The Statistical Accounts of Scotland 1791−1845 describes him as "a singular instance of an uncommon variety of talents, being a tailor, a joiner, a clock and watch-mender, a blacksmith, and a physician." He was also, at various times in his life, a farmer, fisherman, and weaver. A self-taught physician, Williamson developed and administered smallpox inoculations for an estimated 3,000 patients during the late 18th century, primarily during the decade before Edward Jenner developed his smallpox vaccine in 1796. The procedure Williamson used, known as variolation, had been used for thousands of years, and it involves taking live virus from a smallpox patient and “inoculating” that virus onto the skin of a recipient who did not have the disease. The wig-stretching block appearing on this month’s cover is thought to be the only surviving work from John Williamson, who lived on the Shetland Islands of Scotland. Williamson, perhaps better known by his nickname “Johnnie Notions” (sometimes spelled Johnie), proved to be skilled at many trades. The Statistical Accounts of Scotland 1791−1845, which provides this account of Williamson’s work: Ian D. Conacher, a physician and researcher, and Brian Smith, an archivist and author with the Shetland Archives, have each authored detailed accounts of Williamson’s life and his work as an inoculator. Both authors provide historical context for how smallpox was introduced from the mainland of Scotland to Shetland and the devastating consequences resulting from a series of smallpox outbreaks, curiously occurring in 20-year intervals . Both also draw from “Williamson was a local pioneer in the use of vaccines against smallpox. Unassisted by education, and unfettered by the rules of art. He is careful in providing the best matter and keeps it a long time before he puts it to use - sometimes 7 or 8 years. And, in order to lessen its virulence, he first dries it in peat smoke, and then puts it under the ground covered in camphor....by a small knife, made by his own hand, he gently raises a very little of the outer skin of the arm ....then puts in a very small quantity of the matter....The only plaister he uses, for healing the wound, is a bit of cabbage leaf.”Conacher noted that there is no evidence that Williamson caused any cases of smallpox or deaths among the people he inoculated. However, it is difficult to determine whether his techniques to reduce virulence of the virus were always successful. Variolation was inherently risky, in that, for some, the process could result in a full-blown case of smallpox. Regardless of the resulting severity, the individual recipient could then transmit the virulent smallpox virus to others. According to the Shetland Museum and Archives, Williamson created the wig block for the Cheyne family, lairds at Tangwick Haa, Northmavine, Shetland, and “The face is modelled on an old man suffering from smallpox, the wood chosen because it was naturally marked with similar scarring.” The archivist Smith wrote that “There is a strong tradition in Shetland that ‘Johnie Notions’ used as his model for this stretcher an old man from Hillswick whose face was marked with smallpox.” A careful observer might conclude that, given the few pockmarks on the face, the man had experienced a relatively mild case of smallpox. During that time in Europe, wigs were often favored by the wealthy and aristocratic classes but not simply to signal wealth and social status. Syphilis, which was widespread across Europe, caused hair loss and skin sores, so wigs helped disguise those symptoms. Blocks such as the one Williamson created from a piece of pine were essential for preening, shaping, and maintaining wigs. On the Origin of the Vaccine Inoculation, Jenner wrote, “the annihilation of the smallpox, the most dreadful scourge of the human species, must be the final result of this practice.” During the last years of Williamson’s life, Jenner developed the first vaccine against smallpox and envisioned the elimination of smallpox. In his 1801 treatise A good measure of control was achieved in many developed countries, largely via mass vaccination campaigns. However, because of poor health infrastructures in most developing countries, mass campaigns were not very successful, and smallpox circulation continued into the 20th century. In the 1960s, the deployment of an approach known as surveillance and containment—in which cases of smallpox were identified and traced and all those exposed and nearby were vaccinated—proved successful. Consequently, in 1980, the World Health Organization declared smallpox to be eradicated. Although the smallpox virus (variola virus), the best known of the poxviruses affecting humans, no longer exists naturally, other poxviruses, including monkeypox virus, orf virus, molluscum contagiosum, yatapoxviruses, and parapoxviruses, can infect humans or other animals. The recent epidemic of mpox highlights the importance of continued vigilance, response, prevention, and innovation in public health efforts to control and treat diseases caused by poxviruses.


To investigate the seasonal, monthly, and daily distributions of the incidence of Stanford type-A acute aortic dissection (TAAAD) and identify seasonality in the duration of hospital stay and in-hospital mortality of TAAAD in south-eastern China.2 test was used to compare in-hospital mortality among the four groups. Non-parametric methods were used for all comparisons of the duration of hospital stay. Univariate logistic and multivariable logistic regression analyses were performed to assess the duration of hospital stay.We enrolled patients diagnosed with TAAAD between 1 June 2017 and 31 May 2021. Participants were divided into seasonal, monthly, and daily groups according to the need for analysis. Analysis of variance was applied to compare the number of TAAAD in different seasons, months, and days. χP = 0.04, P < 0.01, and P < 0.01, respectively). This study did not identify any significant decrease in maximal, mean, or minimum temperatures between the three days before TAAAD and the day of TAAAD. No seasonal variations associated with in-hospital mortality was observed (P = 0.89). However, significant differences were observed in the seasonal distribution of the duration of hospital stay for TAAAD . Multiple factor analysis showed that winter was the independent risk factor for the increased duration of hospital stay. The odds ratio for winter was 2.21 .Of the 485 patients, 154 were diagnosed in winter (31.8%), 115 in spring (23.7%), 73 in summer (15.1%), and 143 in autumn (29.5%). The daily, monthly, and seasonal distributions of TAAAD were significantly different (Our study confirmed that the incidence of TAAAD exhibits seasonal, monthly, and daily variations in south-eastern China. Moreover, the daily incidence of TAAAD is higher on weekdays than that on weekends.The online version contains supplementary material available at 10.1186/s13019-023-02222-4. Type-A acute aortic dissection (AAD) (TAAAD) involves the ascending aorta, and is a life-threatening disease, with an incidence of approximately 2.0 cases per 100,000/year . TherefoThis study included all consecutive patients admitted to our department with a diagnosis of TAAAD between 1 June 2017 and 31 May 2021. We included patients who (1) came from Fujian province and nearby areas and (2) had a diagnosis of TAAAD, as confirmed by computed tomography angiography or magnetic resonance angiography. We excluded patients if (1) the exact date of TAAAD symptom onset was unknown, (2) they had a clear aetiology such as iatrogenic AD secondary to cardiac surgery, Loeys-Dietz syndrome, Marfan syndrome, or a history of surgery for AD, and (3) they had a history of other cardiac surgery or chronic aortic dissections. This retrospective study was approved by the Ethics Committee of our Hospital and was conducted in accordance with the Declaration of Helsinki. The Strengthening the Reporting of Observational studies in Epidemiology guidelines were followed in this retrospective study , laboratory inspections, and hospital admission . We gathered data on in-hospital outcomes from medical records.The patients in our study were divided into seasonal groups in south-eastern China, , monthly groups, or daily groups according to our need for analysis.This study was conducted in Fuzhou (south-eastern China in Fujian province). This area has a subtropical monsoon climate. The mean daily temperatures range between 3 and 10 °C in winter and between 22 and 32 °C in summer. Daily regional meteorological data were retrieved from the Fuzhou Meteorological Service. The following parameters were assessed each day: minimum, mean, and maximum air temperatures (°C).2 test was used to compare baseline characteristics among the seasonal groups. χ2 test was used to analyse the differences in in-hospital mortality among the seasonal groups. Non-parametric methods were used for all comparisons of the duration of hospital stay. The cut-off point was considered according to the average duration of hospital stay of all patients, and variables with P<0.20 on univariate analysis were selected to perform multivariate logistic regression analysis on the related risk factors of patients, using generalized additive models to explore changes in weather and aortic dissection. The statistical significance of all tests was defined as p < 0.05 (two-sided). Statistical analyses were performed using SPSS V.22.0 and R software V.3.4.2. For individual cases of lost data, we used the mode principle of statistics to replace the missing values. The cases with several missing values (more than three) were omitted.Continuous variables were reported as mean±standard deviation (SD) or median and interquartile range (IQR) according to whether they followed a Gaussian distribution. Analysis of variance or χP < 0.01).Between 1 June 2017 and 31 May 2021, 485 consecutive patients diagnosed with TAAAD in our department were enrolled in this study; of these, 154 cases of TAAAD occurred in winter (31.8%), 115 in spring (23.7%), 73 in summer (15.1%), and 143 in autumn (29. 