Residues within the Foot-and-Mouth Disease Virus 3Dpol Nuclear Localization Signal Affect Polymerase Fidelity.

Many RNA viruses encode a proof-reading deficient, low-fidelity RNA-dependent polymerase (RdRp), which generates genetically diverse populations that can adapt to changing environments and thwart antiviral therapies. 3Dpol, the RdRp of the foot-and-mouth disease virus (FMDV), is responsible for replication of viral genomes. The 3Dpol N terminus encodes a nuclear localization signal (NLS) sequence,MRKTKLAPT, important for import of the protein to host nucleus. Previous studies showed that substitutions at residues 18 and 20 of the NLS are defective in proper incorporation of nucleotides and RNA binding. Here, we use a systematic alanine scanning mutagenesis approach to understand the role of individual residues of the NLS in nuclear localization and nucleotide incorporation activities of 3Dpol We identify two residues of 3Dpol NLS, T19 and L21, that are important for the maintenance of enzyme fidelity. The 3Dpol NLS alanine substitutions of T19 and L21 results in aberrant incorporation of nucleoside analogs, conferring a low fidelity phenotype of the enzyme. A molecular dynamics simulation of RNA- and mutagen (RTP)-bound 3Dpol revealed that the T19 residue participates in a hydrogen bond network, including D165 in motif F and R416 at the C terminus of the FMDV 3Dpol and RNA template-primer. Based on these findings and previous studies, we conclude that at least the first six residues of theMRKTKLAPT sequence motif play a vital role in the maintenance of faithful RNA synthesis activity (fidelity) of FMDV 3Dpol, suggesting that the role of the NLS motif in similar viral polymerases needs to be revisited.IMPORTANCE In this study, we employed genetic and molecular dynamics approaches to analyze the role of individual amino acids of the FMDV 3Dpol nuclear localization signal (NLS). The NLS residues were mutated to alanine using a type A full-genome cDNA clone, and the virus progeny was analyzed for defects in growth and in competition with the parental virus. We identified two mutants in 3Dpol, T19A and L21A, that exhibited high rate of mutation, were sensitive to nucleotide analogs, and displayed reduced replicative fitness compared to the parental virus. Using molecular dynamics simulation, we demonstrated that residues T19 and L21 played a role in the structural configuration of the interaction network at the 3Dpol palm subdomain. Cumulatively, our data suggest that the T19 and L21 3Dpol amino acids are important for maintaining the fidelity of the FMDV polymerase and ensuring faithful replication of the FMDV genome.

Health Care, Family, and Community Factors Associated with Mental, Behavioral, and Developmental Disorders and Poverty Among Children Aged 2-8 Years - United States, 2016.

Childhood mental, behavioral, and developmental disorders (MBDDs) are associated with adverse outcomes that can persist into adulthood (1,2). Pediatric clinical settings are important for identifying and treating MBDDs (3). Early identification and treatment of MBDDs can promote healthy development for all children (4), especially those living in poverty who are at increased risk for MBDDs (3,5) but might have reduced access to care (6). CDC analyzed data from the 2016 National Survey of Children's Health (NSCH) on MBDDs, risk factors, and use of federal assistance programs (e.g., Supplemental Nutrition Assistance Program [SNAP]) to identify points to reach children in poverty. In line with previous research (3,6), compared with children in higher-income households, those in lower-income households more often had ever received a diagnosis of an MBDD (22.1% versus 13.9%), and less often had seen a health care provider in the previous year (80.4% versus 93.8%). Among children living below 200% of the federal poverty level (FPL) who did not see a health care provider in the previous year, seven of 10 were in families receiving at least one public assistance benefit. Public assistance programs might offer collaboration opportunities to provide families living in poverty with information, co-located screening programs or services, or connection to care.

Systemic immune response and virus persistence after foot-and-mouth disease virus infection of naïve cattle and cattle vaccinated with a homologous adenovirus-vectored vaccine.

