To describe the temporal and geographical distribution of Zika virus infection and associated neurological disorders, from 1947 to 1 February 2016, when Zika became a Public Health Emergency of International Concern (PHEIC).We did a literature search using the terms “Zika” and “ZIKV” in PubMed, cross-checked the findings for completeness against other published reviews and added formal notifications to WHO submitted under the International Health Regulations.From the discovery of Zika virus in Uganda in 1947 to the declaration of a PHEIC by the World Health Organization (WHO) on 1 February 2016, a total of 74 countries and territories had reported human Zika virus infections. The timeline in this paper charts the discovery of the virus (1947), its isolation from mosquitos (1948), the first human infection (1952), the initial spread of infection from Asia to a Pacific island (2007), the first known instance of sexual transmission (2008), reports of Guillain-Barré syndrome (2014) and microcephaly (2015) linked to Zika infections and the first appearance of Zika in the Americas (from 2015).Zika virus infection in humans appears to have changed in character as its geographical range has expanded from equatorial Africa and Asia. The change is from an endemic, mosquito-borne infection causing mild illness to one that can cause large outbreaks linked with neurological sequelae and congenital abnormalities. Aedes, was discovered in 1947 in Uganda.Zika, a flavivirus transmitted mainly by mosquitos in the genus Aedes mosquitos in the Americas, WHO declared that Zika infection associated with microcephaly and other neurological disorders constituted a Public Health Emergency of International Concern (PHEIC). By the start of February 2016, local transmission of Zika infection had been reported from more than 20 countries and territories in the Americas and an outbreak numbering thousands of cases was under way in Cabo Verde in western Africa. Beyond the range of its mosquito vectors, Zika virus infections are expected to be carried worldwide by people as they travel and be transmitted by travellers to sexual partners who have not been to places where the virus is endemic.In February 2016, as infection moved rapidly through the range occupied by ,To illustrate the spread of Zika virus and associated neurological complications, we did a literature search in PubMed using “Zika” and “ZIKV” as the search terms and cross-checked our findings for completeness against other published reviews.Aedes (Stegomyia) africanus, caught on a tree platform in the forest.http://www.who.int/bulletin/volumes/94/9/15-171082). The first reported case of Zika virus dates to 1947 when the virus was isolated in samples taken from a captive sentinel rhesus monkey by scientists conducting routine surveillance for yellow fever in the Zika forest of Uganda.As shown in Aedes. Sporadic human cases were identified, mostly by serological methods, but such cases were rare and the disease was regarded as benign. No deaths or hospitalizations were reported and seroprevalence studies consistently indicated widespread human exposure to the virus.,,–,–Between 1960 and 1999, Zika virus was being detected in mosquitos and sentinel rhesus monkeys used for field research in a narrow band of countries that stretch across equatorial Africa . Altoget–In 2007, Zika virus spread from Africa and Asia to the Pacific island of Yap, in the Federated States of Micronesia, and caused the first large outbreak in humans. Before this event, only 16 cases of human Zika virus disease  had been documented worldwide .46 House,Although wind-blown mosquitoes can travel distances of several hundred kilometres over the open ocean, introduction of the virus by travel or trade involving an infected person or an inadvertently imported mosquito is considered the most likely source of this outbreak, especially as no monkeys were present on the island.-,Between 2010 and 2015 sporadic cases of Zika virus infection were reported from several countries in south-east Asia.,,,–,,,In 2012, researchers published genetic sequences of Zika virus strains collected in Cambodia, Malaysia, Nigeria, Senegal, Thailand and Uganda, and constructed phylogenetic trees to assess the relationships between them.During the 2013–14 outbreak of Zika virus in French Polynesia, two mothers and their newborn babies had Zika virus infection confirmed by PCR done on serum samples collected within four days of birth. Because they were able to show evidence of infection in the first week of life, the researchers concluded that Zika virus had been acquired by transplacental transmission or during delivery.On 1 February 2016 the Director-General of WHO declared that the recent association of Zika infection with clusters of microcephaly and other neurological disorders constituted a PHEIC. Before this declaration, three epidemiologic alerts were issued by the Pan American Health Organization (PAHO) and WHO. The first epidemiological alert had been issued on 7 May 2015.Results of previous studies in which Zika virus was confirmed by serological surveys need to be interpreted with some caution, as they used different serological methods with varying degrees of specificity. These methods include hemagglutination inhibition, complement fixation and enzyme-linked immunosorbent assays, in addition to more specific sero-neutralization tests. The fact that Zika virus cross-reacts immunologically with dengue, other flaviviruses and chikungunya, which frequently co-circulate with Zika and have the same vectors, complicates the interpretation of serological results. Nonetheless, the results of serological surveys, supported by the isolation of Zika virus from mosquitoes and vertebrate hosts, suggest that the virus is endemic in several African and Asian countries.Travellers who returned with Zika infections from Malaysia, the Maldives, Senegal and Thailand in 2012, were sentinels for virus circulation that may otherwise have gone undetected. Reasons why the virus may not always be detected include widespread immunological protection in the local population, the asymptomatic or mild and self-limiting nature of most Zika infections, clinical symptoms that overlap with dengue and chikungunya, the weakness of surveillance systems and the lack of specific diagnostic tests in many settings.Therefore, additional research is needed to understand whether population immunity in endemic countries will confer protection against the epidemic strains of Zika currently circulating in Latin America and the Caribbean.Aedes vectors. Person-to-person transmission, both vertically, from mother to fetus, and horizontally through sexual transmission, is also expected to continue, and we anticipate that infections will be carried widely by international travel.Zika virus infection in humans appears to have changed in character while expanding in geographical range. The change is from an endemic arbovirus causing mild illness across equatorial Africa and Asia. From 2007 onwards, Zika virus caused large outbreaks in previously unexposed populations, and from 2013 onwards, outbreaks linked with neurological disorders including Guillain-Barré syndrome and congenital malformations, for reasons that are not yet known. The future transmission of Zika infection is likely to coincide with the global distribution of


Am18w). These results demonstrate that organosilicone spray adjuvants that are considered biologically inert potentiate viral pathogenicity in honey bee larvae, and guidelines for OSS use may be warranted.Honey bees are highly valued for their pollination services in agricultural settings, and recent declines in managed populations have caused concern. Colony losses following a major pollination event in the United States, almond pollination, have been characterized by brood mortality with specific symptoms, followed by eventual colony loss weeks later. In this study, we demonstrate that these symptoms can be produced by chronically exposing brood to both an organosilicone surfactant adjuvant (OSS) commonly used on many agricultural crops including wine grapes, tree nuts and tree fruits and exogenous viral pathogens by simulating a horizontal transmission event. Observed synergistic mortality occurred during the larval-pupal molt. Using q-PCR techniques to measure gene expression and viral levels in larvae taken prior to observed mortality at metamorphosis, we found that exposure to OSS and exogenous virus resulted in significantly heightened Black Queen Cell Virus (BQCV) titers and lower expression of a Toll 7-like-receptor associated with autophagic viral defense ( Apis mellifera) add an estimated 15 billion dollars of value to the crops they pollinate in the United States alone25467As generalist pollinators, honey bees  from 2013 report a total use of approximately 0.3 lbs. per acre of OSS adjuvants in California almonds4We hypothesize that the use of organosilicone adjuvants in combination with exposure to common viruses present in the hive environment and transmitted during pollination events can result in the interrupted development and brood mortality observed in hives used for almond pollination. Although there are limited data regarding the spread of diseases during almond pollination, shared floral resources and drift between colonies occurring more often due to the close proximity of the colonies30in vitro assay. Sylgard 309 is more uniform in composition than other spray adjuvants analyzed, being almost 80% pure acetyl-capped trisiloxane surfactant , Deformed Wing Virus (DWV) Israeli Acute Paralysis Virus (IAPV) and Sacbrood Virus (SBV)2 = 30.35, p < 0.0001, df = 3, 2 = 12.37, p < 0.0004), and chronic exposure to OSS resulted in a positive trend toward higher numbers of individuals exhibiting this symptom . Additionally, the two terms interact, resulting in a similar positive trend .The highest significant mortality was seen in larvae exposed to both OSS and the viral inoculum (OSS + V)  with over 68.4% (+/−4.32%) of the population failing before adult eclosion . The higIn the larvae that successfully pupated, all treatment groups experienced a significant delay in pupal eclosion relative to Control larvae (Ctrl) 2 adj. = 0.63, df = 7, p < 0.0001).To test our second hypothesis, that the mortality observed in response to OSS and virus exposure together were related to elevated virus titers and compromised immunity, we examined the relative abundance of four honey bee viruses, Israeli Acute Paralysis Virus (IAPV), Black Queen Cell Virus (BQCV), Deformed Wing Virus (DWV) and Sacbrood Virus (SBV), and the expression of immune genes in larvae prior to pupation see . We explExposure to OSS was a significant predictor of higher BQCV titers (p ≤ 0.01134) but exposure to virus alone did not result in significantly higher viral titers (p ≤ 0.08411). However, a significant interaction between exposure to OSS and viruses resulted in significantly higher BQCV titers in treatment OSS + V (p ≤ 0.00053) ; but, exposure to OSS depressed this expression . IAPV infection levels and exposure to OSS interacted to increase BQCV titers .In the larvae sampled, IAPV titers correlated with BQCV titers , hymenoptaecin , and defensin 1 . Similar to 18-wheeler, the