,Bos taurus(9913); dog,Canis lupus familiaris(9615); cat,Felis catus(9685); rabbit,Oryctolagus cuniculus(9986); mouse,Mus musculus(10090); rat,Rattus norvegicus(10116); pig,Sus scrofa(9823); and bat,Pteropus alecto(9402).The primary aa sequences of the pathogen proteins/proteomes were dissected into heptapeptides offset by one residue, that is, MIRAYEQ, IRAYEQN, RAYEQNP, and so on. Then, each pathogen heptapeptide was analyzed for occurrences within mammalian reference proteomes, that is, proteomes that have been selected because they cover well-studied model organisms and other organisms of interest for biomedical research and phylogeny F. tularensis,T, gondii, variola virus, andY. pestis, respectively, and two pathogen proteomes, namely, Ebola virus proteome, and HIV-1 proteome for heptapeptide sharing with the mammalian proteomes described under Methods. The heptapeptide sharing is quantitatively reported inWe analyzed four protein antigens derived frommn/N, wheremis the number of heptapeptides present in the first protein,nis the number of heptapeptides present in the second protein, and N is 20mandn<< N, the probability of sharing only one heptapeptide is 0.00000000078125, that is infinitesimal.As a preliminary observation, it is noteworthy, as already underscored elsewhere,Then,On the whole, the data exposed into say that efficacy trials of HIV vaccines and microbicides have, to date, been disappointing is something of an understatement.” Today, in 2020, Thomas' observation is still valid and the promised effective vaccine that had to cure HIV has not yet been found,Thus,Recently, it was shown that heptapeptides from poliovirus, measles virus, dengue virus, and SARS-CoV-2 occur in the human proteome, but not in proteomes from primates and domestic animals.F. tularensis,T. gondii, variola virus,Y. pestis, Ebola virus, and HIV-1, and the human proteome. Such high molecular mimicry is practically absent when proteomes from primates, domestic animals, and cattle are analyzed. Hence, this study might explain not only the wave of autoimmune diseases that are increasingly burdening the human population worldwide,The present study confirms such previous dataAlso, it has to be considered that the present data underestimate the cross-reactivity potential by two orders of magnitude. Indeed, if one considers that a minimal immune determinant corresponds to five aa residues,In light of these additional caveats and of the consequent higher cross-reactivity risk, it appears to be mandatory to investigate the molecular mechanisms that underlie the different extents of molecular mimicry between pathogens and mammals. Possible objects of investigation might be, for example, alterations of gene transcription/translation potentially involved in the different shaping of human and primate genomes/phenomes.In this regard, studies by Puente et alIn addition, segmental duplications in the genome and transposable elements are important sources of genetic/phenetic differences between humans and primates. Segmental duplications are blocks of highly homologous duplicated sequences that define hotspots of chromosomal rearrangement and act as mediators of normal variation as well as genomic diseases.In sum, it is not surprising that primates are not good models for many major human diseases/conditionsFinally, as a conclusive note, it appears pertinent to recall the basic concept first stated in 2000


In humans, coronaviruses can cause infections of the respiratory system, with damage of varying severity depending on the virus examined: ranging from mild-to-moderate upper respiratory tract diseases, such as the common cold, pneumonia, severe acute respiratory syndrome, kidney failure, and even death. Human coronaviruses known to date, common throughout the world, are seven. The most common—and least harmful—ones were discovered in the 1960s and cause a common cold. Others, more dangerous, identified in the early 2000s and cause more severe respiratory tract infections. Among these the SARS-CoV, isolated in 2003 and responsible for the severe acute respiratory syndrome , which appeared in China in November 2002, the coronavirus 2012 (2012-nCoV) cause of the Middle Eastern respiratory syndrome (MERS) from coronavirus, which exploded in June 2012 in Saudi Arabia, and actually SARS-CoV-2. On December 31, 2019, a new coronavirus strain was reported in Wuhan, China, identified as a new coronavirus beta strain ß-CoV from group 2B, with a genetic similarity of approximately 70% to SARS-CoV, the virus responsible of SARS. In the first half of February, the International Committee on Taxonomy of Viruses (ICTV), in charge of the designation and naming of the viruses , thus definitively named the new coronavirus as SARS-CoV-2. This article highlights the main knowledge we have about the biomolecular and pathophysiologic mechanisms of SARS-CoV-2. Coronaviruses show a notable propensity to transmit themselves to new hosts, and this propensity has emerged on several occasions in the evolutionary history of this viral family; suffice it to recall the human coronavirus HCoV-OC43, which has close genetic correlations with the bovine coronavirus (BCoV), and which seems to derive from this or the human coronavirus HCoV-229E which after originating from a bat Alphacoronavirus has passed back to an animal host generating the swine epidemic diarrhea virus (PEDV), or the most important human coronavirus, SARS-CoV and SARS-CoV-2, which seems to have its reservoir in bats. In fact, the sequences of the Wuhan beta coronavirus shows