Imprinted genes are peculiar in that expression of the two copies differs depending on whether the copy was maternally or paternally inherited. The discovery of this striking pattern of gene expression inspired myriad evolutionary theories, the most successful of which identify scenarios that create an asymmetry between the maternally and paternally inherited gene copies that favors silencing of one of the copies. Most notably, imprinting can evolve when gene dosage affects kin interactions  or when silencing enhances coadaptation by coordinating traits expressed by interacting kin. Although we have a well‐established theory for the former process (the “Kinship Theory”), the coadaptation process has only been explored for the specific case of interactions between mothers and offspring. Here, we fill this critical gap in our understanding by developing a general “Coadaptation Theory” that explains how imprinting can evolve to coordinate interactions between all types of relatives. Using a simple model in which fitness of an individual is determined by an interaction between its own phenotype (and hence genotype) and that of its social partner(s), we find that when the relatedness of interactants differs through their maternally versus paternally inherited gene copies, then selection favors expression of the allele through which relatedness is higher. The predictions of this Coadaptation Theory potentially apply whenever a gene underlies traits that mediate the outcome of conspecific interactions, regardless of their mechanism or the type of organism, and therefore provide a potential explanation for enigmatic patterns of imprinting, including those underlying adult traits. By providing simple testable predictions that often directly contrast with those derived from alternative theories, our model should play an important role in consolidating our understanding of the evolution of imprinting across genes and species, which will ultimately provide crucial insights into imprinted gene function and dysfunction. For most genes, no distinction is made between the copy inherited from mothers versus fathers, but for a peculiar subset of genes that show “genomic imprinting,” expression of each gene copy depends on its parental origin. Why did such an odd pattern of gene expression evolve? We address this fundamental question using a simple model where an individual's success in social interactions with relatives depends on the combination of traits that they and their social partners express. This “Coadaptation Theory” demonstrates that genomic imprinting can evolve because it leads to more successful social interactions by coordinating the traits expressed by interacting individuals. More specifically, imprinting benefits an individual because it enhances the compatibility between the gene copy that they express and the gene copy (or copies) expressed by their social partner(s). Understanding the conditions that favor genomic imprinting is important because it provides critical insights into the evolutionary processes shaping a key epigenetic feature of genomes. Such insights are broadly important because theories for the evolution of genomic imprinting have been used as central organizing principles for understanding the nature of imprinted genes across essentially all research areas. Thus, in addition to the value to evolutionary biologists, the Coadaptation Theory has potential utility for myriad problems in biology. For example, evolutionary theories for genomic imprinting have provided critical insights into the properties of imprinted genes in areas such as developmental biology by providing an explanation for the types of traits that they affect and the pathological consequences of loss of function mutations or epigenetic changes (such as changes that lead to a loss of imprinting at a gene). The Coadaptation Theory provides a predictive, testable framework that can be applied in such scenarios to understand why certain genes are imprinted, and more broadly, the utility of this critical genomic feature.Genomic imprinting is an epigenetic phenomenon where the maternally and paternally inherited copies of a gene . The four ordered diploid genotypes  occur in Hardy–Weinberg proportions .We consider a locus (the “A locus”) that has two alleles, Di) and also affects some trait expressed in the individuals with whom the focal individuals interact . From the perspective of the focal individuals, the social trait can be considered to be a component of the social environment they experience. The model applies equally to the case where there is one trait, such that the direct trait also influences the social environment , or there are two distinct traits, one direct and one social .We assume that the locus directly affects some trait possessed by “focal” individuals  for the direct trait and J (‒1 ≤ J ≤ 1) for the social trait. Positive values of I and J indicate expression biased toward the matrigenic copy  and negative values indicate a bias toward the patrigenic copy for the relevant trait. When I or J = 0 there is normal biallelic expression and when |I| or |J|= 1 one copy is silenced, resulting in uniparental expression. This model structure allows the locus to potentially show different patterns of expression for the different traits (or for the same trait at different times in life), which is consistent with the empirical observation that imprinted genes can show different expression patterns in different contexts  would therefore have the phenotypic values of Di =  and Si = , matching the pattern expected for the classic quantitative genetic model  for the direct trait and (1 + J) for the social trait, while the effect of the patrigenic copy is weighted by (1 – I) and (1 – J) for these traits, respectively ad + (1 – I)ad], which simplifies to ad for all patterns of imprinting, since the A1 allele is always the allele being expressed. Likewise, the phenotypic value of the direct trait associated with genotype A1A2 is ½[(1 + I)ad – (1 – I)ad], which simplifies to Iad, while the direct trait phenotypic value associated with A2A1 is ½[–(1 + I)ad + (1 – I)ad], which simplifies to –Iad. This difference between the reciprocal heterozygotes highlights the impact of imprinting on trait expression, where a heterozygote will have the phenotype associated with the matrigenic allele under maternal expression, but the patrigenic allele under paternal expression. Overall, the phenotypic values of the four genotypes at the A locus  for the direct trait are given by the vector AD = , and for the social trait by AS = .To connect the allelic variation to trait expression, we build from the classic additive quantitative genetic model, where the two alleles in a genotype have independent effects on trait expression  is determined by an interaction between the phenotypic value of its own direct trait (Di) and the phenotypic value of the social trait of its social partner (Sj). The effect of the interaction on the fitness of the focal individual is given by the coefficient ψ wij=wA=1+ψ vec (SADAT)(where the operation “vec” achieves vectorization).These fitness values defined by equation (1) are given by the vector rMM, rMP, rPM, and rPP (following Jacquard M) or patrigenic (P) gene copies in the focal individual are identical by descent (IBD) with each copy in its social partner . The offspring‐mother case provides a simple example: Offspring inherit their matrigenic gene copy from their mother, and there is an equal probability that the offspring inherits its mother's own matrigenic or patrigenic copy, making the genetic identity coefficients rMM = rMP = ½ (where the first subscript indicates the offspring's gene copy and the second the mother's). In a randomly mating population, offspring are not related to their mother through their patrigenic copy, therefore rPM = rPP = 0. The average of the four genetic identity coefficients gives the coefficient of kinship for the pair [ϕGG′ = ¼(rPM + rPP + rMM + rMP)], which is half the coefficient of relatedness  for the direct trait and by (1 + J) in social partners for the social trait, making the expression weighted identity coefficient for the matrigenic copies ρMM=(1+I)(1+J)rMM. It follows that ρMP=(1+I)(1−J)rMP, ρPM=(1−I)(1+J)rPM, and ρPP=(1−I)(1−J)rPP. The average of the four expression weighted identity coefficients gives the expression weighted coefficient of kinship of interactants:We measure relatedness using four genetic identity coefficients, Jacquard . Thus, f Queller  we need  Queller . For exaφGG′), which averages the four types of relatedness. However, when there is imprinting, the expression weighted coefficient of kinship only averages over the gene copies being expressed. For example, if only the matrigenic copy is expressed for the direct and social traits (I = J = 1) then φEE′=rMM, demonstrating that only matrigenic kinship is important. Likewise, if only the patrigenic copy is expressed for the direct trait but there is no imprinting for the social trait , then φEE′=12(rPM+rPP), so only the patrigenic copy in the focal individual matters when measuring kinship.When there is no imprinting, this equation reduces to the coefficient of kinship  and allele frequencies (Table S1). The frequencies of these pair‐wise allelic combinations determine the frequencies of interactions between each of the focal‐partner diploid genotype combinations, fij , which are given in Table S2. These frequencies can be used along with the vector of fitness to calculate population mean fitness as: w¯=wA·FA.The frequency of each of the possible pair‐wise allelic combinations in interactants is a function of the four relatedness terms . We assume that the B1 allele causes some degree of imprinting (denoted δ) of the A locus for its effect on the direct trait while the B2 allele does not. Therefore, it is the B locus genotype that determines the value of the imprinting parameter I for a given genotype (and hence has the same consequences for trait expression and fitness). The overall influence of a given B locus genotype on imprinting of the A locus for its effect on the direct trait is determined by the average influence of the pair of alleles, so the imprinting effects of the four B locus genotypes  on expression of the A locus for the direct trait are δ, ½δ, ½δ, and 0. As with the imprinting parameter I, −1≤ δ ≤ +1 with positive values indicating expression biased toward the matrigenic copy  and vice versa. When the imprinting modifier allele is segregating at the B locus, the population imprinting parameter I corresponds to the average degree of imprinting of the A locus for its effect on the direct trait , such that I would simply equal x1. Further details of the properties of the A and B two‐locus system (phenotypes and genotype frequencies) are given in the Supplementary Methods.The B locus has two alleles, B1) allele, Δx1. This