expression of these genes appears to be depressed in the OSS + V treated bees relative to those in bees from other treatments, but the expression levels for these immune genes are not by themselves significantly related to BQCV levels or related to treatments  and the neonicotinoid pesticide thiacloprid results in additive larval mortality and an increase in BQCV titersBQCV was the only virus studied here that was significantly affected by the treatments. Little research has reported impacts of BQCV on worker pupal development or colony survival, even though it is one of the most prevalent viruses detected in hives in the United States15585960586462626566The relationship between BQCV and IAPV suggests that the co-infection of IAPV with BQCV may enhance BQCV infections. Likewise, the predictive relationship between IAPV titers and OSS exposure on BQCV suggest that the IAPV in the virus inoculum may have influenced the virulence of BQCV in larvae that had been immuno-compromised via OSS exposure. Recent work reveals that exposure to different virus combinations results in different viral dynamics within the honey bee host46The parallels between the symptoms observed in hives following almond pollination and the symptoms observed in this study are striking and support the hypothesis that OSS use in combination with other stressors can lead to the brood mortality observed following almond pollination. A recent survey of virus prevalence in hives before, during and after almond pollination found that the highest abundance of viral pathogens occurs following almond pollination, implying that infections are spread or worsened during the event22Apis mellifera ligustica  and two splits (derived from hive 2 and 3) from a Pennsylvania State University apiary  were selected based on relative health and surveyed for the presence of viruses. To ensure uniform age among larvae, the queen was caged on a frame for 24 hours and then excluded from the frame until it was collected for grafting three days later. The experiments commenced in June 2015, and the last frame was taken in late July 2015.Three original colonies of et al.Sylgard 309, lot no. 0006018786 was obtained from the Dow Corning Corp., Midland, MI, USA. It is not currently possible to obtain purified strains of honey bee viruses, therefore, a semi-purified virus solution was prepared using a protocol described in Singh 2010 TM Genesig kit for the IAPV genome, the solution contained 940 genome equivalents of IAPV/μL of inoculum  Sylgard 309 (OSS) and two with an equivalent volume of Milli-Q purified water (Ctrl). This concentration was selected following an oral communication with an industry representative suggesting a thousand fold dilution from the tank mix concentration of 1% as a field realistic dose of OSS. To one of the Ctrl plates (Ctrl + V) and one of the OSS plates (OSS + V), virus inoculum (approx. 47 copies of virus per larva) was added as a one-time exposure. An equivalent volume of saline was added to the Ctrl and OSS diets. Additionally, a positive control treatment group was present in each experimental replicate. See Immature bees were photographed upon death and visible symptoms  and dateOn day six of the larval rearing, 24 hours following the last scheduled feeding and immediately prior to transferring larvae to pupation plates, five larvae were removed from each plate and frozen at −80 °C for future RNA extraction. Larvae selected were healthy in appearance and had consumed all of the provided diet. q-PCR was performed using primers for IAPV, BQCV, DWV, SBV and immune and detoxification genes, including 18-wheeler, hymenoptaecin, defensin-1, and prophenoloxidase see . Novel pSurvivorship analysis , standard least squares regression, and contingency analysis were performed and graphs were prepared using JMP Version Pro 12. Significance was evaluated using a 0.05 probability threshold. Survivorship analysis was performed across all hives and reps (n = 171), and Wald tests were used to evaluate differences. Tukey’s HSD test or Student’s T-test were used to compare least squared means of response variables between groups. Nominal logistic regression was evaluated using a likelihood ratio test. All symptoms are displayed in How to cite this article: Fine, J. D. et al. An Inert Pesticide Adjuvant Synergizes Viral Pathogenicity and Mortality in Honey Bee Larvae. Sci. Rep.7, 40499; doi: 10.1038/srep40499 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The World Health Organization (WHO) stated in March 2016 that there was scientific consensus that the mosquito-borne Zika virus was a cause of the neurological disorder Guillain–Barré syndrome (GBS) and of microcephaly and other congenital brain abnormalities based on rapid evidence assessments. Decisions about causality require systematic assessment to guide public health actions. The objectives of this study were to update and reassess the evidence for causality through a rapid and systematic review about links between Zika virus infection and (a) congenital brain abnormalities, including microcephaly, in the foetuses and offspring of pregnant women and (b) GBS in any population, and to describe the process and outcomes of an expert assessment of the evidence about causality.The study had three linked components. First, in February 2016, we developed a causality framework that defined questions about the relationship between Zika virus infection and each of the two clinical outcomes in ten dimensions: temporality, biological plausibility, strength of association, alternative explanations, cessation, dose–response relationship, animal experiments, analogy, specificity, and consistency. Second, we did a systematic review (protocol number CRD42016036693). We searched multiple online sources up to May 30, 2016 to find studies that directly addressed either outcome and any causality dimension, used methods to expedite study selection, data extraction, and quality assessment, and summarised evidence descriptively. Third, WHO convened a multidisciplinary panel of experts who assessed the review findings and reached consensus statements to update the WHO position on causality. We found 1,091 unique items up to May 30, 2016. For congenital brain abnormalities, including microcephaly, we included 72 items; for eight of ten causality dimensions , we found that more than half the relevant studies supported a causal association with Zika virus infection. For GBS, we included 36 items, of which more than half the relevant studies supported a causal association in seven of ten dimensions . Articles identified nonsystematically from May 30 to July 29, 2016 strengthened the review findings. The expert panel concluded that (a) the most likely explanation of available evidence from outbreaks of Zika virus infection and clusters of microcephaly is that Zika virus infection during pregnancy is a cause of congenital brain abnormalities including microcephaly, and (b) the most likely explanation of available evidence from outbreaks of Zika virus infection and GBS is that Zika virus infection is a trigger of GBS. The expert panel recognised that Zika virus alone may not be sufficient to cause either congenital brain abnormalities or GBS but agreed that the evidence was sufficient to recommend increased public health measures. Weaknesses are the limited assessment of the role of dengue virus and other possible cofactors, the small number of comparative epidemiological studies, and the difficulty in keeping the review up to date with the pace of publication of new research.Rapid and systematic reviews with frequent updating and open dissemination are now needed both for appraisal of the evidence about Zika virus infection and for the next public health threats that will emerge. This systematic review found sufficient evidence to say that Zika virus is a cause of congenital abnormalities and is a trigger of GBS. In a systematic review, Nicola Low and colleagues use a causality framework to examine the evidence for zika virus infection as a cause of congenital brain abnormalities and Guillain–Barré syndrome. In 2015, the mosquito-borne Zika virus caused epidemics of a mild viral illness for the first time in Brazil and then other countries in Latin America and the Caribbean.In mid to late 2015, clinicians in northeastern Brazil reported unexpected increases in the numbers of babies born with abnormally small heads  and of adults with Guillain–Barré syndrome (GBS), a paralytic condition triggered by certain infections.In February 2016, the World Health Organization (WHO) declared a Public Health Emergency of International Concern and called for research about the causal relationship between Zika virus and congenital brain abnormalities, including microcephaly, and GBS.We developed a causality framework for Zika virus and (a) congenital brain abnormalities, and (b) GBS. For each outcome, we developed specific questions in ten different dimensions of causality: temporality; biological plausibility; strength of association; exclusion of alternative explanations; cessation; dose–response relationship; animal experimental evidence; analogy; specificity; and consistency of findings.We did a systematic review of published and unpublished evidence up to May 30, 2016. We summarised the evidence descriptively. A panel of experts assessed the findings and reached a consensus about causality.For congenital brain abnormalities, we assessed 72 studies that addressed questions in one or more causality dimensions. Reports of pregnancies affected by Zika virus have come from countries with circulating Zika virus in the Americas, the Pacific region, and West Africa. Clinical reports have documented Zika virus infection in pregnant women followed by foetal abnormalities, particularly with infection in the first trimester. These women did not have any other congenital infection or dengue virus infection. The risk of congenital brain abnormalities could be around 50 times higher in mothers who had Zika virus infection in pregnancy compared with those who did not. In laboratory studies, Zika virus has been shown to cross the placenta and replicate in human brain cells.For GBS, we assessed 36 studies that addressed questions about one or more causality dimensions. In several countries in the Americas and the Pacific region, a temporal association has been found, with symptoms of Zika virus infection preceding the onset of GBS. In these countries, surveillance reports of cases of GBS followed the pattern of reports of Zika-like illness. During a Zika virus outbreak in French Polynesia in 2013–14, scientists estimated that around one in 4000 people with Zika virus infection developed GBS. The odds of having had a recent Zika virus infection were more than 30 times higher in patients with GBS than those without in a hospital-based study in French Polynesia. Several other infections that can trigger GBS were excluded.This systematic review found sufficient evidence to conclude that Zika virus is a cause of congenital abnormalities and is a trigger of GBS.Systematic reviews of evidence about emerging public health threats need to be updated frequently. Campylobacter jejuni or cytomegalovirus ..7]. SincThe level of certainty required before judging that Zika