similarities to the Betacoronaviruses found in bats. Thus, bats are the natural reservoir of numerous Alphacoronaviruses and Betacoronaviruses .Tab.Tab13].TBetacoronavirus and others) [In general, viral evolution can follow two paths: (a) viral population coevolves with the host following a single fate; if the host thrives, the virus thrives too. However, since there are no host alternatives for the virus, a factor may arise that prevents replication of the virus in that host , and therefore, the viral population dies out (more common for DNA viruses); (b) viral population expands into multiple biological niches by infecting different hosts. If a factor arises that prevents the virus from replicating in one host, the virus replicates in another and the population does not become extinct (more common for RNA viruses such as the  others) .Selection is the process by which a genotype is fixed or not in the population through the evaluation made by the environment of the phenotype it encoded. For evolutionary purposes, the main characteristics of the virus-receptor interaction are: (a) a virus can use different receptors and co-receptors; (b) the same receptor can be used by different viruses and other microorganisms; (c) the type of receptor used by a virus cannot be predicted on the basis of its phylogenetic position or biological properties; and (d) one or a few mutations of the viral genome can modify the receptor used, the cell tropism, and the pathogenicity –15.By coevolution, we mean a process in a dynamic equilibrium (which varies over time) of reciprocal adaptation, by means of genetic modification, between two or more species. In this process, the genetic modifications of one species are the result of the selection made by the other species and vice versa. In the co-evolutionary process, a pathogen and its host are closely related and mutually adapt in opposite directions, just think of some characteristics such as the infectivity of the pathogen and the resistance of the host, or the response of the host to infection and immune evasion by the pathogen. In this context, virulence, e.g., the damage manifested by the host in response to the infection given by the pathogen, is an ambiguous manifestation of the state of coevolution between the two, since it manifests a state of poor fitness of the host that is not always accompanied by an efficient completion of the pathogen replication cycle; it often happens that the adaptation of a virus in a host leads to an attenuation of its virulence , 17.The genetic variation is the process, devoid of specific and predetermined purposes, which underlies the differentiation and therefore the evolution of viruses. The set of viral variants that originate because of different molecular mechanisms allow the virus to adapt to the environment it will act by selecting the form, or viral type, with the winning characteristics. Viruses use the same molecular mechanisms of genetic variation as other life forms: mutation, recombination, and gene reassortment .−3–10−5 nt/site, while that of DNA viruses is at least 300 times lower, 10−8–10−9 nt/site). [Mutation is a copy error of the nucleic acid by RNA or DNA polymerase, action of enzymes of the host cell; chemical-physical damage of the nucleic acid and can be divided into three main types: (a) transitions: replacement of a purine with a purine or of a pyrimidine with a pyrimidine; (b) transversions: replacement of a purine with a pyrimidine or vice versa; (c) insertions or deletions (indel): addition or subtraction of one or more nucleotides. The mutation, understood as an event that occurs on a population (set of organisms or genes or traits of them), can be quantitatively expressed through two parameters, the mutation rate, and the mutation frequency. The mutation rate quantifies the number of mutations per single copied nucleotide, regardless of the fate of the error produced, so it is a measure of a biochemical event. The mutation rate of viruses is influenced by (a) whether the viral DNA polymerase has proofreading activity; (b) whether the virus codes for restorative proteins; (c) replicative mechanism of the virus; (d) intracellular site of virus replication; (e) genome length . , 15, 18 t/site). .Recombination is a widespread mechanism of genetic variation that occurs for both RNA and DNA viruses and often with the participation of the replicative complex of the virus . To haveThe gene reassortment is the exchange of genome fragments between two or more viruses (RNA or DNA) with a segmented genome that infect the same host cell. It is an evolutionary phenomenon that allows major changes and is greatly exploited by these viruses, just think of the frequent generation of new variants that occurs in this way for the flu virus. These different molecular mechanisms of genetic variation make it possible to distinguish two types of viral evolution which can be differentiated precisely by the molecular mechanisms involved, the times with which they occur and the effects they have on the virus. The “genetic drift,” is the progressive and scaling modification over time, by small successive steps, of the viral genome; it is the genetic diversity resulting from single mutations (due to duplication errors) that occur each time the genome replicates and from the subsequent selection made by the environment. The “genetic shift” is the massive and single-stage modification of the viral genome as a consequence of recombination or reassortment; it is the genetic diversity resulting from a relatively rare event that