change is determined by the covariance between the proportion of an individual's B locus alleles that are B1 with its relative fitness (wij/w¯) w¯=Δx1w¯ (see Supplementary Methods).Evolution of imprinting for the effect of the A locus on the direct trait is determined by the change in the frequency of the imprinting modifier (¯) Price :  cov  are σ, ½σ, ½σ, and 0. These define the value of the imprinting parameter J for each C locus genotype (with the properties of σ following that of J), and hence have the same consequences for trait expression and fitness. Note that when the imprinting modifier allele is segregating at the C locus, the imprinting parameter J corresponds to the average degree of imprinting for the effect of the A locus on the social trait , such that J would simply equal y1.The basic properties of the C locus follow those of the B locus except that it modifies imprinting of the A locus for its effect on the social trait. The two alleles, C1) allele, Δy1. This change is determined by the covariance between the proportion of an individual's C locus alleles that are C1 with its relative fitness (wij/w¯) w¯=Δy1w¯ (see Supplementary Methods).As with the B locus, evolution of imprinting of the A locus for its effect on the social trait is determined by the change in the frequency of the imprinting modifier (¯) Price :  cov (CDi) and the value of a trait expressed by their social partner(s)  (see equation 1). The direct and social traits can potentially be completely different traits linked by pleiotropy  the value of a trait they express  by their expression pattern. For example, if individuals share alleles that are IBD but at least one of those alleles is not expressed due to imprinting, then that genetic relationship would not contribute to the overall weighted coefficient of kinship, which is logical since that component of relatedness has no fitness consequences.When the fitness effect of social interactions depends on the combination of traits that individuals express (as in our model), individuals will have higher fitness when they experience “compatible” social environments . Consequently, selection is expected to favor mechanisms that lead to adaptive coordination between traits expressed by interactants :The overall pattern of social coadaptation between the traits expressed by interactants can be captured by the phenotypic covariance of the traits they express in the interaction (cov2p1p2adas) and the expression weighted coefficient of kinship (φEE′) of interactants (see equation 2). To elucidate the role of imprinting, we can rewrite this equation rMM > 0) then maternal expression for the direct and social traits  increases the contribution of relatedness to the social covariance by a factor of four . Hence, such a pattern can be said to “coordinate” the interaction by enhancing the compatibility of the gene copies that interactants express.The RHS of this equation has two components, the additive genetic covariance between the direct and social traits :The first term inside the brackets simply reflects expected fitness under random interactions, which is not influenced by imprinting. Hence, the only term in this equation that is affected by imprinting or relatedness is the social covariance  plays in the expected fitness of individuals in social interactions  on a modifier allele with a given pattern of imprinting is:We can formally examine the conditions under which imprinting is favored by considering the conditions under which selection favors some modifier that changes the imprinting status of a locus affecting traits that mediate social interactions. The pattern of selection is captured by selection gradients on modifier alleles at loci where one allele causes imprinting and one does not (see Methods and Supplementary Methods). These selection gradients give both the conditions for invasion by the modifier and the fixation conditions. For the direct trait, the selection gradient (δ) while negative values would favor a modifier causing paternal expression . The selection gradient can be simplified by combining the effect parameters  that mediate each of the steps in the connection between allelic variation at the locus and fitness, into a single selection parameter, θ. This selection parameter can be viewed as a sort of “synergism” , and a set of expression weighted relatedness differentials (the terms in brackets). Importantly, this selection gradient not only describes the conditions favoring an imprinting modifier, but more generally describes the conditions under which selection favors imprinting for the direct trait, regardless of the mechanism . This can be demonstrated by partial differentiation of population mean fitness , the amount of allelic variation at the locus (ness eq.  with resβJ) on a modifier causing a given pattern of imprinting of the A locus is:For the social trait, the selection gradient (σ) while negative values favor a modifier causing paternal expression . The basic form of this selection gradient matches that of the direct effect (eqs. φGG′). Relatedness of interactants is critically important for the evolution of imprinting of the effect of the A locus on the social trait because the value of the social trait expressed by an individual influences the fitness of relatives, not their own fitness. Consequently, imprinting of the A locus for its effect on the social trait evolves by kin selection (Haig J), ∂w¯/∂J, which has the same basic form as equation As with the selection gradient on a modifier causing imprinting of the A locus for its effect on the direct trait (eq. 5), positive gradients indicate conditions that favor a modifier causing maternal expression of the A locus for its effect on the social trait (rMM−rPM) and (1−J)(rMP−rPP). The first of these terms gives the difference in the relatedness of the focal individual's matrigenic (rMM) and patrigenic (rPM) copies to the matrigenic copy of their partner, while the second term gives the difference in relatedness of these copies to the partner's patrigenic copy (rMP and rPP). Maternal expression of the locus for its effect on the direct trait is favored when the matrigenic copies in focal individuals are more related to the alleles in their social partners than are the patrigenic copies (rMM>rPM and rMP>rPP) and vice versa. However, these terms have to be weighted by the pattern of imprinting for the effect of the locus on the social trait because this determines which copy in the partners is actually expressed (and hence which copy matters). If only the matrigenic copy is expressed for the social trait (J = 1), then logically only the matrigenic copy in social partners matters (since the patrigenic copy would be silenced) and hence only the first of the relatedness differentials would matter. In general, these asymmetries demonstrate that selection favors expression of the gene copy that has higher relatedness with the relevant copy in their social partner(s) (which is perhaps most likely when one type of relatedness is zero).Given a particular fitness effect of the locus (θ), the patterns of imprinting that are favored for the direct and social traits depends on a set of relatedness asymmetries eqs.  and 6. Fp1p2 in eqs. 5 and The selection gradients  that mediate social interactions between conspecifics with consequences for fitness, regardless of the mechanism or the type of organism. For example, behavioral interactions between parents and offspring have well‐documented consequences for fitness, particularly in the context of resource provision and demand in organisms with high levels of parental care such as many mammals  in mammals, where selection from kin interactions appears a likely explanation for the occurrence of genomic imprinting  the direct trait (the “B” locus) and (b) the social trait (the “C” locus) in interacting individuals in a randomly mating population.Table S4. Frequencies of social interactions between each of the genotype combinations at loci that modify imprinting of the effect of the A locus on (a) the direct trait (the “B” locus) and (b) the social trait (the “C” locus) in a randomly mating population.Click here for additional data file.


The efficacy and safety of antibiotic treatment for uncomplicated acute appendicitis has been established at long-term follow-up with the majority of recurrences shown to occur within the first year. Overall costs of antibiotics are significantly lower compared with appendectomy at short-term follow-up, but long-term durability of these cost savings is unclear. The study objective was to compare the long-term overall costs of antibiotic therapy versus appendectomy in the treatment of uncomplicated acute appendicitis in the APPAC (APPendicitis ACuta) trial at 5 years.This multicentre, non-inferiority randomized clinical trial randomly assigned 530 adult patients with CT-confirmed uncomplicated acute appendicitis to appendectomy or antibiotic treatment at six Finnish hospitals. All major costs during the 5-year follow-up were recorded, whether generated by the initial visit and subsequent treatment or possible recurrent appendicitis. Between November 2009 and June 2012, 273 patients were randomized to appendectomy and 257 to antibiotics. The overall costs of appendectomy were 1.4 times higher (p<0.001)  compared with antibiotic therapy  resulting in cost savings of €1545 per patient  in the antibiotic group. At 5 years, the majority  of antibiotic group patients did not undergo appendectomy.At 5-year follow-up antibiotic treatment resulted in significantly lower overall costs compared with appendectomy. As the majority of appendicitis recurrences occur within the first year after the initial antibiotic treatment, these results suggest that treating uncomplicated acute appendicitis with antibiotics instead of appendectomy results in lower overall costs even at longer-term follow-up. Out of the more than 200 million annual surgical procedures performed globally, appendectomy is one of the most common incurring significant health care costs.