Viruses cause important yield losses in kiwifruit. Here, we studied the occurrence and population structure of the major kiwifruit viruses in the Sichuan province of China. RT-PCR results showed the presence of Actinidia virus A (AcVA), Actinidia virus B (AcVB), Actinidia chlorotic ringspot-associated virus (AcCRaV), and the cucumber mosaic virus (CMV). AcCRaV was widely distributed, followed by CMV. These two viruses were often detected in co-infection with AcVA and AcVB. The virus detection rate was positively correlated with vine age. Four phylogenetic groups of AcVA and AcVB were identified, with AcVA isolates clustering mainly in subgroup I, and AcVB isolates clustering mainly in subgroups II, III, and IV. All CMV isolates clustered in subgroup II, and AcCRaV isolates clustered in subgroup IA. The genome of AcVA and AcCRaV was under negative selection pressure, while the genome of AcVB and CMV was under positive selection pressure. All the viruses, except AcVB, were in a state of expansion. The full-length genome of the most widely distributed AcCRaV isolate in kiwifruits in the Sichuan province was characterized by sequencing. Unique eight-nucleotide (TTTTTGAT) repeats were found in the 5′-terminal non-coding region of the AcCRaV RNA3 in a possible association with reduced disease symptoms. This is the first study of kiwifruit viruses in Sichuan. The Sichuan and Shaanxi provinces produce about 75% of China’s kiwifruit. Kiwifruit is highly susceptible to infection by fungi, bacteria, and viruses . Most prBetaflexiviridae family, such as Actinidia virus A (AcVA), Actinidia virus B (AcVB), and the Actinidia seedborne latent virus (ASbLV) [Viruses that specifically infect kiwifruit are mostly from the  (ASbLV) ,4,5. Inf (ASbLV) . The cit (ASbLV) ,8,9. Act (ASbLV) . Later,  (ASbLV) ,12,13. A (ASbLV) .Other kiwifruit viruses include cucumber mosaic virus (CMV) , apple sThe genetic diversity of virus populations is affected by many factors, including selection pressure and gene flow ,30,31,32Kiwifruit leaf samples with vesicular shrinkage, ringspots, leaf roll, chlorotic spots, narrow and long deformities, vein yellowing, necrosis, and other symptoms were collected from Dujiangyan City, Qionglai City, Ya’an City, Cangxi County, and Pujiang County in the Sichuan province, China. Leaf samples were stored in dry bottles with anhydrous calcium chloride, transported in an ice box at 4 °C, and preserved in a freezer at −80 °C.2O were added to bring the reaction system up to 10 μL for cDNA synthesis. PCR was performed as follows: 94 °C for 3 min for denaturation, followed by 35 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min. The CP sequences of the virus were compared by DNAMAN software. Primers were designed using Premier 5.0 software according to conserved virus genome regions. The primers were synthesized by Shenggong Bioengineering Co., Ltd. . The primer sequences used for amplifying the viruses are listed in Total RNA was extracted from leaves using a previously described method ; 4 μg ofThe PCR products were analyzed by gel electrophoresis, purified by a DNA gel extraction kit  and sequenced by TSINGKE Biological Technology Company . For each sequence, to ensure accuracy, additional PCR and sequencing experiments were performed at least three times.Escherichia coli strain JM109 and sequenced by TSINGKE Biological Technology Company . For each CP gene, three clones were sequenced in both orientations to confirm the consensus sequence, then all sequences were deposited in the GenBank database. The accession numbers of the virus sequences and information on the sampling sites are listed in Purified PCR products of four viruses  were cloned and analyzed to study the molecular variability of the viruses. Samples that were RT-PCR-positive for AcVA, AcVB, AcCRaV, and CMV were selected and used for amplification of the complete coat protein (CP) gene using the CP gene primers  and the The classification of disease severity referred to the 9-grade classification standard. The kiwifruit virus disease classification standards are: 0, asymptomatic; Grade 1, an area of chlorosis less than 10% of the total leaf area; Grade 3, an area of chlorosis 11–25% of the total leaf area, and the leaves are slightly atrophic; Grade 5, an area of chlorosis 26–40% of the total leaf area; Grade 7: an area of chlorosis 41–65% of the total leaf area, and leaf atrophy is more serious; Grade 9, an area of chlorosis over 65% of the total leaf area, leaves are curled and/or necrotic. The chlorotic areas on the collected kiwifruit leaves were selected using Photoshop software to take the average of the total area and the mosaic area.CP genes were aligned using MUSCLE, as implemented in the MEGA 5.0 software. Six methods  were performed using the RDP4 software package to detect possible recombination events in the CP genes of the virus populations. Predictions were considered reliable when the results obtained by four or more of the six methods were supported by p-values of <10−6. Phylogenetic trees of the CP genes of the viruses were reconstructed using the neighbor-joining method with the Kimura two-parameter model implemented in the MEGA 5.0 software. To test the phylogeny, 1000 bootstrap replications were performed in total. Branches with bootstrap values less than 50% were removed [We analyzed the CP gene fragment of 96 virus samples from different vines. There were 22 samples of AcVA, 28 samples of AcCRaV, 16 samples of AcVB, and 30 samples of CMV . The seq removed .Haplotype diversity (Hd) and nucleotide diversity (π) were used to estimate the genetic diversity . The extTo explore variations in the evolutionary constraints in the CP genes of the viruses, the ratio of the non-synonymous substitution rate (dN) to the synonymous substitution rate (dS) was calculated in DnaSP 5.0. Tajima’s D, and Fu and Li’s D and F tests, implemented in DnaSP 5.0, were used to explore the demographic history of the virus populations . Tajima’https://www.ncbi.nlm.nih.gov/orffinder/ accessed on 12 September 2022) for analyzing the sequence alignments.We adopted MEGA 5.0 for a genome-wide comparison of AcCRaV, using the website viewer ORFfinder of the NCBI  at Among the single infections, AcCRaV had the highest detection rate (41.94%), followed by CMV. The percentages of AcVA + AcVB, AcVA + AcCRaV, and AcCRaV + CMV were 5.16%, 7.10%, and 8.39%, respectively . AcCRaV Four kiwifruit viruses were tested. Leaves infected with AcVA showed mild chlorosis and mild atrophy A. LeavesLeaf samples infected by AcCRaV and CMV showed localized local deformities and large chlorotic spots with irregular edges. With the appearance of red patches, this was similar to the symptoms of CMV F. The syAt the same altitude (600 m), the virus detection rate was related to the age of the kiwifruit vines . As the AcVA CP gene sequences identified in this study and 28 AcVA CP gene sequences identified in ‘Xuxiang’ kiwifruit. Zhao Lei et al. divided the AcVA CP gene sequence into two subgroups, namely subgroups I and II in ‘Xuxiang’ kiwifruit [Phylogenetic analysis was performed on the 23 iwifruit . AcVA isAcVB CP gene sequences analyzed in this study and the 16 AcVB CP gene sequences [AcVB CP gene sequences of Sichuan , and that from the Qionglai isolate was close to that from JY6 (an isolate from Jiangyou lettuce) (Cucumber mosaic virus (CMV) was divided into subgroups I and II, and subgroup I was divided into subgroup IA and subgroup IB. Phylogenetic analysis was performed on 30 lettuce) A.AcCRaV CP gene sequences obtained in this study and 24 sequences [Phylogenetic analysis was performed on the 28 equences  of ShaanAmong the AcVA isolates, high haplotype diversity occurred in isolates from Zhouzhi, Hanzhong, Yangling, Guangyuan, Qionglai, and other places. The Guangyuan isolate had the highest nucleotide diversity, while the Zhouzhi isolate had the lowest nucleotide diversity. AcVA may have first appeared in Guangyuan City, Sichuan province .Among the AcVB isolates, high haplotype diversity was found in Meixian, Yangling, Guangyuan, Ya’an, and Dujiangyan. The haplotype diversity was lowest in Qionglai. The diversity of nucleic acids was highest in Dujiangyan isolates and lowest in Yangling isolates. AcVB may have first appeared in Dujiangyan City, Sichuan province .Among the CMV isolates, high haplotype diversity was found in isolates from Meixian, Zhouzhi, Hanzhong, and Yangling. The haplotype diversity was lowest in the broad-element population. The diversity of nucleic acids was highest in Yangling isolates. CMV-infecting kiwifruit may have first appeared in the Yangling District, Xianyang City, Shaanxi province .Among the AcCRaV isolates, high haplotype diversity was found in isolates from Zhouzhi, Hanzhong, Yangling, and Ya’an. The haplotype diversity of the Guangyuan isolate was the lowest. Nucleic acid diversity was highest in the Zhouzhi isolates and lowest in the Ya’an isolates. AcCRaV may have first appeared in Zhouzhi County, Shaanxi province .AcVA CP gene, the FST values of the populations between Ya’an and Pujiang, between Ya’an and Meixian, between Ya’an and Hanzhong, between Ya’an and Zhouzhi, between Ya’an and Yangling, between Pujiang and Meixian, between Pujiang and Hanzhong, between Pujiang and Zhouzhi, and between Pujiang and Yangling were greater than 0.25000, indicating that there were large genetic differences in the populations from these regions. The FST values between groups from Ya’an and Qionglai, and between groups from Ya’an and Guangyuan were between 0.15000 and 0.25000. The FST value between the populations from Pujiang and Guangyuan was less than 0.15000, indicating that there was a moderate degree of genetic differentiation in the CP genes of the AcVA populations from Pujiang and Guangyuan. The FST value between Pujiang and Zhouzhi was the highest, namely 0.67050. The second highest FST value was that between Ya’an and Zhou Zhi, namely 0.65041. The FST value between the populations of Pujiang and Guangyuan was the lowest at 0.12429. In general, the CP genes of the AcVA populations had a moderate degree of genetic differentiation among many regions. The Nm values between the populations of Ya’an and Qionglai, between those of Ya’an and Guangyuan, between those of Ya’an and Meixian, between those of Ya’an and Yangling, between those of Pujiang and Qionglai, and between of those of Pujiang and Guangyuan were all > 1.00, indicating that there was significant gene flow between the populations of these places. The results showed that the gene flow of CP genes in the AcVA populations was not influenced by the geographical location . The populations of Pujiang and Qionglai had the smallest FST value of 0.10212. The Nm values between populations from Pujiang and Qionglai, between populations from Dujiangyan and Meixian, between populations from Dujiangyan and Meixian, between populations from Meixian and Yangling, between populations from Hanzhong and Zhouzhi, and between populations from Zhouzhi and Yangling were all higher than 1.00. This result suggested that there is gene flow between these places. The largest Nm value was between the populations from Pujiang and Qionglai (4.40). There was no significant gene flow between other populations . Gene flow between other regions was not obvious  and other places. The flow of the AcVB CP gene mainly occurred between Meixian County and other regions. The flow of the CMV CP gene mainly occurred in the Sichuan province or the Shaanxi province. CMV is a typical virus transmitted by aphids [AcCRaV-CP gene mainly occurred in Pujiang and other places. Through investigation, it was found that Pujiang is an important production base for kiwifruit germplasm resources. AcCRaV is the most prevalent virus in