occurs only in certain circumstances .When we refer to emerging viruses, we mean the causative agent of a new or never previously encountered viral infection in the population such as SARS-CoV-2. The emergence of the virus leads to an increase in the incidence of the disease caused by it in a new host, or in the traditional host following events that alter the epidemiology; in the latter case, it would be more correct to speak of re-emergence. Viruses undergo a process of random evolution resulting from the biological interactions they establish with the host, and, in conjunction with certain events or factors, this evolution can lead to the emergence of a disease. The emergence of new viral diseases can therefore affect the passage of a pathogen from one animal to another, or the passage of a pathogen from an animal to man. The emergence of new zoonoses represents the main threat to public health and are events that have repeated themselves with some frequency in the last decades, suffice it to recall the past SARS virus, the influenza, Nipah, Hendra, Ebola, and today the pandemic SARS-CoV-2 viruses –22.To have a pandemic emergence of a disease (such as COVID-19), two elements are required: (a) the introduction of the pathogen into the population and (b) its consequent spread and maintenance in the population itself. The factors that can lead to the emergence of infectious diseases are different and genomic adaptability plays an important role . For thia stable, keeps the virus in the environment , characteristic behavior of the virus in the primary host or reservoir; b evolving, passage of the virus from the traditional population to a new one, belonging to the same host species or to different species, with progressive adaptation of the pathogen; c dead-end or dead-end, passage of the virus from the traditional host to a new one in which the virus is unable to transmit effectively and therefore does not adapt, it may also be due to the high lethality of the virus which causing death too quickly the new host does not allow the transmission; d resistant: the infected host completely blocks the replication cycle of the virus (non-receptive host). To infect a new host, a virus must be able to overcome the species barriers and carry out its replication cycle in its cells; this process can be limited at various levels, including receptor binding, cell entry, replication, gene expression, and the spread of an infecting progeny [In general, the interactions between virus and host can therefore be divided into four types:  progeny , 25.The various factors that influence the ability of the virus to transmit from one host species to another, such as the ability to bind to the cell receptor of the new host, are generally influenced by the phylogenetic constraints existing between the host species in question; in the sense that the more the original host and the new host are phylogenetically related, the higher the probability that host barriers will be successfully overcome  , 27. TheUltimately, the probability that the adaptation of the virus to the new host takes place successfully and that the pathogen therefore remains in it depends on four factors : (a) the3 replacements/site/year [The RNA viruses, such as the coronaviruses, show a much higher evolutionary speed than DNA viruses, because of the high susceptibility to replication error mediated by RNA polymerase or reverse transcriptase, by the considerable size of the viral population and by the higher replication speed. These rapid evolutionary modifications allow RNA viruses to quickly generate mutations that can allow them to adapt to a new environment, including a new host species. If RNA viruses show an extreme variability, even the most complex DNA viruses still show a certain ability to generate different variants; the latter do not exhibit high mutation rates like RNA viruses since this would affect their functionality, but are subject to variation of specific sites called “hot spots.” Viruses with genetic material composed of a single small DNA strand which, probably, due to a high replication error rate, can reach an evolutionary speed equal to that of RNA viruses are an exception. The quasispecies theory is suitable for explaining the cloud of viral variants that created in the host infected with viruses characterized by high mutation rates; RNA viruses are therefore the main viruses to follow this dynamic, together with some DNA viruses with small genomes .The evolite/year . In an Rite/year , 17, 29.Betacoronaviruses, that manifest more than other viruses, probably thanks to their greater genomic flexibility, mutation, recombination, and reassortment phenomena that allow them to overcome species barriers, adapt to different hosts, and give rise to new variants which will have isolated events. On the other hand, the primary host will always be needed to have the infection of the new one, while, in the second case, the pathogen will stabilize in the new population without needing the primary host and an event will develop epidemic that may become endemic [R = 4, the number of cases will quadruple [In the case of viruses, emergence with pandemic effects is more frequently associated with RNA viruses, such as the  endemic . The mai endemic , 26. Thiuadruple , 26–28.Coronavirus (CoV), order Nidovirales and family Coronaviridae, are a group of viruses equipped with single-stranded, linear not segmented genome, positive sense RNA (ssRNA +) and provided with envelope, which have a helical symmetry core and a characteristic crown