–3 AppendThe fundamental differences between antibiotic therapy and surgery as the primary treatment options for uncomplicated acute appendicitis result in the challenge of using comparable definitions of treatment success. The assessment of the optimal treatment paradigm for uncomplicated acute appendicitis cannot be solely based on outcome definition of treatment success defined by risk of recurrent appendicitis as antibiotics will never reach the definitive treatment efficacy of appendectomy in that respect. Future appendectomy may not be a valid primary outcome measure either since about 30% of patients treated with antibiotics will get recurrence and potential protocol stated surgery as with the standard appendectomy approach, 100% of patients will get surgery. Therefore, the comprehensive assessment of the best possible treatment option should also include outcomes independent of the compared treatment strategies, i.e. treatment related morbidity, time to recovery, post-intervention pain, along with patient related factors including patient preference at her or his current situation, and also treatment costs. The economic evaluation of the APPAC trial 1-year follow-up showed 1.6 times higher overall costs for appendectomy taking into account all costs whether generated by the initial visit and subsequent treatment or possible recurrent appendicitis.Appendicitis recurrence after initial successful antibiotic therapy at long-term follow-up is an important question, as the costs accrued from surgical treatment of the recurrences will reduce the original cost advantage of antibiotics versus surgery. Using a decision tree model in the US context, Wu et al. estimated in their report that a recurrence rate as high as 56% for antibiotic treated patients would be the cut-off point after which initial operative treatment becomes the most cost effective treatment strategy for uncomplicated acute appendicitis. In the ATo our knowledge, there are no available long-term results of previous randomized trials comparing the overall costs of antibiotic therapy versus appendectomy in the treatment of uncomplicated acute appendicitis. This study reports the 5-year overall costs for all the patients enrolled in the original APPAC (APPendicitis ACuta) trial.Details of the study design, rationale and methods have been published previously., 18 The https://clinicaltrials.gov/ct2/show/NCT01022567Patients aged 18 to 60 years admitted to the emergency department with a clinical suspicion of uncomplicated acute appendicitis confirmed by CT were enrolled in the study after giving written informed consent. CT criteria for acute appendicitis included appendiceal diameter exceeding 6 mm with wall thickening accompanied with at least one of the following features: abnormal contrast enhancement of the appendiceal wall, inflammatory edema, or minor fluid collections around the appendix. Exclusion criteria included complicated acute appendicitis defined as the presence of an appendicolith, perforation, abscess, or suspicion of a tumor on the CT scan. Other exclusion criteria were contraindications for CT, peritonitis, unable to co-operate and provide informed consent, and the presence of serious systemic illness.For patients randomized to operative treatment, the predefined surgical procedure in the trial protocol was open appendectomy performed using a McBurney right lower quadrant muscle-splitting incision technique. Laparoscopic appendectomy was performed in 15 patients (5.5 per cent). Prophylactic antibiotics (single dose of cefuroxime 1.5g and metronidazole 500mg intravenously) were administered approximately 30 minutes before incision.For patients randomized to antibiotic therapy, intravenous ertapenem sodium (1g/day) was administered for 3 days followed by 7 days of oral levofloxacin (500mg once daily) and metronidazole (500mg 3 times daily).The primary endpoint was treatment success predefined to be assessed at one-year follow-up., 18 In tThe predefined secondary endpoints included late recurrence (after 1 years) of acute appendicitis after antibiotic treatment, overall postintervention complications, length of hospital stay, the amount of sick leave, postintervention pain scores , and treatment costs. AccordinAll cost estimates were based on the cost levels of 2016. In the base case of analyses annual discount rate of 5 per cent was applied to all costs. Hospital charges were recorded based on diagnosis-related group codes as overall hospital costs and registered in all participating hospitals. The hospital charges were a bulk sum including all the cost components  incurred by the treatment and patient up-keep during hospitalization, thus representing the true costs used to charge the final payer. In the Finnish system, the community of residence of the patient pays the diagnosis-related group-based bulk costs charged by the hospitals and accurate proportion of each separate component cannot be reliably identified. In Finland the health care system is organised by communities, based on central government guidance and hospital costs are charged from communities in full.The treatment of acute appendicitis in Finland is practically entirely carried out in the public hospital setting, and the role of occasional rare involvement of private health care providers or primary health care organizations is marginal and has insignificant economic impact. In our analysis, all major hospital costs were recorded, whether generated by the initial visit and subsequent treatment or possible complications or recurrent appendicitis during the 5-year follow-up period, and an intention-to-treat analysis was performed.Two antibiotic group patients undergoing subsequent surgery during the long-term follow-up were lacking sufficient data regarding operative treatment or productivity loss information based on surgical treatment abroad in one patient and change in hospital cost recording system in the other patient, but they were also included in the analyses. Their follow-up costs were estimated by using age and sex standardized linear regression models, based on complete initial operation and productivity loss data from the hospital district where they received their initial treatment.When estimating the costs of absence from work, the human capital approach was applied. The days spent in hospital were all considered sick leave days and additional sick leave prescribed at discharge was also recorded. The costs of productivity losses were based on the average monthly gross salaries for working Finnish adults in 2016, €3075 for women and €3675 for men. The per day productivity loss estimate was computed by dividing the gross monthly salary by 21, the number of average monthly working days.The cost for imaging, laboratory, and medicine used during hospitalization or prescribed at hospital discharge were marginal and non-significant at 1-year follow-up. Therefort- test. The data on hospital charges, productivity costs and overall costs had very acceptable skewness  and kurtosis  values and the Student’s t-test was concluded robust enough to minor violation of the normality assumption. Differences between groups in length of hospital stay and sick leave were tested using Mann-Whitney U–test because of very skewed distributions. Normality of the distributions was evaluated using Kolmogorov-Smirnov–test, skewness and kurtosis of the distributions and visual evaluation.Categorical variables were described using frequencies and percentages, and continuous variables with means and 95% confidence intervals (95% CI) or in case of skewed variables medians with 95% CI. Statistical analysis of the data on average costs was based on Student’s Sensitivity analyses were performed to determine whether the final outcome was sensitive to certain crucial factors. The values of selected components were changed and the effect on the outcome estimate was evaluated. The role of the costs of absence from work days was determined in two directions, i.e. by decreasing the days of prescribed sick leave and increasing the salary costs with 10% intervals up to 50% lower and higher values. When estimating the sensitivity of sick leave days, the days in hospital were not reduced, only the sick leave days prescribed when the patient was discharged. The effect of discount rate was evaluated by performing the analyses using also 0%, 3%, 7% and 10% annual rates. Two-sided p-values less than 0.05 were considered statistically significant. All analyses were performed using SPSS software version 23 .Between November 10, 2009, and June 20, 2012, 530 patients were enrolled, and 273 patients were randomized to the appendectomy group and 257 patients to antibiotic treatment. Higher costs in the surgical group were observed both in hospital charges and productivity losses. In both groups productivity losses formed the slightly larger proportion of the overall costs compared with hospital charges. Almost equal relative differences in the costs between the two study groups could be observed in hospital charges, productivity losses as well as in overall costs. The cost distribution is presented in detail in The findings of this study were not sensitive to any of the selected components studied. The sensitivity analysis of the costs for the two groups after 5-year follow-up is presented in The overall costs at 5-year follow-up were 1.4 times higher in the appendectomy group than in the antibiotic group when all major costs were taken into account, whether generated by the initial visit and subsequent treatment or possible recurrent appendicitis. After the first year of follow-up, overall costs in the appendectomy group were 1.6 times higher and despThese results corroborate the findings from the decision tree model by Wu et al. and earlSurgeons have very different views of the optimal number of sick leave days for the same patient cases. From a sAs we now know that antibiotic therapy is safe, 25–27, A limitation of the APPAC trial is the open approach in the surgery group as the current gold standard of laparoscopic appendectomy has been shown to shorten both the hospital stay and sick leave., 31 OpenThe Finnish cost estimates used in this study may differ from those observed in other societies with different salary levels or structures. However, even when the salary levels were increased by 50% in the sensitivity analysis, the differences between the groups remained similar. As the increase in the overall costs was almost equal in proportion to increases in the salary costs, these findings can be assumed to be generalizable also to societies with different salary levels or structures. People and societies tend to have a positive time preference—we prefer to have the benefits sooner and carry the costs later. Thus, costs that accrue in later years need to be valued differently than those that are formed earlier. This valuation is done by discounting, a method that produces net present values where costs accruing in different years are made comparable. Discount rate of 5% has become a standard in economic analysis over the years, although there is no scientific basis for using that particular rate. Thus, a sensitivity analysis is justified, where the discount rate is varied and the effect of changing it on the outcome measure is estimated. In the present study changing the discount rate had a non-significant and marginal effect mainly due to the low number of patients having recurrent appendicitis after the first year of follow-up.In the APPAC trial, the treatment costs at 1-year follow-up were 1.6 times higher in the appendectomy group and remaS1 FileAPPAC study protocol and statistical analysis plan.(DOCX)Click here for additional data file.


