Although semen cryopreservation has facilitated the diffusion of artificial insemination and in vitro fertilization in cattle, it still represents a major factor affecting sperm fertility. It is known that cryopreservation induces the loss of fertility-associated proteins, while the effect on metabolites has not been evaluated, although several compounds affect sperm physiology and fertility. The aim of the present work was to study the metabolome in bovine sperm and seminal plasma after cryopreservation and to correlate the metabolic profile of high- and low-fertility bulls in order to identify fertility markers. The analysis, carried out by liquid chromatography–mass spectrometry methods, revealed differences in metabolite contents between fresh and cryopreserved semen, both at cellular and plasmatic levels. Interestingly, metabolites showing variation have important functions related to fertility. In addition, the study highlighted the differences in lipid profile between high- and low-fertility bulls. The identification of new potential fertility markers is of high economic impact. In addition, it opens the way for the development of corrective strategies to improve the fertility of low-fertility bulls.l-glutaminyl-l-glutamine were observed in cryopreserved compared to fresh spermatozoa. The fresh seminal plasma of high-fertility bulls showed an over-expression of l-acetylcarnitine, glycerol tripropanoate, 2,3-diacetoxypropyl stearate and glycerophosphocholine, and an under-expression of lysophosphatidylcholine and butyrylcarnitine, compared to low-fertility bulls. Higher levels of glycerophosphocholine and lysophosphatidylcholine (16:0/0:0) were recorded in fresh spermatozoa from high-fertility bulls. In high-fertility bulls, a greater content of glycerophosphocholine and lower levels of butyrylcarnitine, glycine betaine and l-carnitine were found in cryopreserved seminal plasma, and lower levels of glycine betaine were detected in cryopreserved spermatozoa. In conclusion, cryopreservation affects bovine semen metabolome at both plasmatic and cellular compartments, and metabolic profile differs between high- and low-fertility bulls.Semen cryopreservation determines several sperm damages, including the loss of fertility-associated proteins. The purpose of the study was to compare the metabolite contents in bovine sperm and seminal plasma before and after cryopreservation, and between high- and low-fertility bulls in vitro. Forty-eight ejaculates, collected from eight bulls (six per bull), were analyzed by liquid chromatography–mass spectrometry. Cryopreservation resulted in an over-expression of lysophosphatidylcholine ) in seminal plasma. In addition, higher levels of glycine betaine and pyro- Fertility is a major factor influencing the sustainability of livestock breeding. It is known that reproductive failure in cattle is in great part due to the poor fertilizing ability of sperm ,2. SemenAnother important limiting factor is the bull effect  ,11. DespMetabolomics characterizes the downstream of biology systems and has been recently applied to study reproductive processes ,15,16. TTo our knowledge, the variation in metabolite contents following semen cryopreservation has still not been investigated in cattle. Metabolites are reliable indicators of phenotypic traits, as they are the end products of metabolic pathways . The ideThe aim of this study was to evaluate the differences in the metabolites present in seminal plasma and spermatozoa between fresh and cryopreserved bovine sperm by a metabolomic approach (LC–MS). A further objective of this work was to compare the metabolomes of seminal plasma and spermatozoa of high-fertility (HF) and low-fertility (LF) bulls, as assessed in vitro, to identify new reliable fertility markers.6 spermatozoa per mL in 0.5 mL French straws, which underwent combined cooling with an equilibration period of 3 h at 5 °C. The straws were kept in an automatic programmable biological cell freezer  until the temperature of straws reached −145 °C. Then, the straws were plunged into liquid nitrogen (−196 °C) for storage. The straws were thawed at 37 °C for 40 s in a water bath, and the seminal plasma and spermatozoa were separated by centrifugation  and stored at −20 °C (for approximately two months) until further analysis . In order to distinguish HF and LF bulls, sperm viability, morphology, membrane integrity, DNA fragmentation, as well as in vitro fertilizing ability, were evaluated after thawing.In this work both the seminal plasma and spermatozoa of fresh and cryopreserved semen were analyzed. Eight healthy Holstein (Bos Taurus) bulls (4–6 years of age), maintained at an authorized National Semen Collection Center , under uniform management conditions and routinely used for semen collection, were selected for the trial. For this study, a total of forty-eight ejaculates were collected weekly from eight bulls (six ejaculates per bull) by artificial vagina . On fresh semen, motility was evaluated by phase contrast microscopy and only ejaculates containing more than 80% motile sperm were used in the study. After the initial semen assessment , each ejaculate was split into two aliquots. An aliquot was centrifuged  to separate seminal plasma and spermatozoa and were immediately processed for analysis . The other aliquot was cryopreserved by standard method with commercial extender BioXcell , to have a final concentration of 30 × 10n = 48) used for metabolite extraction were thawed and utilized for the assessment of sperm viability, morphology, membrane integrity and DNA fragmentation. Cleavage and blastocyst rates were evaluated after in vitro fertilization with frozen–thawed spermatozoa from three randomly selected ejaculates/bull (n = 24).Cryopreserved semen straws from the same ejaculates (Sperm viability (percentage of acrosome intact live sperm) and morphology were assessed by the Trypan Blue/Giemsa technique, as previously described . The funl-glutamine and 50 µg/mL gentamycin (25–30 COCs/400 µL) in four well plates  for 22 h at 39 °C and 5% CO2 in the air. In vitro, matured COCs were washed and transferred into 300 µL of Tyrode’s modified medium without glucose and bovine serum albumin (BSA), supplemented with 5.3 SI/mL heparin, 30 µM penicillamine, 15 µM hypotaurine, 1 µM epinephrine and 1% BS (IVF medium). The sperm selected by BoviPure gradients  were diluted with IVF medium and added in the fertilization wells at a concentration of 1 × 106 sperm/mL. Gametes were co-incubated for 20 h at 39 °C in 5% CO2 in the air, after which presumptive zygotes were vortexed for 2 min to remove cumulus cells in Hepes–TCM with 5% BS, washed twice in the same medium and randomly distributed into 400 µL Synthetic Oviduct Fluid (SOF) medium with 30 µL/mL essential amino acids, 10 µL/mL nonessential amino acids and 5% BS and incubated in a humidified mixture of 5% CO2, 5% O2 and 88% N2 in the air at a temperature of 39 °C. The cleavage and blastocyst rates, calculated out of the total COCs, were recorded, respectively, on days two and seven after in vitro fertilization.To assess in vitro fertilizing ability, abattoir-derived bovine oocytes were matured and fertilized in vitro with frozen–thawed sperm from different bulls, then cultured in vitro to the blastocyst stage, according to our standard procedure . BrieflyMetabolites extraction was performed by adding 900 µL methanol to 300 µL seminal plasma, vortexed for 1 min and stored at −20 °C for 2 h, in order to precipitate the proteins. The samples were then centrifuged at 12,000 rpm for 15 min at 4 °C and the supernatant was filtered with 0.22 µm membrane syringe filters. Spermatozoa were lysed in methanol according to a protocol previously described , consist® Express C-18 column , held at a constant temperature of 40 °C. The mobile phase consisted of A: 0.1% (v/v) formic acid (FA) in water (H2O) and B: 0.1% formic acid (FA) in acetonitrile (ACN). Elution was done at a flow rate of 0.6 mL/min and the gradient was as follows: starting condition 5% B, held for 1.10 min, ramping to 95% B until 9.30 min, held at 95% B for 1.10 min, lowering to 5% B in 2.30 min, and 5% B for 3 min as equilibration time. The injection volume was 4 µL. UV spectra were collected by DAD, setting the detection wavelength at 210, 250 and 280 nm. Both HPLC and MS and their parameters were set using the Agilent MassHunter Data Acquisition Software, rev. B.05.01 (Agilent Technologies). The system operated in positive ion mode and MS spectra were recorded in the m/z 50–1000 range as centroid spectra, with a speed of 3.3 spectra/s. The capillary was maintained at 2000 V, fragmentor voltage at 180 V, cone 1 (skimmer 1) at 45 V and Oct RFV at 750 V. The gas flow rate was set at 11 L/min at 350 °C and the nebulizer was set at 45 psig. A standard solution was infused by using an Isocratic pump  in order to perform the real-time lock mass correction. The solution consisted of two reference mass compounds: purine  and hexakis –phosphazene . The flow rate was set at 0.06 mL/min, while the detection window and the minimum height were set at 1000 ppm and 10.000 counts, respectively, for reference mass correction.Analyses were done on an Agilent high performance liquid chromatograph (HPLC) 1260 Infinity Series  equipped with a DAD (Diode Array Detector) system (Agilent Technologies) and coupled to a quadrupole-time of flight (Q-TOF) mass spectrometer model G6540B (Agilent Technologies) with a Dual ESI source (Agilent Technologies). The column used for separation was an AscentisData from the LC–MS analysis were first subjected to the extraction of peak components and to chromatographic alignment, performed by using MassHunter Profinder, version 06.00. The batch recursive feature extraction was used to extract and align the molecular features in all biological and analytical replicates for each group of samples. The parameters for molecular feature extraction included a peak height of ≥ 5000 counts and the extraction of possible ions inclusive of the protonated ion, or the sodiated