5%) . Moreover, the frequency of TAAAD in January was three times higher than that in July (P < 0.01), and the daily distribution in TAAAD was significantly different (P = 0.04). The frequency of TAAAD was higher on weekdays than that on weekends (P < 0.01). The generalized additive models showed that months and average temperature have a non-linear relationship with the incidence of TAAAD. The lower the average temperature, the more the cases of TAAAD. Similarly, the colder the months (winter), the more the cases of TAAAD , as presented in Fig. Significant variations in TAAAD symptom onset were investigated; the onset of TAAAD was 2.11 times higher in winter than that in summer (The variations in atmospheric temperatures during the three days preceding TAAAD are presented in Table P = 0.89). However, the seasonal distribution of the duration of hospital stay for TAAAD revealed significant differences (P < 0.01). The duration of hospital stay for TAAAD was higher in winter than that in spring (P = 0.04) and autumn (P = 0.03). Univariate analysis of the duration of hospital stay showed that winter, autumn, coronary malperfusion, cerebral malperfusion, acute renal failure, acute aortic regurgitation, and serum creatinine are risk factors for the increased duration of hospital stay. Multiple factor analysis showed that winter was the independent risk factor for the increased duration of hospital stay. The odds ratio of winter was 2.21 , P < 0.01 (Table The in-hospital mortality and duration of hospital stay in patients with TAAAD onset in different seasons are presented in Tables 01 Table .Table 3IOur findings confirm that the incidence of TAAAD varies seasonally, monthly, and daily in south-eastern China. In this study, the occurrence of TAAAD was independent of the rapid change in atmospheric temperature. Moreover, the distribution of in-hospital mortality in patients with TAAAD did not show seasonal variations. However, the seasonal distribution of the duration of hospital stay for TAAAD was inhomogeneous in this study.A series of studies showed that the fall and winter months represent a high-risk temporal frame for the occurrence of aortic dissections , 21, 22,In the present study, we found a distribution of the daily incidence of AAD, with a significantly higher incidence on weekdays than that on weekends. This finding is similar to that reported by Karangelis et al. . A similBenouaich et al. suggested that the relative change in temperature, rather than absolute temperature, is a mechanistic factor of TAAAD . HoweverPrevious studies investigated whether the onset season acted as a trigger in the prognostic value of patients with AAD; however, no association with seasonal variability could be found on the patients’ outcome and in-hospital mortality , 29. In Our analysis is the first to suggest that the duration of hospital stay is affected by the season of disease onset. Compared with spring and autumn, the duration of hospital stay in winter was the lowest. The in-hospital mortality was the highest in winter, and many patients were in serious condition and were extremely late to be cured; this is in accordance with the duration of hospital stay in winter, which was the lowest. However, there was no significant difference in the duration of hospital stay between winter and summer. There is a possibility that the number of hospitalized patients in summer is relatively less compared to that during other seasons, meaning that the duration of hospital stay is lesser. Further studies are needed to identify the factors that affect seasonal changes in hospitalization duration.This study has some important limitations. A major limitation of our study is that our meteorological data did not include atmospheric pressure, humidity, and other specific climate data, which may provide more details regarding the reason for seasonal variability. Second, the number of preceding days that were investigated may be insufficient, meaning that the influence of rapid alterations in atmospheric temperature on TAAAD were not accurately investigated. Third, our study had a single-centre design in one province, and the sample size of this study was small. Additionally, the findings might not necessarily be reflected in other countries or cities with different climates. China has a large land area; thus, follow-up studies on the differences between the south and north and the east and west, and a multi-centre study are our targets. At present, the research period is four years, which may lead to some bias in the research results.Our study confirmed that the incidence of TAAAD exhibits seasonal, monthly, and daily variations in south-eastern China. Moreover, the daily incidence of TAAAD is higher on weekdays than that on weekends. This could promote the formulation of effective prevention strategies during the colder months, and further studies should seek to clarify the pathophysiology of seasonality in the incidence of TAAAD.Additional file 1. STROBE Guidelines.


HighlightsSimian Haemorrhagic Fever Virus (SHFV) poses a great threat to African primates by causing fatal haemorrhagic fever.Researchers have reported CD163, as an intracellular receptor for SHFV.Replication competency of SHFV in human monocytes further increases the concern.Dear Editor,The global population is witnessing the emergence of infectious zoonotic diseases due to altered environmental homeostasis by humans at the peak of evolution. Remarkably, primitive microbes mutate and adapt according to the need or turmoil. Anthropogenic activities, such as civilisation, industrialisation, and deforestation, have brought humans and wild animals in close proximity, resulting in the spillover of new pathogens. Diseases like Influenza, Ebola, Marburg, Zika, Boca, SARS-CoV (severe acute respiratory syndrome coronavirus), MERS-CoV (Middle East respiratory syndrome coronavirus), Lassa, Nipah, and Hendra, originating in response to natural infringement, have spread globally. Additionally, many pathogens are on the verge of spillover to humans after a long and continuous adaptation process, owing to continuous contact with human cells followed by a series of mutations. Many distinctive pathogens circulate among domestic and wild animals worldwide without causing symptoms. An increasing number of unique pathogens, like animal viruses, have jumped to humans and wreaked havoc on naive immune systems by unravelling, gaining access to cells, multiplying, and escaping the body’s defence mechanisms. The Simian Haemorrhagic Fever Virus (SHFV), endemic in monkeys and some African primates, poses a great risk of spillover in the near future.Arteriviridae, first isolated in 1964 from rhesus macaques. Arteriviruses include equine arteritis virus, porcine reproductive and respiratory syndrome virus, SHFV, lactate dehydrogenase-elevating virus, and wobbly possum disease virus, and their hosts include horses, pigs, possums, nonhuman primates, and rodents. Various African nonhuman primates, such as patas monkeys, vervets, Guinea baboons, and olive baboons, have been implicated as potential carriersSHFV is a single-stranded, positive-sense RNA virus within the family SHFV poses a great threat to African primates by causing fatal haemorrhagic fevers and jumping species barriers. Interestingly, researchers have reported CD163 as an intracellular receptor for SHFV, which serves as a rare mode of virus entry shared with other haemorrhagic fever viruses such as Ebola virus and Lassa virushttps://www.news-medical.net/news/20220930/Obscure-family-of-viruses-already-endemic-in-African-monkeys-poised-for-spillover-to-humans.aspx). These findings suggest that it is necessary to monitor SHFV to avoid another possible pandemic.Remarkably, profound similarities have been reported in SHFVs and simian viruses responsible for the human immunodeficiency virus (HIV) pandemic. Like HIV and simian immunodeficiency virus (SIV), SHFV has also been reported to successfully evade human cells by disabling protective immune mechanisms, thus taking long-term hold in the body (Although SHFV infections have not been reported in humans, studies on simian arteriviruses must be prioritised by global health communities, considering its spillover potential. High arterivirus viral loads have been reported in asymptomatic African monkeys, which frequently interact with human populations. HIV has been reported to originate from SIVs in African nonhuman primates and jumped to humans during the early 1900s. It caused fatalities in humans in the 1980s in the United States, but no serological tests or treatments were available. Thus, although jumping the species barrier in SHFV is uncertain, more animal viruses are likely to spread to humans. As humans are immunologically naive to SHFV, the development of serological tests for serosurveillance of high-risk populations in close contact with animal carriers is needed. Moreover, raising awareness and continuously monitoring viruses with zoonotic potential is critical for the rapid containment of possible future spillovers.Globalisation, human settlements in forests, and increased international travel have enabled the rapid spread of pathogens than ever before. Historically, it took years or even decades for diseases to spread between countries and continents, allowing many previously unexplored regions of the world to remain disease-free; for example the Black Death happened over 150 years before Columbus’s first expedition to the Americas, leaving these continents largely unaffected. Based on the ‘One Health’ perspective and experiences from past epidemics and pandemics, drivers of spillover events can be impeded by strategic planningNot applicable.Not applicable.None.All the authors substantially contributed to this work.There are no conflicts of interest.Not commissioned, externally peer-reviewed.