BACKGROUND: In order to investigate host factors associated with the establishment of persistent foot-and-mouth disease virus (FMDV) infection, the systemic response to vaccination and challenge was studied in 47 steers. Eighteen steers that had received a recombinant FMDV A vaccine 2 weeks earlier and 29 non-vaccinated steers were challenged by intra-nasopharyngeal deposition of FMDV A24. For up to 35 days after challenge, host factors including complete blood counts with T lymphocyte subsets, type I/III interferon (IFN) activity, neutralizing and total FMDV-specific antibody titers in serum, as well as antibody-secreting cells (in 6 non-vaccinated animals) were characterized in the context of viral infection dynamics. RESULTS: Vaccination generally induced a strong antibody response. There was a transient peak of FMDV-specific serum IgM in non-vaccinated animals after challenge, while IgM levels in vaccinated animals did not increase further. Both groups had a lasting increase of specific IgG and neutralizing antibody after challenge. Substantial systemic IFN activity in non-vaccinated animals coincided with viremia, and no IFN or viremia was detected in vaccinated animals. After challenge, circulating lymphocytes decreased in non-vaccinated animals, coincident with viremia, IFN activity, and clinical disease, whereas lymphocyte and monocyte counts in vaccinated animals were unaffected by vaccination but transiently increased after challenge. The CD4(+)/CD8(+) T cell ratio in non-vaccinated animals increased during acute infection, driven by an absolute decrease of CD8(+) cells. CONCLUSIONS: The incidence of FMDV persistence was 61.5 % in non-vaccinated and 54.5 % in vaccinated animals. Overall, the systemic factors examined were not associated with the FMDV carrier/non-carrier divergence; however, significant differences were identified between responses of non-vaccinated and vaccinated cattle.

Chronic conditions, functional difficulties, and disease burden among American Indian/Alaska Native children with special health care needs, 2009-2010.

The purpose of this study was to determine the prevalence of chronic conditions and functional difficulties of American Indian/Alaska Native (AIAN) children with special health care needs (CSHCN). We conducted bivariate and multivariable analysis of cross-sectional data on 40,202 children from the 2009-2010 National Survey of Children with Special Health Care Needs aged birth through 17 years, including 1,051 AIAN CSHCN. The prevalence of AIAN CSHCN was 15.7 %, not significantly different from the prevalence of US white CSHCN (16.3 %). As qualifiers for special needs status among AIAN children the use of or need for prescription medication was the most frequent (70 %), compared to the lower rates of need for elevated service use (44 %) and emotional, mental, or behavioral treatment/counseling (36 %). Asthma (45 %), conduct disorder (18 %), developmental delay (27 %), and migraine headaches (16 %) were significantly more common chronic conditions among AIAN CSHCN compared to white CSHCN, as were functional difficulties with respiration (52 %), communication (42 %), anxiety/depression (57 %), and behavior (54 %). AIAN CSHCN were also more likely to have 3 or more chronic conditions (39 vs. 28 %, respectively) and 3 or more functional difficulties (70 vs. 55 %, respectively) than white CSHCN. Results indicated a greater impact on the daily activities of AIAN CSHCN compared to white CSHCN (74 vs. 63 %). Significantly greater disease burden among AIAN CSHCN suggests that care must be taken to ensure an appropriate level of coordinated care in a medical home to ameliorate the severity and complexity of their conditions.

Family perceptions of shared decision-making with health care providers: results of the National Survey of Children With Special Health Care Needs, 2009-2010.

The Maternal and Child Health Bureau recently revised its measure of family-provider shared decision-making (SDM) to better align with parents' views and the intent of SDM. We sought to assess achievements in meeting the revised measure; examine socio-demographic/health correlates; and determine the relationships between SDM and access to quality health care. We analyzed data for 40,242 children with special health care needs (CSHCN) from the 2009-2010 National Survey of CSHCN and assessed the prevalence of SDM and association with other US CSHCN socio-demographic/health characteristics using bivariate and multivariate methods. Logistic regression was used to determine associations between SDM and having a medical home and preventive medical/dental visits. Approximately 70% of families of CSHCN perceived themselves as shared decision-makers in their child's care. Families of CSHCN with greater functional limitations had twice the odds of lacking SDM than those never affected. Disparities in attainment rates were noted for families with low versus high income (61 vs. 77%), less versus more than high school education (59 vs. 73%), privately insured versus uninsured (76 vs. 57%), and minority versus white race (63 vs. 74%). CSHCN with medical homes had 6 times greater odds of perceived SDM and as much as one and a half times the odds of receiving preventive care than CSHCN without a medical home. Major differences in family SDM perceptions are associated with having a medical home, particularly when characterized by family-centered care. Populations of concern are those with more functionally limited children and increased socio-economic challenges.