adduct. At least two ions were required for a single molecular feature. For binning and alignment, a tolerance of 0.3 min and 20 ppm was set for the retention time window and mass window, respectively. The post-processing filters applied included the requirement for the molecular feature to be present in at least three out of six replicates in one group.t-test (p > 0.05) with Tukey–HSD post hoc and Benjamini–Hochberg correction was applied to assess the differential significance of samples. Eventually, fold change > 2.0 was applied. The results obtained were then subjected to principal components analysis (PCA) and hierarchical clustering in order to compare the metabolome of seminal plasma and spermatozoa between HF and LF bulls to detect differences in metabolome induced by the cryopreservation.Statistical analysis was carried out using Mass Profile Professional, version 13.1.1 (Agilent Technologies). Aligned data on seminal plasma and spermatozoa from fresh , and cryopreserved  groups were filtered based on the quality of their presence  in the samples and conditions. After this operation, only entities in which at least 100% of the values in any one of two conditions have acceptable values, were retained. The remaining entities were further filtered based on their frequency of occurrence among the samples and conditions. In particular, only entities that appeared in at least 100% of the samples, in at least one condition, were retained. An unpaired Student Statistically relevant compounds were identified using the freely available electronic database, Human Metabolome database (HMDB) and by comparison with standard compounds or with data present in the literature. Among the detected molecules, only those with a mass error below 10 ppm and a sufficient score (≥95%) were reported.The metabolic profile was different between fresh (F-P) and cryopreserved (CRY-P) seminal plasma. The separation is shown in PCA scores plot, as shown in l-glutaminyl-l-glutamine), were higher in content in the cryopreserved samples, as shown in Differences between fresh and cryopreserved spermatozoa were detected, as shown in The fertility-associated parameters of HF and LF bulls are shown in l-acetylcarnitine, glycerol tripropanoate, 2,3-diacetoxypropyl stearate and glycerophosphocholine (GPC), and lower levels of butyrylcarnitine, lysoPC (P-16:0/0:0) and piperidine in the F-P samples. In the CRY-P samples, higher levels of GPC and lower levels of l-carnitine, butyrylcarnitine and glycine betaine were found in HF bulls.Principal Component Analysis (PCA)and hierarchical clustering showed a distinct separation between HF and LF bulls, according to the metabolic content in fresh and cryopreserved seminal plasma, as shown in Both PCA and hierarchical clustering differentiated HF and LF bulls according to the metabolite contents in fresh and cryopreserved spermatozoa, as shown in This study analyzed, using LC–MS techniques, the changes in metabolite contents in bovine sperm and seminal plasma occurring during cryopreservation. A further objective was to compare the metabolite contents in fresh and cryopreserved sperm and seminal plasma between HF and LF bulls, in order to identify fertility markers. To our knowledge, this is the first report showing a variation in the metabolome of bull seminal plasma and spermatozoa due to cryopreservation.2+ concentrations [The results of the study showed that cryopreservation influences the metabolome of seminal plasma. Indeed, in the diluted seminal plasma obtained from semen cryopreserved by conventional freezing (CRY-P), some compounds were in higher amount, compared to fresh seminal plasma (F-P) among which LysoPC ) was putatively identified. LysoPC ) is a lysophospholipid characterized by the presence of one chain of linoleic acid at the C-2 position. It is formed by the hydrolysis of phosphatidylcholine by the enzyme phospholipase A2 as part of the de-acylation/re-acylation cycle. Lysophosphatidylcholine has proinflammatory properties, cell signalling functions and is known to induce sperm capacitation and acrosome reaction by increasing Catrations ,28. Thertrations . In thistrations . This istrations  that maytrations . Howeverl-glutaminyl-l-glutamine) were higher in CRY-S. This suggests that conventional semen cryopreservation is efficient to protect sperm from cryoinjuries to a certain extent. Among the two putatively identified metabolites, glycine betaine is of particular interest. Glycine betaine is a methyl group donor that functions in the normal metabolic cycle of methionine—transforming the harmful homocysteine into methionine [The metabolome of spermatozoa derived from cryopreserved semen (CRY-S) did not show many differences compared to fresh spermatozoa. Indeed, only two metabolites  and butyrylcarnitine.The fresh seminal plasma of HF bulls showed a different lipid profile compared to LF bulls, with an accumulation of l-acetylcarnitine, the most abundant and powerful of the acylcarnitine derivates. l-carnitine is an hydrosoluble amino acid that plays a critical role as a cofactor essential for fatty acid metabolism, by transporting fatty acids into the mitochondria for subsequent β-oxidation and hence energy generation. This process results in the esterification of l-carnitine to produce acylcarnitines, among which the l-acetylcarnitine is the most predominant. In addition, l-carnitine plays a critical role in modulating the intracellular CoA homeostasis. Carnitine is present in high concentration in the epididymis and in the spermatozoa of several species [l-carnitine may act as a carrier system of acetyl groups between the mitochondria and cytoplasm via a translocase present on the mitochondrial membrane, playing an important role for sperm metabolism [l-acetylcarnitine levels and a high l-acetylcarnitine:free l-carnitine ratio in human spermatozoa were associated with motility [l-acetylcarnitine in the fresh seminal plasma of HF bulls suggests that it can be considered a male fertility biomarker.Particularly interesting is the accumulation in fresh seminal plasma of HF bulls of  species ,44,45,46 species . Acetylctabolism . It was motility ,50. Sememotility . Finallymotility . The impmotility ,54, as wmotility . It follIn addition, the triacylglycerols, glycerol tripropanoate and 2,3-diacetoxypropyl stearate, were accumulated in HF bulls. It was previously demonstrated that sperm can utilize triglycerides for their metabolism ,56. It iTherefore, differences in the metabolites in fresh seminal plasma between HF and LF bulls allowed for the identification of potential fertility markers. However, in earlier studies, other fertility markers were identified in bovine fresh seminal plasma, such as 2-oxoglutaric acid and fructose , or citrl-carnitine contents were lower in the CRY-P of HF bulls. Metabolic relationships exist among the differentially expressed metabolites between HF and LF bulls, as GPC, glycine betaine and l-carnitine are involved in the same metabolic pathway. In addition, these metabolites have been proven to influence male fertility, as previously mentioned [In this study, changes in metabolome between HF and LF bulls were also observed in diluted seminal plasma from cryopreserved semen (CRY-P). Some of the metabolites followed the same pattern described for fresh seminal plasma samples. Interestingly, the CRY-P of HF bulls also contained higher levels of GPC and lower levels of butyrylcarnitine, as in fresh seminal plasma. In addition, glycine betaine and entioned ,52,62,63l-glycerol 3 phosphate, also known to regulate sperm metabolism on capacitation [The differences in metabolite content in fresh spermatozoa between HF and LF bulls were limited to two metabolites. Specifically, sperm from HF bulls contained higher levels of GPC and lysoPC (16:0). Glycerophosphocholines derive from unsaturated fatty acids that are the main components of the sperm membrane, essential for sperm viability and motility. It is known that sperm, during epididymal transit, incorporate GPC that is involved in sperm capacitation and metabolism . Indeed,citation . The othcitation . Lysophocitation ,28, contcitation . In a prcitation .Finally, differences in cryopreserved spermatozoa between HF and LF bulls were limited to glycine betaine, whose content was lower in the former bulls. This finding is difficult to interpret, and future in vitro studies are needed to better understand the reasons. The significantly higher proportion of live spermatozoa undoubtedly accounts for a more physiological sperm metabolism in HF bulls. In addition, spermatozoa from HF bulls exhibited improved membrane integrity, assessed by the HOS test that measures the cell ability to counteract osmotic stress. This may suggest a lower capacity of spermatozoa from LF bulls to respond to the stress conditions related to cryopreservation, to which they react with a compensatory increase in glycine betaine. On the other hand, we cannot rule out that the higher amount of glycine betaine recorded in LF bulls may be due to reduced utilization, in line with the lower proportion of viable spermatozoa. It is, however, interesting to note that higher levels of glycine betaine were also recorded in cryopreserved vs. fresh spermatozoa, with the former known to exhibit lower viability .In conclusion, cryopreservation affects bovine semen metabolome at both plasmatic and cellular compartments, resulting in the variation of metabolites that counteract oxidative stress and regulate sperm capacitation and motility. Furthermore, the metabolic profiles differ between HF and LF bulls, particularly in fresh seminal plasma, with several lipids differentially expressed, indicating the way for the identification of potential fertility biomarkers. Differences in few metabolites between HF and LF bulls are also present in cryopreserved seminal plasma and spermatozoa. Due to the high individual variability in fertilizing ability among bulls, it is thought necessary to increase the sample size in order to validate these findings in future studies.


