Few studies have estimated the economic burden of chronic diseases  attributable to unhealthy eating. In this study, we estimated the economic burden of chronic disease attributable to not meeting Canadian food recommendations. We first obtained chronic disease risk estimates for intakes of both protective  and harmful  foods from the Global Burden of Disease Study, and food intakes from the 2004 Canadian Community Health Survey 24-hour dietary recalls . We then calculated population attributable fractions (PAFs) for all relevant food-chronic disease combinations by age and sex groups. These PAFs were then mathematically combined for each disease for each age and sex group. We then estimated attributable costs by multiplying these combined PAFs with estimated 2014 annual direct health care  and indirect  costs for each disease. We found that not meeting recommendations for the eight foods was responsible for CAD$13.8 billion/year . Nuts and seeds and whole grains were the top cost contributors rather than vegetables and fruit. Our findings suggest that unhealthy eating constitutes a tremendous economic burden to Canada that is similar in magnitude to the burden of smoking and larger than that of physical inactivity which were estimated using similar approaches. A status quo in promotion of healthy eating will allow this burden to continue. Interventions to reduce the health and economic burden of unhealthy eating in Canada may be more effective if they are broad in focus and include promotion of nuts and seeds and whole grains along with vegetables and fruit rather than have a narrow focus such as primarily on vegetables and fruit. In Canada, chronic diseases  were the leading causes of death in 2013 . This formula makes the assumption that the risk factors are independent (presence of one risk factor does not affect the probability of having another) and the joint effects of these risk factors are multiplicative . This approach has also been previously used to combine dietary risk factors for other outcomes  [We calculated separate PAFs for each food for each chronic disease by sex for each of the following age groups: ≤14 years, 15–34 years, 35–54 years, 55–64 years, 65–74 years, and ≥75 years. The PAF values for each chronic disease were then combined for each sex and age group. Because some of the risk factors may be overlapping  and it is unclear what this overlap entails, it is important that we do not combine PAF values by adding them together as this would result in double counting. As suggested by Krueger et al [rtality) ,54,55.The calculated PAF outcomes were used to estimate the direct health care and indirect costs attributable to unhealthy eating. Multiple different sources released in different years were used to estimate costs. First, the proportion of direct health care costs  associated with each relevant chronic disease by sex for different age groups  were extracted from the 2008 EBIC . Second,A sensitivity analysis was also conducted similar to Krueger et al . This anCombined PAF values for each disease by age and sex are presented in Not meeting recommendations for the eight foods that have established recommendations was estimated to be responsible for an economic burden of CAD$13.8 billion . The estimated direct health care costs that were attributable to not meeting these food recommendations represents approximately 3.9% of all hospital, physician, and drug costs in Canada in 2014. About 1/3 of the estimated costs were attributable to females and 2/3 were attributable to males . Over 50% of these estimated direct health care and indirect costs were attributable to ischemic heart disease ; type 2 diabetes was also a substantial contributor . Fewer of these estimated costs were attributable to cancer, stroke, and chronic kidney disease . Our sensitivity analysis that used the relative risk 95% confidence interval boundaries in the PAF calculations revealed 95% confidence interval for total estimated costs to be $6.9-$18.5 billion. For estimated direct health care costs this was $2.6-$6.8 billion, and for estimated indirect costs this was $4.3-$11.7 billion.We also calculated the estimated cost savings if Canadians who do not meet recommendations for any of the eight foods improved their dietary choices by consuming half a serving closer to the recommendations for fruit, vegetables, milk, whole grains, nuts and seeds, red meat, and processed meat, and everyone who did not meet recommendations for sugar-sweetened beverages consumed one serving closer to the recommendations. In this scenario, the estimated economic burden would be $8.9 billion , which is $4.9 billion  less relative to the burden without this assumption. Because severe allergies to nuts are a concern in Canada , we repeWe estimated that CAD$13.8 billion in direct health care and indirect costs (which could be as low as CAD$6.9 billion and as high as CAD$18.5 billion) in 2014 were associated with not meeting Canadian food recommendations. Our analysis revealed that the largest contributors to this economic burden were inadequate intakes of nuts and seeds and whole grains. These estimates are insightful for public health professionals when considering the range of potential policy options and programs that aim to reduce the health and economic burden of chronic diseases.Our estimate of CAD$13.8 billion for the economic burden of not meeting food recommendations suggests that investments in promotion of healthy eating have the potential of substantial savings in direct health care and indirect costs in Canada. Krueger et al [Several previous studies conducted both in Canada ,24,31,32The estimated economic burden associated with the consumption of the eight different foods are the result of a combination of their relative risk of disease, and the percentage of the population not meeting recommendations. These estimates help to draw attention to dietary intakes that are associated with the greatest burden of disease . These estimates also illustrate that relatively small positive changes in dietary choices at the population level can lead to substantial cost savings. However, our estimates do not provide decision makers with actionable solutions on how to decrease the economic burden of disease. Although the estimated costs of inadequate nuts and seeds and whole grains exceeds that of vegetables and fruit, decision makers will also have to consider the possibility that it may be more difficult to increase intakes of nuts and seeds and whole grains because fewer Canadians meet those intakes compared to vegetables and fruit. The estimates from this study are useful to help guide the direction of future intervention and simulation studies examining the cost-effectiveness and cost-benefits of various programs and policy options  to improve dietary intake. These studies, once completed, will provide guidance on how to best move forward with programs and policies to best decrease the burden of chronic diseases associated with unhealthy eating.This study has several strengths. Dietary intakes were assessed using 24-hour dietary recalls of a representative sample of 33,932 Canadians. As well, only foods that cause chronic disease as identified by the GBD were included. A limitation is that our calculated value is likely an underestimate as the costs of certain chronic diseases and also costs associated with nutrients  were not included. In addition, dietary assessment is a difficult process and underreporting unhealthy foods and over reporting of healthy foods is a common phenomenon. However, even with this limitation, the estimated costs associated with not meeting food recommendations were substantial. We also assumed that the proportion of costs associated with different diseases and ratio of direct health care to indirect costs remained consistent over time which may not be the case; however, these assumptions have been used in other similar Canadian studies ,7,13,24.The economic burden of not meeting food recommendations in Canada was estimated to be $13.8 billion in 2014. This estimate allows the impact of our food choices to be compared to estimates associated with other behavioral lifestyle choices  determined using similar approaches. Although making dietary changes is a complex process which requires strong support and major investments from government and society, policies and programs which have the ability to make small positive changes at the population level have the potential to reduce the currently tremendous economic burden.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


The prevalence of genital piercing among women is increasing. As the popularity increases, the number of complications from infection, injury, and retained jewelry is likely to rise. Techniques to remove embedded jewelry are not well described in the literature. The purpose of this report was to describe a case of a patient with a retained clitoral glans piercing, discuss a simple technique for outpatient removal, and review current evidence regarding associated risks of clitoral piercings. A 24-year-old female presented to the emergency department with an embedded clitoral glans piercing. Local anesthetic was injected into the periclitoral skin and a small superficial vertical incision was made to remove the ball of the retained barbell safely. In conclusion, among patients with retained genital piercing, outpatient removal of embedded jewelry is feasible. While the practice of female genital piercing is not regulated, piercing of the glans of the clitoris is associated with increased injury to the nerves and blood supply of the clitoris structures leading to future fibrosis and diminished function compared to piercing of the clitoral hood. Genital piercing is increasing in frequency among many demographic groups. Genital piercings are unique as they may have a functional role in addition to aesthetical value, with some women reporting heightened sexual arousal and increased orgasm potential after clitoral piercings . In womeA 24-year-old female presented to the emergency room with pain at the site of her clitoral piercing for 1 day. She reported that earlier that day, she noted that the piercing did not move freely on with touching the area. The patient was sexually active one day prior. The patient's own attempts to remove the piercing were unsuccessful due to pain. She had also returned to the tattoo parlor where the piercing was performed 8 months earlier who recommended she seek medical care. On examination, the labia were normal without lesions. There was a straight metal barbell protruding at a perpendicular angle from the glans of the clitoris. On closer inspection, one of the removable balls was missing from the barbell externally and on palpation, the other ball attached to the straight barbell was appreciated underneath the glans of the clitoris. There was no drainage, bleeding, or evidence of infection. There was no evidence of scarring or keloid formation at the site of the piercing. The patient was unable to tolerate initial attempts to remove due to pain. She gave consent for attempted removal of the piercing in the emergency department. She was premedicated with 0.2 milligrams of intramuscular hydromorphone and the area was thoroughly cleansed with betadine. Then, 3 cc of 1% lidocaine without epinephrine was injected into the periclitoral skin and tested to ensure analgesia. A #11 scalpel was used to make a 2-millimeter superficial incision within the nonkeratinizing squamous epithelium overlying the clitoral glans over the embedded ball of the piercing . The embGenital piercing is not a new phenomenon. There are historical records of male genital piercing from as early as the 1st century B.C., and this practice has been alluded to in writings from the Mayan, Victorian, and World War II eras . While gThe practice of female genital piercing is not standardized. Common anatomical locations for female genital piercings include the labia majora, labia minora, and clitoris. The clitoris may be pierced through the prepuce, either horizontally or vertically or through the glans of the clitoris. Alternative female genital piercing locations that have been described include a vertical clitoral piercing exiting superiorly on the mons pubis, placement of a ring entering through the urethra and exiting between the urethral and vaginal openings (“Albertina Piercing”) , 8, and For clitoral piercings specifically, piercings through the clitoral hood or prepuce are associated with decreased rates of infection, injury to the clitoris, and fibrosis. Genital piercing of the clitoral glans has been associated with higher rates of loss of sensation and diminished sexual function due to increased risk of damage to the nerves and vascular structures that supply of clitoris  .Traumatic injuries occurring at the time of insertion or afterwards, such as during sexual intercourse, are not uncommon , 8. SurgEmbedded jewelry from piercings has been reported in other areas of the body, most commonly for lingual piercings. Piercings of the tongue and clitoris are alike as barbell jewelry is commonly used in both which consists of a central stud with two peripheral balls , 8, 10. Healthcare providers should be aware of the increasing prevalence of genital piercing and the complications inherent to this practice. Complications after genital piercings are common, reported in over 50% of patients who had genital piercings. While the practice of female genital piercing is not standardized or supported by a wealth of evidence based literature, prior studies do suggest that piercing of the glans of the clitoris is associated with higher rates of injury to the neurovascular structures leading to future fibrosis, decreased function, and injury , 8. Ther


