Previously, we showed using phylogenetics that during this period of decline, six amino acid residues appeared to be positively selected. To assess more directly the effect of these mutations, we utilized reverse genetics and competition fitness assays in the enzootic host and vector . We observed that the mutations contemporary with reductions in WEEV circulation and disease that were non-conserved with respect to amino acid properties had a positive effect on enzootic fitness. We also assessed the effects of these mutations on virulence in the Syrian-Golden hamster model in relation to a general trend of increased virulence in older isolates. However, no change effect on virulence was observed based on these mutations. Thus, while WEEV apparently underwent positive selection for infection of enzootic hosts, residues associated with mammalian virulence were likely eliminated from the population by genetic drift or negative selection. These findings suggest that ecologic factors rather than fitness for natural transmission likely caused decreased levels of enzootic WEEV circulation during the late 20th century.Understanding the circumstances under which arboviruses emerge is critical for the development of targeted control and prevention strategies. This is highlighted by the emergence of chikungunya and Zika viruses in the New World. However, to comprehensively understand the ways in which viruses emerge and persist, factors influencing reductions in virus activity must also be understood. Western equine encephalitis virus (WEEV), which declined during the late 20 Equally important to understanding how arboviruses emerge is understanding the conditions in which they experience reductions in activity. Western equine encephalitis virus (WEEV) provides a unique case study due to its reduction in equine and human incidence and wildlife transmission activity during the second half of the twentieth century. Despite those reductions, we identified six amino acid substitutions that appeared to increase fitness in avian hosts and/or mosquito vectors. We also found no effect of these mutations on mammalian virulence. Our results suggest that ecological factors likely explain the reduction in WEEV activity, even in the face of adaptive evolution. Understanding the evolutionary and ecological circumstances in which arthropod-borne viruses (arboviruses) emerge, often into naïve geographical regions, is critical for the development of proactive, targeted control and prevention strategies. The need for this understanding has been highlighted by the recent emergence of chikungunya and Zika viruses in the Americas –3. HowevAlphavirus, family Togaviridae  from day 10 post-bloodmeal to determine changes in the virus ratio during infection, replication and dissemination in the vector; 95% of these salivary glands were WEEV-positive  with 4 logHamsters inoculated with IMP181 tolerated infection well with 100% survival and no significant difference in weight compared to sham-infected animals. IMP181-6X exhibited the same pattern of virulence as compared to IMP181. In contrast, all but one BFS932-infected animal became moribund , with sith century, the late-20th century was characterized by a marked reduction in WEEV activity with the last human case documented in 1998 [Although WEEV caused many major outbreaks of human and equine encephalitis in western North America throughout the early- to mid-20 in 1998 , and con in 1998 , 26. We  in 1998 .in vivo experiments because previous studies with other alphaviruses indicate that in vitro fitness infrequently correlates with in vivo findings [To reliably determine the fitness effect of the six nonsynonymous mutations, competition assays were conducted in enzootic hosts and vectors. We chose to use competition assays to capitalize upon their high degree of sensitivity and efficiency as demonstrated previously for several viruses including alphaviruses –43. In afindings , 44–46.Cx. tarsalis, which is highly competent for WEEV in part due to its colonization since 1958 [Our choice of competition assay also mitigated the limitation of our use of the Bakersfield colony of nce 1958 , 47; notC. tarsalis and HOSPs. Thus, the three non-conservative amino acid substitutions that occurred coincident with the reduction in WEE during the second half of the 20th century conferred a fitness advantage. The lack of a significant fitness difference between IMP181 and BFS932, which differ in the same mutations, in C. tarsalis and HOSPs could reflect genetic difference between these strains other than the 6X, and possibly epistatic, that confer enzootic fitness. Indeed, when IMP181 and BFS932 are analyzed for non-synonymous mutations there are 26 and 23 non-synonymous mutations present in their non-structural and structural ORFs, respectively. Competition assays in C. tarsalis and HOSPs indicate that the non-conservative, nonsynonymous synapomorphic mutations that were fixed in the WEEV populations during the mid-20th century conferred fitness gains for transmission by C. tarsalis mosquitoes, consistent with their positive selection.Our results show that IMP181 outcompeted both IMP181-6X and IMP181-3X-Nonconservative in Our hamster experiment showed IMP181-6X and IMP181 produced no discernable disease, indicating that these six historic mutations have no effect on this model for mammalian virulence, while BFS932 was significantly more virulent. This finding suggests that unidentified mutations in BFS932 affecting virulence were eliminated from the WEEV the population via negative selection or drift. Future experiments using chimeras between BFS932 and IMP181 should be used as a first step to identify these putative virulence determinants.th century. The prolonged fixation of deleterious mutations in the population would be inconsistent with the fitness differences in C. tarsalis and HOSPs that were indicated by our competition of the recent IMP181 strain with IMP181-6X. Furthermore, the competitions between the natural isolates IMP181 and BFS932 showed that IMP181 is likely more fit in C. tarsalis and at least equally fit in HOSPs. Our previous phylogenetic findings indicating that the WEEV population is in decline and experiencing purifying selection cannot be completely discounted. However, purifying selection is typically the dominant form of selection identified in phylogenetic studies of many arboviruses, inducing those undergoing emergence [Our results argue against a sustained, dominant Muller’s Ratchet or drift effect to explain the reduction in WEEV circulation during the late 20mergence –51. Thisth century as a result of mechanized agriculture [Ecological factors accounting for WEEV’s population decline also deserve consideration. One is that equids (possibly juveniles) at one time participated in epizootic transmission of WEEV. However, even if equids served as amplification hosts during the past, they were likely eliminated from this role via their vaccination beginning in the 1940s, combined with their drastic population reductions during the 20iculture . This maiculture , 18.C. tarsalis change over time? 2) what is the vector competence for individual virus strains within each WEEV lineage? 3) what is the specific adaptive pathway taken by WEEV during the 20th century? and 4) do non-synapomorphic  mutations that we did not examine interact epistatically with the nonsynonymous synapomorphic  residues that we tested? These questions will require additional, extensive experiments.The limited scope of our experiments did not allow us to address the following issues possibly related to the decline in WEEV circulation and disease incidence: 1) did the vector competence of th century. Previous phylogenetic analyses demonstrated that, during the 20th century, WEEV followed an influenza virus-like pattern of lineage emergence and purification [th century were positively selected for enzootic transmission. The evidence of positive selection is a bit surprising in the context of our previous phylogenetic findings indicating a decline in the WEEV population and a possible sustained Muller’s Ratchet effect [C. tarsalis of West Nile virus in western North America since 2002, at the time of WEEV decline, suggest that reductions in vector populations are not an explanation.Taken together, our results support the hypothesis that WEEV, despite undergoing a population decline, underwent fitness gains for enzootic transmission during the 20fication . This pafication . Using tt effect , 25. Howt effect . Other st effect , 53, 54.th century, suggesting that shifts in arbovirus ecology can have profound impacts on human and equine exposure even in in the face of adaptive evolution. The specific ecological circumstances that precipitated this putative downward evolutionary trajectory remain cryptic. The potential role of equids in previous WEEV amplification and the effect of equine vaccination on enzootic circulation also deserve further study [In summary, our results provide a unique perspective on the evolution of WEEV through the 20er study , 52. Fin2 in a humidified incubator.Vero cells  were maintained in DMEM (Dulbecco’s Modified Eagle Medium) , 10% (vol/vol) fetal bovine serum (FBS), 1% (vol/vol) 100X Non-Essential Amino Acids (Sigma-Aldrich), 1% (vol/vol) Sodium Pyruvate (100mM) (Gibco) and 1% (vol/vol) Penn-Strep (Penicillin & Streptomycin)  (Gibco). Cells were incubated at 37°C with 5% COInfectious clones, containing all or half of the mutations previously identified , were geStocks of infectious clones were generated, sequences were verified, and RNA was transcribed as previously described . RNA wasin vitro transcribed RNAs was used to generate stocks of all other wild-type and mutant WEEV strains, and the sequence of output virus was verified [Stocks of WEEV/BFS932 (BFS932) were generated by picking a plaque of the virus and subsequently using the picked plaque to infect Vero cells and generate a stock of virus, the sequence of the resulting stock was verified . Electroverified . Stock tverified .In order to have a better understanding of the effect of the discovered mutations, competition assays between mutant virus and wild-type IMP181 were conducted in various models with the appropriate controls, with virus ratios determined by pyrosequencing. One important note concerning the competition assays we conducted was that we mixed by infectious units (PFUs) and measured the ratio of the viruses by relative genome equivalents. The PFU:genome ratio was fairly consistent between all of our constructs, as indicated by the inoculum and blood meals measured for the HOSPs and mosquitoes.C. tarsalis, isolated in 1958, was used for the following studies [10 PFU/ml mixed at a 1:1 ratio based on plaque forming units (PFU). Proper titers were verified by conducting plaque assays on each diluted virus before they were mixed. Virus mixtures were subsequently mixed at a ratio of 1:1 with artificial blood meal formulated with PBS-washed chicken blood  as previously described [The Bakersfield colony of  studies . Triplicescribed . Aliquot10 PFU/ml and mixed at a 1:1 ratio. HOSPs were subcutaneously inoculated (SC) with 100μl diluted virus mixture. Titers of viruses before mixing were taken to verify proper concentrations. Also, an aliquot of the inoculum was reserved for testing the initial ratio of viruses. HOSPs were bled on days 1 and 2-post infection via the jugular vein. Serum was collected from blood via centrifugation at 12,000 rcf for 5 minutes, and stored at -80°C until analysis. Pyrosequencing analysis was conducted as described below.HOSPs were caught using mist nets in Larimer County, CO and transferred to the ABSL3 at Colorado State University (CSU). Previous exposure to WEEV was determined by hemagglutination-inhibition (HI) and only naïve HOSPs were used. Mixtures of viruses were diluted to 4 logPrior to pyrosequencing analysis, virus positive samples were determined by CPE (cytopathic effect) assay as previously described .Aliquots of mosquito lysates and serum samples were prepared in Roche  external lysis buffer IVD (200μl) and deposited into individual wells of 96 deep-well processing plates (Roche Applied Science). Nucleic acids were subsequently extracted in high-throughput fashion using a Magna Pure 96 instrument employing large-volume Cellular RNA extraction kits (Roche) according to the manufacturer’s protocol for fresh/frozen biological samples. After extraction, a portion of the RNA was immediately converted to cDNA and the remaining material was archived at -80°C.cDNA was synthesized from extracted RNA using an iScript synthesis kit . 