A new gnotobiotic pig model of P[6] human rotavirus infection and disease for preclinical evaluation of rotavirus vaccines

Human rotavirus (HRV) is a leading cause of gastroenteritis in children under 5 years of age. Licensed vaccines containing G1P[8] and G1-4P[8] strains are less efficacious against newly emerging P[6] strains, indicating an urgent need for better cross protective vaccines. Here, we report our development of a new gnotobiotic (Gn) pig model of P[6] HRV infection and disease as a tool for evaluating potential vaccine candidates. The Arg HRV (G4P[6]) strain was derived from a diarrheic human infant stool sample and determined to be free of other viruses by metagenomic sequencing. Neonatal Gn pigs were orally inoculated with the stool suspension containing 5.6 × 105 fluorescent focus units (FFU) of the virus. Small and large intestinal contents were collected at post inoculation day 2 or 3. The virus was passaged 6 times in neonatal Gn pigs to generate a large inoculum pool. Next, 33–34 day old Gn pigs were orally inoculated with 10−2, 103, 104, and 105 FFU of Arg HRV to determine the optimal challenge dose. All pigs developed clinical signs of infection, regardless of the inoculum dose. The optimal challenge dose was determined to be 105 FFU. This new Gn pig model is ready to be used to assess the protective efficacy of candidate monovalent and multivalent vaccines against P[6] HRV.Instituto de VirologíaFil: Nyblade, Charlotte. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Hensley, Casey. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Parreño, Gladys Viviana. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Parreño, Gladys Viviana. Instituto Nacional de Tecnología Agropecuaria (INTA). INCUINTA. Instituto de Virologia e Innovaciones Tecnologicas (IVIT); ArgentinaFil: Zhou, Peng. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Frazier, Maggie. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Frazier, Annie. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Ramesh, Ashwin. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Lei, Shaohua. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Degiuseppe, Juan Ignacio. Administración Nacional de Laboratorios e Institutos de Salud (ANLIS). Instituto Nacional de Enfermedades Infecciosas “Dr. Carlos G. Malbrán” (INEI). Laboratorio de Gastroenteritis Virales; ArgentinaFil: Tan, Ming. Cincinnati Children’s Hospital Medical Center. Division of Infectious Diseases; Estados UnidosFil: Tan, Ming. University of Cincinnati College of Medicine. Department of Pediatrics; Estados UnidosFil: Yuan, Lijuan. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados Unido

Establishment of a gnotobiotic pig model of Clostridioides difficile infection and disease

Clostridioides difficile (C. difficile) is a gram-positive, spore-forming, anaerobic bacterium known to be the most common cause of hospital-acquired and antibiotic-associated diarrhea. C. difficile infection rates are on the rise worldwide and treatment options are limited, indicating a clear need for novel therapeutics. Gnotobiotic piglets are an excellent model to reproduce the acute pseudomembranous colitis (PMC) caused by C. difficile due to their physiological similarities to humans and high susceptibility to infection. Here, we established a gnotobiotic pig model of C. difficile infection and disease using a hypervirulent strain. C. difficile-infected pigs displayed classic signs of C. difficile infection, including severe diarrhea and weight loss. Inoculated pigs had severe gross and microscopic intestinal lesions. C. difficile infection caused an increase in pro-inflammatory cytokines in samples of serum, large intestinal contents, and pleural effusion. C. difficile spores and toxins were detected in the feces of inoculated animals as tested by anaerobic culture and cytotoxicity assays. Successful establishment of this model is key for future work as therapeutics can be evaluated in an environment that accurately mimics what happens in humans. The model is especially suitable for evaluating potential prophylactics and therapeutics, including vaccines and passive immune strategies.Instituto de VirologíaFil: Nyblade, Charlotte. Virginia Polytechnic Institute and State University. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Parreño, Gladys Viviana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología e Innovaciones Tecnológicas; ArgentinaFil: Parreño, Gladys Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Parreño, Gladys Viviana. Virginia Polytechnic Institute and State University. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Zhou, Peng. Virginia Polytechnic Institute and State University. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Hensley, Casey. Virginia Polytechnic Institute and State University. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Oakes, Vanessa. Virginia Polytechnic Institute and State University. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Mahsoub, Hassan M. Virginia Polytechnic Institute and State University. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Mahsoub, Hassan M. Virginia Polytechnic Institute and State University. Center for Emerging, Zoonotic, and Arthropod‑Borne Pathogens; Estados UnidosFil: Kiley, Kelsey. Virginia Polytechnic Institute and State University. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Frazier, Maggie. Virginia Polytechnic Institute and State University. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Frazier, Annie. Virginia Polytechnic Institute and State University. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Zhang, Yongrong. University of Maryland at Baltimore. Department of Microbial Pathogenesis; Estados UnidosFil: Feng, Hanping. University of Maryland at Baltimore. Department of Microbial Pathogenesis; Estados UnidosFil: Yuan, Lijuan. Virginia Polytechnic Institute and State University. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Yuan, Lijuan. Virginia Polytechnic Institute and State University. Center for Emerging, Zoonotic, and Arthropod‑Borne Pathogens; Estados Unido

