This is a retrospective chart review of 55 persons  diagnosed with fetal alcohol spectrum disorder (FASD) in one Finnish central hospital. The aim was to determine typical problems and interventions and estimate their costs during different periods of childhood between ages 0 and 16. During the first year, 29/38 (76.3%) were treated in the neonatal intensive care unit, 29/43 (67.4%) received physiotherapy, 15/43 (34.9%) were diagnosed with congenital malformation, 8/43 (18.6%) had heart defects. Between 1 and 6 years, 39/45 (86.7%) received occupational therapy, 25/45 (55.6%) speech therapy, and 12/45 (26.7%) were diagnosed with strabismus. Between 7 and 12 years, 25/37 (67.6%) were diagnosed with ADHD and special education was recommended for 30/37 (81.1%). Learning disorders and the need for psychiatric care increased with age. Between 13 and 16 years, 12/15 (80%) were treated in the psychiatric unit, and by this age, 8/15 (53.3%) were diagnosed with intellectual disability. Before 16 years, 44/55 (80%) were placed out of home, which caused 78.5% of the estimated cumulative mean extra costs of EUR 1,077,000 in 2022 currency. Except for psychiatric costs, health care costs were highest during early years. Charting typical patterns of problems may help in identifying children with FASD and planning follow-ups, content of assessments, and interventions. FASD is a spectrum of structural, neurobehavioral, and developmental disorders that are primarily caused by harmful effects of prenatal alcohol exposure , but somThere are multiple different diagnostic criteria for FASD used in the world with subtle differences that are beyond the scope of this study . In FinlIn this study population, the IOM’s 2005 criteria were used for those diagnosed before 2017, and for those diagnosed 2017 or later, the IOM’s 2016 criteria were used. The main difference between the use of IOM 2005 and IOM 2016 criteria in Finland is that the 2016 criteria allow diagnosing FAS and PFAS even when documented maternal alcohol use during pregnancy is not available, which sometimes is the case in adopted or fostered children.Using school-based active-case-ascertainment studies, the prevalence of FASD was determined to be 1–5% in countries and areas with similar female alcohol consumption amounts and patterns as in Finland ,7, but cFASD has been considered the leading preventable cause of developmental disabilities in the world . Many diAdolescents and adults with FASD have a high risk for disrupted education and being institutionalized either in prison or facilities for persons with psychiatric and substance use disorders, but an early diagnosis of FASD and a stable living environment may have the potential to reduce the risk for these adverse outcomes ,10. It hThere is currently a lack of knowledge and understanding regarding the emergence and evolution of problems associated with FASD during an individual’s development and what the needed interventions are, their cost, and when they should be introduced.Because there is a severe problem of underdiagnosis, there is a need for knowledge of the typical age-specific patterns of problems associated with FASD to be able to better identify persons with possible FASD for evaluation and planning of interventions. Because early intervention and prevention of secondary problems is typically more cost-efficient than trying to mitigate already emerged and chronic problems, we also need to anticipate the trajectories of typical problems. To reduce the societal costs associated with FASD, we also need to know the cost structure.In Finland, there is an extensive and reliable unified electronic patient register within each hospital district. In addition, the Patient Data Repository (Kanta) is a national information system service for archiving electronic patient data produced in the healthcare service. The Client Data Act obliges all public and private healthcare providers to join the Kanta. Furthermore, all medication prescriptions are saved in the Prescription Centre, which is a centralized database for prescription data as a part of the Kanta. The data stored in the Kanta archive are accessible by healthcare professionals and can be used for gathering a patient’s health history during the clinical evaluation. This enables performing detailed studies based on electronic patient registers.In Finland, FASD is typically diagnosed in pediatric neurology outpatient clinics in hospitals and sometimes in pediatric or adolescent psychiatric outpatient clinics or inpatient wards in hospitals. In both pediatric neurology and psychiatric clinics, patient entries are typically very detailed and include a wide range of information concerning patients’ health and family history, developmental history, results of all assessments and evaluations by a multidisciplinary team, as well as reports from therapies, day care, and schools. They also include recommendations for rehabilitation, disability allowance, and special school arrangements. Patient register data are therefore not limited to health information but allow for more complete analysis of problems, interventions, and their costs.What were the most common diagnoses and problems in everyday life for those diagnosed with FASD within four age groups: infancy, preschool age, primary school age, and adolescence?What were the typical interventions in different age groups?What were the estimated additional costs associated with problems and interventions in different age groups?In this detailed retrospective descriptive electronic patient register-based chart review study of all patients diagnosed with FASD in one Finnish central hospital, we wanted to discover the following:-P04.3 ;-Q86.0 ;-F83  or F81.3 (Mixed disorder of scholastic skills) or F90 (ADHD) and had FASD or ARND listed as a written sub-diagnosis, or maternal alcohol use during pregnancy was listed as the etiology for developmental disorder.In Päijät-Häme Central Hospital, which is a secondary-level hospital in southern Finland with a catchment population area of 220,000, we included all persons who had been patients  in the Päijät-Häme Central Hospital between 2010 and 2022 and had one of the following ICD-10 diagnoses:Available information was collected from the patient register between 1 October 2021 and 30 September 2022. A total of 55 persons were included. All those with the initial diagnosis of P04.3 had later been diagnosed as having some type of FASD.There was not enough reliable information about the quantities or timing of alcohol exposure to include it in analysis. In eight patients, alcohol exposure was reported to be accidental due to late recognition of pregnancy, and in them the reported exposure varied from heavy exposure in early pregnancy to repetitive moderate exposure during the whole pregnancy because the pregnancy was not recognized before labor began. In others, the diagnosis of maternal alcohol use disorder was considered as the reason for alcohol exposure. In some patients, the prenatal alcohol exposure had been recognized and addressed already during prenatal care, but the information concerning timing of recognition, interventions provided during pregnancy and their effectiveness were not available. In others, the information about alcohol use during pregnancy or maternal alcohol use disorder was postnatally retrieved from the mother, close relative or social worker. We did not have access to maternal health records.In Finland, out-of-home placement is considered as the last resort when all other support interventions for the family have failed, or when there is a severe acute crisis in the family and milder interventions are not sufficient. Still, the majority of the patients diagnosed with FASD were in foster care or had been adopted by the time of the first evaluation in the hospital. Maternal alcohol use disorder is typically at least one of the reasons why the child is taken into custody by child protection services and placed out of home.We determined that the data collected from hospital records were limited because full hospital records were available only for the period when the patients were living at the Päijät-Häme hospital district, leading to varying lengths of follow-up times . Data coThe electronic patient register was introduced in 2010, which means that full information before 2010 was available only in paper records. The most important cause of missing information was out-of-home placements and the subsequent moving of patients from one hospital district to another. Even with the national Kanta records, some patients were unavailable for follow-up because they moved out of the hospital district and the research permit did not allow searching the Kanta for information of patients who were no longer living in the hospital district. The Kanta was introduced between 2010 and 2014 and the use of electronic prescriptions was not mandatory before 2017. This means that even for patients living in the hospital district, older information from other hospital districts was not available. For those who moved from other districts or countries to the research district, referral information was often incomplete. Further, foster parents could not help with missing history, because medical information is typically not disclosed to the foster parents by the authorities.There was very limited information available during adolescence, especially between ages 16 and 18, because pediatric neurology clinics and pediatric clinics treat and follow patients only up to 16 years of age. To have more complete information, we decided to limit analysis to 0–16 years of age.From the unified electronic health record system used in the Päijät-Häme Central Hospital, of those 55 persons diagnosed with FASD, we collected information from birth and all hospital visits either as inpatient or outpatient, developmental assessments, rehabilitation interventions, long-term medications for chronic conditions, referrals from basic health care, support arrangements and their levels at kindergarten and school. Each type of information was analyzed descriptively divided for age groups to determine typical problems and interventions at each age. The data were collected mainly by one person (LMJ); some information was collected by HS.The collected birth information included gestational age, birth measurements of length, weight, head circumference and their standard deviation Z-scores, Apgar scores, pH of umbilical artery, treatment in neonatal intensive care and surveillance unit (NICU), and neonatal diagnoses. Available information was collected concerning maternal use of tobacco, drugs—both legal and illegal—and alcohol during pregnancy.We collected all hospital outpatient and inpatient examination and treatment data including main diagnoses, expensive examinations such as brain MRI, sleep–wake EEG, molecular karyotype/chromosomal microarray and fragile-X-examination, patient age at the visits and