40μl reactions were assembled in 96 well PCR plates (Thermofisher Scientific) through the addition of 8μl iScript reaction mix, 2μl reverse transcriptase and 30μl of extracted RNA. RT was completed using a Bio-Rad C1000 thermocycler using the following protocol: 1) 25°C, 1.5 min; 2) 42°C, 30 min; 3) 85°C, 5 min; 4) indefinite hold at 4°C. Generated cDNA was analyzed immediately then stored at -20°C.WEEV pyrosequencing (a sequencing by synthesis technology) was performed using PCR-based pyrosequencing. Primers were designed using Pyromark Assay Design 2.0 software for SNP analysis. Initial PCR was carried out as follows: 12.5μl of iQ supermix  was mixed within a 25μl PCR reaction containing 200nM of both forward and reverse primers, 3μl cDNA and nuclease-free water. The specific primers used in each assay and the sequences analyzed are listed on Initial validation of the PCR assay was performed using a WEEV infectious clone plasmids that had their sequences confirmed using conventional Sanger-based dideoxynucleotide sequencing methodology. Preparation of PCR amplicons for pyrosequencing was performed using a Qiagen Pyromark Q96 vacuum workstation. Pyrosequencing was run in SNP mode on the Pyromark ID using Pyromark Gold reagents.10 PFU. Hamsters were infected with individual virus strains  in order to compare pathogenesis and virulence. Proper inoculum doses were verified by plaque assay. Body weights were measured each day with blood harvested alternatively every other day in each subject. On days 2 through 5, three hamsters from each group (one from mock) were perfused with PBS as previously described [58). Major organs were harvested and half of each organ, or the contralateral organ, were placed in 10% neutral buffered formalin and the remaining halves were placed in DMEM containing 5% FBS for measurement of viral load by plaque assay (amount of media was adjusted for each sample according to its weight to obtain a uniform limit of detection). Histopathological analysis and viral burden plaque assays were conducted as previously described [Female Golden Syrian hamsters 5–6 weeks of age were purchased from Charles River . Cohorts  were infected IP with 4 log0/i0), where w is relative fitness, f0 is the proportion of virus in the sample, and i0 is the proportion of virus in the inoculum. Relative fitnesses were evaluated graphically to determine consistency between replicates and pooled for analysis. The relative fitness values calculated for each virus in competition with IMP181 were analyzed by two-tailed Wilcoxon tests, testing the relative fitness values against a null value of 1 . This type of analysis is commonly used to statistically analyze competition assay experiments for viruses [For virus competition experiments in both mosquitoes and house sparrows, relative replicative fitness values for viruses in each sample were analyzed according to w =  transforming the dependent variable  and analyzing by ANOVA. Day post-exposure and virus group were included as independent variables. For blood viremia data, 3 repeated measurements were removed prior to analysis. Significant effects for by virus group were analyzed by post-hoc Tukey’s Honest Significant Difference method to make pairwise comparisons between virus groups, correcting for multiple comparisons using the Holm’s procedure.Work with Syrian Golden Hamsters was approved by UTMB’s IACUC under protocol 0209068B. Hamsters were anesthetized using isoflurane and sacrificed via perfusion. Work with House Sparrows was approved by CSU’s IACUC under protocol 16-6420A. House Sparrows were euthanized at the end of the study with carbon dioxide. All studies were conducted according to the NIH guidelines on the care and use of laboratory animals. Additionally, both universities are AAALAC accredited and our studies were conducted according to those guidelines as well. House Sparrows were caught in Larimer County, Colorado. State or federal permits are not required to catch House Sparrows.S1 FigWEEV infection rates in the salivary glands. Day 3 n = 5 per replicate, day 5 n = 5 per replicate, day 10 n = 5 per replicate. Error bars indicate standard error. No groups were significantly different from one another on each day as determined by a one-way ANOVA with Tukey post-tests.(TIF)Click here for additional data file.S2 FigPanels show viral burden in the A) spleen, B) lung, and C) liver. No statistical significance was detected between groups.(TIF)Click here for additional data file.S3 FigImages taken at 20X. Images indicate: perivascular cuffing, inflammation & nuclear dust, and hemorrhage, marked arrows. Images were taken at day 5 post-infection.(TIF)Click here for additional data file.S4 FigHeart images taken at 20X. Yellow arrows indicate foci of myocarditis. BFS932 image is from day 4 post-infection. IMP181-6X, IMP181, and MOCK images were taken at day 5 post-infection.(TIF)Click here for additional data file.


This study aimed to provide a detailed overview of the health care and productivity costs of non-fatal road traffic injuries by road user type. In a cohort study in the Netherlands, adult injury patients admitted to a hospital as a result of a traffic accident completed questionnaires 1 week and 1, 3, 6, 12 and 24 months after injury, including the iMTA Medical Consumption and Productivity Cost Questionnaire. In-hospital, post-hospital medical costs and productivity costs were calculated up to two years after traffic injury. In total, 1024 patients were included in this study. The mean health care costs per patient were € 8200. The mean productivity costs were € 5900. Being female, older age, with higher injury severity and having multiple comorbidities were associated with higher health care costs. Higher injury severity and being male were associated with higher productivity costs. Pedestrians aged ≥ 65 years had the highest mean health care costs  and motorcyclists the highest mean productivity costs (€ 9000). Bicycle injuries occurred most often in our sample  and accounted for the highest total health care and productivity costs. Considering the high proportion of total costs incurred by bicycle injuries, this is an important area for the prevention of traffic injuries. Road traffic accidents also cause up to 50 million non-fatal injuries each year ) with a 95% Confidence Interval (CI) was reported. Age was categorized as 18–34 years, 35–64 years and ≥ 65 years and the ISS was categorized as 1–3, 4–8, 9–15 and ≥16. The number of comorbidities was categorized as having no comorbidities, one comorbidity or two or more comorbidities. A p-value <0.05 was considered statistically significant. All analyses were conducted with SPSS version 24.0 .2 = 73.1, p < 0.001) . The characteristics of participants by road user type are shown in In total, 1024 out of 2281 people admitted to a hospital because of a traffic-related injury (44.9%) were included in the BIOS study. The distribution of road user groups differed significantly between responders and non-responders (chi< 0.001) . The majMean productivity costs were highest for motorcyclists (€ 9000) and lowest for (electric) bicyclists (€ 5200) and unspecified road users (€ 5100). The number of injuries per road user type differed widely. For example, injuries of pedestrians occurred in 3.5% (n = 36) of the cases in this study, while bicycle injuries were most common (n = 554 (54.1%)). Although mean health care and productivity costs were lower for (electric) bicyclists compared to other road users, their total health care and productivity costs were by far the highest because of the high number of injuries due to traffic accidents involving bicyclists, as shown in Health care costs were 1.21 (1.06–1.34) times higher for females than males . Yet, prPattern of costs by age group vary widely for the different road type users. Within the road user categories, health care cost differed across age groups . Post-hoThis study estimated the costs of traffic injury of six road user types. On an individual level, health care costs were highest for older pedestrians and productivity costs were highest for younger motorcyclists. Bicycle injuries occurred most frequently in our sample and accounted for the highest total health care and productivity costs in our study sample. Our study showed that female sex, increasing age and injury severity and having multiple comorbidities is associated with higher health care costs for traffic-related injuries. This corresponds to the results of a previous study from the Netherlands from 2017, that also showed higher health care costs for females and older adults for the entire injury population, but did not specifically show which factors were associated with traffic-related injury . The stuThe distribution of road user types in our study was similar to the findings of the previous study from the Netherlands . They alA strength of this study is that it provided a detailed overview of in-hospital, post-hospital and productivity costs for the total traffic injury population and by road user type. Therefore, we were able to compare these components across road user types. There are several limitations to this study. Firstly, people with minor injuries treated by their general practitioner or at the ED followed by discharge to the home environment were not included in this study. Minor traffic injuries were therefore not represented in this study and therefore it is probable that the mean costs per patient of the entire traffic injury population in the Netherlands are lower than presented in this study. Secondly, the patients included in this study differed by age and road user type from the patients who did not participate. It should be taken into account that the proportion of older patients and patients with bicycle injuries was higher in respondents compared to non-respondents. Since older age was found to be associated with higher health care costs, mean health care costs of the entire traffic injury population in Brabant, the Netherlands, may be lower than reported in this study. Thirdly, in the calculation of productivity costs, the average wage rates of males and females in the Netherlands were used. Since we did not use age-specific wage rates, it is possible that productivity costs were underestimated for prime working age adults and overestimated for young adults and adults approaching retirement age . Productivity costs are based on Dutch capital incomes and cannot be directly compared to the costs in other countries. However, the magnitude of cost of traffic injuries and the variation between road user is useful information for other countries.More than 50% of all traffic injuries in our sample were sustained by (electric) bicyclists. Therefore, these road users carry a high proportion of the health care costs related to traffic injuries. In the Netherlands, there is a cycling culture among both young and old and across social economic status groups . A studyThis study estimated both the health care and productivity costs of traffic injuries and compared these costs between road user types. Being female, older age, with higher injury severity and having multiple comorbidities were associated with higher health care costs. Higher injury severity and being male were associated with higher productivity costs. Both health care costs and productivity costs varied wildly across road user type and age groups. Older pedestrians had the highest health care costs and motorcyclists had the highest productivity costs. However, more than half of all injuries were due to a bicycle accident, resulting in the highest total health care and productivity costs. Therefore, (electric) bicyclists are a priority in the prevention of traffic injuries in the Netherlands.