morphology. Until a few years ago, all the viruses belonging to the family Coronaviridae were subdivided in only two genera: Coronavirus and Torovirus, and all the viruses classified in the genus Coronavirus were subdivided in their turn in only three antigenic groups . The recent discovery of a wide variety of new coronaviruses in different host species has prompted the Coronavirus Study Group of the International Committee for Taxonomy of Viruses (ICTV) to propose the reclassification of the Coronaviridae family into two subfamilies: Coronavirinae and Torovirinae. In particular, the subfamily Coronavirinae now includes three genera: Alphacoronavirus, Betacoronavirus, and Gammacoronavirus [Sarbecovirus subgenus has been found in bats (such as horseshoe or other species). Therefore, it is possible that these bats could be the origin of SARS-CoV-2 [The onavirus , 28, 30.RS-CoV-2 . SARS-CoSARS-CoV-2 shows projections on its surface (about 20 nm long) formed by the glycoprotein S (spike) that join in groups of three that thus make up a trimer that together with the others form the structures that resemble a crown that surrounds the virion. The main differences of this new Coronavirus compared to SARS virus appear to be localized precisely in this spike protein and that glycoprotein S is the one that determines the specificity of the virus for epithelial cells of the respiratory tract, that is, to be able to bind the ACE2 receptor (angiotensin converting enzyme 2), expressed by the cells of the capillaries of the lungs, and studies have shown that SARS-CoV-2 has a higher affinity for ACE2 than SARS-CoV. Instead, the membrane protein M crosses the lining (envelope) interacting within the virion with the RNA protein and the protein E helps glycoprotein S to attach to the membrane of the target cell. The dimer-hemagglutinin esterase (HE) is a coating protein, smaller than glycoprotein S, and plays an important role during the release phase of the virus replication within the host cell. The envelope, which is the coating of the virus, consists of a membrane that the virus “inherits” from the host cell after infecting it. Coronaviruses are enveloped viruses that carry a genome of positive (+) sense RNA. RNA consists of a single strand of positive RNA of large size (27 to 32 kb in different viruses) and are not known larger RNA viruses. RNA originates seven viral proteins and is associated with protein N (nucleoprotein), which increases their stability  to generate fusion-catalyzed and unlocked forms on the cell surface. The gRNA will be the model for the translation of two polyproteins (pp1a and pp1ab), which in turn are broken down to form NSPS. Subsequently, the NSPS induces the reattachment of the cell membrane to form a double-membrane vesicle, where the viral RTCs (replication-transcription complexes) are anchored. The gRNA (full length) is replicated via a-gRNA intermediate, and a nested set of sgRNA species is synthesized by discontinuous transcription and that will encode the structural and accessory viral proteins. Subsequently, the viral products will be assembled in the ER/Golgi intermediate compartment where a smooth-walled vesicle will be created and will be directed towards the plasma membrane to exit via exocytosis and so on; again, another cycle will be created . The meaA new variant of the spike protein D614G that increases infectivity and transmissibility has been immersed. Thus, D614G is associated with potentially high viral loads in the patients but not with disease severity. The mutation that modifies the amino acid D614G is passed on as part of a conserved haplotype defined by four mutations that almost always follow together , 36. RecCoronaviruses infect numerous animal and human hosts and are characterized by a remarkable frequency of recombination, which, together with the high rate of mutation, encourage their adaptation to new hosts and ecological niches [l niches . In mostl niches –42. Respl niches , 42–47. l niches , 40.The virus escapes the control of the immune system and decreases the number of lymphocytes so that they do not eliminate the virus, and monocytes/macrophages are not regulated and start pumping “cytokine storms” (neutrophil-to-lymphocyte ratio (NLR) is the leading indicator of hypercytokinemia). The virus’ direct pro-inflammatory effect response can cause acute damage to lung tissue and, subsequently, the hyper-inflammation leads to the acute respiratory distress syndrome. The development of SARS-CoV-2 infection towards the lower airways depends on the damage caused to the bronchioles which disrupts the protective coatings of the surfactant released by type II pneumocytes. This leads to alveolar alteration and desquamation of the endothelial cells and therefore impaired gas exchange and subsequent hypoxemia which will lead to acute respiratory distress syndrome (ARDS). But ARDS itself will in turn cause an overactive immune defect , which will lead to an increase in free radicals, cell debris and proteases, and edema resulting from the increase in proteins in the interstitial space, and the consequent vasoconstriction through platelet activation that further alters alveolar gas exchange with severe hypoxemia and tissue hypoxia which will lead to multi organ failure (MOF) , 43, 48.Human pathogenic coronaviruses (SARS-CoV and SARS-CoV-2) bind to their target cells through ACE2, which is expressed by the epithelial cells of the lung, intestine, kidney, and blood vessels (SARS-CoV-2 binds ACE2 with higher affinity than SARS-CoV).The ACE2 is a monocarboxypeptidase