prescribed long-term medications. We collected the type of FASD  and age at the diagnosis. Main diagnoses in each age group were analyzed descriptively to determine most common diagnoses indicating typical age-specific problems in the study population.Developmental information that was collected included the test results of the assessments by occupational therapists, speech therapists and neuropsychologists, as well as rehabilitation recommendations at each age. Developmental and rehabilitation history before first assessment in the pediatric neurology clinic was collected from parents and referrals from primary health care services.We collected educational special needs support arrangements and the level of support recommended and received separate information for kindergarten, primary school grades (7–12 years) and middle school grades (13–16 years). The frequencies of each support level in each age group were analyzed. The learning objective level according to the type of syllabus used  was collected separately as an indicator for severity of learning difficulties.We also collected the family situation history according to information collected in patient record texts, often by a hospital clinic’s social worker. In case of out-of-home placement, we extracted the age at first known out-of-home placement and, when relevant, the age when the child was returned to home or adopted, as well as the number and types of placements , when available.We divided the collected information about problems and interventions into four different age categories: infancy (0–11.99 months), preschool age (1.0–6.99 years), primary school age (7.0–12.99 years) and adolescence (13.0–15.99 years).For cost estimations, we included only additional costs that are not part of a regular childhood. That is, we considered that health care, education, and social assistance costs estimated for healthy children with normal development living at home with birth parents represent the baseline costs for comparison to children with FASD, thus excluding those costs in cost estimation. We used regular health economic macro-costing methods with available average prices for each cost type. These included Diagnosis-Related Group (DRG) prices and statistical average costs and prices in the hospital catchment area for out-of-home placements and rehabilitations and for each type of special needs education costs. We estimated costs for medication and disability allowance using available national prices, because these are not area-dependent. In Finland, we have publicly available detailed statistics of cost and prices in all areas of public expenditure.Because DRG prices lack accuracy in individual cases, it has been recommended that additional cost analysis should be performed for specific therapeutic areas with high costs . That isBecause we did not have access to actual unit costs  except for out-of-home placements, we used the available pricelists from 2021. First, to obtain an estimated nominal cost of each service for each year, we corrected the unit cost based on the price index of public expenditure for social services and healthcare of local government finances by function area . The ind(1)Hospital costs, including both outpatient clinic visits and inpatient ward stays;(2)Out-of-home placement costs, both in foster families and in institutional placements ;(3)Disability allowance costs, which is money paid by the Social Insurance Institution when a child has a chronic illness or a disability and has greater than normal need for care and attention;(4)Rehabilitation costs including physiotherapy, speech therapy, occupational therapy, neuropsychological rehabilitation and psychiatric therapies;(5)Special needs education costs (average schooling costs were subtracted);(6)Long-term medication costs for regularly used prescription medication .Estimated costs included:See Hospitalization and hospital visit costs were estimated using available DRG prices for each main diagnosis listed in hospital clinic outpatient visit or hospital ward inpatient stay during each age group. We used the 2021 price list for services in the Päijät-Häme area . All adoOut-of-home placement costs were estimated by extracting the years a child was placed out of home and considering the sum of their nominal out-of-home placement costs in the hospital district during 2006–2020 ,19. For Disability allowance costs were estimated by multiplying the presence and level of disability allowance and time in years in each age period. Because foster parents do not typically know the disability allowance level their fostered child is granted, we estimated the disability allowance levels according to diagnoses, rehabilitations, and clinical experience from other patients with similar problems, using the Social Insurance Institution of Finland’s  disability allowance criteria and amounts for 2021 [Rehabilitation costs were estimated using the typical rehabilitation schemes in our hospital district, because the exact number of realized rehabilitation sessions was not easily available. In Finland, rehabilitation for severe developmental disorders  is financed via SII and requires a diagnosis by hospital clinic indicating a specific developmental disorder. Rehabilitation for mild or intermediate difficulties and early interventions before having specific developmental disorder diagnosis are arranged via either hospital or basic health care and financed by the hospital district. If a patient underwent a rehabilitation listed in patient records without a diagnosis of specific developmental disorder, it meant that the rehabilitation was financed by hospital district, and cost was estimated using the pricelist for services in Päijät-Häme area 2021  and averEntry in specialty school for disabled with extended compulsory education and individualized syllabus; very high adult/student ratio; at this support level, taxi transfers and morning and afternoon care are included in total cost;Extended compulsory education in a small special needs class and with individualized syllabus, but situated in regular school; high adult/student ratio;Special needs education and special support in small class without extended compulsory education; general or modified syllabus; higher than average adult/student ratio;Special support with inclusion and integration to mainstream classroom;Intensified support in mainstream classroom.Special needs educational support costs were estimated according to the level of special support listed in kindergarten (5–6 years), primary school (7–12 years) and middle school (13–16 years). Kindergarten-age children had included costs only in case of compulsory extended education, which means two years of kindergarten with special education, and only the costs of additional kindergarten year were included. The mean costs for a typical student without extra support was subtracted from all special needs education costs. Costs were retrieved from the report on financing of educational and cultural provision 2020 from the Finnish National Agency for Education  and costAdditionally, we extracted the information and estimated costs for possible morning and afternoon care for intellectually disabled students after first grade for those attending regular schools. Further, costs for taxi transfer were estimated according to the same guidelines. We considered special needs educational cost information missing if the patient was not old enough to have attended one full year of the educational stage in the age group.Long-term medication costs were estimated using the prices for a typical dosage and medication for each medication type according to patient’s age and typical medication follow-up costs including laboratory tests and medication control health checks for each type of medication. Diagnosing costs were not included because they were included in the hospital visit DRG price. In Finland, prescription medications have the same prices in all pharmacies. Only medications that were prescribed in hospital clinics and used for at least a year were included. Because medications for asthma are also very common in the general population and prescribed in primary health care for older children, they were not included.For each cost category, we calculated the sum of the known estimated costs for each patient within the age groups. If the patient was living in the hospital district during certain age group, but did not have hospital visits, known rehabilitation or known long-term medication, known special needs education services or out-of-home placement mentioned in patient register data during that age period, we assumed that they did not have those services during that period, thus resulting in zero costs for the patient within the age group. If the patient was not living in the hospital district during a certain age and the entry was missing or stated not available, we set the costs as not available.Because the out-of-home placement data and special education data were not always synchronous with hospitalization data, we recorded the corresponding follow-up time for them separately for each patient according to the entries in the hospital records. To accommodate the left and right censoring, we also calculated the follow-up time at each age group separately. If a patient did not have a follow-up at a certain age, the follow-up time at the respective age was 0. Thus, even when the age group size varied at different calculations, the annual costs were consistent.For the mean annual costs, we first aggregated the age-group-specific costs by the cost category. Next, the sum of each cost category was divided by the sum of the follow-up time for the respective age group and the cost category. Similarly, we calculated the total mean annual cost by summing all costs within the age group and dividing the sum by the follow-up time. We also separately calculated mean annual costs for those with complete follow-up through each age group.To determine the cumulative costs, we calculated the sum by two methods: First, we selected the patients with at least 15 years of continuous follow-up time and complete records at each cost category. In this group, we calculated the cumulative sum directly for each person through the age groups. The other method consisted of selecting at each age group the patients with complete follow-up time through the respective age. We calculated the cumulative sum by adding the mean costs of each age group to the mean cost of the previous age group.Study population descriptive statistics can be found in The main interventions  during iEstimated mean annual costs, their medians and interquartile ranges are presented in In this age group, developmental delays were typical reasons for hospital outpatient clinic visits. . In addiNearly all  had received at least one form of therapy, and occupational therapy was the most common . For those 40 assessed by an occupational therapist, fine motor