Sichuan. Some kiwifruit branches with viruses are used for sale. Therefore, human factors also lead to the spread of the virus.In terms of gene flow, most of the flow of the y aphids . Previouy aphids . The difAcVA CP gene was subjected to negative selection pressure. Most AcVB CP and CMV CP genes were subjected to positive selection pressure, while most AcCRaV CP genes were subjected to negative selection pressure. According to the aforementioned analyses of the genetic differences and selection pressure, the CP genes of AcVB and CMV have been greatly influenced by the host and the natural environment [The ironment .From the perspective of kiwifruit viruses’ population dynamics, except for AcVB, the other three viruses showed the trend of population expansion . OngoingAcCRaV is widely distributed in Sichuan and was selected for full-length analysis. The 5′-terminal non-coding region plays an important role in viral transcription and replication. AcCRaV has very long non-coding regions at the 5′-terminal ends of RNA3 and RNA4, ranging from 289 bp to 645 bp. The non-coding region repeats at the 5′-terminal of AcCRaV RNA3 make the symptoms of the host milder. Viruses that are missing repetitive sequences in the 5′-terminal non-coding region aggravate the symptoms in kiwifruit leaves. Our previous study also found that recombination in the soybean mosaic virus could also alleviate the symptoms of the host . More reIn conclusion, this study identified the main viruses infecting kiwifruit in Sichuan, China, and determined the incidence, distribution, and genetic diversity of these viruses. This is the first report of the molecular variability of AcVA, AcVB, AcCRaV, and CMV in Sichuan. These results provide important data for the study of kiwifruit viruses, especially the genetic evolution of the four viruses. Our study provides a basis for the development of guidelines for the prevention of the spread of kiwifruit viruses in the Sichuan province, China. At present, the effect of the viruses on kiwifruit production in China is unclear; few studies have been conducted. Consequently, there is an urgent need for further studies to determine the impact of viruses and recommend appropriate management practices.


The global burden of plant viruses is significantly affecting the world’s food production. The impact is more localized to the regions where there is a very high population density with limited access to resources. This situation is further worsened by the impact of global climate change . CombattXie et al. are looking at wheat streak mosaic virus (WSMV); one of the major diseases that affect wheat production globally. They were able to confirm that the Wsm2 locus is the major locus that controls the WSMV resistance in wheat. Then, they looked at the genetic variations in the locus and came up with potential candidate genes that can be used in future breeding programs.cis-regulatory elements on gene promoters can determine the spatiotemporal gene expression against virus attacks. Ke et al. are doing a deep dive into the regulation of Nicotiana bentamina argonautes 5(NbAGO5); a major gene that is involved in plant defense responses. They highlight the role of NbNAC42 and NbZF3 in the fine regulation of NbAGO5 mediated via the NbAGO5 promoter.The distribution and frequency of Buoso et al.). Guo et al. show that the boron-induced reactive oxygen species (ROS) signaling pathways are responsible for the Cucumber green mottle mosaic virus (CGMMV) resistance using N. benthamina and watermelon plant systems. Their results have a wide application in the fields where farmers can achieve virus resistance by modifying their agronomic practices.External environmental factors can greatly influence plant responses to viruses. Boron is known to enhance plant defense against viruses (Tan et al. demonstrate the functional characterization of UDP-Glycosyltransferases which are known for their antiviral properties. Their findings lay the groundwork for mass production of the UDP-Glycosyltransferases using novel synthetic biology approaches.VH wrote the article; VH and JV have revised the manuscript. All authors contributed to the article and approved the submitted version.


Regional differences in general practitioner consult costs remain significant even after taking into account health, lifestyle, loneliness, and mastery. Regional differences in costs for mental, pharmaceutical, and specialized care are less pronounced and can be explained to a large extent. For total healthcare costs, regional differences are mostly explained through the factors included in this study. Hence, addressing lifestyle factors, loneliness and mastery can help policymakers in restraining healthcare costs. In this study, the region of Zuid-Limburg represents the reference region. Use compare regions for health and healthcare costs (Regiovergelijker gezondheid en zorgkosten) in order to select all other Dutch regions as reference region.Healthcare costs in the Netherlands are rising and vary considerably among regions. Explaining regional differences in healthcare costs can help policymakers in targeting appropriate interventions in order to restrain costs. Factors usually taken into account when analyzing regional differences in healthcare costs are demographic structure and socioeconomic status (SES). However, health, lifestyle, loneliness and mastery have also been linked to healthcare costs. Therefore, this study analyzes the contribution of health, lifestyle factors , loneliness, and mastery to regional differences in healthcare costs. Analyses are performed in a linked dataset (The online version of this article (10.1007/s12508-022-00369-4) contains supplementary material, which is available to authorized users. Zuid-Limburg) in particular. In order to reduce the increase in healthcare costs, explanations for regional differences are needed to provide insight into possible leads.Healthcare costs in the Netherlands continue to increase annually . Before A study based on relocations in the Netherlands between 2006 and 2013 showed that 70% of regional differences in total healthcare costs are explained by demand factors . In thesJournal for Health Sciences (Tijdschrift voor gezondheidswetenschappen), we also show that lifestyle, loneliness, and mastery partly explain regional differences in general and mental health  [In this national study, we aimed to explain regional variations in healthcare costs based on lifestyle factors, loneliness, and mastery, after adjusting for demographic factors, SES, and general and mental health. In an accompanying article in a special issue of the istress) .The research question for the current study was: “Do lifestyle factors, loneliness, and mastery contribute to explaining regional differences in healthcare costs in addition to age, gender, SES, and health status?” The results can direct future preventive interventions and policies to help reduce regional differences in healthcare costs.Data were extracted from a linked dataset provided by the Dutch Public Health Services, Statistics Netherlands, and the healthcare claims–based database Vektis. The Health Survey 2016 provides information about demographic factors, SES, lifestyle, loneliness, mastery, and physical and mental state of health of the respondents . RegistrThe datasets were linked using pseudonymized RIN numbers provided by Statistics Netherlands in a secured digital environment. After data linkage and exclusion of missing data, the sample consisted of 334,721 persons. To ensure the representativeness of the sample, weighting factors were added to the Health Survey .The dependent variables were the following five cost categories: total costs , general practitioner (GP) consultation costs, pharmaceutical costs, specialized care costs , and mental healthcare costs . For inpatient mental healthcare, costs are covered by the Healthcare Insurance Act for up to three years. After three years, these costs are covered by the Dutch Long-term Care Act. The costs covered by the Long-Term Care Act were not included in the Vektis data.The independent variables were region, demographic characteristics, SES, general and mental health, lifestyle, loneliness, and mastery. For the variable “region”, we used the regional classification of the Public Health Survey 2016, with the region of Zuid-Limburg as the reference group. Zuid-Limburg was chosen because this is the region with the highest healthcare costs, the highest number of adults with a chronic disease , the low2 was considered “underweight”, 18.5–25 kg/m2 was “normal”, 25–30 kg/m2 was “overweight”, and > 30 kg/m2 was “obese”), smoking history , alcohol consumption  and (in)sufficient physical activity. Loneliness was assessed with the De Jong-Gierveld scale [Demographic factors were age, gender, migration background , and marital status . SES consisted of the highest attained level of education , standardized household income quartile, and income inadequacy  . To asseld scale   was assessed with Poisson regressions. The IRR represented the change in costs per change of the particular independent variable. For the cost categories of GP consultations, pharmacy, specialized care, and mental healthcare, zero-inflated binomial (ZINB) distributions were used, because most people do not need all these types of healthcare and these cost data therefore contain many zeros Tab. . The ZINFor Model 1, only region was included. In Model 2, the demographic characteristics and SES were added to region. Self-perceived health was added in Model 3a, self-reported chronic disease in Model 3b, and psychological distress in Model 3c. In Model 4, the correlation of region with healthcare costs was corrected for demographic factors, SES, self-perceived health, chronic illness, and psychological distress. Lifestyle was added in Model 5a, loneliness in Model 5b, and mastery in Model 5c. In Model 6, all beforementioned factors were included.Marginal average costs were calculated based on the unadjusted costs (Model 1) and the adjusted costs (Model 6). Marginal costs per region represented the average costs per person in a particular region if the population of this region shared the same demographic factors, SES, general and mental health, lifestyle, loneliness, and mastery as the entire Dutch population. All analyses were performed in Stata 15 . MultiplSlightly over half of the sample was female (52.4%), and the mean age was 59.2 years (standard deviation (SD): 16.9) . The maHollands Noorden) (IRR: 0.93) and South Holland–South (Zuid-Holland-Zuid) (IRR: 0.92) incurred significantly lower total healthcare costs than residents of Zuid-Limburg. Residents of the region of Amsterdam incurred significantly higher healthcare costs (IRR: 1.14)  and the most comprehensive model (Model 6) are shown in Tab.  