Combined live oral priming and intramuscular boosting regimen with Rotarix® and a nanoparticle-based trivalent rotavirus vaccine evaluated in gnotobiotic pig models of G4P[6] and G1P[8] human rotavirus infection

Human rotavirus (HRV) is the causative agent of severe dehydrating diarrhea in children under the age of five, resulting in up to 215,000 deaths each year. These deaths almost exclusively occur in low- and middle-income countries where vaccine efficacy is the lowest due to chronic malnutrition, gut dysbiosis, and concurrent enteric viral infection. Parenteral vaccines for HRV are particularly attractive as they avoid many of the concerns associated with currently used live oral vaccines. In this study, a two-dose intramuscular (IM) regimen of the trivalent, nanoparticle-based, nonreplicating HRV vaccine (trivalent S60-VP8*), utilizing the shell (S) domain of the capsid of norovirus as an HRV VP8* antigen display platform, was evaluated for immunogenicity and protective efficacy against P[6] and P[8] HRV using gnotobiotic pig models. A prime–boost strategy using one dose of the oral Rotarix® vaccine, followed by one dose of the IM trivalent nanoparticle vaccine was also evaluated. Both regimens were highly immunogenic in inducing serum virus neutralizing, IgG, and IgA antibodies. The two vaccine regimens failed to confer significant protection against diarrhea; however, the prime–boost regimen significantly shortened the duration of virus shedding in pigs challenged orally with the virulent Wa (G1P[8]) HRV and significantly shortened the mean duration of virus shedding, mean peak titer, and area under the curve of virus shedding after challenge with Arg (G4P[6]) HRV. Prime–boost-vaccinated pigs challenged with P[8] HRV had significantly higher P[8]-specific IgG antibody-secreting cells (ASCs) in the spleen post-challenge. Prime–boost-vaccinated pigs challenged with P[6] HRV had significantly higher numbers of P[6]- and P[8]-specific IgG ASCs in the ileum, as well as significantly higher numbers of P[8]-specific IgA ASCs in the spleen post-challenge. These results suggest the promise of and warrant further investigation into the oral priming and parenteral boosting strategy for future HRV vaccines.Instituto de VirologíaFil: Hensley, Casey. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Nyblade, Charlotte. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Zhou, Peng. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Parreño, Gladys Viviana. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Parreño, Gladys Viviana. Instituto Nacional de Tecnología Agropecuaria (INTA). INCUINTA. Instituto de Virologia e Innovaciones Tecnologicas (IVIT); ArgentinaFil: Parreño, Gladys Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ramesh, Ashwin. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Frazier, Annie. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Frazier, Maggie. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Garrison, Sarah. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Fantasia-Davis, Ariana. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Cai, Ruiqing. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Huang, Peng-Wei. Cincinnati Children’s Hospital Medical Center. Division of Infectious Diseases; Estados UnidosFil: Xia, Ming. Cincinnati Children’s Hospital Medical Center. Division of Infectious Diseases; Estados UnidosFil: Tan, Ming. Cincinnati Children’s Hospital Medical Center. Division of Infectious Diseases; Estados UnidosFil: Tan, Ming. University of Cincinnati College of Medicine. Department of Pediatrics; Estados UnidosFil: Yuan, Lijuan. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados Unido