Moreover, analyses found no impact of naming on anxiety, risk aversion, beliefs about contagiousness of the virus, or beliefs about mortality rate, with mild to strong support for the null hypothesis across outcomes. Exploratory analyses also found no evidence for the effect of naming being moderated by political affiliation. In conclusion, results provide no evidence that virus naming impacted individual’s attitudes toward Chinese individuals or perceptions of the virus, with the majority of analyses finding strong support for the null hypothesis. Therefore, based on the current evidence, it appears that the importance given to naming infectious diseases might be inflated.When labeling an infectious disease, officially sanctioned scientific names, e.g., “H1N1 virus,” are recommended over place-specific names, e.g., “Spanish flu.” This is due to concerns from policymakers and the WHO that the latter might lead to unintended stigmatization. However, with little empirical support for such negative consequences, authorities might be focusing on limited resources on an overstated issue. This paper empirically investigates the impact of naming against the current backdrop of the 2019–2020 pandemic. The first hypothesis posited that using place-specific names associated with China  leads to greater levels of sinophobia, the negative stigmatization of Chinese individuals. The second hypothesis posited that using a scientific name  leads to increased anxiety, risk aversion, beliefs about contagiousness of the virus, and beliefs about mortality rate. Results from two preregistered studies [ This can have serious consequences for people’s lives and livelihoods.”—Keiji Fukuda, Assistant Director-General, World Health Organization .In the face of a pandemic, one of the key decisions that scientists and policymakers face is how to name the infectious disease. While this decision might seem mundane relative to other urgent matters, international bodies, such as the World Health Organization (WHO) have expressed concern about potential unintended negative consequences of disease names ,b. The pWuhan Virus. This is a place-specific name derived from the likely emergence of the virus in Wuhan, China. As mentioned above, WHO guidelines warn against such place-specific names  and the place-specific names (Wuhan Virus or China Virus). Empirical research suggests that names play an important role in how we perceive phenomena , althougsinophobia, the negative stigmatization of Chinese individuals. More specifically, if the above reasoning is correct, we would expect to find more negative views of Chinese people  when the pandemic is referred to by a place-specific name, i.e., Wuhan Virus or China Virus.The first research question this paper investigates is whether using a place-specific name leads to increased xenophobia toward individuals from that country. As discussed, the names Wuhan Virus and China Virus are generally shunned in media circles, and their use has been anecdotally linked to acts of violence against ethnically Chinese individuals living abroad . PsycholMoreover, we examine whether political affiliation moderates this naming effect. The theoretical rationale for such a moderation lies in political affiliation being related to ingroup favoritism, with conservatives showing stronger ingroup bias than liberals during times of threat . This inThe second research question this paper investigates is the potential negative effect of using the official scientific name on attitudes toward the pandemic. Research has found that scientific concepts can lead to greater feelings of stress and increased aversion . FurtherN = 504) investigating the names Wuhan Virus, COVID-19, and Coronavirus and two countries—US and India—in Study 2 (N = 412) adding the name China Virus.In sum, this paper tests two hypotheses with respect to naming: (a) that the place-specific names (Wuhan Virus/China Virus) lead to increased sinophobia relative to other names (Hypothesis 1) and (b) that the scientific names (COVID-19/Coronavirus) lead to more negative attitudes—in the form of increased levels of anxiety, risk aversion, and beliefs about contagiousness and mortality—relative to other names (Hypothesis 2). Also, we explore political affiliation as a potential moderator of this effect. Two separate studies were conducted during the early outbreak of the pandemic (April 2020) and at its later stages (August 2020). The entire set of study materials and analysis plans for both studies were pre-registered before data collection (see osf.io/9s4jk). Given the global nature of the pandemic, we collected data from three countries—US, Canada, and India—in Study 1 , Canada, and India. All participants were recruited through the online surveying platform Amazon Mechanical Turk. Following the best practices in ensuring participant quality , we screN = 504) shows a mean age of 36.09 (SD = 10.71), 29.96% female, 43.06% Caucasian , and 52.18% having an undergraduate degree .A demographic breakdown across the total sample  state anxiety, (b) domain-specific risk aversion, (c) beliefs about contagiousness and mortality of the virus, and (d) attitudes toward Chinese individuals.We manipulated one factor, virus name, across three levels: COVID-19, Coronavirus, and Wuhan Virus. We did so by using the respective name in the article . This was coded as a categorical variable (1–3).Demographic variables were measured using single items for age (18–100 +), gender , and education level .Exclusions were applied in line with the OSF pre-registration. First of all, given the relatively subtle nature of the intervention, we excluded participants who failed an instructional manipulation check . Second,10). For example, a Bayes Factor of 10.00 indicates that the data are 10 times more likely to occur under the alternative hypothesis compared to the null hypothesis; alternatively, a Bayes Factor of 0.1 indicates that the data are 10 times more likely to occur under the null hypothesis compared to the alternate hypothesis  using analysis of variance (ANOVA). We followed up with Bayesian analyses to evaluate the null hypothesis of no naming effect. Please note that all the Bayesian factors reported in this paper compare the likelihood of the data occurring under the alternative hypothesis vs. the null hypothesis  = 0.78, p = 0.46, η = 0.00; (BF10)Bayes Factor = 0.047]. In sum, in evaluating Hypothesis 1, there was strong evidence for the null hypothesis, i.e., place-specific naming did not increase sinophobia.We compared means across conditions for sinophobia using the three measures outlined by Payne and colleagues : warmth,M = 1.96, SD = 0.89). Analyses found no evidence of a significant interaction with condition with very strong support for the null hypothesis, F = 0.71, p = 0.59, η = 0.01; BF10 = 0.000842. This suggests that political affiliation did not moderate sinophobic responses to different virus names.Next, we conducted exploratory analyses to see if any differences emerged depending on political affiliation  = 0.05, p = 0.95, η = 0.00; BF10 = 0.023. Similarly, no significant differences emerged for the measure of domain-specific risk aversion with mild support for the null, F = 1.77, p = 0.17, η = 0.01; BF10 = 0.119. Next, we detected a marginal effect on beliefs about contagiousness although Bayesian analyses still found weak support for the null, F = 2.66, p = 0.07, η = 0.01; BF10 = 0.271. Finally, there was no support for an effect on mortality beliefs, with very strong support for the null, F = 0.03, p = 0.97, η = 0.00; BF10 = 0.023.First, we compared means across the three naming conditions for anxiety, risk aversion and beliefs about contagiousness/mortality separately. Below, we report results pooled across the three samples ; note that similar patterns were seen within country samples see . We founThe result of the first study provided consistent support for a null effect of virus naming on sinophobia and attitudes toward the virus. To further corroborate these results, which ran counter to our pre-registered hypotheses, a follow-up study was conducted. The aims of this study were twofold. First, the study sought to address whether the impacts of naming perhaps only emerge after increased exposure to all of the names by examining the same hypotheses at a second time point much later after the initial outbreak (August 2020). Additionally, given the null effects of the place-specific name “Wuhan Virus” on impacting sinophobia, we sought to investigate whether using a name more explicitly linking China with the pandemic, i.e., “China Virus,” might impact sinophobia. As in the previous study, all materials and analysis plans were pre-registered at osf.io/9s4jk.N = 412) shows a mean age of 37.63 (SD = 12.48), 37.62% female, 48.06% Caucasian , and 58.01% having an undergraduate degree .In the second study, we obtained samples from the US and India. All participants were again recruited through the online surveying platform Amazon Mechanical Turk with the same pre-qualifications as in Study 1 for ensuring participant quality. A demographic breakdown across the total sample  = 1059.12, p < 0.001], indicating that most people gave the correct response and that the manipulation was effective. After the study, participants were debriefed and thanked for their participation.The study procedure was identical to Study 1 with the addition of another moniker (China Virus) as the fourth experimental condition and a different measure of coronavirus-specific risk perceptions. At the time of conducting this second study, a scale for coronavirus specific risk perceptions had been validated by All measures from Study 1 were included in this study and multi-item scales showed similar internal consistencies .A modified version of a coronavirus related risk perceptions scale  was incl2. This was to capture the reality of how widespread the virus had become by the time this second study was conducted (August 2020).Similar to the first study, we excluded 103 participants who failed an instructional manipulation check. However, in contrast to the first study, we did not exclude 149 individuals who (a) had the virus, (b) were in physical contact with someone who had the virus, or (c) had close family and friends who had the virusThe analysis plan remained unchanged from the first study and we compared means across conditions (COVID-19 vs. Coronavirus vs. Wuhan Virus vs. China Virus) using analysis of variance (ANOVA). Similar to Study 1, exploratory analyses explored the moderating effects of political affiliation.Descriptive statistics and correlations are provided in F = 1.21, p = 0.30, η = 0.01, BF10 = 0.051]  = 0.56, p = 0.76, η = 0.01; BF10 = 0.002.Replicating the results of Study 1, but extended to the new China Virus condition, we found no differences across conditions on sinophobia, with strong support for the null hypothesis .Similar to Study 1, we first compared means across the four naming conditions for anxiety, risk aversion, and beliefs about contagiousness/mortality separately. Below, we report results pooled across the two samples (US and India). Note that similar patterns were seen within place-specific samples see . We founGovernments, policymakers, and international bodies must decide how to refer to an infectious disease. As such, significant amounts of effort and consideration go into the process of naming an infectious disease including guidelines being made and international conferences held . FurtherThe null effect of using a place-specific name (“Wuhan Virus” or “China Virus”) on xenophobia is striking and contrary to the prevalent assumption in public policy discourse and the media ,b. In adnot lead to increased sinophobia.The initial name for an infectious disease is typically one that is associated with its location of origin. A similar trend is seen across several infectious diseases, e.g., Spanish Flu, Middle-East Respiratory Syndrome, Zika, or Ebola. This is likely due to the origin being salient in the early outbreak, making it an easy name to generate for the public discourse . There iThese findings speak to a broader literature on the use of racist language in public discourse. Increasing scholarly attention has been given to the impact of racist language since the rise of social media , which gives racist individuals a platform to share and spread their views online . The defA second empirical finding from this research is the lack of evidence for an effect of naming on anxiety, risk perceptions, or beliefs about the virus. Further, Bayesian analysis showed mild to strong evidence for the null hypothesis across all outcomes. This finding is one of the few cases showing a null effect of naming on psychological outcomes cf. . Insteaddecrease their focus on, as opposed to the general recommendations of what they should increase their focus on  can be found below: osf.io/9s4jk.The studies involving human participants were reviewed and approved by the Singapore Management University Ethics Committee. The patients/participants provided their written informed consent to participate in this study.All authors contributed to the methodological design, data analysis, and writing of the manuscript.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.