IRR: 0.9 incurredThe results for GP consultation, mental healthcare, pharmaceutical, and specialized care costs are twofold and are shown in Appendix Tables A4–A7. In addition, the marginal costs were calculated based on both parts of the ZINB model. These are visualized in Appendix Figures A1–A4.By adjusting for demographic factors, SES, and general and mental health, the observed cost differences between regions could be largely explained. The differences that persisted after these corrections could be partly explained by lifestyle, loneliness, and mastery (Appendix Tables A3–A7). Lifestyle (Model 5a) mainly contributed to  regional differences in total, GP consultation, pharmaceutical, and specialized care costs. Loneliness (Model 5b) contributed to explaining the  regional differences in GP consultation, mental healthcare, and specialized care costs. Mastery (Model 5c) contributed to the  differences in mental healthcare and pharmaceutical costs.The marginal costs for total healthcare are shown in Fig. We observed large regional differences in healthcare costs in the Netherlands. Based on total healthcare costs, regional differences could be explained. In addition to the most common factors (demographic factors and SES), health status, lifestyle, loneliness, and mastery contributed to these variations in different ways. General and mental health explained a large part of the regional differences in healthcare costs. Lifestyle, loneliness, and mastery contributed directly and to a small extent to the explanation of regional differences in healthcare costs. Our other published study showed that lifestyle, loneliness, and mastery contributed to regional differences in health and thus indirectly to regional differences in healthcare costs as well . This stWith regard to various healthcare cost categories, a number of findings stand out. First, residents of Zuid-Limburg incurred the highest GP consultation costs, even in the most comprehensive model. In other words, with the same demographic factors, SES, general and mental health, lifestyle, loneliness, and level of mastery, residents of Zuid-Limburg still had more GP consultations. This corresponds with earlier, albeit anecdotal, evidence from research into the health inequalities in Limburg . Even thSecond, regional differences in mental healthcare costs  were less frequently observed, but they were more persistent than regional differences in other healthcare cost categories. Even when we included all factors in this study, regional differences remained. Further research is needed to determine which factors can further contribute to explaining regional differences in mental healthcare costs. A further breakdown of mental healthcare costs  possibly provides more insight into regional cost differences. In addition, combining cost data from the Healthcare Insurance Act and the Long-Term Care Act offers opportunities for improved analyses of long-term, inpatient mental healthcare costs. Moreover, differences between urban and less urban areas within regions or between areas on a smaller geographic scale, such as at the municipal, district, or neighborhood level, may also contribute to explaining regional differences in healthcare costs in future research.Our findings help health insurance companies and policymakers justify investments in basic conditions for health, lifestyle, loneliness, and mastery. Although the direct contribution of lifestyle, loneliness, and mastery seems limited, these contributions still result in large variations in healthcare costs between regions. The differences in GP consultation costs, for example, became significantly smaller in 23 regions, and the differences in mental healthcare costs were significantly smaller in three regions. At the population level , we were able to explain € 1.6 million in GP consultation costs and € 4 million in mental healthcare costs (see Appendix Tables A8 and A9).In addition to this direct contribution, the three factors also contributed to the explanation of regional health differences . These hA second implication is extension of the set of individual characteristics in determining healthcare budgets. For example, in order to prevent risk selection and premium differentiation, health insurance companies receive a contribution from the Health Insurance Fund in addition to the premiums collected. This contribution, the risk equalization, is calculated based on, among other things, demographic and socioeconomic characteristics and zip code area . HoweverA potential limitation of this study is the composition of the sample. Selection bias may occur as certain groups of people are less likely to participate in research, such as those with a lower SES and/or poorer health . The samThe association between mastery and healthcare costs is increasingly important as the role of the citizen (or patient) is becoming more important as society and the healthcare system are increasingly complex . In addiwith education, housing, and public spaces that may affect health skills and self-reliance. In addition, the most vulnerable regions benefit from reconsidering distribution models for risk equalization, since a wider range of individual factors contribute to health, and thus healthcare costs, than the factors that are currently taken into account.In conclusion, the factors included in this study could largely explain regional differences in total healthcare costs. This offers leads for preventive investments aimed at a wide range of individual factors in conjunction. This does not imply an approach that is merely focused on combating an unhealthy lifestyle but also on combating loneliness and improving mastery. This calls for a comprehensive approach and further cooperation between the social and medical sector and Appendix


Whether or not individuals with pauci-symptomatic or asymptomatic Ebola virus infection and unrecognised Ebola virus disease develop clinical sequelae is unknown. We assessed current symptoms and physical examination findings among individuals with pauci-symptomatic or asymptomatic infection and unrecognised Ebola virus disease compared with Ebola virus disease survivors and uninfected contacts.Between June 17, 2015, and June 30, 2017, we studied a cohort of Ebola virus disease survivors and their contacts in Liberia. Surveys, current symptoms and physical examination findings, and serology were used to characterise disease status of reported Ebola virus disease, unrecognised Ebola virus disease, pauci-symptomatic or asymptomatic Ebola virus infection, or no infection. We pre-specified findings known to be differentially prevalent among Ebola virus disease survivors versus their contacts . We estimated the prevalence and incidence of selected clinical findings by disease status.Our analytical cohort included 991 reported Ebola virus disease survivors and 2688 close contacts. The median time from acute Ebola virus disease onset to baseline was 317 days (IQR 271–366). Of 222 seropositive contacts, 115 had pauci-symptomatic or asymptomatic Ebola virus infection and 107 had unrecognised Ebola virus disease. At baseline, prevalent findings of joint pain, memory loss, muscle pain, and fatigue were lowest among those with pauci-symptomatic or asymptomatic infection or no infection, higher among contacts with unrecognised Ebola virus disease, and highest in reported survivors of Ebola virus disease. Joint pain was the most prevalent finding, and was reported in 434 (18%) of 2466 individuals with no infection, 14 (12%) of 115 with pauci-symptomatic or asymptomatic infection, 31 (29%) of 107 with unrecognised Ebola virus disease, and 476 (48%) of 991 with reported Ebola virus disease. In adjusted analyses, this pattern remained for joint pain and memory loss. Survivors had an increased odds of joint pain compared with unrecognised Ebola virus disease contacts ; unrecognised Ebola virus disease contacts had an increased odds of joint pain compared with those with pauci-symptomatic or asymptomatic infection and uninfected contacts . The adjusted odds of memory loss was more than four-times higher among survivors than among unrecognised Ebola virus disease contacts  and two-times higher among unrecognised Ebola virus disease contacts than in those with pauci-symptomatic or asymptomatic infection and uninfected contacts . By 12 months, prevalent findings had decreased in the three infected groups.Our findings provide evidence of post-Ebola virus disease clinical sequelae among contacts with unrecognised Ebola virus disease but not in people with pauci-symptomatic or asymptomatic Ebola virus infection.National Cancer Institute and National Institute of Allergy and Infectious Diseases of the National Institutes of Health. In addition to those with pauci-symptomatic or asymptomatic infection, unrecognised Ebola virus disease survivors might have also had, on average, less severe acute disease than those diagnosed and treated at Ebola treatment facilities.7 Both groups of individuals—those with pauci-symptomatic or asymptomatic infection and those with unrecognised Ebola virus disease—might have developed post-infectious clinical sequelae.Individuals with suspected Ebola virus disease are typically categorised as survivors if they were diagnosed as PCR-positive for Ebola virus, discharged alive from a health-care facility or Ebola treatment facility, and listed in an Ebola virus disease registry.21 These studies raised the question of whether a dose–response relationship exists between the severity of acute Ebola virus disease and subsequent clinical sequelae. However, the small size of previous Ebola virus disease outbreaks and their occurrence mostly in settings with under-resourced health systems prevented systematic study of this hypothesis in individuals with pauci-symptomatic or asymptomatic infection and unrecognised Ebola virus disease.Little evidence exists linking acute Ebola virus disease illness and its severity to clinical sequelae; however, studies have shown that particular symptoms  or higher viraemia, or both during acute Ebola virus disease correlated with subsequent clinical sequelae.During the 2013–16 Ebola virus disease outbreak in Liberia, we identified a large cohort of Ebola virus disease survivors and contacts, inclusive of individuals with pauci-symptomatic or asymptomatic infection and unrecognised Ebola virus disease who can be assessed for evidence of clinical sequelae. Since individuals with pauci-symptomatic or asymptomatic infection and unrecognised Ebola virus disease probably had less severe acute disease than reported survivors, we hypothesised that individuals with less severe acute disease experience post-Ebola virus disease clinical sequelae to a lesser extent than reported Ebola virus disease survivors, and that a viral load-dependent relationship exists between the severity of acute illness and clinical sequelae.NCT02431923) of Ebola virus disease survivors and close contacts in Liberia, implemented through a partnership between the Ministry of Health in Liberia and the National Institute of Allergy and Infectious Diseases (NIAID). A primary objective of the PREVAIL III study is to determine the sequelae of Ebola virus infection. Enrolment occurred at three research sites in Liberia  from June 17, 2015, to June 30, 2017. The methods and findings of the primary study have previously been published.7 In brief, PREVAIL III enrolled Ebola virus disease survivors who were listed in the Liberian Ministry of Health Registry and had a documented diagnosis of the disease. These survivors listed their close contacts, who were then eligible to be enrolled. A close contact was an individual selected by an Ebola virus disease survivor as someone with whom the survivor had contact during acute Ebola virus disease or with whom they had sexual contact following acute illness. Survivors and close contacts underwent study visits every 6 months that included a symptom checklist, physical examination, and collection of blood. A subset of participants was referred for a separate eye examination.7We used data from a longitudinal cohort study  per mL was used to determine seropositivity with 94·4% sensitivity and 96·7% specificity.7Serum samples were analysed at the Liberian Institute for Biomedical Research, as previously described22 with responses selected as a binary (yes or no). We compiled these responses and classified each contact participant as either asymptomatic or symptomatic. Participants who were seropositive and asymptomatic and responded no to all 16 questions were classified as having had a pauci-symptomatic or asymptomatic infection. We selected pauci symptomatic or asymptomatic as the description