Aedes aegypti and viruses is dynamic and related to the genetic background of each vector population and virus variant. There is little understanding of the genetic basis for SGIB and SGEB. Here, nested, half-sib families of Aedes aegypti were used to estimate genetic and environmental variances, in which daughters from an individual dam differ in their phenotype due to the sire. We found that SGIB has a strong genetic basis with dengue virus infections but not with Zika or chikungunya virus infections, in which all salivary glands became infected. SGEB has a moderate genetic basis for Zika or chikungunya infections but not with dengue virus infections.Mosquito salivary glands are infected with an arbovirus prior to entering saliva. The virus in the mosquito’s saliva can then infect a vertebrate host when the vector acquires its next bloodmeal. Salivary gland infection and escape barriers  have been identified that modulate arbovirus transmission. SGIB are manifest as the absence of virus in the salivary glands of mosquitoes having a disseminated infection, while SGEB are evident as an absence of virus in the saliva of mosquitoes even though salivary glands are infected. The interaction between Aedes aegypti half-sib families  infected with three dengue 2 (DENV2), two chikungunya (CHIKV), and two Zika (ZIKV) genotypes. We determined virus titer per salivary gland and saliva at seven days post-infection and virus prevalence in the half-sib population. CHIKV or ZIKV genotypes did not present SGIB, whereas DENV2 genotypes showed low rates of SGIB. However, virus titer and prevalence due to additive genetic factors in the half-sib family displayed a significant narrow-sense heritability (h2) for SGIB in two of the three DENV2 genotypes and one CHIKV and one ZIKV genotype. SGEBs were detected in all seven virus strains: 60–88% of DENV2 and 48–62% of CHIKV or ZIKV genotype infections. SGEB h2 was significant for all CHIKV or ZIKV genotypes but not for any of the DENV2 genotypes. SGIBs and SGEBs exhibited classical gene-by-gene interaction dynamics and are influenced by genetic factors in the mosquito and the virus.Arthropod-borne viruses (arboviruses) infect mosquito salivary glands and then escape to saliva prior to virus transmission. Arbovirus transmission from mosquitoes can be modulated by salivary gland infection barriers (SGIBs) and salivary gland escape barriers (SGEBs). We determined the influence of SGIBs and SGEBs by estimating the quantitative genetic contributions of  Vector competence (VC) for an arbovirus is affected by intrinsic (genetic) factors and mechanisms that control the ability of a vector mosquito to acquire, maintain, and transmit an arbovirus. There are several tissue barriers in the mosquito that the virus must overcome in order to establish a productive infection: the midgut infection barrier (MIB), midgut escape barrier (MEB), salivary gland infection barrier (SGIB), and salivary gland escape barrier (SGEB). In mosquito vectors of disease, the salivary gland is the organ leading to transmission of an arbovirus from an infected mosquito . In all Culex tritaeniorhynchus ..Culex trAe. aegypti, whereas SINV does not seem to infect the median lobes of Aedes albopictus or Ae. aegypti . After 4, 6, or 10 days incubation at 37 °C , cells were stained with 150 µL/well of 3 mg/mL MTT  solution and incubated for at least 4 h ,42. ViraA nested half-sib design was used to provide estimates of genetic and environmental variances and covariances ,43,44. A2P) is a function of three observed sources of variance: the average variance among offspring of a single sire is σ2S, the average variance among full-sib families from dams mated to a common sire is σ2d, and the average variance within members of a full-sib family is σ2w. Additive genetic variance (Va) is calculated as:Restricted maximum likelihood (REML) was used to estimate quantitative genetic parameters ,45. REMLThe variance among dams (Vd) bred to a common sire is calculated as:The variance among full-sibs is (Vw):Summing Equations (1)–(3),2) is:Narrow-sense heritability ) was subtracted from 2 restricted log likelihood for the Va = 0 modeSee SAS lines 5–10 for SGIB, and lines 24–30 for SGEB in See SAS lines 12–23 for SGIB and lines 31–42 for SGEB in p < 0.05.All statistical analyses were performed with GraphPad Prism . A two-tailed Fisher’s exact test was used to compare infection rates for SGIB and SGEB and determine any correlation among virus titers in saliva and salivary glands (Pearson correlation). Analysis of variance (one-way and two-way ANOVA) was used to determine the statistical significance, and CORREL function  was used for determining the correlation coefficient. Significance was defined as Our breeding design was established with a minimum number of sires (15–20), a minimum of 2–3 half-sib families (dam/sire) and a minimum of ten F1 females/half-sib families. Under this criterion, we obtained 24 half-sib families for DENV2 American genotype. We also established 21 half-sib families for DENV2 Asian and Cosmopolitan genotypes, ZIKV Asian and African genotypes, and 22 half-sib families for CHIKV genotypes. n = 20–30) by plaque assay for each viral strain at 7 dpi . Significant differences were also detected in virus titer between CHIKV genotypes (p < 0.0001). CHIKV ECSA-IOL virus titers were higher (9 × 105 ± 9 × 104 PFU/mL) than CHIKV Asian virus titers (1.7 × 105 ± 1.9 × 105 PFU/mL). We observed no significant differences in virus titer between the two ZIKV genotypes (p = 0.8931) or between DENV2 Asian and Cosmopolitan genotypes (p = 0.6971)  .Ae. aegypti salivary glands were uninfected with the DENV2 American, Asian, and Cosmopolitan genotypes, respectively. All salivary glands, on the other hand, were infected with CHIKV or ZIKV. Average viral titer (log10 (PFU/mL)) per salivary gland was calculated for all seven virus genotypes. Means and 95% confidence intervals are shown in CHIKV or ZIKV genotypes did not present SGIB, whereas DENV2 genotypes showed low rates of SGIB . Specifip < 0.0001). Of all viruses tested, DENV2 American had the lowest virus titer (2.699 ± 0.58 log10 (PFU/mL)), while ZIKV African had the highest titer (5.723 ± 0.331 log10 (PFU/mL)). DENV2 Cosmopolitan had the highest virus titer (4.742 ± 0.23 log10 (PFU/mL)) between DENV2 genotypes, while DENV2 Asian (3.607 ± 0.0421 log10 (PFU/mL)) was the second lowest of all viruses tested. CHIKV ECSA-IOL had slightly higher titers (4.921 ± 0.206 log10 (PFU/mL)) as compared to CHIKV Asian (4.687 ± 0.711 log10 (PFU/mL)). CHIKV genotypes had the same titers as DENV2 Cosmopolitan (p = 0.3294). Both ZIKV genotypes ; African, 5.723 ± 0.331 log10 (PFU/mL)) exhibited the highest titers. SGIB did affect the quantity of virus present in the salivary gland. It is very clear that different half-sib families can, and do, vary greatly in the amount of virus in salivary glands , genotype titer means for DENV2 and ZIKV were significantly different (y glands .10 (PFU/mL) means/sire occurred with DENV2 American (2) for SGIB were calculated to characterize each viral infection  for log10 (PFU/mL)/salivary gland were obtained . However, for DENV2 Cosmopolitan genotype, the variance among sire means were small (0.0082) and non-significant (p = 0.3238). The narrow-sense heritability h2 was 0.0685 (p = 0.3274).SGIB exhibited strong genetic bases with DENV American and Asian genotypes, but not with DENV2 Cosmopolitan genotype. For DENV2 American genotype and DENV2 Asian genotype, both the estimate of the variance among sire means and the h2 were significantly different . For CHIKV Asian genotype, the variance among sire means and the h2 were not significantly different . For ZIKV Asian genotype, the variance among sire means was 0.008 and not significant (p = 0.0918), and h2 was 0.1526 and marginally significant (p = 0.0416). There was a positive correlation between the variance among sire means and the narrow-sense heritability (h2). As predicted by Equation (5) (h2 = Va/Vp), the greater the variance in sire means, the larger the heritability. In other words, when the amount of virus was low . If ZIKV African was included, Pearson correlation coefficient was −0.24150 (p = 0.6019).Variation among sire families  suggesteervation . Pearsonp < 0.0001). DENV2 Cosmopolitan and CHIKV Asian did not significantly differ in SGEB% (p = 0.9447), nor did CHIKV ECSA-IOL or ZIKV Asian (p = 0.7332).In contrast to SGIB, all seven virus strains showed SGEB . SGEB% (10(PFU/mL)) per saliva were calculated for all seven viral genotypes, and are shown in p <0.0001; CHIKV, p = 0.0129; ZIKV, p < 0.0001). Of all viruses tested, DENV2 American had the lowest average virus titer (0.2442 ± 0.039 log10 (PFU/mL)), while ZIKV African had the highest titer (1.538 ± 0.1302 log10 (PFU/mL)) in saliva. DENV2 Cosmopolitan had the highest virus titer (0.6324 ± 0.071 log10 (PFU/mL)) of the three DENV2 genotypes tested, while DENV2 Asian (0.4038 ± 0.055 log10 (PFU/mL)) was the second lowest of all viruses tested. CHIKV ECSA-IOL had higher titers (0.922 ± 0.085 log10 (PFU/mL)) as compared to CHIKV Asian (0.6191 ± 0.0.079 log10 (PFU/mL)). CHIKV Asian had the same titer as DENV2 Cosmopolitan (p = 0.9013). ZIKV Asian and CHIKV ECSA-IOL both had similar titers (p = 0.7030) (10 (PFU/mL)/saliva was not significantly different between all three DENV2 genotypes tested  (10 (PFU/mL)/saliva for CHIKV and ZIKV genotypes were all significantly different  , while ZIKV African had the highest (67.9%) . As with 0.7030) B. Moreov= 0.136) A–C. In c 0.0173) D–F.2) for SGEB were also calculated to characterize each viral infection as SGIB. The observational components σ2S, σ2d, and σ2w and the causal components of variance  for log10 (PFU/mL)/saliva were obtained (10 (PFU/mL)/saliva) were not significant for any of the three DENV2 genotypes tested . Except for ZIKV African, the heritabilities for SGEB were likewise significant . Clearly, genetic factors in the mosquito do influence the amount of virus in the saliva for CHIKV and ZIKV Asian (Narrow-sense heritabilities (hobtained . In contKV Asian .2. The average log10 (PFU/mL) virus in the saliva was not correlated with heritability . Once the virus has infected the salivary gland, SGEB appears to have a major effect on the presence or absence of virus in the saliva /salivary gland and log10 (PFU/mL)/saliva to determine whether the amount of virus in the salivary glands affected the amount of virus in the saliva. We calculated Pearson’s correlation coefficients between the log10 (PFU/mL)/salivary gland and the average log10 (PFU/mL)/saliva across all individual mosquitoes .We tested for a statistical correlation between logsquitoes  for all 10 (PFU/mL)/salivary gland and the average log10 (PFU/mL)/saliva across all sire families for a genotype. When combined across all genotypes, a correlation of R = 0.6533 (p < 0.0001) was obtained.An additional correlation analysis was completed between the average logp = 0.0095), DENV2 Asian (p = 0.0124) and ZIKV African (p = 0.0016). This suggests that the overall correlation is an artifact of combining results from different viral genotypes. In general, the titer of virus in the salivary glands was not strongly correlated with the titer of virus in the saliva.However, significant correlation was only obtained for DENV2 American  based on observed volume , using t10 (PFU/mL)) for all virus strains tested in