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the COVID-19 pandemic that has been spreading around the world since December 2019. More than 10 million affected cases and more than half a million deaths have been reported so far, while no vaccine is yet available as a treatment. Considering the global healthcare urgency, several techniques, including whole genome sequencing and computed tomography imaging have been employed for diagnosing infected people. Considerable efforts are also directed at detecting and preventing different modes of community transmission. Among them is the rapid detection of virus presence on different surfaces with which people may come in contact. Detection based on non-contact optical techniques is very helpful in managing the spread of the virus, and to aid in the disinfection of surfaces. Nanomaterial-based methods are proven suitable for rapid detection. Given the immense need for science led innovative solutions, this manuscript critically reviews recent literature to specifically illustrate nano-engineered effective and rapid solutions. In addition, all the different techniques are critically analyzed, compared, and contrasted to identify the most promising methods. Moreover, promising research ideas for high accuracy of detection in trace concentrations, via color change and light-sensitive nanostructures, to assist fingerprint techniques (to identify the virus at the contact surface of the gas and solid phase) are also presented. Since the novel coronavirus disease 2019 (COVID-19) appeared for the first time in Hubei Province, China, in December 2019, cases of the virus have been reported in almost every country in the world within three intervening months ,4,5—can SARS-CoV-2 is transmittable from human to human at the rate of more than three people per a confirmed infected person; the virus can spread dramatically in the globalized world, and, apparently, no detection method is available in clinical facilities that can inhibit that. Therefore, the necessity of home-based and point-of-use diagnosis techniques are highly desirable at this juncture. Since the virus, in most cases, does not show very acute symptoms, and the similarity of these indications to viruses, such as influenza, is rather high, these indicators cannot be emphasized enough for home-based diagnosis techniques. However, there is a characteristic of this virus that could help scientists find an optimized, home-based virus detection method. That trait is its ability to survive in aerosols with a size of less than 5 µm for more than 3 h, and its possible movement in the cloud, up to 8 m ,17,18,19The search for means to prevent, diagnose, and treat the virus continues, but so far, investigations into the diagnosis and treatment of the virus have not yet reached the Food and Drug Administration (FDA) approval stage ,21. The In the meantime, scientists, besides improving expeditious prevention and diagnostics while enhancing their quality and accuracy, are striving for creative solutions; viability of the virus in the air, and size and surface morphology of the virus, need to be exploited. Based on recent studies, the diameter of the SARS-CoV-2 is ~60 to 140 nm, which includes its genetic material, as well as the envelope protein spikes on the surface; four structural proteins present will be discussed in the sections below ,28,29,30In this article, we focus on a new and fully adaptable technique based on the MOFs and analytical methods, especially colorimetry, to provide a handy, smart, cost-effective, and simple diagnosis technique for home-based prevention and diagnosis actions. For comparison purposes, all other detection methods are also illustrated in The SARS-CoV-2 virus was initially discovered in a patient sample in Wuhan, China, and the related epithelial cells were cultured with the SARS-CoV-2, which have been isolated from the patients . The med2-, -L-Cys-A-Y-Cys-Cys-N-, -COOH, NH2, and -S-Cys-G-S-Cys-Cys-K-, respectively [There are three structural proteins on the surface of SARS-CoV-2, including small envelope protein (E), spike surface glycoprotein (S), and matrix protein (M), in which the S protein is more accessible than the others in the face of external chemical structures, and media, and one structural protein inside the membrane of SARS-CoV-2, which is termed nucleocapsid protein (N). Based on the newer findings, the S protein is accountable for more than 80% interaction of the virus with the cellular membrane and is also responsible for infecting the cells through receptor-binding mechanisms. Consequently, a smart solution for prevention, early diagnosis, or even treatment would be targeting this protein via chemical, physical, or biological approaches. Generally, the structural analysis of the E and S proteins shows the chemical structure encompassing NHectively ,41,42. IRegarding the interaction of the SARS-CoV-2 with different range of cell lines, there are a few studies that revealed the virus does interact with the angiotensin-converting enzyme 2 (ACE2) ,43,44,45Many methods and kits have been reported in the literature to detect SARS-CoV-2 after isolation from patients and identification of its sequence with China’s whole genome sequencing technique. RT-PCR, chest CT imaging is the first and most common diagnostic techniques in the detection of COVID-19 . RT-PCR NP and ORF1ab genes have been developed to detect the virus in samples [With the high prevalence of COVID-19, many companies, such as ScienCell , Thermo  samples . Despite samples ,55.Loop-mediated isothermal amplification (LAMP) technique is a nucleic acids amplification assay that has been studied widely by many researchers. The emergence of this method is due to its specific primers, a Bst DNA polymerase enzyme with chain displacement activity under isothermal conditions, without needing thermocycler or electrophoresis equipment ,57. The Several immunoassay methods have been developed for the diagnosis of serum antibodies and viral proteins (N and S) of SARS-CoV-2. Most commercial kits use enzyme-linked immunosorbent (ELISA), rapid lateral flow immunoassay (LFIA) assays, as well as IgM and IgG detection from the second week of viral infection . There aThe loop-mediated isothermal amplification (LAMP) method entails the amplification of DNA and RNA molecules, and is a technique with high efficiency, specificity, and rapidity. This technique uses four specially designed primers and a DNA polymerase enzyme with strand displacement activity to mark six sequences on targeted genetic material . If the Furthermore, field-deployable microchambers lead to the identification of 0.4 genome copies, among other acute respiratory disease viruses, such as H1N1 and H3N2 . There iThe analysis of results from the LAMP reaction usually is through gel electrophoresis or a turbidimeter (an instrument can quantitatively measure the intensity loss of light scattered from the suspended particles) ,80. HoweAs an example, a study engaged quenching probes in a fluorescent RT-LAMP assay to detect MERS-CoV. Quenching probes could solve the problem of solution turbidity during monitoring the process because a positive signal in this method just relates to the primer-reaction . AdditioRolling circle amplification is another enzymatic isothermal approach to detect the viruses. The final product of rolling circle amplification (RCA) is a concatemer, including hundreds of tandem repeats, which are complementary to the circular template. Since the RCA specifically amplifies the probe signal, it has higher sensitivity than PCR-based methods. PCR tests have the risk of amplicon cross-contamination because of the exponential amplification process, while RCA can be performed with minimal reagents and under isothermal conditions . Based oMicroarray assay is a microfluidic approach that can rapidly detect the virus RNA. After reverse transcription, the viral RNA produces cDNAs labeled with a specific probe to load in each well, followed by hybridization between oligonucleotides fixed on the microarray. Finally, free DNAs are removed, and the virus detected through specific probes . RecentlProkaryote cells remember an adaptive immunity memory by keeping several genetic elements of pathogenic agents in genomic loci called CRISPR arrays. So the application of CRISPR-Cas biology can help assist in the early diagnosis of infectious agents . Based oThere is a wide range of difference in the symptoms reported in patients, in a typical study, 44% of the patients in China had a fever before being under any therapies, but over 80% of them had developed trends in their fever after starting drug therapies. In that study, about 20% of the patients had shortness of breath; also, 68% of the patients had cough ; therefoThere are some approaches for point-of-care testing of the SARS-CoV-2 and related viruses, wherein most of them are based on the detection based on the lateral flow antigens. However, the advancements in this field are hard to achieve and optimize; therefore, using a different approach is highly suggested. However, for clarification purposes, generally, in the lateral flow antigens-based techniques, there is a paper-like membrane strip that has been chemically coated with metallic NPs conjugated with antibodies that also capture antibodies. The metallic NPs generally possess the active optical features, including Surface-Enhanced Raman Scattering (SERS) or Chelation Enhanced Fluorescence (CHEF) or other types of optical and fluorescence activity. Consequently, in commercial point-of-care testing methods that are based on lateral flow antigens, gold (Au) NPs have been used widely ,103,104.Herein, we want to provide practical ideas for the design and preparation of a highly sensitive coupled with higher accuracy systems for the point-of-care detection of SARS-CoV-2 even before infection. This method is based on using MOFs, which have been synthesized via a size-selective approach. A typical synthesis entails MOF-5@Au-nanorods , which h2O2 , with a virus specificity of 90%. After that, they demonstrated by using the SERS-based techniques, the abundance of viral-specific reads significantly increased from 4.1 to 31.8% for parainfluenza and from 0.08 to 0.44% for the influenza virus. This enrichment method, coupled with Raman techniques, constitutes an innovative system that could be used to quickly track and monitor viral outbreaks in real-time [Chemical interactions between analytes and substrate and magnetic field enhancement are effective in increasing the intensity of the Raman signal from 10 to 1014 . The SER to 1014 ,121,122.eal-time . Furthereal-time .Functionalized Magnetic NPs (MNPs) can be used as a potential candidate to extract viral RNA. Isolation of the virus RNA from several samples within 20 min by a rapid method via performing lysis and elution, all in one step, is one of the advantages of these NPs . The eliSuspended MNPs used in RNA extraction, and MNPs-based thin films have been suggested as potential substrates in SERS-based diagnostic techniques for COVID-19 RNA. In this assay, after binding the RNA virus to the substrate, the detection of the virus is possible by analyzing the Raman signals from the MNPs-substrate interaction with RNA viruses that can help develop diagnostic POC systems.2 [2 [Obviously, each technique has pros and cons, and SERS is no exception. Some of the most important disadvantages of SERS include the need for a homogenous sample, the substrate destruction over time that reduces the signal, as well as the ability to repeat SERS substrate signal in a substrate . However2 , and MnO2 [2 , etc., o2 [2 . HoweverToday, many research groups are working on diagnosing the Coronavirus, and several techniques have been proposed based on analytical chemistry principles. The great competition among scientists on the treatment and detection of this virus has had amazing results; however, the routine techniques require special laboratory facilities. More importantly, diagnosing the virus outside the human body and any living organisms can help mass prevent infection. In this regard, nanotechnology can help scientists to detect the virus outside the living organisms with high accuracy and selectivity. The purpose of this review article is to have a literature survey, with the purpose of providing a logical and scientific solution for detecting the virus at different levels using optical techniques. In this regard, the use of porous nanostructures that are capable of absorbing/adsorbing the virus is very important. For example, scientists can use different Au or Ag NPs and optimize them based on their size and surface functional groups. In the next step, the color of these NPs or modified nanostructures change as per the presence of a virus, or even the concentration of the virus in the air can be measured using different color spectra and fingerprint techniques ,132,133.Au NPs can be used as detection probes in virus surveillance because of their optical/electrical features ; they caThe optical properties of silver (Ag) NPs are similar to those of Au NPs. Silver NPs have been widely used in the SERS system in combination with different metal nanoarrays, which can be able to improve the plasmonic activity. Essentially, by using plasmonic techniques in assistance of Raman labeling with active components and applying electromagnetic field, different viruses such as HBV can be able to detect with a detection limit of 50aM . Moreove2O4 magnetic beads conjugated with influenza “A” antibody was produced to detect the target viruses. The final detection was performed visually based on fluorescence intensity by a fluorescence microscope evaluating R-phycoerythrin as fluorescence-dye; LOD described was as low as 0.007 hemagglutination units [Magnetic bead-based immunoassay is a technique centered on attaching antibodies to a magnetic bead, then separating the target from the sample by exerting a magnetic field. This process can be completely designed to ensure on a chip. For example, MnFeon units . Moreoveon units . Anotheron units .Based on the above-mentioned discussions, porous nanomaterials can be used for the detection of different pathogens. In this regard, the analyte, pathogen, does not need to be absorbed by the porous nanomaterials; however, the pathogen just needed to interact with the surface of the MOF that is modified by different NPs. By this interaction, different Off–On or On–Off optical mechanisms can be optimized to detect the pathogen, and in this case, different optical active components can be used as quenchers or activators ,146,147.However, there are other classes of MOFs composites which perform as a size-selective filter, followed by SERS to detect the low change of concentrations . A bimetA very important and often overlooked aspect that has not been addressed in this field and that can revolutionize the detection and prevention of the virus is its detection on masks or special clothes using optical techniques. Such a strategy can easily detect carriers and greatly prevent people with a low immune system from being infected by the virus. With that objective in mind, different coating layers can be used on the surface by using MOFs that have active metal NPs such as Au NPs and which in the presence of the SARS-CoV-2, changes the color of the NPs considerably, namely from red to blue, thus detecting even trace concentrations of the SARS-CoV-2. This hypothesis is based on recent research that reveals the sustenance of the SARS-CoV-2 in the air for more than 3 h and its movement on the aerosol particles up to 8 m. MOFs with different range of porosity, various sizes, and interconnected volumes can be synthesized. All of them have the ability to interact on the surface with cysteine-sensitive molecules, the active component of the SARS-CoV-2 proteins that are being targeted, as well as loading of different metals with active optical properties.As the COVID19 pandemic is exponentially spreading and affecting millions of innocent people around the world, this article aims to stimulate researchers to think creatively to generate new nanosystems for the prevention, diagnosis, and treatment, and significantly reduce the spread of the disease. To deal with such globally damaging virus, a well-coordinated, timely, fast, and effective response is the essence of mitigation efforts by humans.