of individuals who reported being asymptomatic because of the potential for mild symptoms with recall error. Contact participants who were seropositive and symptomatic were defined as having had unrecognised Ebola virus disease.The explanatory variable included four groups: Ebola virus disease survivors (seropositive); contacts with unrecognised Ebola virus disease (seropositive); contacts with pauci-symptomatic or asymptomatic Ebola virus infection (seropositive); and uninfected contacts (seronegative). We assumed that individuals who were seropositive had an Ebola virus infection following exposure; we then used the serostatus and self-reported post-exposure symptoms of each participant to determine group membership. Self-reported symptoms were determined with the following question: “Did you develop any of the following symptoms within 21 days of the survivor’s Ebola event?” The checklist of self-reported symptoms included 16 items from the WHO Ebola virus disease case definition,7Severity of acute Ebola virus disease illness was ranked (less severe first) by the following categories of disease: no infection; pauci-symptomatic or asymptomatic infection; unrecognised Ebola virus disease; and patients with reported and confirmed Ebola virus disease who survived. The individuals with unrecognised Ebola virus disease did not receive testing or medical care during their illness, were more likely to remain at home through the duration of their infection, and reported, on average, fewer symptoms than reported Ebola virus disease survivors during their post-exposure period.7 Symptoms were urinary frequency, headache, fatigue, muscle pain, memory loss, and joint pain. Physical examination findings were neurological findings, chest (respiratory) findings, muscle findings, joint findings, abdominal findings, and uveitis.Outcomes were defined as current symptoms and physical examination findings reported at each study visit. The current symptoms and physical examination findings reported in this analysis were limited to those found to be statistically significantly more or less prevalent between survivors and close contacts at baseline (p<0·0001) in the parent study by Sneller and colleagues.Categorical baseline factors were compared between groups using χ2 tests, age at enrolment was compared between groups using one-way ANOVA, and baseline antibody concentrations were compared between survivors and close contact groups using linear regression models with generalised estimating equations (GEE) that adjusted for relationships between survivors and close contacts with an independent correlation. Comparisons of the prevalence of self-reported symptoms and abnormal findings on physical examination at the baseline and 12-month study visits were analysed using GEE logistic regression models. All models were adjusted for age at PREVAIL III enrolment, sex, and enrolment site, except for models comparing uveitis at the 12-month visit, which was only adjusted for age and sex. These GEE models adjusted for potential correlation of outcomes within groups of survivors and associated close contacts.We did time-to-event analyses for symptoms, physical examination findings, hospitalisation, and death within 1 year of enrolment. Incidence rates per 1000 person-years of symptoms and physical examination findings are reported for individuals without symptoms or findings at baseline. Time-to-event for symptoms and hospitalisation was calculated as the number of days from enrolment to the date of the 6-month or 12-month follow-up visit: either the first at which a symptom or hospitalisation was reported, or, if neither were reported, the last of these follow-up visits to occur. Survival time was calculated as the number of days from enrolment to death, follow-up discontinuation date, or the 1-year anniversary of enrolment, whichever occurred first. The occurrence of symptoms, findings on physical examination, hospitalisation, and death within 1 year of enrolment were compared between groups using Cox proportional hazard models, which adjusted for age, sex, and enrolment site, except the model for uveitis which only adjusted for age and sex. GEE were used to adjust for potential correlation of outcomes within groups of survivors and associated close contacts. We estimated hazard ratios from these Cox models. All analyses were done using R version 3.3.2, and p values less than 0·05 were considered statistically significant.The sponsor of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.Among 3679 participants, 991 (27%) were reported Ebola virus disease survivors, 107 (3%) were contacts with unrecognised Ebola virus disease, 115 (3%) were contacts with pauci-symptomatic or asymptomatic infection, and 2466 (67%) were uninfected contacts. The median time from acute Ebola virus disease onset to baseline was 317 days (IQR 271–366). Among all 3679 participants, 2048 (56%) were female, and the median age was 25 years (IQR 15–36). The baseline characteristics of the analysis cohort are shown in We determined the prevalence of selected symptoms and physical examination findings and created a graphical representation for each group at study baseline . Table 2vs 14 [12%] of 115 pauci-symptomatic or asymptomatic contacts vs 31 [29%] of 107 contacts with unrecognised Ebola virus disease vs 467 [47%] of 991 Ebola virus disease survivors), memory loss (113 [5%] vs 6 [5%] vs 11 [10%] vs 284 [29%]), muscle pain (242 [10%] vs 10 [9%] vs 16 [15%] vs 227 [23%]), and fatigue , while contacts with unrecognised Ebola virus disease had a similarly increased odds of joint pain compared with contacts with pauci-symptomatic or asymptomatic infection and uninfected contacts , each finding was most prevalent in Ebola virus disease survivors and observed at similar prevalence among the other three groups. For example, urinary frequency was observed in 143 (14%) of 991 reported Ebola virus disease survivors but only in three (3%) of 107 unrecognised Ebola virus disease survivors, five (4%) of 115 pauci-symptomatic or asymptomatic contacts, and 83 (3%) of 2466 uninfected contacts. For one symptom (headache), the finding had similarly high prevalence among reported and unrecognised Ebola virus disease survivors but was equally low among pauci-symptomatic or asymptomatic and uninfected contacts. In adjusted analyses, these patterns remained for urinary frequency, muscle pain, and headache, but not uveitis .From baseline to the 12-month visit, the selected symptoms and clinical findings generally decreased in prevalence . As a reDuring the same 12-month study period, participants reported the new occurrence of selected symptoms and clinical findings that were not reported at baseline. These incident findings occurred among fewer participants than prevalent findings , 4. We c24 Our findings were consistent for multiple symptoms , which adds strength to the evidence that post-Ebola virus disease sequelae occur among unrecognised Ebola virus disease contacts. Once contacts with post-exposure Ebola virus disease symptoms are identified as seropositive in future outbreaks, the Ebola virus disease response community should screen this group for post-Ebola virus disease clinical sequelae and offer clinical care and support services.In this longitudinal cohort in Liberia following the 2013–16 Ebola outbreak, we found evidence of post-Ebola virus disease clinical sequelae in contacts with unrecognised Ebola virus disease but not in contacts with pauci-symptomatic or asymptomatic Ebola virus infection. Previous cohort studies of Ebola virus disease survivors were smaller in size, so even if they had identified a group of contacts with pauci-symptomatic or asymptomatic infection and unrecognised Ebola virus disease, they were underpowered, did not use a control group, and were unable to reliably identify the presence or absence of post-Ebola virus disease symptoms and examination findings considered to be clinical sequelae.21 so our findings support this growing body of evidence. We also extend the evidence for a viral load-dependent association between acute illness and clinical sequelae,21 by showing its occurrence across the spectrum of clinical manifestations, particularly in individuals with unrecognised Ebola virus disease (a group identified as having fewer symptoms than reported Ebola virus disease survivors during acute illness7). Our proof-of-concept study offers insight into the types of post-Ebola virus disease clinical sequelae potentially observed in the clinical setting; however, we need natural history studies that prospectively enrol individuals with asymptomatic infection and mild illness during the acute phase and follow them into the convalescence phase in order to confirm our findings.We found patterns that more severe acute illness has the potential to cause specific types of post-Ebola virus disease clinical sequelae. In particular, memory loss, joint pain, headache, and urinary frequency were observed at higher prevalence across groups . Other studies have shown that specific features of acute illness (symptoms associated with more severe illness or degree of viraemia) can lead to post-Ebola virus disease sequelae, including uveitis and joint pain,7 robust over time,26 and have been used in several other high-impact studies.27 In terms of external validity, Ebola virus vaccines were introduced at the end of this outbreak ,28 so our findings represent the potential for post-Ebola virus disease clinical sequelae in an unvaccinated population. Our study also had several strengths, including the use of a control group to demonstrate between-group differences and sufficient power to draw reliable conclusions across most groups. However, we did not have sufficient power to compare unrecognised Ebola virus contacts against contacts with pauci-symptomatic or asymptomatic infection, even though to our knowledge this study was the largest of unrecognised Ebola virus disease and pauci-symptomatic or asymptomatic Ebola virus-infected contacts so far.We acknowledge several limitations to our study. We do not have data  from the pre-enrolment period, including acute illness, so there is potential unmeasured confounding. Given the absence of within-participant measurements starting from the acute illness, we cannot definitively consider the reported current symptoms or physical examination findings as post-Ebola virus disease clinical sequelae. Enrolment started nearly 1 year after survivors were discharged from an Ebola treatment facility, and those individuals who were sicker may have been more likely to participate, which might be a source of selection bias. Our classification of contacts as seropositive or seronegative cannot be used to confirm infection because of potential cross-reactivity and measurement error, but the test performance characteristics of the immunoassay used in this study are highly accurate,29 In conclusion, contacts with unrecognised Ebola virus disease can suffer from post-Ebola virus disease clinical sequelae and are in need of equitable access to care and support services.This paper emphasises the public health and clinical care value in identifying contacts with unrecognised Ebola virus disease, who accounted for a substantial population (8·7% of our cohort). Our proof-of-concept study strongly suggests the need for widespread testing of contacts during an Ebola virus disease outbreak so that the prevalence of unrecognised Ebola virus disease can be reduced and post-outbreak surveillance of remaining individuals with unrecognised illness can lead to their identification and linkage to care. The full clinical spectrum of acute viral infections such as SARS-CoV-2 is increasingly recognised to cause post-infectious clinical sequelae.