Senecavirus A (SVA) is a positive-sense single-stranded RNA virus that belongs to the Senecavirus genus within the Picornaviridae family. The virus has been silently circulating in pig herds of the USA since 1988. However, cases of senecavirus-associated vesicular disease were reported in Canada in 2007 and in the USA in 2012. Since late 2014 and early 2015, an increasing number of senecavirus outbreaks have been reported in pigs in different producing categories, with this virus being detected in Brazil, China, and Thailand. Considering the novel available data on senecavirus infection and disease, 2015 may be a divisor in the epidemiology of the virus. Among the aspects that reinforce this hypothesis are the geographical distribution of the virus, the affected pig-producing categories, clinical signs associated with the infection, and disease severity. This review presents the current knowledge regarding the senecavirus infection and disease, especially in the last two years. Senecavirus epidemiology, pathogenic potential, host immunological response, diagnosis, and prophylaxis and control measures are addressed. Perspectives are focused on the need for complete evolutionary, epidemiological and pathogenic data and the capability for an immediate diagnosis of senecavirus infection. The health risks inherent in the swine industry cannot be neglected. Senecavirus A species in the new genus Senecavirus. Since then, until the end of 2014, little progress has been made regarding the virus features and infection in swine, including its epidemiology, transmission, pathogenesis, pathology, clinical signs, and diagnosis. The disease was considered exotic and limited in terms of geographic distribution. Senecavirus infection in swine has not been extensively studied by North American researchers or by worldwide research groups, even though it is a vesicular disease, likely due to the low frequency of infection, with the only reported cases being in North American countries.A new virus named Seneca Valley virus was identified in 2002, and classified as the only representative strain of the In early 2015, different isolates of senecavirus were first reported in vesicular disease outbreaks outside of North America in Brazil, China, and Thailand. In the same year, the virus infection was associated with novel clinical manifestations in newborn pigs. Simultaneously, new cases occurred in the USA. These events were responsible for an increase in the number and the diversity of research approaches based on the senecavirus infection.More knowledge has been accumulated and more unknown points have been clarified on the etiological agent and the virus infection, in approximately the last 24 months, than have been in all of the previous years. The scientific community was able to comply promptly with the demand for objective and clear answers to issues raised by pig producers and consultant veterinarians in private and public sectors who suddenly faced outbreaks of a completely unknown vesicular infectious disease. The currently available data were generated in a short timeframe, which is rarely seen in similar cases of viral diseases in animals without a public health impact. The goal of this review is to present the step-by-step evolution of senecavirus-based studies, particularly in the last two years, and to describe the features of the etiological agent and infection in pigs.Seneca Valley virus was incidentally isolated in Gaithersburg, Maryland, USA in 2002 from the PER.C6  cell line. The virus had evidently been introduced into the cell culture through the use of contaminated fetal bovine serum or porcine trypsin . The virSenecavirus A (SVA), genus Senecavirus within Picornaviridae family [Until 2014, only three complete genomic sequences of Seneca Valley virus were available in public databases . Therefore, few studies were based on the complete genome of the virus, with the predominant prototype being SVV-001 (GenBank accession number DQ641257) [SVV-001 has the typical genome of other picornaviruses, with the standard L-4-3-4, layout, namely Leader—4 polypeptides of the P1—3 polypeptides of P2—and 4 polypeptides of P3. The SVV genomic RNA consists of approximately 7200 nucleotides (nt), with additional 666 nt in the 5′UTR portion and 71 nt in the 3′UTR portion and a poly(A) tail. The virus genome has a single open reading frame (ORF) that encodes a polyprotein of approximately 2180 amino acids (aa) [Flaviviridae family), suggesting that the IRES of senecaviruses is type IV [An analysis of the nt sequence within 5′UTR of the SVV-001 prototype revealed high levels of secondary structures . The 5′U type IV ,8,9.Cardiovirus genus, especially in relation to the polypeptides P1, 2C, 3C, and 3D. However, SVV-001 differs from cardiovirus in polypeptides 2B and 3A and the IRES type. Genomic regions 2A, 2B, 3A, and 3B of SVV-001 differ considerably from those of all other picornaviruses [The genomic characteristics of SVA are very similar to the members of Initially, senecaviruses were not associated with any specific pathology. However, many studies have focused on the potential oncolytic activity of senecavirus in human cancer therapy ,10. The Sclerotinia sclerotiorum [Cases of vesicular diseases of unknown etiologies were reported in pigs in New Zealand ,15, Austrotiorum ,15 and trotiorum . In the Senecavirus was not investigated in any of these cases. In New Zealand, Australia, and the USA, the cases of senecavirus infection were not investigated due to the event dates, since the virus was not known at that time. However, the reasons that the virus was not investigated in the United Kingdom and Italy cannot be ascertained due to the limited data available on the outbreaks in these countries . In 2004, outbreaks of vesicular disease were reported in pigs of different production categories in Indiana, USA. Clinical manifestations resembled those of vesicular foreign animal diseases, which were investigated and not detected . Since tPasma, Davidson, and Shaw  in 2007 Although only two reports of senecavirus detection in symptomatic animals were available, a 20-year (1988–2008) retrospective serological study conducted with serum samples of asymptomatic pigs from different states in the USA showed the wide temporal and geographical distribution of senecavirus , revealiConsidering these studies and the indistinguishable feature of the vesicular lesions associated to senecavirus infection with those caused by the vesicular foreign animal diseases, especially FMD, the United States Animal Health Association elaborated a resolution in which senecaviruses and PIVD are the main subjects, with the aim of developing and implementing plans to minimize the consequences of vesicular lesions not associated with foreign animal diseases found in pigs of that country , reinforAt the end of 2014 and the beginning of 2015, outbreaks of vesicular disease in weaned and adult pigs were reported in different geographical regions of Brazil. Simultaneously, increasing mortality rates of newborn pigs at 1 to 4 days of age were recorded in the main Brazilian pig-producing regions. The reported clinical signs in affected piglets included lethargy, cutaneous hyperemia, diarrhea, neurological signs, and/or sudden death ,23,24,25These reports were the first describing senecavirus outside of North American countries. Whether the virus was circulating in Brazil before this period was unknown. However, a 10-year (2007–2016) retrospective serological study conducted in Brazil from serum samples of pigs at different ages suggested that senecavirus was not circulating in the country before 2014, the same year in which senecavirus-associated vesicular disease outbreaks were first reported in the country . Senecavirus was first reported outside of the American continent in China in 2015 ,30. PigsSince July 2015, an increased number of senecavirus-vesicular disease outbreaks was reported in the USA ; but forBased on all of these events, 2015 can be considered a turning point for the epidemiology of the senecavirus infection, with many important features of senecaviruses being identified. Until that year, only two cases of senecavirus-induced vesicular disease were reported with a limited number of affected pigs ,12. HoweIn newborn pigs, morbidity and mortality rates are considerably higher, especially in one- to four-day-old piglets, with morbidity rates that can reach 70%, but the mortality rates vary from 15 to 30% ,33,34,37To the authors’ knowledge, no re-breaks were reported in previously affected herds, but this is not a permanent condition. The infection likely becomes endemic when most of the animals present asymptomatically and/or with subclinical infection and when clinical manifestations occur in pigs that have not been previously infected, are seronegative, or have low titers of senecavirus-specific antibodies. Furthermore, the declining immunity or the introduction of naive gilts in affected herds and the persistence of the virus in the animal and in the environment may trigger a new outbreak in previously affected herds .7 to 1.2 × 1011 genomic copies/mL) [After many outbreaks of senecavirus infection were reported in 2015 in the USA, an investigation of 2033 oral fluid samples of asymptomatic pigs from herds located in 25 states of that country revealed that only 1.2% of the samples were RT-PCR-positive for the virus RNA . In contpies/mL) , suggestSenecavirus shedding was demonstrated in fecal samples from piglets that presented multisystemic clinical signs, including diarrhea ,24, and Senecavirus RNA and viable infective virus particles were isolated in cell cultures from fecal and small intestine samples of mice from clinically affected pig herds in the USA. Houseflies collected from both affected and non-affected herds tested positive for the virus . The detThe possibility that senecavirus can be vertically transmitted was raised after the virus was detected by both RT-PCR and immunohistochemical assays from different tissue/organ samples of piglets aged one to two days old . Howevern = 42) and partial (n = 15) ) genomic sequences were established from senecavirus strains in most of these countries. Therefore, advances were also possible in terms of the molecular epidemiology of the virus. The 42 complete genomic sequences of senecaviruses from 2015 to 2016 showed high nt (95.8 to 99.9%) similarities among each other and lower nt (93.8 to 94.6%) identities with the prototype strain SVV-001. The exception was the Canadian 11-55910-3 strain (GenBank accession number KC667560), which shares 95% and 96–98.2% nt similarities with the prototype SVV-001 and contemporary senecavirus strains, respectively. The grouping of senecavirus strains into three temporal clades was also observed [After the occurrence and/or the increasing incidence of senecavirus-associated vesicular disease in Brazil, China, the USA, Canada, and Thailand ,41,42,43observed . Clade Iobserved ,40.6.5/mL) was detected in serum on the third day post-inoculation (dpi) with a subsequent progressive reduction up to the 10th dpi from when the virus was no longer detected [The definitive association of senecavirus with porcine vesicular disease was shown in experimental infections of 9-week- and 4-month-old pigs with contemporary senecavirus strains ,48. In bdetected . Senecaviruses induce an acute, self-limiting vesicular disease in pigs . InoculaTissue microscopic alterations in experimentally infected pigs were restricted to the tonsils, the spleen, lymph nodes, and the lungs and consisted of mild to moderate lymphoid hyperplasia in the lymphoid tissue and multifocal mild atelectasis and occasionally diffuse congestion with multifocal mild perivascular accumulation of lymphocytes, plasma cells, and macrophages in the lungs . A wide According to the experimental studies, senecavirus shedding lasts up to 28 days. The virus can be shed by oral and nasal secretions and feces. The virus excretion peak occurs between 1 and 5 dpi, especially in oral secretions, which presented higher virus loads relative to the nasal secretions and feces . AdditioCurrently there are only a few studies that have evaluated the host immunological response against senecavirus infection. These studies indicate that the senecavirus infection induces early immunological response in clinically affected and non-affected sows under field conditions  and in eThe maximum concentration of senecavirus-specific immunoglobulin (Ig) M antibodies lasts for approximately 10 days (from 5 to 15 dpi) followed by a decreasing serological IgM concentration to undetectable titers by 21 dpi . In contDifferent senecavirus antibody titers have been reported in naturally infected pigs and may vary from 160 to 2880 by indirect fluorescent antibody (IFA) or virus neutralization (VN) test ,50; but Many diagnostic tests are currently available for the detection of senecavirus infection and/or its association with lesions and disease. Conventional ,27,40,42The other diagnostic tools currently available include immunohistochemical and in situ hybridization, which are assays that identify specific virus antigens and nucleic acid in tissue samples, respectively. Both of these techniques have been successfully used by different study groups for senecavirus-associated disease diagnosis ,25,32,55The antibody detection methods available for senecavirus screening in pigs are indirect ,49,56 anRegardless of the diagnostic method, senecavirus-associated disease in pigs is clinically indistinguishable from classical viral vesicular infections, especially FMD. The clinical history and/or the presence of lesions that indicate the possibility of vesicular disease should always lead to a complete vesicular foreign animal disease investigation, primarily in the major pig-producing countries, since the emergence of senecavirus infections could adversely affect the prompt reporting of FMD in swine .Currently, no vaccines or specific treatments are available for senecavirus infections. Therefore, sanitary practices should include prophylactic and control measures that include the herd, the animals, and the environment. Measures