Insurance coverage of medical foods for treatment of inherited metabolic disorders.

PURPOSE: Treatment of inherited metabolic disorders is accomplished by use of specialized diets employing medical foods and medically necessary supplements. Families seeking insurance coverage for these products express concern that coverage is often limited; the extent of this challenge is not well defined. METHODS: To learn about limitations in insurance coverage, parents of 305 children with inherited metabolic disorders completed a paper survey providing information about their use of medical foods, modified low-protein foods, prescribed dietary supplements, and medical feeding equipment and supplies for treatment of their child's disorder as well as details about payment sources for these products. RESULTS: Although nearly all children with inherited metabolic disorders had medical coverage of some type, families paid "out of pocket" for all types of products. Uncovered spending was reported for 11% of families purchasing medical foods, 26% purchasing supplements, 33% of those needing medical feeding supplies, and 59% of families requiring modified low-protein foods. Forty-two percent of families using modified low-protein foods and 21% of families using medical foods reported additional treatment-related expenses of $100 or more per month for these products. CONCLUSION: Costs of medical foods used to treat inherited metabolic disorders are not completely covered by insurance or other resources.

Rapid and transient activation of γδ T cells to IFN-γ production, NK cell-like killing, and antigen processing during acute virus infection.

γδ T cells are the majority peripheral blood T cells in young cattle. The role of γδ T cells in innate responses against infection with foot-and-mouth disease virus was analyzed on consecutive 5 d following infection. Before infection, bovine WC1(+) γδ T cells expressed a nonactivated phenotype relative to CD62L, CD45RO, and CD25 expression and did not produce IFN-γ ex vivo. Additionally, CD335 expression was lacking and no spontaneous target cell lysis could be detected in vitro, although perforin was detectable at a very low level. MHC class II and CD13 expression were also lacking. Following infection with foot-and-mouth disease virus, expression of CD62L and CD45RO was greatly reduced on WC1(+) γδ T cells, and unexpectedly, CD45RO expression did not recover. A transient increase in expression of CD25 correlated with production of IFN-γ. Expression of CD335 and production of perforin were detected on a subset of γδ T cells, and this correlated with an increased spontaneous killing of xenogeneic target cells. Furthermore, increased MHC class II expression was detected on WC1(+) γδ T cells, and these cells processed protein Ags. These activities are rapidly induced, within 3 d, and wane by 5 d following infection. All of these functions, NK-like killing, Ag processing, and IFN-γ production, have been demonstrated for these cells in various species. However, these results are unique in that all these functions are detected in the same samples of WC1(+) γδ T cells, suggesting a pivotal role of these cells in controlling virus infection.

Child, family, and neighborhood associations with parent and peer interactive play during early childhood.

To examine national patterns of peer and parent interactive play opportunities that enhance early learning/socialization. Bivariate and multivariable analyses of cross-sectional data on 22,797 children aged 1-5 years from the National Survey of Children's Health 2007 were performed to determine the child, family, and neighborhood factors associated with four parent-initiated activities. Outcomes measures included time (days/week) children spent: participating in peer play; being read to; sung to/told stories; and taken on family outings. Covariates included race/ethnicity, poverty, TV watching, childcare, child and maternal physical and mental health, family factors (structure, size, language, stress, education), and neighborhood factors (amenities, support, physical condition, safety). According to adjusted regression models, minority children from lower income, non-English-speaking households with limited education, poorer maternal health and greater parenting stress were read to/told stories less than children without these characteristics, while neighborhood factors exerted less influence. In contrast, significant reductions in days/week of peer play were associated with unsupportive neighborhoods and those with the poorest physical conditions and limited amenities. Likewise, reductions in outings were associated with fewer neighborhood amenities. The findings of this study indicate that a variety of child, family, and neighborhood factors are associated with parent-initiated behaviors such as reading, storytelling, peer interactive play, and family outings. Appropriate evidence-based home visiting interventions targeting child health, parenting skills, early childhood education, and social services in at-risk communities would appear to be appropriate vehicles for addressing such parent-initiated play activities that have the potential to enhance development.

Federal expenditures on maternal and child health in the United States.