Long-acting injectable (LAI) antipsychotics and community treatment orders (CTOs) are used in patients with schizophrenia to improve treatment effectiveness through adherence.Understanding healthcare resource utilization (HRU) and associated costs, and medication adherence in patients with schizophrenia overall and by CTO status before and after second generation antipsychotic (SGA) LAI initiation may guide strategies to optimize health.A retrospective observational single-arm study using administrative data from Alberta was performed. Adults with schizophrenia who initiated SGA-LAI (index date) were included. Medication possession ratio (MPR) was determined; paired t-tests were used to examine differences in HRU and costs ($CDN) between the 2-year pre-index period and 2-year post-index period. Stratified analysis by presence or absence of an active CTO during the pre-post periods was performed.Among 1,211 patients who initiated SGA-LAIs, MPR was greater post-index (0.84) compared with pre-index . All-cause and mental health-related HRU and costs were lower post-index versus pre-index (p<0.001); total all-cause HRU costs were $33,788 lower post- versus pre-index , and total mental health-related HRU costs were $34,198 lower post- versus pre-index . Forty-three percent had ≥1 active CTO during the study period; HRU and costs varied according to CTO status.SGA-LAIs are associated with improved adherence, and lower HRU and costs however the latter vary according to CTO status.The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this abstract: SD and MS have no competing interest to declare. LR, SK, KW, and KM are members of the Real-World Evidence


Monkeypox is a zoonotic Orthopoxvirus called human Monkeypox. It has symptoms that resemble or are pretty similar to smallpox. Monkeypox virus belongs to the genus Orthopoxvirus, which also includes cowpox, vaccinia, and variola viruses. The World Health Organization confirmed in 1970 that the primary virus is the Orthopoxvirus infecting humans after smallpox elimination. Clinically distinguishing the condition from varicella and smallpox is challenging for a clinician. Although the mortality rate of this disease is low, new tests are being tried and studied, which are required for a more accurate and quick diagnosis because the lab diagnosis is the key to the detection of illness and its monitoring. The illness or the virus is endemic to parts of western and central Africa. Surveillance in underdeveloped rural regions is challenging but manageable with evidence-based techniques and training materials for public health professionals. However, as in the present scenario, the disease is having a worldwide outbreak in various countries, and recently India detected its first case on 15 July 2022 in New Delhi. The widespread disease is due to trading exotic pets and international travel. Since smallpox vaccinations are not administered to people regularly, epidemiological studies are required. New medications and vaccines provide hope for treating and preventing Monkeypox; however, further study is required before they can be used effectively. Also, there is a requirement for advanced scientific studies in the etiology, epidemiology, and biological structure of the virus in the endemic zones to know and halt the spread of infection to humans. Monkeypox belongs to the family: Poxviridae and species: Monkeypox virus. It is a type of zoonotic disease which means that it spreads from animals to humans. The spread of the virus can take place via various media. Monkeypox virus was first isolated when monkeys sent from Singapore to a research center in Denmark became ill and were identified in 1958 . The disThe current ongoing outbreak of the Monkeypox viral disease was first confirmed on 6 May 2022. The primary group of the cases was found in the United Kingdom, where the first outbreak case was identified with a traveling history linked to Nigeria, where the disease is already endemic. The recent outbreak of the monkeypox virus among humans occurred mainly in bisexual men or gays. Therefore, male sex transmission is a significant risk factor for the spread of the infection. Henceforth, it is assumed that the monkeypox virus can occur via sexual contact and seminal fluids, including oral or non-penetrative contact. Therefore, having multiple sexual partners can also increase the overall risk of infection. The size of the monkeypox virus under electron microscopy is substantial (less than 250 nm). Viruses are brick-shaped and have a linear ds DNA enclosed in a lipoproteinecious sheath . PoxviruEpidemiological variants of Virus There are two different epidemiological variants of monkeypox viruses: those in West Africa and those in Central Africa. On a general note, West African variant infections show a low severity disease in humans and other primates . A groupHuman-generated cells from previously infected monkeypox patients cannot produce inflammatory cytokines because central African Monkeypox blocks T-cell receptor-mediated T-cell activation. According to these findings, Monkeypox might create a modulator that inhibits host T-cell responses . The CenIn addition, compared to West African strains of Monkeypox, Central African strains selectively suppress host responses, including apoptosis in the host body. The observed differences in pathogenicity could be caused by several loci ,10. AddiPathophysiology The virus multiplies at the site of the inoculation after virus entry by any media , then spreads to nearby lymph nodes. Salivary secretions, oronasal droplets, or direct contact with exudate of the lesion or crust material are thought to be the main routes of transmission ,13. AnotEvidence suggests that the members of the house or health care providers who provide care for a monkeypox patient are at high risk for contracting an illness, even though the man-to-man spread of the virus is less than smallpox. However, the rate of man-to-man transmission occurs up to 11.7% of house members of the cases who had not previously received a vaccine for smallpox . The sprThe onset of symptoms is related to a secondary viral infection causing 1 to 2 days of prodromal symptoms. The symptoms can be fever and lymphadenopathy. Symptoms precede the lesions, and the infected patients can remain highly infectious. Lesions begin in the oro-pharyngeal region and then appear on the skin. When lesions appear, serum antibodies are often detected . Clinical featuresThe incubation period of Monkeypox ranges from five days to three weeks ,16, and The lesions progress at 1- to 2-day intervals through macule, papule, vesicle, and pustule phases throughout the ensuing 2 to 4 weeks. Lesions range in size from 2 to 10 mm, are hard, and undergo synchronous change. Before crusts form, lesions are in the phase of pustules for almost a week or seven days. Most often, the illness clears itself 3 to 4 weeks after the commencement of symptoms, with crusts developing and desquamating during the following 7 to 14 days. Patients are no longer considered to be infective once all crusts fall off. After healing, the lesion may leave pale marks before turning into dark scars . Monkeypox is a self-limited disease. The severity of the disease is related to the period of contact with the virus and the patient's immune status. In the recent 2022 outbreak, many cases of the monkeypox outbreak presented with genital and peri-anal lesions, fever, swollen lymph nodes, and pain when swallowing, with some patients manifesting only single sores from the disease. In a study, the involvement of the anorectal mucosa was reported as the presenting symptom in 61 persons; this involvement was associated with anorectal pain, proctitis, tenesmus, or diarrhea (or a combination of these symptoms) as mentioned in the following figure ComplicationUnvaccinated patients (74%) had more severe problems and sequelae than patients who had received vaccinations (39.5%). Indicative of a secondary lung infection, patients are also seen with respiratory discomfort or bronchopneumonia, a frequent late manifestation of the disease. By the end of the second week of the illness, vomiting or diarrhea can start, which may significantly worsen the dehydration. One patient had encephalitis, while another with more than 4500 lesions had septicemia . Ocular The widespread, long-term consequence of infection survivorship is pitted scarring. Unvaccinated patients have an average case-fatality rate of up to 11% and can lead to death , and youDiagnosisThe diagnostic assay remains the most critical part of identifying the Orthopoxvirus. Conventional tests, including virus isolation from a clinical specimen, electron microscopy, and IHCs, are still reliable, but they need complex lab equipment and highly skilled technicians.Real-time polymerase chain reaction (PCR) specimen analysis can determine whether Monkeypox or Orthopoxvirus is present in a lesion sample -27. All Antibody-based diagnostics are required to identify the root cause of instances discovered retroactively. Orthopoxvirus cross-reactivity makes anti-Orthopoxvirus immunological assays effective in situations when the virus that is causing sickness has already been identified. For the individuals who came in contact with an orthopoxvirus, including by vaccination, anti-Orthopoxvirus immunoglobulin G (IgG) alone cannot offer a conclusive diagnosis. As an alternative, serological assays which measure anti-Orthopoxvirus immunoglobulin M (IgM) are better suited to identify acute retrograde illness, even in people who have previously had vaccinations . Utilizing lesion samples from acute orthopoxvirus infections, a recent Tetracore Orthopox BioThreat Alert pilot produced encouraging results. In preparations with 107 plaque-forming units/mL, this technique consistently recognized the vaccinia and monkeypox viruses, and 5 out of 6 evaluated clinical specimens were correctly identified . Cases of the monkeypox virus frequently want diagnosis and treatment at outlying clinics or healthcare setups devoid of electricity; as a result, there is a mandatory need for the creation of assays that can be used to conduct testing in rudimentary settings with no staff training. The various differential diagnosis of the Monkeypox Virus can be as follows: Smallpox; Chickenpox; Generalized vaccinia; Disseminated zoster; Disseminated herpes simplex; Syphilis; Rickettsial