Identification of the Active-Site Residues of the 3C Proteinase of Foot-and-Mouth Disease Virus

AbstractTo identify the active-site residues of the 3C proteinase of foot-and-mouth disease virus (FMDV), we introduced mutations into the 3C coding region and examined the activity of mutant enzymes on various substrates. Based on alignment of FMDV 3C with other picornavirus 3C proteinases and with the trypsin family of serine proteinases, mutations were introduced at residues presumed to be part of the catalytic triad, involved in substrate binding, or present in nonconserved regions. Wild-type and mutant 3C proteins were expressed inEscherichia coliand tested for their ability to cleave synthetic substrates corresponding to different portions of the viral genome. Substitutions at His-46 (catalytic triad), Asp-84 (catalytic triad), or His-181 (substrate binding) produced enzymes unable to process P1, P2, or P3 substratesin trans,whereas a change in the conserved Asp-98 had no effect on enzyme activity. Substitution of Ser for Cys-163 (catalytic triad) yielded an enzyme that retained activity on some substrates, while a substitution of Gly at this position resulted in a completely inactive enzyme. The kinetics oftransprocessing of translation products from a transcript encoding the P1 and P2 coding regions and the 2C/3A cleavage site with wild-type 3C or a transcript encoding P1 with 3C mutants revealed that the order of cleavage was VP3-VP1, VP0-VP3, VP1-2A, 2C-3A, and 2B-2C. Mutations in 3C that resulted in a partially active enzyme were individually introduced into full-length FMDV cDNA and RNA transcripts were translated in a cell-free system and used to transfect cells. In all cases the virus that was rescued had reverted to the wild-type 3C codon

Identification of optimal regions for phylogenetic studies on VP1 gene of foot-and-mouth disease virus: analysis of types A and O Argentinean viruses

An analysis of the informative content of sequence stretches on the foot-and-mouth disease virus (FMDV) VP1 gene was applied to two important viral serotypes: A and O. Several sequence regions were identified to allow the reconstruction of phylogenetic trees equivalent to those derived from the whole VP1 gene. The optimal informative regions for sequence windows of 150 to 250 nt were predicted between positions 250 and 550 of the gene. The sequences spanning the 250 nt of the 3$'$ end (positions 400 to 650), extensively used for FMDV phylogenetic analyses, showed a lower informative content. In spite of this, the use of sequences from this region allowed the derivation of phylogenetic trees for type A and type O FMDVs which showed topologies similar to those previously reported for the whole VP1 gene. When the sequences determined for viruses isolated in Argentina, between 1990 and 1993, were included in these analyses, the results obtained revealed features of the circulation of type A and type O viruses in the field, in the months that preceded the eradication of the disease in this country. Type A viruses were closely related to an Argentinean vaccine strain, and defined an independent cluster within this serotype. Among the type O viruses analysed, two groups were distinguished; one was closely related to the South American vaccine strains, while the other was grouped with viruses of the O3 subtype. In addition, a detailed phylogeny for type A FMDV is presented.Identification des régions optimales pour les études phylogénétiques du gène VP1 du virus de la fièvre aphteuse : analyse des virus argentins de types A et O. Une analyse du contenu informatif de parties de séquences du gène VP1 du virus de la fièvre aphteuse (VFA) a été appliquée à deux sérotypes viraux importants : A et O. Plusieurs régions de séquences ont été identifiées afin de permettre la reconstruction d'arbres phylogénétiques équivalents à ceux obtenus à partir de gène VP1 entier. Les régions informatives optimales pour des fenêtres de séquences de 150 à 250 nt ont été supposées être comprises entre les positions 250 et 550 du gène. Les séquences recouvrant les 250 nt de l'extrémité 3$'$ (positions 400 à 650), qui ont été largement utilisées pour les études phylogénétiques du VFA, avaient un contenu moins informatif. Malgré cela, l'utilisation de séquences de cette région a permis d'obtenir des arbres phylogénétiques pour les VFA de type A et O, qui ont montré des topologies similaires à celles précédemment décrites pour le gène VP1 entier. Quand les séquences déterminées pour les virus isolés en Argentine entre 1990 et 1993 ont été incluses dans ces analyses, les résultats obtenus ont révélé des caractéristiques de circulation des virus de type A et O sur le terrain, dans les mois qui ont précédé l'éradication de la maladie dans ce pays. Les virus de type A étaient étroitement apparentés à une souche argentine de vaccin et définissaient un groupe indépendant à l'intérieur de ce sérotype. Parmi les virus de type O analysés, nous avons pu distinguer deux groupes : l'un était étroitement apparenté aux souches vaccinales sud-américaines, l'autre était proche des virus du sous-type O3. Une phylogénie détaillée du VFA type A est également présentée

Bioinformatics and Molecular Analysis of the Evolutionary Relationship between Bovine Rhinitis A Viruses and Foot-And-Mouth Disease Virus

National audienc

Identification of Cellular Genes Affecting the Infectivity of Foot-and-Mouth Disease Virus▿

Foot-and-mouth disease virus (FMDV) produces one of the most infectious of all livestock diseases, causing extensive economic loss in areas of breakout. Like other viral pathogens, FMDV recruits proteins encoded by host cell genes to accomplish the entry, replication, and release of infectious viral particles. To identify such host-encoded proteins, we employed an antisense RNA strategy and a lentivirus-based library containing approximately 40,000 human expressed sequence tags (ESTs) to randomly inactivate chromosomal genes in a bovine kidney cell line (LF-BK) that is highly susceptible to FMDV infection and then isolated clones that survived multiple rounds of exposure to the virus. Here, we report the identification of ESTs whose expression in antisense orientation limited host cell killing by FMDV and restricted viral propagation. The role of one such EST, that of ectonucleoside triphosphate diphosphohydrolase 6 (NTPDase6; also known as CD39L2), a membrane-associated ectonucleoside triphosphate diphosphohydrolase that previously was not suspected of involvement in the propagation of viral pathogens and which we now show is required for normal synthesis of FMDV RNA and proteins, is described in this report