Compared to Group 1, Group 3  and Group 4  were adversely associated with favorable outcomes. FPR was associated with functional outcome in a time-dependent manner. Even for patients who have achieved FPR, their functional outcome might not be favorable if the P-to-R time is >30 min.Clinical benefit can be time-dependent even after first-pass recanalization (FPR) in endovascular treatment of acute stroke. This study aimed to evaluate the association between favorable outcome and FPR under a specific time frame. Patients who underwent mechanical thrombectomy were retrospectively reviewed. Recanalization status was categorized into four groups based on FPR and dichotomized time from groin puncture to recanalization (P-to-R time). Favorable outcomes were compared between groups. A total of 458 patients were included. As the cutoff of P-to-R time for favorable outcome was 30 min, recanalization status was categorized into FPR (+) with a P-to-R time ≤ 30 min (Group 1), FPR (–) with a P-to-R time ≤ 30 min (Group 2), FPR (+) with a P-to-R time > 30 min (Group 3), and FPR (–) with a P-to-R time > 30 min (Group 4). Favorable outcomes in Group 3 (37.5%) were significantly less frequent than those in Group 1 (60.4%,  Achieving significant recanalization is an ultimate goal of endovascular treatment of acute ischemic stroke . A higheHowever, not all FPRs are beneficial. Although a single pass in FPR is a surrogate for rapid recanalization, FPR cannot be always rapid. For example, a single pass of FPR can be delayed due to various clinical situations such as a patient’s agitation, operator’s experience, and any technical difficulties including a severely tortuous artery. Thus, FPR theoretically might not be quicker than multiple-pass recanalization. Considering that FPR is derived from the basis of rapid recanalization, the time to achieve FPR could be quite critical for clinical benefit from FPR. In other words, the clinical benefit of FPR can be time-dependent. However, the relationship between FPR and time to recanalization has not been reported yet. From a practical viewpoint, if one can determine the time limit to guarantee a clinical benefit from FPR, it would be so helpful to set an optimal endovascular strategy.Accordingly, we hypothesized that favorable outcomes by FPR would be time-dependent, which meant that favorable outcomes would be not guaranteed if the time to achieve FPR was delayed. We aimed to evaluate the association between favorable outcomes and FPR under a specific time frame.We retrospectively reviewed consecutive patients with acute intracranial large vessel occlusion who underwent endovascular treatment between 2010 and 2021 in a tertiary stroke center. Endovascular treatment was generally considered for patients who met the following criteria: (1) a computed tomography (CT) angiography-determined endovascularly accessible intracranial vessel occlusion associated with neurological symptoms; (2) age ≥ 19 years; (3) baseline National Institutes of Health Stroke Scale (NIHSS) score ≥ 4; (4) time from stroke onset to groin puncture < 24 h; (5) preprocedural CT–Alberta Stroke Program Early Computed Tomography Score (CT-ASPECTS) ≥ 6; and (6) for patients with time from stroke onset > 6 h, eligibility criteria of DWI or CTP Assessment with Clinical Mismatch in the Triage of Wake-Up and Late Presenting Strokes Undergoing Neurointervention with Trevo (DAWN) and Diffusion and Perfusion Imaging Evaluation for Understanding Stroke Evolution (DEFUSE 3) trials were also considered. We preferably performed endovascular treatment in patients with a premorbid modified Rankin Scale (mRS) score ≤ 3. Patients eligible for intravenous tissue-type plasminogen activator (tPA) treatment were treated with 0.9 mg/kg tPA.As this study focused on endovascular responses to mechanical thrombectomy, we excluded patients who had a specific occlusion etiology  and those who did not undergo a mechanical thrombectomy. Posterior circulation strokes were also excluded because clinical outcomes would be quite disparate between anterior and posterior circulation strokes. To evaluate the particular role of FPR in mechanical thrombectomy, this study included only patients with successful recanalization. The Institutional Review Board approved this study and waived the requirement of informed consent owing to the retrospective nature of this study.All endovascular procedures were performed under local anesthesia. Conscious sedation was administered when necessary. The choice between a stent retriever and a contact aspiration thrombectomy was made at the discretion of the operator. However, in most cases, a stent retriever was used as a front-line endovascular modality. An 8- or 9-F balloon guide catheter (BGC) was routinely used. A distal access catheter was hardly used. It was only used for severely tortuous arteries. A mechanical thrombectomy procedure was performed according to common recommendation ,13. BrieData of all variables used in this study were basically collected from a registry of patients with acute stroke. Successful recanalization was defined as a final mTICI grade 2b or 3 without further reocclusion during the procedure. FPR was defined as near-complete or complete revascularization (eTICI grade 2c or 3) after the first pass of the thrombectomy device ,11. For Functional outcome was assessed based on the mRS score at 3 months after stroke onset. Favorable outcome was defined as mRS scores of 0, 1, or 2. These mRS scores were primarily evaluated by stroke neurologists during patients’ routine clinic follow-up at 3 months (±2 weeks). If a patient could not visit the clinic, a stroke neurologist or trained nurse interviewed the patient or their family via telephone to determine the mRS score.p-values derived from multiple comparisons were adjusted by the Benjamini–Hochberg procedure. Third, to observe the independence of recanalization status for favorable outcome, multivariable logistic regression analysis was performed by adjusting for other variables with p < 0.1 in univariable analyses. Raw values of P-to-R time or time from stroke onset to recanalization  time) and the number of passes of the thrombectomy device were not entered into the multivariable model because they were already incorporated into the concept of recanalization status.First, to set a new recanalization status by FPR and the time from groin puncture to recanalization  time), we tried to find a relevant P-to-R time associated with a favorable outcome. For this, we performed receiver operating characteristic (ROC) curve analysis and determined an optimal P-to-R time as the cutoff to predict favorable outcomes using the Youden index. The P-to-R time was then dichotomized based on the cutoff value. Recanalization status was categorized into four ordinal groups using a combination of FPR and the dichotomized P-to-R time. Second, to evaluate the association between the recanalization status and functional outcome, frequencies of favorable outcome were compared between four groups of recanalization status, along with a variety of clinical and endovascular variables. For trend analysis, Pearson or Spearman correlation tests and Cochran–Armitage trend tests were performed for continuous and categorical variables, respectively. To observe particular differences in P-to-R time and favorable outcome across the recanalization statuses, they were compared between the four individual groups of recanalization status using the Mann–Whitney U test and χ2 test. All p < 0.05 with a 95% confidence interval (CI). All statistical analyses were performed using the R software .Statistical significance was set at A total of 458 patients  were finally included . The inip < 0.001). Among patients with FPR, 70.6% (96 of 136) could have recanalization within 30 min (P-to-R time ≤ 30 min). However, it was 26.1% (84 of 322) for patients who did not have FPR  value, 0.636 ; sensitivity, 51.9%; specificity, 71.2%; have FPR . Using ap-value for trend < 0.001 in p < 0.001; p < 0.001) different between Group 3  and Group 4 . Likewise, O-to-R time became longer with increasing group number from Group 1 to Group 4 . However, despite FPR, patients in Group 3 had less favorable functional outcomes than those in Group 2 (p = 0.033) and Group 1 (p = 0.029). In the case of FPR, probability of favorable outcome was decreased as P-to-R time increased . However, Group 3  and Group 4  were adversely associated with favorable outcome.Recanalization status was significantly associated with favorable outcome. Functional outcome was less favorable in Group 3 and Group 4  even if FPR was achieved, favorable outcome might not be guaranteed if the P-to-R time was > 30 min, such as those in Group 3; and (2) for patients who had a rapid recanalization without FPR, functional outcome might be as favorable as those with FPR if the P-to-R time was ≤ 30 min, such as those in Group 2. Accordingly, the effect of FPR on favorable outcome seems to be time-dependent. For better functional outcomes, as always, one might need to focus on the P-to-R time in addition to achieving FPR.Beyond the traditional endovascular endpoint, FPR has recently been highlighted to achieve the best endovascular performance. The conceptual relevance of FPR has also been demonstrated in several studies as it is significantly associated with superior clinical outcomes ,10,11,16For practical purposes, a continuous time frame can be segmented into the number of passes of the thrombectomy device . Among tOne interesting finding of this study was that FPR was not associated with functional outcome if only the P-to-R time was controlled by a relevant threshold. As observed, functional outcomes were comparable in early groups (between Group 1 and Group 2) and late groups (Group 3 and Group 4). In subgroup analyses, FPR and P-to-R time were not associated with favorable outcome for early groups  or late Although this study broke the common belief that FPR would be omnipotent for better clinical outcome, we still think that FPR should be achieved. Under the common practical condition, as shown in this study, more than 70% of patients with FPR could have their recanalization with a P-to-R time ≤ 30 min. Achieving a rapid FPR might be the only modifiable procedural factor for better clinical outcome. Moreover, without a time frame, lone FPR was still significantly associated with favorable outcome.It is obvious that first-pass recanalization is a reliable and achievable goal to represent the better endovascular performance in acute stroke. However, a single pass of thrombectomy device is only a surrogate for a segmented time frame. Rapidity inherent in first-pass recanalization seems to have been overrated. This study highlighted the necessity of additional effort for more rapid recanalization even in a single pass of a thrombectomy device. In fact, an endovascular endpoint has evolved over the years to make the clinical outcome better, from simple reperfusion grade  to first-pass recanalization. This study might arouse a specific condition to maximize the clinical benefit of first-pass recanalization.® was exclusively used. Thus, FPR, a chief variable in this study, did not appear to be seriously affected by the retrospective study design.This study had limitations. First, as this study was performed retrospectively, endovascular procedures were not protocolized. Many were decided at the operator’s discretion. This might have affected FPR in this study. However, endovascular procedures were performed homogeneously in a single center. We strictly followed the general common methods for mechanical thrombectomy and attempted to minimize variations, such as combining a stent retriever and an aspiration catheter. Moreover, stent retriever thrombectomy was chosen as the front-line treatment technique in most cases and SolitaireSecond, the generalizability of recanalization status derived in this study could be limited. The P-to-R time to predict favorable outcome was 30 min in this study. However, the cutoff could be different for other study populations. It would be affected by the characteristics of the study population and the eligibility of endovascular treatment. After all, a specific P-to-R time to be achieved in FPR would be changed, although the concept of rapid FPR is still valid. To utilize the study findings in real practice, the cutoff should be constant. To derive recanalization status with a constant P-to-R time and overcome the limited generalizability, multicenter-based research of a large study population is necessary. Based on the constant P-to-R time, we might be able to make a general rule in achieving proper FPR.Recanalization status by FPR and P-to-R time was significantly and independently associated with functional outcome after mechanical thrombectomy. FPR was associated with functional outcome in a time-dependent manner. Even for patients who achieved FPR, their functional outcome might not be favorable if the P-to-R time was > 30 min. To secure the benefit from FPR, recanalization should be as rapid as possible even in cases with FPR.