pox; Measles; Drug-associated eruption table  17]..17].Therapy and vaccinationsVarious compounds have shown promising action against the monkeypox virus with the advancement in therapeutics. Cidofovir, an antiviral agent, acts efficiently against various viruses by blocking viral DNA polymerase. The nephrotoxicity associated with cidofovir has been reduced in the modified cidofovir molecule known as CMX-001. Numerous orthopoxvirus species have been used to demonstrate the antiviral activity of CMX-001. The medication ST-246 has demonstrated good efficacy against several orthopoxvirus species, including the variola virus. It prevents the release of the virus from the cell (intracellularly). These substances have been utilized in various combinations to treat severe vaccine-related adverse effects, including with vaccinia immune globulin . In a feAdditionally, clinical studies have demonstrated that modified vaccinia Ankara is safe and promotes the formation of antibodies in individuals with atopy and weakened or low immunity status -conditions that are said to be contraindicated for the injection of live vaccinia . The CDCDisinfectants and biocidal agents used against MonkeypoxIn suspension experiments, 70% ethanol (for 1 minute), 0.2% peracetic acid (for 10 minutes), and 1-10% of a probiotic cleanser (for 1 hour) could cause all inactive vaccine viruses by at least four log10, which was most frequently demonstrated with various types of organic load. In suspension experiments, hydrogen peroxide (14.4%) and iodine (0.04-1%) worked well. Artificially polluted surfaces responded well to sodium hypochlorite (0.25-2.5%) and 2% glutaraldehyde (10 min). In 3 minutes, copper (99.9%) was equally efficient against the monkeypox and vaccinia viruses. Evolution Of Poxvirus The evolution of zoonotic infections to become more contagious or virulent in humans is a big worry. One concern with orthopoxviruses like monkeypox virus is the likelihood that they could develop into diseases that could cause another pandemic similar to smallpox. As a viral DNA polymerase enzyme replicates the DNA genome with 3'-5' exonuclease proofreading activity, xenovirus has a lower mutation rate than RNA viruses . According to molecular clock studies, poxviruses have substitution rates ranging from 2 × 10−6-1 × 10−5 substitutions of nucleotides per site each year , which m Many of the mutants have tandem repeats of a viral gene in their genomes that is either the direct drug target or a near companion of the drug target ,36, enabGene accordions, a multi-step evolutionary process, may hasten the evolution of poxviruses . RecombiIn order to prevent the Monkeypox infection and the spread of the disease following steps are to be followed: Stay at home and avoid contact with the infected person or those with symptoms; Practice safe sex, avoid multiple sexual partners, and avoid close physical contact with anonymous; Use masks to maintain good hand hygiene and respiratory practices, like covering your mouth with a handkerchief or tissue while coughing and sneezing; Avoid touching contaminated surfaces; clean and disinfect them; Use a personal protective kit if caring for an infected person; Eat thoroughly cooked food items that contain animal meat or parts.The spread of Monkeypox has been endemically seen in more than 20 countries since May 2022. The degree of infectivity of Monkeypox is relatively less, and its chances of becoming a pandemic are meager. It is crucial to educate humans and healthcare professionals in areas where the monkeypox virus has already been affected or in areas that are already at high risk of exposure. The best protection against global spread is local confinement. Enhancing patient awareness of the illness, reporting accuracy, and accessibility to diagnostic tools are essential for gathering the data required to comprehend Monkeypox more thoroughly and develop a more vigorous defense against it. The civil war between the countries and forced migration raise worries about the spread of the virus into a monkeypox-free region or about people moving to more heavily forested areas where they are more likely to come into contact with wildlife and a variety of zoonoses. In order to better comprehend the variety of elements involved in the transfer of the disease and distribution, the recorded increase in the frequency of human disease requires deeper investigation and analysis. Improving our knowledge of these essential microorganisms will assist in better direct preventive tactics and lessen human disease. There are still numerous mysteries concerning human disease, animal reservoirs, and the virus. Easy access to medical facilities, improved testing methods, and it is efficiency. The construction of healthcare setups restricts the capability of making the best decisions about dealing with this ignored tropical disease. Hence, there is a scope for improvement that can make this disease easily treatable and eradicate it in the future. We should strengthen the fundamental research on zoonotic poxvirus disease and pay close attention to scientific organization. It is crucial to increase awareness and surveillance to stop the spread of Monkeypox. Early detection is crucial to stopping the monkeypox outbreak, and international technical cooperation is crucial to lowering the danger of Monkeypox.


R genes Tm‐1, Tm‐2, and Tm‐22 in tomato and L1 and L2 alleles in pepper. Currently, no commercial ToBRFV‐resistant tomato cultivars are available. Integrated pest management‐based measures such as rotation, eradication of infected plants, disinfection of seeds, and chemical treatment of contaminated greenhouses have achieved very limited success. The generation and application of attenuated variants may be a fast and effective approach to protect greenhouse tomato against ToBRFV. Long‐term sustainable control will rely on the development of novel genetic resistance and resistant cultivars, which represents the most effective and environment‐friendly strategy for pathogen control.Tomato brown rugose fruit virus (ToBRFV) is an emerging and rapidly spreading RNA virus that infects tomato and pepper, with tomato as the primary host. The virus causes severe crop losses and threatens tomato production worldwide. ToBRFV was discovered in greenhouse tomato plants grown in Jordan in spring 2015 and its first outbreak was traced back to 2014 in Israel. To date, the virus has been reported in at least 35 countries across four continents in the world. ToBRFV is transmitted mainly via contaminated seeds and mechanical contact . Given the global nature of the seed production and distribution chain, and ToBRFV's seed transmissibility, the extent of its spread is probably more severe than has been disclosed. ToBRFV can break down genetic resistance to tobamoviruses conferred by Tomato brown rugose fruit virus belongs to the genus Tobamovirus, in the family Virgaviridae. The genus also includes several economically important viruses such as Tobacco mosaic virus and Tomato mosaic virus.The ToBRFV genome is a single‐stranded, positive‐sense RNA of approximately 6.4 kb, encoding four open reading frames. The viral genomic RNA is encapsidated into virions that are rod‐shaped and about 300 nm long and 18 nm in diameter. Tobamovirus virions are considered extremely stable and can survive in plant debris or on seed surfaces for long periods of time.Leaves, particularly young leaves, of tomato plants infected by ToBRFV exhibit mild to severe mosaic symptoms with dark green bulges, narrowness, and deformation. The peduncles and calyces often become necrotic and fail to produce fruit. Yellow blotches, brown or black spots, and rugose wrinkles appear on tomato fruits. In pepper plants, ToBRFV infection results in puckering and yellow mottling on leaves with stunted growth of young seedlings and small yellow to brown rugose dots and necrotic blotches on fruits. This pathogen profile summarizes current knowledge about ToBRFV, highlights recent research progress, discusses future research directions, and proposes short‐run and long‐term control strategies. We retrieved these sequences and conducted a phylogenetic analysis to explore their possible relationship and diversity. A phylogenetic tree was constructed based on nucleotide sequences Figure . All ToB3Because ToBRFV is highly transmissible and poses an emerging threat to the global tomato industry, rapid and accurate detection and diagnosis are particularly critical for the implementation of timely measures to control its spread. PCR (for detection of DNA viruses) or reverse transcription PCR  and enzyme‐linked immunosorbent assay (ELISA) are the most commonly used methods to detect viruses. Transmission electron microscopy (TEM) and next‐generation sequencing (NGS) followed by data analysis have also been used to investigate the presence of ToBRFV. Due to similar virion morphology and serological cross‐reactivity , the TEM and commercial ELISA kits currently available cannot distinguish ToBRFV from other tobamoviruses. NGS approaches have been used successfully to detect ToBRFV and have been instrumental in determining full genomic sequences of ToBRFV, and useful for understanding evolutionary relationships and the spread of ToBRFV worldwide. However, NGS methods are not the preferred choice for ToBRFV detection due to the costs  and processing time required compared to amplification‐based approaches.Several nucleic acid amplification‐based protocols, including RT‐quantitative PCR (RT‐qPCR) and droplet digital PCR (ddPCR), have been developed to detect ToBRFV in leaf and seed samples  contains a detailed protocol for seed sampling, RNA extraction, and a one‐step duplex RT‐qPCR  ISF, . As SE‐q4R genes, including N, Tm‐1, Tm‐2, Tm‐22, L1, L2, L3, and L4, have been identified and deployed into solanaceous crop species  through breeding programmes to control tobamoviruses  barrel‐like protein, and all the other seven R genes encode nucleotide‐binding domain leucine‐rich repeat proteins (NB‐LRRs). Tm‐2, Tm‐22, and the four L genes encode coiled‐coil NB‐LRR (CC‐NB‐LRR), whereas N codes for Toll interleukin‐1 receptor‐NB‐LRR (TIR‐NB‐LRR). On tobamovirus infection, these R‐encoded proteins recognize specific proteins of the target virus, termed avirulence effectors (Avr), to activate effector‐triggered immunity response (ETI) to restrict virus infection. The