should be taken to avoid the introduction of the etiological agent into the pig herds and in those where the infection is established to avoid virus dissemination among the animals of the different pig-producing categories present in the same herd. The introduction of senecavirus and other infectious agents can be prevented by adopting strict biosecurity measures. The entry of vehicles, equipment, people, animals, and food into the pig production unit must be strictly controlled ,40,58. LBiosecurity measures for people movement events should also be addressed. On-farm employees should shower-in or shower-out of the facility, change clothing and boots prior to entry, and observe a period of downtime after contacting other swine . In the p-tertiary-amilphenol (4%); o-benzyl-p-chlorophenol (10%); o-phenylphenol (12%)), and quaternary ammonium-aldehyde compounds ; Glutaraldehyde (7%)) that have been used against senecavirus were evaluated at different temperatures (4 °C and ≈25 °C) on five different surfaces including cement, rubber, plastic, stainless steel, and aluminum. The results showed that 1:20 diluted household bleach was the most effective disinfectant for the inactivation of the virus at any temperature and on any of the five surfaces evaluated. The disinfectant based on phenolic derivatives was not effective against the virus at any temperature, and the results obtained from the disinfectant based on quaternary ammonia and aldehyde were between those of sodium hypochlorite and phenolic derivatives [In senecavirus-positive farms, in addition to the measures previously mentioned, strict cleaning and disinfection of the facilities and the equipment, an in-pen downtime period, and the all in-all out system have to be adopted. The effectiveness of disinfectants against senecavirus is not yet well-known. Since the clinical signs caused by the senecavirus infection are very similar to those caused by the FMD virus, control measures should be adopted that consider the possibility of FMD virus circulation, including disinfection protocols. This includes the use of sodium hydroxide (2%), sodium carbonate (4%), citric acid (0.2%), acetic acid (2%), sodium hypochlorite (3%), potassium peroxymonosulfate/sodium chloride (1%), and chlorine dioxide . Three divatives . Accelerated hydrogen peroxide (AHP) is another chemical compound that has been tested as a broad-spectrum disinfectant against bacteria, fungi, and viruses. Disinfectants containing AHP showed virucide activity against some human and animal viruses, such as bovine viral diarrhea virus (BVDV), feline calicivirus, and poliovirus, which also belongs to the Picornaviridae family. In 2016 AHP was shown to be an effective disinfectant against FMD virus, SVD virus, and senecavirus; but its efficacy is dependent on the dilution (1:20) and the contact period (10 min), which are greater than the manufacturer's recommendations . Monitoring of the senecavirus circulation should be performed by means of periodical diagnostic examinations conducted on biological samples of symptomatic and asymptomatic pigs from different sites of the production unit. In addition to serological tests, molecular, immunohistochemical, and/or in situ hybridization tests can be performed. Depending on the laboratory methodology to be used, a variety of biological samples such as vesicular and oral fluids and organ/tissue fragments of pigs at different ages, especially tonsils, lymph nodes, spleen, heart, lungs, liver, kidneys, and urinary vesicles, can be analyzed ,24,32. TImportantly, the adoption of these measures does not rule out the officially recommended procedures. In 2016, the United States Department of Agriculture (USDA) released a list of recommendations, procedures, and responsibilities for the management of diagnosed or suspected cases of animal vesicular diseases to ensure that investigations into exotic animal diseases occur properly, such that precautions can be taken to prevent the spread of communicable diseases . In BrazThe studies focusing on the etiological agent and viral infection have considerably expanded the knowledge related to senecavirus and its associated vesicular disease in a short period of time. New study methodologies and tools, particularly the molecular techniques that are notable for the speed, specificity, and sensitivity of their results, have been quickly developed and evaluated. In these studies, some features of senecavirus infection were elucidated in adult animals, and infection in neonates was also described, providing important information on the epidemiology of senecavirus infection, the likely forms of transmission, the pathogenesis, clinical signs, anatomopathological changes, immunity, and a number of suggestions for prophylaxis and control strategies. However, many questions remain to be answered regarding the (i) specific features of the virus, including its biological and molecular evolution, pH stability, receptors and co-receptors, cellular macromolecules, replication cycle, environmental survival; (ii) senecavirus epidemiology, such as how the virus has been disseminated to other countries, non-swine species reservoirs and/or vectors, likely through vertical transmission; (iii) pathogenicity in piglets; and (iv) host immunological response, including the protective antibody titers in colostrum.Currently, the swine industry, specifically the pig farming sector, is increasingly challenged in terms of animal health and biosecurity. Emerging viral diseases in swine have significantly increased in the past two decades in the global swine population and pose risks to animal health and, consequently, to the production chain . Good exEqually important is the capability of the country to detect and diagnose the disease. The rapid establishment of the etiological agent of a disease enables the rapid adoption of control measures, mitigating and/or preventing the local, regional, and/or international dissemination of the infectious agent. Despite the technological advances in the diagnostic field, weaknesses in the veterinary infrastructure of many countries make disease control even more difficult .


Despite the progress that has been made in research on autism spectrum disorders (ASD), the understanding of the biological basis of ASD to identify targets for novel, effective treatment remains limited. One of the leading biological theories of autism is a model of cortical hyperexcitability. While numerous genetic and epigenetic studies support this model, how this particular biological alteration relates to known phenotypes in ASD is not well established. Using examples of sensory processing alterations, this review illustrates how cortical excitability may affect neural processes to result eventually in some core clinical phenotypes in ASD. Applications of the cortical excitability model for translational research and drug development are also discussed. Frequent seizures and sensory hyperreactivity in autism spectrum disorders (ASD) suggest cortical hyperexcitability in this population. Accordingly, an elevation in cortical excitability has been proposed to be a fundamental neurobiological characteristic for many patients with ASD ,2. WhileSeveral lines of genetic and epigenetic evidence suggest an elevation in cortical excitability in ASD by documenting gamma-aminobutyric acid (GABA) ,6,7,8,9 Second, the reduced expression of GABA-related genes, such as glutamic acid decarboxylase (GAD) 65 and GAD67 proteins has been observed in the cerebellum and parietal cortex in histological studies of ASD ,20 and pFourth, macrolevel analyses using magnetic resonance spectroscopy (MRS) are also consistent with elevated excitability in ASD. Lower GABA concentrations have been reported in the motor , somatosWhile there is increasingly robust evidence for an elevation in cortical excitability in ASD, cortical excitability has many mechanisms to affect cortical networks, via changes in synaptic formation, neural plasticity, and neural communication ,31,32. TOne of the best-documented impacts of increased cortical excitability is on neural communication within brain networks. Control of excitability affects neural activity in both phasic and tonic fashions ,33. The Past electroencephalogram (EEG) studies have shown reduced neural oscillation in ASD ,41,42, w(BOLD) responses during MRI sensory studies, and this is not accounted for by general signal-to-noise ratio differences [Reduced temporal control over neural activity also predicts more variable neural response timing across trials. Many neurons need to fire in synchrony to produce large enough physiological signal changes to be detected by techniques such as EEG and magnetic resonance imaging (MRI). Reduced neural synchrony results in more variable responses when the system is repeatedly stimulated. ASD participants are known to have greater intertrial variability in Blood-oxygen-level dependent ferences ,50. Simiferences .Reduced temporal control over the timing of neural activity is also likely to have effects on behavioral phenotypes in ASD. Because conscious perception of stimuli depends on the duration of cortical sensory responses ,53, precIn addition to the fast phasic influence, cortical excitability can affect gain control of pyramidal cell activity in a more tonic fashion. This occurs through the generation of inhibitory currents via drive from extrasynaptic GABA receptors, which are located on soma, dendrites, and axons that are distant from synaptic neurotransmitter release sites ,32. ThesWe have shown that abnormal response scaling under visual stimulus contrast manipulation is present in ASD participants who are seizure-free . We measAdditional evidence indicating the difficulty in regulating neural output includes enhanced activation in the visual cortex in functional MRI (fMRI) studies, where suprathreshold, high-contrast stimuli were used ,65. EnhaPsychophysical studies in ASD are consistent with a disproportionately elevated neural scaling when a wide range of suprathreshold stimuli are used. Greater sensitivity to middle- to high-intensity stimuli has been documented by lower thresholds for blink startle responses to loud sounds ,67 and cProblems with the dysregulation of neural response amplitude may be exacerbated when the sensory system is repeatedly stimulated. Repeated stimulation typically results in habituation. Habituation, or repetition suppression, refers to a reduction of neural activity when stimuli are repeated. This is an important neural mechanism to differentiate “old” and “new” stimuli to code history with the particular stimulus. ASD individuals are known to show less neural habituation , or evenAlthough habituation is a complex phenomenon that is probably related to both perceptual and postperceptual processes, reduced habituation could contribute to both hyper- and hyposensitivity to sensory events that are observed in ASD . ReducedASD individuals also show reduced adaptation ,74,75,76Sensory adaptation has been well studied in the context of visual motion perception, and we have shown that sensory adaptation to visual motion is reduced in ASD . In a tySimilar principles based on disruption in mutual inhibition apply to adaptation to other stimulus features, such as orientation ,90,91, aWe have discussed how cortical excitability influences neural synchronization, output scaling, habituation, and adaptation, and how these neural processes are altered in ASD. These neural changes are consistent with documented perceptual changes in ASD and are consistent with sensory abnormalities observed clinically. It is worth noting that alterations in cortical excitability seem to exist to varying degrees in individuals with ASD, and only a subset of affected individuals may have neuronal hyperreactivity ,30,63. IIn addition to individual differences in overall levels of cortical excitability, further examining specific aspects of cortical excitability may clarify which potential neural mechanisms contribute to cortical hyperexcitability in ASD and possibly inform us about biological heterogeneity in the disorder. Different aspects of excitability control can result from distinct genetic and epigenetic factors, as well as atypical developmental trajectories ,32. For It has been shown that many preclinical models of ASD have abnormal sensory processing and altered balances for excitation and inhibition. The similar elevation of cortical excitability to the human data warrants well-controlled cross-species studies using identical tasks to seek parallel neurophysiological characteristics, such as changes in neural synchronization and neural output scaling. In particular, EEG and event related potentials (ERP) provide excellent tools for the investigation because homologues of human ERPs and oscillatory activity have been identified in rodents. This provides a particularly strong framework to link findings from rodent models to studies of human diseases and to develop novel translational research programs. ERP and oscillatory activity have a high reliability, which is suited for such investigations ,97,98,99Elevation in cortical excitability is observed in ASD at genetic, epigenetic, neural, and behavioral levels. While changes in cortical excitability affect general cognitive function, cortical excitability has especially profound effects on sensory phenotypes in ASD. The effect on sensory phenotypes is complex, affecting sensory processes through multiple different neural processes, which may contribute to the heterogeneous expression of symptoms. Despite the complex effect, close homologues in human and animal models offer great promise for developing models of cortical excitability and their neural mechanisms and creating novel therapeutic strategies. When combined with the ability to capture individual differences, these measures of cortical excitability have high potential for predicting and evaluating therapeutic outcomes to such therapies.