virus is a cause of microcephaly and GBS is contentious . Most asWe describe three linked components: the causality framework, the systematic reviews, and the expert panel assessment of the review findings. The WHO Zika Causality Working Group convened the expert panel of 18 members with specialist knowledge in the fields of epidemiology and public health, virology, infectious diseases, obstetrics, neonatology, and neurology (membership of the expert panel is provided in the Acknowledgments).In February 2016, we developed a causality framework for Zika virus by defining specific questions for each of ten dimensions, modified from Bradford Hill’s list : temporaOur protocol was registered on March 21, 2016 in the database PROSPERO (CRD42016036693)  in S1 TeTo report our findings, we use the term item for an individual record, e.g., a case report. Occasionally, the same individuals or data were reported in more than one publication (item). To avoid double counting, we organised these items into groups. We chose a primary publication (the item with the most complete information) to represent the group, to which other items were linked .We included studies of any design and in any language that directly addressed any research question in the causality framework , p3.We searched multiple electronic databases and websites . We included 106 items from 87 groups  and the Caribbean  and French Polynesia have reported an increase in GBS cases during outbreaks of Zika virus infection , 109. SuWe reviewed six items , 116 in We reviewed seven items , 122 in C. jejuni, Mycoplasma pneumoniae, HIV, Epstein–Barr virus, and herpes simplex virus. No single infectious trigger would have resulted in GBS outbreaks in multiple locations.We included ten items , 116–121Eight items , 122 in We did not find any relevant studies.C. jejuni gastroenteritis [C. jejuni  has grown more quickly than that for GBS (36 items). Research efforts might have concentrated on congenital abnormalities because clusters of affected infants were so unusual, especially in Brazil, where rubella has been eradicated. In contrast, GBS is an established postinfectious neurological disorder, and some commentators have already assumed Zika virus as another cause . Whilst Comparative studies from French Polynesia suggest that the risk of both microcephaly or of GBS is at least 30 times higher in people who had Zika virus infection compared to those who did not , 94, 112Current evidence does not show which specific environmental and host factors interact with Zika virus. A cofactor that increases the risk of neurological damage could help to explain why surveillance reports show clusters of microcephaly or GBS in some geographical areas but not others. Dengue virus has been suggested as a possible cofactor or another component cause . One majThe strengths of our study are that we appraised evidence of causality systematically but rapidly and transparently within a structured framework. We searched both published and unpublished sources. The systematic review process could not eliminate publication bias but reduced the risk that only positive reports in favour of causation would be evaluated. There were limitations to the process, too. Our search strategy did not cover the literature about analogous conditions or cofactors systematically. We did not select studies or extract data in duplicate, but additional reviewers checked the extracted data independently. The included studies used a variety of case definitions for microcephaly and GBS, and we could not standardise these, so misclassification is possible, but this limitation did not change the overall conclusions. Our rapid assessment of quality was not quantitative; we did not find a tool that covered all review questions and study designs appropriately and were not able to standardise the GRADE tool across study designs in the time available .The conclusions of the expert panel facilitate the promotion of stronger public health measures and research to tackle Zika virus and its effects. The evidence gaps that we identified provide researchers with research questions, and WHO has published a Zika virus research agenda . Better Our systematic review deals with multiple neurological disorders and more detailed questions about causality than other reviews. We reached the same conclusion as Rasmussen et al. , but theOur review will quickly become outdated because the pace of new publications is outstripping the time taken for the review process. The concept of a “living systematic review” has been proposed as a way to combine rigour with timeliness for intervention research  through In summary, rapid and systematic reviews with frequent updating and open dissemination are now needed, both for appraisal of the evidence about Zika virus infection and for the next public health threats that will emerge. This rapid systematic review found sufficient evidence to conclude that Zika virus is a cause of congenital abnormalities and is a trigger of GBS.S1 PRISMA Checklist(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.S2 TextFrom PAHO Epidemiological Update 28 April 2016 .(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.


Injury-associated deaths have substantial economic consequences in the United States. The total estimated lifetime medical and work-loss costs associated with fatal injuries in 2013 were $214 billion , lifetime costs (in 2014 U.S. dollars), and lifetime costs per capita (in 2014 U.S. dollars) varied widely among the 50 states and DC for each of the four intents. Overall, total injury-related mortality rate and lifetime costs per capita ranged from 101.9 per 100,000 and $1,233, respectively (New Mexico) to 40.2 and $491 (New York) . The ratWest Virginia had the highest lifetime costs per capita for fatal unintentional injuries ($815), the highest unintentional injury mortality rate among males (95.2), and the highest unintentional injury mortality rate among persons aged 25–64 years (88.5) . MarylanAlaska and New Jersey had the highest and lowest lifetime suicide costs per capita  . MontanaThe highest and lowest lifetime homicide-related mortality costs per capita were in DC ($273) and Hawaii ($24), respectively .† DC hadEconomic burdens of fatal injuries varied widely in the 50 states and DC for each of the four categories of intent. Across all the four fatal injury intents, some states consistently had lower lifetime costs per capita than most other states. For example, New York, New Jersey, and California ranked among the five lowest states in terms of lifetime costs per capita for injuries of all intents, unintentional injuries, and suicides. In contrast, New Mexico ranked among the five highest states in terms of lifetime costs per capita for injuries of all intents, unintentional injuries, and suicides. Varying economic burdens of fatal injuries in the 50 states and DC might be attributed to the different injury mortality rates, the different medical costs resulting from different medical procedures, and the different demographic characteristics of injury decedents, such as sex and age.Implementation of effective injury prevention strategies is needed to help reduce the substantial lifetime medical and work-loss costs associated with fatal injuries. The differing state-level lifetime costs per capita for fatal injuries suggests an urgent need in some states to prevent injuries. States that consistently have lower lifetime costs per capita across different intents of injuries might have successful injury prevention experiences that could be shared with states with higher per capita costs.The findings in this report are subject to at least four limitations. First, the costs account for medical and work-loss costs associated with decedents. Other societal costs, such as criminal justice costs and the pain and suffering of family members, were not considered. Second, work-loss costs, based on the mean earnings of the general population by sex and specific age groups, might be over- or underestimated because the mean earnings of decedents might differ from those of the general population. Third, intent of fatal injury, as determined from the manner of death assigned on death certificates by coroners or medical examiners, might differ across jurisdictions  and for each of four injury intent categories  for 2014. Economic burdens varied widely among the states and DC. Lifetime costs per capita ranged from $1,233 (New Mexico) to $491 (New York) among fatal injuries of all intents, from $815 (West Virginia) to $261 (Maryland) among unintentional injuries, from $338 (Alaska) to $107 (New Jersey) among suicides, and from $273 (DC) to $24 (Hawaii) for homicides.States can engage more effectively and efficiently in injury prevention if they are aware of the economic burden of injuries, identify areas for immediate improvement, and devote necessary resources to those areas. States that consistently have lower lifetime costs per capita across different intents of injuries might have successful injury prevention experiences that could be shared with states with higher per capita costs.


Picornaviridae family, is a small non-enveloped RNA virus, that is linked to porcine idiopathic vesicular disease (PIVD). SVV infection in swine results in vesicular disease and epidemic transient neonatal losses (ETNL). The first case of SVV infection was reported in Guangdong, South China in 2015.Seneca valley virus (SVV), a member of the We isolated and characterized an SVV HB-CH-2016 strain from vesicular lesion tissue specimens from piglets with PIVD in Hubei, Central China. The complete genome sequence of SVV HB-CH-2016 strain shares high nucleotide identities (94 to 99 %) with all previously reported SVV genomes, moreover, the polyprotein accounts for 98–99 % of amino acid sequence identity. Therefore, the SVV HB-CH-2016 strain is closely related to the SVV CH-01-2015 strain.The case reported in this paper is the second SVV infection case in China. Our findings demonstrate that sporadic SVV infection has occurred in Central China, and therefore, active surveillance on the swine population is important. Moreover, veterinarians must pay attention to this vesicular disease and reinforce biosecurity measures and prevent SVV spread.The online version of this article (doi:10.1186/s12985-016-0631-2) contains supplementary material, which is available to authorized users. Senecavirus genus of the family Picornavirus [Seneca valley virus (SVV), a small, non-enveloped virus with a single-stranded and positive-sense RNA that 7.2 kb long, belongs to the rnavirus . Its genrnavirus . SVV hasrnavirus . In the rnavirus . In thisIn March 2016, an outbreak of vesicular disease occurred among piglets in a swine farm in Hubei province. To determine etiology, vesicular lesion swab specimens were collected and detected using VSV, FMDV and SVDV specific primers. Total RNA from the specimens was isolated using the TRIzol reagent  and 1.0 μg of total RNA was reverse-transcribed using a First Strand cDNA Synthesis Kit  according to the manufacturer’s instructions. PCR amplification was performed using various virus-specific primers . The strain consists of 7300 nucleotides (nt) in length, including a 668-nt 5′ UTR, an opern reading frame (ORF) encoding a 2181 amino acid polyprotein and a 86-nt 3′ UTR cantains a partial poly(A) sequence. Analysis of the full-genome sequence of SVV HB-CH-2016 strain indicated that it shares high nucleotide identities (94 to 99 %) with all previous SVV genomes in the GenBank Fig. , and theSVV infection in piglets cuases neonatal losses and the mortality rate of 1 ~ 4-day-old pigs is approximately 30 ~ 70 % , 11. Cur