Knowledge of the survival and stability of a pathogen is important for understanding its risk, reducing its transmission, and establishing control measures. Lassa virus is endemic in West Africa, causes severe disease, and is an emerging pathogen of concern. Our study examined the survival of Lassa virus in blood and tissue culture media at two different temperatures. The stability of Lassa virus held within a small particle aerosol was also measured. In liquids, Lassa virus was found to decay more quickly at 30 °C compared to room temperature. Sealed samples protected from environmental desiccation were more stable than samples open to the environment. In a small particle aerosol, the decay rate of Lassa virus was determined at 2.69% per minute. This information can contribute to risk assessments and inform mitigation strategies in the event of an outbreak of Lassa virus. Viruses frequently cause outbreaks of severe disease in humans. These outbreaks can be geographically limited or can quickly spread to infect an entire region or spread globally. There are a range of factors that facilitate the ease by which viruses can spread through a population. One such factor is the stability of the virus on surfaces or in an aerosol. Understanding virus survival is important for decision making to identify the risk from these pathogens and potential mitigation strategies. Lassa virus, a member of the Arenaviridae family, causes Lassa (haemorrhagic) fever and is responsible for approximately 5000 deaths a year. The disease, endemic in West Africa, is spread by rats; the main route of transmission is direct contact with rat excreta or items contaminated with rat faeces or urine ,2. NosocAlthough the main route of transmission is via contact with rodent excreta, there have been instances of nosocomial spread during outbreaks ,3,6,7. TTo determine virus stability on surfaces, Lassa virus was incubated either in Dulbecco’s Modified Eagle tissue culture medium (TCM) or rat’s blood  over a 48 h time period within polypropylene tubes. The virus was incubated at 30 °C or room temperature (RT: 19–22 °C); at both temperatures, virus was either left exposed (open tube) or sealed to the environment. Small volumes (20 µL) were used to mimic real-life scenarios in clinical settings, such as droplets generated by coughing or blood splatter. Droplets held at 30 °C or in open tubes at room temperature all dried. Virus in TCM and blood within sealed tubes at room temperature remained as droplets over 48 h. The titres of viable Lassa virus within blood or TCM maintained at 30 °C open, or sealed to the environment, decreased rapidly over 24 h. No virus or only viral titres at the limit of detection could be detected after 48 h B,D. The Survival data were fitted to the one-phase exponential decay model so the decay constant could be determined . Decay c-1. Viable titres of Lassa virus could still be quantified after 90 min in a dynamic aerosol . The biological decay rate of Lassa virus was defined by the decay constant, K, and determined to be −0.02726; this is equivalent to a biological decay rate of 2.69 % min aerosol . Under s aerosol  and indi aerosol .In small volumes of blood and tissue culture media, samples representative of common hospital and laboratory matrices, Lassa virus did not survive for long periods of time at an elevated temperature or when exposed to the open environment . Compared to other Hazard Group 4 viruses studied under similar conditions, Lassa virus was found to be less stable than Nipah virus, which could be detected after three days under identical conditions . When dr10 PFU/mL was reported [6 PFU/mL was reported in the blood of crab-eating macaques [3.6 TCID50/mL are associated with a case fatality rate of over 70% [High levels of viremia have been reported in non-human primates infected with Lassa virus ,14. In mreported  and >106over 70% . The titover 70% ,17 and tover 70% . InclusiIn an experimentally generated small particle aerosol held in dark conditions at 20 °C and 50–60% relative humidity, viable Lassa virus could still be detected after an hour. The stability of Lassa virus (Josiah strain) in an aerosol has been reported with a biological decay rate of 3.2% per minute and biological half-life of 21 min at 24 °C and 55% relative humidity . This waThe decay rate of Lassa virus in a small particle aerosol was similar to that of Influenza virus with a half-life of 32 min . Lassa vMany factors including environmental conditions and the phenotype of the virus (for example whether it is enveloped) as well as physical properties of surfaces contribute towards the survival of viruses on various surfaces (reviewed in ) and in 2 and was enumerated using the endpoint fifty percent tissue culture infectious dose (TCID50) assay. Briefly, Lassa virus was ten-fold serially diluted in 96 well plates previously seeded with Vero C1008 cells. After 5–7 days of incubation at 37 °C/5% CO2, all wells were observed under the microscope and scored for presence or absence of cytopathic effects. The 50% end-point was then calculated using the method of Reed and Muench [6 TCID50/mL. Lassa virus GA391 from a case in Nigeria  was kindd Muench . All wor50 assay being performed on the resuspended droplet. The experiment was performed on two separate occasions from the same initial starting stocks. Analysis was performed in GraphPad Prism v8. To determine the biological decay rate, a line of best fit was added using the one phase decay equation: Y = (Y0 − Plateau) * e−KX + Plateau, where Y0 is the Y value when X (time) is zero, Plateau is the Y value at infinite times and K is the rate constant. Studies were similar to those described previously with Nipah virus . Briefly50 assay. Data were corrected for the dilution effect of make-up air per time-point. To determine the biological decay rate, a line of best fit was added using the exponential decay equation (Y = Y0*ekX) where Y0 is the Y value when X (time) is zero and K is the rate constant. Harvested cell culture supernatant of Lassa virus was aerosolised using a Collison nebuliser and AeroMP system  to produce small particle aerosols of 1–3 µm diameter. A rotating Goldberg drum methodology was similar to that described previously ,21. Brie


Moreover, 9 of the 11 NIH model viruses with published in vivo data were detected by HTS with an equivalent or better sensitivity . Our general-purpose HTS method is unbiased and highly sensitive for the detection of adventitious viruses, and has a large breadth of detection, which may obviate the need to perform in vivo testing.High-throughput sequencing (HTS) is capable of broad virus detection encompassing both known and unknown adventitious viruses in a variety of sample matrices. We describe the development of a general-purpose HTS-based method for the detection of adventitious viruses. Performance was evaluated using 16 viruses equivalent to well-characterized National Institutes of Health (NIH) virus stocks and another six viruses of interest. A viral vaccine crude harvest and a cell substrate matrix were spiked with 22 viruses. Specificity was demonstrated for all 22 viruses at the species level. Our method was capable of detecting and identifying adventitious viruses spiked at 10 There have been a number of instances over previous decades where evidence for the presence of adventitious virus contamination in a marketed vaccine product has threatened public trust in immunization programs3. Examples include the detection of simian virus 40 (SV40) in the early polio vaccine in the 1960s, the finding of reverse transcriptase in measles and mumps vaccines in 1995 and of porcine circovirus (PCV) nucleic acid sequences and/or infectious circovirus in rotavirus vaccines in 20104.The potential contamination of biopharmaceuticals with adventitious  viruses poses a serious safety risk and threatens public confidence in the use of biopharmaceuticals. This is particularly true in the case of vaccines administered to large numbers of healthy people, including children6.These viruses could be unintentionally introduced at various manufacturing stages and may originate from multiple sources including raw materials, the cell substrate or the environment. Implementation of Good Manufacturing Practise and close monitoring of the manufacturing process can help reduce the likelihood of viral contamination. Therefore, adventitious virus testing at various stages of the manufacturing process is an integral part of the safety assessment for vaccines and other biological products, and there are well-defined regulatory requirements to ensure that these products are absent of adventitious viruses9. Non-specific adventitious virus testing of biological materials has typically included in vivo tests and cell culture-based in vitro tests, the breadth and sensitivity of which are presumed from historical experience rather than from systematic assay validation9. In addition, routine testing for adventitious viruses following the compendial requirements generally requires testing the cell substrate and the viral crude harvest (at seed lot and/or bulk levels). Both of these manufacturing stages present complex matrices for in vivo and in vitro testing. Targeted PCR-based virus detection methods require prior knowledge of the adventitious virus with respect to primer design/selection to target specific nucleic acid sequences. As such, there is a need for a broad-range detection method for both anticipated viruses, as well as unanticipated viruses.Gaps exist in the current compendial adventitious virus testing package where some viral families are not detected or incompletely detected by the compendial methods9. The results from the NIH study support the use of tests for broader detection of adventitious viruses, particularly where no suitable animal models or appropriate culture methods for detection exist. The World Health Organization 10 and the European Pharmacopoeia 13 recommend or require that prior to the implementation of new alternative methods to detect adventitious agents, the specificity and sensitivity of the new and existing methods must be compared; and whether the new method has at least the same sensitivity as in vivo methods should be determined  in 2014 compared the sensitivity of in vivo assays and in vitro cell culture tests using a panel of 16 viruses and found the in vitro tests to be more sensitive for detecting most of the tested viruses17. High-throughput molecular biology methods  had succeeded in detecting the contamination of Rotarix vaccine by a porcine circovirus18. HTS may also be more sensitive than quantitative PCR (qPCR)20; however the method may be overly sensitive to the detection of background and cross-contaminating viral nucleic acids originating from the