The goals of this study are to estimate federal maternal and child health (MCH) expenditures and identify their sources. This analysis is intended to provide a broad view of MCH funding appropriations and a basis for discussion of whether funds could be better utilized for the benefit of the population served. Data on federal maternal and child health expenditures for fiscal year (FY) 2006 were derived from examining federal legislation, department/agency budgets, and various web-based program documents posted by federal agencies. Based on selected criteria, we identified programs targeting children under 21 or pregnant/parenting women within the United States. The funding levels of agency programs for maternal and child health activities were determined and the programs briefly summarized. The identifiable funding for maternal and child health programs in FY 2006 approached $57.5 billion dollars. Funding sources for maternal and child health were concentrated within the U.S. Department of Health and Human Services, but spread across several different agencies within the department and in the Departments of Defense, Education, Agriculture, Housing and Urban Development, and the Environmental Protection Agency. Multiple agencies and offices often funded related activities, without evidence of a common underlying strategy. Federal maternal and child health funding mechanisms may lead to a fragmentation in maternal and child health activities. The funding and service delivery apparatus would benefit from an integrative MCH infrastructure approach to pediatric research, service delivery, and data collection/access that incorporates life-course and social/environmental determinants perspectives.

Children and Youth With Special Health Care Needs: A Profile.

BACKGROUND AND OBJECTIVES: The National Survey of Children's Health (NSCH) is the nation's primary source for data on children and youth with special health care needs (CYSHCN) and the only source for state-level estimates. We provide the latest estimates of CYSHCN in the United States, describe population characteristics, as well as the proportion that are served in a well-functioning system of care. METHODS: Data from the 2016, 2017, 2018, and 2019 NSCH were appended, resulting in a final analytic sample of 30 301 CYSHCN. Bivariate associations between the covariates and SHCN status as well as the 6 core outcomes that comprise a well-functioning system of care were examined using χ2 tests. Multivariable logistic regression was used to identify factors independently associated with SHCN status and a well-functioning system of care. RESULTS: The overall prevalence of CYSHCN was 18.8% and ranged from 13.6% in Hawaii to 24% of individuals included in the NSCH in West Virginia. The most reported type of SHCN was prescription medication use (29.3%), whereas 25.9% of CYSHCN had functional limitations. Over one-third reported no daily activity impacts, whereas 18.9% reported consistent or significant daily impacts. CYSHCN were more likely than non-CYSHCN to be male, older, non-Hispanic Black, live in poverty, and have public insurance but disparities by race and ethnicity and income were no longer significant after adjustment. Only 14.9% of CYSHCN were reported to receive care in a well-organized system. Rates were substantially lower among older and more heavily affected children with adjusted rate ratios for access to a well-functioning system of care, indicating a 72% reduction for adolescents (12-17), compared with young children (0-5) and a 24% to 53% reduction for those with more than a prescription medication qualifying need. CONCLUSIONS: CYSHCN remain a sizable and diverse population with distinct challenges in accessing well-functioning systems of care, particularly for those with the greatest needs. Our results provide a profile of the population designed to inform future surveillance, research, program, and policy priorities showcased in this Special Issue.

Systems of care among children and youth with special health care needs with and without adverse childhood events: National Survey of Children's Health 2016-2017.

BACKGROUND: Knowledge of adverse childhood events (ACEs), the associated access to healthcare and unmet needs among children and youth with special health care needs (CYSHCN) is limited. OBJECTIVES: Our objectives were to compare the likelihood of CYSHCN versus non-CYSHCN experiencing one or more ACEs and document differences in receipt of recommended and needed health care among CYSHCN with and without ACEs. METHODS: We combined two years of data from the 2016 and 2017 National Survey of Children's Health (N = 71,181), providing a sample of 16,304 CYSHCN. Our primary outcome measures included eight adverse childhood events, compared to singular and aggregated ACEs among non-CYSHCN. We calculated associations between ACEs and secondary outcome measures for six components of well-functioning systems of care and unmet need for different types of health care using bivariate and multivariate analyses. RESULTS: CYSHCN were more likely to have each of the ACEs measured and were likelier to experience aggregated levels of ACEs compared to non-CYSHCN. The likelihood of CYSHCN having a medical home with family-centered and coordinated care decreased with increased ACEs, while one or more ACES increased the likelihood of having unmet needs for mental health care. CONCLUSION: The findings of the current study extend our understanding of the additional adverse event burden associated with special health care needs status, the accompanying limitations in access to family-centered and coordinated care in a medical home and unmet need for mental health care, indicating that much work remains in establishing appropriate care systems for this very vulnerable population.