that acts on many molecules within the renin-angiotensin system and other substrates, such as apelin (molecule that regulates blood sugar and increases insulin sensitivity). Expression of ACE2 has been reported in type 2 pneumocytes. Functionally, there are two forms of ACE2: (a) “full-length” ACE2 contains a transmembrane domain capable of attaching its extracellular domain to the plasma membrane. The extracellular domain is a receptor that binds to protein S from both SARS-CoV and SARS-CoV-2. The new coronavirus is evolutionarily related to Bat-SARS, which similarly uses membrane-bound ACE2 as a receptor and (b) the soluble form of ACE2 lacks membrane anchorage and circulates in small quantities in the blood. Some authors suppose that this soluble form can act as an interceptor of SARS-CoV-2  which by acting can prevent the link between S and ACE2 at the cell surface as shown by some in vitro studies. Furthermore, ACE2 combined with a certain portion of Fc immunoglobulin was neutralizing for SARS-CoV-2 in vitro , 42, 50.Coronaviruses (at least the 4 coronaviruses that cause flu in humans) and following the SARS-CoV epidemic and studies led to the belief that cross-immunity between the common cold virus and SARS-CoV-2 is very likely directed against the antigens common to all coronaviruses and not the specific antigens of SARS-CoV-2 [The virus can be detected by PCR in whole blood and in stool samples ), and the serological blood test looks for antibodies created by the immune system , 59. SomRS-CoV-2 . AccordiRS-CoV-2 , 59–62. RS-CoV-2 , 63–66.13 cells colonized by 1014 microbes. These cells maintain the immune homeostasis for defense that is the eubiosis condition. In some cases, as in viral infections, arises a dysbiosis that creates an imbalance not only locally but also to other systems [Lactobacillus, Bifidobacterium, Streptococcus, Clostridium, and Firmicutes were over represented and the genera Coprococcus, Parabacteroides, Roseburia, Faecalibacterium, and Bacteroidetes were less. Firmicutes have a potential influence on intestinal ACE2 expression and Bacteroidetes have shown an inverse correlation with SARS-CoV-2 fecal load and protective effect against inflammation. Firmicutes have a potential influence on intestinal ACE2 expression and Bacteroidetes have shown an inverse correlation with the fecal load of SARS-CoV-2 which has a potential protective role, and this may be a predictor responsible for the course and severity of the infection. In fact, recent studies show that gastrointestinal pathology in patients with infections that are due to rhinoviruses, adenoviruses, infectious mononucleosis viruses, and coronaviruses (such as the new COVID-19 pandemic) can be more frequent and lasting than pulmonary ones. This can be linked precisely to the imbalance of the intestine/lung axis, or from bacterial dysbiosis, or to an ischemic damage of the central nervous system, but also from interleukin-6 which can also be facilitated by nasopharyngeal dysbiosis [Human colonization by microbes in different numbers and compositions begins immediately after birth in any area of the body that comes into contact with the environment, such as the skin; the oral cavity; the rhinopharynx; the lungs; the intestine; the breast; the urogenital system; and, based on recent studies, the placenta and uterine. The microbiota are an organized community of microorganisms, bacteria, fungi, protozoa, which are found in a network of polysaccharides and are attached to a living or inactive surface. An adult human being consists of about 10 systems . These p systems –70. The ysbiosis –75. The ysbiosis , 76–81.The SARS-CoV-2 pandemic emergency has shown the world that only an international scientific effort can stop a threat of this magnitude; also because in some cases, it has shown the whole world the weakness of local health systems. The research effort of the scientific community continues to understand how the new virus works and thus to have an early diagnosis, better management, and adequate therapy. The virus is now well-researched in a short time but the victims around the world continue to be a lot. It causes no or mild symptoms, such as flu-like and taste and smell disorders, but also serious diseases such as the diffuse alveolar damage (DAD), acute respiratory distress syndrome (ARDS), multi organ failure (MOF), and thromboembolic phenomena that make it necessary to study the virus pathophysiology continuously. However, it seems that the virus is “changing” and probably this will make it less pathogenic because it shows the deletion in non-structural proteins (such as NSP1), the most important determinants of the pathogenicity of SARS-CoV-2. On the other hand, cross-immunity and immunity questioned at a certain time that a patient can develop and maintain over time. However, there are no certainties about the future evolution of the virus and its possible disappearance or, on the contrary, its persistence in the population with an enhancement of its virulence. This is important for the risk of reinfection in recovered patients, documented by various cases around the world, and reinforces the need for research for an effective vaccine. Finally, based on the recognized ability of probiotics to stabilize and enhance the resident microbiota and host immune system, as well as on the basis of positive data emerging from ongoing clinical trials on the potential role of probiotics in therapy for SARS-CoV-2, these could be considered both for prevention and to improve the course of the disease.