this study . It is very clear that the amount of virus in saliva varies greatly in samples of the same volume ). This is true for all DENV, CHIKV, and ZIKV genotypes tested.If all mosquitoes produce the same concentration of virus, then we would expect the amount of virus to be proportional to the amount of saliva. The amount of live virus would also be a function of the volume of saliva released into the host, which is also a continuous variable. Alternatively, if the saliva contains clusters of viruses, then there should be no relationship between the amount of saliva and the amount of virus. It has been reported that saliva often contains clusters of viruses rather than a uniform distribution ,22. There volume . Interes10 (PFU/mL)/saliva and saliva volume for all viruses (p = 0.0018). In general, saliva titers were not correlated with saliva volumes.An additional correlation analysis was completed between the log viruses . These cAe. aegypti from Mapastepec, Mexico. Half-sib analysis allows for an assessment of whether there is a genetic basis for a phenotypic trait that varies in expression in nature or in the laboratory. The critical question addressed by this study is whether there is a genetic basis for SGIB and SGEB in Ae. aegypti. The answer is affirmative, but with the qualification that the heritability of these traits in the mosquito is dependent upon the viral genotype, or more specifically, on the growth rate of the virus in the salivary gland. In addition, many of the significant h2 estimates are small, suggesting that most of the phenotypic variation is still unexplained, and that many uncontrolled factors in the environment contribute to the Vp exhibited in SGIB and SGEB. For each half-sib analysis, we assumed that the variance contributed by the virus should be minimal, because high-passaged virus in cell culture were used in this study. Usually there is abundant genetic variation present in Ae. aegypti field collections [2 suggests that this is due to the segregation of genetic factors that affect viral reproduction among and within half-sib families. In contrast with this idea, a DENV2 genotype (Cosmopolitan) and a CHIKV genotype (Asian)  C,E exhib (Asian) A, and th3 PFU/mL) were significantly higher (p = 0.0003) than at 7 dpi (1.7 × 104 PFU/mL). However, the percentage of salivary glands infected and the percentage of saliva infected was similar, at 7 dpi and 14 dpi.A low percentage of mosquitoes injected with DENV2 genotypes remained uninfected, but all mosquitoes injected with CHIKV or ZIKV genotypes were infected . The ext2 will approach zero. The distribution of phenotypes among the progeny of half-sib families will appear as in 2. The distribution of phenotypes among the progeny of half-sib families appear as in Ae. albopictus infected with different CHIKV genotypes [2 estimates should have been significant. However, in fact, four of the seven half-sib analyses of virus titer/salivary gland had a significant h2, and in three of the seven half-sib analyses of virus titer/saliva, h2 was significant. Thus, despite the random contribution of saliva volume and the potential presence of attached tissues during salivary gland dissections, significant heritabilities were still estimated. Further, it is unlikely that we may have underestimated h2 for SGEB as a result of problems associated with determining the volume of saliva produced by individual females and other uncontrolled factors. It is very clear that the amount of virus can vary greatly in similar saliva volumes , then Ve will be large, Va will be small, and henotypes . A simil volumes .2 (2 will approach zero (10 PFU/mL) among dams within sire is less than the average variation among all sires–families. The genetic explanation for this involves either (1) dominance wherein variance among heterozygotes is not intermediate in value between parental types or (2) common environmental variance arising when progeny of a single female is all raised in the same larval container. This leads to two types of environmental variance: variance arising from a common environment , and variance arising from half-sibs raised under different, albeit unapparent, conditions. Because Vp = Va + Vd + Ve and h2 = Va/Vp, a negative dominance reduces the denominator and increases h2. Raising half-sib larvae in a common environment could reduce Ve and inflate the covariance among siblings, and this also could inflate h2. There is abundant evidence for gene by gene (G × G) interactions between arboviral genotypes and Ae. aegypti [The heritabilities for DENV2 SGEB were not significant, nor were the variances among sires , Table 32  is consiach zero . This paach zero , and Vd ach zero . Mathema aegypti . The gen aegypti ,55,56 imAe. aegypti from Mapastepec, Mexico. The critical question addressed by this study is whether there is a genetic basis for SGIB and SGEB in Ae. aegypti. The answer is affirmative, but with the qualification that the heritability of these traits in the mosquito is dependent upon the viral genotype, or more specifically, on the growth rate of the virus in the salivary gland. This pattern suggests that when the amount of a virus is low, genetic factors in the mosquito may have an influence on viral titer in the salivary glands. However, once viral titers reach a high enough level in the salivary glands, the mosquito’s genetic contribution may no longer have any effect. Once the virus reaches a certain threshold, the effects of mosquito genes in determining the amount of virus declines, as does the variance in viral titers among sires. In addition, many of the significant h2 estimates are small, suggesting that most of the phenotypic variation is still unexplained, and that many uncontrolled factors in the environment contribute to the Vp exhibited in SGIB and SGEB.Herein, we report the results of a half-sib quantitative genetic analysis of SGIB and SGEBs in a freshly colonized strain of


Henipavirus and the Paramyxoviridae family. It is an endemic most commonly found at South Asia and has first emerged in Malaysia in 1998. Bats are found to be the main reservoir for this virus, causing disease in both humans and animals. The last outbreak has occurred in May 2018 in Kerala. It is characterized by high pathogenicity and fatality rates which varies from 40% to 70% depending on the severity of the disease and on the availability of adequate healthcare facilities. Currently, there are no antiviral drugs available for NiV disease and the treatment is just supportive. Clinical presentations for this virus range from asymptomatic infection to fatal encephalitis.  Nipah belongs to the genus  This study is aimed at predicting an effective epitope-based vaccine against glycoprotein G of Nipah henipavirus, using immunoinformatics approaches. Results and Conclusions. The two peptides TVYHCSAVY and FLIDRINWI have showed a very strong binding affinity to MHC class I and MHC class II alleles. Furthermore, considering the conservancy, the affinity, and the population coverage, the peptide FLIDRINWIT is highly suitable to be utilized to formulate a new vaccine against glycoprotein G of Nipah henipavirus. An in vivo study for the proposed peptides is also highly recommended. Glycoprotein G of the Nipah virus sequence was retrieved from NCBI. Different prediction tools were used to analyze the epitopes, namely, BepiPred-2.0: Sequential B Cell Epitope Predictor for B cell and T cell MHC classes II and I. Then, the proposed peptides were docked using Autodock 4.0 software program.  Henipavirus within the family Paramyxoviridae and has first emerged in Malaysia in 1998, gaining its name from a village called Sungai Nipah where it was isolated from the cerebrospinal fluid (CSF) of one of the patients [Pteropus seem to act as a natural reservoir of NiV based on the isolation of Hendra virus which has showed the presence of neutralizing antibodies to the Hendra virus on the bats [Nipah virus (NiV) is an RNA virus that belongs to the genus patients . NiV is patients –9. Largethe bats , 11. Altthe bats . The casthe bats , 13, 14.Laboratory diagnosis of Nipah virus infection is made using reverse transcriptase polymerase chain reaction (RT-PCR) from throat swabs, cerebrospinal fluid, urine, and blood analysis during acute and convalescent stages of the disease. IgG and IgM antibody detection can be done after recovery to confirm Nipah virus infection. Immunohistochemistry on tissues collected during an autopsy can also confirm the disease , 16. CurNipah virus invades host cells by the fusion of the host cell membranes at an optimal physiological pH for cleavage without requiring viral endocytosis. Cell-cell fusion is a pathological lineament of Nipah virus infections, resulting in a cell-to-cell spread, inflammation, and destruction of endothelial cells and neurons . Both NiThe production of peptide vaccines is extremely safe and cost-effective, especially when they are compared to conventional vaccines. Traditional vaccines that prevent emerging infectious diseases (EIDs) are very difficult to produce because they require the need to culture pathogenic viruses in vitro. However, epitope-based peptide vaccines do not require any means of in vitro culturing which makes them biologically safe, allowing a large scale of bioprocessing to be carried out rapidly and economically. Finally, their selectivity allows a precise activation of the immunological responses by means of selecting immunodominant and conserved epitopes , 28. Thehttps://www.ncbi.nlm.nih.gov/protein) [http://www.iedb.org/) [The amino acid sequences of glycoprotein G (Glycoside hydrolase family) for a total of 21 strains of Nipah virus were retrieved from the NCBI database (protein)  in a FASdb.org/)  were theConservation regions were determined using multiple sequence alignments with the help of Clustal-W in the BioEdit software version 7.2.5 . Epitopehttp://tools.iedb.org/bcell/result/) [http://tools.iedb.org/bcell/result/) were also proposed to determine the sites of antigenic epitopes with a default threshold value of 1.030 and a window size 6.0 [Candidate epitopes were analyzed using several B cell prediction methods to determine their antigenicity, flexibility, hydrophilicity, and surface accessibility. The predicted linear epitopes were obtained from the Immune Epitope Database (result/)  using a result/) . Kolaskasize 6.0 .http://tools.iedb.org/mhcI. This tool employs different methods to determine the ability of the submitted sequence to bind to a specific MHC class I molecule. The artificial neural network (ANN) method was used to calculate IC50 values of the peptide binding to MHC class I molecules. For both frequent and nonfrequent alleles, the peptide length was set to 9 amino acids prior to the prediction. The alleles having a binding affinity of IC50 that are equal to or less than 500 nM were considered for further analysis [The binding peptide was assessed by the IEDB MHC I prediction tool at analysis .http://tools.iedb.org/mhcII provided by the Immune Epitope Database (IEDB) Analysis Resource consisting of human allele references sets was used [To predict the peptide binding to MHC class II molecules, the MHC II prediction tool was used . The arthttp://tools.iedb.org/tools/population/iedb_input). This tool was used in order to determine the fraction of individuals predicted to respond to a given set of epitopes, with known MHC restrictions [The population coverage of each epitope was calculated by the IEDB population coverage tool at (rictions . For evehttp://raptorx.uchicago.edu/), where the reference sequence was submitted in a FASTA format on 14/9/2018 and the structure was received on 15/9/2018 [The 3D structure of glycoprotein G of Nipah virus was predicted using the RaptorX web portal  was downloaded from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do), which was in a complex with an azobenzene-containing peptide [In order to estimate the binding affinities between the epitopes and molecular structures of MHC I and MHC II, we have carried out an in silico molecular docking. Sequences of proposed epitopes were then selected from the Nipah virus reference sequence using Chimera 1.10 and saved as a (pdb) file. The obtained files were then optimized and energy minimized. The HLA-A peptide .All water molecules and heteroatoms in the retrieved target file 4UQ3 were then removed. The target structure was further optimized and energy minimized using Swiss PDB viewer V.4.1.0 software .Molecular docking was performed using AutoDock 4.0 software, based on the Lamarckian genetic algorithm, which combines energy evaluation through grids of affinity potential to find the suitable binding position for a ligand on a given protein , 45. PolThe physicochemical properties of the Nipah virus glycoprotein G protein was assessed using BioEdit software version 7.0.9.0. The protein length was found to be 602 amino acids, and the molecular weight was at 67035.54 Daltons. The amino acids that form the Nipah virus glycoprotein G protein are shown in The ref sequence of the Nipah virus glycoprotein G was subjected to a Bepipred linear epitope prediction. Emini surface accessibility and Kolaskar and Tongaonkar antigenicity methods in IEDB were used to determine bindings to the B cell and in testing its surface and immunogenicity. The results are shown in Figures 150) ≤ 500; the least most promising epitopes that had a binding affinity with the class I alleles along with their positions in the Nipah virus glycoprotein G are shown in The Nipah virus glycoprotein G sequence was analyzed using the IEDB MHC class I binding prediction tool based on ANN-align with half-maximal inhibitory concentration (IC50) ≤ 1000. The list of the epitopes and their correspondent bindings to MHC class II alleles, along with their positions in the Nipah virus glycoprotein G, while the list of the most promising epitopes that had a strong binding affinity to MHC class II alleles and depending on the number of their binding alleles is shown in The Nipah virus glycoprotein G sequence was analyzed using the IEDB MHC class II binding prediction tool based on NN-align with half-maximal inhibitory concentration  [In our present work, potential peptides were suggested to design an epitope-based vaccine for Nipah virus, using the latest amino acid sequences of glycoprotein G (glycoside hydrolase family) for a total of 21 strains of Nipah virus that were retrieved from the NCBI database (protein)  on July protein) .  Figure50 ≤ 500 [∗02:01, HLA-A∗02:03, HLA-A∗02:06, HLA-A∗68:02, HLA-C∗03:03, HLA-C∗06:02, HLA-C∗07:01, and HLA-C∗12:03), while FSWDTMIKF with 8 alleles  and TVYHCSAVY with 11 alleles .Various literatures were surveyed to define the antigenic part of the virus. Glycoprotein G was found to be on the outer surface of the virus which was chosen as our target. Initially, we have evaluated the binding affinity of the virus to MHC alleles. This was done by submitting the protein reference sequence to IEDB MHC, a binding prediction tool, based on the ANN align method with IC50 ≤ 500  for MHC The reference sequence of Nipah virus glycoprotein G was reanalyzed using the IEDB MHC II binding prediction tool based on NN-align with half-maximal inhibitory concentration (IC50) ≤ 1000 . The anaPopulation coverage results for the total peptides found and the proposed peptides binding to MHC classes I and II alleles are summarized in Tables ∗02:01, in which it has been specifically chosen in relation to their contribution to several immunological and pathological diseases [The selected peptides were further subjected to both MHC I- and MHC II-based population coverage analysis in the whole world, Southeast Asia, South Asia, North Africa, and Sudan as shown in diseases –56, althdiseases , 56.Calculations of the root mean square deviation (RMSD) between coordinates of the atoms and formation of clusters based on RMSD values have computed the resemblance of the docked structures. The most favorable docking is considered to be the conformation of the lowest binding energy. The least energy predictions of the peptide FLIDRINWI  and the 3D structure of the allele and its peptide are shown in As a result of these interesting outcomes, formulating a vaccine using the suggested peptide is highly promising and encouraging to be highly proposed as a universal epitope-based peptide vaccine against Nipah virus.The present study proposed a very promising epitope-based peptide vaccine against glycoprotein G of Nipah virus. It is expected to be highly antigenic with a minimum allergic effect. The proposed peptide FLIDRINWI has a strong binding affinity to both MHC class I and MHC class II alleles. Moreover, it shows an exceptional population coverage result for both MHC class I and MHC class II alleles in the whole world, Southeast Asia, South Asia, North Africa, and Sudan.Despite having to validate the findings of the current study, an in vivo assessment of the most promising peptides, namely, FLIDRINWI, TVYHCSAVY, and FAYSHLERI, is highly recommended and will serve as the ground data for such work as shown in Figures 5


Rhabdoviridae, Aspiviridae, Fimoviridae, Tospoviridae, and Phenuiviridae. They include both emergent pathogens that infect a wide range of plant species, and potential endophytes which appear not to induce any visible symptoms. As a consequence of biased sampling based on a narrow focus on crops with disease symptoms, the number of NSR plant viruses identified so far represents only a fraction of this type of viruses present in the virosphere. Detection and molecular characterization of NSR viruses has often been challenging, but the widespread implementation of HTS has facilitated not only the identification but also the characterization of the genomic sequences of at least 70 NSR plant viruses in the last 7 years. Moreover, continuing advances in HTS technologies and bioinformatic pipelines, concomitant with a significant cost reduction has led to its use as a routine method of choice, supporting the foundations of a diverse array of novel applications such as quarantine analysis of traded plant materials and genetic resources, virus detection in insect vectors, analysis of virus communities in individual plants, and assessment of virus evolution through ecogenomics, among others. The insights from these advancements are shedding new light on the extensive diversity of NSR plant viruses and their complex evolution, and provide an essential framework for improved taxonomic classification of plant NSR viruses as part of the realm Riboviria. Thus, HTS-based methods for virus discovery, our ‘new eyes,’ are unraveling in real time the richness and magnitude of the plant RNA virosphere.The use of high-throughput sequencing (HTS) for virus diagnostics, as well as the importance of this technology as a valuable tool for discovery of novel viruses has been extensively investigated. In this review, we consider the application of HTS approaches to uncover novel plant viruses with a focus on the negative-sense, single-stranded RNA virosphere. Plant viruses with negative-sense and ambisense RNA (NSR) genomes belong to several taxonomic families, including  Viruses are the most numerous biological entities on Earth, but the number of reported and formally described virus species, the known virosphere, is exiguous. This underestimated global virus landscape has led to a distorted view of virus diversity and function. Some aspects of virus biology including their small size, rapid rate of evolution, or lack of universally conserved viral genetic markers are likely the major cause for the lack of knowledge of most of the viruses present on our planet . Thus, aa priori knowledge. Thus, when combined with specific bioinformatics tools, HTS provides a powerful, efficient and economical alternative that has enabled not only the untargeted detection of both known and unknown viruses that inhabit a particular organism or environment but also a rapid characterization of their genomes (High-throughput sequencing (HTS) technology, also referred to as next generation sequencing, has revolutionized the nucleic acid characterization process since it allows the parallel sequencing of millions of nucleotides in a short period of time at a very high redundancy (depth of sequencing) without any  genomes . Thus, tHigh-throughput sequencing is also widely used to advance the molecular characterization not only of those viruses which have a poorly characterized genome, but also of preserved historic virus isolates which had been subjected to studies of their biological properties some decades ago. For instance, sterile stunt disease of maize, characterized by severe stunting and top necrosis of susceptible maize genotypes, was first reported in 1977 in Australia, but the genome of its etiological agent, maize sterile stunt virus was only characterized by HTS and recently reported . Similarin situ virus particle detection by electron microscopy or immunoassays, to name a few.In spite of the many advantages of HTS, there are some limitations and constraints that should be considered when using this platform for virus discovery. Given the high sensitivity of HTS, this technology may detect contaminant viral sequences or viruses that may not be actually replicating in the sampled plant tissue where they were found . In thisonly on their genomic sequences which constitutes a paradigm shift in their taxonomical classification  total plant RNA extracts, usually with a ribosomal depletion step (ii) virion-associated nucleic acids (VANA) extracted from purified viral particles, (iii) double-stranded RNA (dsRNA), enriched through cellulose chromatography or monoclonal antibody pull-down, and (iv) small interfering RNAs (siRNAs)  . These d(siRNAs) .Recent technological advances have led to the appreciation of exciting novel aspects of negative-sense and ambisense RNA (NSR) viruses, as recently reviewed by Solanum species resulted in the identification of viruses belonging to 20 different families, but only one was a NSR virus . Wuhan insect viruses 4–6 may be plant viruses based on their clear phylogenetic relationship with plant rhabdoviruses in the genus Cytorhabdovirus and that their genomes encode a P3 protein that most resembles plant rhabdovirus cell-to-cell movement proteins.Most plant NSR viruses are transmitted by arthropods in a persistent-circulative and propagative manner, thus they are adapted to infect both arthropod and plant cells. In fact, it has been suggested that plant NSR viruses may have originated from arthropod viruses that evolved to also infect plants . TherefoSince most NSR viruses replicate in both the plant host and the arthropod vector, the true virus host origins cannot be unambiguously discerned from the HTS data. Some of the newly discovered NSR viruses