Tobamovirus genus. We confirmed Koch’s postulates for the disease followed by partial host range determination and revealed that tomato cultivars certified to harbor the Tm-22 resistance gene are susceptible to the new viral disease. We further characterized the viral source of the disease using a range of antisera for serological detection and analyzed various virus genera and families for cross-reactivity with the virus. In addition, next generation sequencing of total small RNA was performed on two cultivars grown in two different locations. In samples collected from commercial cultivars across Israel, we found a single virus that caused the disease. The complete genome sequence of the new Israeli tobamovirus showed high sequence identity to the Jordanian isolate of tomato brown rugose fruit virus.An outbreak of a new disease infecting tomatoes occurred in October-November 2014 at the Ohad village in Southern Israel. Symptomatic plants showed a mosaic pattern on leaves accompanied occasionally by narrowing of leaves and yellow spotted fruit. The disease spread mechanically and rapidly reminiscent of tobamovirus infection. Epidemiological studies showed the spread of the disease in various growing areas, in the South and towards the Southeast and Northern parts of the country within a year. Transmission electron microscope (TEM) analysis showed a single rod-like form characteristic to the  Solanum lycopersicum) with edible fruit are important vegetables in the world diet [Tobamoviruses, Potexviruses and Pospiviroids. According to the 2015 release of the International Committee on Taxonomy of Viruses (ICTV) http://www.ictvonline.org/virustaxonomy.asp, the Tobamovirus genus is the largest genus (35 species) among the seven genera in the family Virgaviridae. The Tobamovirus genus includes the well-known species the type member Tobacco mosaic virus (TMV) [Tomato mosaic virus (ToMV) as well as Tobacco mild green mosaic virus (TMGMV) and Pepper mild mottle virus (PMMoV) among the viruses capable of infecting Solanaceae crops [Tobamoviruses are characterized by a typical rod-shaped particle morphology encapsulating a single stranded RNA (+ssRNA) sense genome of 6.2 to 6.4kb encoding four ORFs. ORF1 and ORF2 are separated by a leaky stop codon and encode non-structural proteins that form the replicase complex. ORF3 on the large subgenomic RNA encodes the non-structural movement protein (MP). ORF4 on the small subgenomic RNA encodes the coat protein (CP) of 17 to 18 kDa. Tobamoviruses are transmitted by mechanical contact: through workers' hands, clothes, tools, and are capable to preserve infectivity in seeds and contaminated soil [Tm-2 and Tm-22 (Tm-2a) respectively [Tm-2 and Tm-22 resistances share the viral MP as the Avirulence protein (Avr). However, different domains in the MP and different protein structure requirements are necessary for each resistance [Tm-22 resistance has been more durable than the Tm-2, which has been broken [Tm-22 resistance rises since new tobamoviruses infecting tomatoes were identified. In Mexico, a tobamovirus named Tomato mottle mosaic virus (ToMMV) [Tobamovirus genus.Tomato plants  . ToMMV cSymptomatic tomato fruit and leaves were collected from infected symptomatic plants. Virions were purified from 100 g of symptomatic plants, as described previously . Leaf-diTm-22 resistance gene. Symptomatic tomato plants from the initial experiments served for sap-mechanical inoculation assays. Tomato fruit and leaves were ground in 0.01 M phosphate buffer (pH 7.0) and inoculated to a variety of potential host plants (pre-dusted with carborundum) for extended host range determination. Host range testing was performed in three sets of experiments (> four plants per species). Inoculation of a range of commercial tomato varieties harboring the Tm-22 resistance gene, as certified by the 'Tomato Genetic Resource Center' (TGRC) http://tgrc.ucdavis.edu/Data/Acc/dataframe.aspx?start=AccSearch.aspx&navstart=nav.html, was performed with 8 replicates.Purified viral particles served for mechanical inoculation of laboratory test plants and tomato plants harboring the Tm-22 resistance gene) cv’s: Ikram, Odelia, Bruno, Olympiacos, Magi, Tory which served as propagation hosts. In addition to the registered resistance to ToMV by the seed-companies, the authenticity of the presence of Tm-22 was further confirmed by DNA sequencing of the resistance gene in Odelia and Ikram cultivars. All virus-infected plants were kept in an insect-proof growth chamber inside a glasshouse and sprayed regularly with insecticides to prevent any infestation.The virus was maintained on systemically infected tomato plants . Pre-immune blood was collected from two New Zealand white rabbits three months old. Native purified virus preparation (with no SDS or boiling) was diluted with saline, combined with complete Freund’s adjuvant was injected subcutaneously to the rabbits to create enhanced immune response. Three sequential injections followed, at 21 day intervals in which the antigen was mixed with incomplete Freund’s adjuvant. Ten to thirteen days following the fourth injection the rabbits were bled and sera (40 ml) was separated from blood cells and served for evaluation by direct ELISA and Western blot analysis prior to IgG purification.Direct and Double Antibody Sandwich (DAS) ELISA were performed on various plant preparations as previously described  using laTomato spotted wilt virus (TSWV) was performed using TSWV specific antibodies in a commercial kit (Agdia). The optical density (O.D) readings of hydrolyzed substrate of Alkaline-Phosphatase (Sigma) were measured at 405 nm.The diagnosis of For RNA extraction, purified viral preparations served as the source material as described previously  with mod5’-CAATCCTTGATGTGTTTAGCAC-3’ reverse complement primer was used for the RT reaction. The resulting cDNA was amplified by PCR using Taq DNA polymerase JMR PCR mix (JMR Holdings) and the designed general primer set F-3666: 5’-ATGGTACGAACGGCGGCAG-3’ and R-4718. Additional primer sets that were used for genome amplification for validation of the NGS obtained contigs are described in 5’…GATCATGTCTAAGGTTAAGC…3’ followed by PCR amplification with the supplement of the forward primer F-Tswv-NC-1980 5’…CAGCTGCTTTCAAGCAAGTTC…3’. The resulting amplicons were cloned into pGEM-T-easy vector and sequenced in both orientations. Sequence homology was determined using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) algorithms.For the RT reaction viral RNA served as a template using the Maxima Reverse Transcriptase cDNA kit (Thermo Fisher Scientific). General tobamovirus primer sets were designed according to consensus sequences that we identified from complete genome sequences of tomato-infecting tobamoviruses from the GenBank. The R-4718: For NGS analysis, two different symptomatic tomato plants samples positively detected by ELISA, western blots and RT-PCR were selected. The first sample of cv. Mose exhibited the typical disease symptoms, was collected in October 2014 from Ohad village in Southern Israel. The second sample of cv. Odelia, which exhibited distinct disease symptoms, was collected in May 2016 from Sde-Nitzan village also in Southern Israel. Symptomatic samples were subjected to total RNA extraction using TRI reagent (Sigma-Aldrich), followed by mirVana miR Isolation kit (Invitrogen) for enrichment of small RNA molecules. The obtained small RNA fraction was used to construct a small RNA library using the TruSeq Small RNA Sample Preparation kit (Illumina). Library fragment size and quality of sample purification were analyzed using the Agilent 2100 Bioanalyzer (Agilent Technologies). Size selection of the complete library was performed using E-Gel EX 4% agarose gels (Invitrogen/Life Technologies). The library was sequenced using the MiSeq Sequencer . Phylogenetic tree analysis was carried out for concatenated ORF amino acid sequences. First, the MAFFT program was used to align nucleotides or ORF . Then, ahttps://github.com/dzerbino/velvet/tree/master/contrib/AssemblyAssembler1.3). Generated contigs were then BLASTed using blastx algorithm against the non-redundant NCBI protein database .The 3' adapter sequence was removed and sequences were filtered for size selection. The size range selected was between 19 and 30 bp in length using the software UEA sRNA Workbench version 2.5 . After sThe 5’- and 3’-UTR sequences of the viral genome were obtained using a RACE kit (Invitrogen) according to the manufacturer’s protocol, on a template of viral RNA extracted from purified virus preparations. The RACE procedure was repeated three times on different virus preparations to ascertain the accuracy of the UTRs.Nicotiana tabacum cultivars (bioassay) (The Israeli "Plant Protection and Inspection Services” (PPIS) survey was conducted in February 2015. Sampling for disease detection was executed on a third of each farmer's growing area up to three structures per grower. From each structure, thirty sub-samples were collected and divided into three pools of ten samples each for ELISA. Selected ELISA-negative and all the ELISA-positive samples were tested by RT-PCR amplification followed by amplicon sequencing for validation. In addition, for selected samples, negative and positive ELISA and RT-PCR analysis were further tested by inoculation of susceptible ioassay) .Tobamovirus genus was observed  of a single farm at Ohad village in Southern Israel. The disease symptoms were mild and severe mosaic of the leaves with occasional narrowing of the leaves. Yellow spotted fruit that amounted to 10 to 15% of the total fruit occurred on symptomatic plants . The natobserved . In viraobserved  and 18nmobserved . SDS-PAGobserved .Tm-22 resistance (Tm-22 resistance as certified by the Tomato Genetic Resource Center (TGRC)  developed on the inoculated leaves at 4 dpi, and then at 7 dpi leaf yellowing symptoms occurred, followed by leaf fall at 9–12 dpi  and Chenopodium murale were identified as potential reservoirs for the virus. S. nigrum was asymptomatic for virus infection while C. murale shows a hypersensitive response (HR) by necrotic leaf yellowing followed by local necrotic lesions prior to symptomless systemic infection.Partial host range analysis of the new Israeli tobamovirus isolate (the details of virus identification are below) was carried out in two steps. At first, viral particles purified from the source tomato plants were inoculated to healthy tomato plants cv. Ikram and tobacco plants. Secondly, the inoculated tomato plants served for sap-mechanical inoculation of additional commercial tomato varieties carrying the sistance  and of lsistance . In all r (TGRC) . Variati9–12 dpi . When pe9–12 dpi . In petu9–12 dpi . Local a as well . The S. At 21 dpi newly emerged leaf samples were collected