laboratory environment or from other sources21. Viral genome size can also influence the sensitivity of HTS23 as sensitivity is expected to be proportional to the mass ratio of nucleic acids in a given matrix. Although HTS results for adventitious virus testing may differ between laboratories24, the development of well-characterized model virus stocks would support standardization and validation of the different HTS platforms22. Currently there are no established viral reference standards with corresponding in vivo data, for assessing new techniques for the detection of adventitious viruses.High-throughput sequencing (HTS) is a non-specific technique with the potential to detect both known and unknown adventitious agents including viruses9. Here, we describe the development of a general-purpose HTS-based test for the detection of adventitious viruses and its performance using virus stocks equivalent to the 16 NIH model viruses, plus another six viruses of interest. Detection of these viruses was assessed in a live Yellow Fever virus vaccine crude harvest matrix and a Vero cell substrate matrix to define the sensitivity (limit of detection [LOD]) and to demonstrate the specificity of the HTS test for adventitious virus testing.The panel of 16 virus stocks from the NIH is instrumental in our efforts to develop, qualify and validate the HTS adventitious virus test, by providing an important baseline against which new techniques for the detection of adventitious viruses can be comparedNucleic acid recovery for both the Yellow Fever Virus vaccine crude harvest and the Vero cell substrate matrix are shown in Supplementary Note 1.3 to 104 genome copies per mL of Yellow Fever Virus vaccine crude harvest. Specificity was assessed for all 22 viruses by determining whether the bioinformatics tool PhyloID was able to unequivocally detect each of the 22 spiked-in viruses and that none of these viruses was detected in the negative control (unspiked Yellow Fever virus vaccine crude harvest). Specificity was verified for all 22 viruses at the species level while specificity was verified for 20 of the 22 viruses at the strain level. For mammalian reovirus type 3 and coxsackievirus B3, the reads from the virus used for the spike were spread over two (for coxsackievirus B3) or three  reference sequences of the correct viral species.The number of sequence reads detected for each of the spiked viruses is listed in Table 5 to 103 genome copies per mL, most viruses were consistently detected at least at 104 genome copies per mL in viral vaccine crude harvest  at a spiking level of 106 cells per mL, 19 of the 22 viruses were detected at least at 104 genome copies per mL (0.01 genome copies per cell) in the cell substrate matrix, with potentially better sensitivity for some viruses. Influenza A virus, minute virus of mice and reovirus 3 were not detected at this highest spike level of 0.01 genome copies per cell.The number of sequence reads detected for each of the spiked viruses is listed in Table Preliminary evaluation indicates that the adventitious virus detection test using HTS can potentially achieve a limit of detection that is at or below 0.01 genome copies per cell in a Vero cell substrate matrix Table . 19 of t4 genome copies per mL. Of the viruses assessed, 21 were detectable at 104 genome copies per mL, with 16 of these at 103 genome copies per mL in a matrix containing 109 viral vaccine genome copies per mL . The LOD for a panel of 22 model adventitious viruses was assessed in a viral vaccine crude harvest matrix and a cell substrate matrix. Based on the results, the adventitious virus test using HTS technology is capable of detecting adventitious viruses at least at 104 genome copies per mL in the viral vaccine crude harvest matrix. Detection in the cell substrate matrix was at least at 0.01 viral genome copies per cell (104 genome copies per mL) for 19 of the 22 viruses. In a similar study evaluating the performance of HTS for the detection of adventitious viruses by spiking model viruses into a cellular matrix containing whole cells, all of the model viruses used  were detected when spiked at 3 genome copies per cell; only EBV and RSV were detected at 0.1 genome copies per cell22. This study showed that HTS can detect almost all viral contaminants evidenced by the large panel that was investigated, i.e., 22 different viruses that were enveloped/non-enveloped, single-stranded/double-stranded, RNA, DNA and of varying genome sizes. The use of the method for different sample types was also demonstrated, i.e., the nucleic acid recovery from the viral vaccine crude harvest matrix was higher than the cell substrate matrix, however a high proportion of contaminants was detected for both samples. The difference in detection levels observed for some of the viruses is likely attributed to a higher amount of background host nucleic acids from the cell substrate. Compared to the vaccine viral harvest, where even if the cells are lysed during manufacturing, the host cell nucleic acids are limited within the viral harvest, which is very different when testing the cell substrate matrix. These cells are lysed during sample preparation, releasing their nucleic acids, and will therefore make up a higher proportion of the sequence data leading to a poorer signal  to noise ratio and a decrease in the number of reads detected for the viral spikes. Overall, the successful detection of the spiked-in viruses in these complex backgrounds demonstrates the applicability of HTS for adventitious virus detection.Various reviews of HTS for adventitious agent detection have shown that there is a need for unbiased extraction methods, relevant controls, the use of spike recovery experiments, and quality control measures during library preparation11. In addition, such well-characterized materials can be used as a reference to help ensure satisfactory test performance in many matrices and thus, enable more meaningful comparisons between laboratories24. Importantly, the use of HTS is in-line with the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 with the aim, and the need, to reduce the number of animals used for scientific purposes25.Methods for the detection of adventitious agents continue to evolve and improve. The use of viral stocks equivalent to the well-characterized viral stocks from the NIH-supported studies allows us to compare our HTS performance against compendial in vivo testing, as proposed in the European Pharmacopoeia Chapter 5.2.1426. However, resolution at the strain level can be more difficult when counts are low and when strains are similar in their sequences, as the reads may get assigned to any of the reference genomes within that species. We are, however, working on improving the strain resolution of PhyloID.The approach to this study is based on European Pharmacopeia chapter 5.2.14 , a contract organization for Sanofi Pasteur, using plasmids containing synthetic nucleic acids of the targeted sequence to establish a standard curve; the resultant GC was used in addition to the infectious titer to convert the in vivo and in vitro LOD values established in the previous NIH study9 (expressed in infectious units) into equivalent genome copy numbers (the unit used for HTS detection)  were spiked at levels of 104 and 103 genome copies per mL . Replicate spikes were made at each spike level.Spiking experiments were designed based upon previous internal and collaborative studies32 and was undertaken with the Invitrogen PureLink Viral RNA/DNA Mini kit (Life Technologies cat #12280050) and the Wako® DNA Extractor Kit (WAKO cat #295-50201). cDNA synthesis was undertaken with the SuperScript® Double-Stranded cDNA Synthesis kit (Invitrogen cat #11917-010), followed by nucleic acid purification using the QIAquick PCR Purification Kit (Qiagen cat #28104). The nucleic acid extractions were assessed using the Agilent 2100 Bioanalyzer system.A schematic overview of the viral nucleic acid extraction protocol is presented in Fig. Sequencing library preparation was performed using the Illumina Nextera XT DNA Library Prep kit. Sequencing was carried out on the Illumina HiSeq1500. Paired-end sequencing was carried out at 2 ×151 bp. Sequence data were converted from the Bcl to FASTQ formats using the Illumina Bcl2Fastq2 software.26. Sequence data were trimmed, assembled into contigs and identified using a phylogenomic approach based on the profile of matches against reference data. Sensitivity was determined as the lowest concentration that a virus was conclusively positive .Data analysis was undertaken using PhyloID (a Sanofi Pasteur in-house bioinformatics tool developed for adventitious virus detection) and has been described previously9. The LOD achieved by the in vivo test methods, as described in the NIH study, was converted to genome copies based on a determination of genome content of the NIH stocks by Clean Cells to facilitate the comparison with the HTS studies described here.The limit of detection of adventitious viruses using HTS was compared to the data obtained from the NIH studyThe genome copies at LOD for the in vivo test were calculated using the formula:For example, for Herpes Simplex Virus Type 1:9, †As determined by Clean Cells from the NIH stock.*All obtained from the NIH stock(The limit of detection for viruses not detected in vivo is specified as greater than the genome copy number after conversion from the titer of the NIH stock virus [Supplementary Tables Supplementary Information