We present the case of an elderly male patient with known multiple myeloma who was hospitalized with profuse watery diarrhea and abdominal pain after a course of induction chemotherapy. Intestinal intramural gas was found on imaging and the diagnosis of pneumatosis intestinalis was confirmed by colonoscopy. We propose counterperfusion supersaturation as the etiology for this patient's pneumatosis coli via disruption of homeostasis between nitrogen and hydrogen normally present in the bowel. His condition was successfully treated with antidiarrheal medications and inhaled oxygen as well as intravenous hydration, and he eventually completed multiple myeloma directed chemotherapy with an excellent response. In this report, we discuss how clinicians can improve management of pneumatosis intestinalis by understanding the proposed pathophysiology. Pneumatosis intestinalis (PI) is a radiographic finding, which describes intestinal intramural air. It is frequently benign and does not typically require intervention , 2. It iAn 86-year-old male with a past medical history significant for IgG lambda light chain multiple myeloma, congestive heart failure, and atrial fibrillation on anticoagulation presented for evaluation of abdominal pain, increasing abdominal distention, and inability to pass flatus or bowel movements for two days. Prior to his ileus-like symptoms, he experienced anorexia, rigors, and severe watery diarrhea for several days in correlation with the completion of his second cycle of Revlimid , Velcade (bortezomib), and dexamethasone induction chemotherapy for multiple myeloma.∧9/L, hemoglobin of 8.1 g/dl, platelets of 15 x10∧9/L, creatinine of 2.35 mg/dL (baseline of 0.9 mg/dL), and lactic acid of 2.3 meq/L. A noncontrast abdominal computerized tomography (CT) scan was performed, which demonstrated diffuse colonic distention with submucosal and intraluminal gas, distended fluid-filled distal small bowel loops, and innumerable small submucosal lesions covering the surface of the colon (Figures On presentation, he was afebrile and hypotensive to 72/42. Physical exam demonstrated a pale man with dry oral mucosa and a mildly tender, tympanitic abdomen without peritoneal signs. On laboratory examination he was found to have a white blood cell count of 4.6 x10Initially, the diagnosis of bortezomib-induced ileus was entertained; however, the patient's diarrhea quickly resumed once he had been fluid resuscitated. He continued to have watery stools requiring rectal tube placement without improvement on piperacillin-tazobactam and metronidazole. The gastroenterology service was consulted to determine the etiology of his diarrhea and abdominal pain, at which point the diagnosis of pneumatosis coli was considered. Repeat CT scan with IV contrast was requested and revealed bowel wall thickening of the transverse and proximal descending colon as well as multiple mildly thickened mid and distal small bowel loops. Based on the updated CT findings, concern for underlying typhlitis arose and the patient was maintained on empiric antibiotic therapy. Due to continued voluminous watery diarrhea, hypotension requiring constant fluid administration, severe hypokalemia, and little to no improvement on antibiotics, the patient was prepped for colonoscopy for direct visualization of the abnormal appearing bowel on imaging.On colonoscopy, the colonic mucosa was normal appearing except for the presence of diffuse intramural gas consistent with pneumatosis coli Figures –5. BiopsPneumatosis intestinalis is the term used to describe pockets of gas found within the wall of the intestine. Determination of the origin of the deposited gas can help us understand the mechanisms by which pneumatosis intestinalis can occur. Gas can be found in the intestinal lumen due to ingestion of air, production by flora, production by invasive pathogenic gas-producing bacteria; can diffuse from nearby mesenteric blood vessels; or can be of pulmonary origin. One theorized mechanism for gas deposition within the luminal wall involves intraluminal air dissecting into the wall of the intestine via mucosal defects. Bacterial invasion of the intestinal wall by gas-producing organisms can also lead to deposition of gas in the submucosa of the intestine. Gas from ruptured lung parenchyma can dissect into the mediastinum to the retroperitoneum, from where it can eventually track along perivascular tissue planes to deposit in the wall of the bowel . FinallySymptomatology of PI is largely nonspecific and is attributed to the underlying condition causing pneumatosis intestinalis. Frequently, patients present with diarrhea, abdominal pain, abdominal distention, nausea, vomiting, and weight loss if the diarrhea is protracted and mucous in the stool. Interestingly, PI is one of the differential diagnoses associated with sterile pneumoperitoneum .Diagnosis of pneumatosis intestinalis is largely imaging based. It can be diagnosed on X-ray, ultrasound, CT, and endoscopy among other modalities. Abdominal radiographs can demonstrate pneumatosis intestinalis in two-thirds of cases . UltrasoComplications occur in about 3% of patients with pneumatosis intestinalis and include volvulus, bowel obstruction, gastrointestinal hemorrhage, and intestinal perforation manifested as pneumoperitoneum [In general, inhaled oxygen therapy has been cited as early as the 1970s as a treatment for PI . StudiesWe believe the pneumatosis intestinalis in this patient formed via counterperfusion supersaturation for a number of reasons. First, his initial presentation with significant hypotension likely resulted in a disruption of the normal nitrogen and hydrogen homeostasis of the intestine by impairing diffusion of nitrogen from the blood to the gut due to low blood pressure. Additionally, chronic disruption of mesenteric vascular permeability as a result of amyloid vasculopathy may have contributed to the formation of pneumatosis intestinalis in this patient. Finally, there was likely an increase in hydrogen in the lumen of the intestine due to reduction in gut hydrogen absorption by intestinal bacteria as a result of antibiotic administration. Since the mucosa was not disrupted on colonoscopy, we do not believe the PI was formed as a result of amyloid deposition throughout the GI tract, despite the presence of amyloid on biopsy.The differential for pneumatosis intestinalis is broad and includes both benign and life threatening conditions. Any disease state causing damage to the intestinal blood supply or disruption of the gut microbiome can potentially cause PI. Lack of knowledge of this condition and its proposed pathophysiology may limit timely diagnosis and as a result could cause patient harm. Specifically, patients are at risk for unnecessary surgery, extreme volume depletion, and electrolyte abnormalities due to uncontrolled diarrhea, side effects from antibiotic therapy, and increased hospital length of stay, all of which ultimately can result in poor outcomes and increased healthcare costs.Pneumatosis intestinalis is a condition caused by the deposition of gas into the bowel wall. Air can make its way into the bowel by a number of pathologic mechanisms, all of which take into account either mucosal injury or pressure/diffusion gradients between body compartments. The diagnosis is imaging based and is best performed with MDCT scan due to its ability not only to identify the finding, but also to potentially reveal the underlying disease process. Treatment of pneumatosis intestinalis itself is supportive consisting of intravenous hydration and inhaled oxygen therapy. Clinical and laboratory evaluation to diagnose the underlying cause of the pneumatosis intestinalis is paramount and careful consideration is necessary prior to initiating aggressive interventions.


Segmentation of viral genomes allows exchange of intact genes between related viruses when they coinfect the same cell . This exIn theory, any virus with a segmented genome can undergo reassortment. Among viruses that infect vertebrates, those that carry segmented genomes belong to the Arenaviridae, Birnaviridae, Bunyavirales, Orthomyxoviridae, Picobirnaviridae, and Reoviridae. Reassortment has been documented to occur in nature for each of these viral taxa –6. NeverSince reassortment takes place in coinfected cells, a critical factor governing reassortment is the frequency of coinfection . When thOf course, reassortment has a greater impact on viral genotype if coinfecting viruses are not derived from the same population but rather represent two distinct lineages. The likelihood of such a mixed infection occurring depends on numerous factors, including prevalence of the viral lineages in circulation, likelihood of dual exposure, and the spatial dynamics of the two viruses within a coinfected host. Another important factor is the active exclusion of a second virus as a result of superinfection interference ,13. ThisIn theory, coinfection does not necessarily lead to reassortment. The efficiency of reassortment within a coinfected cell depends on (i) the extent to which viral replication is compartmentalized within the cell and (ii) the stringency of genome packaging and compatibility of packaging signals between coinfecting viruses. The first of these factors determines the level of mixing between coinfecting viral genomes, while the second dictates whether or not segments derived from differing parental strains can be coincorporated into nascent virus particles.Most viral life cycles are characterized by a compartmentalization of viral functions into localized areas. These can take the form of cytoplasmic inclusion bodies, viral replication organelles associated with host cell membranes, or punctate accumulations of viral components within the nucleus. The concentration of viral genetic material, viral proteins, and necessary host factors within these inclusions is thought to increase the efficiency of viral functions. However, reassortment is predicted to be limited by inclusions. If each incoming parental virus generates its own inclusion, the resultant constraint on the physical mixing of genome segments would restrict reassortment. Reoviruses (family Reoviridae) give an excellent example of a highly compartmentalized viral lifecycle. Reovirus components accumulate in distinct inclusion bodies within the cytoplasm, which are the sites of viral transcription, translation, replication, and particle assembly . In thisViral mechanisms that evolved to ensure the coordinated packaging of genome segments into virus particles may also place constraints on reassortment. Incorporation of viral genomes into virions is typically directed by specific nucleic acid sequences, viral protein motifs, or a combination of both. For segmented viruses, these packaging signals must be present on all gene segments. In addition, some segmented viruses enforce a high fidelity of genome packaging to ensure that one of each segment is present, a process that relies on segment-specific signals and selective incorporation. Divergence among related viruses in packaging signals may limit the potential for reassortant genotypes to form. For IAV, this form of constraint has been demonstrated experimentally ,22. AlthIn contrast to other forms of recombination, reassortment does not rely on template switching during replication and does not result in the formation of chimeric genes. Reassortment therefore does not give rise to nonfunctional genes. Nevertheless, most reassortment events are deleterious, even between similar viruses  Fig 2)..Fig 2). Although usually deleterious, reassortment is very important in the evolutionary history of many segmented viruses because of the rare occasions when a reassortant virus is successful on a population scale. A striking example from the Bunyavirales is that of Ngari virus. This virus is a reassortant that derives its large and small gene segments from Bunyamwera virus and its medium gene segment from Batai virus . The conReassortment in nature is best documented for the viruses of the Orthomyxoviridae, owing to the relatively thorough surveillance and full genome sequencing efforts focused on influenza viruses circulating in human and nonhuman hosts. The extent to which reassortment contributes to emergence of novel viruses belonging to other segmented virus families is less clear, and further research is needed on this topic. In addition, the significance to virus evolution of reassortment among related variants of a single virus population is understudied. Most segmented viruses have RNA genomes and therefore have high mutation rates. Thus, even a clonal virus population is not characterized by a single sequence but is rather a cloud of related variants. By analogy to sexual reproduction in eukaryotes, reassortment within a viral population is expected to accelerate adaptive change by combinatorial shuffling of beneficial and deleterious mutations ,33. WhilIn summary, for viruses with segmented genomes, reassortment is an important means of genetic diversification. Its prevalence is governed by a number of stochastic effects, like the probability of dual exposure, but also by fundamental aspects of a given virus’ replication strategy. Viruses formed through reassortment of heterologous parental strains usually suffer fitness defects but occasionally possess a selective advantage and therefore can have a major impact on the evolution and epidemiology of circulating viruses.