coordination was documented as below normal (<−2 SD) in 36/40 (90%), sensory processing was below normal in 34 (85%), and static balance was below normal in 33 (82.5%). Gross motor abilities were below normal in 19 (47.5%).At this age, 17/45 (37.8%) were placed out of home in addition to the 23 placed already before. Three children returned to their birth families and two were adopted during this age. At the age of 6 years, 35/45 (77.8%) were in out-of-home placements. All those who were returned home continued to be surveilled by child protection services. As many as 17 (37.8%) needed child psychiatric care Although rehabilitation costs were at their highest level before school age, total mean annual costs were at their lowest during preschool age. For those with complete follow-up time, 1–6 years mean annual costs were approximately EUR 49,300 , Figure.During primary school, attention problems were described in patient records in 36/37 (97.3%) while 25 (67.6%) were diagnosed with attention-deficit hyperactivity disorder (ADHD). At this age, 25 (67.6%) had a diagnosis indicating impaired learning abilities —see Psychiatric diagnoses other than ADHD were diagnosed in eight (21.6%) patients, of which two had reactive attachment disorder (F94.1) and two had disinhibited attachment disorder (F94.2).Psychostimulant medication was prescribed for 24 (64.9%) patients. Regarding the support received or planned for school , only onCosts for special needs education and disability allowance were high at primary school age  Figure . SomaticOf the middle-school aged adolescents, 12/15 (80%) had a diagnosis indicating impaired learning abilities, either Mild intellectual disability (F70) or Developmental disorders of scholastic skills F81. The majority  had dual diagnoses of cognitive impairment and mental health problems, and the other 6 (40%) were diagnosed with either cognitive impairment or mental health problems. The most common main diagnosis in psychiatric unit visits was mild intellectual disability with significant impairment of behavior (F70.1) in five patients. They had symptoms that are typical for conduct disorder, but that was not diagnosed because of intellectual disability. Other non-unique psychiatric diagnoses were depressive episode (F32.9) in three patients, anxiety disorder (F41.9) in two, and reactive attachment disorder (F94.1) in two.The majority  were patients in the adolescent psychiatric unit. Those diagnosed with reactive attachment disorder, depressive episode and one with eating disorder (F50) all received long inpatient psychiatric treatments. Most  teenagers used at least one regular medication, but only 5 (33.3%) continued psychostimulant medication for ADHD symptoms, although 10 (66.7%) still had ADHD listed as a diagnosis during this age.Support level in school  was highPsychiatric hospitalizations and institutional out-of-home placements increased costs and medication costs were at their highest level during adolescence  Figure . Mean anBrain MRI was performed in 18/55 (32.7%) cases: 12/32 (37.5%) with FAS, 5/17 (29.4%) with PFAS and 1/6 (16.7%) with ARND. Of those who had brain MRIs taken, 10/18 (55.6%) had findings listed in their neuroradiological reports, of which some could be classified as incidental findings or normal variants. There were 4/18 (22.2%) cases of wide/enlarged cisterna magna or prominent retro cerebellar cerebrospinal fluid space, 2 (11.1%) with agenesis or dysgenesis of corpus callosum, 2 (11.1%) had hypoplastic midbrain structures , 2 (11.1%) had apparent microcephaly without structural anomalies, and 1 (5.6%) had wide perivascular spaces.Sleep–wake EEG registration was performed on 15/55 (27.3%): 10/32 (31.3%) with FAS and 5/17 (29.4%) with PFAS. The reasons for EEG were suspected seizures or suspected developmental regression. Of those EEG registrations, 9/15 (60%) were classified as abnormal. Interictal epileptiform discharges were detected in 6 (40%), but only 2 (3.6%) were diagnosed with epilepsy that required medication. Three (20%) had slow background activity.Infections were common even after infancy; 16/55 (29.1%) had at least one hospital visit for recurrent or complicated middle ear infections, 6 (10.9%) had three or more urinary tract infections, 1 had chronically elevated creatinine and cystatin c levels indicating kidney dysfunction but normal kidney ultrasound results, and 5 (9.1%) had tonsillectomy/adenoidectomy performed. Some endocrinological and autoimmune problems were also noticed. Three received growth hormone treatment for short stature and growth hormone deficiency. One had juvenile diabetes mellitus, and two had recurrent hypoglycemic and reactive hyperglycemic episodes without diabetes. One had celiac disease, one had hyper-IgA without apparent cause, and two had significant hyperprolactinemia associated with risperidone medication.Those 8/55 (14.5%) who were adopted more often had a full FAS 6/8 (75%) diagnosis compared with the others, lower average birth weight (2180 g) and smaller latest average head circumference Z-score . All but one of the adopted lived in their adoptive families since infancy; three first as foster children. Mean age at the time of data collection for the adopted was 13.6 years . Comparing those who were adopted to others who were not adopted but were also at least 7 years old at the data collection , fewer adopted  had had psychiatric contact before the age 16 years than non-adopted  . Those wThe majority (78.5%) of the estimated mean cumulative total costs of EUR 1,077,000 for 0 to 16 years using those with complete follow-up within each age group  occurred from long-term out-of-home placements . For thoThis study clarified the phenotype of FASD and its evolving pattern of health care needs and their associated costs during childhood. Typical problems in each age group and high costs among children with FASD were in line with many previous findings. There is a need for a syndromic approach to diagnosis and multidisciplinary follow-up in FASD. When we know to search for typical problems at each age, we can move from treating symptoms towards prevention of secondary problems. Early diagnosis, counseling for parents and caregivers, psychological support and realistic expectations for the children have been shown to improve their long-term prognosis ,27.High incidence of neonatal problems, including prematurity and congenital malformations in children who later become diagnosed with FASD, has been described many times before . It undeLearning during early development happens by observing, interacting, and experimenting with the environment. For that, a young child typically uses eyesight, hearing and other sensory input, and their ability to manipulate objects by hand. Importantly, this study showed that eyesight, hearing, sensory processing and fine motor skills are often at least temporarily impaired in small children with FASD. Early recognition, treatment and adequate timely rehabilitation of these problems may improve the developmental trajectory that now often seems poor.The high prevalence 31.1%) of early onset visual problems, especially strabismus (26.7%) and amblyopia, was lower than in a study on internationally adopted persons with FASD .1% of ea. A spectRecurrent or complicated middle ear infections that cause intermittent conductive hearing impairment in prenatally alcohol-exposed children has also been described before ,38. OtitAlmost universal sensorimotor problems before school age—sensory processing was below normal in 85% and fine motor skills in 90%—support the finding that prenatal alcohol exposure alters sensorimotor network in the brain . In addiAlthough the majority of the study population required physiotherapy during infancy and speech therapy during preschool age, significant catch-up in both gross motor skills and speech was common with rehabilitation. Inverse trajectory was seen in learning abilities. Although 79.1% started school according to the regular syllabus, increasing support was gradually needed, and in middle school, only 31.2% was able to follow the regular syllabus and all received extra support. Learning problems became more significant when demands for executive skills and abstract thinking increased after the first primary school years. Learning support should be provided from early on, instead of waiting for the child to fail before providing the necessary support.In addition to cognitive difficulties in FASD, adaptive problems that impair everyday functioning tend to become more apparent when socioenvironmental requirements increase with age . The preSecondary psychiatric problems increased with age, especially during puberty, and 80% of adolescents received psychiatric treatment indicating deficient early support and possibly a poor postnatal living environment, although 41.8% had been placed out of home already before the age of one year. Being adopted appeared to be associated with lower secondary psychiatric problems, supporting earlier findings that a stable family environment from early age is beneficial for mental wellbeing , becauseMRI findings among those who had brain MRI scans may overestimate the prevalence of abnormal MRI results, because the majority had not been scanned. However, the findings indicated that 22.2% had prominent retrocerebellar CSF space indicating cerebellar and midbrain hypoplasia and 11.1% had dysgenesis of corpus callosum, which is in line with previous research ,46 showiThis study confirmed the high societal cost of FASD during childhood, although only part of all societal costs, was possible to collect or estimate from hospital records. The aim was to include only those additional costs that are not present during a typical childhood. We included and estimated costs for special health care, meaning hospital inpatient stays and outpatient clinic visits, rehabilitation, out-of-home placements, regular long-term medications, disability allowance and extra support needed for school. Naturally, all children cause significant societal costs, such as primary health care, day-care, short-term medications for common ailments, and schooling costs, and these also may differ in children with FASD compared to the whole population, but those costs were not part of this study and considered as baseline.Mean annual costs were estimated using the year 2022 prices. They should be interpreted as the costs that significant health and social care needs and interventions for a child with FASD would currently cause for the society in one year in Finland. Mean annual additional costs were age-dependent and varied from EUR 49,000 in the preschool age to EUR 91,000 during adolescence. The mean annual calculated costs for those with full consecutive information available were EUR 93,300 between ages 0 and 16 and significantly higher than for corresponding age group averages. Our totals were close to the EUR 76,000/year estimated in a Swedish study . The