Optical spectroscopy can be used to quickly characterise the structural properties of individual molecules. However, it cannot be applied to biological assemblies because light is generally blind to the spatial distribution of the component molecules. This insensitivity arises from the mismatch in length scales between the assemblies (a few tens of nm) and the wavelength of light required to excite chromophores (≥150 nm). Consequently, with conventional spectroscopy, ordered assemblies, such as the icosahedral capsids of viruses, appear to be indistinguishable isotropic spherical objects. This limits potential routes to rapid high-throughput portable detection appropriate for point-of-care diagnostics. Here, we demonstrate that chiral electromagnetic (EM) near fields, which have both enhanced chiral asymmetry  and subwavelength spatial localisation (∼10 nm), can detect the icosahedral structure of virus capsids. Thus, they can detect both the presence and relative orientation of a bound virus capsid. To illustrate the potential uses of the exquisite structural sensitivity of subwavelength superchiral fields, we have used them to successfully detect virus particles in the complex milieu of blood serum. A technique that uses twisted light fields to detect biomolecular structures could find application as a low-cost clinical tool for screening viruses. The protein coatings around many viruses, such as the turnip yellow mosaic virus (TYMV), have complex polyhedral shapes that are difficult to resolve with conventional optical microscopes. Malcolm Kadodwala from the University of Glasgow and other colleagues in the United Kingdom now report that ‘superchiral’ light — localized fields generated by metal nanostructures that spiral as they travel — are sensitive to the asymmetric polyhedral of TYMV. By spectroscopic measurements of particle rotations in superchiral light at different frequencies, the team identified specific asymmetric signals that correlated to virus alignment on gold photonic substrates. This approach was then used to determine TYMV levels in human blood serum spiked with the virus. Thus, characterisation of the static and dynamic structural properties of biological assemblies is achieved through alternative techniques, diffraction and NMR, which lack the advantages of ease of use and rapidity of optical spectroscopy. In this work, we seek to span this length scale gap in the spectroscopic toolbox. We show that electromagnetic (EM) near fields of subwavelength extent that have enhanced chiral asymmetry  can probe the structure of biomolecular assemblies. To validate this hypothesis, we used chiral near fields to sense the chiral structure of a model biological assembly, a plant virus with an icosahedral capsid (turnip yellow mosaic virus (TYMV)) and thus detect its relative alignment on a surface.One of the markers of the transition from chemistry to biology is when individual molecular building blocks self-assemble into complex biological architectures. Optical spectroscopy provides a means of characterising the static and dynamic structural properties of individual molecules through probing of quantised states. However, optical spectroscopy cannot generally do the same for molecular assemblies3. In general, when molecules aggregate into larger assemblies, the electronic states of the monomer are not perturbed, and therefore, the spectroscopic response reflects the monomer and not the aggregate structure4. There are exceptions to this, such as J-aggregates, which have a different spectroscopic response than the individual component monomer4. However, this requires wavefunction mixing, which occurs for relatively simple aromatic molecules, that creates new electronic states correlated with the structure of the aggregate that provide a spectroscopic fingerprint. This does not occur for structurally and compositionally more complex biological assemblies (e.g. virus capsids formed by the assembly of protein molecules). In the absence of electronic perturbations upon aggregation, spectroscopy can discriminate between molecular assemblies that are strongly anisotropic (e.g. rod-like structures with high aspect ratios) and other assemblies. This arises because anisotropic aggregates have molecular polarisabilities with respect to the molecular reference frame that are not equivalent. Consequently, polarisation-dependent spectroscopic techniques, such as oriented circular dichroism (CD)6, linear dichroism (LD)9 and polarised Raman10, can be useful. However, in the general case of aggregates that are not strongly anisotropic, alignment does not provide a route to additional information. For example, spectroscopy is insensitive to the details of the icosahedral structures adopted by a vast array of viruses. This is because the size of the capsid is much smaller than the wavelength of light required to excite the chromophores of the coat proteins (IR/Vis/UV). Consequently, the electric field is uniform throughout the virus particle, and the spectroscopic response is insensitive to the spatial distribution of the coat proteins (CPs) in the capsid. Effectively, to an IR/Vis/UV photon, icosahedral capsids are spherical objects. Hence, spectroscopic characterisation of virus capsids is limited to fingerprinting the secondary structure and folds of the coat protein subunits with techniques such as UV/VIS CD11, vibrational CD (VCD)12 and Raman optical activity (ROA)16.Spectroscopy is sensitive to the structure of individual free floating  molecules in solution because they have electronic and vibrational states that provide structurally sensitive spectroscopic fingerprints20. These chiral near fields display strong spatial variations of both the intensity and chiral asymmetry on length scales ≤ the size of the virus capsid. It is this combination of subwavelength localisation and enhanced chiral asymmetry to which the enhanced structural sensitivity is ascribed.Near fields are localised nonpropagating EM fields created by light scattering from nanostructures. They vary spatially on a length scale 1–2 orders of magnitude smaller than the wavelength of light from which they are generated. Light scattering from chiral nanostructures creates near fields that in local regions of space, possess chiral asymmetries greater than the incident light, a property sometimes referred to as superchiralityTo validate the structural sensitivity of chiral near fields, we demonstrate a dependency of the interaction of a superchiral near field on the alignment of the TYMV particle. Any dependency on alignment provides definitive evidence that the superchiral near fields are sensitive to the structural details of the icosahedral virus capsid. Our results presage a spectroscopic approach for characterising the static and dynamic structural properties of viruses.T = 3, assembled from 180 subunits of an identical sequence (with 60 subunits forming 12 pentamers and 120 subunits forming 20 hexamers) coat protein (CP) with a molecular mass of 20,600 kDa and a diameter of 28 nm  is derived from the mirror symmetry breaking of the protein subunits.Plant viruses are ideal for this study because they are readily available in large quantities, are non-pathogenic to humans, have well characterised structures, and can be immobilised onto a surface with relatively well-defined orientations using biochemical techniques. TYMV has an unenveloped icosahedral capsid with a quasi-symmetry of Measurements were carried out using TYMV particles adsorbed from solution directly onto the substrate. It is assumed that TYMV will nonspecifically bind to the substrate, producing random orientations, hence creating an overall isotropic distribution. Varying levels of alignment of TYMV have been achieved using two surface immobilisation strategies Fig. . The fir23. They were ~100-nm thick and consisted of either left-handed (LH) or right-handed (RH) “shuriken” shaped indentations 25D is the displacement field, B is the magnetic induction and D and B are their respective time derivatives. In free space, the optical chirality density is conserved; however, in localised regions of space, C can be higher than that of equivalent circularly polarised light (CPL), a property that has been referred to as superchirality20. Numerical simulations have been performed to calculate the C of the near fields, as shown in Fig. The chiral asymmetry of a near field of frequency ω can be parametrised by the optical chirality density parameter (εr) εo is the (relative) permittivity of free space, and (μr) μ0 is the (relative) permeability of free space. E is the complex electric field, and H is the magnetic field. ξ(λ) is a wavelength-dependent second rank complex tensor describing chiral molecular properties, the sign of which is dependent on the handedness, and it is zero for achiral media. In the case that electric dipole–magnetic dipole interactions are the sole contributor to optical activity, ξ(λ) takes the formThe interaction of EM fields with chiral dielectrics  can be understood through the following constitutive equations:a = x, y, z) are the effective tensor elements of the virus particle, and n = 180. In the case of TYMV, both T = 3 icosahedron.For the TYMV particle, the chiral response is derived from a combination of the assembled coat proteins and the RNA,For the case of interaction with light,29. Constitutive Eqs.   29. Cons31 and modelling28 studies, which has been parametrised usingLH/RH are the shifts induced (compared to a reference) in the position of the bisignate ORD peaks for left-handed (LH) and right-handed (RH) structures by the introduction of a chiral dielectric (TYMV). If there is a nonchiral change in the dielectric environment of the near field region, then ΔΔλ = 0. In this study, we have derived ΔΔλ from the two extremes of the bisignate line shape referred to as peaks 1 and 2, which are labelled ΔΔλ1 and ΔΔλ2, respectively.In the current study, we have focussed on the asymmetry induced in the ORD spectra, an approach used in previous experimental24. Figure 1,2 parameters derived from these data are displayed in Fig. The optical properties of the TPS are sensitive to chiral materials, displaying equal and opposite asymmetries in optical properties when exposed to molecular enantiomers30.Turning first to the nonspecific case of TYMV binding directly to Au Fig. , greaterThe binding of TYMV-Thiol Fig.  is simil31.The mixed-Fab’ layers in isolation produce asymmetries in the ORD spectra, which is indicative of the expected well-defined orientation of the immobilised Fab’  interaction, defined by the equilibrium dissociation constant (Kd). The typical Kd values for antigen-antibody binding fall in the range of 10–100 pM32. Because the concentrations of TYMV used here, 10–100 nM, are much greater than Kd, saturated layers are achieved on the surface in this study. The different signs of ΔΔλ1,2 for TYMV-Thiol and TYMV-Fab’ can be attributed to the relative contributions of the RNA and protein terms in Eq. . When a functionalised TPS is immersed in serum spiked with TYMV, the virus particle will displace the nonspecifically bound layer. TPSs functionalised with the mixed-Fab’ layers were exposed to serum spiked with TYMV  plus a control of nonspiked serum for 120 min, after which ORD spectra were collected. Subsequently, to remove any potentially nonspecifically bound TYMV, the TPSs were washed in copious amounts of nonspiked serum, and then, ORD spectra were collected in the presence of nonspiked serum. The ORD spectra for these two experiments are shown in Figs. 1,2 parameters, shown in Fig. To illustrate a potential application of the enhanced structural sensitivity of superchiral near fields, we used them to detect TYMV spiked into blood serum. Serum is a complex biological fluid comprising all the components of blood apart from blood cells and clotting agents. This complex milieu contains >1000 components, spanning nine orders of magnitude in concentration. It includes many different types of chiral molecules, such as serum proteins, antibodies, antigens, hormones