are highly divergent which makes it even more difficult to unequivocally determine if they are plant or plant-associated viruses, and it will require a significant amount of research to confirm their status, which is rarely carried out. This is exemplified by the recently discovered coguviruses and rubodviruses, where the host assignment based on their phylogenetic relationships is only preliminary. Thus, further biological studies will be required to determine if they are plant, plant-associated, or insect viruses .Negarnaviricota, which includes members characterized by (i) negative-sense or ambisense single-stranded unsegmented or segmented genomes, (ii) the presence or absence of a lipid membrane enveloping the capsid, and (iii) a diverse host range (Orthomyxoviridae), Zaire ebolavirus (Filoviridae) or Crimean-Congo hemorrhagic fever virus (Nairovirus). Most of the genera contain NSR viruses that infect vertebrates and only about 10% contain phytoviruses , Muvirales, Articulavirales, among others, have been created to accommodate diverse viruses which have been placed in the major phylogenetic gap between the two large groups of NSR viruses  belonging to five different families  include species of phytoviruses (Coguvirus and Rubodvirus) were recently created to accommodate novel species of NSR viruses related to members of the Phenuiviridae family , and Dichorhavirus and Varicosavirus (bi-segmented genomes) (The family genomes)   . The virgenomes)   .The increased application of HTS has seen a significant rise in the number of novel plant rhabdoviruses , as wellHigh-throughput sequencing was used successfully to complete the genome sequences of previously reported plant rhabdoviruses where only partial sequence fragments were available, such as Iranian citrus ringspot-associated virus , ivy veiLamprocephalus sp. and kalanchoe were analyzed by HTS  . FurtherFinally, partial fragments of RNAs1, 2 and 4 of a putative novel emaravirus infecting alfalfa in Australia, named alfalfa ringspot-associated virus were recently identified using HTS . PCR ampOphioviridae, contains only one genus, Ophiovirus, which is exclusively composed of members that have plants as their hosts  and their biological properties were known . DiffereAt the time of this review, only partial genome sequence of four other reported orthotospoviruses are available: groundnut yellow spot virus (S RNA), lisianthus necrotic ringspot virus (S RNA), pepper necrotic spot virus (S RNA) and tomato necrotic ringspot virus (S and M RNAs) ; HTS couPhenuiviridae includes 19 ICTV-recognized genera (Tenuivirus and likely two recently accepted new genera Coguvirus and Rubodvirus (2019.026M.A.v1.Phenuiviridae_4gen79sp.xlsx) contain species of plant-infecting viruses (Most phenuiviruses have enveloped particles with helical morphology, except tenuiviruses that have non-enveloped filamentous particles, and their genomes are comprised of two to four single-stranded, linear RNA segments. As of 2019, the family d genera . Only th viruses . Phyloge viruses .This genus is composed of two previously unknown viruses that have bi-segmented genomes, which have been discovered using HTS of citrus , while tMoreover, partial segments of a cogu-like virus were discovered when the virome of ornamental flowers was analyzed by HTS . HoweverThis genus is composed of species for two previously unknown viruses that have tri-segmented genomes which have been discovered using HTS in apple , while tTenuiviruses have a segmented genome of four to eight single-stranded RNA segments with negative-sense or ambisense polarity . The appThe number of NSR plant viruses identified so far represents a negligible fraction of the potential number of NSR viruses present in the virosphere. The discovery and in-depth molecular characterization of these viruses has been challenging given their extensive divergence, their outstanding diversity in terms of genomic architecture, and mostly, the negligible share of plant species studied for virus discovery. However, the widespread implementation of HTS, a cost-effective and efficient technology platform, allowed the discovery and molecular characterization of the genome sequences of at least 70 NSR plant viruses in the last few years. Thus, we predict that the increasing use of HTS, not only for plant samples but also in arthropod vectors, will allow the identification of many novel NSR phytoviruses which will be crucial to unravel the evolutionary landscapes of many NSR virus clades that are poorly characterized today. Nevertheless, as many more novel NSR viruses are being discovered, careful analysis will be essential to confirm their correct ecological context, to determine whether the new viral agents are plant-infecting viruses, fungi- or arthropod-infecting viruses whose RNA was co-purified along with the plant RNA, or just contamination-inadvertently sampled environmental viruses.Riboviria. In conclusion, HTS-based methods for virus discovery, our ‘new eyes,’ are unraveling in real time the richness and magnitude of the plant RNA virosphere.As soon as the virome characterization of multiple samples of cultivated and wild plants becomes more routine, more viruses with unique features and novel phylogenetic relationships will likely be discovered. As we assess the viral ‘dark matter,’ we will start to grasp the evolutionary history of plant viruses, eventually leading us to untangle the diversity of the NSR virosphere and gain increased knowledge about its complex evolutionary and phylogenetic relationships. A rich picture of the plant virome landscape will also provide an essential framework for improved taxonomic classification of plant NSR viruses as part of the realm All authors wrote the manuscript and approved it for publication.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.


Electron microscopy (EM) should be used in the front line for detection of agents in emergencies and bioterrorism, on accounts of its speed and accuracy. However, the number of EM diagnostic laboratories has decreased considerably and an increasing number of people encounter difficulties with EM results. Therefore, the research on viral structure and morphologyant in EM diagnostic practice. EM has several technological advantages, and should be a fundamental tool in clinical diagnosis of viruses, particularly when agents are unknown or unsuspected. In this article, we review the historical contribution of EM to virology, and its use in virus differentiation, localization of specific virus antigens, virus-cell interaction, and viral morphogenesis. It is essential that EM investigations are based on clinical and comprehensive pathogenesis data from light or confocal microscopy. Furthermore, avoidance of artifacts or false results is necessary to exploit fully the advantages while minimizing its limitations.


Camelpox virus (CMLV). The disease is characterized by mild, local skin or severe systemic infections and may have a major economic impact due to significant losses in terms of morbidity and mortality, weight loss, and low milk yield. Prevention of camelpox is performed by vaccination. In this study, we investigated the composition of a CMLV-based, live-attenuated commercial vaccine using next-generation sequencing (NGS) technology. The results of this analysis revealed genomic sequences of Modified Vaccinia virus Ankara (MVA).Camelpox is a viral contagious disease of Old-World camelids sustained by  Camelpox virus (CMLV), a double-stranded (ds) DNA virus belonging to the genus Orthopoxvirus (OPXV) of the family Poxviridae, subfamily Chordopoxvirinae. Twelve species are recognized within this genus, and the most notable include Variola major virus (VARV), Vaccinia virus (VACV), and Cowpox virus (CPXV). The Orthopoxvirus genome is composed of several structures. Two inverted terminal repeats (ITRs), identical in sequence, are present at the ends of the dsDNA genome and form covalently closed hairpin structures [Vaccinia virus Ankara (MVA), used during the last years of the WHO smallpox eradication campaign, shows a significantly smaller genome compared to extant VACV strains likely as a consequence of the massive passaging (over 570 times) in chicken embryo fibroblasts [Camelpox is a viral contagious disease of Old-World camelids and is characterized by mild, local skin or severe systemic infections. This disease may have a major economic impact due to significant losses in terms of morbidity and mortality, weight loss, and low milk yield . The cauructures . In addiructures . Howeverructures . Such deing over 70 times roblasts . The virroblasts ,7. Unlikroblasts . In this6.5 TCID50/mL of strain CaPV298-2, which was isolated in the United Arab Emirates (UAE) and attenuated by 120 serial passages into Vero cells [The live-attenuated Ducapox vaccine  contains 10ro cells  and prodDNA purification was performed starting from 200 µL of reconstituted sample using the High Pure Viral Nucleic Acid Kit  according to the manufacturer’s guidelines. DNA was then quantified by Qubit dsDNA HS assay , and one nanogram was used for library preparation by Nextera XT DNA Library Prep kit  according to the manufacturer’s protocol. Deep sequencing was performed on the NextSeq 500 platform  using the NextSeq 500/550 Mid Output Reagent Cartridge v2, with 300 cycles and standard 150 bp paired-end reads .http://evomics.org/resources/software/molecular-evolution-software/modeltest/) was used to identify the most appropriate model of evolution for the entire dataset and for each gene individually. The identified program settings for all partitions, under the Akaike Information Criteria, included six character states , a proportion of invariable sites, and a gamma distribution of rate variation across sites.Phylogenetic analysis was performed based on the nine concatenated open reading frames (ORFs) ,14, namede novo assembly by SPAdes software v. 3.11.1 [Vaccinia virus strain Acambis 3000 Modified Virus Ankara (accession No. AY603355), and this sequence was used to perform a reference-based assembly by Bowtie 2 (v2.1.0) [The sequencing run of Ducapox vaccine produced a total number of 1,202,012 reads. Quality controls and trimming carried out by FASTQC (v0.11.5) and trimmomatic-0.36 ,19, main. 3.11.1 ,22 that (v2.1.0)  and depoThe nine ORFs  of the Ducapox vaccine sequence were concatenated and aligned with homologous sequences of selected OPXV strains. Phylogenetic analysis was performed using Bayesian inference. Posterior probability percentages were consistently high (>95%) for all clades on phylograms, which supported inferred phylogenetic relationships.Vaccinia virus strain Acambis 3000 Modified Virus Ankara  and Vaccinia virus strain Ankara (U94848) strains. Nt identity of Ducapox vaccine strain with different VACV strains ranged between 98.2 and 99.9%.In the phylogenetic tree , DucapoxVaccinia virus Ankara (CVA) after continuous serial passages in chicken fibroblast tissue culture [In this study, the genome composition of Ducapox vaccine, a commercial CMLV vaccine produced by the Central Veterinary Research Laboratory  starting from the CMLV CaPV298-2 field strain, was investigated by NGS. The vaccine sequence content revealed the highest nt identity with an MVA strain (AY603355) of VACV and the absence of CMLV-unique regions including ORFs CMLV185, CMLV186, and CMLV187 . Interes culture . Accordi culture . In fact culture ,27.


