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

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Gamma-irradiated rotavirus: A possible whole virus inactivated vaccine.

Rotavirus (RV) causes significant morbidity and mortality in developing countries, where children and infants are highly susceptible to severe disease symptoms. While live attenuated vaccines are available, reduced vaccine efficacy in developing countries illustrates the need for highly immunogenic alternative vaccines. Here, we studied the possible inactivation of RV using gamma(γ)-irradiation, and assessed the sterility and immunogenicity of γ-irradiated RV (γ-RV) as a novel vaccine candidate. Interestingly, the inactivation curve of RV did not show a log-linear regression following exposure to increased doses of γ-rays, and consequently the radiation dose required to achieve the internationally accepted Sterility Assurance Level could not be calculated. Nonetheless, we performed sterility testing based on serial passages of γ-RV, and our data clearly illustrate the lack of infectivity of γ-RV preparations irradiated with 50 kGy. In addition, we tested the immunogenicity of 50 kGy γ-RV in mice and our data illustrate the induction of strong RV-specific neutralising antibody responses following administration of γ-RV without using adjuvant. Therefore, whilst γ-RV may not constitute a replacement for current RV vaccines, this study represents a proof-of-concept that γ-irradiation can be applied to inactivate RV for vaccine purposes. Further investigation will be required to address whether γ-irradiation can be applied to improve safety and efficacy of existing live attenuated vaccines

Inactivation curves of RV and SFV following exposure to γ-irradiation.

<p>SFV and RV samples were exposed to increased doses of γ-irradiation on dry-ice, and the reduction in virus titre was determined by (A) plaque forming assay for SFV, or (B) fluorescent focus assay for RV. All samples tested in triplicate and data presented as mean ± SEM.</p

RV-specific antibody responses.

<p>Mice were primed twice with live RV or γ-RV, 2 weeks apart. Serum samples harvested on Day 14 post-2^nd priming and analysed for RV-specific IgG using ELISA. (A) Serial dilutions of serum samples and absorbance readings at 450/620 nm for total IgG. (B) IgG titres in primed groups calculated relative to cut-off value (dotted line), determined using OD values of serum from control mice. Data presented as mean ± SEM (n = 6), and analysed by unpaired t-test (n.s., not significant) (C) Absorbance at 450/620nm for total IgG, IgG1, and IgG2c in serum at 1:200 dilution by ELISA. Data presented as mean ± SEM (n = 4), analysed by One-Way ANOVA (****, p < 0.0001).</p

Sterility testing of 50 kGy γ-irradiated RV.

<p>Monolayers of MA104 cells were incubated for 24h with live RV or γ-RV at 4 x 10⁵ FFU-equivalent/well. Culture supernatant was harvested and used to infect new MA104 cell monolayers, and previously infected monolayers stained for RV infection by FFA. DAPI channel (blue) indicates cell nuclei, and FITC channel (green) indicates RV infection. Stained monolayers visualised using Nikon TiE inverted fluorescence microscope. Scale bar = 100 μm. Images representative of 5 replicates per sample for each passage.</p

Neutralising antibody responses induced by γ-RV.

<p>Mice were primed with live RV or γ-RV twice, 2 weeks apart. Serum samples harvested on Day 21 post-2^nd priming, and neutralising ability of immune serum determined by <i>in vitro</i> neutralisation assay. (A) FFU/well determined following incubation of MA104 cells with sera treated-RV at MOI 0.005. RV treated with serial dilutions of HI control or immune sera. PBS-treated RV used to indicate the baseline level of infection. Data presented as mean ± SEM (n = 2), and analysed by One-Way ANOVA (****, p < 0.0001 compared to naïve control sera for each dilution. #, p < 0.05, when directly comparing immune sera groups). (B) Representative fluorescence images of RV infection after treatment with live or γ-RV sera at 1:1280 dilution. DAPI channel (blue) indicates cell nuclei, and FITC channel (green) indicates RV infection. Scale bar = 100 μm.</p

The ability to detect very low FFU of live RV.

<p>Monolayers of MA104 cells were incubated for 24h with 0.2 to 2000 FFU live RV. Uninfected MA104 cells were used as a negative control. Culture supernatant was used to infect new MA104 monolayers, and previously infected monolayers were visualised by FFA. DAPI (blue) indicates cell nuclei and FITC (green) indicates RV infection. Stained monolayers were visualised using Nikon TiE inverted fluorescent microscope. Scale bar = 100μm. Images representative of 3 replicates per MOI for each passage.</p