and sugars. When a TPS with mixed-Fab’ layers is exposed to serum, some serum proteins will nonspecifically interact with the Fab’ componentThe primary and most significant result of this work is the sensitivity of chiral near fields to the higher order (quaternary) structure of the virus capsid, established by the correlation between the level of orientational order and the magnitude of the optical asymmetries. The inherent novelty of the current work lies in the ability of chiral near fields to detect the icosahedral shape of a virus, a complex self-assembled biological aggregate, which is invisible to conventional spectroscopic phenomena that utilise CPL. This goes beyond the previous examples of using chiral near fields to probe/detect the lower-order structure of simpler biomolecules. Clearly, the reported phenomenon does not provide the rich structural information of high-resolution crystallography. However, the icosahedral TYMV capsid is sensed by the chiral fields, rather than appearing as an isotropic spherical object as in optical spectroscopic techniques. In effect, the chiral near field can detect not only the presence of a virus particle but also its orientation. This unique capability can be used to enhance the effectiveness of immunoassays for pathogenic virus detection. A virus particle binds to an immobilised antibody element via an epitope on the capsid surface. Consequently, the relative orientation of the virus is determined by where the epitope is on the capsid surface. The novel structural sensitivity of chiral near fields provides an additional discrimination mechanism that could offer a means of differentiating between, for instance, two closely related virus pathogens that may either bind to a recognition element via different epitopes or produce specific and nonspecific binding. The combination of the novel structural sensitivity with the binary binding/nonbinding functionality of conventional immunoassays offers the potential to mitigate false positive results.17. However, given the typical spectral acquisition times of ∼10 min, combining our low-cost disposable polycarbonate substrates with portable reflectance polarimetry33 would create an effective field diagnostic technology.If one wished, the reported experiments could be performed using a combination of expensive quartz substrates fabricated using electron beam lithography and commercially available spectrometersIn summary, using TYMV as a model, it has been demonstrated that superchiral EM fields with subwavelength spatial extent are, in contrast to normal light, sensitive to the higher order icosahedral symmetry of the virus capsid. We believe that in the context of the biophysical toolbox, the sensitivity of superchiral near fields is best exploited as a spectroscopic “triaging” tool, providing speedy low-resolution assessment of materials, which, if required, can subsequently be studied in more detail with costly low-throughput and high-resolution techniques. The ability of superchiral near fields to detect virus particles within serum provides a proof-of concept of the potential applications, presaging a novel label-free simple spectroscopic measurement for immunoassays for detecting viruses. The enhanced structural incisiveness of superchiral fields provides an additional parameter for discriminating between target and off-target interactions with an immobilised recognition element.ORD spectra were collected using a custom-made polarimeter that measures the reflected light from our samples. The design is similar to a basic reflected light microscope with a tungsten halogen light source (Thorlabs), Glan-Thompson polarisers (Thorlabs) and a ×10 objective (Olympus). A camera (Thorlabs) is used to position the sample, and spectra are collected using a compact spectrometer (Ocean optics USB4000). ORD spectra are obtained using the Stokes method, and the intensity of light is measured at four analyser angles . LH and RH pairs of ORD spectra are collected in 10 min.Electromagnetic (EM) simulations were performed using a commercial finite-element package . Periodic boundary conditions were used to emulate the metafilm arrays. Perfectly matched layer conditions were used above and below the input and output ports. Linearly polarised EM waves were applied at normal incidence onto the films. COMSOL uses the finite-element method to solve Maxwell’s equations for a specified geometry with the fields and optical chirality being measured at predefined surfaces above, within, and below the films.Brassica rapa var. pekinensis plants were grown under greenhouse conditions at 21 °C. Turnip yellow mosaic virus (TYMV) in crude sap extract mixed with abrasive celite was rub inoculated onto a true leaf of each Brassica rapa pekinensis plant grown to the first two true leaf stage of development. After 4 weeks, the plants developed strong systemic symptoms and were harvested, and total TYMV was isolated according to the protocols established by Leberman34 with the modifications suggested by Katouzian–Safadi and Berthet–Colominas35, with high speed centrifugations being carried out in a Beckman SW 41Ti rotor at 28,000 rpm for 3 h. The pelleted virus was resuspended in 10 mM Tris HCI pH 7.5 with 0.1 M EDTA and was subjected to CsCl density centrifugation in a gradient to obtain samples with densities of 1.26, 1.36 and 1.46, which were loaded into a 13.2 ml Thinwall Ultra-Clear tube (Beckman Coulter) and centrifuged in a SW 41Ti rotor at 28,000 rpm for 3 h. The upper band corresponding to the natural top component (empty virus) and the lower band corresponding to the bottom component (virus containing genome) were visualised under white light and isolated using a needle attached to a syringe. The samples were diluted and centrifuged in an SW 41Ti rotor as before to a pellet and were resuspended in an appropriate buffer, centrifuged to a pellet and resuspended in the buffer again to remove CsCl. The concentrations of the TYMV forms were estimated according to the protocols of Tamburro et al.36.The thiol groups were attached to the lysines at the virus surface by means of N-hydroxysuccinimide (NHS) ester chemistry, also called amine-reactive cross linker chemistry. At physiological pH, the amines of a protein are positively charged and therefore sit at the protein surface. These amines then become available for conjugation reagents. A fluorometric thiol assay was used to confirm virus thiolation.2 fragments were generated using the Pierce™ F(ab’)2 preparation kit (Thermo Fisher Scientific: 44988) following the manufacturer’s instructions. Briefly, a TYMV-specific IgG sample was loaded onto a prewashed Zeba Spin Column using digestion buffer  and centrifuged at 5000 × g for 1 min (Eppendorf Centrifuge 5415D) to collect the desalted sample. The immobilised pepsin resin was prepared by adding 65 µL of the 50% slurry placed into the 0.8 µL spin column, followed by centrifugation at 5000 × g for 1 min. The buffer was discarded, and the resin was washed with 130 µL of digestion buffer, followed by centrifugation at 5000 × g for 1 min. The flow through was discarded, and 125 µL of desalted TYMV-IgG was loaded onto the spin column with a capped bottom containing the pepsin resin. The sample was briefly mixed using a vortex. The digestion reaction was incubated for 2 h using an end-over-end mixer in a static 37 °C incubator. Next, the bottom cap was removed, and the spin column was placed into a microcentrifuge tube. The sample was centrifuged at 5000 × g for 1 min to separate the digest from the immobilised pepsin. The resin was washed twice with 130 µL of PBS  by centrifugation at 5000 × g for 1 min. The flow through containing F(ab’)2 fragments was collected, and the immobilised pepsin was discarded. To remove any undigested IgG, the F(ab’)2 fragments were further purified using a Nab protein A Plus Spin Column. The equilibrated column was washed twice with 400 µL of PBS by centrifugation at 5000 × g for 1 min. The bottom of the column was capped, and the sample was added to the column. The samples and resin were resuspended by inversion followed by a 10 min incubation at room temperature with an end-over-end mixer. Next, the column was placed in a new collection tube, and the flow through containing F(ab’)2 was collected by centrifugation at 5000 × g for 1 min. The column was washed twice with 200 µL of PBS by centrifugation at 5000 × g for 1 min, and both wash fractions were added to the sample. The protein concentration was determined using the Pierce™ BCA Protein assay kit (Thermo Fisher Scientific: 23225) following the manufacturer’s instructions. To assess digestion and purification, the samples were analysed by SDS-PAGE using non-reducing loading dye and NuPAGE™ 4–12% Bis-Tris protein gels (Thermo Fisher Scientific: NP0336BOX) and SimplyBlue™ SafeStain Invitrogen™.TYMV-specific rabbit polyclonal IgG was purchased from DSMZ AS-0125), and antibody specificity against the TYMV coat protein (CP) was confirmed by western blot analysis 2 fragments, with a small portion of the digest representing Fab’ fragments, most likely due to over-digestion 41. EG-thiol is a neutral spacer molecule with biorepellent properties, so any interactions between Fab’ molecules and a surface will be minimised42. This layer will subsequently be referred to as a mixed-Fab’ layer. When tested against buffered solutions of single proteins, similar mixed-Fab’ layers were observed to not only retain specificity to the original target but also display nonspecific interactions with other types of protein molecules30. The Fab’ will bind to a limited range of epitopes (or a single epitope) of TYMV, thereby generating a narrow distribution of orientations relative to the nonspecific interactions of the unfunctionalised surface.Polyclonal antibodies are an ensemble of antibodies that can recognise multiple epitopes on an antigen. A monoclonal antibody, by contrast, displays greater specificity, binding uniquely to a single epitope of a macromolecular antigen such as a protein. In the current study, we immobilised onto the TPS a fragment derived from polyclonal rabbit IgG that had been produced against TYMV, referred to as poly anti-TYMV-IgG. Western blot analysis was used to confirm antibody specificity for the 20 kDa TYMV-CP  in distilled water. Serum (both nonspiked and spiked) solutions with a concentration ranging from 1 mg ml−1 were produced by diluting the stock serum solutions with 10 mM PBS buffer, pH = 7.4. Spiked serum samples were produced by adding the relevant amount of TYMV to the 1 mg ml−1 serum solution.The TPSs were enclosed in a 100 μL cell, with TYMV and TYMV-Thiol adsorbed from a 10 mM PBS buffer, pH = 7.4. Stock serum solutions with a total protein concentration of 60 mg mlSupplementary Information