Geminiviruses and their diseases are a considerable economic threat to a vast number of crops worldwide. Investigating how and where these viruses replicate and accumulate in their hosts may lead to novel molecular resistance strategies. In this study, we used the Rep-inducible In Plant Activation (INPACT) expression platform, based on the genome of tobacco yellow dwarf virus (TYDV), to determine where this model mastrevirus replicates in its host tobacco. By developing an infectious clone of TYDV and optimizing its delivery by agroinfiltration, we first established an efficient artificial infection process. When delivered into transgenic tobacco plants containing a TYDV-based INPACT cassette encoding the β-glucuronidase (GUS) reporter, we showed the virus activates GUS expression. Histology revealed that reporter gene expression was limited to phloem-associated cell types suggesting TYDV replication has a restricted tissue tropism. Geminiviridae and Nanoviridae families) infect, replicate, and move throughout their hosts and the strategies they use to subvert cellular processes in their favor. Replication of the cssDNA genome is a key step in the proliferation of these viruses and occurs throughout the infection cycle, requires host cell factors, and is carried out exclusively in the nucleus [Understanding virus–host interactions and the processes that occur during pathogenesis is fundamental to the development of virus control measures and ultimately resistance. Over the past twenty years, significant advances have been made in determining how circular, single-stranded (css)DNA plant viruses  and French beans (Phaseolus vulgaris L.), respectively [Raphanus spp. and Amaranthus spp. [Orosius orientalis (formerly Orosius argentatus), which has been reported to feed on over 60 plant species and is widely distributed in Australia, Asia, and the Pacific region [Tobacco yellow dwarf virus (TYDV) is a dicot-infecting mastrevirus belonging to the family  in size . The vir in size ,21. A la in size . A smallectively ,22,23, hhus spp. ,25,26. Tc region .Nicotiana species. The INPACT expression platform is unique in that the gene of interest is split and only reconstituted and expressed in the presence of the cognate TYDV Rep/RepA proteins. In the above cases, Rep/RepA expression was placed under the control of the alcohol-inducible alcA:AlcR gene switch and the proteins provided in trans within the same transgenic plant. In most transgenic tobacco lines, expression of the gene of interest was negligible in the absence of Rep/RepA but could be rapidly activated in their presence. This led us to investigate whether the INPACT system could also be utilized as a means of precisely tracking TYDV replication during an infection. In this scenario, Rep/RepA derived from the infecting virus would be responsible for activating transgene expression from the INPACT platform. Here, we describe a strategy which uses the β-glucuronidase (GUS) enzyme as a visual reporter and show Rep/RepA expressed by TYDV during an infection can activate GUS expression from an integrated INPACT platform and that replication of this virus is highly restricted to phloem-associated cell types.While not a significant threat to global food and fiber production, its wide host range and simple genome structure has established TYDV as a useful model for geminivirus research, both as a tool to better understand viral pathogenesis and as a molecular vehicle for gene silencing  and manuN. tabacum) leaves infected with TYDV were obtained from Myrtleford, Victoria. Full-length TYDV genomic DNA was isolated from this material using the cetyltrimethylammonium bromide (CTAB) method [GCGGCCGCATTAAGGCTCAAGTACCGTACGATG-3′) and Not-R (5′- GCGGCCGCATGCCTTCAGCCCCCCAGAAAACCAA-3′) and an EcoRI fragment (at position +1194) using primer pairs Eco-F (5-GAATTCTTCCACTGGTGATGTTGCTG-3′) and Eco-R (5′- GAATTCTTCCACTCTGTGCTAACCCCTA-3′). Both genomic products were initially cloned into pGEM®-T.Easy  and Sanger sequenced . The first TYDV NotI genome fragment was ligated into NotI digested binary plasmid pART27. The second TYDV EcoRI genome fragment was then ligated into the unique EcoRI site located within the first complete viral genome. The resulting dimer infectious clone was called pART-TYDV-2mer (Tobacco  method  and PCR.) method . A greatYDV-2mer . AssemblAgrobacterium tumefaciens strain LBA4404 and pART-TYDV-2mer into A. tumefaciens strains GV3101, C58, and AGL1 by electroporation using the method of [N. tabacum (cv. Samsun) was transformed using the leaf disk method of [pINPACT-35S-GUS was mobilized into ethod of . N. tabaethod of . Tissue 2:MES:Acetosyringone (MMA) buffer alone.Tobacco leaves were agroinfiltrated essentially as described by . As a moLeaves were histochemically assayed for β-glucuronidase (GUS) expression according to . Leaves 2 sections and fixed in 4% formaldehyde solution for 48 h at room temperature and then cleared in 100% ethanol. Fixed leaf tissues were dehydrated through a graded ethanol series and embedded in paraffin wax. Transverse and longitudinal sections of leaves were cut using a Leica RM2245 rotary Microtome set to 5 µm. Samples were deparaffinized with xylene, and then gradually hydrated through a decreasing ethanol series  and then placed in distilled water. Leaf sections were then counter-stained in 0.5% safranin-O in 50% ethanol. After staining, slides were dehydrated using an increasing ethanol series  and then placed in xylene. Slides were mounted with dibutylphthalate polystyrene xylene (DPX) , scanned with a 3D Histech slide scanner and images viewed using CaseViewer software.GUS stained leaves were cut into 5–7 mmmp) gene. As a control, primer pair NtAct-F (5′-CTATTCTCCGCTTTGGACTTGGCA-3′) and NtAct-R (5′-AGGACCTCAGGACAACGGAAACG-3′) were used to amplify part of the tobacco actin housekeeping gene. PCR cycles were as follows: 95 °C for 30 s, followed by 30 cycles of 95 °C for 10 s, 55 °C for 30 s, 72 °C for 30 s, and a final extension of 72 °C for 2 min. PCR amplicons were electrophoresed through a 1.5% agarose gel.Total DNA was extracted from the upper leaf of tobacco plants using the CTAB method of . ApproxiAgrobacterium strains  harboring pART-TYDV-2mer were compared for their ability to initiate virus infection via agroinfiltration. To determine whether culture density and plant age influenced infection rates, agrobacterium cultures were infiltrated at two different optical densities and plant growth points. In the first experiment, five soil-acclimatized eight-week-old tobacco (N. tabacum cv. Samsun) plants were infiltrated with each recombinant agrobacteria strain  = 0.1). Plants were monitored for symptom development and TYDV infection rates assessed by PCR at four weeks post infiltration using primers designed to amplify a 300 bp fragment of the TYDV mp gene  of agrobacteria strains C58 and GV3101 harboring pART-TYDV-2mer. Plants were monitored for symptom development and TYDV infection tested by PCR, four weeks post infiltration. Infection rates of 80% and 60% were observed in the plants infiltrated with strains C58 and GV3101, respectively , and absence of “leaky” GUS expression, four lines  were selected for further analysis. The four lines were multiplied in tissue culture and acclimatized to soil for subsequent infection assays.Ten independent transgenic events were generated following tobacco leaf disk transformation with pINPACT-35S-GUS and grown on Murashige and Skoog media containing the antibiotic, hygromycin. Based on their growth rates, presence of the A. tumefaciens strain C58 (OD600 = 0.05) harboring the infectious clone pART-TYDV-2mer. As a control, a single biological replicate of each line was mock inoculated with MMA buffer only. In one case, an inoculated leaf was detached from a transgenic pINPACT-35S-GUS plant five days post agroinoculation and GUS stained for 48 h then cleared with 100% ethanol. Intense blue-stained cells were evident only in the leaf zone agroinoculated with the TYDV infectious clone . Upon Rep expression, but in the absence of virus infection, the cassette was released from the integrated T-DNA and amplified extra-chromosomally with resultant GUS expression only in those cells undergoing episomal replication. Transgenic tobacco plants containing the cassette displayed a distinctive speckled pattern of blue staining that was often associated with leaf veins, following the addition of the GUS substrate. Here, we used the Rep-inducible INPACT expression platform to definitively prove where TYDV replication occurs during an infection. The INPACT system differs from the deconstructed virus vector strategies of [TYDV has long been considered phloem-limited based on the cytopathic effects observed in the phloem tissue of infected plants . Later, egies of  and thosegies of ,17,18. IOrosius orientalis, however, rearing and maintaining these insects in a laboratory is both difficult and time-consuming. An alternative strategy to introduce a virus into a plant is by agroinfiltration, whereby Agrobacterium is used to deliver cloned viral DNA into the plant cell, resulting in the reconstitution of the complete virus genome and initiation of an infection. This approach has been successfully adopted for many geminiviruses; it is simple and cheap and can be used to inoculate large numbers of plants in a short period of time. However, agoinfiltration can be unpredictable and its effectiveness can vary between Agrobacterium strains, culture densities, plant species, type of delivery vector, age of plant, and transgene [Agrobacterium, plant age and culture density, we developed a relatively efficient artificial infection strategy for TYDV that resulted in an 80% infection rate using four-week-old soil-acclimatized tobacco plants infiltrated with Agrobacterium strain C58 at a culture density of OD600 = 0.05.TYDV is naturally transmitted from plant-to-plant by the leafhopper, ransgene ,41,42. BTransgenic tobacco plants containing the TYDV-based pINPACT-35S-GUS cassette were generated and infected with the virus by agroinfiltration. Histochemical GUS staining of infected leaves showed that TYDV-derived Rep/RepA was capable of activating the integrated pINPACT-53S-GUS cassette as blue-stained cells were clearly visible throughout the leaves of infected plants only. Interestingly, these cells were always located near the leaf veins and most abundant in the fully expanded mature leaves compared to the younger leaves. This may suggest virus replication or the Rep/RepA proteins are most abundant in the older tissue types, a finding similar to that of , and mayUnder high magnification, GUS-expressing cells were primarily phloem-associated and not in the xylem. Transverse sections showed these cells were located in vascular bundles and not in non-phloem domains such as mesophyll and epidermal cells. This would suggest TYDV is primarily phloem-restricted, much like abutilon mosaic virus, squash leaf curl virus, and tomato yellow leaf curl Sardinia virus ,12,16. HThe Rep-inducible nature of the INPACT expression platform makes it a particularly useful and versatile system for the study of cssDNA virus replication in plants. By simply replacing the origins of replication flanking the INPACT cassette, the platform could be adapted to any geminivirus or nanovirus. Further, with additional refinements, such as the use of a non-destructive reporter gene , the platform could potentially be used to track virus replication in real time throughout the plant, during the course of a natural infection. The utility of the INPACT system to be adapted into a virus-inducible resistance strategy, whereby the gene of interest encodes a toxic or anti-viral product, also remains to be further pursued from transient studies  to whole