The study of virus disease emergence, whether it can be predicted and how it might be prevented, has become a major research topic in biomedicine. Here we show that efforts to predict disease emergence commonly conflate fundamentally different evolutionary and epidemiological time scales, and are likely to fail because of the enormous number of unsampled viruses that could conceivably emerge in humans. Although we know much about the patterns and processes of virus evolution on evolutionary time scales as depicted in family-scale phylogenetic trees, these data have little predictive power to reveal the short-term microevolutionary processes that underpin cross-species transmission and emergence. Truly understanding disease emergence therefore requires a new mechanistic and integrated view of the factors that allow or prevent viruses spreading in novel hosts. We present such a view, suggesting that both ecological and genetic aspects of virus emergence can be placed within a simple population genetic framework, which in turn highlights the importance of host population size and density in determining whether emergence will be successful. Despite this framework, we conclude that a more practical solution to preventing and containing the successful emergence of new diseases entails ongoing virological surveillance at the human–animal interface and regions of ecological disturbance. Prediction is very difficult, especially about the future.—Niels Bohr1.Emerging infectious diseases have the potential to wreak havoc on agricultural industries and native flora and fauna, and can pose a significant challenge to the health and economic status of both developed and developing countries. The broad-scale drivers of the apparent increase in the number of emerging diseases are well documented, involving such factors as climate change, environmental disruption, an increasingly centralized agricultural system and rapid global transportation, as well as high densities of humans, animals and crops. Combined, these factors have created new opportunities for viruses and other pathogens to change their host range and cause epidemic disease. In humans, this confluence of factors has led to the emergence of a number of high-profile viral infections over the last 40 years, including the ongoing global epidemic of human immunodeficiency virus (HIV), pandemic influenza A viruses such as H1N1/09, SARS coronavirus (CoV), MERS-CoV, and the more recent outbreaks of Ebola virus in West Africa and Zika virus throughout the Americas.Importantly, the study of disease emergence has moved from making lists of emerging diseases and the proximate ecological factors responsible for their appearance (e.g. see ), to estHowever, accurate predictions of disease emergence must be based on a correct and rigorous understanding of how viruses jump between hosts and adapt to new transmission cycles, including the time scale over which these processes occur. We show here that a more meaningful understanding of virus emergence requires us to shift the focus away from the broader processes of virus evolution and towards the short-term factors that influence the probability of the successful establishment of a virus in a host population. In other words, if the goal is to develop a meaningful predictive model of disease emergence, there may be considerable value in tuning out the background ‘noise’ of virus evolution rather than building the model around long-term evolutionary processes. More fundamentally, we will argue that a more practical approach to the challenge of virus emergence will involve abandoning prediction in favour of genomic surveillance at the ecological ‘fault-lines’ of emergence.2.The successful emergence of a virus in a new host will often entail a significant adaptive challenge. Indeed, one of the most important observations in disease emergence is that not all viruses that jump species boundaries successfully evolve onward transmission in the new host. Rather, many such viruses appear as transient ‘spill-over’ infections that soon die out, even in the absence of infection control. For example, despite repeated spill-over events from birds to humans, H5N1 avian influenza virus has not been able to evolve sustained human-to-human transmission . Other vThere has been a great deal of experimental research in a number of systems directed towards identifying those specific viral genomic mutations responsible for successful host adaptation , althougR0 > 1) to allow its spread within these confined spaces. However, CIV has failed to ignite a wider epidemic in dogs, probably because contact heterogeneity in the domestic dog population is much greater than in dog shelters such that there is an insufficient density of susceptible hosts for the outbreak to take hold [An informative example concerns the recent emergence of the A/H3N8 subtype of canine influenza virus (CIV). Although this virus was first recorded in dogs in the USA in the early 2000s, with horses acting as the reservoir host , it has ake hold . This inake hold ,8,16.3.Predicting emergence has become one of the highest stakes topics in the study of infectious disease. The multi-host dynamics of virus emergence, from reservoir to recipient hosts, requires us to consider the interplay of host ecology and virus genetics. At the ecological level, emergence risk has been associated with such factors as climate change, population demographics , geograpA central goal of research in this area has been to reveal the ‘rules’ that underpin disease emergence, on the implicit assumption that predictive accuracy will follow. A more ambitious scheme was established in 2016 in the guise of the Global Virome Project (GVP). Through a global partnership the GVP aims to identify and characterize 99% of zoonotic viruses with epidemic potential to better predict, prevent and respond to future viral threats . To achiSurveys of virus biodiversity have already revealed remarkable levels of genomic and phylogenetic diversity ,29, and The idea of a virosphere so expansive is supported by the vast numbers of viruses discovered by recent studies of viral biodiversity that have been stimulated by advances in metagenomics, particularly the use of bulk RNA sequencing ,29,31. Irare events to predict rare events.Although vertebrates, particularly mammals, may carry a smaller number of viruses, the number is still so very large as to make any detailed experimental follow-up of even the vertebrate virosphere impractical, particularly as the rapid nature of RNA virus evolution means that any individual virus species will harbour a wide diversity of ever-changing variants. This impracticality is augmented when one considers our lack of knowledge of whether this vast set of viruses can replicate in human cells, and even this trait will not guarantee that a virus will be able to successfully transmit between hosts. In this context it has been proposed that machine learning may help in pandemic prediction, for instance by using sequence data to predict which cell receptors a virus might use ,36. HoweParadoxically, then, the more we sample animal populations, the less frequently virus cross-species transmission to humans seems to occur. For example, when SARS coronavirus (CoV) was revealed to have its origin in bats , the totIt is also important to recall that the most recent viruses to achieve epidemic spread in humans—Ebola and Zika—were caused by known and well described human pathogens, with the first descriptions of Zika virus going back to the 1940s . Yet ourPrevious attempts to predict aspects of virus emergence have met with limited success. There has been considerable interest in trying to predict the geographical locations where viruses may emerge in the future, based on identifying the locations where such emergence events have occurred in the past—the so-called ‘hotspots’ of emergence . AlthougIt has also been suggested that wildlife host species richness is an important predictor of disease emergence . ConversAlthough cross-species virus transmission sits at the heart of virus emergence, phylogenetic studies of the frequency with which different virus families are able to jump species boundaries also offer little predictive power as all exhibit a strong tendency to jump hosts. Indeed, it now appears that the evolutionary history of most virus families comprises a complex mix of cross-species transmission and virus–host co-divergence, and that trying to disentangle the respective contributions of each process will be challenging . In addiWhile other comparative analyses have revealed those virological factors that increase the transmissibility of emerging viruses in humans , these aGiven these uncertainties, and the fact that elements of the evolutionary process that underpins emergence are inherently unpredictable, we suggest that there is no simple algorithm that will enable an accurate prediction of what viruses might emerge in the future. Hence, it is necessary to lower our expectations about disease emergence as a predictive science. In particular, although metagenomics undoubtedly has major implications for our understanding of virus evolution, it also probably undermines biodiversity-based attempts to predict the virus source of the next major disease pandemic . There aPredictions also sit uneasily with most aspects of evolutionary biology. Even relatively simple traits like virulence, which have generated considerable evolutionary theory, have proven difficult to predict because of myriad unknown forces that shape their evolutionary trajectory . Althoug4.At face value it seems obvious that evolutionary ideas and analyses will help predict the emergence threat posed by different viruses. However, a major limitation is that evolutionary processes, particularly those reliant on phylogenetic or other comparative analyses, often occur on a markedly different time scale than the epidemiological processes relevant to pandemic prediction. Indeed, one of the most important conclusions of recent work in the study of RNA virus evolution is that the time scale over which these viruses have evolved, including cross-species transmission events, is probably far longer than previously imagined. This realization comes from both phylogenetic studies of virus biodiversity and branching patterns ,52, partAlthough central to understanding evolutionary processes, these time scales are irrelevant for predicting the next pandemic within an epidemiological time scale (i.e. 1–10 years). The same caveat applies to studies that have used the taxonomic span covered by viral families as a way of determining which have the greatest propensity to jump hosts . These tOrthomyxoviridae [An informative example of this fundamental disconnect between evolutionary and epidemiological time scales is provided by the hepadnaviruses, which