We report a case of monkeypox in a man who returned from Nigeria to Israel in 2018. Virus was detected in pustule swabs by transmission electron microscopy and PCR and confirmed by immunofluorescence assay, tissue culture, and ELISA. The West Africa monkeypox outbreak calls for increased awareness by public health authorities worldwide. Monkeypox is a zoonotic disease caused by monkeypox virus, an orthopoxvirus closely related to variola virus, the causative agent of smallpox. Human cases were first described in 1970; in subsequent decades, sporadic outbreaks were reported in Africa. Mortality rates are 1%–10%  and chills and started self-medicating with nonsteroidal antiinflammatories and oral penicillin. On October 1, an erythematous rash appeared first on his face and later on his trunk and extremities. Examination at Shaare-Zedek Medical Center on October 4 revealed that the patient was febrile and had a nonblanching maculopapular rash on his face , panel ARickettsia conori, Rickettsia typhi, Brucella spp., Treponema pallidum, and antigen/antibody combination for HIV. Pustular samples were negative for herpes simplex virus by PCR. Because of the rash characteristics and the patient’s travel history, monkeypox was suspected.At a follow-up visit 2 days later (October 7), he was afebrile. The rash was locally synchronous and had progressed from maculopapular to vesicular and pustular; some lesions displayed black umbilication and crusting (https://www.beckman.com) before negative staining with phosphotungstic acid. Samples were sent to the Israel Institute for Biological Research, Ness-Ziona, Israel, and processed in Biosafety Level 3 laboratories. The pustule sample was processed for PCR analysis and transmission electron microscopy. Vero cells were infected for immunofluorescence assay and monitored for cytopathic effect. For transmission electron microscopy, particles were enriched by using a Beckman Airfuge  embedded in skin tissue and as single virions (PCR diagnosis was based on specific primers to discriminate between the West African (581 bp) and the Congo-Basin (832 bp) clades by product size  and were shed (All of the patient’s contacts in Israel (5 household members and 11 HCWs) were offered smallpox vaccination, but only 1 HCW agreed. All contacts were followed up for 21 days; no virus transmission was detected.Since the first documented case of human monkeypox in 1970, sporadic outbreaks have been reported, especially in the Congo Basin and West Africa. Contributing to the increased frequency of such occurrences were discontinued vaccination against smallpox, increased interaction with wildlife because of deforestation and population movement, consumption of bushmeat, and increased population density  have involved the West African clade of the virus (For this study, we used multiple diagnostic approaches. The virus was detected in pustule swab specimens by transmission electron microscopy and PCR within 3 hours of sample arrival and confirmed by immunofluorescence assay, tissue culture, and ELISA for orthopoxvirus antigens.The very high virus titers contained by pustules and scabs, as demonstrated in this case, increase the risk for human-to-human transmission and environmental spread. To prevent further transmission, HCWs should implement safety practices and local authorities should map contacts and consider use of smallpox vaccines or antiviral drugs (


Hylocereus) fruit, followed by fructose and sucrose. Expression levels of two sucrose-hydrolyzing enzyme genes HpINV2 and HpSuSy1 obviously increased during fruit maturation, which were correlated well with the elevated accumulation of glucose and fructose. A WRKY transcription factor HpWRKY3 was further identified as the putative binding protein of the HpINV2 and HpSuSy1 promoters by yeast one-hybrid and gel mobility shift assays. HpWRKY3 was localized exclusively in the nucleus and possessed trans-activation ability. HpWRKY3 exhibited the similar expression pattern with HpINV2 and HpSuSy1. Finally, transient expression assays in tobacco leaves showed that HpWRKY3 activated the expressions of HpINV2 and HpSuSy1. Taken together, we propose that HpWRKY3 is associated with pitaya fruit sugar accumulation by activating the transcriptions of sucrose metabolic genes. Our findings thus shed light on the transcriptional mechanism that regulates the sugar accumulation during pitaya fruit quality formation.Sugar level is an important determinant of fruit taste and consumer preferences. However, upstream regulators that control sugar accumulation during fruit maturation are poorly understood. In the present work, we found that glucose is the main sugar in mature pitaya ( Hylocereus) fruit, also called dragon fruit, is generating considerable consumer attention in many tropical and subtropical countries with exotic appearance, striking colors, high nutrients, and a delicious taste  motif in the promoter sequences of their downstream targets ,34. An iromoters  were bioromoters , causingromoters B. Additiromoters B. The repI value of 6.71 (2H2 (C–X4–5–C–X22–23–H–X1–H) and C2HC (C–X5–7–C–X23–H–X1–C), are usually observed at the C-terminal of WRKY proteins [2H2 zinc-finger motif. The phylogenetic tree also showed that HpWRKY3 together with AtWRKY3 and AtWRKY33, belong to group I (HpWRKY3 has an open reading frame (ORF) of 1515 bp length, encoding a protein with 505 amino acid residues, calculated molecular weight of 54.89 kDa, and  of 6.71 . The mosproteins . WRKY prproteins ,36. As sproteins ,36, HpWR group I B. HpWRK3 during pitaya fruit maturation was analyzed by qRT-PCR. As shown in HpWRKY3 was steadily increased from 16 to 30 DAAP, reaching the peak at 30 DAAP, following the initiation of glucose and fructose accumulations. WRKY TFs are known to be localized to nuclei [HpWRKY3 was fused to GFP and transiently expressed in tobacco leaves. Microscopic observation of tobacco leaf epidermal cells revealed that GFP alone was expressed in the cytoplasm as well as the nucleus, whereas the signal for HpWRKY3-GFP was exclusively detected in the nucleus /renilla luciferase (REN) ratio  ratio D. Based HpINV2 and HpSuSy1 as the potential downstream targets of HpWRKY3, and establishing HpWRKY3 as a transcriptional activator, we were curious whether HpWRKY3 could directly activate the transcriptions of HpINV2 and HpSuSy1. To address this particular question, we performed the transient dual-luciferase reporter assay in tobacco leaves. For this assay, HpINV2 and HpSuSy1 promoters were fused upstream of the LUC gene to serve as reporters, along with the REN gene driven by CaMV35S serving as an internal control. As presented in HpINV2 and HpSuSy1 promoters significantly elevated LUC/REN ratios, suggesting that HpWRKY3 indeed enhances the transcriptions of HpINV2 and HpSuSy1 in vivo. After figuring out SUT2 and SUT4, and tonoplastic monosaccharide transporter TMT2 are found to be positively correlated with fruit sucrose accumulation [MdTMT1, MdSUT2, and MdTMTs have been shown to be involved in the accumulation of fruit soluble sugars [CitSUT1-overexpressed transgenic citrus lines [PR genes in the disease response [FaSPS3 expression during sucrose accumulation [Besides INVs and SuSys, other sucrose metabolic enzymes including hexokinase, fructokinase, sucrose phosphate synthase, and UDP-glucose pyrophosphorylase, as well as sugar transporters, are also important factors determining fruit yield and quality by stimulating sugar accumulation. For example, transcripts of peach sucrose transporter mulation . Inducede sugars ,10. A nous lines . Intriguus lines ,39. Thusus lines . For exaresponse , while tresponse . A recenmulation . TherefoHylocereus polyrhizus cv. Guanhuahong) with red peel and pulp was selected for this work. Plants were grown in Jinsuinong commercial orchard  received standard horticultural practices, and disease and insect control. Based on the change of pulp color, fruits were harvested at six important stages ) from October to December 2017. At each sample point, fruits were cut into two portions for photograph. Pulps from three fruits from three different plants were sliced and ground into powder in liquid nitrogen. Thereafter, all samples were stored at −80 °C until future physiological and molecular assays. A pitaya cultivar  at 45  °C, and the residue was dissolved in 0.5 mL distilled water and filtered with water syringe filter. The filtered solution of 20 μL was used for sugars analysis by the high-performance liquid chromatography (HPLC) , and quantity of individual sugars was calculated using peak areas of standards, as described previously [The extraction of sugars  was performed as previously described . One graeviously .HpWRKY3 gene was isolated from our transcriptome database reported previously [pI) and mass values were assessed on the website (http://web.expasy.org/compute_pi/). WRKYs were aligned by CLUSTALW (version 1.83) and viewed using GeneDoc software. The neighbor-joining phylogenetic tree of WRKYs was obtained using MEGA program (version 5.0). Total mRNA was extracted from pitaya pulp using Quick RNA Isolation Kit . cDNAs were synthesized from 1 μg of total RNA using the reverse transcriptase M-MLV  according to the supplier’s instructions. The full-length of eviously . TheoretActin [cDNAs from each sample were subjected to quantitative real-time PCR (qRT-PCR) assays. qRT-PCR was performed using the step one plus a CFX96 Touch™ real-time PCR detection system  and GoTaq qPCR master mix kit . The expression levels of target genes were normalized by using housekeeping gene Actin . HpINV2 and HpSuSy1 were isolated from pitaya genomic DNA by nested PCR. Cis-elements present in the promoters were analyzed through Plant-CARE online platform (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Gene promoters of HpINV2 and HpSuSy1 was inserted into the reporter plasmid pAbAi and transformed into yeast strain Y1H Gold. To construct the cassette expressing the effector, the coding sequence of HpWRKY3 was cloned into pGADT7. The resulting vector was then introduced into reporter strains. The transformed reporter strains were grown on SD/−Leu media containing 500 ng/mL Aureobasidin A (AbA) for 3 days at 28 °C to test the possible interaction. Yeast one-hybrid assay was performed by using Matchmaker Gold Yeast One-Hybrid System , as described previously . About 6E. coli strain Transetta (DE3) was transformed with GST-HpWRKY3-C, and 5 mL of overnight-grown transformed cells were used to inoculate 500 mL of LB medium, which was incubated with shaking at 37 °C. Reaching an OD600 nm of 0.4–0.6, 0.3 mM isopropyl-β-D -thiogalactopyranoside (IPTG) was added to the cells to induce the expression of the HpWRKY3 protein for 3 h at 37 °C. After the incubation period, the cells were centrifuged and disrupted by sonication. Equilibrating with lysis buffer, the induced proteins were then affinity-purified using a Glutathione-Superflow Resin (Clontech) according to the manufacturer’s protocols, following SDS-PAGE and Coomassie Brilliant Blue staining to confirm protein size and purity.The HpINV2 and HpSuSy1 oligonucleotides were biotin-labeled at the 5′ end. The purified recombinant HpWRKY3 was incubated with biotin-labeled probes in binding buffer for 25 min at 30 °C. Competitions were carried out by adding cold probes with unlabeled DNA fragments with the same or mutant sequences. The reaction mixtures were separated on 6% native polyacrylamide gels. After electrophoresis, the DNA-protein complexes were transferred onto a nylon membrane. The signals from the labeled DNA were detected by using the LightShift chemiluminescent EMSA kit  in a ChemiDoc™ MP Imaging System (Bio-Rad).Electrophoretic mobility shift assay (EMSA) was performed as described previously ,46. The Agrobacterium tumefaciens strain EHA105 strains harboring HpWRKY3-GFP constructs were infiltrated into tobacco (Nicotiana benthamiana) leaves by using a 1-mL disposable syringe as described previously [To investigate