Human rotavirus replicates in salivary glands and primes immune responses in facial and intestinal lymphoid tissues of gnotobiotic pigs

Human rotavirus (HRV) is a leading cause of viral gastroenteritis in children across the globe. The virus has long been established as a pathogen of the gastrointestinal tract, targeting small intestine epithelial cells and leading to diarrhea, nausea, and vomiting. Recently, this classical infection pathway was challenged by the findings that murine strains of rotavirus can infect the salivary glands of pups and dams and transmit via saliva from pups to dams during suckling. Here, we aimed to determine if HRV was also capable of infecting salivary glands and spreading in saliva using a gnotobiotic (Gn) pig model of HRV infection and disease. Gn pigs were orally inoculated with various strains of HRV, and virus shedding was monitored for several days post-inoculation. HRV was shed nasally and in feces in all inoculated pigs. Infectious HRV was detected in the saliva of four piglets. Structural and non-structural HRV proteins, as well as the HRV genome, were detected in the intestinal and facial tissues of inoculated pigs. The pigs developed high IgM antibody responses in serum and small intestinal contents at 10 days post-inoculation. Additionally, inoculated pigs had HRV-specific IgM antibody-secreting cells present in the ileum, tonsils, and facial lymphoid tissues. Taken together, these findings indicate that HRV can replicate in salivary tissues and prime immune responses in both intestinal and facial lymphoid tissues of Gn pigs.Instituto de VirologíaFil: Nyblade, Charlotte. Virginia Polytechnic Institute and State University. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Zhou, Peng. Virginia Polytechnic Institute and State University. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Frazier, Maggie. Virginia Polytechnic Institute and State University. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Frazier, Annie. Virginia Polytechnic Institute and State University. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Hensley, Casey. Virginia Polytechnic Institute and State University. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Fantasia-Davis, Ariana. Virginia Polytechnic Institute and State University. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Shahrudin, Shabihah. Indiana University. Department of Biology; Estados UnidosFil: Hoffer, Miranda. Indiana University. Department of Biology; Estados UnidosFil: Agbemabiese, Chantal Ama. Indiana University. Department of Biology; Estados UnidosFil: LaRue, Lauren. GIVAX Inc.; Estados UnidosFil: Barro, Mario. GIVAX Inc.; Estados UnidosFil: Patton, John T. Indiana University. Department of Biology; Estados UnidosFil: Parreño, Gladys Viviana. Virginia Polytechnic Institute and State University. Virginia-Maryland College of Veterinary Medicine. Department of Biomedical Sciences and Pathobiology; Estados UnidosFil: Parreño, Gladys Viviana. Instituto Nacional de Tecnología Agropecuaria (INTA). INCUINTA. Instituto de Virologia e Innovaciones Tecnologicas (IVIT); ArgentinaFil: Parreño, Gladys Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Yuan, Lijuan. Virginia Polytechnic Institute and State University. Center for Emerging, Zoonotic, and Arthropod‑Borne Pathogens; Estados Unido