smaAn important finding was that some problems remain undiagnosed even when acknowledged in rehabilitation or patient register texts, which makes register-based or only diagnosis-based research prone to show less issues than there are in reality. For example, only 31.6% had the diagnosis of fetus and newborn affected by maternal use of alcohol (P04.3), although all had been exposed to alcohol. During infancy, only 13.9% had a diagnosis of microcephaly (Q02), although 43.2% were microcephalic at birth. Attention problems were described in all but one during school age, but just 67.6% had a diagnosis of ADHD. In addition, the number of specific psychiatric diagnoses was lower than the number of children who had been treated in pediatric or adolescent psychiatric units. There are probably different reasons for not entering a formal diagnosis in the patient register even when the problem is acknowledged in text. One reason could be not wanting to stigmatize the child or unsettle parents; another reason could be a habit of just entering one diagnosis, even when there are many recognized problems.Patients were examined in the same unit and the hospital’s full comprehensive electronic patient record information was available. We were able to obtain detailed individual age-specific information of the sequence of problems and interventions including not only diagnoses and medications but also rehabilitations, out-of-home placements, social benefits and special schooling arrangements.The study population was ethnically homogenous, all but two were of Finnish or other northeastern European origin.It was possible to estimate typical interventions and their associated costs fairly accurately because the main author (LMJ) of this article conducts clinical work with these and similar patients and is familiar with rehabilitation and the school system in the hospital district.The majority of those patients in this study who were diagnosed in our hospital during the last five years had been diagnosed with FASD by two of the authors (HS and LMJ), ensuring that the diagnostic protocol was uniform, and patients met all the IOM’s diagnostic criteria for FASD.All the estimated costs are additional costs that occur in addition to regular childhood costs. This means that all costs estimated in this study are related to significant problems that are not a part of a regular childhood and thus can be interpreted as costs that are sequelae of FASD. For typically developing children living with their birth families without significant chronic medications or health problems that require hospital visits, there would not be any of these estimated costs. No basic health care costs nor basic schooling costs nor easily accessible community support services were included, because they were considered to be part of the baseline.Combining in a single study clinical data concerning problems and interventions with their estimated costs  enables us to see a more comprehensive picture of the multifaceted effects associated with FASD in different periods of childhood.The retrospective nature of the study precludes assessments of record accuracy and completeness. Furthermore, the data were originally recorded for treatment and/or administrative purposes, without considering secondary use, such as cost estimation, and therefore was by nature incomplete for our study purposes. This is especially pertinent to estimates derived from coding text, which can therefore be assumed to be conservative.The study population was small and derived from a single hospital in Finland, and follow-up time and age of patients at the time of neurological assessments and data collection time varied. Because out-of-home placements and adoptions were common, parental and prenatal information were incomplete. We did not have access to maternal health records and there was not enough reliable information about the quantities or timing of alcohol exposure to include in the analysis. Patients often moved in and out of the hospital district and there were changes in living arrangements, which caused a paucity in records. There was very limited information about teenagers. Incomplete follow-up times even within an age group are likely to underestimate the real prevalence of problems and interventions.The high percentage of children in out-of-home placements suggests that it has probably been easier to diagnose FASD in children who are not living and accompanied by their birth mothers. Most children with undiagnosed FASD are probably living with their birth families and have been diagnosed with a variety of symptoms and comorbidities. The problems observed in early childhood can be linked directly to prenatal alcohol exposure, but with increasing age, the environment may have a more important role. Children who have been placed out of home often experienced inadequate parenting and maltreatment and some of their problems might be secondary to a traumatic postnatal life.In a clinical data set without a control group, there is always a possibility of bias towards excessive problems, because only those with significant problems are referred for a hospital clinic assessment and are tracked for longer periods of time.There is a need for repeated studies with larger sample sizes from multiple hospital areas and, if possible, additional data sources, such as national health and social care registers.As all cost and comorbidity prevalence estimates are based on small numbers, they are subject to substantial random sampling bias and must be treated as approximates. Cost calculations are also conservative estimates because of the retrospective study design; in addition there are types of additional costs that cannot be estimated using patient register data. There is always a significant amount of uncertainty and potential inaccuracy when using macro-costing methods, which tend to underestimate real costs.We did not have access to actual nominal prices of the past except for out-of-home placements, and using current price lists to estimate past nominal prices may have caused inaccuracy and bias, although the used services were similar during the years.We had no access to registers of social services. There are some additional societal costs associated with FASD that could not be included, although they also are rare in the regular non-FASD population. There are typically other extensive services provided for a family before out-of-home placement is considered and also when a child has been returned home after an out-of-home placement. Costs occurring because days off work or reduced working hours of parents or caregivers could not be included. Further, neither costs caused by damaging objects nor criminal activities could be included, because of the lack of specific information in patient records, although there were some mentions of criminal or antisocial behavior during adolescence. Juvenile delinquency has been shown to cause high costs in the FASD population in Canada, for example .Services and their related costs differ between countries and even areas. This study represents Finnish health care, social services, and public education services. It is fairly similar to other Nordic countries and comparable to other countries with public health care and social welfare systems. It differs significantly from countries with mainly insurance-based and private services. However, all the problems and needs revealed in this study would cause significant costs in any country. Finland has high efficiency in health care production and a sustainable healthcare system in comparison with other OECD countries ,49. In oChildren with FASD have significant health, psychiatric, cognitive, adaptive and social burdens that are associated with high costs for society. The revealed age-specific pattern of problems in this study could be used as an aid for recognizing persons with undiagnosed FASD and constituting a follow-up protocol. Early diagnosis is important to make sure that they have the necessary assessments and support in all phases of childhood to minimize secondary problems and high long-term costs. Young children with suspected FASD need to be examined by a pediatric neurologist, cardiologist, ophthalmologist and otorhinolaryngologist, and they also need a developmental assessment by an occupational and speech therapist and psychologist. Both cognitive and adaptive functioning should be evaluated in person with FASD. Because many problems become apparent only with age, and because the pattern of difficulties evolves, it is important to keep following those with known or suspected prenatal alcohol exposure, even when initially they might seem to do well or catch up with normal development. Support for a stable living environment is important for as smooth a development as possible.


Zoonotic pathogens that are vector-transmitted have and continue to contribute to several emerging infections globally. In recent years, spillover events of such zoonotic pathogens have increased in frequency as a result of direct contact with livestock, wildlife, and urbanization, forcing animals from their natural habitats. Equines serve as reservoir hosts for vector-transmitted zoonotic viruses that are also capable of infecting humans and causing disease. From a One Health perspective, equine viruses, therefore, pose major concerns for periodic outbreaks globally. Several equine viruses have spread out of their indigenous regions, such as West Nile virus (WNV) and equine encephalitis viruses (EEVs), making them of paramount concern to public health. Viruses have evolved many mechanisms to support the establishment of productive infection and to avoid host defense mechanisms, including promoting or decreasing inflammatory responses and regulating host machinery for protein synthesis. Viral interactions with the host enzymatic machinery, specifically kinases, can support the viral infectious process and downplay innate immune mechanisms, cumulatively leading to a more severe course of the disease. In this review, we will focus on how select equine viruses interact with host kinases to support viral multiplication. Zoonotic diseases, which constitute a large number of (re)emerging viral infections globally, are transmitted to humans from their natural animal reservoirs by intermediate vector species, including other animals such as bats and swine, and insects such as mosquitoes and sandflies . It is cEquine viruses represent a major concern for human disease because of the potential for equines to serve as intermediate hosts . The virA step towards further understanding how equine viruses play a role in human disease is to elucidate how the host machinery is engaged by these viruses, both in the equine and the human hosts. Host kinases constitute the cellular enzymatic machinery that aids in the transferal of phosphate groups from ATP to other molecules that results in the functionalization of the phosphorylated