Parental Concerns About Child Weight Among 10-17-Year Olds With Overweight/Obesity: A Family Ecological Model.

OBJECTIVES: The purpose of this study was to identify child, family/household, organization (provider), and neighborhood/community factors associated with parental concern about weight among children with overweight/obesity in order to inform effective interventions for improving health in this pediatric population. METHODS: Prevalence of parental concern about child weight was estimated and factors identified within an adapted family ecological framework. Using cross-sectional data from the 2018 National Survey of Children's Health, we conducted bivariate and multivariable analyses of 10 to 17-year olds (N = 15,427) for whom height and weight information was reported by parents or primary caregivers. RESULTS: There were 4287 children, aged 10 to 17 years, with overweight/obesity (31%). Approximately 34% of parents of children with overweight/obesity reported being concerned about their child's weight, with the remainder being not concerned. In adjusted analyses, 23% of children with overweight and 45% of children with obesity had parents who reported being concerned. Factors associated with parental concern among children with overweight/obesity included child weight status, female gender, peer social difficulties, the extent of the child's daily activities affected by health conditions, poorer parental coping, and having been told the child was overweight by a provider. CONCLUSIONS: Only one in three parents of children with overweight/obesity reported being concerned about their child's weight, although parental concern was more common among children affected by obesity more so than overweight. A combination of child, family, and organization (provider) factors were associated with parental concern. Provider feedback about child overweight may improve parental awareness of a weight-related health issue.

Prevalence of Parent Reported Health Conditions Among 0- to 17-Year-Olds in Rural United States: National Survey of Children's Health, 2016-2017.

PURPOSE: This study's purpose was to determine the prevalence of physical, mental, and developmental health conditions among US children and assess the association with urban versus rural residence. METHODS: Bivariate/multivariable analyses were conducted with cross-sectional data for children aged 0-17 years (N = 71,811) from the 2016-2017 National Survey of Children's Health. Prevalence estimates of excellent/very good health were derived from parents' qualitative judgments. Parent-reported health conditions were aggregated by condition type (physical, mental, developmental). Prevalence was determined for condition type and severity. Adjusted risk ratios assessed the effect of residence on having physical, mental, or developmental conditions. RESULTS: Among rural children in the general population, we found lower crude rates of excellent/very good overall health and higher rates of ≥1 physical condition(s) and ≥1 mental condition(s), as well as these 2 conditions in combination with ≥1 developmental condition(s). Rural children in the general population were also more likely to have physical and mental conditions that parents rated as moderate/severe in unadjusted analyses. To a lesser extent, these differences held true for the children with special health care needs. Risk ratios for rural residence were largely nonsignificant in adjusted analyses. CONCLUSIONS: While rural children had lower crude rates of parent-reported excellent/very good health and higher crude rates of parent-reported or doctor-diagnosed physical and mental health conditions compared to urban children, the same pattern of urban-rural differentials was not evident in the adjusted analyses. Compositional and contextual differences in the urban/rural populations suggest that social determinants of health may have accounted for rate disparities in child health conditions.

CSHCN with hearing difficulties: Disparities in access and quality of care.

BACKGROUND: Hearing difficulties (HD) affect a sizable minority of children in the United States and can have a significant impact on child development. Children with HD may face barriers around language development, communication abilities, learning, and social interactions, which in turn can affect multiple aspects of their lives. OBJECTIVE/HYPOTHESIS: Little is known about the extent to which children with HD have access to a system of care and we hypothesize that this population may experience significant unmet needs. METHODS: In this cross-sectional study, using bivariate and multivariable regression models, we analyzed data from the 2009/2010 National Survey of Children with Special Health Care Needs (NS-CSHCN). Selected survey items were used to assess health and healthcare characteristics of CSHCN with some level of HD (n = 2,315) compared to those without HD (n = 37,851). The study sample included 40,242 CSHCN aged 0-17 years, whose caregivers responded to the survey. RESULTS: Approximately 694,000 CSHCN in the U.S. were reported to have any level of HD, representing 6.3% of the CSHCN population. Compared to CSHCN without HD, those with HD were less likely to have access to a medical home, community services, or have adequate health insurance. CONCLUSIONS: Significant deficits in medical home access, adequate insurance, and accessibility of community-based services are evident for CSHCN with HD. Families having knowledge of how and when to access community services and partner with providers in their child's health care is important for the overall health and wellbeing of this subpopulation of CSHCN.