Torque Teno virus (TTV) in humans is characterized by ubiquitous occurrence in peripheral blood (PB), without any related disease described to date. Several studies reported a significant increase of TTV plasma DNA levels in allogeneic transplant recipients, and suggested a correlation of elevated virus titers with immunosuppression and transplant-related complications. However, the site of viral replication in this setting has remained unclear. We have studied TTV in serial plasma specimens derived from 43 pediatric allogeneic hematopoietic stem cell transplantation (HSCT) recipients by RQ-PCR, and found increasing TTV-DNA levels in all patients post-transplant, with a peak around day +100 and maximum virus copy numbers reaching 4 × 10E9/ml. To assess whether the virus replicates in PB-cells, leukocyte subsets including granulocytes, monocytes, NK-cells, T- and B-lymphocytes were serially isolated by flow-sorting for TTV analysis in 19 patients. The virus was undetectable in most cell types, but was identified in granulocytes in all instances, revealing a median DNA copy number increase of 1.8 logs between days +30–100 post-transplant. Our data therefore provide evidence for TTV replication in granulocytes in this setting. In a control cohort of immunocompetent children and in HSCT recipients before day +30, TTV positivity in granulocytes was less common (33%), and the copy numbers were considerably lower. However, rising TTV replication about 2 weeks after granulocyte engraftment (>500 cells/μl) was observed suggesting that granulocyte recovery might be required for TTV expansion in severely immunosuppressed transplant recipients. Torque Teno Virus (TTV) was first discovered in 1997 in a patient with hepatitis of unknown etiology , and wasIn the present study, consecutive plasma samples derived from 43 pediatric allogeneic HSCT recipients were monitored for TTV levels over a time period of more than 1 year post-transplant. The potential site(s) of TTV replication in individual blood cell types were assessed by investigating the virus copy numbers in various leukocyte subsets isolated by flow-sorting. The study performed in pediatric HSCT recipients provides evidence for TTV replication in granulocytes which culminated around 100 days post-transplant. The onset and kinetics of virus replication appeared to correlate with the level of immunosuppression and the time course of granulocyte engraftment.Archived plasma, stool, PB, and bone marrow (BM) specimens derived from pediatric allogeneic stem cell transplant recipients treated at the St. Anna Children’s Hospital, Vienna, Austria, were used for the study. The specimens had been collected as part of routine diagnostic virus screening at consecutive time points starting before conditioning followed by collection at 7-day intervals until day +100, thereafter at 2 to 3 week intervals until day +360, and at less regular intervals after the first year post-transplant. Written informed consent was obtained from each of the 43 patients and/or the parents. The patient characteristics are listed in Table +, CD8+, CD19+, CD56+, CD33+, and CD15+ as part of routine diagnostics in HSCT recipients at the St. Anna Children’s Hospital, Vienna, Austria. Cell sorting was performed on a FACSAria instrument (BD Biosciences) using the FACSDiVa software, as described previously .Individual leukocyte subsets were isolated from PB or BM via staining for specific cell surface antigens including CD4eviously . The purThe QIAamp DNA Mini Kit (Qiagen) was employed for the isolation of DNA from plasma, PB and individual cell subsets. Extraction of DNA from stool specimens was performed by the QIAamp DNA Stool Mini Kit (Qiagen) according to the manufacturer’s recommendations, and viral RNA was isolated with the RNeasy Kit plus (Qiagen), with subsequent DNA digestion with DNase Q1 (Promega). For further real-time quantitative (RQ)-PCR analyses, the RNA was transcribed into cDNA using the MLV reverse transcriptase (Promega).RQ-PCR targeting the UTR region of the TTV genome was used for virus detection. The PCR assay previously described by RQ-PCR assays exploiting SYBR green were used to analyze the TTV RNA expression. The Maxima SYBR green Master Mix (Thermo Fisher Scientific) with the following oligonucleotides was used: TTV1 forI 5′-GACACAGAACTCACAGCCC-3′, TTV1 forII 5′-GACACTGACGTGACAGCCG-3′, TTV1 rev 5′-GTTAGTGGTGAGCCGAACG-3′. The PCR was performed with 10 pmol of each oligonucleotide targeting the VP1 region of the TTV genotype 1, at an annealing temperature of 60°C, according to the protocol recommended for the Maxima SYBR green Master Mix.Spearman correlation was employed for analysis of the correlation coefficient between TTV levels in plasma and lymphocyte as well as granulocyte counts on days +30, +60, and +100 post-transplant. The calculations were performed by employing the GraphPad Prism 5 software.in vivo T cell depletion (involving primarily ATG), day of engraftment, and grade of GvHD, if present. In TTV positive patients, the median virus load detected before transplantation and until day +30 post-transplant was 1 × 10E4 DNA copies per ml plasma, with rapidly rising levels after day +30. The peak was generally reached around day +100, with a median of 7 × 10E7 (range: 7 × 10E3 – 4 × 10E9) virus copies per ml plasma .Elevated TTV titers were previously described primarily in adult solid organ transplant (SOT) recipients and in a small number of recent studies in patients undergoing HSCT . In the a Figure . The TTVa Figure . After da Figure . Monitora Figure . A signi+, CD8+ cells and granulocytes increased by more than one log within this time interval. Subsequently, after day +30 post-transplant, following T cell and neutrophil engraftment, rapidly rising TTV DNA copy numbers were observed, with peak levels around day +100 . However, the TTV plasma levels correlated significantly with the granulocyte counts on days +30 and +60  isolated either from PB or BM tested invariably positive in post-transplant samples of all patients analyzed . To assess the presence of TTV in various leukocyte subsets, T and B lymphocytes , NK-cells, monocytes and granulocytes were isolated from PB of all 30 patients by flow-sorting, and 4,000 cells of each subset were used for DNA extraction and RQ-PCR analysis. Based on the observation of TTV positivity in granulocytes and monocytes of HSCT recipients, an additional 100,000 cells of these leukocyte subsets and T cells were collected to increase the sensitivity of virus detection. Of the cell types analyzed, only granulocytes tested positive for TTV, and the virus was detected in DNA samples derived from two additional patients in whom TTV was undetectable in DNA isolated from 4,000 cells. However, only 10/30 (33%) patients from the control cohort had TTV detectable in granulocytes, and the virus copy numbers were lower than in transplant recipients . In line with this observation, 5 of the 19 transplant recipients investigated (26%) were positive for TTV in granulocytes already within the first month post-HSCT, and the intracellular virus copy numbers were similar in both indicated settings  of the PB specimens and in 22/30 (73%) of the plasma specimens analyzed. In all instances, the virus loads detected in whole PB samples were considerably higher than in plasma samples of the same patients, suggesting common TTV association with cellular elements Figure . The med Figures .In order to address the notion that granulocytes are not the only TTV reservoir in PB of immunocompetent children, erythrocyte and thrombocyte preparations were investigated for presence of the virus. Erythrocytes isolated from ten patients of the control cohort, seven of whom had tested TTV-positive in leukocyte preparations, tested negative for the virus. Moreover, platelet concentrates from 14 different healthy donors were analyzed for the presence of TTV, and the virus was detected in six instances (43%). The median virus copy number per platelet determined by RQ-PCR was 0.6 range 0.2–7.5). To determine whether the TTV positivity found in platelet concentrates might be attributable to residual plasma present in the preparations, densely packed platelets derived by centrifugation of the concentrates were tested in parallel by RQ-PCR and revealed identical results in three of the six positive samples, suggesting that platelets can carry either internalized or adherent particles of the virus. The distribution and quantity of TTV in different PB cells are summarized in Table –7.5. To Several studies suggested a correlation of increasing TTV levels in PB and plasma with the immune status of patients after transplantation, and indicated the possibility of exploiting post-transplant quantitative TTV analyses in SOT and HSCT recipients particularly as a surrogate marker for immune reconstitution . HoweverIn agreement with earlier studies, our analysis of plasma specimens derived from HSCT recipients showed increasing TTV loads during the first 3 to 4 months post-transplant , followe+ cells representing neutrophilic granulocytes, the most abundant fraction of the granulocytes, and expansion of the virus generally starts 1 month after transplantation, upon engraftment of these cells. This finding might suggest that sufficiently high granulocyte numbers might be a prerequisite for efficient TTV expansion post-transplant, and could therefore provide a possible explanation for the delayed onset of rapid replication of the virus. Indeed, TTV copy numbers revealed a significant correlation with the number of neutrophils at +30 and +60 days after HSCT. A previous study reported a drop of TTV levels in patients treated with anti-thymocyte globulin (ATG) which was more pronounced than in patients treated with basiliximab, a weaker T lymphocyte inhibiting antibody. The possible effect of anti-T lymphocyte drugs on TTV levels provided the basis for the suggestion that T lymphocytes are the host cells for TTV replication (10 lower levels in other hematopoietic cells. Our findings indicated not only the presence of the virus in granulocytes but revealed a clear increase of TTV copy numbers in these cells with peak levels around 100 days post-transplant. It is necessary to consider that the life span of peripheral granulocytes is very short, generally within the range of a few days only, but our analyses of myeloid cells derived from the bone marrow also showed evidence for the virus. Although granulocytes are an important site of TTV replication, as demonstrated by the data presented, they may not serve as host cells for persistence of the virus. The exploitation of diverse cell types for persistence and replication of individual viruses is a well-known phenomenon. Viral persistence requires the infection of cells with a long life span, whereas replication, which is commonly