include human hepatitis B virus (HBV). There is strong evidence for hepadnavirus–host co-divergence stretching back for effectively the entire time-span of vertebrate evolution ,54. Crosoviridae , it is sThose cases in which viruses have been deliberately released as biological controls also highlight the disconnect between evolutionary and epidemiological time scales. These natural experiments proceed over epidemiological time scales which in many ways parallel the natural emergence and spread of a novel virus in a new host. Most notably, both myxomavirus (a poxvirus) and rabbit haemorrhagic disease virus  have been successfully released as biological controls into populations of European rabbits in Australia and Europe, in the 1950s and 1990s, respectively . What is5.The study of virus emergence represents a synthesis of two different types of scientific enquiry: virology, which aims to determine, usually experimentally, the mutations that enable a virus to infect a new host; and epidemiology, which primarily seeks to identify the ecological factors responsible for viruses crossing the species boundary and spreading in a new host.We contend that these approaches can be synthesized within a single population genetic framework. Specifically, the cross-species transmission and emergence of a virus in a new host can be envisioned as a simple form of the adaptive process, in which the subject under consideration is the acquisition of mutations that facilitate replication and transmission and hence increase viral fitness. Although they may be of myriad form, the ecological factors that dictate whether such an emergence event will be successful are directly analogous to the random sampling effects that necessarily impact the spread of any new allele in a population, increasing the likelihood of genetic drift that will in turn result in stochastic loss. For example, the extensive contact heterogeneity (i.e. lack of susceptible hosts) that prevents CIV spreading in the domestic dog population is equivalent to the fate of an advantageous allele in a small host population. That is, although the virus (mutation) may be host-adapted (advantageous), it will not spread far because the host population is so small/sparse that genetic drift dominates substitution dynamics .We suggest that this new population genetic view of the process of cross-species transmission and emergence can be achieved by making the move away from thinking about viruses spreading horizontally through a population (by host-to-host transmission), which is the realm of epidemiology, and towards thinking about virus alleles/genes being inherited vertically, which is the domain of population genetics. A well-understood framework is that successful cross-species transmission requires three steps: (i) encounter a new host species, (ii) infection of that new host and (iii) propagation in the new host population . AdaptatImportantly, however, this adaptive process must also occur within the background of random sampling. Because of a lack of host contacts, or descendants, genetic drift will reduce sampling of the fittest virus and decrease the probability of emergence. Hence, as the host population increases in size, the probability that a virus will be sampled increases , althougThe importance of genetic drift can also be seen in the transmission bottlenecks that will routinely occur as a virus moves between hosts , which p6.If the accurate prediction of virus emergence is impractical, or even unattainable, what can be done to help prevent the emergence of major epidemic or pandemic disease in humans? Cross-species transmission events highlight the public health threat of wildlife trade and consumption, and heightened contact between animal hosts and humans has probably facilitated these events. It is therefore critical to direct our attention to the animal–human interface  as this can be thought of as the ‘fault-line’ at which most disease emergence events occur.We therefore propose that a more effective practical strategy for managing emerging and re-emerging epidemic or pandemic disease is the targeted surveillance of viromes at the human–animal interface. The vast biodiversity of viruses in the animal world makes their analysis prior to any emergence in humans a Sisyphean exercise. Rather, humans are the best sentinels: a virus discovered in humans very obviously can replicate in that host, which will not be the case for myriad viruses identified through biodiversity surveys of other animal taxa.We therefore urge regular genomic surveillance at the fault-line of disease emergence that captures this human–animal interface . ExampleThis virome surveillance should be ongoing and performed simultaneously on multiple human populations globally, with existing serological data perhaps helping to determine which geographical locations harbour human populations most frequently exposed to animal viruses, and hence where virome surveillance will be most informative. In addition, while metagenomics is hugely powerful in characterizing the viromes of individual organisms, including the discovery of new species, it requires active infection (replication) and that samples be taken from tissues that contain the virus. For this reason meta-serological surveys will also be of importance as they enable the identification of infections that have occurred in the recent past.7.after emergence occurs, such that they are likely to be of little predictive power. In partial response to these problems, we suggest that the field may be advanced by using a population genetic framework that melds genetic and ecological studies of virus emergence, and which highlights how the effective susceptible population size of a new host plays a major role in dictating the chance of successful emergence. In this manner we identify the possibility of a meaningful theoretical framework for the study of emergence that is grounded in evolutionary theory, but that tunes out the ‘noise’ of virus macroevolution.Predicting virus emergence has risen to become a key goal of the study of infectious disease. The study of virus evolution has revealed much about the nature of virus emergence and its history over evolutionary time scales. However, due to the fundamental differences between evolutionary and epidemiological time scales, a focus on virus evolution may in fact be a distraction when it comes to predicting the next virus pandemic. Similarly, while virological features that increase the likelihood of virus emergibility can be identified, these features cannot be treated as hard and fast rules determining which viruses will successfully emerge. Further, many of these features are only capable of being observed Despite such a framework, the inconvenient truth for all those working in the realm of disease emergence is that the vastness of the unknown virosphere and the diverse range of viruses that have achieved endemic transmission in humans means that any attempt to predict what virus may emerge next will face substantial, and probably crippling, difficulties. In light of this we suggest it may be of more benefit to public health to target, via surveillance, the fault-line of disease emergence that is the human–animal interface, particularly those shaped by ecological disturbance. Once a virus is identified as being of interest in this manner, other analyses may be able to assess its impact and pandemic potential. Such a shift in focus, away from being able to make predictions of emergence based on fundamental rules and towards the better assessment of emergence impact, is both more achievable and more likely to provide positive public health outcomes.


E. coli, P. syringae and L. monocytogenes) and one enzyme (Taq DNA polymerase) were investigated. Results show that while some ILs were virucidal, no clear structure activity relationships (SARs) could be identified for the non-enveloped viruses P100 and MS2. However, for the first time, a correlation has been demonstrated between the effects of ILs on enveloped viruses, bacteria and enzyme inhibition. These identified SARs serve as a sound starting point for further studies.For three decades now, ionic liquids (ILs), organic salts comprising only ions, have emerged as a new class of pharmaceuticals. Although recognition of the antimicrobial effects of ILs is growing rapidly, there is almost nothing known about their possible virucidal activities. This probably reflects the paucity of understanding virus inactivation. In this study, we performed a systematic analysis to determine the effect of specific structural motifs of ILs on three different biological test systems . Overall, the effects of 27 different ILs on two non-enveloped and one enveloped virus , two Gram negative and one Gram positive bacteria ( Ionic liquids (ILs), organic salts composed entirely of ions, have been successfully established over the past few decades as alternatives to organic solvents for various applications ,2. On acHowever, the “green solvent” attribute for ILs is now obsolete due to the growing database of (eco)toxicity data obtained from various biological systems, such as algae, fungi, plants, mammalian cells and bacteria ,10,11,12The limitations of today’s antibiotics and disinfectants, however, not only apply to resistant pathogenic bacteria and fungi, they also extend to viruses. However, not only do today’s antibiotics have limited or no efficacy against viruses, many commonly used disinfectants are useless, especially against non-enveloped viruses ,24,25. SAlthough ILs have already been utilized successfully for various antibacterial and antifungal applications, their virucidal potential has until now been neglected. To the best of our knowledge, our research group has been the only one thus far to investigate ILs specifically as possible virucides. In a previous study, we investigated the efficacy of various ILs against the non-enveloped viruses P100 and MS2, based on already known structure activity relationships (SARs) . To our Pseudomonas syringae (P. syringae) phage is an enveloped, double-stranded-RNA (ds-RNA) virus of the family Cystoviridae [Cl], [DODMA][Cl] and [TOMA][Cl]  was tested  exhibited no effect. A so-called “cut-off” effect was already observed in other organisms [Cl] to [TOMA][Cl] on the enveloped virus Phi6, this could be due to a “cut-off” effect, again.10C10im][Cl], a concentration of merely 100 mg/L was required to result in a reduction of ≥4 log10 units. This result could be due to the longer side chains with the increasing number of C-atoms [I]. This is hardly surprising, as iodine is well known to be a potent antibacterial agent itself [Cl] and [C4mim] were provided by Merck KGaA . [TMC8A][Cl], [DODMA][Cl], [C1mim][Cl], [C4mim][I and TCA] were synthesized using the CBILS® (is a registered trademark of Proionic GmbH) route [8A] and [C4mim][MC]) were provided by Proionic GmbH . Hydrochloric acid and trichloroacetic acid were obtained from Merck KGaA , iodic acid was purchased from Sigma-Aldrich Chemie GmbH . [C12–C16mim][Cl] and [C10C10im][Cl] were purchased from Iolitec . All other ILs were provided by Proionic GmbH . IL-solutions were prepared at concentration of 100,000 mg/L, 50,000 mg/L, 20,000 mg/L and 2000 mg/L, 200 mg/L, 