The ebola epidemic that raged in West Africa between 2013 and 2016 was the largest since the discovery of the virus in 1976. During this epidemic, more than 11,000 cases were notified with a lethality of over 67%. Several means of transmission have been described. The great difficulty noted during the epidemic was the estimation of the number of asymptomatic and pauci symptomatic cases, however there is evidence that this population has been in contact with the virus for some time. Thus, they could be a source for the spread of the epidemic. In this paper, we report in Guinea-Conakry three stories of probable pauci-symptomatic form of ebola disease that would have been the cause of massive infection in a population sorely tried by the epidemic between 2014 and 2015 in Guinea. Ebola virus causes serious disease, which is fatal in most of case. The virus infectivity, lethality, the rapidity of human-to-human transmission and the lack of an effective treatment captured the world's attention. The last outbreak in West Africa with 28 652 cases and 11 325 deaths reported, is the largest and most complex ebola outbreak since 1976 [Indeed, studies conducted near contacts of ebola cases have shown a very high EBOV seroprevalence . In SierOctober 8, 2014: the first wife infected with ebola virus developed symptoms . She was admitted to the Ebola Treatment Center (ETC) in Conakry. Tested positive for ebola Zaire by RT-PCR. She died the same day. The second wife (MD) and the husband (LC) known as contacts were monitored few days and they escaped the monitoring system.October 15, 2014 a week later: LC has a fever associated with headache and asthenia. Quickly considered as uncomplicated malaria. After self-medication with antimalarial, antibiotic, analgesic and anti-inflammatory drugs, the symptoms disappear in 3 days without ever being admitted to a ETC or healthcare facilities.October 28, 2014: MD, 18 years old, second wife of LC had a headache associated with fever, abdominal pain and vomiting. After 2 days´ self-medication with unknown drugs, she was admitted in a military healthcare facility in Conakry. She was treated for both severe malaria and typhoid fever. Its symptoms disappear after 4 days without tested for ebola virus infection.The case that elucidated the history of ebola virus circulation in this family: MC, 7-month-old (son of LC and MD) has at October 28, 2014, fever associated with diarrhea. He was admitted in the paediatrics service in a Conakry University Hospital. In the first time, he was treated for malaria and acute gastroenteritis. After one week and the occurrence of hematemesis (4 November 2014), he was transferred in Donka ETC and tested positive for ebola virus by RT-PCR but died the same day. The looking for the source of infection has led to perform serology and RT-PCR in parents. They have been positive in ELISA serology and negative in RT-PCR. LC and MD were positive for ebola IgM and IgG antibodies. Despite these medical evidences, the parents remained reluctant and accused the devil. However, a total of 21 individuals were followed by contact tracing teams and none developed symptoms of the ebola virus disease and all were tested negative 2 times in RT-PCR for ebola virus.On November 7, 2014: AT, 72 years old have attended the funeral of his son MT; 49 years old (considered as a probable case of infection with the ebola virus).On November 19, 2014: AT developed a fever associated with headache, abdominal pain and joint pain. He has been seen by a physician at the emergency service in an university hospital in Conakry. Known diabetic, her blood sugar was 6.83 mmol/l with a temperature at 39.7°C. He receives antimalarial treatment and an antibiotic. The symptoms disappear after 5 days without ever tested for ebola and without having been admitted to an ETC.On November 30, 2014: the AT´s daughter, FT developed a fever over 40°C, diarrhea, vomiting and physical asthenia. She was admitted at the Donka ETC, tested positive for the ebola Zaire virus by RT-PCR. Unfortunately, she died 11 days later. In total, 72 individuals have been registered as contacts around them. They were followed, released after 2 negative PCR and none developed any symptoms related to ebola disease.History 3: fear and promiscuity: AB, 25 years old and his friend ZM 33 years old, are living in the same bedroom.On March 26, 2015: AB: developed a fever associated with headache, dyspnea, joint pain and physical asthenia. After five days, he has been seen by a physician in cardiology department in an university hospital in Conakry. He received as an outpatient antimalarial treatment, antibiotics and anti-inflammatory. Given the persistence of signs, he has been seen by a traditional healer without success. Frightened by the epidemic context, he