The Doctor of Philosophy Seminar and Examination of Charlotte Nyblade

Biography

Charlotte’s interest in the intersection of veterinary medicine and research began as a child when she was able to observe goat parasites under a microscope. Since then, she has continued to develop her interest in both topics. She attended Brown University for her undergraduate education, graduating in 2021 with a BA in Health and Human Biology. During that time she discovered her love for virology research in particular, first studying Japanese encephalitis virus and later plant viruses at Utah State University via summer research programs. She also performed research at Brown, working for two years under the supervision of Dr. Walter Atwood on human polyomaviruses. Concurrently, she shadowed small animal veterinarians in medicine and surgery. She entered the DVM/PhD program at Virginia Maryland College of Veterinary Medicine in Fall 2021 to further her studies in veterinary research. Under the mentorship of Dr. Lijuan Yuan, Charlotte worked to establish gnotobiotic pig models of enteric viruses and bacteria. These models were then used to elucidate replication and pathogenesis features of the pathogens. After completing her PhD, Charlotte will complete the DVM portion of her program. Long term, she is interested in working in veterinary vaccine research and development. Outside of school, Charlotte enjoys running, reading, and spoiling her cat Marble.

Funded by

• Virginia Tech Institute for Critical Technology and Applied Science
• National Institute of Allergy and Infectious Disease
• The Office of Research and Graduate Studies

Awards and Academic Achievements

• BMVS Travel Award, Spring 2024
• Omicron Delta Kappa Honor Society, Spring 2024
• Student Travel Award for American Society of Virology Conference, Spring 2023
• Travel award for Calicivirus Conference 2023, Spring 2023
• CEZAP Student Mini Grant Competition Grant Recipient, Spring 2023
• Graduate Research and Development Program (GRDP) grant recipient, Fall 2022
• CEZAP Student Mini Grant Competition Grant Recipient, Spring 2022

Lay Language Abstract 

Clostridioides difficile (C. difficile) and human rotavirus (HRV) cause gastrointestinal related symptoms when they infect humans. Treatments available for C. difficile and HRV both have significant drawbacks. This represents a gap in knowledge which we aimed to fill by developing germ-free (gnotobiotic [Gn]) pig models of C. difficile and HRV infection and disease. Animal models that mimic the outcomes of disease seen in humans are essential for evaluating protectiveness of new therapeutics. The more similar the disease presentation, the more likely the treatment results will be translational to humans. We began with C. difficile; pigs were orally fed C. difficile and monitored for a week post infection for development of signs of infection. Inoculated pigs lost weight and developed diarrhea. Bacterial cells and toxins were isolated from fecal samples collected on various days post infection. Multiple changes were observed in infected pigs’ large intestinal tissues, including severe bleeding, tissue distension, and fluid buildup. Infected pigs also had significant upregulation of pro-inflammatory cytokines, indicating activation of the immune response. We performed a similar procedure for the establishment of the HRV model. Gn pigs were orally challenged with differing doses of G4P[6] HRV and followed for several days post infection. Consistent with HRV infection in children, the pigs developed watery diarrhea that lasted for multiple days. Small intestinal tissues collected at necropsy had several signs of damage, including blunted villi, fluid buildup, and immune cell invasion. These lesions were also consistent with HRV infection in humans. Taken all together, these results indicated successful establishment of both C. difficile and HRV models. While the primary goal of generating these models was to evaluate new treatments, a secondary goal was to use them to better our understanding of pathogen replication dynamics. For example, the small intestine was thought to be the primary site of HRV infection. Using a pig model of HRV, we expanded on this knowledge to show that HRV can replicate in the nose and salivary glands as well. Additionally, we found HRV infection to induce immune responses near the sites of infection, including the intestine, tonsils, and facial lymph nodes. Overall, these studies demonstrate the utility of germ-free pig models and are an important first step in generating more effective treatments for bacterial and viral infections. 

Publications

Nyblade, C.; Yuan, L. Virus Shedding and Diarrhea: A Review of Human Norovirus Genogroup II Infection in Gnotobiotic Pigs. Viruses 2024, 16, 1432. https://doi.org/10.3390/v16091432

Hensley C, Roier S, Zhou P, Schnur S, Nyblade C, Parreno V, Frazier A, Frazier M, Kiley K, O'Brien S, Liang Y, Mayer BT, Wu R, Mahoney C, McNeal MM, Petsch B, Rauch S, Yuan L. mRNA-Based Vaccines Are Highly Immunogenic and Confer Protection in the Gnotobiotic Pig Model of Human Rotavirus Diarrhea. Vaccines (Basel). 2024 Mar 1;12(3):260. doi: 10.3390/vaccines12030260. 

Nyblade C, Zhou P, Frazier M, Frazier A, Hensley C, Fantasia-Davis A, Shahrudin S, Hoffer M, Agbemabiese CA, LaRue L, Barro M, Patton JT, Parreño V, Yuan L. Human Rotavirus Replicates in Salivary Glands and Primes Immune Responses in Facial and Intestinal Lymphoid Tissues of Gnotobiotic Pigs. Viruses. 2023 Aug 31;15(9):1864. doi: 10.3390/v15091864. 