target. Such functionalized targets directly result in phosphosignaling cascades that control key cellular events such as growth, differentiation, and maturation. Kinases phosphorylate a subset of host targets: tyrosine, serine/threonine residues on proteins, or lipids ,14. SeveViruses have in turn developed mechanisms that can evade, moderate, and/or nullify the host-based innate immune mechanisms by controlling the host processes such as transcription and translation. Such virus-based control of host innate immune mechanisms often involves interactions between viral and host proteins. There are a wide variety of zoonotic viruses that have evolved escape mechanisms from PKR to enable and sustain robust infection . One sucTogaviridae. These single-stranded RNA viruses are the causative agents for encephalitic disease in equines and humans in the Americas [In this section, we will describe the interactions of two closely related EEVs with host-derived kinases, specifically focusing on the Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV). VEEV and EEEV are new-world alphaviruses belonging to the family Americas ,10,11,19Americas . Birds aAmericas . The treVEEV and EEEV are major concerns for human disease not only because of the potential for transmission to humans through infected mosquitoes but also because these viruses are highly stable and infectious in aerosols form. In the context of aerosol transmission, there is a significant involvement of the central nervous system (CNS) and a high potential for encephalitic disease and death. In the context of human disease, several studies have been conducted about the interaction of VEEV proteins with human kinases and how they contribute to the establishment of a productive infection. For example, the human IKKβ kinase was shown to play an important role in the ability of VEEV to multiply in the cells of CNS origin through a series of studies involving small-molecule inhibitors and targeted depletion using siRNAs, as well as through additional proteomic studies ,23,24. TArteriviridae family [Equine arteritis virus (EAV) is an enveloped, single-stranded, positive-sense RNA virus belonging to the e family . EAV is e family . A smalle family . The vire family .While not fully elucidated mechanistically, viral entry is likely to be clathrin-dependent ,33. ChroFlaviviridae family, is an enveloped, single-positive stranded RNA virus that is transmitted to humans through infected mosquitoes [West Nile virus (WNV) belonging to the squitoes . First isquitoes . WNV possquitoes . There asquitoes . WNV remsquitoes . WNV hassquitoes . In addisquitoes . Estimatsquitoes . Among the kinases that are implicated in WNV infection, AMP-activated protein kinase (AMPK) degradation mediated by the WNV C protein was through ubiquitin-mediated protein degradation. AMPK is a stress-activated protein that inhibits key responses, including autophagy. The ubiquitin-mediated degradation of AMPK is thought to be relevant to the development of the CNS phenotype in WNV infection . CalciumRetroviridae family. Acute infection with EIAV usually presents with recurrent high fever, depression, anorexia, and anemia [Equine infectious anemia virus (EIAV) is an enveloped, RNA virus belonging to the d anemia . Spontand anemia . Recurred anemia . Althougd anemia . Mosquitd anemia . New evid anemia . Humans d anemia . While Ed anemia . Limited information exists on the interactions of EIAV with host kinases. The presence of equine lentivirus receptor 1 (ELR1) on equine cells appears to make cells susceptible to EIAV infection . ELR1 haReoviridae. RVA infection is the leading cause of diarrhea-associated morbidity and mortality globally among infants and children under the age of 5. Although RVA-associated deaths are preventable via vaccine, the hospitalization rates of children under the age of 5 due to RVA are reported to be 30–40% worldwide. Rotavirus was first discovered in the 1950s from anal swaps of monkeys, and in the 1960s, it was seen again in the gastrointestinal tracts of mice. In 1973, Ruth Bishop and colleagues had a breakthrough in identifying RVA in samples from children with acute diarrhea. The distinct appearance gave its name rotavirus (adapted from the Latin word “rota” meaning wheel) [Group A rotavirus (RVA) is a double-stranded RNA virus of the family g wheel) . The trag wheel) . While RVA has gained significant attention as a human pathogen due to the devastating impact it has on unvaccinated children, equine RVA is also a major pathogen that causes diarrhea in foals . MortaliFrom the perspective of kinases that play integral roles in the infectious process, an extensive body of literature is available due to their critical relevance to human and animal health and their impact on gastrointestinal health. Only a salient subset of kinases are included in this article. Detailed reviews of the engagement of host kinases with RVs are available ,63,64,65Paramyxoviridae family and emerged in 1994 as a zoonotic disease, typically spilling over from bats of the genus Pteropus, although other bat species have been implicated as well [Hendra virus (HeV) is a rare but deadly infection that causes fatal diseases in equines . HeV bel as well ,69. HeV  as well . During  as well . From th as well ,73. Orthomyxoviridae family with a segmented negative RNA genome. Equine infection through EIV is characterized by elevated body temperature and dry cough, with severely infected horses presenting with inspiratory and expiratory wheezing and crepitations. Anorexia, lethargy, and nasal discharge are also characteristic features of EIV infection. In the context of vaccinated animals, animals usually recover within 7–10 days, while morbidity can reach 100% in non-immunized populations [Equine influenza virus (EIV) is a highly contagious virus belonging to the ulations ,75. Consulations ,76,77. Although the majority of documented cases of EIV are limited to equids, there has been evidence of rare spillover incidents of EIV into other species such as dogs, pigs, and foxhounds ,79,80,81Currently, the available information about kinase activation in EIV-infected host cells is limited. RIPK1/3 and MLKL proteins have been shown to be associated with necroapoptosis in the context of EIV infection ,86. StreAlphaherpesvirinae  or Gammaherpesvirinae  subfamilies [EHV was first isolated as being associated with causing equine abortions ,90. Currfamilies ,91,92. Afamilies . In aligfamilies ,91. Virufamilies . EHV-1 ifamilies . Horse-tfamilies . Infectefamilies . A clinifamilies ,96,97,98families ,99.EHVs have been shown to activate several cellular kinases and signaling pathways, one of which is the NF-κB pathway. Specifically, the vCLAP protein was involved in activating NF-κB in an IKK-gamma-dependent pathway . In expeMany non-equine herpesviruses and other DNA viruses  encode for viral thymidine kinases (TKs), which play important roles in the viral life cycle, specifically of significance in neuropathogenicity ,105,106.Although the cases of EHV in humans have not been fully documented, it has been demonstrated that the potential for a crossover of species exists. In 2007, EHV-9 infection was documented in a 12-year-old polar bear with progressive encephalitis in a zoological garden located in San Diego, California . It is bRhabdovirus family [Rabies virus (RABV) is an enveloped, negative-sense RNA virus belonging to the s family ,114,115.s family . RABV wis family . Rabies AP-2-associated kinase 1 (AAK1), a serine–threonine kinase, has been shown to mediate rabies viral entry via the phosphorylation of AP2M1 ,113. WhiBornaviridae within the order Mononegavirales [Borna disease virus (BDV) is a non-segmented negative-strand RNA virus that is the etiological agent of Borna disease (BD), belonging to the family avirales ,118. BDVavirales ,119. BDVavirales ,121,122.avirales ,126,127.avirales ,129. Mosavirales ,132,133.avirales ,134,135.avirales ,137. Disavirales ,137. In avirales ,139. In In regard to the effects of BDV on human health, there has been much debate on the topic for decades. Due to the detection of the neurological effects of BDV infection on behavior and psyche in animals, many scientists suspected this to occur in humans as well. Therefore, early studies focused primarily on psychiatric patient cohorts, which led to debatable results. However, in 2015, a close relative of Borna disease virus 1 (BoDV-1), variegated squirrel bornavirus 1 (VSBV-1), was discovered ,141. FouMononegavirales have phosphoproteins that play important roles as transcription factors. In the case of BDV, the phosphoprotein P is believed to be phosphorylated by protein kinase C (PKC) and casein kinase II [Because BDV-induced behavioral impairments are hallmark clinical manifestations of BD, many studies have aimed at studying the effects of BDV infection on neurons. It has been shown that when considering synaptic vesicle (SV) recycling as a measure of synaptic activity, BDV specifically blocks the enhancement of SV recycling and causes defects in long-term potentiation . This isinase II ,148. In inase II . Becauseinase II ,150. FurEquine viruses constitute an important group of potential vector-transmitted zoonotic viral pathogens that are highly relevant to the One Health concept due to the disease burden and symptomology in animals and humans. Several factors contribute to the persistence and global distribution of these pathogens, with fairly limited attention paid to the development of robust therapeutic strategies to treat human exposures. There are vaccine-induced protection processes for equines for several diseases discussed here, and the possibility of spillover into humans continues to pose a significant challenge. When evaluating the interactions of several of the equine virus proteins with the host kinase machinery, some common trends emerge, including the role played by highly conserved innate immune phosphosignaling mechanisms, in the establishment of a productive infection. This suggests that the repurposing of FDA-approved therapeutics to treat human cases of equine virus infections is a viable option, with a greater chance of achieving broad-spectrum protection. However, much work is still needed to identify such broadly effective solutions to treat infections and ascertain any potential for the development of resistant variants.