associated with cell lysis, can occur in short-lived cell types providing the environment conducive for viral multiplication. In line with this notion, similar studies on human adenoviruses performed by us and others showed persistence of the virus in tonsillar and mucosal T lymphocytes (We observed elevated TTV copy numbers not only in plasma but also in stool specimens tested at the same time points, which might be attributable to the shedding of leukocytes carrying the virus into the intestines. This finding supported the possibility that TTV replication occurs in specific leukocyte subsets, and this notion was assessed by testing individual cell types isolated by flow-sorting at different time points after HSCT. Our data obtained in pediatric patients show that replication of the virus occurs in CD15lication . This cophocytes , whereasphocytes . AlthougThe observation of rather constant TTV plasma levels during the first month post-transplant, the detection of rising plasma virus levels with documented replication in granulocytes during the ensuing 2 to 3 months, and the subsequent gradual return of the virus copy numbers to pre-transplant levels in the pediatric HSCT recipients investigated prompted us to compare the findings to a control cohort of immunocompetent children. The occurrence of TTV positivity in the plasma of patients prior to HSCT reflecting the state of persistence was very similar to the control group, revealing 79 and 73% of virus-positive individuals, respectively. However, TTV was identified in granulocytes in only one third of the children within the control cohort, while all HSCT recipients invariably tested positive for the virus in granulocytes by day +50 post-transplant. This implies that the virus is also carried by other cells in the immunocompetent control patients. However, in agreement with the findings in HSCT recipients, we could not detect the virus in isolated lymphocytes. In search for a carrier of TTV in PB, we identified presence of the virus in plasma-depleted platelet concentrates, raising the possibility that TTV may be taken up or adhere to thrombocytes. This notion is supported by the fact that several platelet receptors are known to bind viruses such as Epstein-Barr virus, hepatitis virus C, adenovirus, rotavirus, or human immunodeficiency virus , but litKK designed the study and carried out the experiments, analyzed and interpreted the data, and wrote the manuscript. SK carried out the experiments and analyzed the data. HP, AL, and VW provided patient samples and were involved in data collection. RG was involved in data analysis and interpretation. TL designed the study, was involved in data interpretation and manuscript preparation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Potyvirus, Allexivirus and Carlavirus have been reported. Aiming for an unbiased identification of viruses from a garlic germplasm collection in Brazil, total RNA from eight garlic cultivars was sequenced by high-throughput sequencing (HTS) technology. Although most viruses found in this study were previously reported, one of them did not belong to any known genera. This putative new virus was found in seven out of eight garlic cultivars and phylogenetic data positioned it as representative of an independent evolutionary lineage within family Betaflexiviridae. This virus has been tentatively named garlic yellow mosaic-associated virus (GYMaV), sharing highest nucleotide identities with African oil palm ringspot virus (genus Robigovirus) and potato virus T (genus Tepovirus) for the replicase gene, and with viruses classified within genus Foveavirus for the coat protein gene. Due to its high frequency in garlic cultivars, GYMaV should be considered in upcoming surveys of pathogens in this crop and in the development of virus-free garlic plants.Plant vegetative propagation strategies for agricultural crops cause the accumulation of viruses, resulting in the formation of virus complexes or communities. The cultivation of garlic is based on vegetative propagation and more than 13 virus species from the genera  Allium sativum L.) is one of the most consumed vegetables in the world with triennial world production (2011–13) of over 23 million tons  has drawn attention for infecting both garlic and onion , garlic virus X (GVX), and garlic mite-borne mosaic virus (GMbMV)  are oftend onion . IYSV isnd onion .Betaflexiviridae .In this study, we identified viruses present in different garlic cultivars from the germplasm collection of EMBRAPA Hortaliças, Brazil. The majority of the viruses found in these samples were previously reported, except for a new virus putatively classified as a member of new genus in the family Branco Mineiro, Cateto Roxo, Amarante, Gigante Lavinia, Moz 2014 Africa, Ito, San Valentin, and Chonan. All of them are planted commercially in Brazil and are classified in three main groups  according to their climate requirements for bulbification. Temperatures around 20 °C, below 15 °C, and below 10 °C are required for proper bulbification of early , medium , and late  garlic cultivars, respectively. In addition, all these plants displayed yellowish mosaic in their leaves during vegetative development.The eight garlic cultivars analyzed in this study are part of the germplasm collection of the Brazilian Agricultural Research Corporation on Vegetables , Brazil. These cultivars are known as ® Mini kit  according to the manufacturer’s instructions. For high-throughput sequencing, the RNA samples were combined together (RNA pool). cDNA libraries and sequencing (2 × 100 bp read length) on the HiSeq 2,000 platform were performed at Macrogen Inc. . The generated reads were trimmed and de novo assembled using CLC Genome Workbench 6.5.2 . Contigs related to viruses were retrieved using Blastx against a RefSeq virus database. To determine whether the assembled contigs corresponded to complete virus genomes, they were compared with complete virus genomes deposited on public databases using Geneious 7.1.8  reaction followed by polymerase chain reaction (PCR) amplification. Complementary DNA sequences (cDNAs) were synthesized using SuperScript III reverse transcriptase  and random hexanucleotides. Then, PCR reactions were performed using PCR Master Mix  and specific primer pairs for each one of the detected viruses . NucleotBetaflexiviridae was built based on the deduced amino acid (aa) sequences of the replicase and coat protein (CP) genes. For the cophylogeny trees, aa sequences of both replicase and CP were used. Multiple alignments were performed using the MAFFT method , Carlavirus (GCLV and GLV) and Potyvirus (OYDV and LYSV). Surprisingly, a new virus genome sequence which had close relationship to viruses of the family Betaflexiviridae was also found , was deposited on the GenBank database under the accession number MH120170 .Betaflexiviridae  and African oil palm ringspot virus . On the other hand, the GYMaV CP shares 64%, 62%, and 61% nt identity, respectively, with peach chlorotic mottle virus (EF693898), apple stem pitting virus (D21829), and apricot latent virus (HQ339956), all members of genus Foveavirus. These values are well below the accepted species discrimination level of 72% nt identity for both CP and replicase , and a CP . The leniviridae , a pairwiviridae . GYMaV replicase . Even thBetaflexiviridae (Robigovirus and the unassigned banana mild mosaic virus (AF314662) using replicase proteins. In contrast, GYMaV clustered within genus Foveavirus in CP phylogeny as suggested by pairwise comparisons  of ICTV recognized species in the family iviridae . Despiteparisons  and S3. parisons , bringinAiming the identification of garlic-infecting RNA viruses following an unbiased approach, total RNA from eight garlic cultivars was high-throughput sequenced. Overall, virus isolates taxonomically classified in ten virus species were identified, nine of them having been previously reported in garlic VCs . The bioBetaflexiviridae encompasses two subfamilies (Trivirinae and Quinvirinae) that together include eleven genera . GYMaV shares the highest nt identities with African oil palm ringspot virus (genus Robigovirus) and potato virus T (genus Tepovirus) for the replicase  and with viruses classified in the genus Foveavirus for the CP (61–64% nt identity). According to the ICTV, viruses of suggested new genera are supposed to be less than 45% nt identical in those genes with viruses already reported , was found in seven out of eight garlic cultivars tested. The presence of GYMaV in most cultivars indicates that it has likely been spread by vegetative propagation. However, its transmission by an insect vector should be not ruled out. Currently, the family reported . Howeverxviridae , and thexviridae .Trivirinae or Quinvirinae, respectively. Although the replicase and CP genes are used for genus demarcation, our analyses reinforce the concept of modular evolution, showing that these genes and protein products are phylogenetically incongruent. Thus, either one or another should be used for taxonomical purpose. Our analyses also suggest that either GYMaV underwent recombination or that these genes have different mutation rates due to different selection pressures.GYMaV virions may be shaped as flexuous filaments as observed for other betaflexiviruses . With a GYMaV as a component of garlic VCs should be considered in the development of virus-free garlic varieties. Many surveys of garlic viruses previously reported were based on target specific methods, since specific detection tools were utilized . AlthougBetaflexiviridae, cophylogeny analysis pointed that these genes sort out the betaflexiviruses differently.GYMaV is a putative new betaflexivirus found in virus complexes of several garlic cultivars. Based on its high frequency in these plants, GYMaV is likely to be vegetative propagated like other viruses previously reported in such complexes. Although the replicase and CP genes are used as taxonomical criteria for genus demarcation of the family 10.7717/peerj.6285/supp-1Figure S1Abbreviations: triple gene block protein (TGB), Movement protein (MP), and Coat protein (CP).Click here for additional data file.10.7717/peerj.6285/supp-2Figure S2The numbers indicate the percentage of identical nucleotides or amino acids upon pairwise alignment.Click here for additional data file.10.7717/peerj.6285/supp-3Figure S3The taxa included CTV recognized species of different genera apart from viruses that are unassigned or unclassified as seen on NCBI. This tree was built on the CP predicted amino acid sequences of betaflexiviruses that have their ORFs completely sequenced.Click here for additional data file.10.7717/peerj.6285/supp-4Supplemental Information 1E-mail messages regarding submission of a taxoomic proposal of new plant virus genome sequence to ICTV.Click here for additional data file.