tobamoviral CP is the Avr effector of L‐mediated resistance to tobamoviruses and the LRR domain of L proteins is the determinant of recognition spectra  through its TIR domain that directly interacts with the N receptor‐interacting protein NRIP1 and the P50 domain to trigger HR and restrict TMV infection  without visible HR (necrotic lesion)    evaluated the risk of ToBRFV and proposed a rating A for this virus CDFA, . In JuneAs ToBRFV is a seedborne virus and the introduction of this virus into uninfected areas is believed to occur through contaminated seeds, it is highly recommended to use virus‐free tomato and pepper seeds that are harvested from healthy parental plants   highly resistant to ToBRFV , LA0716 (S. penellii), LA4117 (S. chilense), and LA2747 (S. chilense), that are tolerant to ToBRFV  Hall, . These gPotato virus X, Potato virus Y, and Potato virus S as a 600‐bp inverted repeat showed nearly 100% resistance to these three viruses . PDR is mediated by RNA silencing , a conserved defence mechanism triggered by double‐stranded RNA (dsRNA) in eukaryotes and the primary antiviral response in plants  encodes a seven‐pass transmembrane protein that interacts with tobamoviral replication proteins and is indispensable for efficient multiplication of tobamoviruses  and catechinic acid , have anti‐ToBRFV activity (Iobbi et al., In addition to the above control strategies, other novel approaches are also under investigation. For example, Iobbi and co‐workers reported that autoxidation products of the methanolic extract from 6The development of attenuated ToBRFV variants to control ToBRFV through cross‐protection to provide a fast and effective solution in the short term.Screening for resistance in tomato germplasms and subsequent introgression of identified resistance genes into elite cultivars. Although this approach usually takes a very long time to succeed, positive outcomes of durable resistance will benefit the industry more profoundly.Induction of mutagenized tomato populations and screening for novel resistance. The advanced technologies available may accelerate the integration of identified resistance into elite cultivars through breeding programmes.Tm‐22‐mediated durable resistance by tobamoviruses are of particular interest.New control strategies such as host factor‐based resistance. Research is needed to better understand the ToBRFV infection process and the molecular plant–ToBRFV interaction. The search for ToBRFV host factors required for ToBRFV multiplication and research on mechanics under breakdown and counterbreakdown of L alleles to ToBRFV inoculation/infection require further evaluation. The mechanisms underlying these responses are to be investigated.Impact of ToBRFV on pepper production. So far, most ToBRFV research has been done in tomato and very little has been done in pepper. Differential responses of pepper plants harbouring different Plant viruses are a major constraint to agriculture, accounting for nearly 50% of newly emerging plant diseases and causing an estimated economic loss greater than $30 billion annually (Yang et al., The authors declare that no competing interests exist.


This treatment caused the infectious virus titer to be reduced to below detectable limits. The Galahad™ -inactivated influenza preparation without adjuvant or preservative was given to BALB/c mice using a variety of routes of administration and dosing regimens. The most protective route of administration and dosing regimen was when mice were given the vaccine twice intranasally, the second dose coming 14 days after the primary vaccine dose. All the mice receiving this vaccine regimen survived the virus challenge while only 20% of the mice receiving placebo survived. This suggests that a Galahad™-inactivated influenza virus vaccine can elicit a protective immune response even without the use of an adjuvant. This technology should be investigated further for its potential to make effective human vaccines.Galahad™ is a proanthocyanidin complexed with polysaccharides that inactivates viruses and indicates potential for an innovative approach to making protective vaccines. The polysaccharide portion of Galahad™ consists mainly of arabinan and arabinogalactan. In a seven-day toxicity study in rats, it was not toxic even when tested undiluted. Galahad™ inactivated a wide range of DNA and RNA viruses including adenoviruses, corona viruses such as SARS-CoV-2, and influenza viruses. Electron microscopy studies showed that exposure to Galahad™ caused extensive clumping of virions followed by lack of detection of virions after longer periods of exposure. Based on the viral inactivation data, the hypotheses tested is that Galahad™ inactivation of virus can be used to formulate a protective inactivated virus vaccine. To evaluate this hypothesis, infectious influenza A virus  with a titer of 10 •Discovery of novel virus-inactivating agent: polysaccharide/catechin .•Non-toxic: inactivates/lowers titers in 25 viruses, including Avian H5N1, COVID-19.•Agent used to make whole virus vaccine; tested in 400 animal mouse model.•Galahad™-inactivated vaccine 100% successful intranasally vs. lethal H5N1 challenge.•Platform offers possible benefits—in time, cost, & mass distribution. Vaccine; Polysaccharide; Catechin; Influenza; H5N1; SARS-CoV-2 (COVID-19). However, formalin modifies vaccine antigens to such a great extent that immunogenicity of the inactivated virus decreases. In some cases, that modification by formalin, when used to inactivate virus, may not protect the immunized individual from disease and in some cases may even exacerbate disease upon infection by infectious virus . For exaβ-propiolactone is also commonly used as a virus inactivation agent to produce inactivated whole virus vaccines and is not as problematic as formalin for making inactivated whole virus vaccines. In contrast to formalin, β-propiolactone does not need to be removed from inactivated virus preparation, since it is rapidly hydrolyzed . HoweverThus, alternative inactivating agents that are less toxic and that do not need a process to remove the inactivating agent to create an efficacious vaccine should be evaluated as possible agents to make inactivated whole virus vaccines.Therefore, a grape seed extract designated as Galahad™ (Patent: US8629121B2) was evaluated for inactivation of multiple viruses. The Galahad™-inactivated influenza preparation was developed as an intranasal vaccine to achieve vaccine efficacy against Influenza A H5N1 virus. This vaccine needs no preservative for storage to be efficacious.The hypothesis to be tested was that mice would be protected from a lethal influenza virus infection when immunized with Galahad™-treated virus.22.1Many sources of grape seed from various growing seasons were used over time to extract pure, consistent batches of pure Galahad™. Various concentrations of Galahad™ in distilled water were prepared from 5% grape seed extract . For cell culture experiments and in vivo vaccine experiments, Galahad™ was filtered through a micropore filter  to provide material free of bacterial contaminants. Methods for characterizing Galahad™ are found in supplementary section [IV-IX]. Final product of each batch was tested for concentration using a photospectrometer (Genesys 10S UV-Vis) at 439NM (linear equation y = 0.5900995 x -8.583618E-03).2.22.2.1thermofisher.com, Fisher Scientific, Waltham, MA, USA) supplemented with 5% fetal bovine serum (FBS) from Thermo Fisher Scientific  and 0.1% NaHCO3. Human lung carcinoma cells (A-549) were obtained from ATCC and were grown in Dulbecco's MEM (Gibco BRL) supplemented with 0.1% NaHCO3 and 10% FBS. Madin-Darby canine kidney cells (MDCK) were obtained from American Type Culture Collection .Vero 76 cells from American Type Culture Collection  were grown in minimal essential medium (MEM) from Thermo Fisher Scientific-Gibco  obtained from Dr. Robert Webster of St. Jude Hospital, Memphis, was prepared in MDCK cells. Medium used to prepare influenza virus stocks was MEM without serum, 0.18% NaHCO3, 20 μg trypsin/ml, 2.0 μg EDTA/ml, and 50 μg gentamicin/ml.Adenovirus 1 (Chicago) was obtained from World Reference Center for Emerging Viruses and Arboviruses (WRCEVA). This virus was propagated in Vero 76 cells. The medium used to create virus stocks for Vero 76 cells was minimal essential medium (MEM) supplemented with 2 mM L-glutamine, 2% FBS, 0.1% NaHCOThis influenza A H5N1 virus was adapted to mice by passaging it through mice until virus induced pneumonia-associated death in mice exactly as described by Sidwell et al .50 units (50% tissue culture infectious dose).Viruses in samples were quantified using the method described by Reed and Muench . Cell cu2.3For the seven-day toxicity study, approximately 42-day-old male and female Sprague Dawley rats were obtained from Harlan Sprague Dawley, Inc. Animals were housed one animal per cage during the study.For vaccine studies, specific pathogen-free 18–21 g (5–6 weeks old) BALB/c mice  were obtained from Charles River Laboratories .2.3.1These studies were conducted in accordance with approval of Institutional Animal Care and Use Committees at various institutions where experiments were done. All are AAALAC-accredited laboratories. Animal experiments were also done in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (Assurance no. A3801-01).2.410 dilutions of Galahad™ or PSS for 10 min at room temperature. Virus was titered as described above.Influenza A H5N1 virus was exposed to an equal volume of six ½ logRepresentative viruses from other genera, including SARS-CoV-1 and SARS-CoV-2 were also treated in the same manner as above with various dilutions of Galahad™ for 10 min (See Supplementary Tables S5–S7).2.5T. ni pupae as described in the supplementary section V .Dr. Parastoo Azadi was supported by Data will be made available on request.TM. The other 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. Research on GalahadTM did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors, but was financed by companies with ownership interest in GalahadTM without role in experimental design, data analysis, collection and interpretation.The authors declare the following conflict of interests: Thomas Konowalchuk owns shares of Galaxy Force Technologies, LLC that has patent rights to produce vaccines using GalahadNo additional information is available for this paper.