from inoculated host plants and subjected to ELISA for virus detection. The susceptibility of the hosts to the virus inoculation was analyzed again at 30 dpi . LaboratTm-22 resistance were susceptible to virus inoculation  and Western blot analyses . In ELISA the average O.D readings following 20 minutes of substrate hydrolysis (color development) at room temperature were 0.7 ±0.2 O.D which is >50 times the negative control samples that were 0.015 ± 0.02 O.D. In Western blot, the antiserum allowed specific identification of a ~17.5 kDa viral CP in extracts of infected plants, characteristic of tobamoviruses . AnalysiSmall RNA was extracted from symptomatic leaf samples of two different tomato cultivars collected from two separate locations in the Bsor area (Southern Israel). cv. Mose samples were collected in October 2014 from Ohad village. In May 2016, in Sde-Nitzan village, samples of cv. Odelia demonstrating unique fruit symptoms were collected  comparedde-novo assembly of sRNA raw data resulting in two large assembled contigs: contig 1 (928 nt), and contig 2  demonstrating the highest nucleotide sequence identity , and amino acid sequence identity  to the TMV Ohio strain complete genome (accession number FR878069.1). Notably, even though there were significant differences in disease symptoms between the tomato cvs. Mose and Odelia , a total of 2,594,213 and 148,765 reads respectively remained; 51,414 and 9,630 of these were viral. The reads were mapped on the TMV genome (accession number EF392659.1), yielding 46% and 28% coverage of the TMV viral genome respectively, excluding the 5' and 3' ends. The distribution of the sRNA along the assembled and validated genome of the new tobamovirus isolate   . This prT383474) .The complete genome sequence of the new Israeli isolate comprised of 6,392 nucleotides was submitted to GenBank (accession no. KX619418). This genome sequence includes putative four ORFs of 1117, 474, 267 and 160 amino acids and two untranslated regions (UTRs) at the 5’ and 3’ ends of the viral genome . The 5’ The outbreak of the new tomato tobamovirus disease was an isolated event that was not treated, while detection of healthy tomato plants was received from various areas in the country. According to official inspectors of 'The agricultural extension services of Israel' there were no reports of disease symptoms prior to the September-October outbreak. However, in February 2015, in the four months following the outbreak, the disease spread to new tomato growing areas in the South of Israel , probably through the practice of visiting agronomists and professional inspectors or by transfer of non-tested contaminated seeds or seedlings. An official survey conducted by the Israeli PPIS started in February 2015 showed the isolated occurrence of the disease in the Bsor area in Southern Israel. While the remote areas of Ramat Negev and the Arava valley were detected negative for the virus . At thisAfter 7 months, the disease spread to the Ramat Negev region, where growers specialize in the tomato cherry varieties . Later oTm-1, Tm-2 and Tm-22 [Tm-22 was introduced by introgression from L. peruvianum to L. esculentum [Tm-22 resistance would reduce viral virulence [Tm-22 resistant tomato plants, which occurred from October to November 2014 at Ohad village in Southern Israel. An outbreak of tobamiviruses requires immediate and full response at a very early stage, which includes quarantine of the contaminated area to prevent the disease from spreading. Successful eradication of new pathogen is challenged with limited success, although it may postpone disease establishment [Tomato plants are grown worldwide in open fields, which often expose the plants to insect pests and vectors of plant viruses –32. Grownd Tm-22 . Tm-22 wculentum . Althougirulence . Here welishment , 37. UnfTm-22 resistance-conferring allele against ToMV in tomato plants has been jeopardized by the newly discovered tobamovirus isolate. Interestingly, the observed length range of the virus particles is wide, reminiscent of ToMV-R [Hibiscus latent fort pierce virus (HLFPV) [It is quite clear now that the durability of the f ToMV-R  and Hibi (HLFPV) . While sRehmannia mosaic virus (ReMV)[S. lycopersicum cv. Candela, which resemble the unique symptoms described above for the 'Odelia' variety. Phylogenetic analysis showed that the Jordanian isolate originated from a branch leading to TMV clade [ToMMV that causes tissue necrosis on leaves of tomato seedlings and mosaic and leaf distortion of mature plants has been reported in Brazil  and Mexius (ReMV). The latus (ReMV), 42. Folus (ReMV)–45, on pus (ReMV) and on tus (ReMV) and in Sus (ReMV). In contus (ReMV), 50. In us (ReMV). This neMV clade . The higSolanaceae family, under certain circumstances the Israeli isolate is capable to infect pepper plants harboring the L1,3,4 resistance genes . N. tabacum cv. Rustica. (B).N. tabacum cv. Samsun.Local lesions developed on (TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(A-B) Distribution of viral particles lengths as imaged by TEM showing variability in particle sizes. Arrows indicating larger than 300 nm long particles.(TIF)Click here for additional data file.S4 Fig(D) Necrotic lesions followed by dried apoptotic leaves. (E) HR symptoms developed following root inoculation demonstrating dried spots on stems leading to plant growth inhibition.(TIF)Click here for additional data file.S5 FigTomato mosaic virus ; line 2: Tomato mottle mosaic virus ; line 3: Tomato brown rugose fruit virus ; line 4: Tobacco mosaic virus ; line 5: Israeli isolate of tomato brown rugose fruit virus . Arrows represent the borders of the conserved regions, which served as a template for RT-PCR amplification. A designed general tobamovirus primer set: F-3666(TobGen) and R-4718(TobGen), encompassing the variable nucleotide sequences was used for species identification followed by amplicon sequencing using Sanger analysis.Line 1: (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Merkel cell polyomavirus is the primary etiological agent of the aggressive skin cancer Merkel cell carcinoma (MCC). Recent studies have revealed that UV radiation is the primary mechanism for somatic mutagenesis in nonviral forms of MCC. Here, we analyze the whole transcriptomes and genomes of primary MCC tumors. Our study reveals that virus-associated tumors have minimally altered genomes compared to non-virus-associated tumors, which are dominated by UV-mediated mutations. Although virus-associated tumors contain relatively small mutation burdens, they exhibit a distinct mutation signature with observable transcriptionally biased kataegic events. In addition, viral integration sites overlap focal genome amplifications in virus-associated tumors, suggesting a potential mechanism for these events. Collectively, our studies indicate that Merkel cell polyomavirus is capable of hijacking cellular processes and driving tumorigenesis to the same severity as tens of thousands of somatic genome alterations. A variety of mutagenic processes that shape the evolution of tumors are critical determinants of disease outcome. Here, we sequenced the entire genome of virus-positive and virus-negative primary Merkel cell carcinomas (MCCs), revealing distinct mutation spectra and corresponding expression profiles. Our studies highlight the strong effect that Merkel cell polyomavirus has on the divergent development of viral MCC compared to the somatic alterations that typically drive nonviral tumorigenesis. A more comprehensive understanding of the distinct mutagenic processes operative in viral and nonviral MCCs has implications for the effective treatment of these tumors. MCPy2–APOBEC3B , 7. AnotAPOBEC3B –12. To dHigh-throughput sequencing has been highly beneficial to many fields, including cancer biology and virology. Sequencing shows individual mutations as well as mutation patterns or signatures, which implicate distinct mutational processes acting within tumors over time. These processes are responsible for intratumoral genetic heterogeneity and provide the necessary substrate for evolution, survival, and metastasis –16. Addi–To determine if a tumor expressed viral genes, transcriptome sequencing (RNA-seq) reads obtained from 6 MCC tumor specimens were aligned to a reference containing both the human (hg19) and MCPyV (NCBI) genomes . Merkel We performed high-coverage (~100×) whole-genome sequencing of two virus-positive MCC tumors and one virus-negative MCC tumor and analyzed somatic mutations, copy number variants (CNVs), and structural rearrangements compared to the normal somatic DNA isolated from peripheral blood mononuclear cells PBMC) isolated from the corresponding patients. What was exceptionally striking was that the virus-negative MCC tumor had over 30-fold-more somatic mutations with a total of 127,236 mutations in addition to many copy number alterations and interchromosomal translocations compared to the two virus-positive tumors MC isolat. This mu18–CBFA2T3, CHEK2, FANCC, FLI1, ITPR1, MUC16, NF1, NUTM, PTPRB, PTPRR, SETX, and STK11IP in the virus-negative tumor, which may further promote tumor survival and evolution.By functionally annotating the mutations overlapping genes, the virus-positive tumors had a cumulative total of 12 missense and nonsense mutations targeting genes implicated in cancer as annotated by COSMIC, whereas the virus-negative tumor harbored 51 missense and nonsense mutations targeting COSMIC-annotated genes. Of these 51 mutations, 34 were predicted by either SIFT or PROVEAN to be deleterious to the function of the primary protein product. The effects of these mutations on all potential protein products are detailed in 10.1128/mBio.02079-16.1Table S1 Table S1, XLSX file, 0.02 MB.Functional implications for somatic nonsense and missense mutations detected in MCC tumors. Missense and nonsense mutations targeting genes implicated in cancer as annotated by COSMIC and predicted by either SIFT or PROVEAN to be deleterious to the function of the primary protein product. Download Copyright © 2017 Starrett et al.2017Starrett et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the MDM4 and single-copy deletions of PTEN and SUFU. In stark contrast to the virus-positive tumors, the virus-negative tumor-050 had single-copy amplifications of EGFR and JUN and single-copy deletions of APC, ATM, BIRC, BRCA1, BRCA2, FANCA, FANCD2, CDKN2A, MLH1, PAX5, PBRM1, RB1, and VHL. RB1 function may be absent in sample-050, as there was a somatic G-to-A transition mutation in the remaining allele at position chr13:49047495. This base substitution is predicted to interfere with the splice acceptor for the adjacent exon 20 with the potential to produce a nonfunctional protein. Of the detected interchromosomal translocations in these tumors, none