The FDA-approved Adenovirus Type 4 and Type 7 Vaccine, Live, Oral is highly effective and essential in preventing acute respiratory diseases (ARDs) in U.S. military recruits. Our study revealed the presence of a previously undetected mutation, not found in the wild-type human adenovirus type 4 (HAdV-4) component of the licensed vaccine, which contains an amino acid substitution (P388T) in the pre-terminal protein (pTP). This study demonstrated that replication of the T388 HAdV-4 vaccine mutant virus is favored over the wild type in WI-38 cells, the cell type utilized in vaccine manufacturing. However, results from serial human stool specimens of vaccine recipients support differential genome replication in the gastrointestinal tract (GI), demonstrated by the steady decline of the percentage of mutant T388 vaccine virus. Since vaccine efficacy depends upon GI replication and the subsequent immune response, the mutation can potentially impact vaccine efficacy. Adenoviridae, genus Mastadenovirus) are non-enveloped, double-stranded DNA viruses found worldwide in humans. Greater than 80 human Ad types, belonging to species A-G, cause a diversity of illnesses, where species B, C and E can cause upper and lower respiratory tract infections and lead to severe acute respiratory disease (ARD) -3′) differing only at the 3′ terminal nucleotides corresponding to their respective nucleotide polymorphism, i.e., G or T at nucleotide position 9171 of the HAdV-4 genome. The qPCR assay has a limit of detection (LOD) of 50 genome-equivalent (GE) copies. Non-extracted viral culture clear supernatant and extracted fecal suspensions were diluted at either a 1:5 or 1:10 ratio in water, respectively, to reduce PCR inhibitors, then combined with PowerUp SYBR Green Master Mix (Thermo Fisher) prior to being subjected to 40 cycles of quantitative PCR (qPCR) with either an ABI 7500 or ABI QuantStudio 7 Flex in duplicate. Each reaction well was quantified against an amplicon standard curve with known molecular concentration. The nucleotide percentage frequency was calculated by dividing the number of SNP-specific molecules by the total number of molecules in both SNP-respective wells ).Statistical analysis was completed using SAS system software. A Student’s t-test was used to compare in vitro multiplication kinetics for pTP wild-type P388 and mutant T388 viruses in WI-38 by day for each virus independently, followed by multiple comparisons through a general linear model with virus type (wild type vs. mutant), days and their interaction as predictors on the natural log value of genome equivalents (Ln GE/mL). Additionally, the over-adjusted Ln(GE/mL) average of the viruses over the 10-day period was estimated by the least-square mean. Similarly, an initial Student’s t-test, followed by a general linear model using the same parameters listed above and least-square mean, was used for the multiple comparisons of in vitro multiplication kinetics of the viruses following passaging.p < 0.05 was considered significant.A non-linear regression model by adding the square term of the percentage of input was used to evaluate the association between the percentage of the input of the pTP wild-type P388 vaccine virus and the output of the pTP wild-type P388 vaccine virus in the stool of vaccine recipients with number of days post-vaccination as predictors. Additionally, we performed multiple comparisons of the least-square mean of pTP wild-type P388 percentage by days for virus detected in stool. For the analyzed data, a value of HAdV-4 vaccine virus production primary seed stock Lot 089, manufactured in 1980 by Wyeth and also used by Teva, and several commercial vaccine tablets lots were subjected to whole-genome NGS, unpassaged, to determine genetic homogeneity . The assResults of SNP genotyping by qPCR confirmed the G/T variation frequency estimates based on sequence alignment depth of NGS data. Wyeth seed Lot 089 (1980) was determined to contain only nucleotide G9171 by SNP genotyping with no detectable T9171, while the evaluated tablets from both Wyeth and Teva contained both G9171 and T9171 .To further confirm the presence of two distinct viruses in the vaccine tablets and evaluate them independently, a Teva HAdV-4 vaccine tablet  was subjected to plaque purification of viruses. Two independent HAdV-4 virus strains containing pure G9171 and T9171, respectively, were isolated and replicated stably in ten repetitive passages with genomic homogeneity confirmed by both NGS deep sequencing and SNP genotyping.The 9171 G/T variation is within the gene for pTP, corresponding to a Proline (P)-to-Threonine (T) substitution at amino acid position 388. Only G9171 was found in Lot 089 (1980), and comparison of all HAdV-4 and other related Ad sequences available in GenBank clearly showed that G9171 is highly conserved in related Ad  The T9171 variant is a novel nucleotide polymorphism among published Ad sequences and only present in the HadV-4 vaccine. Therefore, in this study, G9171 is referred to as wild-type P388, while T9171 is referred to as mutant T388.Together, our results indicate that a G9171T mutation led to generation of a novel distinct mutant vaccine virus in the HAdV-4 vaccine. The mutant T388 vaccine virus is not detected in the early production lots of Wyeth tested in this study but was detected in all tested licensed vaccine tablets of Wyeth and Teva, at levels ranging from 35 to 65% of the viral population.The pTP and subsequent protease-cleaved intermediate terminal protein (iTP) and mature terminal protein (TP) play multiple roles in Ad replication ,21. The p < 0.001). Compared to the wild-type P388 vaccine virus, the overall growth rate of the mutant T338 vaccine virus is significantly higher across all time points evaluated; the average daily increase was 1.02 units of Ln(GE/mL) for the mutant T388 vaccine virus and 0.78 units of Ln(GE/mL) for the wild-type P388 vaccine virus per day (p = 0.003). The difference in genome produced between the two viruses calculated from the general linear model showed an increase over time by 0.24 × (number of days), ranging from 1.02 to 0.78 units of Ln(GE/mL). By Day 10 post-infection, the least-square mean of the viral titer for the wild-type P388 vaccine virus was 11.85 Ln(GE/mL) and 14.75 Ln(GE/mL) for the mutant T388 vaccine virus with a significance of p < 0.0001. The difference in viral genome production reached almost 1000-fold by the end of the ten-day experiment.DNA production of the wild-type P388 and mutant T388 vaccine viruses was evaluated in WI-38 cells individually each day for ten days and compared overall after ten days . Signifip = 0.92, which was not a statistically significant increase in viral genomes after each passage. However, the natural logarithm of viral titer for the mutant T388 vaccine virus increased by 1.94 units per passage; this increase rate was found to be significant (p < 0.0001) and also significantly higher than that of the wild-type P388 vaccine virus (p < 0.0001) over the passage series.Similarly, genome replication after each passage for both the wild-type P388 vaccine virus and mutant T388 vaccine  and overTogether, these results demonstrate that the P388T mutation confers a genome replication advantage for virus cultured in WI-38 cells. The T388 mutant vaccine virus reproduced more efficiently in WI-38 cells than the wild-type P388 vaccine virus.A competition assay of the wild-type virus and the mutant T388 virus was conducted to determine if WI-38 cells favored replication of the mutant virus in the presence of the wild-type virus. Starting with varying reciprocal percentages (ranging from GE ratio 5 to 95% of input virus), the final percentage (output virus) of the wild-type P388 to the mutant T388 vaccine virus following 10-day infection in WI-38 cells was determined by SNP qPCR .When starting with 5% of mutant T388 virus in the mixture (GE ratio of T5:P95), the mutant T388 virus increased to 32%, while the wild type decreased from 95% to 68% (output GE ratio of T32:P68). Similarly, the input mixtures of T10:P90 and T25:P75 resulted in output ratios of T40:P60 and T56:P44, respectively. When starting with an equal mix of the wild-type and the mutant T388 viruses (T50:P50), mutant T388 clearly outcompeted, ending with a ratio of T84:P16. Interestingly, when starting with an input GE ratio of only 5% wild-type P388 virus, limited replication was observed and the proportion dropped to less than 1% wild-type P388 recovered.p = 0.0078) and 21 (p = 0.0004) post-vaccination was significantly different when compared to the initial percentage of the wild type in the HAdV-4 tablet administered to vaccines (p = 0.0001) per day.The Wyeth and Teva Ad live vaccines are administered orally; the virus replicates in the GI tract of vaccine recipients and is shed in stools for as long as 28 days. Replication in the GI tract is a critical step for a protective immune response. With the discovery that the Wyeth and Teva HAdV-4 vaccines comprise a mixture of wild-type and mutant strains, we investigated whether the differential replication that we demonstrated in vitro with plaque-purified viruses also occurs in the human GI tract. Archived stool specimens from volunteers who received the Wyeth HAdV-4 and HAdV-7 vaccine were subjected to SNP genotyping . The pervaccines B. DuringLicensed Adenovirus Type 4 and Type 7 Vaccine, Live, Oral have been in use by DoD for over 50 years, during which time two different manufacturers, Wyeth and Teva, produced essentially the same vaccine ,11,13. TA seed-lot system was used during vaccine virus propagation in which WI-38 cells were infected with stock vaccine virus and cell supernatant was collected, then passaged multiple times to generate aliquots of working seed virus stocks, which then undergo additional passages to produce vaccine drug substance lots for tablet production ,28. ExpeInterestingly, although the mutant T388 vaccine virus outcompeted the wild type in vitro, we found that its replication in the GI tract of human vaccines was diminished relative to the wild-type P388 vaccine virus. The drastic increase in the P388-to-T388 ratio in fecal specimens collected post-vaccination suggests that the wild-type virus established a more robust infection and prolonged replication in the GI tract of vaccines, outcompeted the T388 mutant virus, and eventually became the predominant strain. The differential genome replication observed in vitro and the concomitant decline in the percentage of T388 in the stool of vaccine recipients could indicate decreased production of infectious virus particles; further investigation is needed to fully explore this hypothesis.The oral HAdV vaccines produce transient asymptomatic infection of the GI tract, thereby inducing a type-specific protective immune response that is highly correlated with the presence of serum broadly neutralizing antibody titers ,27,28,29Our discovery of the significant functional impact of the P388T mutation raises questions about its mechanism of action and underscores the need for further investigation of the role and structure of the pTP. Furthermore, this study highlights the importance of verifying findings from in vitro studies in relevant tissues and organs of human vaccine recipients. More systematic comparative analyses are needed to shed light on how a single-amino-acid substitution at this residue results in a drastic phenotypical change and what factors modulate the differential replication in an in vivo versus in vivo environment.