A partial deletion in non-structural protein 3A can attenuate foot-and-mouth disease virus in cattle

AbstractThe role of non-structural protein 3A of foot-and-mouth disease virus (FMDV) on the virulence in cattle has received significant attention. Particularly, a characteristic 10–20 amino acid deletion has been implicated as responsible for virus attenuation in cattle: a 10 amino acid deletion in the naturally occurring, porcinophilic FMDV O1 Taiwanese strain, and an approximately 20 amino acid deletion found in egg-adapted derivatives of FMDV serotypes O1 and C3. Previous reports using chimeric viruses linked the presence of these deletions to an attenuated phenotype in cattle although results were not conclusive. We report here the construction of a FMDV O1Campos variant differing exclusively from the highly virulent parental virus in a 20 amino acid deletion between 3A residues 87–106, and its characterization in vitro and in vivo. We describe a direct link between a deletion in the FMDV 3A protein and disease attenuation in cattle

Engaging caregivers and providers of children with sickle cell anemia in shared decision making for hydroxyurea: Protocol for a multicenter randomized controlled trial

Background: Sickle cell anemia (SCA) is a genetic blood disorder that puts children at a risk of serious medical complications, early morbidity and mortality, and high health care utilization. Until recently, hydroxyurea was the only disease-modifying treatment for this life-threatening disease and has remained the only option for children younger than 5 years. Evidence-based guidelines recommend using a shared decision-making (SDM) approach for offering hydroxyurea to children with SCA (HbSS or HbS/β0 thalassemia) aged as early as 9 months. However, the uptake remains suboptimal, likely because caregivers lack information about hydroxyurea and have concerns about its safety and potential long-term side effects. Moreover, clinicians do not routinely receive training or tools, especially those that provide medical evidence and consider caregivers\u27 preferences and values, to facilitate a shared discussion with caregivers. Objective: The aim of this study is to understand how best to help parents of young children with sickle cell disease and their clinicians have a shared discussion about hydroxyurea (one that considers medical evidence and parent values and preferences). Methods: We designed our study to compare the effectiveness of two methods for disseminating hydroxyurea guidelines to facilitate SDM: a clinician pocket guide (ie, usual care) and a clinician hydroxyurea SDM toolkit (H-SDM toolkit). Our primary outcomes are caregiver reports of decisional uncertainty and knowledge of hydroxyurea. The study also assesses the number of children (aged 0-5 years) who were offered and prescribed hydroxyurea and the resultant health outcomes. Results: The Ethics Committee of the Cincinnati Children\u27s Hospital Medical Center approved this study in November 2017. As of February 2021, we have enrolled 120 caregiver participants. Conclusions: The long-term objective of this study is to improve the quality of care for children with SCA. Using multicomponent dissemination methods developed in partnership with key stakeholders and designed to address barriers to high-quality care, caregivers of patients with SCA can make informed and shared decisions about their health

Engaging Caregivers and Providers of Children With Sickle Cell Anemia in Shared Decision Making for Hydroxyurea:Protocol for a Multicenter Randomized Controlled Trial

BACKGROUND: Sickle cell anemia (SCA) is a genetic blood disorder that puts children at a risk of serious medical complications, early morbidity and mortality, and high health care utilization. Until recently, hydroxyurea was the only disease-modifying treatment for this life-threatening disease and has remained the only option for children younger than 5 years. Evidence-based guidelines recommend using a shared decision-making (SDM) approach for offering hydroxyurea to children with SCA (HbSS or HbS/β0 thalassemia) aged as early as 9 months. However, the uptake remains suboptimal, likely because caregivers lack information about hydroxyurea and have concerns about its safety and potential long-term side effects. Moreover, clinicians do not routinely receive training or tools, especially those that provide medical evidence and consider caregivers’ preferences and values, to facilitate a shared discussion with caregivers. OBJECTIVE: The aim of this study is to understand how best to help parents of young children with sickle cell disease and their clinicians have a shared discussion about hydroxyurea (one that considers medical evidence and parent values and preferences). METHODS: We designed our study to compare the effectiveness of two methods for disseminating hydroxyurea guidelines to facilitate SDM: a clinician pocket guide (ie, usual care) and a clinician hydroxyurea SDM toolkit (H-SDM toolkit). Our primary outcomes are caregiver reports of decisional uncertainty and knowledge of hydroxyurea. The study also assesses the number of children (aged 0-5 years) who were offered and prescribed hydroxyurea and the resultant health outcomes. RESULTS: The Ethics Committee of the Cincinnati Children’s Hospital Medical Center approved this study in November 2017. As of February 2021, we have enrolled 120 caregiver participants. CONCLUSIONS: The long-term objective of this study is to improve the quality of care for children with SCA. Using multicomponent dissemination methods developed in partnership with key stakeholders and designed to address barriers to high-quality care, caregivers of patients with SCA can make informed and shared decisions about their health. TRIAL REGISTRATION: ClinicalTrials.gov NCT03442114; https://clinicaltrials.gov/ct2/show/NCT03442114 INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/2765