the subcellular localization of HpWRKY3, its coding sequence was cloned into the binary vector pEAQ-GFP. eviously . After 4HpWRKY3 was cloned into the vector pGBKT7. The resulting constructs were then transformed into the yeast strain Y2HGold. The MATCHMAKER GAL4-based Two-Hybrid system 3 (Clontech) was used for the transactivation activity assay according to the manufacturer’s protocol. Each yeast liquid culture was diluted serially to OD600 = 0.6, and 5 μL of each dilution was inoculated onto various synthetic dropout (SD) medium, and the trans-activation activity was indicated by the yeast growth and α-galactosidase activity, as described previously [The coding sequence of eviously .firefly luciferase (LUC) gene was used as a reporter, and the renilla luciferase (REN) gene in the same plasmid was used as an internal control. To determine the transcriptional regulation activity of HpWRKY3 in plant cells, the CDS of HpWRKY3 was inserted into pBD to construct pBD-HpWRKY3 as effector. The positive control (pBD-VP16) was constructed by fusing VP16, a herpes simplex virus-encoded transcriptional activator, to pBD. pBD itself was used as a negative control. To determine how HpWRKY3 activate HpINV2 and HpSuSy1 expression, the CDS of HpWRKY3 was introduced into pEAQ vector to generate the effector. HpSuSy1 and HpINV2 promoters were inserted into the dual luciferase vector pGreenII 0800 as reporters. The reporter and effector constructs were co-infiltrated into tobacco leaves mentioned above. After 48 h of infiltration, luciferase assays were performed using dual-luciferase reporter assay kit (Promega) and quantified by a Luminoskan Ascent Microplate Luminometer . The trans-activation ability of HpWRKY3 was defined by the ratio of LUC/REN. Assays were carried out as described previously . The GALt-test was applying for statistical analysis . Correlation analysis between transcripts of sucrose metabolic genes and amounts of soluble sugars was performed by SPSS Statistics 22.0. Linear regressive analysis and scatter plots were prepared with Origin8.0.All assays in this study were performed as at least three independent biological replicates. Values are means ± SE of three or six biological repeats. The Student’s All primer sequences used in this study are listed in HpINV2 and HpSuSy1 are correlated well with the elevated accumulation of glucose and fructose during pitaya fruit maturation. One nuclear-localized transcriptional activator HpWRKY3 is identified. More importantly, HpWRKY3 directly bind to HpINV2 and HpSuSy1 promoters, subsequently activating their transcriptions. Our findings shed novel light on the transcriptional regulatory mechanisms underlying sugar accumulation during fruit maturation, thereby laying the foundation for exploring new effective techniques to improve fruit quality.We find that expressions of two sucrose metabolic genes


Due to the intense selection pressure against inbreeding, humans are expected to possess psychological adaptations that regulate mate choice and avoid inbreeding. From a gene’s-eye perspective, there is little difference in the evolutionary costs between situations where an individual him/herself is participating in inbreeding and inbreeding among other close relatives. The difference is merely quantitative, as fitness can be compromised via both routes. The question is whether humans are sensitive to the direct as well as indirect costs of inbreeding. Using responses from a large population-based sample , we found that human motivations to avoid inbreeding closely track the theoretical costs of inbreeding as predicted by inclusive fitness theory. Participants were asked to select in a forced choice paradigm, which of two acts of inbreeding with actual family members they would want to avoid most. We found that the estimated fitness costs explained 83.6% of participant choices. Importantly, fitness costs explained choices also when the self was not involved. We conclude that humans intuit the indirect fitness costs of mating decisions made by close family members and that psychological inbreeding avoidance mechanisms extend beyond self-regulation. From a gene’s-eye perspective, there is little evolutionary difference between the costs of selecting fitness-jeopardizing sexual partners oneself and the costs when other family members do the same . The likEach instance of inbreeding carries multiple types of fitness consequences: There are (i) direct and (ii) indirect fitness consequences to each individual in the inbreeding pair. In addition, there are (iii) indirect fitness consequences to third parties related to the individuals in the inbreeding pair . Take, fExtending outward from an inbreeding pair  is a web of related individuals – mother, father, other siblings etc.–each with a unique perspective on the magnitude of costs associated with the siblings producing an inbred child. For this reason, adapted mechanisms that reliably estimate the costs of inbreeding, not only for the purpose of regulating one’s own mating behavior, but also for regulating the mating behavior of close genetic relatives are hypothesized to exist. This generates the testable prediction that for a given event of inbreeding, the possible costs to a focal person’s fitness should map on to the intensity of opposition to the event of inbreeding. That is, by hypothesis, proximate aversions should track estimated ultimate costs. Moreover, this should hold both when individuals assess the costs of inbreeding themselves and the costs of inbreeding between two close relatives.The fitness costs of a given event of inbreeding for a focal individual  can be estimated as:xr and yr signify the relatedness (r) between the focal person and individual X and individual Y, engaging in inbreeding, and δ is the fitness compromise for a given inbred offspring as compared to a non-inbred offspring. The magnitude of δ is determined by the relatedness between the two persons, X and Y. If, for example, a focal person him/herself engages in inbreeding with a full sibling, the focal person is X and the full sibling is Y. Because the focal person is 100% related to him/herself, xr is 1; yr is 0.5 for the full sibling. If δxy = 0.3 in offspring from inbreeding full siblings, where xy = 0.5, this means that the total cost of inbreeding to the focal person is 0.45, that is, 0.3(1 + 0.5) in this case. If, instead, a sibling of the focal person engages in inbreeding with their common sibling, the relatedness between the focal person and both individuals in the inbreeding union is 0.5. In this case, both xr and yr are 0.5. Also in this case, the fitness decrease in their offspring is 0.3, but the cost to the focal person is 0.3, that is, 0.3(0.5 + 0.5). As the relatedness between persons X and Y decreases, the fitness compromise in the inbred offspring also decreases. For example, in the case of inbreeding between cousins δxy = 0.075.where r, is purely theoretical. Rather than following the theoretical r, natural selection will in this case operate through the perceived degree of relatedness, that is, an internally estimated, psychological variable. Indeed, prior work on kin detection in humans suggests that humans do not possess exact representations of the true degree of relatedness between two individuals  factors, which weight the r by subjective certainty of relatedness to X and Y, respectively:In general, expression (1) is modeled to be an approximation of the costs associated with a given event of inbreeding. However, the exact relatedness term, ividuals . Humans xy(Cxx + Cyryr) (2)δxr = 0.5 and yr = 0.25, respectively) can be weighted downward according to perceived (un)certainty in relatedness. For example, if the certainty regarding the full-sibling was 75% and the half-sibling was 95%, the perceived relatedness values would be 0.375 and 0.2375, respectively. In this way, we can more accurately model how the mind might perceive the costs of inbreeding and determine whether these costs track hypothetical decisions regarding sex with family members.While the relatedness term to self is unaffected by this, the relatedness terms to, for example, a full and a half-sibling (1.We designed a study to investigate whether human motivations to avoid inbreeding reflect the following predictions derived from inclusive fitness theory: (i) the strength of opposition to an event of inbreeding is tightly knit to the inclusive fitness cost of inbreeding; (ii) inclusive fitness costs apply similarly to decisions regarding both 1st and 3rd party inbreeding; and (iii) that the perception of these costs should be influenced by subjectively perceived certainty in relatednessMage = 33.9, SD = 9.2, range 18–57) living in Finland completed an on-line study , subsequent questions concerned only one of these. Participants were at a later stage asked to make pairwise forced choices between different inbreeding scenarios selecting the alternative they wanted to avoid more. For each participant, the number of forced choices was limited to include only scenarios involving the participant’s own actual relatives. Only relatives older than 13 years of age were included in the scenarios. We used the names of their actual relatives in the scenarios presented.  and Y (yr) in a scenario, and the degree of relatedness between person X and Y (xyr). We then combined these components into the following expression: δxy(x + ryr). Independently of whether the participant him/herself was or was not involved in the scenario, this expression weights δ by the degree of relatedness between the two individuals engaging in inbreeding. This then reflects the decreased biological fitness in an inbred child. The decreased biological fitness of this child is then weighted by its relatedness to the respondent via both of the two persons engaging in the inbreeding . We used a value 0.6 for δ, meaning that the average fitness compromise in offspring to inbreeding siblings or parents and their children  would be 30%  and ranged from 0 to 100.Perceived certainty in relatedness was measured by asking the participants to report, on a scale from 0 to 100 , how certain they were in the biological relationship to each of their actual relatives. Participants were not asked to report this for themselves, neither were mothers asked to report this with respect to biological children. In both cases, full certainty was assumed and values of 100 were imputed. The mean kinship certainty was 78.2  could then be compared to the estimated fitness costs. This was done using a zero-order correlation (CORREL function in Excel) analysis between the aggregated choice probabilities and the fitness costs.For analyses, we used Excel for Mac 16.11.1 and xr and yr) in both of the two scenarios, and the relatedness between the two individuals included in the two scenarios . From these variables, the relative difference between the fitness compromises of the two scenarios in each trial was calculated as (δxy(x + ryr))1 – (δxy(x + ryr))2. To analyze whether this relative difference between fitness compromises explained the outcome of each choice, we used a multi-level binary logistic regression. To consider the clustering of choices within respondents, participant was added as a random intercept. This was done using the package lme4 .r = 0.883, p < 0.001). Although women have a stronger inbreeding aversion, the relative difference between different types of inbreeding scenarios should be similar for men and women. Indeed, the correlations were similar for male  and female respondents   that included two 1st person scenarios . The second group consisted of all trials  that included two 3rd person scenarios . The third and final group consisted of all trials  that included one 1st person and one 3rd person scenario . We added the term Type as a predictor alongside the term for the difference in fitness compromise. The association between difference in fitness compromise and choice remained statistically significant, z = -72.95, p < 