Rapid genomic changes during a coevolutionary arms race highlight the reciprocal effects of ecology and evolution. Species interactions and coevolution are integral to ecological communities, but we lack empirical information on when and how these interactions generate and purge genetic diversity. Using genomic time series data from host-virus experiments, we found that coevolution occurs through consecutive selective sweeps in both species, with temporal consistency across replicates. Sweeps were accompanied by phenotypic change (resistance or infectivity increases) and expansions in population size. In the host, population expansion enabled rapid generation of genetic diversity in accordance with neutral processes. Viral molecular evolution was, in contrast, confined to few genes, all putative targets of selection. This study demonstrates that molecular evolution during species interactions is shaped by both eco-evolutionary feedback dynamics and interspecific differences in how genetic diversity is generated and maintained. Host-pathogen interactions are commonplace and play a major role in many ecological and evolutionary processes  and initially isogenic double-stranded DNA (dsDNA) virus (PBCV-1) in replicated chemostat systems (n = 3)  to identify time points when resistance and infectivity evolved. In intervals with a phenotypic match, selection for host resistance was strong, while we expect virus populations to increase in size. On the other hand, a phenotypic mismatch warrants pressure on the virus populations to evolve new means of infectivity, while it allows the host to grow without antagonistic restraints. Experiments started with a phenotypic match, and then match and mismatch alternated, followed by a phenotypic mismatch after the generalist has evolved.To follow evolutionary change on the genome level, we sequenced whole genomes of samples from 12 time points for each replicate to obtain population data (Pool-seq) for both species simultaneously. For analysis, we exclusively focused on single-nucleotide polymorphisms (SNPs) absent from the ancestral population, hereafter called derived alleles (table S1). To unambiguously distinguish variants from sequencing errors, we only report SNPs that reached appreciable frequencies at more than one time point. Because of differences in sequencing coverage between the species, variants never or only once reaching a frequency >0.05 in the virus or >0.25 in the host were excluded . Because the experiments were started with an isogenic culture grown from a single cell, we cannot exclude that some of the observed variants were already present at a frequency below our detection limit in the starting culture. It is, however, unlikely that low-frequency variants are particularly common in a freshly grown culture. Hence, the genomic change presented excludes various technical artifacts and rare and low-frequency variants but adequately represents the variation governed by selection and selective interferences.Dynamics of molecular change play out differently between the two species. The total number of derived alleles per replicate was 388 ± 83 in the host and 9 ± 2  in the virus populations, respectively. In the host populations, more than half of the observed mutations (211 ± 55) were synonymous or occurred in intergenic regions . The majOur temporal resolution allowed us to detect the signature of a selective sweep in the host populations at two time points. In every replicate, within-population diversity  markedlyChlorella .For each time point, we compared the distribution of variant allele frequencies (VAFs) in the host to a neutral null model. As such, the classic Luria-Delbrück model predicts that the distribution of VAFs can be described by a simple power law distribution, assuming a neutral population expansion . The probability of observing the same mutation more than once by chance is small (table S2), hence suggestive of the action of natural selection. More than half of the repeated mutations (219 ± 35) were already observed before the introduction of the virus at day 12, and they might provide a benefit in the abiotic environment or during competitive growth rather than confer resistance. We expect variants in genes affecting resistance to increase in frequency at time intervals leading up to those time points where sweeps were detected, and we evaluated which SNPs fit this criterion. Potential candidate genes for the evolution of resistance are genes affecting outer cell wall composition, alterations of which would prevent viral recognition of the host cell. However, we found no primary candidate genes among the high-frequency and repeatedly found SNPs at days 27 and 64 (tables S3 and S4). This could be because resistance was encoded by a larger structural variant . All five were nonsynonymous, and three of them—among which the one mutation observed in every replicate—altered the protein structure of the gene Adaptation by natural selection is based on the premise that genetic variation, which enhances reproductive success, increases in frequency over generations. Here, we have documented frequency change on a fine temporal scale, in an experimental system comprising two species that are each other’s primary selective agent. Recent experimental studies on molecular evolution in host-virus systems showed that coevolution accelerates molecular evolution ], our chemostat setup allowed the coevolutionary interaction to unfold without interference with population sizes  as previously described . For virus enumeration, samples were fixated with 1% glutaraldehyde, flash frozen in liquid nitrogen, and stored at −80°C. Virus particles were counted using flow cytometry  following  was compared with the growth of the algal clone in the absence of the virus. Growth rates were calculated on the basis of optical density measurements  measured at time point 0 and after 72 hours. A host clone was deemed susceptible when the mean growth rate plus 2 SDs of four technical replicates in the presence of the virus was lower than mean growth rate minus 2 SDs of the control or when mean growth rate in the presence of the virus was negative. Resistance and infectivity ranges were calculated as the proportion of virus populations a host individual was resistant to and the proportion of host individuals a virus population could infect, across sampled time points. Growth rates of algal clones measured in the absence of virus were correlated to resistance range to estimate trade-offs between resistance and growth rates.We defined phenotypic match and mismatch intervals based on resistance and infectivity evolution over time. Resistance evolution occurred when hosts from a given time point became resistant against virus from their past and contemporary time points . Infectivity evolution occurred when virus became able to infect previously resistant hosts, i.e., when contemporary virus or virus from the next time point could infect hosts, whereas the same hosts could not be infected by virus from past time points . As we do not have continuous data, we marked these transitions between the different stages by arrows in the infection matrix. Note that we only took observations into account when the phenotype was maintained in the next time step. Because we were only interested in identifying when resistance and infectivity evolved, we marked transitions at the time point when a phenotype was first observed and did not take into account the frequency at this time point.DNA was extracted from frozen and concentrated samples using the DNeasy Blood and Tissue Kit (Qiagen) with minor modifications. First, 100 μl of buffer ATL + 30 μl of Proteinase K were added to 200 μl of concentrated sample, followed by an incubation at 56°C for 4 hours, continued by adding 600 μl of 1:1 buffer AL + ethanol mix and proceeding by following the standard column-based protocol. Paired-end libraries were prepared using a Nextera XT library preparation kit. Twelve time points from one replicate (replicate 3) were sequenced in four runs of an Illumina NextSeq (Illumina NextSeq 500 high output) machine. Twelve time points from the other two replicates of coevolution were first sequenced in five runs. The amount of DNA obtained for host and virus was correlated to their relative population sizes, which, for some time points, led to comparatively low genome-wide coverage for the host. Six more NextSeq runs were performed to mitigate this issue and increase coverage for selected time points where we anticipated low host coverage.https://github.com/jstjohn/SeqPrep) to merge read pairs in case forward and reverse reads overlapped and subsequently remove reads shorter than 70 base pairs (bp). Reads were mapped with bwa mem v0.7.12 with slightly increased accuracy setting (-r 1) (http://broadinstitute.github.io/picard), removed alignments with quality 0 with awk, and merged data per time point (across lanes and sequencing runs) using SAMtools v1.3  obtain only those alleles with patterns driven by selection, following other genomic time series analyses  have comparable growth rates , the expected distribution of VAFs generated by the underlying stochastic process of de novo mutation follows a Landau distribution  = 2.4 × 10−9 substitutions/position/cell division. While there are no good estimates of the mutation rate in Chlorella, such a rate is plausible. As the virus genome is orders of magnitude smaller (~330 kb), observing comparable power law dependencies would require a mutation rate of 1/(330 × 103 × 10) = 3.0 × 10−7 substitutions/position/cell division. Although such rates have been observed for DNA viruses, no increases in genetic diversity after sweeps were observed. We attribute this to the fact that small virus genomes are densely packed with protein-coding regions; hence, neutral positions are rare. Because of the low number of variants, we did not attempt to do statistics on the viral VAF distribution.Considering a selective sweep, when a single highly adaptive cell grows exponentially, then to reach a frequency of 10% by hitchhiking alone, any neutral mutation needs to occur during the first 10 cell divisions this cell undergoes. This means that with a genome size of ~42 Mb, observing power law dependencies and rapid neutral allele frequency increases of 10% during sweeps are feasible if the We performed a logistic regression of time points where the VAF distribution matches the expectation under neutral expansion  on host population growth  using the R package lme4 (P < 10−5). Because of, i.a., random variation in sequencing coverage and complex genomic regions, not every position of the reference genome can be reliably evaluated; hence, reference genome size is an overestimate of vase size. To get an idea of how large the pool of mutable sites need to be to reproduce the empirically observed overlap, we repeated the simulations for a grid of vase sizes using the empirical number of observed mutations (minimum across three replicates) as the total number of marbles drawn and reported the number of marbles drawn more than once  in table S2.Assuming that mutations evolve de novo, are neutral, and are uniformly distributed across the genome, every genomic position has the same probability of acquiring a mutation. Under these assumptions, observing a mutation in our experiments is equivalent to randomly drawing marbles from a vase without replacement. The size of the vase is equivalent to the number of genomic positions we evaluated with our sequencing approach, and the sample size (number of marbles drawn) equals the number of mutations observed per replicate. To assess how likely the empirically observed overlap is, we simulated such a sampling procedure using R and found that the probability of the observed overlap  is extremely small (