of them reflected known annotated translocations in cancer. To further define and consolidate the impact of the sheer number of somatic alterations in the nonviral tumor, we performed pathway analysis on the abovementioned variants. This analysis predicted significant inhibition of p53, ATM, and BRCA1 signaling and inhibition of DNA damage checkpoint regulation, all of which would contribute to the observed severe genome instability and the ability of the tumor to survive the corresponding stress  signaling was also predicted to be significantly impacted, it was neither activated nor inhibited due to inactivation of ATM and ITPR1 and amplification of EGFR and JUN.The relative abundance of structural variants in each tumor genome mimicked the profiles of the abovementioned somatic mutations. Tumor-088 had no amplifications or deletions corresponding to known copy number variations in cancer. The other virus-positive tumor, tumor-076, had a single-copy amplification of g stress . ActivatRecent studies have highlighted and classified the multitude of mutation processes critical for shaping tumor development and evolution across cancers –23. Upon–At this time, signatures 5 and 16 currently have no known etiologies. However, it was recently reported that the mutations commonly observed in liver cancer, corresponding to signature 5, exhibit a bias for mutations on the nontranscribed strand of genic DNA, termed transcription-coupled damage (TCD) . To addr27–To further characterize the mutational processes in MCC, we evaluated the density and distribution of mutations across the entire genome by calculating intermutational distance (IMD) for each somatic base substitution and plotting the values by position . As antiMSH2 and MLH1, and Fanconi anemia family genes, FANCA and FANCC, suggestive of a potential mechanism for the accumulation of the large amount of somatic mutations identified in the MCPyV-negative genome and the low number of somatic mutations in the MCPyV-positive tumors  software (Qiagen) to study differentially expressed genes. These results indicated that virus-negative MCC tumors were significantly enriched for genes associated with basal cell carcinoma signaling pathways, and many of these genes are associated with the WNT signaling pathway, which is consistent with results inferred previously for MCC from microarray data  31). Ma. Ma31). r growth –34. InteVirus integration has the potential to disrupt or alter the function of genes as well as produce novel fusion transcripts. To identify the integration sites in our virus-positive whole-genome alignments, we used a custom pipeline to discover reads that map to both the host and viral genomes. Tumor-076 revealed two integration sites on chromosome 1 that are approximately 40 kb apart. Discordant read pairs show that these insertional breakpoints are linked to the C-terminal end of large T antigen . Tumor-0A previous publication reported that HPV integrants frequently coincide with focal copy number alterations in cancer cell lines and head and neck squamous cell carcinoma (HNSCC) primary tumors . To detede novo assembled all of the read pairs that mapped to the host and viral genomes into fusion contigs. The reads were remapped to these contigs to identify their original positions from the viral and host genomes . The observed contig indicates a deletion between coordinates 1560 and 2754 of the viral genome, causing a frameshift after V311 that results in a 321-amino-acid (aa) amino-terminal truncation of large T antigen, which is also supported by the integration analysis from the WGS reads. Analysis of tumor-076 , indicating a chimeric transcript antisense to the FLJ46066 gene. The other three chimeric junctions mapped within the ECH1 (at host positions chr19:39307703 and chr19:39307378). The viral detection pipeline resulted in two MCPyV contigs. One contig shows a deletion occurring between coordinates 1330 and 1877 of the viral genome. This results in a frameshift after codon P234, resulting in a 240-aa amino-terminal truncation of large T antigen. The other contig aligns to VP1 and VP2.For tumor-090, the read depth graph shows expression of the full-length large T antigen transcript and truncated variants . One chiumor-146 . One chiBy combining the analyses of point mutations, copy number alterations, structural variants, and viral integration sites of primary MCC tumors at the single-nucleotide-resolution level for both the transcriptome and whole genome, we identified numerous common features and pathways manipulated in virus-associated MCC and distinct features from non-virus-associated MCC. First, the distinct dichotomy between the number of mutations and the mutation signatures of the virus-positive and the virus-negative tumors is surprising, since UV damage has generally been thought to be a significant contributing factor to both types of MCC , 37. AltConversely, viral MCC has a low mutation load and is enriched for signature 5, which has been identified previously in many cancers but best defined in hepatocellular carcinomas (HCCs) . AlthougAPOBEC3B expression and a dominant proportion of APOBEC signature mutations  virus HPV and exhibit high utations , 39–41. tions 39–. This saIt is curious that the continued expression of viral genes in patients appears to associate with the maintenance of the host genome integrity compared to virus-free tumors, considering the ability of MCPyV to integrate into the host genome and the apparent necessity of this event to establish cancer. From the standpoint of the virus, less DNA damage is beneficial for continued proliferation of the host cell and the virus, as integration is not part of the normal viral life cycle and results in a replication-deficient virus. This provides a potential explanation of why MCC is an exceptionally rare cancer despite upward of a 90% prevalence of MCPyV infection in the human population . As seen43–SUFU in our virus-positive tumor-076, which mirrors a recent report that identified an inactivating mutation of SUFU in another MCC tumor. This particular tumor was characterized by an absence of mutations in any of more than 300 cancer-related genes sequenced, which, based on our results and others, suggests that it was also a virus-associated MCC  DNA preparations (10 μg) were sequenced by the Beijing Genome Institute (BGI) on the Complete Genomics platform to an average of 100× depth . Alignme51–q value) of less than 0.05 and a 3-fold or greater change in expression in virus-positive versus virus-negative samples were considered significant. To focus on relevant genes, only genes implicated in cancer by the COSMIC cancer gene census, E2F-regulated genes, and leukocyte-related genes were further characterized . The nature of this analysis indicated that pathways enriched for virus-positive tumors were pathways with the highest positive Z-scores as calculated by IPA and virus-negative tumors were pathways with the lowest negative Z-score; only pathways with an enrichment P value of less than 0.05 were considered statistically significant. Principal-component analysis was performed using the R statistical package with all annotated genes.RNA was purified using the Ultra RNA Library Prep kit (New England BioLabs) and was sequenced  on the Illumina HiSeq 2500 platform with paired-end flow cells and 50 cycles in each direction. Sequences were aligned to a combination of the hg19 and MCPyV genomes (NCBI) using TopHat2 . DiffereFlanking 5′ and 3′ bases at the site of each somatic mutation were collected from the hg19 reference genome. The proportion of each mutation in its trinucleotide context was calculated in respect to the total number of somatic mutations. Mutation profiles were plotted using the R statistical software with the SomaticSignatures package . This paMutation clusters (kataegis) were evaluated by taking the distance in base pairs from one somatic single-base substitution to the previous mutation or intermutational distance (IMD). The genomic distributions of mutations were plotted using ggplot2. Clusters of mutations were determined by the same method as that of Alexandrov and colleagues , which tde novo assembled using Velvet with a word size of 11 bp  and coloring them by origin  with ggplot2.Half-mapped read pairs were extracted from the whole-genome alignments using a custom script. Due to the variable, gapped structure of Complete Genomics reads, we used only the 20-bp continuous segment located at the beginning of the read . These rof 11 bp . The disof 11 bp . Using tHM355825.1) and human hg19 genomes. Next, all unmapped reads were input into BLASTN with parameters “-word_size 16” and “-outfmt 6” and compared with the Merkel cell polyomavirus genome. Then, all reads with a BLASTN hit to the viral genome were run through BLASTN against the hg19 genome, using the same parameters. Finally, SummonChimera was run with the BLASTN and SAM report files and generated a report containing all detected chimeric junctions.Identification of viral integration sites follows the pipeline suggested by the SummonChimera  softwarehttp://prinseq.sourceforge.net/). High-quality reads were assembled with CLC Assembler. Contigs of ≥500 bp were masked with Repeat Masker and filtered as described previously (Raw fastq reads were mapped to the hg19 version of the human genome and a human mRNA database (to remove spliced reads) with Bowtie2 using default parameters . Then, ueviously . Then, h