0.001. With 1st vs. 1st trials as the reference category, the difference was statistically significant for 1st vs. 3rd trials, z = -12.33, p < 0.001, but not for 3rd vs. 3rd scenarios, z = 1.10, p = 0.271. The percentage of correctly classified choices in these three groups was 85.2% (1st vs. 1st), 84.2% (3rd vs. 3rd), and 82.6% (1st vs. 3rd).To further test whether the choices followed a gene’s-eye perspective rather than being biased toward avoiding costs to self, we then grouped the trials in three separate groups. The first group consisted of all trials  that may affect these decisions were not considered, our theoretically derived predictions explained a large proportion of the forced-choice decisions respondents made with regards to which of two inbreeding situations to avoid. Critically, we demonstrate that there is little effect of whether the respondent him/herself was involved in the inbreeding scenario or not. Classification accuracy was high for all possible combinations of 1st vs. 3rd perspective inbreeding scenarios.Although the current study does not provide insight into the actual mechanisms that regulate opposition to inbreeding , there is a vast literature suggesting that mating between close kin  is rare in most cultures , and thaPerceived relatedness is also likely to be a function of both the type of biological relationship and the degree of relatedness. For example, whereas a woman can be almost certain a child born by her is a biological child, a man can almost never be fully certain he is the biological father . Also, yHumans tend to react with disgust when asked to contemplate sex with close kin , and thein vivo.Another limitation of the current study is that the method measures mate choices outside of its natural context. The findings should therefore not be taken as evidence of one type of inbreeding actually being more likely than another, although studies suggest this to be real possibility . Actual We conclude that humans perceive the indirect fitness costs of mating decisions made by close family members and that the strength of opposition against inbreeding are explainable in terms of inclusive fitness costs.JA, PS, CH, and DL conceptualized and planned the study. JA, CH, and AA collected the data. JA, PS, CH, and AM analyzed the data. JA, PS, and DL wrote the manuscript. All authors provided their comments on the manuscript. JA and CH prepared figures and tables.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.


Latent herpes simplex virus (HSV) DNA has been detected in the central nervous systems (CNS) of humans postmortem, and infection with HSV has been correlated with the development of neurodegenerative diseases. However, whether HSV can directly reactivate in the CNS and/or infectious virus can be transported to the CNS following reactivation in peripheral ganglia has been unclear. In this study, infectious virus was recovered from both the trigeminal ganglia and the brain stem of latently infected mice following a reactivation stimulus, but a higher frequency of reactivation and increased titers of infectious virus were recovered from the trigeminal ganglia. Viral proteins were detected in neurons of the trigeminal ganglia, but a cellular source of infectious virus could not be identified in the brain stem. These results suggest that infectious virus is transported from the ganglia to the CNS following reactivation but do not exclude the potential for direct reactivation in the CNS. in vivo viral reactivation in the peripheral nervous system, triggered by hyperthermic stress, has been well characterized with respect to frequency and cell type. However, characterization of in vivo reactivation in the CNS is extremely limited. Further, it remains unclear whether virus reactivated in the peripheral nervous system is transported to the CNS in an infectious form, how often this occurs, and what parameters underlie the efficiency and outcomes of this process. In this study, reactivation was quantified in the trigeminal ganglia (TG) and the brain stem from the same latently infected animal using direct assays of equivalent sensitivity. Reactivation was detected more frequently in the TG than in the brain stem and, in all but one case, the amount of virus recovered was greater in the TG than that detected in the brain stem. Viral protein positive neurons were observed in the TG, but a cellular source for reactivation in the brain stem was not identified, despite serially sectioning and examining the entire tissue (0/6 brain stems). These findings suggest that infectious virus detected in the brain stem is primarily the result of transport of reactivated virus from the TG into the brain stem.Herpes simplex virus (HSV) establishes latency in neurons of the peripheral and central nervous systems (CNS). Evidence is mounting that HSV latency and reactivation in the nervous system has the potential to promote neurodegenerative processes. Understanding how this occurs is an important human health goal. In the mouse model, IMPORTANCE Latent herpes simplex virus (HSV) DNA has been detected in the central nervous systems (CNS) of humans postmortem, and infection with HSV has been correlated with the development of neurodegenerative diseases. However, whether HSV can directly reactivate in the CNS and/or infectious virus can be transported to the CNS following reactivation in peripheral ganglia has been unclear. In this study, infectious virus was recovered from both the trigeminal ganglia and the brain stem of latently infected mice following a reactivation stimulus, but a higher frequency of reactivation and increased titers of infectious virus were recovered from the trigeminal ganglia. Viral proteins were detected in neurons of the trigeminal ganglia, but a cellular source of infectious virus could not be identified in the brain stem. These results suggest that infectious virus is transported from the ganglia to the CNS following reactivation but do not exclude the potential for direct reactivation in the CNS. Herpes simplex virus (HSV) infection occurs at the body surface and results in establishment of a latent viral reservoir in neurons of both the peripheral and central nervous systems . The abiDetection of HSV DNA in human CNS tissue postmortem has been variable, ranging from 2 to 100% of samples tested –10. This–in vivo. Previous studies have rarely recovered infectious virus in CNS tissues by traditional explant assays for reactivation competency .C57BL/6 female mice were infected with HSV-1 strain McKrae, as described in Materials and Methods, and viral replication efficiency and the amount of latency established in the TG and brain stem were determined from a minimum of three mice at each time point. Infectious virus titers peaked in the TG on day 4 postinfection (p.i.), achieving titers of ∼10∼104 PFU . Titers ∼104 PFU . At >45 ∼104 PFU . There wIn order to directly compare the frequency of reactivation and the amount of infectious virus produced in the brain stem and TG post-hyperthermic stress (phs), a quantitative assay with equivalent sensitivity to that utilized to quantify virus reactivation in the TG was required to quantify virus reactivation in the brain stem. We anticipated that as in the TG, low levels of virus would be generated from a very limited number of cells in the brain stem. Alternatively, reactivated virus transported from TG into the brain stem would be anticipated to be at or below the levels detected in the TG. One obvious difference between TG and brain stem is the size of the tissue, the weight of the brain stem is ∼6 times that of a TG pair, 0.081 ± 0.021 g versus 0.015 ± 0.005 g, respectively. This difference in tissue size would be expected to dramatically limit recovery of infectious virus and also detection of any rare viral protein positive cells in the brain stem.in vivo .To test the effect of tissue weight on virus recovery, brain stems were harvested from uninfected mice and divided into halves, fourths, or maintained as whole pieces. Each sample size was weighed and samples were spiked with approximately 150, 75, or 20 PFU, representative of the low levels of virus typically associated with a reactivation event in vivo . Spiked in vivo reactivation in the TG. To determine the relative percent recovery of infectious virus in the brain stem compared to the TG when tissue weights were similar, brain stems were harvested from 13 mice and sagitally sectioned into six pieces .These data revealed that tissue weight could influence infectious virus recovery in the range of viral titers observed during To test the possibility that the latently infected and/or post-hyperthermic stress tissue environment would alter recovery, brain stems from five latently infected mice, pre- and post-hyperthermic stress, were spiked with approximately 20 PFU (confirmed titer 21 ± 10 PFU/50 μL) of 17VP16pLZ . Recovern = 5, n = 6), TG and brain stems were harvested at 20 to 24 h phs and directly processed for the detection of infectious virus. Brain stems were divided into six pieces, and each piece was individually homogenized, as described above and in Materials and Methods. Infectious virus was detected in 82% (9/11) of the TG and 45% (5/11) of the brain stems at 20 to 24 h phs  . In two independent experiments ( t test) . Infecti t test) . InfectiIt has previously been reported that recovery of infectious virus in brain stem tissue can be enhanced by culturing homogenized samples in a 3-ml volume for 3 days, harvesting wells, and replating the samples on new monolayers for an additional 3 days . Overlayr = 0.9631; Pearson’s correlation, P < 0.0001). This raised the possibility that the second step of the assay was a result of cross-contamination and not a delayed ability to detect virus generated during the in vivo reactivation.Five C57BL/6 female mice latently infected with HSV McKrae were induced to reactivate using hyperthermic stress . TG and To test the potential for contamination using the two-step assay, virus expressing fluorescent marker during acute infection was utilized. This allowed us to visualize and count plaque development and spread while the assay was ongoing. Two six-well plates were spiked with ∼9 PFU of OK12 fluorescent virus ; one plaDue to the risk for contamination when culturing high titers of PFU in a large volume near negative wells, samples with evidence of CPE during the first culture step were not replated. Across three additional reactivation experiments, 140 samples that appeared reactivation negative during the first culture step were replated. Importantly, infectious virus was not detected in any of these samples (0/140). This demonstrates that if infectious virus is present in the brain stem, it can be detected by 3 days postplating (similarly to the TG), and extensive culturing time does not result in additional reactivation positive samples.Infectious virus in the brain stem could be the result of direct reactivation from latently infected neurons in the brain stem, could result from transport of infectious virus from the TG to the brain stem, or both. The detection of rare reactivating neurons in the TG has been greatly enhanced by a method for immunohistochemical detection of viral proteins in the whole ganglia . HoweverThe presence of even low levels of infectious HSV in the CNS has been proposed to have significant implications for human health  at 24 h phs if reactivation was occurring directly in the CNS. The entire brain stems from six mice latently infected with McKrae and harvested at 20 to 24 h phs were serially sectioned and processed for HSV protein expression. However, in these 160 sections, which included the full 3.2-mm thickness of each brain stem, viral protein-positive cells were not detected. Due to the large size of the brain stem, inherent tissue loss during processing, and the predicted rarity of a reactivation event, this outcome does not exclude reactivation occurring directly in the brain stem, but it does offer support