Culicoides biting midges collected in Lichuan County, Jiangxi Province, China. The virus isolate showed significant cytopathic effects within 48 hours after inoculation with mammalian cells (BHK-21). JXLC1806-2 virus could form plaques in BHK-21 cells, and the virus titer was 1×105.6 pfu/mL. After inoculation with the virus, suckling mice developed disease and died. The nucleotide and amino sequence analysis showed that the JXLC1806-2 virus genome was composed of S, M and L segments. Phylogenetic analysis showed that the S, M and L genes of JXLC1806-2 virus belonged to the Tete serogroup, Orthobunyavirus, but formed an independent evolutionary branch from the other members of the Tete serogroup. The results showed that the JXLC1806-2 virus, which was named as Lichuan virus, is a new member of Tete serogroup, and this is the first time that a Tete serogroup virus has been isolated in China.In July 2018, a virus (JXLC1806-2) was isolated from  This family of viruses contains four viral genera, namely Hebevirus, Orthobunyavirus, Pacuvirus and Shangavirus  in 2017 classified all viruses into 9 orders, 131 families, 46 subfamilies, 803 genera, and 4853 species . Orthobugavirus . The repra virus . Orthobura virus . The traOrthobunyavirus genus, which includes the Tete virus (SAAn3518 virus strain), which was isolated from newborn mice inoculated with a bird  tissue sample collected in South Africa on February 5, 1959. This was the first virus that was isolated of the Tete serogroup viruses (Oriolus oriolus) and ticks samples collected in Egypt and Cyprus in 1966. The I612045 virus was isolated in India in 1961 (Euplectes afer) collected in the Central African Republic in 1970  based on the area of isolation. This is the first Tete serogroup virus isolated in China. The results are reported below.There are many types of orthobunyaviruses, and based on the serological reactions of the viruses, they can be divided into several serogroups . The Tet viruses . The Tet viruses  and Matr viruses  were als in 1961 , the Bat in 1970 , and the in 1970 . China h in 1970 , but the in 1970 . In thise region . The vir22.1Culicoides biting midges “. Collection environment and insect species data were recorded, and the specimens were stored in liquid nitrogen before being transferred to the laboratory for testing . The collection time was from 6pm to 6am. In this study, entomologists separated mosquitoes and midges with the naked eye at the specimen collection site, then selected the midge one by one under a stereoscopic microscope. Since morphological identification of midge species requires complex chemical treatment of an individual midge, which cannot be carried out in the field of specimen collection, the midges are referred to only as “  testing .2.2Aedes albopictus) cells were maintained in our laboratory. BHK-21 cell culture conditions were as follows: 90% Eagle’s medium (laboratory preparation), 7% fetal bovine serum , 1% penicillin and streptomycin (100 U/mL), 1% glutamine (30 g/L), and 1% NaHCO3. The C6/36 cell culture conditions were as follows: 89% RPMI1640 (Invitrogen), 10% FBS (Invitrogen), and 1% penicillin and streptomycin (100 U/mL). BHK-21 and C6/36 cells were cultured in an incubator at 37°C with 5% CO2, and at 28°C, respectively (BHK-21 (golden hamster kidney) and C6/36 , 1% penicillin and streptomycin (100 U/mL), 8% glutamine (30 g/l) and 1% NaHCO3 (75 g/l)] was added to each pool (50–100 mosquitoes or 500 midges) of samples followed by two washes (for three minutes/each wash) for cleaning the body surface of the specimen. Then, 1.5 mL of grinding solution was added (Same with washing solution), and the samples were ground using a glass grinder  under ice bath conditions. The samples were centrifuged after grinding , and 100 μL of the supernatant was inoculated into 80% monolayers of BHK-21 and C6/36 cells on culture plates . BHK-21 and C6/36 cells were continuously cultured in an incubator at 37°C with 5% COiscarded .2.4-1–10-6) was sequentially added to a 6-well culture plate (0.1 mL/well). After adsorption for 1 h in an incubator at 37°C with 5% CO2, 1.3% methylcellulose-MEM semi-solid medium (4 mL/well) containing 2% FBS was added to each well. After 72 hours of culture, when a plaque was obvious under the microscope, the medium was discarded, and the plaque was stained with crystal violet. Finally, the number of plaque-forming units (pfu) expressing the virus was calculated (BHK-21 cells were transferred onto a 6-well culture plate (Corning Inc.). The next day, once cells had grown into a monolayer covering 80% of the plate, a 10-fold dilution of the virus (10lculated .2.5Suckling mice (2-day-old Kunming mice) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All mice were raised under sterile conditions at the animal facility of the Chinese Center for Disease Control and Prevention. All animal experiments were conducted in strict compliance with the regulations set by the Animal Ethics Committee of China CDC(NO.2014112509).Each suckling mice was inoculated with 20 µL of supernatant infected with virus on the fifth passage of BHK-21 cells intracranially. The incidence and mortality of mice were observed and recorded every day. After death, the suckling mice were sacrificed and dissected. The mouse brain was ground, and the ground liquid was centrifuged at 12,000 rpm for 30 min at 4°C, and the supernatant was used to inoculate the next generation of suckling mice. The virus was passaged three times in suckling mice. Animals inoculated with the virus without disease or death were routinely sacrificed after 14 days .2.6The Viral RNA Mini Kit (Qiagen) was used to extract total RNA from the supernatant of virus-infected cells samples according to the manufacturer’s instructions. The extracted RNA was immediately placed in a water bath at 65°C for 10 min and placed on ice for 2 min. Then, 32 μL of RNA was absorbed into the first-strand reaction tube (Ready-To-Go You-Prime First-Strand Beads), which was maintained at room temperature for 1 min before 1 μL of random primers were added (phosphorylated random hexamer primers -pd (N) 6); this was centrifuged immediately and incubated in a water bath at 37°C for 1 h. Moloney Murine Leukemia Virus (M-MuLV) reverse transcriptase was used for reverse transcription. The total volume of the cDNA library was 33 μL, which was used immediately or stored at -80°C for future use .2.7de-novo assembly using the CLC De Novo Assembler and contigs of less than 300 bp were discarded from all assembles. The virus-related contigs were extracted based on the taxonomic information of the BLAST hits. To avoid mis-assembly, reads were mapped back to the virus genome and inspected with CLC Genomics Workbench. The viral genome sequences obtained by NGS was used as template for overlapping PCR primers design for Sanger sequencing confirmation.Total RNA was extracted from the supernatant on the fifth passage of BHK-21 cells infected with the JXLC1806-2 isolate using the QIAamp Viral RNA Mini Kit . A metagenomics shotgun sequencing library was constructed using the TruSeq total RNA library Prep kit  according to the manufacturer’s instructions. The libraries were sequenced on the MiSeq platform  in paired-end 150-bp mode. The sequencing data generated via mNGS were imported to the CLC Genomics Workbench  and were trimmed based on quality and ambiguity. The clean reads were applied for 2.8® Green Master Mix, 2× , and 10 μmol/L of upstream and downstream primers. The PCR reaction conditions were pre-denaturation at 95°C, and the amplification conditions of the virus isolate were 94°C 30s, 50°C 30s, 72°C 60s, and a total of 35 cycles were followed by extension at 72°C for 10min. After the PCR reaction was completed, 5 μL of the gene amplification product was detected by 1% agarose gel electrophoresis. The nucleotide sequences of the amplification products were determined by Sanger sequencing  was carried out in a total volume of 25 μL, and included the cDNA template, GoTaqquencing . JXLC1802.9Primers were designed based on the obtained partial sequences, and the 5’/3’ RACE system for rapid amplification of cDNA ends (Invitrogen) was used to amplify the gene sequences of the 5’ untranslated region (UTR) and 3’ UTR. For the 3’ UTR without a poly(A) tail, the Poly (A) Polymerase Tailing Kit (Epicentre) was used to add the A-tail prior to amplification. The amplified products were detected by 1% agarose gel electrophoresis, purified using a QIAquick Gel Extraction Kit (Qiagen), cloned into the pGEM-T Easy vector (Promega), and 10 clones of each product for each region were sequenced by the Sanger method .2.10Orthobunyavirus isolated from different hosts and countries for different years were downloaded from GenBank. The virus strain information used in the analysis is shown in The sequences of representative strains of 33.1Culicoides biting midges, two genera, and two species of mosquitoes were collected in Lichuan County, Jiangxi Province, which contained 270 Culex tritaeniorhynchus and 230 Anopheles sinensis  Figure 13.4The results showed that four of ten mice inoculated in the first generation gradually developed illness  and died within 96 h of inoculation, five mice died within 108 h of inoculation, with the remaining one mice being maintained to 14 days without illness or death; Two of five mice inoculated in the second generation died 72 h after inoculation, with the remaining three mice died 84 h after inoculation; Three of six mice inoculated in the third generation died 60 h after inoculation, with the remaining three mice died 72 h after inoculation Figure 23.53.5.1Orthobunyavirus genus showed that the JXLC1806-2 virus had 66.5% (Batama virus) to 75.0% (Matruh virus) of nucleotide identity, and 69.8% (Batama virus) to 77.2% (Bahig virus) of amino acid similarity with the S segment of other viruses in this group; 70.2% (Tete virus) to 78.0% (Matruh virus), and 75.1% (Tete virus) to 86.2% (Marruh virus) with the M segment; and 74.5% (I612045 virus) to 80.4% (Matruh virus), and 81.6% (I612045 virus) to 92.2% (Matruh virus) with the L segment. A comparison of JXLC1806-2 or the Tete virus with the Oyo virus outsidethe Tete group revealed differences in the lengths of nucleotide and amino acid sequences of the three segments as well as lower homology than other viruses in the Tete serogroup , belonged to the Orthobunyavirus Tete serogroup, and the JXLC1806-2 virus was independent of the Tete viruses, Matruh virus, and Bahig virus, which formed a separate evolutionary branch. In this paper, the ML method  of the JXLC1806-2 virus strain were 58, 39, and 57 bp, respectively, and the lengths of the 3’ UTR regions of the three segments were 206, 115, and 127 bp, respectively. The 5’ and 3’ ends of the three segments of the S, M, and L segments were all reverse complementary sequences of 8 nucleotides (5’-AGTAGTGT … ACACTACT -3’) (th position of the 5’ UTR (conserved region of the virus non-coding region), the 11th position of the 3’ UTR of the virus L segment, and the 11th position of the S segment 5’ UTR . The results showed that the UTRs on both sides of the three segments of the JXLC1806-2 virus were consistent with the nucleotide sequences of other viruses in the Tete group. There were only three nucleotide variations at the 94Culicoides biting midges specimen collected in the nature of Jiangxi Province in southeastern China was isolated from a virus isolate (JXLC1806-2) that can cause cytopathic effects in mammalian cell, which can cause morbidity and death in suckling mice. Sequencing and analysis of the entire genome of the virus showed that JXLC1806-2 virus was RNA virus with three segments. The results of virus gene sequence homology and molecular genetic evolution analysis showed that the virus is a new member of the Tete virus group in the Orthobunyavirus genus, named Lichuan virus (LICV). This is the first time that Tete virus group has been isolated from Culicoides biting midges specimens collected in nature in China.In this paper, a 4.1Orthobunyavirus contained S, M, and L segmentss, and the S segment was generally 1000 nt in length and encoded two proteins (N and Ns protein)  . However protein . Therefo protein . The S s protein Table 2,Orthobunyavirus.Evolutionary molecular genetic analysis based on the nucleotide sequence of the virus S segment revealed that the Tete serogroup virus was significantly different from the OYO virus, which was placed outside of the serogroup, and was divided into two large evolutionary branches; the first branch contained the Tete, Batama, and I612045 viruses (the I612045 virus was independent of the first two viruses), and the second branch contained the Matruh and Bahig viruses. Evolutionary analysis of the JXLC1806-2 virus S segment and the five Tete serogroup viruses showed that all six of these viruses were divided into two evolutionary branches. The JXLC1806-2 virus was independent of the Matruh and Bahig viruses and formed a separate evolutionary branch Figure 24.2Culicoides biting midges, suggesting that midges were vectors for the Tete serogroup virus. A number of viruses that cause human and animal diseases have been isolated from midges collected from across China , Culicoides oxystoma (12.16%), and Culicoides puncticollis (7.89%). Culicoides crakawai was dominant in the local duck and hen houses, Culicoides oxystoma dominated in the piggery, and Culicoides puncticollis dominated in the sheep pens. The cattle farm had various types of midges  can be found in the article/The animal study was approved by The meeting of ethics committee of national institute for viral disease control and prevention, China CDC. The study was conducted in accordance with the local legislation and institutional requirements.QY, RC, XX and GL involved in drafting the manuscript. QY, RC, XX and GL involved in revising the manuscript. RC, ZX, JW, SF, HX, SZ YH, FL and SX participated in the acquisition of data. QY, RC, XX, ZX, JW, SF, XL and GL participated in the analysis and interpretation of data. SF, HW, BW and GL supported funding for the study. HW, BW and GL contributed to conception and design of the manuscript. All authors contributed to the article and approved the submitted version.