A total of 22 cytokines from all families of four-helical cytokines are S100P binders with the equilibrium dissociation constants, dK, ranging from 1 nM to 3 µM . Molecular docking and mutagenesis studies revealed the presence in the S100P molecule of a cytokine-binding site, which overlaps with the RAGE-binding site. Since S100 binding to four-helical cytokines inhibits their signaling in some cases, the revealed ability of the S100P protein to interact with ca. 71% of the four-helical cytokines indicates that S100P may serve as a poorly selective inhibitor of their action.S100 proteins are multifunctional calcium-binding proteins of vertebrates that act intracellularly, extracellularly, or both, and are engaged in the progression of many socially significant diseases. Their extracellular action is typically mediated by the recognition of specific receptor proteins. Recent studies indicate the ability of some S100 proteins to affect cytokine signaling through direct interaction with cytokines. S100P was shown to be the S100 protein most actively involved in interactions with some four-helical cytokines. To assess the selectivity of the S100P protein binding to four-helical cytokines, we have probed the interaction of Ca The calcium concentration was chosen to be close to the level of free calcium in the serum of 1.1 mM. The temperature of 25 °C was used to ensure consistency with the previous SPR data on S100P interaction with other four-helical cytokines  to 3 µM (IL-10)  , Table 3or, RAGE .Examination of the free energy changes accompanying the S100P–cytokine interactions  shows thExamination of the IntAct  and BioGIt should be noted that the S100P-specific cytokines are evolutionarily distant from each other. The pairwise sequence identities within each of their SCOP families, calculated using Clustal Omega 2.1, as implemented in EMBL-EBI service , lie in 2+-induced structural rearrangement for the efficient S100P binding to the cytokines. Since Ca2+ binding mostly induces solvent exposure of S100P residues of the ‘hinge’ between helices α2 and α3 [The S100P-specific cytokines  coupled 2 and α3 ,34, this2+-loaded S100P is about (64 ± 24) nM [dK values for some of the S100P–cytokine interactions  may reach subnanomolar level or even lower (Considering that the equilibrium dimer dissociation constant for Ca± 24) nM , the SPR± 24) nM , which s± 24) nM ,37. Hencen lower , which iThe average blood concentrations of the most S100P-specific cytokines are normally below 0.1 nM , which iS100P binding could modify signaling of the S100P-specific cytokines, as exemplified by inhibition of the IFN-β-induced suppression of the viability of MCF-7 breast cancer cells by S100A1/A4/B/P proteins ,20. AnotThe promiscuous binding of the wide spectrum of four-stranded cytokines by the S100P protein under the conditions of its quantitative excess indicates the possibility that S100P serves as a buffer for the cytokines, capable of absorbing their excess when the cytokine levels are excessively increased and releasing the cytokines under conditions of their depletion.2+-bound S100P and the wide spectrum of the four-helical cytokines  are shown in dK values exceed 10−4 M. Therefore, the mutagenesis data support involvement of the ‘hinge’ and residue F89 in the recognition of most of the cytokines studied.To validate the modelling results, we have studied by SPR spectroscopy affinity of S100P mutants F89A and Δ42–47 (that lacks PGFLQS sequence in the flexible ‘hinge’) to 11 S100P-specific four-helical cytokines covering all families of this fold . Despite2+-bound S100P dimer via the residues of N-terminus and helices α1 and α3  are locAnalysis of the cytokine tertiary structures in the complexes of some of the S100P-specific cytokines with their respective receptors shows partial overlapping of the receptor-binding sites with the predicted S100P-binding sites for GM-CSF, IL-3, IL-5, IL-15, IL-21, G-CSF, GH, IL-20, and IFN-ω1 . Among tIt should be emphasized that the accuracy of the predictions of tertiary structures of the S100P–cytokine complexes based upon molecular docking is limited, since they do not take into consideration structural flexibility of the interaction partners. This is especially true in the case of the presence of the regions prone for disorder, including S100P (PDB entry 1J55 contains unresolved fragment 46–51), IL-15 (2Z3Q: unresolved fragment 76–80), THPO , G-CSF (DisProt  entry DPE. coli as previously described [Human S100P protein and its F89A and Δ42–47 (lacks PGFLQS sequence) mutants were prepared in escribed . The cytescribed .2, EDTA, and TWEEN 20 were purchased from Sigma–Aldrich Co., . Tricine was from Helicon . Glutaraldehyde was from Amersham Biosciences . Coomassie brilliant blue R-250 was bought from Merck .Sodium acetate and ethanolamine were bought from Bio-Rad Laboratories, Inc.  HEPES, sodium chloride, and SDS were from PanReac AppliChem . Potassium hydroxide, CaCl2, 0.05% TWEEN 20, pH 7.4) was passed over the chip surface at a rate of 30 μl/min for 300 s, followed by flushing the chip with the running buffer for 2.400 s. Each double-referenced SPR sensogram was fitted according to either a one-site binding model (S100P–IL-13 interaction) or a heterogeneous ligand model (1), which assumes existence of two populations of the ligand (L1 and L2) that bind a single analyte molecule (A):a1ka2k1 + AL←→1AL2 + AL←→2AL(1)d1kd1Kd2kd2Kwhere ak and dk refer to kinetic association and dissociation constants, respectively, while dK are equilibrium dissociation constants (dk/ak). dK and dk values were evaluated for each analyte concentration using ProteOn Manager™ v.3.1 software , followed by averaging of the resulting values (n = 3–5). The standard deviations are indicated. The ligand was regenerated by passage of 20 mM EDTA solution pH 8.0 for 100 s. The free energy changes, accompanying the interaction between S100P and the cytokines were calculated as follows: ΔGi = −RT ln(55.3/diK), i = 1,2.Surface plasmon resonance (SPR) measurements of S100P affinity to cytokines at 25 °C were performed using ProteOn™ XPR36 protein interaction array system . Ligand (0.03 mg/ml cytokine) in 10 mM sodium acetate pH 4.5 buffer was immobilized on ProteOn™ GLH sensor chip surface  by amine coupling, according to the manufacturer’s instructions. The remaining activated amine groups on the chip surface were blocked by 1 M ethanolamine solution. Analyte (62 nM–16 µM S100P) in a running buffer . The cy2+-loaded human S100P dimer was extracted from the structure of its complex with V domain of RAGE .The models of tertiary structures of S100P–cytokine complexes were built using ClusPro docking server  mainly aD of PDB  entry 2Must 2022 )  were measured at 20 °C using JASCO J-810 spectropolarimeter , equipped with a Peltier-controlled cell holder . The instrument was calibrated according to the manufacturer’s instruction. Bandwidth was 2 nm, averaging time 2 s, and accumulation 3. The spectral contribution of the buffer was subtracted from the protein spectra. Fluorescence spectra of S100P samples (10 µM) were measured using Cary Eclipse spectrometer , equipped with a Peltier-controlled cell holder (10 × 10 mm quartz cell). The sample temperature was monitored inside the cell. Fluorescence of S100P was excited at 280 nm; excitation and emission bandwidths were 5 nm; photomultiplier voltage of 800 V. Scanning of emission wavelengths from 290 nm to 380 nm, step 2 nm, averaging time 1 s. The emission spectra were corrected for spectral sensitivity of the spectrometer and described by a log-normal function  using LoCrosslinking of decalcified S100P (67 µM) with 0.02% glutaraldehyde was performed at 20 °C for 16 h. The reaction was quenched by addition of SDS sample buffer. The resulting sample was analyzed by SDS–PAGE using 18% resolving gel, 5 µg of protein per lane . The gel® VSL2 computational tool, which combines neural network predictors for short and long disordered regions [Intrinsic disorder propensities of human four-helical cytokines were evaluated by the PONDR regions  and whic regions ,52,53. TImportant information on the global classification of disorder status of query proteins can be obtained using binary disorder predictors , such as the charge-hydropathy (CH) plot ,55 and t2+-bound S100P dimer exceeding that for the V domain of its receptor, RAGE (Among the 41 four-helical cytokines studied to date with regard to their affinity to the S100P protein , only 12or, RAGE . Since tor, RAGE . Meanwhior, RAGE . Neverthor, RAGE , therebyS100P is considered as one of the most promiscuous members of the S100 protein family, able to bind numerous ligands with a high affinity, and sharing interaction partners with other S100 proteins ,61. Ther