for the hypothesis that infectious virus is being transported from a reactivation event in the TG.Although a cellular source for reactivation in the brain stem was not identified, the detection of infectious virus in the CNS postreactivation stimulus confirms an earlier report , strengt2 incubator. Virus stocks of HSV-1 strain McKrae , HSV-1 strain 17syn+ , HSV-1 strain OK12  were maintained in minimal essential medium supplemented with 5% newborn calf serum and incubated at 37°C in a 5% COain OK12 , and 17Vain OK12  were gen5 PFU of McKrae was placed onto each scarified corneal surface , were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg of body weight) prior to inoculation. A 10-μl drop containing 1 × 10 surface . TG and Isolation and quantification of total viral genomes by real-time PCR was performed as detailed previously with some modifications . The triin vivo by the hyperthermic stress method . At 72 h postreplating, the plates were stained with crystal violet.TG pairs and brain stems were harvested and brain stems were sagitally divided into six pieces. Each TG pair and brain stem piece was homogenized in 3 ml of medium. Homogenates were briefly centrifuged, and the supernatant was plated on one well of a six-well plate. Samples were cultured for 3 days at 37°C in a 5% CO2O2 for approximately 5 min. Tissues were cleared in glycerol to aid in visualization of the HSV protein-positive neurons.HSV proteins were detected in whole ganglia as described previously . PrimaryEncephalitic brain stems harvested at 8 dpi, or brain stems harvested at 24 h phs from latently infected mice, were directly placed in 4% formaldehyde overnight. Brain stems were then dehydrated in a graded ethanol series, cleared in xylene, and paraffin embedded. Blocks were serially sectioned at 10 μm, and consecutive sections were placed on Superfrost Plus slides (Fisher Scientific). Slides were deparaffinized, rehydrated, and examined for HSV protein expression using the antibodies described above. Color development was achieved by exposing slides to the solution of diaminobenzidine (Aldrich). After viewing slides for positive reaction product, all slides were subsequently counterstained with cresyl violet (Sigma). All slides were viewed under an Olympus BX40 microscope and photographed with AxioCamHRc (Zeiss).d-galactopyranoside) buffer per ml of PBS for 3 h.When HSV-1 strain 17VP16pLZ was used for determining infectious virus recovery in spiked samples, RSC plates were rinsed in phosphate-buffered saline (PBS) pH 7.2 and fixed in 4% formaldehyde for 5 min. The plates were then rinsed with PBS and exposed to a solution containing 5 mM potassium ferricyanide, 5 mM ferrocyanide, 2 mM magnesium chloride, and 0.1 μg of X-Gal . A


A simple and easy method of concentrating white spot syndrome virus by employing aluminium sulphate, alum as a flocculant was developed and evaluated for field detection. The concentrated virus was detected by a monoclonal antibody based flow-through assay, RapiDot and compared its performance with polymerase chain reaction. The semi-purified virus that was flocculated by 15 and 30 ppm alum in a 50 ml cylinder can be detected successfully by both RapiDot and I step PCR. In addition, alum could also flocculate the virus that is detectable by II step PCR and the concentration of virus was similar to the one observed in water from an infected pond. Furthermore, experimental infection studies validated the successful concentration of virus by alum flocculation followed by rapid detection of virus using monoclonal antibody based RapiDot. Overall, the results obtained in this study indicate that the white spot syndrome virus in water could be easily flocculated by alum for sensitive field detection by monoclonal antibody based RapiDot. The intensification of shrimp culture and its expansion has been largely increased over the decades to meet the global demand and has simultaneously resulted in the increase in number of viral pathogens to the OIE list for penaeid shrimps. Among the OIE list of shrimp viruses, white spot disease (WSD) caused by white spot syndrome virus (WSSV) has the largest impact and continued to be an obstacle to the sustainable shrimp farming . WSSV isSeveral strategies for concentrating virus in water for detection including, the tangential flow filtration (TFF) with fetal bovine serum as stabilizing agent for the detection of infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus by plaque assay , elutionLitopenaeus vannamei showing signs of WSD were collected from shrimp farms of Nellore, Andhra Pradesh, India and brought to the laboratory on ice. WSSV infection in the diseased shrimps was confirmed by MAb (specificity to 28 kDa protein of WSSV) based immunodot [Pacific shrimp, mmunodot . The immmmunodot  and re-cmmunodot . Diseasemmunodot .-10 in TM buffer. One microliter from each dilution was subjected to I step and II step nested PCR [2(SO4)3•16H2O . A 50 ml capacity measuring cylinder containing 50 ml filtered (0.2 µm nitrocellulose membrane filter) seawater was added with alum at the concentrations of 15 and 30 ppm, and no alum addition served as a control. The diluted or original stock of semi-purified WSSV was added to the respective cylinders, mixed thoroughly and kept undisturbed to allow flocculation of WSSV by gravity. At every 2 h interval for the initial 12 h period and followed by 6 h interval till 36 h period, the floc deposited at the bottom in each treatment and control were collected completely using sterile sucker pipettes. The floc samples was centrifuged at 10,000 × g for 15 min at 4°C and the resultant pellet was resuspended in 100 µl PBS, labelled and stored at 4°C for further analysis. All the floc samples were analyzed by RapiDot [To concentrate WSSV by alum through flocculation, freshly prepared semi-purified virus was diluted serially to 10sted PCR . The ori RapiDot , I and I RapiDot .L. vannamei free of WSSV were collected from farms around Kundapur, Karnataka, India and acclimatized to the laboratory condition for a week. Three shrimps (~14 g each) were stocked in aquarium tank containing 25 L seawater  with continuous aeration. The acclimatized shrimps were injected with 100 µg semi-purified WSSV at the lateral position of third abdominal segment using 1 ml insulin syringe fitted with 30G × 5/16 (0.30 × 8 mm) needle. The injected shrimps were fed with commercial diet at 5% body weight and monitored daily for the development of clinical white spots on carapace and mortality. Semi-purified WSSV was prepared from the dead shrimps and added back to aquarium tanks, from which water samples were collected into 2 and 1 L beakers, and a 50 ml measuring cylinder for concentrating the virus by alum flocculation as per the protocol explained above. The white spot syndrome virus in the bottom floc samples was analyzed by RapiDot and II step PCR.L. vannamei was further confirmed by I step PCR . The amplification of an amplicon size 1447 bp in I step PCR products that were electrophoresed on 1% agarose electrophoresis reaffirmed the presence of WSSV in semi-purified virus preparation. The preparations from normal shrimp showed no amplification of WSSV-specific amplicons by II step PCR.The semi-purified virus prepared from the immunodot-positive -2 and 1 × 10-6 by I and II step PCR, respectively . The dilution of 1 × 10-6, in which II step PCR detected the virus was further used in the flocculation experiment and compared the results with original stock of virus flocculation by alum. The WSSV was detectable by RapiDot in floc samples from original stock of virus flocculated by 15 and 30 ppm alum compared to control. In RapiDot analysis, the intensity of dot gradually increased till 12 h flocculation of virus by 30 ppm alum and then decreased over time . However, I step PCR showed that the virus was detectable in all the floc samples, but the gel band intensity lowered after 18 h in alum floc samples compared to control . In addition, the WSSV was also detectable by RapiDot in floc samples from log-diluted virus flocculated by 15 and 30 ppm alum compared to control. The MAb based RapiDot could detect the WSSV very fast after 2 h of flocculation by alum and the dot intensity increased gradually until 24 h . However, I step PCR could detect the virus from 6 h of flocculation by alum and the gel band intensity lowered after 12 h in alum floc samples compared to control . Furthermore, the WSSV was not detectable by RapiDot in floc samples from both the controls added with diluted and original stock of virus, but without addition of alum. Moreover, I step PCR also could not detect the WSSV in floc samples of diluted virus control, with an exception, it detected the virus in floc samples from original stock virus control .Each log dilution of WSSV stock was analyzed by PCR and found the lowest dilutions detectable at 1 × 10Fig. 3A and B). However, the dot intensity in RapiDot increased over the time for 2 L and 1 L beaker floc sample, but decreased with time for 50 ml measuring cylinder floc samples. Although II step PCR detected WSSV at all the time intervals but, the intense bands were observed at 18, 24, 30 and 36 h for 2 L, 18, 24, and 30 h for 1 L beaker samples, and 12, 18, and 24 h for 50 ml measuring cylinder samples. Further, RapiDot could not detect the WSSV in floc samples from control without alum addition to 1L beaker and 50 ml cylinder, with an exception, it detected the virus in floc sample from 2 L beaker with very faint dots. In similar, WSSV was not detectable in floc samples from 1L beaker by II step PCR, but very light bands showed that the virus was detected by II step PCR for floc samples from 2 L beaker and 50 ml cylinder. The WSSV detection by RapiDot and II step PCR in control samples could be due to the improper distribution of viral load from the dead shrimps or may attribute to the human errors during the sampling of water for analysis.Alum at 15 and 30 ppm flocculated the WSSV from experimentally infected shrimp water in different containers . The WSSV was detectable by both RapiDot and II step PCR in all the floc samples at 6 to 36 h flocculation time with varied intensities ([Water is the major route of WSSV transmission into an aquaculture system . WSSV is. Therefo. Althoug, 31, 32.The virus detection in water has always been a challenge due to its low concentration. In this study, we tested the alum that is being routinely employed in drinking water treatment, for concentrating the WSSV in water and its detection by simple MAb based RapiDot. It is known that alum tend to flocculate the organic matter by enabling the aggregation of smaller organic/protein particles to floc that might be easier for the detection . We madeL. vannamei with WSSV injection. RapiDot and II step PCR detected the WSSV in floc samples within the first 6 h of alum addition at 15 and 30 ppm in 2, 1 L and 50 ml infected water. However, the intensity of dot increased over time of flocculation in all water samples with slight variations. The interesting point here is that we could floc the virus by alum in less volume of infected water, which is much lesser compared to the report of >60 L water for WSSV concentration and detection by PCR [Further, this concept of concentrating virus by alum flocculation was evaluated by simulating the white spot disease outbreak in the laboratory aquaria through experimental infection of n by PCR . In addin by PCR , glass-fn by PCR  and TFF n by PCR . However