Caudovirales and Microviridae, were commonly found in pools of samples with a very low abundance; a few sequences for invertebrate, plant and giant viruses were found in some of individuals; however, the remaining 31 viruses mostly reflect extensive contamination from commercial reagents and the work environments. In conclusion, this study is the first comprehensive investigation of blood virome in healthy individuals by metatranscriptomic sequencing of VLP in China. Further investigation of potential false positives representing a major challenge for the identification of novel viruses in mNGS, will offer a systemic idea and means to reveal true viral infections of human.Emerging evidence indicates that gut virome plays a role in human health and disease, however, much less is known about the viral communities in blood. Here we conducted a direct metatranscriptomic sequencing of virus-like-particles in blood from 1200 healthy individuals, without prior amplification to avoid potential amplification bias and with a strictly bioinformatic and manual check for candidate viral reads to reduce false-positive matches. We identified 55 different viruses from 36 viral families, including 24 human DNA, RNA and retroviruses in 70% of the studied pools. The study showed that anelloviruses are widely distributed and dominate the blood virome in healthy individuals. Human herpesviruses and pegivirus-1 are commonly prevalent in asymptomatic humans. We identified the prevalence of RNA viruses often causing acute infection, like HEV, HPIV, RSV and HCoV-HKU1, revealing of a transmissible risk of asymptomatic infection. Several viruses possible related to transfusion safety were identified, including human Merkel cell polyomavirus, papillomavirus, parvovirus B19 and herpesvirus 8 in addition to HBV. In addition, phages in  We can not only focus on pathogenic viruses (eg. emerging viruses), but also go beyond to the human virome in physiologically healthy individuals and their interactions with each other, with other microbes, and with host genetics and immune systems, and how they affect health and disease . In this22.1All blood collection was performed by technicians at the Beijing Red Cross blood Center  during November 2018 to November 2019. Two milliliters of blood were collected from healthy blood donors into sodium heparin vacutainers  and stored at 4°C until processing within 24 hours after blood collection. Written informed consent was obtained from all individuals. The study was approved by the ethics committees of the Beijing Red Cross blood Center.2.2We have combined a series of enrichment methods including filtration and ultra-centrifugation to enrich for virus-like-particles (VLPs) in PBMC and plasma in parallel. Two milliliters of heparin-treated whole blood samples were first centrifuged at 3,000 × g for 10 min at 4°C within 3 hours of sample collection to isolate cell-free plasma and haemocytes . Plasma was transferred to microcentrifuge tubes and mixed in a pool of 12, which was centrifuged at 10,000 × g for 10 min to remove residual cells and further ultracentrifuged at 100,000 × g for 3 hours to concentrate the VLPs in 280μl phosphate buffered saline (PBS). In parallel, the cells in precipitation from each plasma-corresponding blood were subjected to Red Blood Cell Lysis Using ACK Lysing Buffer (Thermo fisher), and PBMCs were collected by centrifugation at 300 x g for 5 minutes and resuspended in 150μl of PBS twice. The clean PBMCs were frozen and thawed twice to release viruses cells into supernatant, then centrifuged at 10,000 × g for 10 min and the supernatant of 12 samples were pooled and subjected to further processing .Fig. 1St2.3A total of 200 pools  were constructed from 1200 healthy donors. The concentrate of VLPs from each pool was treated with a cocktail of Turbo DNase (Ambion), RNase (Promega) and Benzonase (Ambion) at 37 °C for 90 min to degrade nonencapsulated nucleic acids. Finally, each of 200 μl VLP preparation was used for viral nucleic acid extraction using the QIAamp MinElute Virus Spin Kit (Qiagen) immediately and subjected to double-stranded cDNA (ds-cDNA) synthesis SuperScript™ Double-Stranded cDNA Synthesis Kit (ThermoFisher).2.4Nextera XT Samples Prep kit (Illumina). Barcoded libraries were pooled for sequencing. The concentration of the pooled libraries was measured using Qubit (Invitrogen) and the size distribution of the pooled libraries was checked by Agilent Technology 2100 Bioanalyzer using a High Sensitivity DNA chip (Agilent). Sequences were acquired using the HiSeq 2000 . Metatranscriptomic sequencing data is deposited at the Genome Sequence Archive in the BIG Data Center, Beijing Institute of Genomics, Chinese Academy of Sciences, under BioProject accession no. PRJCA008687.Ds-cDNA was used directly for the construction of the shotgun libraries. The DNA concentration was measured using the Qubit (Invitrogen). Libraries were made using an Illumina 2.5Quality control for the blood VLP reads was performed using the in-house bioinformatics pipeline . In brie33.1We performed direct metatranscriptomic sequencing of VLP in 200 plasma pools and PBMC pools  from 1200 healthy individuals in China. Total nucleic acid was extracted from VLP to comprehensively detect both DNA and RNA viruses, and subjected to dsDNA synthesis and further deep sequencing without prior random amplification to avoid potential amplification bias. Illumina sequencing generated a total of 218 GB 101 base pair (bp) single-end reads. After quality control, the remaining high quality, unique, nonhuman reads were then translated and analyzed via protein similarity search (BLASTx) against a comprehensive database of viruses, bacteria, archaea, human, vectors and other eukaryotes. Finally, a total of 5,264,459 nonredundant viral reads  were obtained (Supplementary Table 1). Under an empirical cut-off of read number ≥ 2, we detected a total of 36 viral families-derived sequences that were presented in at least one pool, including sequences from 24 human DNA viruses, RNA viruses and retroviruses in 70% of the study pools.3.23.2.1Anelloviruses dominate the blood virome in healthy individuals. Anelloviruses were detected in each of the pool, and constituted the most of viral communities . The Anelloviridae fraction was composed mostly of viruses from alphatorquevirus , betatorquevirus  and unclassified anelloviruses. In addition, gammatorquevirus , SEN viruses and small anelloviruses were also identified  were identified in 2 PBMC pools and 1 plasma pool by manual check. This virus was reported to be highly seroprevalent and positive by PCR, but not as in this study. We found a few of reads for human mastadenovirus C in both plasma and PBMC pools, and most of which were confirmed as synthetic construct clone Parva T-Ag, and only 1 plasma pool was confirmed to be positive for human adenovirus 2 with 3 reads. (Among the human herpesviruses (HHV), HSV-1 was identified in 8 (8%), CMV was identified in 16 (16%), HHV6A was identified in 3 (3%) and HHV6B was identified in 1 (1%) of the PBMC pools. A few of sequences of HHV6 was also found in 4 (4%) of the PBMC pools, but can't be subtyped to HHV6A or HHV6B. More types of the HHV were detected in plasma pools. HSV-1 was identified in 1 (1%), EBV identified in 3 (3%), CMV was found in 16 (16%), HHV-6 was found in 2 (2%), HHV6A was identified in 1 (1%), HHV-7 was found in 4 (4%), and HHV-8 (KSHV) was found in 8 (8%) of the pools , Fig. 3.3 reads. , Fig. 3.3 reads. , Fig. 3.3.2.3We identified 9 human RNA viruses including human pegivirus-1 (HPgV-1 or GBV-C), parainfluenza virus (HPIV), respiratory syncytial virus (RSV), human coronavirus HKU1 (HCoV-HKU1), enteroviruses  and Kadipiro virus (KDV). The blood-borne HPgV-1 was identified and abundant in 14% of plasma pools with a median of 41,501 reads per pool, while in 8 respective PBMC pools of the 14 plasma pools with a median of 19 reads per pools. Upon validation, we identified a cluster of pools with the HPgV-1 in the same flow cell. The sample with the highest viral load led to contamination of samples sharing the same flow cell, which, although positive, were thus classified as contaminants , Fig. 3.Retroviridae with best match to spleen focus-forming virus. The source of these viruses is likely to be through contamination of cell reagents or the environment. We identified a few of sequences for Circoviridae, most of which, however, cannot be aligned to specific viral species, reflecting contamination occurring during experiment process as reported as previously. A few of reads of influenza A virus (H7N9), HCV and Zika virus were also found. However, the inspection of sequences showed 100% identities against our laboratory strains in use, thus they were identified as synthetic vectors and removed from analysis. A few of sequences for Arenaviridae and Astroviridae were found, however, these were identified as contaminants from environmental contaminants sharing between the flow cell and removed from subsequent analysis. (Supplementary Table 1).We also identified a few sequences for HIV-1 in 2 PBMC pools, and observed the presence of more reads from 3.3Caudovirales  and family Microviridae. At species level, we found 3 shared bacteriophages, including pseudomonas virus EL, tetrasphaera virus TJE1 and bdellovibrio phage phi1422 in more than 50% of healthy individuals. Taxonomic assignment of these viral reads revealed a prevalence of unknown phages belonging to the order Caudovirales and the family Microviridae, indicating that the majority of bacteriophages are unknown, with only a limited subset that can be taxonomically classified. As described previously, although there is a possibility that some phage DNA could translocate from the gut, the presence of phages always is revealing of contamination. Phage DNA can also derive from bacteria contaminating the reagents. Pseudomonas virus PRD1 in Tectiviridae was identified in 34 (29.3%) of PBMC pools, and 29 (20.4%) of plasma pools. We identified a few of sequences of a linear ssRNA phage, acinetobacter phage AP205, in 23 plasma pools with a median of 2 reads per sample, also reflecting environmental contamination (Supplementary Table 1).We observed