Hensley C, Nyblade C, Zhou P, Parreño V, Ramesh A, Frazier A, Frazier M, Garrison S, Fantasia-Davis A, Cai R, Huang PW, Xia M, Tan M, Yuan L. 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. Vaccines (Basel). 2023 May 2;11(5):927. doi: 10.3390/vaccines11050927. 

Nyblade C, Hensley C, Parreño V, Zhou P, Frazier M, Frazier A, Ramesh A, Lei S, Degiuseppe JI, Tan M, Yuan L. A New Gnotobiotic Pig Model of P[6] Human Rotavirus Infection and Disease for Preclinical Evaluation of Rotavirus Vaccines. Viruses. 2022 Dec 15;14(12):2803. doi: 10.3390/v14122803. 

Nyblade C, Parreno V, Zhou P, Hensley C, Oakes V, Mahsoub HM, Kiley K, Frazier M, Frazier A, Zhang Y, Feng H, Yuan L. Establishment of a gnotobiotic pig model of Clostridioides difficile infection and disease. Gut Pathog. 2022 Jun 6;14(1):22. doi: 10.1186/s13099-022-00496-y. 

Presentations

“A GII.6 strain of norovirus is infectious and pathogenic in gnotobiotic pigs”. 43rd Annual Meeting of the American Society for Virology. Columbus, OH. Oral presentation. 6/27/24.  

“Human rotavirus replicates in the salivary glands and induces IgM responses in the facial lymphoid tissues of gnotobiotic pigs.” AppliedResearch&ScienceEcon2024. Virtual. Oral presentation. 6/4/24. 

“A GII.6 strain of norovirus is infectious and pathogenic in gnotobiotic pigs”. 2024 ICTAS Doctoral Scholar Research Poster Symposium. Blacksburg, VA. Poster. 3/29/24. 

“A GII.6 strain of norovirus is infectious and pathogenic in gnotobiotic pigs”. 33rd Annual BMVS Research Symposium. Blacksburg, VA. Poster. 3/18/24. 

“Immunogenicity and replication capacity of recombinant rotaviruses expressing norovirus proteins.” BMVS RIP Seminar. Blacksburg, VA. Oral presentation. 2/2/24.

“Immunogenicity and replication capacity of recombinant rotaviruses expressing norovirus proteins.” 2023 CeZAP Infectious Diseases Symposium. Blacksburg, VA. Oral presentation. 10/6/23.

 “Human rotavirus replicates in the salivary glands and induces IgM responses in the facial lymphoid tissues of gnotobiotic pigs”. 42^nd Annual Meeting of the American Society for Virology. Athens, GA. Oral presentation. 6/23/24. 

“Engineered probiotic yeast strains secreting anti-norovirus VHH transiently reside in the gnotobiotic pig gut and produce high titers of nanobodies”. Calicivirus Conference 2023. Rotterdam, The Netherlands. Oral presentation. 5/11/23. 

2023 Virginia Tech Center for Drug Discover Poster Session, “Engineered probiotic yeast strains secreting anti-norovirus VHH transiently reside in the gnotobiotic pig gut and produce high titers of nanobodies”. Blacksburg, VA. Poster. 4/13/23.

“Human rotavirus replicates in the salivary glands and induces IgM responses in the facial lymphoid tissues of gnotobiotic pigs”. 2023 ICTAS Doctoral Scholars Poster Symposium. Blacksburg, VA. Poster. 3/31/23.

“Human rotavirus replicates in the salivary glands and induces IgM responses in the facial lymphoid tissues of gnotobiotic pigs”. BMVS Research in Progress Seminar. Blacksburg, VA. Oral presentation. 3/15/23.

“Human rotavirus replicates in the salivary glands and induces IgM responses in the facial lymphoid tissues of gnotobiotic pigs”. 32^nd BMVS Graduate Research Symposium. Blacksburg, VA. Oral presentation. 3/14/23.

“A new gnotobiotic pig model of P[6] human rotavirus infection and disease”. 2022 CEZAP Infectious Disease Symposium. Blacksburg, VA. Poster. 10/7/22. 

“Gnotobiotic pig models of Clostridioides difficile infection and disease”. National Association for Veterinary Scientists. Minneapolis, MN. Oral and poster presentations. 8/4/22. 

“Establishment of a gnotobiotic pig model of Clostridioides difficile infection and disease”. 2022 ICTAS Doctoral Scholars Research Symposium. Blacksburg, VA. Poster. 4/22/22. 

Examination Graduate Committee

Major Advisor/Chair:

Lijuan Yuan, PhD
Professor of Virology and Immunology
Department of Biomedical Sciences and Pathobiology

Graduate Advising Committee Members:

Jonathan Auguste, PhD
Associate Professor of Arbovirology
Department of Entomology

Nisha Duggal, PhD
Associate Professor of Virology
Department of Biomedical Sciences and Pathobiology

Kylene Kehn-Hall, MS, PhD
Professor of Virology
Department of Biomedical Sciences and Pathobiology

X.J. Meng, MD, MS, PhD
University Distinguished Professor of Molecular Virology, VMCVM. Professor of Internal Medicine, VTC School of Medicine
Department of Biomedical Sciences and Pathobiology