Peste des petits ruminants (PPR) is a severe infectious disease in both domestic and wild small ruminants. Due to its heavy economic burden and hence social and health impacts on human populations, Food and Agriculture Organization of the United Nations (FAO) and The World Organization for Animal Health (OIE) have targeted PPR for eradication by 2030. In order to plan and implement a successful eradication program, factual status assessments prior to devising disease control strategies is a vital criterion. The aim of this study was to isolate and characterize PPR virus from a rising wave of outbreaks in southern Iran.Twenty-one clinical samples, including blood as well as oral, nasal and ocular swabs were collected from ten sick animals in 4 various herds and were examined with ELISA and RT-PCR for the presence of PPR virus antigen and genome, respectively. The virus was successfully isolated in primary lamb kidney cell culture and identified by RT-PCR. Phylogenetic analysis of the sequenced N genes revealed that, while the earliest reports of Iran’s outbreaks were grouped into clusters with Saudi Arabia, Turkey and Africa, in this study reported sequences were grouped with samples from Pakistan, Tajikistan and China in particular. This observation suggests a shift in PPRV flow from the western borders of the country to the eastern neighboring countries.Lineage IV of PPR virus is presently circulating in Iran, with certain levels of genetic diversity. Present study along with previous reports demonstrates the dispersal patterns and movements of PPR virus, which highlights the reversal pattern of virus flow in recent years. Such information is necessary to understand PPRV molecular epidemiology and to develop more proper control strategies to eradicate the disease in the planned time.The online version of this article (10.1186/s12917-019-1920-y) contains supplementary material, which is available to authorized users. Morbillivirus genus [Peste des petits ruminants, also known as goat plague, is an acute highly contagious viral disease that causes serious economic losses due to high morbidity and mortality rates –3. This us genus . The genus genus . Based ous genus , each wius genus , 9. Receus genus . While dus genus .Located in Eurasia and western Asia, Iran is considered a crossroads of three continents, Asia, Europe, and Africa. According to the latest report of OIE Iran has 10 and 5% of the Middle East sheep and goats herd populations, respectively. Despite the use of the live attenuated vaccine, it is not unexpected that 32% of the reported outbreaks of PPRV over the last 16 years were from Iran with over 5000 outbreaks being recorded between 2005 and 2011. Since the first PPRV outbreak report in 1995 , till noDetermining the nature of circulating strains of PPR virus has a crucial role in devising more appropriate diagnostics and control strategies. Therefore, the aim of the present study was to isolate, characterize and phylogenetically analyze the circulating PPR virus strains in small ruminants’ outbreaks in Fars province, Iran.The characteristic clinical signs of PPR were observed Fig.  in affecAll nasal and oral swabs submitted to the local veterinary service were reported positive for PPR virus antigen using ELISA. These results demonstrate appropriate clinical criteria were applied for the sample collection procedure. RT-PCR of samples also confirmed that all samples were positive for PPR. Further, for molecular detection, RT-PCR was conducted to amplify parts of N gene and to confirm this amplification, electrophoresis of RT-PCR products was performed and the expected band was observed at 352 bp.Morbillivirus infection was observed as aggregates of rounding cells and syncytia formation after nine days of PI, further, cells destruction occ twelve days of PI . The cytopathic effect (CPE) similar to a  PI Fig. .Fig. 2ViIn order to represent a comprehensive picture of PPR epidemiology and its circulating strains in the region, multiple alignments and phylogenetic tree construction were performed using the obtained sequences in this study and a collected list of various sequences reported previously worldwide Fig. . These aHaving 50% of the sheep herd population in the Middle East, Iran is accounted to be the 4th country in sheep herd population worldwide. Most of these small ruminants are either kept by nomad populations or rural villagers with constant trading in between, mostly at traditional and religious events. This situation makes a region vulnerable to recurrent outbreaks of contagious disease as is observed in the case of PPRV epidemics in Iran.Since the first report of PRRV in Iran in 1995  numericaIn previous reports  sandwichMorbillivirus infection in addition to subsequent syncytia formation and cell destruction has exhibited the successful isolation procedure.As a proper amount of virus genetic material was needed for further molecular characterization and sequencing, we conducted virus isolation on primary lamb kidney cell culture. Inoculated nasal and oral swabs taken from live affected animals were used for this purpose. So far, the typified phenotype of CPE of In order to gain insight into the genetics nature of circulating virus in the region, we depicted a phylogenetic tree based on the N gene partial sequence. Recent studies have proved that N gene sequence can more accurately discriminate different strains in comparison to F and H genes. The phylogenic tree shows that the present analyzed PPR viruses are more closely related to reported strains from China, Tajikistan, and Pakistan. Furthermore, this analysis reveals distant clustering of reported sequences in this study with previous reports of this region The possible relationship of Iranian strains resemblance to that of Pakistani strains of PPR virus could be due to the fact that Iran has common borders with Pakistan and Afghanistan where uncontrolled animal trade across borders, along with trans boundary movements encompassing Pakistan, Afghanistan and Tajikistan is quite common. Yet close proximity of reported sequences in this study with recent reports of China, within a short period, suggests virus introduction through importing small ruminants from this country. The grouping of these strains of PPR virus at two different places of the phylogenic tree over time shows that PPR virus isolates may have undergone exceptional evolutionary pathway or this might be as a result of virus introduction from a variety of sources. Considering the latter possibility, this observation suggests a reversal pattern of virus flow in recent years. However, more samples collected from a wider range of outbreaks are needed to evaluate such commentary..In spite of being endemic in Iran, outbreaks of PPRV are regularly occurring, and limited information is available on the genetic nature of PPRV. The sequences described in this study increase the available information on the circulating strains of PPRV in Iran. With these bounded data, we still endeavor to demonstrate the presence of a new population of PPRV, which necessitates future such studies to be performed at the national level to determine the complete picture of circulating viruses in the country. According to the findings of this study, the circulation of the virus from the west to the east of Iran is now reversed, from the east to the west. Therefore, proper understanding of this virus circulation will help PPRV endemic countries such as Iran develop effective control strategiesDuring a period of 14 months (June 2014 to August 2015), samples from twenty-one domesticated small ruminants (5 sheep and 16 goats) with clinical outcomes consistent with PPRV were collected in Fars province of Iran  and detected N antigen, as described .PFLK was prepared in the Department of Virology at Shiraz University for the virus isolation. The cells were incubated at 37 °C with 5% CO2 and monitored daily for CPE caused by the virus. After observation of CPE similar to a Morbillivirus, cell rounding, and syncytia formation, the cells were frozen and thawed 3 times and centrifuged. The supernatants were used for RNA extraction.All samples, as well as supernatant of destroyed cultured cells and PPRV vaccine (as the positive control), were subjected to RNA extraction using RNA-Plus Solution . RNA extracts were stored at − 70 °C until use. The swab obtained from the animal without any clinical sign and negative for Ic-ELISA was used as negative control.The synthesis of cDNA of PPRV was performed according to the manufacturer’s procedures of AccuPower® Cycle Script RT PreMix (dN6) from all samples . RT products were cooled on ice and stored at − 20 °C until used.For RT-PCR, oligonucleotide primers corresponding to N gene sequence were used. These primers were adopted from O.I.E. proposed primers with some modification. Upstream primer sequence: (NP4–19) 5’CCTCCTCCTGGTCCTCCAG3´. Downstream primer sequence: (NP3–17) 5’GTCTCGGAAATCGCCTC3´.This primer pair amplifies 352 bp region of N gene. The PCR was performed by AMPLICON master mix (Denmark) according to the manufacturer recommendation.Four samples of RT-PCR products representing N genes of isolated positive samples in cell culture were submitted to Macrogen Corporation for sequencing.The electropherogram files of resulting sequences were examined and edited using Chromas. Partial N gene sequences generated in other reports were obtained from GenBank, Fig. Additional file 1:Figure S1. Ulcerative stomatitis in a PPRV affected goat. (JPG 58 kb)


The application of High-Throughput Sequencing (HTS), also known as next-generation sequencing, has proven very successful for virus discovery to resolve disease etiology in many agricultural crops. Building on this success, the movement to apply HTS for routine virus detection is gaining momentum. Deployment of HTS as a detection tool comes with the same challenges faced when adopting any new method but with some additional technology-specific issues that are explored here.The application of HTS for virus discovery has been very successful, in part because it has largely been used in the early stages of a diagnostic investigation, to identify putative viral sequences. From that point onwards more familiar techniques such as PCR or ELISA could be used to provide a definitive diagnosis  against which the sequences are compared. Sequence information obtained can also be used to provide insight on the virus population structure, ecology or evolution or to differentiate virus variants that may contribute differently toward disease etiology. HTS has the potential to reduce the time from virus discovery to development of targeted detection assays such as PCR or LAMP and to contribute to the improvement of existing assays, by elucidating sequence variation within virus populations. Another advantage of HTS is that sequence data can be analyzed by multiple end-users or may be re-analyzed as databases are expanded.For the production of propagation stocks, candidate nuclear plants are tested for a range of “targeted or regulated” viruses using a panel of specific methods, such as ELISA, RT-PCR and RT-qPCR, and bioassays  and to the sequencing depth.In the diagnostics field, novel methods may be validated through direct comparisons with existing, validated methodologies. There have been so far few direct comparisons with HTS approaches and plant viruses. Comparisons with RT-PCR  using a panel of isolates, the specificity of HTS methods could be assessed by verifying inclusivity and exclusivity of the database(s) of sequences used in the bioinformatic approach.As obtaining a diagnostic result by HTS is a multi-phasic process, the approach, platform, bioinformatic strategy, interpretation all need to be considered for reproducibility. Thus far there have been only limited investigations into the reproducibility of these various phases. Comprehensive studies testing the same sample through different approaches have not yet been performed. The comparability of different sequencing approaches has been partially investigated by Visser et al.  and PecmHarnessing the diagnostic power and flexibility of HTS for screening applications also brings the inherent tension of how to deal with inadvertent non-target findings. These may be commensal or mutualistic viruses  and diagnostic workflows (e.g., Massart et al., All authors listed have made a substantial, direct and intellectual contribution to the work, 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.