a widespread presence of phage DNA in both of plasma and PBMC pools, mainly derived from the order Inoviridae was found as escherichia virus M13 (vector) and removed from our analysis (Supplementary Table 1).A archaeal virus, acidianus bottle-shaped virus, was identified in 8 PBMC and plasma pools with a median of 14 reads per sample. A fungal virus, penicillium chrysogenum virus, was also identified in 6 plasma pools with a median of 2 reads per sample. it was removed because there is a possibility of cross contamination or from baculovirus vectors. Pseudomonas virus PRD1 was also removed because of the suspicion of misshit of transcripts. Notably, escherichia virus phiX174 was only found in 3 pools with 3 reads and removed from our analysis. A few of reads from 3.4 matched to orf virus by blastx in 2 plasma pools with a median of 3 reads per pool, and in 2 PBMC pools with a median of 2 reads per pool. However, after manual inspection, these reads can be aligned to cattle genome by blastn with higher nucleotide similarity  and represented false positive. We also identified a few reads of invertebrate viruses , and plant viruses . The source of these viruses is likely to be through contamination of reagents or the environment as well as transients from foods. We found a few of reads for Dicistroviridae in 52 plasma pools with a median of 2 reads per pool. Although detection of dicistrovirus RNA in blood of febrile Tanzanian children was reported (∼95%), aphid lethal paralysis virus  and other dicistroviruses with a distance relative , reflecting contamination from reagents or work environment (Supplementary Table 1).We identified a few viral sequences of giant virus, pacmanvirus S19 in 4 PBMC pools with a median of 2 reads per pool, and in 3 plasma pools with a median of 3 reads per pool. However, it was difficult to confirm whether it was truly present or the reagent or laboratory contamination. We also found a few of reads distantly reported , all rea3.5Anelloviridae, Hepadnavirida and Flaviviridae together accounted for most of viral sequences in blood with high ALT (Hepadnaviridae (HBV) were significantly higher than those in normal-ALT plasma (Alanine aminotransferase (ALT) is very sensitive to liver inflammation, and an ALT test is currently demanded for blood donation to ensure transfusion safety . Herein,high ALT . In highT plasma , 5D.Fig.4This study, as we know, is the first study on blood virome in China by direct meta-transcriptomic sequencing of VLP in PBMCs and plasma. Total nucleic acid was extracted from enriched VLP to deeply detect circulating DNA and RNA viruses without being abundant in blood. The libraries were constructed directly from ds-cDNA without prior amplification to avoid potential amplification bias and contamination.We found 55 different viruses from 36 viral families, including 24 human DNA, RNA and retroviruses, however, the remaining 31 viruses of them represented false positive or contamination and a massive effort was required to rule out them. For example, we found HIV-1 in one pool of PBMC with 65 reads, all of which were mapped to synthetic HIV-1 clone pSF256.8 upon further inspection. And a few reads for Zika virus were found in 5 PBMC pools and 2 plasma pools, however, all of them were mapped to a synthetic construct sequence. Thus, they were removed from subsequent analysis. Phage DNA could translocate from the gut, and can also derive from bacteria contaminating the reagents . HoweverThe study found that anelloviruses were dominant in viral communities and widely distributed among healthy individuals. However, more than 55% of sequences assigned to this family were unclassified indicating a lot of novel or unknown viruses remained to be studied as indicated previously. In addition, we identified 23 viruses associated with human infection in 70% of the study pools, which included not only the DNA virome, such as 6 different HHV types, HPV, MCPyV, HAdv, parvovirus B19, HBoV, but also the RNA virome such as HPgV-1, KDV, HEV , HPIVs, RSV and HCoV-HKU1. These viruses generally correspond to those known to be prevalent in the human population. Notably these viral sequences detected in pools of samples may overestimate the prevalence of the specific virus.We identified 6 types of the herpesviruses with the exception of HSV-2 that was sexually transmitted viral infection affecting the skin or mucous embranes of the genitals , and varThis study has the following limits. First, we conducted a complete search by Blastx. This approach takes a lot of time and computing power and not suitable for timely testing. However, it serves to identify viral sequences not only prevalent as we know, but also those distantly related to known viruses in database. Second, the pooling of samples was adapted to enrich VLP as much as possible. However, this may overestimate the frequency of the specific viruses. In preliminary test, we realized that no adequate amount of nucleic acid can be obtained without pre-amplification for sequencing in individual sample and we thus adapted the pooling of sample. Third, as implied in previous reports , relativIn conclusion, this study comprehensively defined the virome in Chinese population through direct metatranscriptomic sequencing of VLP circulating in the blood, providing us for basic data and insight into blood microecology and potential blood-borne infection. It is also emphasized that mNGS is a powerful approach for surveying viral infections, however, there are still several challenges for excluding false positives and identifying real viral pathogen.Bo Feng: Methodology, Resources, Investigation. Bo Liu: Software, Data curation. Min Cheng: Visualization, Writing – original draft. Jie Dong: Methodology. Yongfeng Hu: Conceptualization, Visualization, Writing – original draft, Writing – review & editing. Qi Jin: Conceptualization. Fan Yang: Conceptualization, Supervision.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Acanthamoeba is well known to host a variety of microorganisms such as viruses, bacteria, protozoa, and yeast. Given the recent number of cases of monkeypox infection, we speculate that amoebae may be aiding viral transmission to the susceptible hosts. Although there is no confirmatory evidence to suggest that Acanthamoeba is a host to monkeypox (a double-stranded DNA virus), the recent discovery of mimivirus (another double-stranded DNA virus) from Acanthamoeba, suggests that amoebae may shelter monkeypox virus. Furthermore, given the possible spread of monkeypox virus from animals to humans during an earlier outbreak, which came about after patients came in contact with prairie dogs, it is likely that animals may also act as mixing vessel between ubiquitously distributed Acanthamoeba and monkeypox virus, in addition to the environmental habitat that acts as an interface in complex interactions between diverse microorganisms and the host. Monkeypox is a rare viral disease affecting humans and animals and is caused by an Orthopoxvirus . The disClinical symptoms of monkeypox infection in humans and animals are comparable to those of smallpox infection . Flu-likLegionella pneumophila, was due to the presence of bacteria inside air-conditioning units; however, how L. pneumophila survived in these air-conditioning units was not known [L. pneumophila were able to survive in harsh environments by taking up refuge inside free-living amoebae, i.e., Acanthamoeba. More recently, a double-stranded DNA virus, i.e., mimivirus, was discovered in amoebae. Given the ability of Acanthamoeba to host a variety of microbes  that can serve as a reservoir for bacteria/viruses should also be considered as potential hosts for monkeypox.More importantly, these cases have no established travel links to endemic areas; nonetheless, epidemiological investigations are ongoing . It has ot known . Furtheriewed in ), some olla spp. , and thella spp. . Based olla spp. ). This inditions . BecauseIn the 10 years following the first human case in 1970 ,15, therIn various African nations, including Cameroon, Benin, Gabon, Central African Republic, Democratic Republic of the Congo, Ghana , Liberia, Côte d’Ivoire, Nigeria, Sierra Leone, and South Sudan, monkeypox was thought to be endemic . With moOf concern, in 2022, there was a global resurgence of monkeypox cases, with a high infection incidence in non-endemic nations. According to a recent report , BelgiumAcanthamoeba is known to have an extensive global distribution that spans both aquatic and terrestrial habitats [Acanthamoeba is one of the most prevalent protists in the soil. As significant grazers of the bacterial biomass in these settings, these amoebae are thought to regulate not only the variety but also the abundance and turnover of bacterial communities in the soil and plant rhizospheres [Acanthamoeba also releases nutrients locked in the microbial biomass in the soil microbial loop, ultimately promoting plant growth [habitats ,22. In fospheres ,24,25. At growth .Acanthamoeba can survive in a variety of environments and have been found in swimming pools, bottled water, seawater, ponds, stagnant water, freshwater lakes, saltwater lakes, river water, distilled water bottles, ventilation ducts, the water–air interface, air-conditioning units, sewage, compost, sediments, soil, beaches, vegetables, air, surgical instruments, contact lenses and their cases, the atmosphere (recent decontamination studies), etc., and they have been isolated from the continent of Antarctica [Acanthamoeba spp. have been discovered in a diverse range of animals, including monkeys, dogs, lizards, kangaroos, Indian buffaloes, reptiles, mice etc. [Acanthamoeba have been described in animals; however, prospective studies are warranted to comprehend the prevalence of amoebae in animals [tarctica ,27. Moreice etc. , and eveice etc. ,30,31. F animals .Acanthamoeba is comprised of a vegetative trophozoite stage during which amoebae divides mitotically and an inactive dormant cyst stage [Acanthamoeba is already known to shelter a wide range of viruses such as poliovirus, mimivirus, enterovirus, coxsackievirus, adenoviruses, and echovirus, amongst others [Coxiella, Legionella, Mycobacterium, Helicobacter, Salmonella, Pseudomonas, Escherichia