20 mg/L and 2 mg/L (w/v in water). H) route ,61 as prH) route . The preL. monocytogenes EGDe (ATCC BAA-679) was used for virus P100, E. coli Top 10F’  was used for virus MS2 and P. syringae van Hall 1902  was used for virus Phi6. All bacteria strains were grown overnight in tryptone soya broth (TSB) with 0.6% (w/v) yeast extract . L. monocytogenes EGDe and E. coli Top 10F’ were incubated at 37 °C overnight, whereas P. syringae was incubated at 25 °C overnight. Overnight cultures of E. coli Top 10F’ were diluted ten-fold in fresh medium and incubated at 37 °C for 3–4 h to gain a maximum number of viable cells in the logarithmic growth phase (log phase). For propagation of virus stocks , the virus solutions were used for plaque assays  and shaken overnight at 4 °C. Afterwards the SM buffer was separated and centrifuged at 8000 rpm for 2 min. The supernatant was filtered (0.02 μm), aliquoted and stored at −20 °C.Virus P100, purchased as Listex™ P100 solution , was used as a model for DNA viruses. Virus MS2, kindly provided by Regina Sommer, Medical University of Vienna, was used as a model for RNA viruses. Both viruses, P100 and MS2, were tested as surrogates for non-enveloped viruses, whereas virus MS2 was also used as a representative of human enteric viruses, such as human noroviruses . Virus Psays see  and plat10–1011 Plaque forming units (PFU)/mL. Virus titre of P100 and Phi6 was determined using the Small Drop Plaque Assay [L. monocytogenes EGDe or P. syringae was 10-fold diluted in SM buffer [E. coli Top 10F’ (OD600 nm 0.6–0.8) was added to each virus dilution. Afterwards 200 μL of this solution were mixed with 3 mL overlay medium (prewarmed TSB with 0.3% agar), and spread on a TSA-plate. When the overlay medium was hardened, the plates were incubated overnight at 37 °C. Finally, plaques were counted and compared with the untreated controls.All viruses were used at concentrations of approximately 10ue Assay . In shorM buffer ,65. Subsv/v; solid ILs w/v in water) and were incubated for 30 min at room temperature. Afterwards the viruses were numerated as described above (10 reduction of virus concentration (plaque forming units (PFU)/mL), in comparison to the untreated control, was calculated. All ILs were initially tested at a concentration of 50,000 mg/L (w/v in water). ILs which caused a reduction that reached the detection limit of the assay or exhibited a reducing effect on virus titre concentrations of ≥4 log10 units were additionally tested at concentrations of 25,000, 10,000 and 1000 mg/L. ILs with particularly considerable impact on virus titre were further tested at 100, 10 and 1 mg/L. The VC was defined as the lowest concentration of the respective IL where a virus titre decreased by greater than 4 log10 units or the detection limit was reached.All viruses were mixed with each IL—solution to reach a final concentration of 1–50,000 mg/L (liquid ILs ed above . The log10 reduction (depending upon the virus used and on the ILs and their influence on the bacteria). All experiments were performed in duplicate on at least two different days  or with at least two different dilutions with the Double Agar Overlay Assay on at least three different days. Each experiment included positive and negative controls. The detection limit was at least 4.5 logL. monocytogenes EGDe and E. coli Top 10F’ were diluted 1:10 in 9 mL fresh media and incubated at 37 °C for 3 h to ensure a maximum number of viable cells in the log phase. For P. syringae overnight culture was used. Afterwards each dilution well of the microtiter plates  was inoculated with 5 × 105 CFU/mL of the respective bacterial cells and subsequently measured at a wavelength of 610 nm in a TECAN infinite F200 microplate reader  to determine potential IL interference. After the first measurement the microtiter plates were then incubated for 24 h at the respective temperatures and thereafter absorbance was measured at 610 nm a second time to determine bacterial growth. The MIC was defined as the lowest concentration of the respective IL where no bacterial growth could be observed after 24 h. Each experiment included positive (inoculated growth medium) and negative controls (pure medium) and the experiments were performed at least three times on different days.The bactericidal impact of ILs was investigated by determination of the minimum inhibitory concentration (MIC). MIC of the examined ILs were determined by the serial two-fold dilution microtiter plate method in TSB-Y medium, according to Morrissey et al. . OvernigprfA gene of L. monocytogenes [® . One qPCR reaction of 25 μL final volume contained 2.5 μL of 10 × reaction buffer , 500 nM of each primer , 1.25 μM of probe EvaGreen® , 200 μM of each dNTPs , 1 U of Taq DNA Polymerase , ~150 copies of the internal amplification control L. monocytogenes EGDe and 5 μL of the IL. qPCR was performed in an Mx3000p real-time PCR thermocycler  with initial denaturation at 94 °C for 5 min and amplification over 45 cycles at 94 °C for 15 s and 64 °C for 1 min. A final extension was performed for 1 min at 95 °C, 30 s at 55 °C and 30 s 95 °C. The MIC which was defined as the lowest concentration of the respective IL where no amplification was detected. All ILs were tested at final concentrations of 50,000, 10000, 2000 and 400 mg/L (w/v) in water. For the more active ILs, concentrations of 50, 0.5 mg/L and at least with 0.005 mg/L were tested. All experiments were performed at least twice on different days.in duplicate or triplicate.The qPCR enzyme inhibition assay was used in this study to determine the toxicological potential of ILs at an enzymatic level. The qPCR assay used was modified for the ytogenes , using tIn conclusion, the results of our study demonstrate that while virucidal effects of ILs on non-enveloped viruses occurred, previously described SARs are not applicable and future research must focus on identifying their actual mode(s) of action. In contrast to non-enveloped viruses, previously established SARs from biological model systems can be used as a sound starting point for further investigations of ILs on enveloped viruses. However, in order to facilitate the search for highly virucidal ILs that are universal against all virus types, further research must focus on concrete mechanisms of action.


We investigated an outbreak of exanthematous illness in Maceió by using molecular surveillance; 76% of samples tested positive for chikungunya virus. Genetic analysis of 23 newly generated genomes identified the East/Central/South African genotype, suggesting that this lineage has persisted since mid-2014 in Brazil and may spread in the Americas and beyond. Aedes aegypti mosquitoes, and cause similar clinical symptoms upon infection, complicating epidemiologic surveillance. Brazil harbors the highest diversity of CHIKV in the Americas; both the Asian and the East/Central/South African (ECSA) lineages circulate in the country (Dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV) co-circulate in Brazil, are predominantly transmitted by During March 30–May 3, 2016, ≈12,000 patients visited 2 private hospitals in Maceió; roughly 70% of them had exanthematous illness symptoms compatible with DENV, CHIKV, or Zika virus infection. We analyzed 273 randomly chosen samples by using molecular diagnostics and virus discovery methods. The study was approved by the Faculty of Medicine from the University of São Paulo Review Board, and we obtained informed consent from all participants. t) values for CHIKV RNA–positive samples were lower (average Ct = 24.6) than those for ZIKV (average Ct = 33.5).Analyzed samples were from patients who were on average 37 years of age (range 1–86 years); 175 (64%) were female, and 198 (73%) resided in Maceió municipality. Diagnostic tests for DENV, ZIKV, and CHIKV confirmed that 208 (76%) were positive for CHIKV RNA . In addiWe applied a metagenomics next-generation sequencing protocol to 38 randomly chosen CHIKV RNA–positive samples . We recoOn the basis of available sequences of isolates from the Americas, the Maceió sequences we analyzed fell within a single strongly supported monophyletic clade  that belongs to the ECSA genotype . GeneticThe brief to negligible period of undetected transmission of CHIKV in Alagoas is consistent with past reports Table 2. Ae. aegypti mosquitoes (Ae. albopictus mosquitoes in Brazil are also highly competent in CHIKV ECSA transmission (The unrecognized transmission of the CHIKV ECSA genotype in Northeast Brazil is unique in the Americas. The spread of this genotype in this region will be mediated by several factors, including herd immunity, vector suitability, and human mobility. Serologic and molecular surveys in human and mosquito populations are required to characterize the factors involved in transmission and the extent of cross-protection of the Asian and the ECSA genotypes in the Americas. Although CHIKV ECSA has been found only in Additional information about the testing, sequencing, and analysis of chikungunya virus genomes.


Under these circumstances, conventional strategies can fail to control rapidly evolving and emerging plant viruses. Genome-engineering strategies have recently emerged as promising tools to introduce desirable traits in many eukaryotic species, including plants. Among these genome engineering technologies, the CRISPR /CRISPR-associated 9 (CRISPR/Cas9) system has received special interest because of its simplicity, efficiency, and reproducibility. Recent studies have used CRISPR/Cas9 to engineer virus resistance in plants, either by directly targeting and cleaving the viral genome, or by modifying the host plant genome to introduce viral immunity. Here, we briefly describe the biology of the CRISPR/Cas9 system and plant viruses, and how different genome engineering technologies have been used to target these viruses. We further describe the main findings from recent studies of CRISPR/Cas9-mediated viral interference and discuss how these findings can be applied to improve global agriculture. We conclude by pinpointing the gaps in our knowledge and the outstanding questions regarding CRISPR/Cas9-mediated viral immunity.Plant viruses infect many economically important crops, including wheat, cotton, maize, cassava, and other vegetables. These viruses pose a serious threat to agriculture worldwide, as decreases in cropland area  Among biotic stresses, phytopathogenic viruses cause an estimated 10–15% reduction in global crop yields each year . Thus, iThe mechanisms of virus infection and transmission give many potential targets for controlling viruses in crop plants; however, the diversity of viruses and their rapid evolution make such approaches difficult . Most plGeminiviridae infect important crops of several families including Cucurbitaceae , Euphorbiaceae (cassava), Solanaceae , Malvaceae , and Fabaceae  in different regions of the world  , in a circulative persistent manner and are mostly restricted to the phloem of infected plants , and bipartite . DNA-A and DNA-B are 2.7–2.8 kb and each component has its own ORFs in a bidirectional fashion. Monopartite begomoviruses (or DNA-A of bipartite begomoviruses) have six ORFs, four in complementary sense orientation (AC1/C1–AC4/C4) and two in virion sense orientation . The ORF