This opinion article makes a case for increasing the number of ranks used in virus taxonomy from the current five to ten (as are used to classify cellular life forms) and placing the Baltimore classes in the proposed basal rank of domain. These suggestions aim at initiating the process of accommodation of Baltimore classes in virus taxonomy and extension of the virus taxonomy scale to encompass also the most distant relationships. The International Committee on Taxonomy of Viruses (ICTV) oversees the development of hierarchical virus taxonomy according to the rank structure introduced in 1970 and expanded on several occasions . CurrentThis contemporary taxonomic structure with its modifications has served virology and virologists for fifty years, when the number of established virus families increased from two to more than one hundred and the number of recognized virus species increased by some two orders of magnitude . Also, tThe roots of this increased knowledge and rank expansion could be traced back to the advent of virus genome sequencing and comparative virus genomics, introduced in the end of 1970s and beginning of 1980s , 23, 25.Below, I list several arguments in favor of adopting an expanded version of the rank structure of the modern Linnaean taxonomy for virus taxonomy and adoption of the Baltimore groupings in the rank of domain, which might be seen as a revival of the idea put forward by Lwoff, Turnier, and Horne . It shouGeneral. The virus host imposes major constraints on virus divergence which effectively links virus and host diversities . Due to Domain and Class. Virologists recognize the Baltimore classification of viruses into six  classes Fig.  5] at i at i5] aBirnaviridae and the ssRNA + Permutotetraviridae, which form an inter-class monophyletic group [Unlike currently recognized taxa, Baltimore classes were established using purely functional considerations concerning genome type and its expression. Its broad albeit informal use in taxonomy is due to overall (perceived) good agreement between these classes and taxa that are recognized phylogenetically. However, this correspondence is not universal, as, for instance, was demonstrated for the dsRNA ic group , 30. TheThe formal recognition of Baltimore classes at the basal rank of virus taxonomy would open these newly formed taxa, as any other taxa, for oversight and revision by practitioners under the ICTV auspices. Using the conventional taxonomy proposal framework of creating, dissolving, and moving taxa, and coupling it with evolutionary reasoning and public debate, the placement and composition of the original taxa including Baltimore classes could be refined, and a public record of the revision and its reasoning created. The formal recognition of Baltimore classes as taxa at the basal rank of virus taxonomy would also (re-)define the scale of the entire taxonomy, which is informative for defining scales of other newly created ranks. Combined these changes will contribute to the advancement of virus taxonomy and its use in teaching, research, and practical applications.Picornavirales [Tymovirales [Caliciviridae and Potyviridae, share different number of conserved domains and separated by different distances from the Picornavirales, indicating that more than a single additional level above order would be required to accommodate them fully in an hierarchical structure.Order and above. While the basal rank of virus taxonomy is order, several “super-order” groupings of viruses have been described on phylogenetic grounds. For ssRNA + viruses, they are known as supergroups or superfamilies , 8, 24. avirales  and Tymoovirales , respectovirales , 20, theNidovirales, which comprises a distinct supergroup of ssRNA + viruses [Nidovirales distantly resemble Astroviridae and a subset of Luteoviridae [This example is not an exception, as was shown by our extensive analysis of the order  viruses . This or viruses . They ar viruses . An extroviridae  and all oviridae , furtherReoviridae, Mononegavirales, etc.Clearly, the availability of extra levels above the family rank will facilitate the taxonomy development of other, currently less structured supergroups of ssRNA + viruses of plants and animals, and other classes of viruses, which have highly diverse monophyletic groups, e.g. Coronaviridae and, particularly, Picornaviridae [Family. Using a rigorous method for quantifying the statistical support for clusters and ranks called DEmARC , we partaviridae  familiesaviridae , is usedThe current taxonomic rank structure of five levels was developed to accommodate an ever-increasing but still relatively limited virus diversity known to us, at a time when virus discovery was mostly an annual event to celebrate. Now viruses are discovered daily if not hourly. The number of known virus species is expected increase from the current thousands to zillions in the future and their classification will be driven by comparative genomics. To accommodate the complexity of phylogenetic relationships apparent within this fast growing diversity, the virus taxonomic rank structure must be adjusted accordingly, as has already become evident from bioinformatics analysis of few better characterized groups of viruses. Using an expanded version of the Linnaean taxonomic structure of ten ranks  will contribute to a better description of virus diversity and improve cross-talk between the taxonomies of viruses and their hosts. The number of these ranks could be revisited and, if necessarily, expanded further in the future. Besides the obvious changes to the Virus Code, the formal recognition of the Baltimore classes at the basal level of virus taxonomy could be the first practical steps in this direction.


Here, we isolated two chikungunya virus strains (SZ1050 and SZ1239) from patients infected with chikungunya virus who were returning to China from India and Indonesia, respectively. Strain SZ1050 was classed in the Indian Ocean lineage, and strain SZ1239 belonged to the Asian lineage. Here, we isolated two chikungunya virus strains (SZ1050 and SZ1239) from patients infected with chikungunya virus who were returning to China from India and Indonesia, respectively. Strain SZ1050 was classed in the Indian Ocean lineage, and strain SZ1239 belonged to the Asian lineage. No A226V mutation in the envelope protein E1 was found in either strain. Togaviridae. CHIKV is the causative agent of chikungunya fever (CHIKF), characterized by high fever, rash, arthralgia, and even sometimes death (Chikungunya virus (CHIKV) is an arthropod-borne alphavirus belonging to the family es death . Recentles death . There aes death . Thus, tIn October 2010, a patient back from New Delhi, India, had a fever with chest pain and arthralgia. He was diagnosed with a chikungunya virus infection by real-time fluorescent quantitative PCR. The virus was then isolated in C6/36 cells from his serum sample and named strain SZ1050 . AnotherThe complete genome sequence of SZ1050 was determined to be 11,844 bp, including two ORFs flanked by the 5′ UTR (86 nucleotides [nt]) and 3′ UTR (521 nt). One ORF was responsible for translation of the nonstructural polyprotein , whereas the second ORF contained the structural polyprotein . There are three repeated sequence elements (RSEs) in the 3′ UTR of SZ1050, similar to the RSEs in Ross 79. In add7–MG664850 (SZ1050) and MG664851 (SZ1239).The complete genome sequences of the two CHIKV strains in this study have been deposited in GenBank under the accession no.