Grapevine red blotch virus (GRBV) and grapevine Pinot gris virus (GPGV) are two of the most concerning grapevine viruses in Canada. Currently, the secondary spreading of these viruses (spreading via an insect vector) in Canada is poorly documented. To monitor the spreading of these viruses, we deployed virus-free grapevines onto two virus-infected vineyards with different agronomic practices. One vineyard was organic while the other was conventional. Using two independent diagnostic tools, namely the polymerase chain reaction (PCR) and the high-throughput sequencing technique (HTS), we have found that GPGV is spreading in both vineyards. The spread of GPGV was noticeably faster in the organic vineyard than in the conventional one, possibly due to a higher insect population. There was no evidence of the spreading of GRBV. A small collection of plant species in and around the vineyard was also tested by the two methods above. None of the non-grape species harbored any grapevine viruses but a large portion of the wild grape found on the edge of the vineyard carried GPGV. This study is the first direct proof of insect-mediated spreading of GPGV in North America.Medicago sativa, Trifolium repens, Cirsium arvense and Taraxacum officinale), perennial plants in border areas  and wild grape (unknown Vitis sp.). The HTS result showed that cover crops and perennial plants did not harbor any grapevine viruses, while 4/5 wild grapes tested positive for GPGV but not GRBV. A pairwise sequence identity analysis revealed high similarities between the GPGV isolates found in the established vines on the vineyard and the newly contracted GPGV isolates in the sentinel vines, implicating a recent transmission event. This work provides novel insights into the spread of grapevine viruses in Niagara Region and is also the first direct proof of the spread of GPGV in natural vineyard conditions in North America.This study investigated the transmission of grapevine viruses, specifically grapevine red blotch virus (GRBV) and grapevine Pinot gris virus (GPGV), in vineyards in Niagara Region, Ontario, Canada. Forty sentinel vines that were confirmed free of GRBV and GPGV by both high-throughput sequencing (HTS) and endpoint polymerase chain reaction (PCR) were introduced to two vineyards  that were heavily infected with both GRBV and GPGV. Four months post-introduction, the sentinel vines were relocated to a phytotron. The HTS results from 15 months post-introduction revealed a widespread infection of GPGV among the sentinel vines but did not detect any GRBV. The GPGV infection rate of sentinel vines in the organic vineyard (13/18) was higher than in the conventional vineyard (1/19). The possibility of an alternative viral reservoir was assessed by testing the most abundant plants in between rows ( In recent years, the emergence of grapevine viral diseases is posing a serious threat to the profitability and sustainability of the grape and wine sector . AmongstGrablovirus of the family Geminiviridae ). Vineyard A is a certified organic vineyard. The selected site in vineyard A was a block of French hybrid, cv. Vidal blanc, located at the northwest corner of the vineyard, previously reported to have at least 80% incidence of GRBV and GPGV (data published in the webinar cited above). Approximately 10 m to the north and to the west of this block was a thin strip of unmanaged riparian area, populated by predominantly white ash (Fraxinus americana), elm (Ulmus americana) and buckthorn (Rhamnus cathartica). Wild grape (unknown Vitis sp.) was also found in this area, albeit less frequently than the other species. In the conventional vineyard B, the selected block was also a cv. Vidal Blanc block, located close to the middle of the vineyard and no non-grape perennial plant was found in close proximity to the sampling area (data published in the webinar cited above). This block was reported to have at least 85% incidence rate of GRBV and GPGV. The overall layouts of each vineyard can be found in Two vineyards in Niagara Region, henceforth referred to as vineyard A and B, were selected in this study due to the high prevalence of grapevine red blotch virus (GRBV) and grapevine pinot gris virus (GPGV) . These vines were kept in the phytotron at Brock University for 2 years prior to this study. The phytotron conditions were 26 °C in the day and 15 °C at night, 15 h of combined sunlight and artificial light, humidity 60–80%. The 40 sentinel vines were sampled once for virus testing prior to being introduced to the vineyards on 1 July 2021, in the same protocol described above. Since the sentinel vines were expected to be free of economically important viruses, the 40 pre-introduction sentinel vine samples were combined into eight composite samples, each comprising five sentinel vines and were named CSV-1 to CSV-8 and tested with HTS. To confirm the HTS result, PCR tests for GRBV and GPGV were also conducted for these samples.Forty grafted Sentinel vines were introduced to the two targeted vineyards on 1 July 2021, in 5 litre pots filled with mycorrhizae soil  and watered twice a week. They were placed in the gap under the trellis of the commercial vines, in two adjacent rows in an alternating manner . The distance between two sentinel vines in the same row was approximately two to three meters, and the rows were three meters (10 feet) apart. The sentinel vines were relocated back to the phytotron at Brock University on 31 October 2021. Three vines  died during this period due to mechanical damage. The remaining 37 vines, prior to being replanted in the phytotron, underwent a complete foliar removal and extensive roots washes to ensure that no insect species was brought to the phytotron. In September 2022, 15 months post-introduction, the 37 sentinel vines were sampled again with the same procedure. The long inoculation time was to ensure that GRBV and GPGV, if contracted from the field, had enough time to increase in titre and spread systematically within the vine. These samples were tested with HTS and PCR for GRBV and GPGV on an individual basis.Medicago sativa), white clover (Trifolium repens) and creeping thistle (Cirsium arvense). In vineyard B, they were creeping thistle, white clover and common dandelion . One composite sample containing five individual samples for each species from each vineyard was collected. For these species, four mature leaves facing four different directions were collected as an individual sample. Due to the proximity of the unmanaged riparian area to the study site on vineyard A, perennial plants and wild grape were also considered potential viral reservoirs. One composite sample for each of the three most abundant species  were collected. For these samples, four mature leaves on four branches facing different directions were collected as an individual sample. Since wild grape was previously shown to be a host for GRBV [A survey was conducted at each vineyard to determine the three most abundant plant species on the vineyard floor (henceforth referred to as floor-covering plants). In vineyard A, they were alfalfa . The extracted total RNAs then underwent PCR for GRBV and RT-PCR for GPGV. Quality control was done with Nanodrop 1C  and the cut-off threshold was set to be between 1.6 and 2.2 A260/A280 ratio.For GRBV, we used the primer set GVGF1 (5′-CTCGTCGCATTTGTAAGA-3′) and GVGR1 (5′-ACTGACAAGGCCTACTACG-3′) described in Al Rwahnih, 2013 . For GPGAnother aliquot was used for the HTS pipeline. To enrich the viral titre in samples, a scaled-down version of the dsRNA extraction protocol  described in Tzanetakis and Martin (2008) was used. Nanodrop 1C  was used for quality control. Samples with a A260/A280 ratio outside the range of 1.5 and 2.2 were reextracted. Concentrations of these samples were determined using Qubit 4, high-sensitivity RNA kit .® Stranded mRNA Library Prep Plant (96 samples) and Truseq RNA CD index plate (96 indexes)  following the manufacturer protocol. The prepared libraries were sequenced with MiSeq platform .Extracted dsRNA  with sufhttps://www.virtool.ca/, accessed on 15 March 2023). In Virtool’s pipeline, adapter sequences and ambiguous regions are trimmed off by Skewer 0.0.2 and then subjected to FastQC 0.11.5 for quality assessment [Virtool, a cloud-based bioinformatic pipeline developed by Dr. Mike Rott and CFIA, was used to scan for all viruses and viroid presence in each sample . However, considering that this segment was relatively short in comparison to the ORF-1 (9/5538 nucleotides or 0.16%), WG-1 was also included in this analysis. To better visualize the relationship among the GPGV isolates found in this study with GPGV isolates from other regions of the world, six additional ORF-1s from published sequences on NCBI were also included in the sequence identity analysis. These additional sequences were BC-1 and ON-1 from Canada (KU194413 and OK558797), S148 and S149 from the USA (MK514531 and MK514532) and fvgIs-12 and fvgIs-13 from Italy (MH087443 and MH087444). The total data pool of 14 ORF-1s were imported to Sequence Demarcation Tool version 1.2 (SDTv1.2), where they were aligned using the MUSCLE algorithm. The option to clusters isolates together via a neighbor joining tree was rejected for better visualization. Their pairwise distances were calculated using default parameters. A time-tree was constructed by first building a maximum likelihood model . From that maximum likelihood model, the time-tree was constructed using RelTime method by Takamura et al., 2012 and choosing apple chlorotic leaf spot virus (ACLSV), which belongs to the same Trichovirus genus as GPGV, as the outgroup. ACLSV was chosen because the accuracy of the time-tree is improved if the phylogenetic distance between the ingroups and outgroup is not too large [No GRBV was detected in the sentinel vines by either HTS or PCR. The only newly contracted grapevine virus detected in the sentinel vines was GPGV. To determine the relationship among GPGV isolates found in the sentinel vines, commercial vines and wild grape, a sequence identity analysis was conducted. HTS-generated reads from all samples that were found to be positive with GPGV with HTS in the previous section were imported to CLC Genomic Workbench 20.0.4 (CGW). They were then trimmed off with the threshold of Q20 . We first attempted to extract GPGV genomes from each sample using the reference assembly on “Grapevine Pinot gris virus, complete genome” (NCBI accession number: NC_015782.1). However, because none of the samples contained a full GPGV genome, we opted to construct a consensus sequence of the first open reading frame (ORF-1) instead. Starting at nucleotide 95th and ending at nucleotide 5632nd of the reference genome (5538 bp in length), ORF-1 is the longest ORF on the GPGV genome and contains the oo large . No addiVirtool’s Pathoscope detected only two grapevine viruses and a viroid in the eight composite samples from pre-introduction 40 sentinel vines, namely GRSPaV, GTLaV and HSVd. GRSPaV and HSVd are considered ubiquitous in grapevine, with minor or no effect on the vine’s physiology ,37. The Viruses that were present in the pre-introduction sentinel vines, namely GRSPaV, HSVd and GaTLV were also found in the sentinel vines post-introduction. GRSPaV was detected in 26/37 samples and high coverage (>80%) of the genome was obtained in 11 samples. Similarly, HSVd was detected in 15/37 samples and high coverage was obtained in 12 samples. For GaTLV, 21/37 samples were found positive, with high coverage obtained in seven samples. Overall, the viral status of the sentinel vines pre- and post-introduction did not change with regards to GRSPaV, HSVd and GaTLV.No GRBV was detected in any of the 37 post-introduction sentinel vines . HoweverThe endpoint PCR confirmed that the two vineyards targeted in this study have high incidence rates of both GRBV and GPGV. Regarding GRBV, 19/20 (95%) vines from vineyard A and 13/20 (65%) vines from vineyard B tested positive. Similarly, 20/20 (100%) and 14/20 (70%) vines from vineyard A and B, respectively, tested positive for GPGV . The gelIn