Avian avulaviruses serotype 1  are capable of infecting a wide spectrum of avian species with variable clinical symptoms and outcomes. Ease of transmission has allowed the virus to spread worldwide with varying degrees of virulence depending upon the virus strain and host species. The emergence of new virulent genotypes from global epizootics, and the year-to-year genomic changes in low and high virulence APMV-1 imply that distinct genotypes of APMV-1 are simultaneously evolving at different geographic locations across the globe. This vast genomic diversity may be favoured by large variety of avian species susceptibility to APMV-1 infection, and by the availability of highly mobile wild birds. It has long been considered that waterfowls are not sensitive to APMV-1 and are unable to show any clinical signs, however, outbreaks from the 90′s contradict these concepts. The APMV-1 isolates are increasingly reported from the waterfowl. Waterfowl have strong innate immune responses, which minimize the impact of virus infection, however, are unable to prevent the viral shedding. Numerous APMV-1 are carried by domestic waterfowl intermingling with terrestrial poultry. Therefore, commercial ducks and geese should be vaccinated against APMV-1 to minimize the virus shedding and for the prevention the transmission. Genetic diversity within APMV-1 demonstrates the need for continual monitoring of viral evolution and periodic updates of vaccine seed-strains to achieve efficient control and eradication of APMV-1 in waterfowls.The online version of this article (10.1186/s13567-018-0587-x) contains supplementary material, which is available to authorized users. Avian avulavirus 1 (APMV-1), which belongs to the Avulavirus genus within Paramyxoviridae family. All APMV-1 strains can be classified into velogenic (highly virulent), mesogenic (intermediate virulent) and lentogenic (non-virulent) based on the intracerebral pathogenicity index (ICPI) in day old specific pathogen free (SPF) chickens [Newcastle disease (ND) is one of the most devastating and commonly prevalent diseases in the poultry industry, around the world. Owing to immense economic losses, World Organization for Animal Health has categorized the disease as “notifiable” . The dischickens , 5 or nachickens , 6.APMV-1 is a single-stranded, negative-sense, non-segmented and enveloped RNA virus with genome length of 15.2 kb. APMV-1 genome encodes for six co-linear genes that translate into six proteins and two non-structural proteins. Structural proteins include nucleoprotein (NP), matrix (M), fusion (F), hemagglutinin–neuraminidase (HN), phosphoprotein (P) and large RNA-dependent RNA-polymerase (L). The RNA editing of the P gene can result in the expression of V and W only in the virus infected cells –10. ViruGenerally, waterfowls were considered to be the natural reservoir for APMV-1 . TraditiThe purpose of this review is to analyze our current understanding on the host-spectrum, molecular pathobiology of APMV-1 in waterfowls, and host immune responses that may play crucial roles in the disease prevention and control.The Class I APMV-1 isolate was reported in 2006, however, currently these isolates are frequently being reported. Recent epidemiological studies direct that class I APMV-1 are common in domestic waterfowl. All viruses belong to class I are avirulent except JS10-A10 and 9a5b strains , 20 whicMost of our understanding on the surveillance of APMV-1 in wild birds came from epidemiological studies on avian influenza viruses. Thus, it is required to design APMV-1 dedicated studies to effectively assess the true prevalence of the virus in wild birds. It is essential to understand and establish the foundations to devise control strategies, especially in wild-birds populated and highly vulnerable commercial poultry areas.Waterfowls are less susceptible to APMV-1 compared to chickens, such as ducks and geese. A key reason for less susceptibility of waterfowl to APMV-1 is the presence of retinoic acid-inducible gene I (RIG-I). RIG-I is absent in the chicken  whereas APMV-1 infection increases the expression of interleukin (IL) IL-1β, tumor necrosis factor-α-like factor and interferon (IFN)-β in duck embryo fibroblast (DEF) cells. Distinct innate immune responses of the waterfowl against APMV-1 may reason the resistance of waterfowls to these infections . A higheBesides the strong innate immune responses, waterfowl are generally considered long-term carrier of APMV-1 and disease outbreaks have been reported since 1997 –14, and APMV-1 infected ducks and geese show the gross lesions on the immune organs such as bursa, spleen, thymus, mild to severe tracheitis, kidney enlargement, necrosis of pancreas, congestion on the meninx and in the brain and diffuse brain edema, focal hemorrhages in the mucosa of the proventriculus and intestine  , 23, 46.Experimental infection studies are necessary to determine the virulence and pathogenesis, in different bird types, age, species, intervention strategies, evaluation, comparison of vaccines etc. Different scientists propose diverse reasons for experimental infection studies and the design of the experimental infection studies varies greatly with the above-mentioned factors. Experimental infection studies on the pathogenesis, infection route, most susceptible age, bird line and immune responses are limited in waterfowl and available information is summarized in Additional file Kang et al.  have stuIntranasal inoculation of the Japanese commercial ducks and chicken males with artificially made APMV-1 class I virus 9a5b results in the higher IFN-β in the duck compared to chicken. This study also demonstrated that replication, distribution, tissue damage and apoptosis were more in the immune organs of the chicken compared to duck . IntramuIn conclusion, these studies indicate that different viruses affect the immune organs and innate immune genes in diverse mechanisms. Waterfowls are more susceptible to APMV-1 infection at an early age, and through natural route results in less damage to immune organs. More comprehensive and detailed studies are warranted for the control of ND in waterfowl.Waterfowl are naturally infected with large number of the viruses which are avirulent and do not cause the diseases in domesticated poultry. Waterfowl are commonly considered to be the natural host as well as carrier of APMV-1 . APMV-1 Waterfowl and shorebirds are not only the host, but are also infected by the APMV-1 and these viruses can also cause the disease in domesticated poultry , 56, 64.APMV-1 infected waterfowls shed the virus for an extended period of time  whereas,In conclusion waterfowl plays a vital role in the transmission and re-emergence of ND in terrestrial poultry. It is recommended that rearing facilities of the waterfowl should be separated from commercial poultry.Cumulating evidences indicate that genetic resistance to APMV-1 exists in various breeds of waterfowl . ComprehAlthough constant outbreaks of ND have been reported in waterfowl from China and other East Asian countries, however, vaccination against ND in waterfowl is still a matter of debate. There are two consortia of scientists; one favors the vaccination and, others think that domestic waterfowl should not be vaccinated. These veterinarians have their own views, scientist, in the favors of vaccination argue, that it will decrease the chances of outbreaks and lessons the virus shedding. But, scientists, which are against vaccination argue that it will increase the virus burden on the birds, consequently increase the variation rate. The annual rate of change of virulent viruses could be as high as ten times the rate of change of low virulence APMV-1, suggesting that other selective pressures such as vaccination may accelerate the rate of evolution of virulent viruses , 72. TheWaterfowls are not only the carrier but also susceptible to APMV-1. Strong innate immune responses may attribute to the less susceptibility of ND to waterfowl. Viral shedding by waterfowl for prolonged duration may increase the transmission, evolution and emergence of new viral strains. A number of APMV-1 isolates from the waterfowl are reported with high mutation rate, which is an alarming matter and may cause the endemics in future. Pathogenicity of ND is affected by different factors such as type, dose, and inoculation route of the virus, and species and age of birds.Further studies are needed to explore the mechanism, and its intervention to prevent the virus shedding by the waterfowl for a long period. Continuous studies are also required to monitor the APMV-1 in the waterfowl, which may be the future threat to commercial poultry industry. Studies on the humoral immune responses of waterfowl are crucial to develop better intervention strategies. It is recommended that rearing facilities of the waterfowl including ducks and geese should be separated from chicken and turkey flocks to prevent the virus transmission.Additional file 1.Experimental infection studies of ducks and geese with Avian Avulavirus 1. Table summarizes the pathobiological findings and immune responses of waterfowl after experimental challenge with Avian Avulavirus 1.