Costs of reproduction are expected to be ubiquitous in wild animal populations and understanding the drivers of variation in these costs is an important aspect of life-history evolution theory. We use a 43 year dataset from a wild population of red deer to examine the relative importance of two factors that influence the costs of reproduction to mothers, and to test whether these costs vary with changing ecological conditions. Like previous studies, our analyses indicate fitness costs of lactation: mothers whose calves survived the summer subsequently showed lower survival and fecundity than those whose calves died soon after birth, accounting for 5% and 14% of the variation in mothers' survival and fecundity, respectively. The production of a male calf depressed maternal survival and fecundity more than production of a female, but accounted for less than 1% of the variation in either fitness component. There was no evidence for any change in the effect of calf survival or sex with increasing population density. The eneLong-term, individual-based studies provide an excellent opportunity to explore the costs of reproduction in wild animals ,9,10. He2.The unmanaged population of red deer in the North Block of the Isle of Rum, Scotland, has been studied since 1971, with survival and reproductive history of individuals known from regular censuses ,11. Feman = 2888 observations of 636 females), and fecundity by whether she gave birth to a calf the following year, conditional on her survival . Calf sex and calf survival to 1 October of the year they were born were included in the models as 2-level fixed factors. Maternal age (linear and quadratic), population size  and calf birth date (days since 1 May) were included as fixed covariates. Year was fitted as a random multi-level factor in both models, and maternal identity was fitted as a random term in the fecundity model . We tested for differential costs of male and female calves depending on whether they survived to 1 October by including an interaction between calf sex and calf survival in each model, and for changes in costs across varying population densities by including an interaction between either variable and population size.We examined the effects of calf survival and calf sex in a given year on the subsequent survival and fecundity of the mother. Maternal survival was assessed as survival to 1 May the following year . Marginal R2 indicates the percentage of variance explained by the fixed effect component of a model, and can be estimated for GLMMs as the variance of the fixed effects divided by the total variance (×100), calculated on the link scale [R2 (ΔR2) when each fixed effect was dropped from the model in turn to estimate the variance in maternal survival and fecundity explained by each of the fixed effects.Maternal survival and fecundity were modelled as binary traits in Bayesian generalized linear mixed models (GLMMs) with the R package MCMCglmm , using tnk scale . We used3.a; PMCMC < 0.001) or to breed again the following year  than mothers whose calves died during the course of the summer; this cost explained a substantial proportion of variation in maternal survival and fecundity , and less likely to give birth the following spring if they did survive . However, calf sex explained less than 1% of the variation both in survival probability and fecundity , there is no indication that the costs associated with calf survival or sex varied with increasing density . A possible explanation of this effect is that maternal investment in lactation is adjusted to the mother's food intake so that variation in food availability affects the growth and survival of calves rather than the survival and fecundity of their mothers [We found evidence of density-dependence in maternal survival and fecundity . However mothers .Several other studies of sexually dimorphic mammals have shown that mothers invest more energy in sons than daughters  but relaIn summary, we found considerable costs of successful reproduction for female red deer in terms of future survival and fecundity. We found evidence for a significantly higher cost of sons than daughters, although this difference was smaller than the effects of juvenile survival. Despite density-dependence in both aspects of maternal performance, we found no indication that reproductive costs varied with ecological conditions. Our analyses illustrate the value of long-term datasets in affording tests of the generality of life-history patterns across changing environments.


Secoviridae are non-enveloped viruses withmono- or bipartite (RNA-1 and RNA-2) linear positive-sense ssRNA genomes withthe size of the RNAs combined ranging from 9 to 13.7 kb.They are related to picornaviruses and are classified in the orderPicornavirales. The majority of known members infectdicotyledonous plants and many are important plant pathogens . This is a summary of the currentInternational Committee on Taxonomy of Viruses (ICTV) report on the taxonomy ofthe family Secoviridae available at www.ictv.global/report/secoviridae.Members of the family  NTP, nucleotide triphosphate.Virions are non-enveloped, 25–30 nm in diameter and exhibit icosahedralsymmetry . Many viThe genome consists of one or two molecules of linear positive-sense ssRNA that arecovalently linked to a small protein  at their 5′ end and have a 3′-terminal poly(A)tract. Each RNA encodes, in the majority of the cases, a single polyprotein .In the case of viruses with a bipartite genome, neither RNA species alone can infectplants systemically. Viral proteins are usually expressed as large polyproteins,which are cleaved by virus-encoded 3C-like proteinases. The replication blockcontains the domain characteristics of nucleoside triphosphate (NTP)-bindingproteins (NTB or putative helicase), 3C-like proteinases (Pro) and RNA-dependent RNApolymerases (Pol) . ReplicaComovirinae). Comoviruses usuallyhave narrow host ranges. Mosaic and mottle symptoms are characteristic.Transmission in nature is exclusively by beetles, especially members of thefamily Chrysomelidae. Beetles retain their ability to transmit virus for days orweeks [Bipartite genome (subfamily orweeks .Comovirinae). Fabaviruses have widehost ranges among dicotyledonous and some families of monocotyledonous plants.Symptoms are ringspots, mottling and wilting. In nature, they are transmitted byaphids in a non-persistent manner.Bipartite genome (subfamily Comovirinae). The genus consists of>35 species that are widely distributed in temperate regions. Ringspotsymptoms are characteristic. Many nepoviruses are transmitted non-persistentlyby longidorid nematodes. Seed and/or pollen transmission are also common. Inherbaceous plants, the symptoms induced are often transient with a so-called‘recovery’ phenomenon. The genus can be divided into subgroups  based on sequence and genome organization [Bipartite genome .Monopartite genome. The natural host range of waikaviruses is usually restrictedto plants within a few families. Field transmission is semi-persistent by aphidsor leafhoppers. Some waikaviruses are helper viruses for the insect transmissionof other viruses; for example, rice tungro spherical virus is the helper virusfor rice tungro bacilliform virus  Report:


Rhabdoviridae, genus Ledantevirus) in nycteribiid bat flies infesting pteropodid bats in western Uganda. Mitochondrial DNA analyses revealed that both the bat flies and their bat hosts belong to putative new species. The coding-complete genome of the new virus, named Kanyawara virus (KYAV), is only distantly related to that of its closest known relative, Mount Elgon bat virus, and was found at high titers in bat flies but not in blood or on mucosal surfaces of host bats. Viral genome analysis indicates unusually low CpG dinucleotide depletion in KYAV compared to other ledanteviruses and rhabdovirus groups, with KYAV displaying values similar to rhabdoviruses of arthropods. Our findings highlight the possibility of a yet-to-be-discovered diversity of potentially pathogenic viruses in bat ectoparasites.Bats are natural reservoir hosts of highly virulent pathogens such as Marburg virus, Nipah virus, and SARS coronavirus. However, little is known about the role of bat ectoparasites in transmitting and maintaining such viruses. The intricate relationship between bats and their ectoparasites suggests that ectoparasites might serve as viral vectors, but evidence to date is scant. Bat flies, in particular, are highly specialized obligate hematophagous ectoparasites that incidentally bite humans. Using next-generation sequencing, we discovered a novel ledantevirus (mononegaviral family  Both megabats and microbats host numerous, taxonomically diverse viruses. Examples of megabat-borne viruses that are highly virulent for humans are Marburg virus and Nipah virus. Severe acute respiratory syndrome coronavirus is an infamous example of a microbat-transmitted human pathogen1. Consequently, characterization of bats, their viromes, and cross-species transmission of bat-borne viruses have become research priorities.Bats (order Chiroptera) represent the second largest order of mammals after rodents (order Rodentia). Classically, bats are divided into two suborders: megabats (Megachiroptera) and microbats (Microchiroptera). Megabats, also referred to as fruit bats, are assigned to a single family, Pteropidae, whereas microbats are taxonomically more diverseMuch less effort has thus far been invested in understanding the role of bat ectoparasites in maintaining viruses in bat populations or potentially transmitting them to humans or mammals of other species. The high degree of specialization and diversity of certain bat ectoparasites suggests that they could, in fact, be reservoirs for certain viruses, maintaining them in their bat hosts. Alternatively, bats could be refractory to infection with ectoparasite viruses, but nevertheless these viruses could be infectious or even pathogenic for other mammals, including humans, and be transmitted through incidental bat ectoparasite bites.3. Bat flies of each family have evolved exquisite morphological and behavioral adaptations to life on bats, reflecting a long history of co-evolution2. Bat flies host a diverse community of bacteria, including bartonellae, some of which are zoonotic5. Bat flies also vector hemosporidian parasites (Plasmodiidae: Polychromophilus melaniferus) that cause “bat malaria”6. On the other hand, evidence for a role of bat flies as reservoirs or vectors of viruses is scant. Only two viruses have been unambiguously identified in bat flies: the putative orthoreovirus Mahlapitsi virus and the putative orthobunyavirus Wolkberg virus, which were both found in the nycteribiid Eucampsipoda africana on pteropodid Egyptian rousettes (Rousettus aegyptiacus)8. In addition, rhabdovirus RNA-like sequences were detected in nycteribiids and bats in Spain, but the sequences were too short (108 nt) to unambiguously substantiate virus infection9.Bat flies are eyeless, wingless, hematophagous dipteran insects (Brachycera: Muscomorpha: Hippoboscoidea) that are obligate bat ectoparasites with off-host breeding life stages. They are assigned to two families, the monophyletic Nycteribiidae and the probably paraphyletic Streblidae, and they infest bats throughout the Old and New WorldsHere, we report the discovery and coding-complete genome sequence of a novel rhabdovirus, Kanyawara virus (KYAV), in a previously unknown nycteribiid bat fly collected from an unclassified megabat in western Uganda. Phylogenetic and genomic analyses of KYAV and its relatives offer new insights into the evolutionary and ecological associations of rhabdoviruses with both bats and arthropods.6 to 1.59 × 106 reads per sample. After quality trimming, rhabdovirus-like sequences were detected in five bat flies, each from a different bat. These sequences mapped with low similarity to conserved regions of rhabdovirus genomes . De novo assembly yielded a contiguous sequence of 10,843 nt in one bat fly sample (MPK004), with five open reading frames matching the canonical rhabdovirus genome organization  of bat flies yielded 0.11 × 106 to 2.97 × 106 reads per