Brain activation abnormalities during speech and non-speech in stuttering speakers.

Although stuttering is regarded as a speech-specific disorder, there is a growing body of evidence suggesting that subtle abnormalities in the motor planning and execution of non-speech gestures exist in stuttering individuals. We hypothesized that people who stutter (PWS) would differ from fluent controls in their neural responses during motor planning and execution of both speech and non-speech gestures that had auditory targets. Using fMRI with sparse sampling, separate BOLD responses were measured for perception, planning, and fluent production of speech and non-speech vocal tract gestures. During both speech and non-speech perception and planning, PWS had less activation in the frontal and temporoparietal regions relative to controls. During speech and non-speech production, PWS had less activation than the controls in the left superior temporal gyrus (STG) and the left pre-motor areas (BA 6) but greater activation in the right STG, bilateral Heschl's gyrus (HG), insula, putamen, and precentral motor regions (BA 4). Differences in brain activation patterns between PWS and controls were greatest in females and less apparent in males. In conclusion, similar differences in PWS from the controls were found during speech and non-speech; during perception and planning they had reduced activation while during production they had increased activity in the auditory area on the right and decreased activation in the left sensorimotor regions. These results demonstrated that neural activation differences in PWS are not speech-specific.

Common neural substrates support speech and non-speech vocal tract gestures.

The issue of whether speech is supported by the same neural substrates as non-speech vocal tract gestures has been contentious. In this fMRI study we tested whether producing non-speech vocal tract gestures in humans shares the same functional neuroanatomy as non-sense speech syllables. Production of non-speech vocal tract gestures, devoid of phonological content but similar to speech in that they had familiar acoustic and somatosensory targets, was compared to the production of speech syllables without meaning. Brain activation related to overt production was captured with BOLD fMRI using a sparse sampling design for both conditions. Speech and non-speech were compared using voxel-wise whole brain analyses, and ROI analyses focused on frontal and temporoparietal structures previously reported to support speech production. Results showed substantial activation overlap between speech and non-speech function in regions. Although non-speech gesture production showed greater extent and amplitude of activation in the regions examined, both speech and non-speech showed comparable left laterality in activation for both target perception and production. These findings posit a more general role of the previously proposed "auditory dorsal stream" in the left hemisphere--to support the production of vocal tract gestures that are not limited to speech processing.

Serological and phylogenetic characterization of foot and mouth disease viruses from Uganda during cross-sectional surveillance study in cattle between 2014 and 2017.

Here, we report the results of a cross-sectional study designed to monitor the circulation and genetic diversity of foot and mouth disease virus (FMDV) in Uganda between 2014 and 2017. In this study, 13,614 sera and 2,068 oral-pharyngeal fluid samples were collected from cattle and analysed to determine FMDV seroprevalence, circulating serotypes and their phylogenetic relationships. Circulation of FMDV was evidenced by the detection of antibodies against non-structural proteins of FMDV or viral isolations in all districts sampled in Uganda. Sequence analysis revealed the presence of FMDV serotypes A, O, SAT 1 and SAT 2. FMDVs belonging to serotype O, isolated from 21 districts, were the most prevalent and were classified into six lineages within two East African topotypes, namely EA-1 and EA-2. Serotype A viruses belonging to the Africa G-I topotype were isolated from two districts. SAT 1 viruses grouped within topotypes I and IV and SAT 2 viruses within topotypes VII, IV and X were isolated from six and four districts respectively. Phylogenetic analysis of SAT 1 and SAT 2 sequences from cattle clustered with historical sequences from African buffalo, indicating possible interspecies transmission at the wildlife-livestock interface. In some cases, Uganda viruses also shared similarities to viral strains recovered from other regions in East Africa. This 3-year study period provides knowledge about the geographical distribution of FMDV serotypes isolated in Uganda and insights into the genetic diversity of the multiple serotypes circulating in the country. Knowledge of circulating FMDV viruses will assist in antigenic matching studies to devise improved FMDV control strategies with vaccination and vaccine strain selection for Uganda.