AV2 is missing in begomoviruses from the New World. All proteins encoded by begomoviruses are multifunctional and are given names according to their functions. AC1/C1 encodes replication-associated protein (Rep), AC2/C2 encodes replication enhancer protein (REn), AC3/C3 encodes transcriptional activator protein (TrAP), and AC4/C4 encodes AC4/C4 protein. The ORF AV1/V1 encodes for the coat protein (CP) while AV2/V2 encodes another protein called pre-coat protein. The DNA-B component of bipartite begomoviruses encodes nuclear shuttle protein (NSP) from the BC1 ORF and movement protein (MP) from the BV1 ORF  (AC1) (The genes in the virion and complementary sense orientations on DNA-A and DNA-B are separated by an intergenic region (IR) containing a common region (CR) of sequences that are conserved between DNA-A and DNA-B. The main topological feature of the CR is a hairpin structure with a conserved nonanucleotide (TAATATT/AC) that spans the virion strand origin of replication (the “/”) . Iterate”) (AC1) . Begomov”) (AC1) . Betasat”) (AC1) .Conventional virus control strategies focus on vector management using pesticides, activating natural predators, or the use of physical barriers like reflective mulches and UV-absorbing sheets . AdditioUtilizing genetic resistance in crop plants by boosting the plant cellular immunity against viruses is rationally the most effective strategy, since above mentioned conventional strategies are expensive, labor intensive and often ineffective, specifically in case of viral diseases . The mosGenome engineering has recently emerged as a ground breaking tool to improve several eukaryotic species, including crop plants, by introducing several traits of interest through the site-specific modification of the genome . In addiFigure 2). These techniques have been harnessed independently to improve crop plant resistance to viruses directly by targeting viral genomes or by targeting host factors (Table 1).Genome engineering refers to the use of site specific nucleases (SSNs), that can be designed to bind and cleave a specific nucleic acid sequence by introducing double stranded breaks (DSBs) at or near the target site . There aThe DSBs generated by the SSNs can be repaired by one of two endogenous mechanisms: non-homologous end joining (NHEJ) or homologous recombination . The mosHere we discuss the utilization of different genome engineering platforms including ZFNs, TALENs, and CRISPR/Cas9 to engineer virus resistance in plants with respect to the technological advancements, limitations, and future prospects.Beet severe curly top virus (BSCTV) or Tomato yellow leaf curl virus (TYLCV) effectively targeted BSCTV and TYLCV, respectively  and Tobacco curly shoot virus (TbCSV)  .AC1 and nonanucleotide, were selected and targeted using TALE. DNA binding efficiencies of TALE were confirmed and transgenic Nicotiana benthamiana plants were developed. Two begomoviruses alone, i.e., TbCSV and Tomato leaf curl Yunnan virus (TLCYnV); and one begomovirus with associated betasatellite, i.e., TYLCCNV with its cognate Tomato yellow leaf curl China betasatellite (TYLCCNB), were tested. Transgenic plants demonstrated partial resistance by developing delayed symptoms and reduced viral DNA accumulation . CRISPR/Cas9 is one of the most widely adapted systems for genome engineering and has been used successfully in several species ranging from simple microbes to complex plants and animals  and phages . Bacterines Cas9   , and Merremia mosaic virus (MeMV). N. benthamiana against Bean yellow dwarf virus (BeYDV) and BSCTV, respectively. Cotton leaf curl Kokhran virus (CLCuKoV) and also showed that targeting the conserved nonanucleotide sequence can target multiple begomoviruses simultaneously , conferring broad-spectrum geminivirus resistance. All of these studies showed that N. benthamiana plants expressing the CRISPR/Cas9 system displayed considerably reduced viral titers, which abolished or significantly reduced disease symptoms.Four recent studies demonstrated the power of the CRISPR/Cas9 system to efficiently confer resistance to geminiviruses in plants (igure 3) . Ali et Since there is an arms race between the invading viruses and their host plants Currently there is no report of directly targeting and cleaving RNA viruses. The limitation of directly targeting RNA viral genomes is that the guide RNA-Cas9 system can only be used to target DNA viruses. This could change in the future, because Cas9 can be programmed to cleave RNA  and the Rather than targeting the virus genome, genome-editing strategies to create resistance to RNA viruses might target the plant genome. In this approach, the CRISPR/Cas9 system can modify plant genes that affect plant responses to viral infection and thus generate virus resistance.eIF4E and eIF(iso)4E, have been demonstrated to be directly involved in the infection of RNA viruses  4E, which is strictly required for viral survival (Cucumis sativus L.) was also demonstrated by disrupting eIF4E, and developing non-transgenic heterozygous eIF4E mutant plants. These non-transgenic plants developed partial resistance to an ipomovirus (Cucumber vein yellowing virus) and two potyviruses (Zucchini yellow mosaic virus and Papaya ring spot mosaic virus-W)  . Therefos (TuMV) . The utisurvival . Developvirus-W) .The translation initiation factors are prime candidates for host genes that can be targeted , but anyeIF4E and eIF(iso)4E have certain additional advantages; for example, many natural sources of Potyvirus resistance rely on loss-of-function mutations in host eIFs In addition to engineering virus resistance, CRISPR/Cas9 can be used to address several basic biological questions. Virus replication within the host cell has been thoroughly studied , but virThe advent of CRISPR/Cas9 technology has revolutionized the field of genome editing. Crucially, the fact that the Cas9 nuclease is guided by RNA rather than protein overcomes the major limitations of TALEN and ZFN technologies. RNA-based guiding is cheaper and easier to engineer and greatly expands the range of possible target sequences, requiring only the commonly occurring NGG PAM sequence and other variants.Although the efficacy of genome engineering techniques has been demonstrated for production of resistance to plant viruses in several studies , it remaR genes, due to lower selective pressures on the virus to evolve counter defense strategies (Notably, the durability of this engineered resistance also remains to be tested. Recessive resistance arising from the loss of a host factor required by the virus is assumed to be more durable than dominant rategies . The resMM and SM provided the outlines of the review and contributed the key ideas. SZ and MT wrote the manuscript and prepared the figures. MM, SZ, MT, and SM worked on and improved the original draft and figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Potexvirus was fully sequenced and characterized. The virus was isolated from babaco (Vasconcellea x heilbornii), a natural hybrid native to Ecuador. The virus contains a 6,692 nt long genome organized in five open reading frames in an arrangement typical of other potexviruses. Sequence comparisons revealed close relatedness with Papaya mosaic virus (PapMV), Alternathera mosaic virus (AltMV) and Senna mosaic virus (SenMV), exhibiting nucleotide identities up to 67% for the polymerase (Pol) and 68% for the coat protein (CP), with deduced amino acid identities of 70% and 72% for the Pol and CP, respectively. The presence of an AlkB domain, in the polymerase region, was observed. Terminal nucleotide sequences were conserved across potexviruses with characteristic motifs and predicted secondary structures at the 3’ UTR. Although serologically undistinguishable from PapMV and AltMV, the new virus showed differences in host range and symptom induction. The name babaco mosaic virus is proposed for this newly characterized Potexvirus. The complete genome sequence of the new virus has been deposited in NCBI GenBank under accession number MF978248.A new member of the genus  Vasconcellea x heilbornii) is a natural interspecific hybrid of V. stipulata and V. pubescens in the family Caricacea. The plant, formerly known as Carica pentagona due to its peculiar pentagon-shaped fruits, is an herbaceous shrub that can grow up to 2.5 meters in height and has morphological characteristics similar to those of its close relative papaya (Carica papaya). Although native to subtropical mountains of southern Ecuador, babaco has been introduced and grown at small scale in Australia, New Zealand, United States, Italy and Spain, among others. In addition to its particularly exotic flavour, the babaco latex has been demonstrated to have lipase activity higher than that of papaya, showing its potential industry use [Babaco  [While several fungal diseases have been identified and characterized in babaco, studies on disease-causing viruses have been overlooked. In 1989, potexvirus-like particles were identified and purified from babaco plants showing yellow mosaic symptoms in Italy . The vir (PapMV) . To the In this study, a new potexvirus was characterized. The virus was isolated from babaco orchards in Ecuador and considering the introduction of Ecuadorean babaco to Italy in the early 80’s, we speculate that the virus is an isolate of the one reported and partially studied in Italy in 1989 .Leaves from babaco plants showing leaf mottling and mosaic were collected, with owners consent, from small orchards in subtropical regions of Azuay and Santo Domingo provinces. Leaves were ground in phosphate buffer and mechanically inoculated onto papaya cv. ‘Sunrise’ plants. Inoculated plants were maintained under standard greenhouse conditions.