decided to hide to the contact tracing team. He has been finally located on April 17, 2015; he was asymptomatic. In Donka ETC, he has been tested positive for ebola Zaire by RT-PCR took care there and released after 2 negative consecutive RT-PCR. His friend ZM presented April 3, 2015 a fever, headache and joint pain. He has been admitted to Donka ETC with abdominal pain and diarrhea. Tested positive for ebola virus by RT-PCR. After treatment, he recovered. A total of 36 contacts have been registered monitored and released after 2 negatives PCR and none of them developed any symptoms related to ebola disease.Zaire ebola virus is the most lethal ebola virus species and one of the most virulent human pathogens, causing a severe hemorrhagic fever syndrome. Fatality rates varied from 25% to 90% in symptomatic patients within a few days , 2. We hIn our knowledge, when we are confronted with epidemiological situations of this nature, the analysis of exposure time, the area of the contact and the number of individuals involved is imperative. In our scenario, we have every reason to believe that through these stories, the epidemic could have been being restarted with no possibility to control. However, some weaknesses in the contact tracing system established, people were finally identified, confined and treated. These stories show once again the difficulties that may be encountered for the control of such an epidemic. These stories raise the question of the sexual transmission of the virus . In the These three case reports raise the problem of investigating and monitoring ebola cases in a health emergency context, but also raise the question of the role of asymptomatic ebola cases in the spread of the disease. It is obvious that there have been shortcomings in the management of the epidemic in Guinea and that not all the exhaustive number of cases is known.Ethical aspects: this study was sponsored by the ethics committee of the public health department of the Abdel Nasser Gamal University in Conakry. Data were collected as part of response activities against the ebola outbreak in Guinea and personal information was not disclosed in this article to protect the anonymity of patients.


Miscanthus sinensis is a grass used for sugarcane breeding and bioenergy production. Using high throughput sequencing technologies, we identified a new viral genome in infected M. sinensis leaf tissue displaying yellow fleck symptoms. This virus is most related to members of the genus Polerovirus in the family Luteoviridae. The canonical ORFs were computationally identified, the P3 coat protein was expressed, and virus-like particles were purified and found to conform to icosahedral shapes, characteristic of the family Luteoviridae. We propose the name Miscanthus yellow fleck virus for this new virus. Miscanthus sinensis Andersson (Chinese silvergrass) is an herbaceous perennial grass native to Eastern Asia. It is naturalized in parts of the United States (X70-80)T[R/K]XGXSG, without conservation of the basic [R/K] that is found among P1s of poleroviruses [ORF1 is expected to encode for P1, a multifunctional protein  with higoviruses , was alsORF1-2 is an expected product combining ORFs 1 and 2 through a predicted -1 ribosomal frameshift at nucleotide position 1650 . The sitORF3A contains a non-canonical start codon CTG and is predicted to encode a polypeptide involved in long distance viral movement  with 70%ORF3 is predicted to encode for the coat protein (P3) , which s5’ ACATAAAA 3’, which is repeated at nucleotide positions 3351–3358. A conserved 5’ ACAAAA 3’ motif is found in many poleroviruses both at the 5’ end and near the beginning of predicted sgRNA sites and is thought to function as a possible transcription enhancer, producing the full genomic RNA and subgenomic RNA species [5’ ACAAAA 3’ motif is not fully conserved in MYFV, exceptions have already been reported, such as 5’ ACTAAA 3’ for WLYaV [The MYFV genome begins with  species , 29, 30.or WLYaV .Tombusviridae and Luteoviridae [MYFV has 3 predicted untranslated regions (UTRs), including a 56 bp 5’UTR, a 272 bp 3’UTR without a polyA tail, and an internal-UTR of 57 bp from nucleotide positions 3357–3413. The internal-UTR forms a predicted pseudoknot structure that could function as an exoribonuclease-resistant RNA element . The preoviridae . The benoviridae .Luteoviridae family, their complete genomes were aligned, and a phylogenetic tree was constructed using Maximum Likelihood  in diameter [To functionally validate the CP sequence, it was heterologously expressed in the model plant  of MYFV . Luteovidiameter , and theMiscanthus sinensis. This virus has all the genomic characteristics of poleroviruses and clearly groups in a phylogenetic tree within poleroviruses. All identified proteins have less