When I worked at the US Centers for Disease Control and Prevention we called them “the diseases of the week,” diseases, newly recognized or reappearing, that suddenly appeared in the United States or elsewhere in the world. That happened more often than one would expect, but should not have surprised us. Diseases of all sorts have been recognized for thousands of years and, recently it seems, more often. Why? Do the media make more of an outbreak than the outbreak deserves? Is the particular situation  of sufficient fascination that everyone wants to know “What is this thing?” or is someone, or more than someone, benefitting  from the media frenzy or want to become famous? When someone dies in a place far from your home and that death does not appear to threaten you or your family, do we think to ourselves that it is not our problem? Given the current exceedingly large human population, the commonality of air travel, and the frequent eruption of wars, civil and otherwise, and the attendant movement of refugees, it is a problem, or a potential problem for people everywhere, as the discovery of this new coronavirus disease has demonstrated for all to recognize.https://www.who.int/csr/don/05-january-2020-pneumonia-of-unkown-cause-china/en/) and now is pandemic. Using increasingly informed models , a global effort now is under way to contain its spread. COVID-19 is caused by the coronavirus known as SARS-CoV-2 . Its containment has not been completely successful anywhere and only partially and belatedly successful even in China, which used heroic and authoritarian means in its efforts to control the virus. The virus appears to have been well controlled but not eradicated from Hubei Province where it was discovered, and perhaps where it originated in bats (The coronaviruses (named for the “crown” [corona] formed by their surface protein spikes when viewed by electron microscopy) comprise a large family of viruses, most of which usually cause mild to moderate upper-respiratory tract illnesses, such as the common cold, in people. There are hundreds of known coronaviruses; most circulate among pigs, camels, bats, and cats. Sometimes those viruses jump to humans (a “spillover” event) and cause disease in them. Seven coronaviruses currently are known to cause human disease. Infections with four of these are mild but three coronaviruses can cause more serious illnesses in humans: severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), which was first recognized in 2002 in China and disappeared two years later; Middle East respiratory syndrome (MERS) coronavirus, which was first recognized in 2012 and remains in circulation in camels, mostly in Saudi Arabia; and this recent nightmare virus, causing what has been named by the World Health Organization COVID-19 (disease name), which emerged in late 2019 in China  and to deliver a packet of salary checks, which had been sent in error to our laboratory in Colorado. When I arrived in Bethesda late in the day, I felt ill and decided to just go to bed in my motel, rather than to dinner. The next morning, I was awakened by someone banging on the door of my room. I opened the door to see two US Government enforcement officers demanding that I turn over the salary packet to them. After they showed me their identifications, I gave them the checks and went back to bed, realizing that I had a severe headache, a fever, and myalgias. I called my sponsor at the NIH, told him what was happening, and asked to be excused from the meeting. He told me that I should report to the Clinical Center, a research hospital on the NIH campus. They took samples from me and insisted that I stay overnight, after which I returned home, still slightly ill. A few days later, a telephone caller from NIH informed me that they had isolated and identified a coronavirus from my throat swab sample. They did not identify the specific virus because of the characterization of my illness and because coronaviruses were and are so common.Other experiences I had were more enjoyable and educational. When we realized that bats had been studied regarding rabies virus, but not much else regarding other viruses, I helped author a paper that reviewed the few known occurrences of viruses of bats , an extrhttps://www.who.int/csr/don/05-january-2020-pneumonia-of-unkown-cause-china/en/). Of course, the disease was not spreading, it was the virus that causes the disease that was spreading – and frighteningly fast, and far. It was obvious then that this might become a global problem, and it has become just that, a pandemic. The government of China and, in particular, the Chinese investigators studying this problem, deserve recognition and praise for the courageous work they did in the early days of this extraordinary event.In late 2019 the Chinese government notified the World Health Organization that it had detected a hitherto unrecognized coronavirus (now named SARS-CoV-2) in people with respiratory distress; some had died and the disease was reported to be spreading . Critically, people in all age groups may become infected with the virus but some of them may shed virus yet do not become ill. It is important to recognize that all people not infected with SARS-CoV-2 themselves comprise a susceptible population, a harbinger of the next round of illnesses caused by this virus. This is a frightening potential situation and one suggesting that total elimination of SARS-CoV-2 is unlikely. Thus, what will occur when the personal controls we humans have self-applied are considered no longer necessary? Will the populace simply be relieved and wander out of their houses to resume life as before? It is likely that life as we knew it will not be as it was before this disease was recognized. There will be no miracle that occurs to make all surfaces safe on “Day One.”In summary, SARS-CoV-2 has led to a disastrous situation and one with which the medical community of each nation must manage in some way, perhaps through World Health Organization protocols, perhaps by a novel approach. Because humans infected with SARS-CoV-2 may shed virus before they become ill, or even if they never become ill, they may serve as reservoirs of the virus. This is a principal reason for patients and non-patients alike not to contact anyone other than a house-mate  for two weeks or longer. This is called “social distancing” but it is actually “physical distancing” because computers, telephones, the mail system, and many other tools allow us to socialize at a distance. Rumors in the popular press have not helped the public understand the situation, and statements by politicians, as always, bend the truth either intentionally or because they do not understand what the truth is.The spike proteins of coronaviruses mediate viral entry into cells by binding to a receptor on the host cell surface and then fusing viral and host membranes . HoweverThis phenomenon has been shown to occur in infections with feline coronavirus, SARS-CoV, MERS coronavirus, influenzaviruses, Zika virus, human immunodeficiency virus, and dengue viruses, and probably occurs with other viral infections, or after certain vaccinations. More relevant to the current pandemic, it may be that older patients produce high affinity IgG antibody and younger patients do not, thus older patients might very well not respond as desired to a vaccine, treatment with monoclonal antibody, or immune serum transfer. As older patients are the ones at higher risk of deadly outcome, vaccination might not be suitable in this instance. I hope I am wrong about this.So, was the SARS experience just “the disease of the week” or month or year? It was discovered in 2003 and disappeared after 2004, after it had spread from Asia to North America, South America, and Europe. As is typical, funding to support research on this disease declined precipitously. That is also typically unfortunate, as it is only research that can maintain and defend preparedness. Huge amounts of money have been spent to gather information we hoped would lead to the prediction of outbreaks. Obviously, we have accomplished little in this regard, a lesson in itself. But have we learned nothing? Perhaps each country, wealthy or poor, should consider putting some proportion of its income into a fund put aside as a contingency in the event of an (inevitable) emergency. That such a fund might have to come from additional taxes is no reason to ignore this possibility; pay it now or pay it later – or pay the consequences. Governments that do not protect their citizens from diseases are simply criminal enterprises.As humans continue to plunder the natural world by using such extractive sources, such as coal, oil, and other forms of non-renewable energy, we scavenge in unexplored areas containing wildlife that likely are infected with presently unrecognized viruses that are potential pathogens for humans and/or livestock and/or other wildlife. Recent cross-species transmission events, particularly those jumping from wildlife infected with genetically malleable RNA viruses have been shown to be the primary source of recently recognized human pathogens .How many more potential human pathogens are there still, and thankfully, unrecognized? What do we do with the possibly predictive information we gather when a previously unknown virus is recognized? Do we continue to repeat over and over again our discoveries and our errors? Are we, as a species, committing suicide?Note: At the time this opinionated column was written, at least 1 000 000 people have been infected with SARS-CoV-2 and nearly 50 000 have died. China, Italy, Spain, and the USA have suffered most. The end is not in sight and the devastation this virus has caused will undoubtedly lead not only to human physical misery and great sadness but to economic turmoil.