® 8.2.1 [Twenty-gram batches were used for double-stranded RNA (dsRNA) extraction using a cellulose-based protocol for detection of RNA viruses in plants . The dsR® 8.2.1 ,10. The ® 8.2.1. For phylogenetic inferences, complete genome sequences for all available potexviruses were obtained from the NCBI database. Sequence alignments were performed using MUSCLE and for phylogenetic inferences, the maximum likelihood method plugged in MEGA® 7 was applied using the most suited substitution models determined also by MEGA 7.Genome organization including identification of characteristic genomic features were done using the open reading frame (ORF) finder available at NCBI and motif search tool from Geneious® reverse transcriptase  following manufacturer’s instructions. PCR was carried out in a 10 μL mixture containing 1 μL 10× buffer, 0.2 uL of 10 mM dNTPs, 1.5 μL cDNA template, 6.8 uL of molecular biology grade water, 0.1 μL of Taq DNA polymerase  and 0.2 μL of each primer (40 μM) designed and selected based on specificity across closest relatives. PCR parameters were as follows: 94°C for 4 min, 40 cycles of 94°C for 45 s, 57°C for 30 s and 72°C for 45 s, and a final extension step of 10 min at 72°C.Virus detection was done by RT-PCR using total RNA as described by Halgren et al. . RT was In addition, a commercial ELISA kit  developed for the detection of PapMV and AltMV was used to test for serological compatibility.Carica papaya), chamburo (Vasconcellea pubescens), tamarillo (Solanum betaceum), Nicotiana benthamiana, Chenopodium quinoa, Chenopodium amaranticolor, Amaranthus dubius, Senna occidentalis, Senna alata, Cucurbita ecuadoriensis, Luffa operculata and Luffa cylindrica.Mechanical inoculations were done by dusting carborundum (600 mesh) on test leaves followed by rubbing infected tissue homogenized in phosphate buffer (0.05 M pH: 7.4). Test plants (5 plants for each species) included the following species: papaya  encodes the putative polymerase. ORF2 , ORF3  and ORF4 , arranged in an overlapping fashion, code for the movement-involved triple gene block (TGB) proteins 1, 2 and 3, respectively. Lastly, ORF5  encodes the putative coat protein (CP) (The genome is organized in five ORFs similar to other potexviruses. ORF1 ein (CP) .Potexvirus such as the overlapping TGB ORFs, highly conserved motifs, and the poly A tail, among others [Flexiviridae the one with more AlkB-containing members discovered thus far [Several genomic signatures have been observed across all members of the g others –14. Howethus far ,16. As sthus far , supportA highly conserved CP motif, involved in RNA-CP binding, has been reported as a signature genomic feature in potexviruses –24. ThisPapaya mosaic virus (PapMV) and Senna mosaic virus (SenMV) in a clade that is related closely to Clover yellow mosaic virus (ClYMV) and other AlkB containing members isolated from cactaceous hosts such as Opuntia mosaic virus, Cactus virus X, Zygocatus virus X, Pitaya virus X and Schlumberga virus X. Interestingly, four other AlkB containing members  were placed in a clade considerably distant from the other AlkB-containing species  is proposed and will be used hereafter for practical purposes.Based on ICTV guidelines for species demarcation in the genus 5’-GAAAAG-3’ present at the 5’end of most potexviruses, was also observed in BabMV; whereas at the 3’ end, the pentanucleotide 5’-TTTCC-3’ showed conservation in BabMV and PapMV. Analysis of the 3’ UTR of BabMV revealed the presence of two conserved motifs nt:6,593AATAAA-3’5’- and nt:6,652ACCTAA-3’5’-, which are putatively involved in signalling for poly (A) tailing and synthesis of negative-sense RNA in potexviruses [Untranslated regions (UTR) of 108 nt and 140 nt were observed at the 5’ and 3’ ends, respectively. The hexanucleotide xviruses –27. In a5-GGATGCACTCATTACATCCAAGC-3 and R: 5’ CCACTCCAAGGCTTCCATGAGC-3’, which amplifies a 647 bp region of the polymerase, was selected based on specificity. The assay was tested on a set of 25 plants (n = 20 symptomless and n = 5 symptomatic) kindly provided by a commercial nursery in Azuay province. BabMV was detected in all symptomatic plants; whereas only one plant from the symptomless group, tested positive for the virus, but developed symptoms after three weeks. Similar results were obtained from ELISA testing, conducted in parallel. The presence of PapMV was not detected by RT-PCR in any of the tested plants, validating the cross-reactivity of PapMV-based antibodies with BabMV.Primers set: F: Two commercial papaya cultivars, ‘Maradol’ and ‘Sunrise’, were used for mechanical inoculations and virus maintenance. Symptoms were prominent in ‘Sunrise’, where a severe mosaic was visible on young leaves at 15 days post inoculation (dpi). Interestingly, as leaf growth progressed, symptoms vanished and plants remained symptomless until new fully developed leaves exhibited the mosaic . In ‘MarVasconcellea pubescens), one of babaco’s parental lines, was also inoculated and showed severe leaf mosaic , Nicotiana benthamiana, Amaranthus dubius, Cucurbita ecuadoriensis, Luffa operculata, Luffa cylindrical and Senna alata.BabMV was not detected on tamarillo  the vegetative propagation nature of this hybrid and ii) the use of BabMV-susceptible rootstock, such as chamburo (V. pubescens), for management of soil-borne pathogens through grafting.Nevertheless, the babaco production in Ecuador has increased significantly in the last two decades; as expected, virus-like diseases have also become conspicuous in the major production areas. Therefore, a certification program for production of virus-free babaco plants needs to be implemented considering The characterization of BabMV is the first attempt to highlight the importance of virus identification and development of effective detection assays for the implementation of virus-clean certification schemes for this crop.S1 FigMaximum likelihood inference based on the coat protein. Virus names abbreviations and NCBI acc. numbers are shown.(TIF)Click here for additional data file.S2 FigTransmission electron microscopy photographs of virus particles observed in plants infected with babaco mosaic virus.(PDF)Click here for additional data file.S1 TableList of potexvirus species used for phylogenetic relationships based on the polymerase. NCBI acc. numbers and corresponding references are shown.(DOCX)Click here for additional data file.


ACE and PR, are significant biomarkers that have served in several genotype–phenotype association studies. In Mexico, studies concerning the frequency of these biomarkers have been conducted mainly in subpopulations from central and southern regions. Here, we screened a population sample of the northwestern region to gain further knowledge regarding the prevalence of Alu polymorphisms within ACE and PR loci.Presently, non-LTR retrotransposons are the most active mobile elements in the human genome. Among these, Alu elements are highly represented in the modern population. Worldwide, distribution of Alu polymorphisms  shows variability between different populations. Two Alu insertion loci, ACE locus, the observed genotype frequencies were 26.5, 51.0 and 22.5% for II, ID, and DD, respectively; and allelic frequencies for I and D were 52 and 48%. Whereas respective genotype frequencies for PR locus were 2.7, 26.5 and 70.8%, and the corresponding allele frequencies were 16 and 84%. Furthermore, the insertion frequency within ACE locus was similar between central, western and northwestern subpopulations, and rather higher in southeastern subpopulation (p < 0.05). Although the occurrence of Alu polymorphisms within PR locus has not been widely examined, the insertion frequency was higher in northwestern subpopulation, as compared with western and southeastern subpopulations (p < 0.05). Based on the frequency of Alu insertions found in ACE and PR loci, subpopulations from the northwestern, western and central regions share a common genetic origin, but apparently not with the subpopulation from the southeastern region, in accordance with the notion that assumes the existence of a broad genomic diversity in the Mexican population. In addition, the high prevalence of Alu insertions reveals their potential application as biomarkers with prognostic value for the associated diseases; e.g., as part of the standard protocols for clinical diagnosis.For  Mobile elements can be classified as either DNA transposons or retrotransposons. DNA transposons are currently not mobilizing in the human genome, while retrotransposons have significant mobility. Retrotransposons can be subdivided into LTR and non-LTR elements, which are distinguished by the presence or absence of long terminal repeats. LTR retrotransposons are endogenous retroviruses with very limited activity. By contrast, non-LTR retrotransposons, typified by L1, Alu, and SVA elements, are presently the most active . TherefoAlu elements are one of the most represented in the human genome, being about one million copies . As active elements in the modern population, new insertions into somatic cells contribute to genomic diversity, gene mutations, and genetic diseases , 8. In aACE) locus is an important genetic biomarker that has served in numerous genotype–phenotype association studies [ACE plays a key role in the regulation of systemic blood pressure and renal electrolyte homeostasis by converting the inactive angiotensin I into the potent vasoconstrictor and aldosterone-stimulator angiotensin II, and by inactivating the pro-inflammatory vasodilator bradykinin [ACE locus, resulting in three different genotypes: II, ID and DD. Moreover, it seems that the serum concentrations of ACE correlate with the I/D polymorphism (DD > ID > II), suggesting that the levels of circulating enzyme may be determined by the genotype at the ACE locus [The insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme  locus comprises an Alu insertion within the intron G (between exon 7 and 8) and two single-nucleotide polymorphisms (SNP): G→T transversion in exon 4, producing a missense mutation (V660L), and C→T transition in exon 5, yielding a silent mutation (H770H). Remarkably, the identification of any of these three alleles uniquely recognizes the presence of the other two [The long-range haplotype, named PROGINS, found in the progesterone receptor  using locus-specific primers  containing 25 pmol of each primer and 100 ± 40 ng of template DNA. Thermal cycling conditions were as follows: an initial denaturation step (2 min at 94 °C), followed by 35 cycles of exponential amplification , and a final elongation step (7 min at 72 °C). PCR products were separated by gel electrophoresis (2% agarose) and stained with ethidium bromide. The I/D polymorphisms were determined by visual discrimination of each fragment length  model. HW equilibrium was verified by the goodness-of-fit test χSince a significant percentage (around 0.5%) of all human genetic disorders might result from Alu-mediated unequal homologous recombination , the disACE and PR loci .We have reported the prevalence of three pharmacogenetic traits in northwestern Mexicans . To gainci Table . For ACEACE locus was quite similar between central, western and northwestern subpopulations, but significantly different in a southeastern subpopulation (p < 0.05), suggesting distinct genetic origins amongst current Mexican subpopulations. Conversely, although the occurrence of Alu polymorphisms within PR locus has not been widely examined, we found that the frequency of insertions was significantly higher in a northwestern subpopulation, as compared with western and southeastern subpopulations (p < 0.05), supporting the previously mentioned notion.Through a comparative analysis Table , we founBecause of their transposition activity, Alu elements represent a significant source of genomic variation . Their iACE and PR loci to determine the occurrence of I/D polymorphism in the northwestern subpopulation. Our results showed that the frequency of Alu insertions found in the examined subpopulation is quite comparable to the observed in western and central subpopulations, but it is significantly different from that reported in southeastern subpopulation (p < 0.05). This is consistent with the notion that presumes the existence of a broad genomic diversity in the Mexican population [In Mexican populations, the ancestral genetic contribution exhibits regional fluctuations . Althougpulation , 34.The high prevalence of Alu insertions in current Mexican population reveals their potential application as biomarkers with a prognostic value for the associated diseases; e.g., as part of the standard protocols for laboratory diagnosis. However, this notion should be properly assessed through genotype–phenotype association studies to adequately fulfill clinical purposes.