Duration of protection and humoral immunity induced by an adenovirus-vectored subunit vaccine for foot-and-mouth disease (FMD) in Holstein steers.

Foot-and-mouth disease (FMD) is a highly contagious viral infection of cloven hooved animals that continues to cause economic disruption in both endemic countries or when introduced into a formally FMD free country. Vaccines that protect against clinical disease and virus shedding are critical to control FMD. The replication deficient human adenovirus serotype 5 (Ad5) vaccine vector expressing empty FMD virus (FMDV) capsid, AdtFMD, is a promising new vaccine platform. With no shedding or spreading of viral vector detected in field trials, this vaccine is very safe to manufacture, as there is no requirement for high containment faciitites. Here, we describe three studies assessing the proportion of animals protected from clinical vesicular disease (foot lesions) following live-FMDV challenge by intradermolingual inoculation at 6 or 9 months following a single vaccination with the commercial AdtFMD vaccine, provisionally licensed for cattle in the United States. Further, we tested the effect of vaccination route (transdermal, intramuscular, subcutaneous) on clinical outcome and humoral immunity. Results demonstrate that a single dose vaccination in cattle with the commercial vaccine vector expressing capsid proteins of the FMDV strain A24 Cruzeiro (Adt.A24), induced protection against clinical FMD at 6 months (100% transdermal, 80% intramuscular, and 60% subcutaneous) that waned by 9 months post-vaccination (33% transdermal and 20% intramuscular). Post-vaccination serum from immunized cattle (all studies) generally contained FMDV specific neutralizing antibodies by day 14. Anti-FMDV antibody secreting cells are detected in peripheral blood early following vaccination, but are absent after 28 days post-vaccination. Thus, the decay in antibody mediated immunity over time is likely a function of FMDV-specific antibody half-life. These data reveal the short time span of anti-FMDV antibody secreting cells (ASCs) and important performance characteristics of needle-free vaccination with a recombinant vectored subunit vaccine for FMDV.

Enhanced sensitivity in detection of antiviral antibody responses using biotinylation of foot-and-mouth disease virus (FMDV) capsids.

Analysis of the immune response to infection of livestock by foot-and-mouth disease virus (FMDV) is most often reported as the serum antibody response to the virus. While measurement of neutralizing antibody has been sensitive and specific, measurements of the quality of the antibody response are less robust. Determining the immunoglobulin (Ig) isotype of the serum antibody response provides a deeper understanding of the biology of the response and more sensitive methods for these assays will facilitate analyses of B cell mediated immunity. We tested the hypothesis that using the virus as the molecular probe could be achieved by adding tags to the surface of the FMDV capsid, and that would enhance sensitivity in assays for anti-FMDV antibody responses. The use of a FLAG-tagged virus in these assays failed to yield improvement whereas chemically biotinylating the virus capsid resulted in significant enhancement of the signal. Here we describe methods using biotinylated virus for measuring anti-viral antibody in serum and antibody secreting cells (ASCs) in blood that are sensitive and specific. Finally, we describe using the biotinylated virus in flow cytometry where such assays should greatly enhance the analysis of anti-virus antibody producing B cells, allowing the investigator to focus on only the FMDV specific B cells when analyzing the development of the B cell response to either infection or vaccination.

Foot-and-mouth disease virus 5'-terminal S fragment is required for replication and modulation of the innate immune response in host cells.

The S fragment of the FMDV 5' UTR is predicted to fold into a long stem-loop structure and it has been implicated in virus-host protein interactions. In this study, we report the minimal S fragment sequence required for virus viability and show a direct correlation between the extent of the S fragment deletion mutations and attenuated phenotypes. Furthermore, we provide novel insight into the role of the S fragment in modulating the host innate immune response. Importantly, in an FMDV mouse model system, all animals survive the inoculation with the live A24 FMDV-S4 mutant, containing a 164 nucleotide deletion in the upper S fragment loop, at a dose 1000 higher than the one causing lethality by parental A24 FMDV, indicating that the A24 FMDV-S4 virus is highly attenuated in vivo. Additionally, mice exposed to high doses of live A24 FMDV-S4 virus are fully protected when challenged with parental A24 FMDV virus.