coli, Vibrio, Listeria, Rickettsia, Shigella, Pasteurella, to name a few . Of notay occur .Acanthamoeba has also been identified as a yaravirus carrier [Acanthamoeba are relatively large, containing 100–1000 genes. In this regard, the genome of the monkeypox virus is around 190 kb and it is an enclosed double-stranded DNA virus [Acanthamoeba has been utilized by other pathogens as a training ground (discussed in the following section) and is also often referred to as a “genetic melting pot”, the speculation that the monkeypox virus may be sheltering in these amoebae is highly plausible. The monkeypox virus is a double-stranded DNA virus, it should thus present with a modest mutation frequency [Acanthamoeba genome may reveal information regarding possible genetic exchanges.For example,  carrier . The maj carrier . The majNA virus . It is iNA virus . FurtherNA virus ,41. Giverequency . The outrequency . It is aMorganella morganii bacteria, isolated from the gut microflora of the Asian water monitor lizard Varanus salvator were able to interact, invade, and survive within Acanthamoeba castellanii. This study provides another example of the ability of Acanthamoeba in acting as a potential vector and aid in the possible transmission of bacteria and/or other microorganisms to their potential hosts, which are found in a variety of places [Acanthamoeba with the amoebae acting as a vector and/or reservoir and aiding in its transmission. Additionally, amoebae cysts may also offer resistance to intracellular viruses from the required levels of biocides. If proven, a symbiosis between amoebae and monkeypox may result in significant challenges to the public health and ought to be investigated. Associations between amoebae and the monkeypox virus may also result in transmission to the central nervous system, resulting in more severe cases.Another example of the highly complex interactions of microorganisms comes from a recent report whereby f places . The speAcanthamoeba as a tool for learning how to avoid the assault of macrophage-mediated death. This is due to the striking similarities observed between how different pathogens such as Mycobacterium or Legionella pneumophila survive inside human macrophages and Acanthamoeba, including the use of similar transcriptional, post-transcriptional, and cellular mechanisms. This suggests that both amoebae and human macrophages may share characteristics that enable the intracellular pathogens to spread infection [It is well known that pathogens may have used their capacity to thrive and proliferate inside nfection ,44.Chlamydiae is not known [Chlamydophila pneumoniae can multiply in free-living amoebae in addition to environmental Chlamydiae [Chlamydiae in this situation. These studies indicate that Chlamydiae may flourish in both amoebae and humans, even though the longevity of this relationship during the amoeba encystment is not yet known [Parachlamydia acanthamoebae can enter and grow in human macrophages and may therefore have the ability to infect humans [Interestingly, the natural reservoir of pathogenic ot known . The dislamydiae  suggestset known . In linet humans ,46,47.Acanthamoeba is capable of taking in and housing Mycobacterium leprae , and the bacteria can keep both their viability and growth characteristics within the amoebae [M. leprae from harmful circumstances such as desiccation and high temperature and pH [In addition,  amoebae . Moreovee and pH . SimilarAcanthamoeba routinely, given the ubiquitous nature of these amoebae and their omnipresence in the environment [Acanthamoeba may be a part of the microbiome of animals, given its presence in diverse environments and the complexity of microbial isolates that have been recovered from amoebae via metagenomic techniques [The need for improved collaboration between the human, animal, and environmental health sectors has been underlined by the apparent inability of global health security to avoid or prepare for the recent COVID-19 pandemic . The monironment . Furtherchniques . This isAcanthamoeba spp., Dictyostelium discoideum, Vermamoeba vermiformis) as potential reservoirs for the plague causing bacteria reservoir Yersinia pestis [To this end, the use of an amalgamation of approaches may well be the way onward in targeting viruses such as the monkeypox virus that may be using amoebae to shelter and persist in the environments. This notion is further strengthened by the findings from a recent study that investigated different amoebae species that are frequently observed in the environment . Using The pathogenesis of monkeypox virus infection is not well understood, but it is believed to involve a complex interplay between viral and host factors. Monkeypox virus is an enveloped, double-stranded DNA virus that belongs to the genus Orthopoxvirus in the family Poxviridae. Members of the Poxviridae family, such as the monkeypox virus, are thought to exhibit diverse spectra of living and surviving in a host cell . The celAcanthamoeba phagocytic killing require experimental investigations, based on genome analysis and earlier investigation using human cells, several viral proteins have been found to be essential for entry, release, and host cell modulation. In this regard, several cell modulatory proteins have been identified that may also play a role in monkeypox evasion of Acanthamoeba intracellular killing. These include: J3R (chemokine binding protein), J2L (cytokine response-modifying protein B), D9L (ankyrin repeat domain containing protein), CP77 (type I interferon (IFN) evasion protein), F3L (RNA-binding protein E3), H1L , D3R (EGFR binding protein), D11L (Protein C6), C7L (Protein F1), B16R , C1L (IFN antagonist K1L), B13R (protein B13), B9R (soluble IFN-γ receptor B8), P1L (protein N1), C6R (protein K7), A37R (MHC modulating protein), A41L (protein A41 (chemokine binding protein), and A47R (TLR inactivating protein); however, studies are needed to determine the role of the aforementioned proteins in evading phagocytic killing of Acanthamoeba [Although the precise mechanisms of monkeypox evasion of thamoeba .Acanthamoeba might be allowing long-term survival and long-distance transmission of the SARS-CoV-2 virus [Acanthamoeba showed the presence of laminin-binding protein as one of the major parasites’ adhesins [Acanthamoeba allowing its entry and facilitating its transmission. Notably, monkeypox virus has a relatively large genome of about 196,858 base pairs, encoding 190 open reading frames needed for viral replication. It is logical that viral replication can only take place during the metabolically-active trophozoite stage of the Acanthamoeba as the virus requires the functional host cell machinery to synthesize its DNA, early and later proteins, virion assembly, trafficking, etc. The cyst stage is inactive or exhibit minimal metabolic activity. For example, viral entry into cells is likely dependent on phagocytic uptake (absent in the cyst stage) and involves actin. Once inside, viral proteins and enzymes promote synthesis of early proteins, DNA replication, transcription factors, late genes, structural proteins, and enzymes and virion genomes are processed and assembled into nascent virions that contain all enzymes, factors, and genetic information needed for a new infectious cycle. Hence, it is likely that, once infected with monkeypox, the metabolism of Acanthamoeba and monkeypox virus are functionally intertwined; however, future experimental studies are needed to determine this.As in the case of other microorganisms, amoebae have been recognized as biological “Trojan horses” for viruses and have been charged with increasing virulence and protecting human viruses against environmental harshness. Amoeba-infecting viruses are not uncommon, and several have been isolated from amoeba cultures, such as adenoviruses  and ente-2 virus . In any adhesins . Given tAcanthamoeba as a model [Acanthamoeba are necessary and will pave the way for the development of disinfectants that may be effective against both the viral agent or agent of interest and the host, Acanthamoeba. A complete understanding of the molecular underpinnings of these interactions, such as variations in the gene expressions or receptors used by microorganisms, may also help to promote a one-health approach and shed light on potential ways to manage pathogenicity and drug resistance. Given this, we suggest that amoebae cysts should be targeted as well to offer robust infection control approaches. The concept of utilizing antiamoebic approaches in containing viruses such as monkeypox should be seriously considered. Nonetheless, prospective research is needed to understand the efficacy of targeting amoebae with disinfectants, as is investigating the interaction of monkeypox with amoebae.As amoebae can harbor different microorganisms and/or pathogens , these pathogens may spread across our ecosystem and cause infections in humans and animals, leading to highlighting the concept of “one health,” in which the wellbeing of humans, animals, and the environment is intertwined. Numerous studies of various metabolic, proliferative, and survival systems in eukaryotic species have been elucidated through the utilization of  a model . Further