Aedes albopictus has increased its spread and resulted in large outbreaks in the Indian Ocean islands. For many alphaviruses commercial diagnostic tests are not available or show cross-reactivity among alphaviruses. Climate change and globalization will increase the spread of alphaviruses and monitoring of infections is necessary and requires virus-specific methods. Method: We established an alphavirus neutralization assay in a 384-well format by using pseudotyped lentiviral vectors. Results: MAYV-specific reactivity could be discriminated from CHIKV reactivity. Human plasma from blood donors infected with RRV could be clearly identified and did not cross-react with other alphaviruses. Conclusion: This safe and easy to use multiplex assay allows the discrimination of alphavirus-specific reactivity within a single assay and has potential for epidemiological surveillance. It might also be useful for the development of a pan-alphavirus vaccine.Background: Alphaviruses are transmitted by arthropod vectors and can be found worldwide. Alphaviruses of the Semliki Forest complex such as chikungunya virus (CHIKV), Mayaro virus (MAYV) or Ross River virus (RRV) cause acute febrile illness and long-lasting arthralgia in humans, which cannot be clinically discriminated from a dengue virus or Zika virus infection. Alphaviruses utilize a diverse array of mosquito vectors for transmission and spread. For instance, adaptation of CHIKV to transmission by  Alphaviruses are predominantly transmitted by mosquitoes and cause a wide range of diseases in humans and animals. The Old-World alphaviruses, such as chikungunya virus (CHIKV), Semliki Forest virus (SFV), and the phylogenetically related Ross River virus (RRV) and Mayaro (MAYV) cause arthralgia in animals and humans. In contrast, New World viruses, such as Venezuelan (VEEV), eastern (EEEV), and western equine encephalitis viruses (WEEV) are encephalitic. SFV can cause encephalitis in rodents, but only mild symptoms in humans . The clinical signs of the acute diseases of Old World alphaviruses are usually indistinguishable from those of infections with the flaviviruses dengue virus (DENV) or Zika virus (ZIKV), which may lead to misdiagnosis. They are characterized by a febrile illness lasting 3–7 days, rash and severe, sometimes long lasting arthralgia ,3,4. Haemagogus mosquitoes which feed on arboreal primates [Aedes aegypti has been described, raising concerns that MAYV may adapt and show urban transmission such as CHIKV, which would result in serious public health problems [Although CHIKV, MAYV and RRV are related and cause similar disease symptoms, these viruses show different geographic distributions, dependent upon, hosts and vector utilization. MAYV was first isolated in Trinidad in 1954; since then, sporadic cases, clusters, and small epidemics of Mayaro fever have been reported from remote regions in Latin America especially the Amazon basin, but also from the city of Manaus in northern Brazil . MAYV reproblems . Ae. aegypti and Ae. albopictus. CHIKV spread was rapidly accelerated through a point mutation in its E1 protein, which improved transmission by Ae. albopictus mosquitoes [Ae. aegypti is confined to the tropics and sub-tropics, however, Ae. albopictus also occurs in temperate and even cold temperate regions and has spread from Asia to moderate climate zones in Africa, Europe and the Americas [In contrast, CHIKV was first described during an outbreak in southern Tanzania in 1953, but since then has been identified in nearly 60 countries worldwide . CHIKV isquitoes . Ae. aegAmericas . RRV is endemic in Australia, Papua New Guinea and islands of the South Pacific and causes disease in humans and horses. RRV’s geographic range was thought to be limited by the distribution of its primary reservoir hosts, initially believed to be marsupials. However, recent evidence suggests that RRV circulates endemically in the Pacific Islands, where marsupials are absent . In addiCurrently, these alphaviruses are nearly globally distributed, and although their distribution is geographically distinct, there is a serious risk of their worldwide spread due to globalization and air traffic. Evidence for such an explosive spread of an arbovirus in a naïve population was provided by the recent infections with ZIKA in Brazil and CHIKV in Latin America ,17. TherTo develop an alphavirus neutralization assay, we extended the use of a pseudotyped lentiviral vector system . Alphavi2. HeLa (ATCC: CCL-2), HEK 293T (CRL-1573), NIH 3T3 (CCL-92), Huh7 (CCL-185), and A549 (CCL-185) cells were grown in Dulbecco’s modified Eagle medium . BHK 21 (CCL-10) and Jurkat (TIB-152) cells were incubated in Roswell Park Memorial Institute medium . All media were supplemented with 10 % foetal bovine serum  and 5 % L-glutamine .All cells used were cultured at 37 °C under 5 % COA human naïve plasma was obtained from a healthy volunteer in Germany. The MAYV plasma was obtained from a patient who had contracted MAYV, presenting with fever, myalgia, maculopapular rash, and polyarthralgias whilst travelling in Bolivia . CHIKV aAll human samples used were taken with consent of the patient for diagnostic purposes according to ethical regulations in Germany. Written informed consent was given.PacI and AscI, blunt ended, and then cloned into the plasmid pIRES2-eGFP (digested with SmaI). This plasmid allows expression of genes as a bicistronic mRNA with the eGFP gene under the control of a CMV promoter. A similar strategy was used to insert the codon optimized ONNV  or BFV (accession number NP_054024) E3-E1 genes. The plasmids pMDLg/pRRE , pRSVrev (encoding HIV-1 Rev) [The gene for the MAYV E3-E1 envelope polyprotein was synthesized by GeneArt  on the basis of the sequence of a virus isolate from the Venezuela outbreak in 2010 . The gene was first cut with V-1 Rev) , pCSII-LV-1 Rev) , pHIT-G V-1 Rev) , and pIRV-1 Rev)  were useV-1 Rev) .® 2000  with the following amounts of plasmid DNA: 10 µg pCSII-Luc, 6.5 µg pMDLg/pRRE, 2.5 µg pRSVrev, 3.5 µg pHIT-G (encoding VSV-G), and 5.3 µg pIRES2-eGFP-CHIKV, pIRES2-eGFP-MAYV E3-E1, pcDNA-RRV, pcDNA-SFV. After 24 h of incubation, the medium was replaced by 5 mL of fresh DMEM per dish. Twenty-four hours later, the supernatant containing vector particle was harvested, sterile filtered with 0.45 µm filters , and ultracentrifuged  to concentrate the volume 100-fold; the particles were resuspended in DMEM and frozen in aliquots at −80 °C. VSV-G-pseudotyped vectors were frozen without concentration by ultracentrifugation.HEK 293T cells in 10 cm dishes were cotransfected using Lipofectamine6 RLU and the neutralization activity was found to be stabile for vector particles corresponding to 30,000 or 5 × 105 RLU/mL. The lowest value was chosen to keep the amount of vector particles as low as possible to save reagents. Plasma dilutions ranged from 1:30 to 1:7290 (1:3 dilutions), and were mixed 1:1 with the vector particles, resulting in a final dilution of 1:60 to 1:14,580 in a volume of 20 µL. Plasma was diluted with DMEM. The vector particle–plasma mixtures were incubated in 96-U-well plates  at 4 °C for 1 h and subsequently added to the 384-well plates. From every dilution/well in the 96-well plate, 20 µL were transferred to three wells each of the 384-well plate (triplicate assay) and resulted in a total reaction volume of 40 µL. After 16 h of incubation, BriteLite  substrate (20 µL) was added to each well, and then incubated for 5 min at room temperature. The luciferase signal was detected with a PHERAstar FS microplate reader . Transduction of cells for luciferase assays was started by seeding 6000 HEK 293T cells per well in a white CELLSTAR 384-well microtiter plate  in a volume of 20 µL DMEM using the MultiFlo Microplate Dispenser . After 24 h incubation at 37 °C, 10 µL of luciferase encoding lentiviral vector particles  plus 10 µL diluted human plasma was added to the cells using a Matrix Multichannel Equalizer Electronic Pipette . The amount of 30,000 RLU was chosen because the upper limit of the readout is 2 × 10The titer of the luciferase-encoding lentiviral vectors was determined by transduction of HEK 293T with serial two-fold dilutions of vector particles in triplicates. After 16 h of incubation, the luciferase activity was measured in each well as described above. The titer was determined as luciferase units/mL. 50 values were performed using the GraphPad Prism 7.04 software  as nonlinear regression and gives as log(inhibitor) vs. response . IC50 values represent the dilution factor of the plasma sample to obtain 50% inhibition of pseudotyped vector transduction and are given without units. The mean values and standard deviations were calculated in Excel.Determination of ICTo determine the immune status of sera against CHIKV, RRV, BFV, DENV, and ZIKV, commercially available ELISA systems were used and performed according to the manufacturer’s protocol. The following ELISA systems were used: NovaLisa Chikungunya Virus IgG qualitative Capture ELISA; EuroImmun Anti-Chikungunya virus ELISA (IgM/IgG); EuroImmun Anti-Mayaro-Virus-ELISA (IgM/IgG); Panbio Barmah Forest Virus IgG/IgM ELISA; Panbio Ross River Virus IgM/IgG ELISA; the EuroImmun Anti-Zika Virus ELISA  and EuroImmun Anti-Dengue Virus ELISA . 8–109 RLU/mL and pseudotyping with the CHIKV E3-E1 protein was superior to RRV, SFV and MAYV. Lentiviral vector particles pseudotyped with VSV-G, or the MAYV, RRV, SFV or CHIKV glycoproteins (E3-E1) were generated by transfection of 293T cells with the following plasmids: a lentiviral vector genome encoding the firefly luciferase gene, the lentiviral packaging vectors and a plasmid encoding VSV-G, or the MAYV, RRV, SFV or CHIKV glycoproteins E3-E1. Two days after the transfection, cell supernatants, except those containing VSV-G pseudotypes, were concentrated by ultracentrifugation and titrated on 293T cells. Since the cellular tropism of CHIKV, RRV and SFV-pseudotyped vectors has been described previously ,27,33, w50 values  were first analyzed by ELISA for antibodies directed against CHIKV, DENV or ZIKV and the outcome is depicted in 50 values of the neutralization assay were determined  and values below 800 for MAYV-pseudotyped vectors (blue bars). The reciprocal IC50 value of the MAYV-specific serum was 4847 when analyzed with MAYV-pseudotyped vectors and only 774 with CHIKV-psudotyped vectors  from Australian blood donors were first analyzed by ELISA for antibodies directed against RRV or Barmah Forest virus (BFV) virus. BFV is another Old World alphavirus only found in Australia with clinical signs similar to the other Old World alphaviruses . For bot50 values were determined and their reciprocal values are plotted in 50 value (The samples were used blinded for the alphavirus neutralization assay. BFV-pseudotyped vectors were additionally included. IC50 value . These d50 value . In cont50 values are indicated in Next, we analyzed plasma samples of three patients from Brazil, of whom one sample was included in the study in Laboratory diagnosis of viral infection is usually done by detection of virus-specific antibodies in the blood, detection of virus antigens or detection of viral nucleic acids. However, for alphavirus infections, nucleic acid detection can only be used in the short viremic phase, which lasts about one week . In addiNext, we established a semi-automated neutralization assay for four different alphaviruses, using pseudotyped lentiviral vector particles and quantification by luciferase activity. This assay can be performed under BSL-2 conditions in contrast to working with the infectious virus, which requires BSL-3. The assay has a small reaction size of 40 μL, which minimizes the amount of sample material required. The automated performance of the assay, including the seeding of target cells, makes it fast and convenient. The co-screening of multiple alphavirus-pseudotyped vectors enables the identification of the specificity of antisera. In contrast, ELISA assays showed cross-reactivity among different alphaviruses . The samThe neutralization assay might be helpful for determining the global distribution of different alphaviruses particularly since commercial ELISAs are currently unavailable for some alphaviruses. However, this, as well as other assays are unable to discriminate very closely related viruses, as shown here for ONNV and CHIKV . Due to The assay can easily be expanded to include more envelope proteins of alphaviruses and will enable screening of neutralizing activities against all alphaviruses within one assay. In addition, the alphavirus-neutralization assay will be helpful for alphavirus vaccine development. As CHIKV infections in immunological naïve populations can cause large epidemics which decline quickly due to the high infection rates and subsequent development of herd immunity, clinical vaccine efficacy trials are almost impossible. Neutralization titers might be suitable correlates of protection for licensure of vaccines directed against CHIKV or other alphaviruses.