the eight commercial vines selected for HTS, seven were found positive with GPGV and five were found positive with GRBV. We also found 100% incidence rate of grapevine Rupestris stem pitting-associated virus (GRSPaV) (8/8) and 75% incidence rate of hop stunt viroid (HSVd) (75%) . GrapeviThe virus scan with Virtool did not detect grapevine viruses from any composite samples of floor covering or perennial plants. White clovers from both vineyards hosted a wide variety of viruses and are similar in overall viral status. White clover mosaic virus (WCIMV) and white clover cryptic virus 1 (WCCV1) were detected in alfalfa. Soybean Putnam virus (SPuV) was detected in creeping thistle from both vineyards, while tomato ringspot virus (ToRSV) was detected in buckthorn and white ash from vineyard A. No virus was detected in elm or dandelion. The follow-up PCR also confirmed the absence of GRBV and GPGV from these samples.The only newly acquired virus found in the sentinel vines in this study was GPGV. With HTS, GPGV was found in 25 samples, of which 14 were sentinel vines post-introduction, seven were established vines and four were wild grapes. In those 25 samples, the reference assembles yielded a complete ORF-1 of GPGV for eight samples. Six of them were sentinel vines  and two were established vines (VA-1 and VA-4). The wild grape sample WG-1, which contained a near-complete OFR-1 with an ambiguous segment of nine nucleotides toward the 3′-end, was also included in this analysis. These nine sequences can be found on NCBI (accession numbers: OQ945219 to OQ945227)The ORF-1 of GPGV isolates found in sentinel and established vines were almost identical, with a pairwise identity of 99% or higher. They also shared very high similarity with GPGV isolates from other locations in Canada and the USA, with pairwise identity in the range of 97% to 100%. In comparison, the Italian GPGV isolates were found to be more genetically distant, as their ORF-1 only shared 95% to 97% sequence identity with the GPGV isolates found in sentinel vines and established vines. Interestingly, the GPGV isolate from wild grape differed even more significantly from the Italian GPGV isolates, sharing only 93% sequence identity. The GPGV isolate in wild grape shared high similarity (97–99%) with the GPGV isolates found in sentinel, established vines and isolate ON-1 , and slightly less similarity to GPGV isolates from the USA  and BC, Canada (BC-1) with around 96% pairwise identity .The primary objective of this study was to investigate if GPGV and GRBV were being transmitted in vineyards in Niagara Region. Forty sentinel vines that were free of GRBV and GPGV were deployed to two vineyards with high-incident rate of both GRBV and GPGV. One vineyard is organic (vineyard A) while the other is conventional (vineyard B). Aside from the sentinel vines, samples from the present commercial vines, floor-covering plants, nearby perennial plants and wild grapes were also collected and tested for GRBV and GPGV using both PCR and HTS.Our results confirmed the high prevalence of GPGV and GRBV in the commercial vines. Using HTS as a viral scanning tool, we also detected high prevalence of ubiquitous virus and viroid GRSPaV and HSVd in the commercial vines, with an occasional presence of other grapevine viruses. GaTLV was detected in two samples, while GLRaV-2, GLRaV-3, GRGV, GSyV and GVB were all detected once. Since GLRaV-2 and -3 are both economically important viruses, it would be beneficial to monitor the spread of these viruses in these vineyards for following seasons.Vitis species in and around these vineyards, the HTS results show a complete absence of grapevine viruses and viroids in the selected floor covering and perennial plants from both vineyards  and seven woody plant species  were tested and none were found to be true host for GRBV, except for Vitis californica x Vitis vinifera [Fraxinus sps.) [Regarding the viral status of non-ineyards . The absvinifera . The absvinifera ,24. A reus sps.) , which mIn the riparian area of vineyard A, wild grape was also present. The HTS viral scan for five wild grape samples showed 100% infection of GRSPaV. None of the wild grape was found positive with GRBV, while four over five wild grape samples were positive for GPGV . Even thWith the viral landscape of each vineyard established , twenty GRBV and GPGV-free sentinel vines were deployed to each vineyard . After four months of exposure followed by 11 months of inoculation in the phytotron, the HTS result revealed that none of the sentinel vines contracted GRBV. Regardless, to conclude that GRBV is not being transmitted in these two vineyards is premature at this point. The transmission of GRBV via an insect vector was previously found to be relatively slow. Even in greenhouse conditions with carefully designed arrangement to facilitate the transmission of GRBV, the rate of infection was quite low ,24. ConsSurprisingly, a large proportion of the sentinel vines (14/37) was found positive with GPGV post-introduction . None ofThe explosive spreading of GPGV in vineyard A was also very similar to another incidence documented in Southern France, in which 70% of GPGV-negative vines got infected within one year . The widTo improve the confidence in our result, all sentinel vines underwent an endpoint PCR test for GPGV and GRBV. PCR and HTS results were in complete agreement, except for the GPGV status of three sentinel vines (SSV-2, SSV-13 and SSV-18 . In prevColomerus vitis, prevent us from a potentially deeper understanding of the role of this insect in spreading GPGV. Hypotheses on GPGV’s vector other than Colomerus vitis have become more plausible since the discovery of a wide range of GPGV alternative hosts [We also acknowledge that there are several limitations for this study. Firstly, the short exposure time of four months does not well represent the conditions that the perennial commercial vines endure. To effectively monitor the movement of plant viruses with low infection rate such as GRBV , a prolove hosts . Lastly,This work provided novel insights on the epidemiology of grapevine viruses in the vineyards in Niagara Region. All non-grape plant species subjected in this study were found free of grapevine viruses. Spread of GPGV was confirmed in both vineyards, serving as the first direct proof of secondary transmission or GPGV in North America. The rate of infection of GPGV in the organic vineyard was substantially higher than the conventional vineyard. Although we did not detect any GRBV in the sentinel vines, the previously reported slow rate of infection of GRBV necessitates longer-term studies.


A separate request for whole sample consumption for FIGG purposes should be reviewed and authorized by the investigative agency, after considering input from the forensic laboratory and the prosecuting agency. All approvals from stakeholders should be retained within the case documentation. Developed policies for whole sample consumption shall be consistent with applicable law. Forensic laboratories may develop policies on consumption with their local prosecuting attorney or equivalent and provide them to submitting agencies.Whole sample consumption is considered a last resort in forensic investigations, so must be given due consideration, have appropriate documentation and authorization, and be in compliance with federal and state laws prior to proceeding with analysis  participating in the [FIGG] shall turn over to the investigator all documentation, records, and materials collected, including material sourced from public records, family trees constructed, and any other genetic or nongenetic data analyses collected” ,7.iv.If court testimony or additional analysis or research is required, appropriate documentation should be provided by the FIGG RA to permit analysis, investigation, preparation, and testimony as necessary.Data storage and transferC.i.The retention and deletion of all FIGG data/documentation/records/trees built during a FIGG investigation must adhere to the corresponding state and/or federal law.ii.The FIGG practitioner/provider or vendor laboratory “may not keep any records or materials in any form, including digital or hard copy records”  unless liii.Ancestry.com or Lucidchart  but may not otherwise use or share the records or materials” ,7.iv.Neither the laboratory conducting SNP or other DNA analysis, nor a law enforcement official or a genealogist may disclose genetic genealogy information or details associated with an ongoing investigation without authorization from the prosecuting agency ,7.iv.ancestry.com set to private are not assured to be so, therefore use of programs that reside on the private computer of a genealogical researcher are preferable.Consideration should be given by genealogical researchers to build family trees outside of public databases and their associated tools, as privacy, security, and confidentiality is not assured. For example, family trees in v.Personally identifiable third-party information may not be included in warrants and other legal documents which could reveal the identity of related individuals  noting tData disclosure and confidentiality7.6A.FIGG discovery must be provided in accordance with relevant law.B.As innocent individuals are documented in family trees through genealogical research that links a SNP DNA profile to biological relatives, who in turn are linked to the suspect, privacy interests of these individuals should be protected in compliance with jurisdictional law.C.If discovery is provided that includes identifying information associated with third-party individuals included in family trees, attempts may be made to protect this information such as with a court order.D.In the event there is no forensic STR DNA profile for a direct match to a suspect reference sample, a SNP panel will be required for comparison between a suspect reference sample and the forensic sample(s). It is anticipated that this situation will more likely require presentation in court and discovery should be provided in accordance with relevant law. For cases that do not require presentation in court, reports shall include sufficient information to be deemed informative by all interested parties.E.Adherence to policies and procedures can be enforced via procedures such as those provided by the FBI Quality Assurance Standards to ensure CODIS compliance .7.7A.Number of FIGG cases investigated ;B.Type of crime for which the FIGG case is being investigated;C.DNA specimen details including type of specimen , DNA quantity and quality;D.Type of DNA typing and analysis applied to the sample specimen;E.Identity of vendor laboratories conducting SNP sequencing analysis and FIGG practitioners/providers conducting genealogical research;F.Number of FIGG cases entered into genetic genealogy databases;G.Number of leads resulting in an identification ,7;H.Costs of FIGG procedures (both cumulative and categorized by laboratory workflow (in-laboratory or vendor), genealogy, investigation ,7;I.“Number of times a third-party DNA sample was requested and collected” ;J.“Number of [FIGG] requests made by defendants and post-conviction attorneys” ; and/orK.The case outcomes of each FIGG investigation ,7.Data that can aid in the continuous improvement of the FIGG program should be collected ,7. Data 7.8The FIGG RA, forensic laboratories, and investigative agencies implementing FIGG may choose to consult with a panel, a commission, or a committee. The panel, commission, or committee shall be comprised of stakeholders deemed appropriate by the relevant jurisdiction.“A panel comprised of judges, prosecutors, defense attorneys, public defenders, law enforcement officials, crime laboratory directors, bioethicists” , genealoThe authors wish to declare the following competing interests: S.A.K is a current employee and shareholder of QIAGEN Inc. C.J.G. and R.A.W. are members of the Investigative Genetic Genealogy Working Group of the Scientific Working Group on DNA Analysis Methods. C.J.G is a member of the Advisory Board of the Investigative Genetic Genealogy Accreditation Board.