Mitochondrial Dysfunction and Apoptosis in Cumulus Cells of Type I Diabetic Mice

Impaired oocyte quality has been demonstrated in diabetic mice; however, the potential pathways by which maternal diabetes exerts its effects on the oocyte are poorly understood. Cumulus cells are in direct contact with the oocyte via gap junctions and provide essential nutrients to support oocyte development. In this study, we investigated the effects of maternal diabetes on the mitochondrial status in cumulus cells. We found an increased frequency of fragmented mitochondria, a decreased transmembrane potential and an aggregated distribution of mitochondria in cumulus cells from diabetic mice. Furthermore, while mitochondrial biogenesis in cumulus cells was induced by maternal diabetes, their metabolic function was disrupted as evidenced by lower ATP and citrate levels. Moreover, we present evidence suggesting that the mitochondrial impairments induced by maternal diabetes, at least in part, lead to cumulus cell apoptosis through the release of cytochrome c. Together the deleterious effects on cumulus cells may disrupt trophic and signaling interactions with the oocyte, contributing to oocyte incompetence and thus poor pregnancy outcomes in diabetic females

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Teaching Conversation Skills to Adults With Developmental Disabilities Using a Video-Based Intervention Package

BACKGROUND: Social skills deficits may hinder learning, terminate relationships, and impede employment. Many individuals with autism and intellectual disability experience difficulties in social judgement, emotional regulation, and interpersonal relationships, all of which can lead to disruptive and aggressive behaviors. Explicit instruction, video modeling, and video feedback are research-based practices that have been used to teach conversation skills to individuals with developmental disabilities and social impairments. OBJECTIVE: This study examined the effects of explicit instruction combined with video modeling and video feedback in teaching conversation-initiation skills to six adults ages 18-20 with autism and intellectual disability in a post-high school transition program. METHODS: A multiple baseline across dyads design was used, with number of correct initiation responses as the independent variable. The independent variable was an intervention package including explicit verbal instruction with interspersed video modeling clips, followed by video feedback. RESULTS: All six participants acquired the skills and were able to initiate a conversation; five of them maintained these skills over time, demonstrating them without the intervention. CONCLUSIONS: Data supported a evidence functional relation between the intervention package and the participants\u27 social initiation skills. Social validity responses indicated that participants enjoyed watching the videos of models and especially enjoyed watching the videos of themselves

mRNA-Based Vaccines Are Highly Immunogenic and Confer Protection in the Gnotobiotic Pig Model of Human Rotavirus Diarrhea

Human rotavirus (HRV) is still a leading cause of severe dehydrating gastroenteritis globally, particularly in infants and children. Previously, we demonstrated the immunogenicity of mRNA-based HRV vaccine candidates expressing the viral spike protein VP8* in rodent models. In the present study, we assessed the immunogenicity and protective efficacy of two mRNA-based HRV trivalent vaccine candidates, encoding VP8* of the genotypes P[8], P[6], or P[4], in the gnotobiotic (Gn) pig model of Wa (G1P[8]) HRV infection and diarrhea. Vaccines either encoded VP8* alone fused to the universal T-cell epitope P2 (P2-VP8*) or expressed P2-VP8* as a fusion protein with lumazine synthase (LS-P2-VP8*) to allow the formation and secretion of protein particles that present VP8* on their surface. Gn pigs were randomly assigned into groups and immunized three times with either P2-VP8* (30 µg) or LS-P2-VP8* (30 µg or 12 µg). A trivalent alum-adjuvanted P2-VP8* protein vaccine or an LNP-formulated irrelevant mRNA vaccine served as the positive and negative control, respectively. Upon challenge with virulent Wa HRV, a significantly shortened duration and decreased severity of diarrhea and significant protection from virus shedding was induced by both mRNA vaccine candidates compared to the negative control. Both LS-P2-VP8* doses induced significantly higher VP8*-specific IgG antibody titers in the serum after immunizations than the negative as well as the protein control. The P[8] VP8*-specific IgG antibody-secreting cells in the ileum, spleen, and blood seven days post-challenge, as well as VP8*-specific IFN-γ-producing T-cell numbers increased in all three mRNA-vaccinated pig groups compared to the negative control. Overall, there was a clear tendency towards improved responses in LS-P2-VP8* compared to the P2-VP8*mRNA vaccine. The demonstrated strong humoral immune responses, priming for effector T cells, and the significant reduction of viral shedding and duration of diarrhea in Gn pigs provide a promising proof of concept and may provide guidance for the further development of mRNA-based rotavirus vaccines