In today's world where the effects of global warming are intense, alternative approaches for a sustainable concrete sector are rapidly increasing. The partial replacement of waste materials, such as bottom ash and marble powder to cement, leads to a significant reduction in carbon dioxide emissions. The expected quality of materials to be used for the concrete sector is very important in terms of building safety. New building materials and binder materials offered as an alternative are often unable to meet this requirement due to existing standards. Therefore, the existing standards should be reviewed and alternative construction materials should be provided for the sector. This dataset would be beneficial for other researchers who design sustainable concrete. Although the data presented herein deals with pure paste, the authors believe that the data can be used for mix design in the concrete construction sector to reduce the cement amount for various civil engineering projects, from low to medium strength concrete applications. The data compiled herein were obtained from the fresh and hardened properties at 7, 28 and 56 days. Physico-mechanical characterization and sulphate resistance were analyzed for laboratory produced marble paste composites. The dataset described here is the pilot study of the “High-volume marble substitution in cement-paste: towards a better sustainability” [1] and “Novel coal bottom ash waste composites for sustainable construction” [2]. Pure paste composites were prepared in a small laboratory, which produced samples. The samples were then assessed on the flow table test, as shown in https://doi.org/10.1016/j.dib.2020.105160.Supplementary video related to this article can be found at Video 13The following is the supplementary data related to this article:22/g. Bottom ash was obtained from local brick factory. The specific gravity was 1.44. Particles that passed through a 0.212 mm sieve was used. The ordinary Portland cement grade 42.5 was used. The specific gravity of cement was 3.15. The fineness of cement was 3650 cm2/g. The produced composites were designed such a way that no natural aggregates were utilized. The water to binder ratio for all groups was kept constant at 36.5%. This value was optimized based on previous studies [The data presented herein were obtained by incorporating a bottom ash and marble powder with cement. To satisfy the sustainability requirement, no treatment was applied to bottom ash and marble powder. The amount of moisture in marble powder were determined and the amount of water used in mixture proportioning were adjusted for every mixture groups. Marble powder was mainly composed of calcium carbonate and classified as limestone grade . The detailed information about chemical composition of marble can be found in Ref. . The spe studies . Four di studies  and ASTM studies ,10. Twel


In seroconversion panels obtained from patients from Brazil, diagnostic testing for Zika virus infection was improved by combining multiple antibody isotypes, techniques, and antigens, but sensitivity remained suboptimal. In contrast, chikungunya virus diagnostic testing was unambiguous. Recurrent recent arbovirus infections suggested by serologic data and unspecific symptoms highlight the need for exhaustive virologic testing. In 2013, Zika virus and chikungunya virus (CHIKV) emerged in Latin America (We prospectively sampled patients in 4 time points up to 90 days post–symptom onset (dpo) . The cohAt enrollment, Zika virus patients most frequently reported fever, rash, and arthralgia (80% each), and CHIKV patients most frequently reported arthralgia (100%), fever (89%), and myalgia (89%) . No co-i4 RNA copies/mL) (6 RNA copies/mL) (t test) from 5 dpo onward, and viral loads in urine were consistently low (Consistent with previous studies (pies/mL) , panel Bpies/mL) , panel CNext, to assess the antibody kinetics of Zika virus and CHIKV, we measured antibody responses over time by commercial and in-house serologic tests. In a widely used nonstructural (NS) protein 1 antigen-based ELISA, Zika virus IgM seroconversion was low (33% [5/15]), whereas CHIKV IgM seroconversion was 100% using an envelope-based ELISA (p<0.0001 by Fisher exact test) , panel A>1 assay (All Zika virus–infected patients showed IgG responses across the 4 time points in >1 assay  Table 1.>1 assay , panel D>1 assay  Figure 5>1 assay  Figure 4We provide pivotal data on Zika virus and CHIKV diagnostic challenges in a Latin American setting. Limitations of our study include the relatively small number of patients, sampling at heterogeneous dpo and heterogeneous numbers of samples per dpo, and lack of acutely DENV-infected patients to assess test specificity. The strengths of our study include rRT-PCR–confirmed infections, waiving the need to define serologic assays prone to cross-reactivity as standards, sampling during Zika virus and CHIKV outbreaks (Our data suggest reliable diagnostic testing for acute CHIKV infections by IgM detection from 5 dpo onward. This finding might enable waiving labor-intense and costly molecular protocols in many patients, minimizing costs for public health systems and cohort studies investigating arbovirus pathogenesis. However, reliability of CHIKV serologic diagnostic tests must be reevaluated for co-circulating genotypes (The difficulties of adequately diagnosing Zika virus infections in areas to which it is endemic have major implications for public health. Reliable testing for flaviviruses in such areas will be key for epidemiologic studies on Zika virus and assessments of the safety of flavivirus vaccination programs, as illustrated by more severe dengue infections in DENV-seronegative individuals who received a live attenuated dengue vaccine (For pregnant women and couples intending pregnancy, accurate diagnosis of acute or past Zika virus infection is crucial. The steep increase in requests for abortion in Latin America illustrates the effect of the Zika virus outbreak on reproductive medicine (Our results highlight that definite exclusion of acute Zika virus infections is challenging in a considerable proportion of patients. However, although limited by a small number of samples, our data highlight the attainability of more accurate Zika virus diagnostic testing by combining molecular and serologic tests using different antibody classes, antigens, and methods and by monitoring an increase of IgG titers in follow-up serum samples. Our data will help clinicians and health authorities build reliable diagnostic algorithms for Zika virus and CHIKV and highlight that exhaustive testing of arboviral infections is required for attributing frequencies of infection-specific symptoms.Additional information on differential shedding and antibody kinetics of Zika and chikungunya viruses, Brazil.


This modification of existing FISH protocols removes numerous chemical wash steps, and thus, reduces protocol time and specimen loss while improving hybridization sensitivity. The presented work is optimized for juveniles of soybean cyst nematode  and has been used to identify both host and associated-microbial  targets. Moreover, through the use of two different long wavelength fluorophores, two probes can be colocalized within one individual. This protocol may be adapted to identify targets-of-interest within other life stages and nematode species.A fluorescence This protocol improves:•Hands-on protocol time (by approximately 1.5 h).•Specimen loss (fewer aspiration steps).•Hybridization . Specification Tablein situ hybridizations (ISH). The primary ISH protocol available for PPN localized gene expression involves chemical fixation, segmenting the specimen, and several permeabilization steps in order to gain probe entry into nematode cells and tissues •Hybridization wash buffer •ProLong Diamond Antifade Mountant (with or without DAPI) (Thermo Fisher Scientific)Prewarm hybridization buffer and hybridization wash buffer at 46 °C and 50 °C, respectively.1g).Pellet hatched cohorts of ppJ2s in a 1.5 ml microcentrifuge tube for 2 min at low speed .Resuspend samples in 50 μl sterile water for each reaction/probe combination (7Pipette 50 μl of nematode water suspension into an electroporation cuvette (0.4 cm gap distance), and then add 150 μm of each probe .8Electroporate nematode/probe sample(s) on an electroporator with a single-pulse square wave at 125 V for 20 ms.9Add 100 μl of pre-warmed (46 °C) hybridization buffer  to the nematode suspension.10Transfer to a 1.5 ml microcentrifuge tube. Incubate 4 h at 46 °C in the dark on a gently rocking platform.[Note: Benzalkonium chloride also acts as a surfactant and reduces adherence of nematodes during protocol.]1g and remove excess liquid.Following hybridizations, centrifuge samples for 1 min at 6000 × 2Perform three incubations at 50 °C in 100 μl of pre-warmed hybridization wash buffer  for 20 min each in the dark on a rocking platform at a slightly higher rocking speed than the hybridization step.3g for 1 min and remove excess liquid from the nematode pellet.Centrifuge at 6000 × 4Pipette treated nematodes onto a glass microscope slide in a small volume of remaining wash buffer (approximately 5 μl).5Let the sample air dry on the slide until only a small film of water surrounds the nematodes. Then quickly (and without introducing bubbles) add 10 μl ProLong Diamond Antifade Mountant (Thermo Fisher Scientific) and seal with a coverslip.6Dry slides horizontally in the dark at 25 °C for 24 h and then store at 4 °C in the dark. Slides can reliably be viewed for up to a month.[Note: ProLong is available with or without DAPI stain incorporated.]Nematodes can be viewed on an appropriate confocal microscope that is capable of separating dye wavelengths without channel bleed-through. For this protocol, a Zeiss LSM 880 confocal microscope with an Apochromat 40x water immersion objective (1.2W Corr FCS M27) was used, and analysis was conducted with Zeiss Zen version 2.3 software.via an alternative permeabilization protocol in which nematodes are electroporated with nucleic acid probes. A comparison of SCN ppJ2s labelled via the two different permeabilization protocol options can be seen in Flock house virus (FHV) which does not naturally infect nematodes. A 20-mer FHV DNA sequence was selected that should not bind to RNA of the nematode or any known associated-microbes. When compared with localization of the host (4G06) SvG probe . Increasing the sodium hypochlorite concentration and wash times dissolved eggshells but resulted in more severe damage to cells and increased autofluorescence at 565 nm above that of untreated samples. Additionally, chitinase treatment had no visible effect without the addition of sodium hypochlorite (tested up to 1.5 h), likely because either the vitelline layer must first be dissolved or this particular type of chitinase from Streptomyces griseus was not as effective against SCN chitin compared to C. elegans [Attempts were made to introduce labeled nucleic acid probes into SCN eggs, but it was discovered that eggshells autofluoresced at both 565 nm and 647 nm in addition to the shorter wavelengths seen in juveniles . Additional attempts to permeabilize the vitelline membrane and chitinous layer of eggshells were conducted in an effort to allow probes to penetrate the egg and be visible above background noise at longer wavelengths. Thus, eggs were washed with sodium hypochlorite followed by incubation with chitinase (isolated from  elegans .The authors declare that there are no conflicts of interest.