Ostreococcus tauri and O. mediterraneus and their viruses (size ca. 100 nm). Ostreococcus sp. is globally distributed and, like other picoplankton species, play an important role in coastal ecosystems at certain times of the year. Further, Ostreococcus sp. is a model organism, and the Ostreococcus-virus system is well-known in marine biology. However, only few studies have researched its evolutionary biology and the implications thereof for ecosystem dynamics. The Ostreococcus strains used here stem from different regions of the Southwestern Baltic Sea that vary in salinity and temperature and were obtained during several cruises spanning different sampling seasons. Using an experimental cross-infection set-up, we explicitly confirm species and strain specificity in Ostreococcus sp. from the Baltic Sea. Moreover, we found that the timing of virus-host co-existence was a driver of infection patterns as well. In combination, these findings prove that host-virus co-evolution can be rapid in natural systems.Marine viruses are a major driver of phytoplankton mortality and thereby influence biogeochemical cycling of carbon and other nutrients. Phytoplankton-targeting viruses are important components of ecosystem dynamics, but broad-scale experimental investigations of host-virus interactions remain scarce. Here, we investigated in detail a picophytoplankton (size 1 µm) host’s responses to infections by species-specific viruses from distinct geographical regions and different sampling seasons. Specifically, we used The online version contains supplementary material available at 10.1007/s00248-023-02243-5. Phytoplankton viruses and hosts are omnipresent in the marine environment. Within the microbial loop, viruses can infect both hetero- and autotrophic microbes and “shunt” carbon away from the classical food web , 2. ConsOstreococcus viruses GC[I]AC[I]GT[I]YTNGA and VpolAAS1 CC[I]GTRAA[I]CCRTA[I]AC[I] SWRTTCAT) . The same-value were at least < 6e-50, percent identity > 90% and the coverage > 90%.All PCR products were cleaned up with a kit (Fisher Scientific PCR cleanup®) prior to Sanger sequencing using the forward primer at EUROFINSGENOMICS®. The 18S rRNA gene sequences were clipped and trimmed for further analysis (sequence length after trimming 551 bp), and taxonomic identification was performed with Codon Code Aligner (Version 9.0.1) and the following reference used: reference genome from , GenBankBoth 18S rRNA and polB gene sequences (55 yielded clean sequences) were then used to build neighbour-joining phylogenetic trees (Codon Code Aligner) to identify relatedness. Default settings of the software for neighbour-joining trees based on number of base-pair differences between sequences were used and missing data considered as deletions. The calculated trees were then exported as newick format and subsequently visualized using the package “ggtree” in R version 4.2.2 . We usedIn addition to the species identity, we added the information of the water sample’s origin (geography and season) to the names of hosts and viruses. Since viruses have an inherent host dependency, they were also named in relation to the water sample of their hosts. This additional information was used to analyse the infection patterns in the following experiment.Ostreococcus sp. strains (2 O. tauri and 15 O. mediterraneus) were infected by all 79 different Ostreococcus viruses. Specifically, we prepared four petri dishes with Ostreococcus culture in semi-solid 0.2% agarose (Thermofisher®) and added 16 virus strains per petri dish. For each virus strain, we pipetted 2 × 2 µL of lysate onto the solidified culture. The multiplicity of infection (MOI) of all infections was 10:1 where the Ostreococcus cultures had a starting concentration of 5*106 cells mL−1 and the virus lysates contained 50*106 viral particles mL−1  and took photographs of the petri dishes using a Canon digital camera (EOS M100). Plaque size was measured via ImageJ® (V 1.53t) , and theWe used the bipartite network analysis to test for nestedness in the infection patterns using ‘computeMod’ in the bipartite package in the R programming environment . To testNext, we analysed the rate of plaque size increase for the infections. Due to non-normalized data, we used Kruskal–Wallis-Tests and subsequent pairwise Wilcoxon-Test with Bonferroni corrections. The following factors were tested: clusters, virus seasonal and geographical origin, host seasonal and geographical origin, and difference between the infection on the isolation and a foreign host. All comparisons were then plotted in boxplots.All distance matrix calculations as well as statistical analyses were carried out using R software (R Version 4.2.2. ) and theOstreococcus tauri and 19 Ostreococcus mediterraneus strains. These strains originated largely from KB and MB, with a higher isolation success during spring and autumn phytoplankton blooms. The isolation success was approximately 7% as we isolated Ostreococcus strains from 8 out of 119 water samples. Then, we were able to isolate 9 Ostreococcus tauri viruses (OtVs) and 70 Ostreococcus mediterraneus viruses (OmVs) from 13 of the Ostreococcus strains using 84 different water samples . The viruses mainly originated from the KB and MB in spring and early summer (March 2018 to May 2020) , O. tauri virus (OtV), and O. lucimarinus virus (OlucV) in names). Note that even though the host of OlucV virus was O. mediterraneus, the polB sequence was most similar to that of an O. lucimarinus virus. Compared to the 18S rRNA gene sequences, we found more nucleotide variability among the viruses forming five strain clusters  identified the host strains to be a et al.  . Similarly, there was a tendency for slower plaque growth for hosts that were isolated at a later time . The opposite was the case when comparing the geographical origins of the water samples: the further to the east the water samples were taken (SW to BB), the faster the plaques grew . The seasonal origin of the virus water sample also had an effect on how fast plaques formed, but no specific time trend could be found .The infection plaques appeared in most cases on day 3 post infection, whereas some only appeared on the 5th or 7th day post infection Fig. . At the Ostreococcus sp. complex from different regions of the Baltic Sea as well as the Swedish Skagerrak and investigated infection patterns within this system. We identified the evolutionary history, and thus the timing of when hosts and their associated viruses coexisted, as the main driver of infection patterns. In addition, host species and strain specificity underline the present understanding of rapid host-virus co-evolution [Over a 2-year sampling period, we successfully isolated 21 hosts and 79 virus strains of the volution , 34, andOstreococcus sp. is present all year round in the Baltic Sea, but its prevalence varies throughout the year and regionally [Ostreococcus strains in spring (March to May) and autumn (October). Sampling success of our focal species likely depends on the composition of the whole phytoplankton community at time of sampling: Spring blooms in the Western Baltic Sea are dominated by large and often chain-forming diatoms [Ostreococcus sp. is favoured over other picoplankton species and diatoms for isolation approaches. In contrast, during summer blooms, the Western Baltic Sea phytoplankton community is dominated by cyanobacteria [Ostreococcus sp. in this season [Ostreococcus populations that are mainly present in early times of the year in the Mediterranean Sea. Based on molecular evidence, Ostreococcus and its viruses were found even in the eastern part of the Baltic Sea [Ostreococcus was also higher in the Western sampling areas with higher salinities. As Ostreococcus are primarily a marine species, the pronounced brackish conditions in the Eastern Baltic Sea could hinder prevalence of algae in that region [Based on metagenomic analyses, gionally . This is diatoms  that canbacteria . These cs season . Our finltic Sea  but at tt region . We concOstreococcus species belonged mainly to the subclade D, also named O. mediterraneus, while two strains belonged to the O. tauri clade C [Ostreococcus sequences were identified as O. tauri. Within the species complex of Ostreococcus, there are very few base pair differences within the 18S rRNA gene [O. tauri was the first species to be isolated and identified within this picoplankton species complex [The 18S rRNA gene sequences revealed that our  clade C , 32. Thi clade C  found th complex , availab complex .O. mediterraneus host did not align with the rest of the sequences because they were more similar to O. lucimarinus pol B sequences based on BLAST comparisons. This was a contradictory result [O. mediterraneus virus. Therefore, the genetic identification of viruses may not be adequate to deduce its host origin or its role in the ecosystem. We will now further discuss the ecology of the infections in this study considering the viruses host origin.The genetic relatedness of the isolated virus strains based on Pol B sequences  shoy result  because Ostreococcus virus system [In previous studies, different virus or host characteristics have been described, ranging from generalist to specialist viruses or highly susceptible to highly resistant hosts. Infection patterns based on the virus or host characteristics have been described in . Clusters system . The seas system , 33, 34.Ostreococcus spp. to flourish [Ostreococcus to take advantage of thriving subpopulations of the host.While it is known that viruses are species-specific, infections can also be specific to environmental conditions . We may flourish . Being aOstreococcus spp. are non-blooming picoplankton species that are adapted to the environmental conditions  that they have experienced in the past . These n et al. ). The vin et al.  showed tFurther infection experiments with changes in salinity or temperature as compared to pure co-evolutionary studies in this system would shed light on these two possible explanations of the infection patterns we found. In addition, finding differences in infection patterns depending on the environmental condition could allow us to make predictions on how phytoplankton would cope under future climate conditions.Ostreococcus populations [The variations in plaque size increase showed that the seasonal and geographical origin and thus evolutionary history are important drivers for the ecology of infections. The differences in plaque size change and when the plaques appear can be used to identify several aspects of the infections: (i) the later a plaque becomes visible, the potentially longer the latency period from the start of infections is; (ii) the faster a plaque increases, the faster the virus spreads; and (iii) the larger the plaque becomes, the stronger the infection is. However, liquid infection assays where host and virus traits are usually identified are needed to validate these assumptions. All of the mentioned characteristics of the infection will affect the infection dynamics within the ecosystem and potential effects on the ulations . TherefoSupplementary file1 (DOCX 1315 KB)Below is the link to the electronic supplementary material.