sample, but no reads mapped to the detected rhabdovirus genome. Application of this method at this sequencing depth is approximately as sensitive as real-time quantitative PCR11; therefore, bat sera could confidently be classified as negative for the virus. For further confirmation, however, we also tested all bat sera by PCR, and results were congruent with NGS results . Oral and urogenital swab samples from all bats also tested negative for the new rhabdovirus by PCR.Sequencing of sera from the bats on which the bat flies were found yielded 1.17 × 10Ledantevirus13. We named this virus Kanyawara virus (KYAV) after the village closest to the roost from which the bats were sampled. Sequence similarity between KYAV and other ledanteviruses based on concatenated, codon-based alignments of the canonical N, P, G, M, and L genes ranged from 62.4% (Mount Elgon bat virus) to 47.6% (Yngjiā tick virus 2) at the nucleotide level and from 59.3% (Mount Elgon bat virus) to 38.7% (Kern Canyon virus) at the deduced amino acid level, respectively. KYAV fulfills four of the five criteria of the International Committee on Taxonomy of Viruses (ICTV) Rhabdoviridae Study Group for classification in the genus Ledantevirus: A) the deduced amino acid sequence of the KYAV RNA-dependent RNA polymerase (L) diverges >7% from that of other ledanteviruses (KYAV:Mount Elgon bat virus = 35.2%); B) the deduced amino acid sequence of the KYAV glycoprotein (G) diverges 15% from that of other ledanteviruses (KYAV:Mount Elgon bat virus = 49.0%); C) KYAV has the same genome organization as other ledanteviruses , with low relative CpG depletion in sigmaviruses, vesiculoviruses, and the Sandjimba virus group accounting for this trend ; these values were comparable to values for the insect-specific sigmaviruses . The coefficient of variation in CpG depletion was lowest for sigmaviruses and lyssaviruses and notably higher for the other virus groups  as are the cyclopdiine bat flies of the species Eucampsiopoda inermis and E. sundaica  and approximately as divergent from those bats as are bats of other species pairs within the genus Myonycteris  are vectored by aphids, leafhoppers, or plant hoppers, and even fish rhabdoviruses transmitted directly through water may have associations with arthropods27. Rhabdovirus genome fragment integration into genomes of arthropods belonging to widely divergent lineages also supports a long history of rhabdovirus-arthropod coevolution28.Viruses of the family et al. searched for the vector of Kumasi rhabdovirus by trapping 1,240 female mosquitoes of six genera close to a large transient breeding colony of African straw-colored fruit bats (Eidolon helvum) in Ghana. No infected mosquitoes were identified29.Despite this family-wide dual adaptation to arthropods and bats, vector-borne transmission of bat-associated rhabdoviruses has proven difficult to confirm. For example, Binger Ledantevirus, sorting within subgroup B, which contains bat-associated viruses  bat flies sampled is consistent with this notion because infection rates of arthropod vectors with vector-borne viruses tend to be much lower than this rate, typically below 10%34.KYAV is a new putative member of the rhabdoviral genus ses Fig. 12. The d35 and within virus groups36. CpG depletion has been used as an index of viral host adaptation38, although a recent study by Di Giallonardo et al. found the measure to be useful only for comparisons of higher taxonomic ranks such as Arthropoda compared to Vertebrata36. CpG frequencies in KYAV and related rhabdovirus genomes , which are transmitted directly between mammals in the absence of arthropod vectors, had higher levels of CpG depletion18. Genomes of ephemeroviruses, hapaviruses, and vesiculoviruses, which infect vertebrates but are vectored by arthropods, had levels of CpG depletion comparable to those of the mammal-specific lyssaviruses, if not somewhat higher. KYAV and Oita virus genomes stand out among ledantevirus genomes by having very high CpG frequencies, similar to insect-specific sigmaviruses. We again caution that dinucleotide composition appears to be shaped more by virus taxon than by host species36; however, this metric remains useful for comparing similar viruses that infect very different hosts 40.The relative CpG dinucleotide frequency in viral genomes varies widely among taxaDipseliopoda . However, our analysis of mitochondrial DNA sequences placed these bats as outgroup to Angolan soft-furred bats. They appear to be novel members of the Epomophorinae in the genus Myonycteris divergent enough to merit consideration as members of a separate species. This assessment is preliminary as it presently relies only on a mitochondrial cytochrome oxidase subunit I and cytochrome C phylogeny  or western blots targeting the major antigenic proteins of KYAV (likely N and/or G) might help elucidate the ecology of this virus in bats and animals of other species. Such studies may also resolve whether the absence of circulating KYAV in the tested bat sera reflects transient viremia, as we speculate, or lack of infection.Overall, our data demonstrate that our understanding of the diversity of megabats, their ectoparasites, and their viruses is still fragmented. Viruses of bats are diverse in part because bats themselves are taxonomically diverse45. Therefore, enigmatic cases of human infection with bat-associated rhabdoviruses may have resulted from incidental bites by bat flies or other bat-associated arthropods. For example, in 1969, Le Dantec virus infected a British dockworker who was bitten by an unidentified insect while unloading peanuts from a ship that had come from Nigeria29. Novel, divergent rhabdoviruses have also been found in apparently healthy people in Africa, suggesting unknown pathways of zoonotic transmission46. Our identification of an unknown rhabdovirus on unknown bat flies of unknown bats suggests further research on the diversity of these insects and their role as disease vectors might prove fruitful.Bat flies occasionally bite people47. Blood, oral swabs, urogenital swabs, and ectoparasites were obtained from bats, and bats were immediately released thereafter. All protocols for animal and sample handling were approved in advance by the Uganda Wildlife Authority, the Uganda National Council for Science and Technology, Makerere University, McGill University, and the University of Wisconsin-Madison, and were performed in accordance with all relevant guidelines and regulations.In February 2010, nine pteropodid bats were mist-netted from an established roost of approximately 12 bats in a peridomestic structure (a storeroom behind a kitchen) at the edge of Kibale National Park, western UgandaBat flies (the only ectoparasites found on sampled bats) were kept separate by bat and were stored whole at −20 °C in RNAlater solution . Swab samples were also stored in RNAlater solution at −20 °C. Blood was separated by centrifugation into components for long-term storage at −80 °C. Single bat flies and swab tips were homogenized by bead beating using a portable homogenizer . RNA was extracted from all sample types and converted to cDNA in the field using lyophilized reagents  and then converted into double-stranded cDNA . DNA was stabilized for long-term storage and transport to the USA at ambient temperature .49; this method is approximately as sensitive as real-time quantitative PCR for detecting viruses11. Briefly, DNA was purified using Agencourt Ampure XP beads . Approximately 1 ng of DNA was prepared for sequencing on a MiSeq instrument  using the Nextera XT DNA sample preparation kit (Illumina). Sequence data were analyzed using CLC Genomics Workbench version 8.5 . Low-quality bases were trimmed , short reads (<75 bp) were discarded, and the remaining reads were subjected to de novo assembly. Assembled contiguous sequences (contigs) were analyzed for nucleotide- (blastn) and protein-level (blastx) similarity to known viruses in GenBank. All sequences generated in this study were deposited into GenBank. All sequences used for analyses and their accession numbers are listed in Supplementary Table DNA was reconstituted, and libraries were prepared for NGS as previously described50. Maximum likelihood phylogenetic analyses were conducted on codon-based alignments of concatenated virus genes, with poorly aligned regions removed. Alignments were created using the MAFFT algorithm51 implemented in Translator X52, with the Gblocks algorithm53 applied to remove poorly aligned regions. Trees were constructed using the maximum likelihood method implemented in PhyML54, with best-fit models of molecular evolution estimated from the data using jModeltest55. Trees were displayed using FigTree56. The same phylogenetic methods were applied to mitochondrial gene sequences of bats (COI and CYTB) and bat flies (COII and CYTB nucleotide sequences) extracted from deep sequence data to investigate the taxonomy of sampled bats and bat flies.Genetic similarity between Kanyawara virus (KYAV) and its relatives was assessed using pairwise sequence comparisons in the computer program MEGA7L) gene. PCR conditions were optimized using bat fly cDNA samples known to be positive and negative for KYAV infection by NGS. The optimized PCR was used to test cDNA extracted from swab samples. PCRs were conducted using the HiFi kit , with 40 cycles at 94 °C for 15 sec, 56 °C for 15 sec, and 72 °C for 15 sec. Amplicons were visualized on 2% agarose electrophoretic gels stained with ethidium bromide.Based on coding-complete virus genome sequences detected in the course of bioinformatics analysis (see Results), PCR primers KYAV-10368 (5′-GCGAACCCGACGATCATAGT-3′) and KYAV-10695 (5′-GCTGTGCATTCCAGTCTCCT-3′) were designed to amplify a 327-bp region of the KYAV RNA-dependent RNA polymerase  from KYAV and related rhabdoviruses using the R Biostrings package57. Rhabdovirus sequences included in this analysis were selected objectively, using all coding-complete genomes from major rhabdovirus clades (genera) with at least four members available in GenBank and the Virus Pathogen Resource database58 as of December 12, 2016. Differences in mean CpG depletion among groups were evaluated for statistical significance using ANOVA, with pairwise differences between groups examined using the Holm post-hoc method, which adjustments for multiple testing59. Variances in CpG depletion among groups were compared using Levine’s test60. Statistical analyses were performed in the computer package R61.The relative frequency of CpG dinucleotide pairs was calculated for each of the canonical rhabdovirus genes (All data generated during the current study are available in GenBank (accession numbers KY385385-385392) or are included in this published article and its Supplementary Information files.Supplementary Information