than 90% identity to those of known poleroviruses, and, therefore, it qualifies as a new virus in the Luteoviridae family per the species demarcation criteria (≥10% difference in amino acid sequence of any gene) [In this paper, we identify and describe a new polerovirus that is associated with yellow flecks on leaves of ny gene) . We subsMiscanthus sinensis is being used in sugarcane breeding, there is a possibility that hybrid miscanes could serve as a common host for these two viruses. Furthermore, identification of MYFV allows for the development of diagnostic tools that are important for successful plant regulatory and quarantine procedures. Interestingly, diagnostic primers developed for ScYLV CP sequence also worked to amplify MYFV CP sequence. Future research should focus on identifying the vector, host range, and genetic diversity for this new polerovirus.MYFV is closely related to the previously characterized poleroviruses ScYLV and WLYaV. ScYLV is a virus of economic importance on sugarcane; since


Paramyxo- and filovirus nucleocapsids (NCs) have bipartite promoters at their 3′ ends to initiate RNA synthesis. The 2 elements, promoter element 1 (PE1) and promoter element 2 (PE2), are separated by a spacer region that must be exactly a multiple of 6 nucleotides (nt) long. Paramyxovirus NCs have 13 nucleoprotein (NP) subunits/turn, such that PE1 and PE2 are juxtaposed on the same face of the NC helix, for concerted recognition by the viral polymerase. Ebola virus (EBOV) NCs, in contrast, have 25 to 28 subunits/turn, meaning that PE1 and PE2 cannot be juxtaposed. However, there is evidence that the number of subunits/turn at the 3′ end of the EBOV NC is variable. We propose a paramyxovirus-like model for EBOV explaining why there are 8 contiguous copies of the PE2 repeat when 3 are sufficient, why expanding this run to 13 further improves minigenome performance, and why there is a limit to the number of hexa-nt that can be inserted in the spacer region. Non-segmented negative-strand RNA virus (nsNSV) genomes are 12 to 20 kB in length and contain 5 to 10 tandemly arranged genes. There are 4 main families: Rhabdoviridae (vesicular stomatitis virus (VSV) and rabies), Paramyxoviridae (Sendai virus (SeV) and measles), Pneumoviridae ), and Filoviridae (Ebola virus (EBOV) and Marburg virus (MARV)). nsNSV genome RNAs are enclosed within a non-covalent polymer of nucleoprotein (NP) to form a helical nucleocapsid (NC), the substrate for all viral RNA synthesis. The NP subunits of paramyxo- and filovirus NCs bind precisely 6 nucleotides (nt) each, with 3 contiguous bases pointing toward the protein core and 3 pointing away, whereas those of pneumo- and rhabdoviruses bind 7 and 9 nt/subunit, respectively. Paramyxo- and filoviruses also differ from the other 2 families in that their mRNAs can be co-transcriptionally edited to express alternate open reading frames (ORFs). EBOV edits its glycoprotein mRNA to make the essential full-length glycoprotein, and MARV (a member of another filovirus genus), which does not need to edit its glycoprotein mRNA, may edit its NP and polymerase (L) mRNAs [5 in NPs number 14–16  for SeV, and 3′-N4GC in NP#13–15 for parainfluenza virus type 5 (PIV5) [4 backbone, and 1, gln202, with an nt base modeled as uracil [cis-acting mRNA editing signal where hexamer phase participates in regulating this process [Paramyxovirus genome replication is governed by the “rule of six” , i.e., a5 (PIV5) ,7. Becau5 (PIV5) –12, thess uracil . In this process .Paramyxo- and filoviruses share many properties, but there are also important differences. EBOV actually acts like a pair of viruses, whose overall genome lengths differ by a single nt because their mRNA editing site can contain a run of either 7 or 8 uridines (EBOV/7U and EBOV/8U); both genomes are present during infection, regardless of which EBOV has initiated the infection . MoreoveIf paramyxo- and filoviruses evolved from a common ancestor and have adopted bipartite promoters to assure genome hexamer phasing (in large part to regulate mRNA editing), we would expect their bipartite promoters to operate similarly to the extent that their biology permits. The EBOV model we recently presented based on a 13 NP/turn helix, for which there is no evidence of its existence, is more than questionable and does not explain the filovirus data ,22, whilRhabdo- and filoviruses form rod-like virions whose length is determined by that of their NC, whereas paramyxo- and pneumovirus NC are contained within spherical envelopes. The 3′ end of the VSV NC, at the pointed end of the bullet-like virion, is itself also pointed or conical. Its tip (the 3′-most turn) likely consists of around 10 subunits/turn and increases to the uniform 33 subunits/turn of the NC trunk after 5 to 6 turns . Remarka