Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001
Performing Department
(N/A)
Non Technical Summary
Equine rotavirus A (ERVA) is one of the most important diseases in young horses under 6 months old. When foals get infected with an equine rotavirus, they can develop severely watery to bloody diarrhea. The disease can become rapidly fatal if left untreated. Due to lack of effective control and prevention methods, rotaviral outbreak happens in foaling season every year, and it causes significant damage to the annual $122 billion U.S. equine industry. ERVA often mutates its complete set of genes, creating many genetic variants. The genetic variability makes the current vaccine ineffective to protect foals against an ERVA infection. All these obstacles emphasize a critical need to study equine rotaviruses, understand their genetic variability, and interpret correlates of protection towards developing a safe and effective equine rotavirus vaccine.Our short-term goal in this project is to develop an innovative toolbox for equine rotavirus research and utilize them to detect and characterize genetic variations in equine rotaviruses. The other short-term goal of this work is to determine how genetic variations affect the abilities of virus-neutralizing antibodies to disarm viral infectivity. Our long-term goal is to study how equine rotaviruses spread and cause disease in foals and develop the next-generation control and prevention strategies to protect horses against infection by different strains or genotypes of equine rotavirus.This project has three objectives. The first objective is to develop a genetic tool for equine rotaviruses. We will isolate equine rotaviruses and determine their full-length gene sequences and then place their set of genes into small DNA molecules. Following the introduction of these DNA molecules into mammalian cells, we will produce equine rotaviruses representative of the well-studied reference variants. The second objective is to use the genetic tool developed from objective 1 to characterize genetic variations of equine rotaviruses and determine how these variations, occurring in equine rotaviruses over time, make the horse's immune system less able to recognize and fight against these variants. The cell culture-based virus-neutralizing antibody analysis will be used to address this important question. The third objective is to isolate and characterize a large panel of equine rotaviruses from diseased foals and determine a relationship between genetic variability and immune protection for equine rotaviruses.Successful completion of the project will allow us to isolate and characterize a large panel of equine rotaviruses. By studying these viruses, we expect to identify a full spectrum of genetic changes naturally occurring in equine rotaviruses over time. Furthermore, with the genetic tool developed from this project, we will demonstrate how genetic changes alter the ability of the horse's immune system to fight against different equine rotaviruses. Finally, using the functional antibody assessment, we anticipate discovering the levels of"protective" antibodies for equine rotavirus disease. These results will advance our knowledge in equine rotavirus, which can be further explored towards development of the next-generation countermeasures against rotavirus infection in horses.We anticipate that successful execution of this project will advance our knowledge concerning the fundamental mechanism equine rotavirus employs to promote its replication, evade the immune protection, and cause the associated disease in horses. This work will also reveal important immune correlates of protection against equine rotaviruses. Furthermore, it will detect the genetic changes in equine rotaviruses and determine their impact on immune evasion of the vaccine-mediated protection. This new knowledge will provide a foundation for developing next-generation broadly protective vaccines against emerging equine rotaviruses as well as other important rotaviral diseases afflicting other agricultural animals such as cattle and pigs. Finally, this project will empower the equine industry with a new toolbox including the genetic tool, functional antibody analysis, virus variants, and reference serum samples that can be used to fight these important enteric diseases in horses. All these efforts will reduce economic losses to the U.S. equine and animal industry.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
Our long-term goal is to develop next-generation prevention and control strategies that can protect horses from infection by various strains or genotypes of equine rotaviruses. This project has three objectives. The first objective is to develop a genetic system representing equine rotavirus G3 and G14 isolates circulating in U.S. horses, while the second objectives is to utilize this platform to elucidate the genetic determinants of antigenic variability among equine rotaviruses. The final objective is to study the antigenic properties of equine rotavirus isolates from a wide geographical range and determine their antigenic and genetic relationship to our reference equine G3 and G14 strains.
Project Methods
The research methods are presented below in the context of each research objective in this equine rotavirus project.Research Objective 1: To develop and characterize reverse genetics systems (RGSs) representing contemporary equine rotavirus G3 and G14 isolates.1.1. Development of reverse genetics systems for equine G3P[12] and G14P[12] viruses.We will follow the standard reverse genetics protocol that was initially described for simian rotavirus 11 (SA11) to develop the reverse genetics systems for two contemporary strains: G3 (ERVA/G3P[12]/2021) and G14 (ERVA/G14P[12]/2021) genotypes. We will first conduct RT-PCR reactions to amplify the full-length cDNAs representing all 11 segments of equine G3 and G14 viruses, respectively. We will then clone each of the 11 cDNAs from each virus into the corresponding plasmids of the reference SA11 RGS, respectively. Upon sequence confirmation, all 11 plasmids together with an accessory pCMV/NP868R plasmid (11+1) will be transfected into BHK-T7 cells. After 24 h, the MA104-N*V cells will be cocultured with BHK-T7 cells in the presence of TPCK trypsin containing DMEM media. Following additional 72 h incubation, co-cultured BHK-T7 and MA-104 N*V cells will be frozen and thawed three times and clarified supernatants will be inoculated into fresh MA-104 N*V cells. We will conduct IFA to confirm the rescue of equine G3 and G14, respectively. Positive staining will indicate the success of virus rescue from reverse genetics systems of equine rotaviruses.1.2. In vitro and in vivo characterization of ERVA G3 and G14 viruses rescued from RGSs.Once successful in rescuing the virus from RGS, we will first take in vitro approaches to examine and compare molecular and antigenic properties of the RGS-derived viruses with their parental viruses: i) full-genome sequencing will be conducted by MiSeq coupled with the Sanger sequencing; ii) viral replication fitness and growth titers will be evaluated in cell cultures including equine primary intestinal epithelial cells; and iii) viral antigenicity will be assessed by the virus-neutralizing antibody assay as well as IFA and ELISA involving a panel of commercial antibodies and a panel of in-house equine antibodies against ERVA G3 and G14.In addition, we will conduct a foal infection experiment to determine whether equine G3 and G14 viruses rescued from RGSs are replication-competent and pathogenic in foals. The successful development of infectious and disease-inducing RGSs should be of great value in identifying viral determinants of pathogenicity and cross-species transmission as well as in delineating infection biology of rotaviruses in horses, which are major gaps in rotavirus virus research. Groups of six foals at 7 days of age will be infected through a sterile nasogastric tube with 10 ml inoculum containing one million infectious particles of each of four viruses (WT G3, WT G14, RGS-derived G3, and RGS-derived G14), respectively. The mock group will receive 10 ml cell culture medium as a control. Infected foals will be observed for 14 days for clinical signs and clinical samples including fecal swabs will be collected daily following infection. Fecal swabs will be used to determine the level of viral replication, while the combination of fecal scoring and clinical symptoms will define virulence. We also will utilize an array of antibody assays to characterize protective antibody responses in foals during the period of 6-months following infection.Because entirely plasmid-based rotavirus RGS has been successfully developed for human, simian, bovine, and swine rotavirus strains, we expect to successfully rescue equine G3 and G14 viruses. Furthermore, we anticipate that the viruses derived from RGSs will behave similarly to their parental viruses in replication, virulence, and antigenic properties.Research Objective 2. To elucidate the genetic determinants of antigenic changes among equine rotaviruses and determine the level of the cross-protective neutralizing antibodies of G3 and G14 viruses.2.1. Production of hyperimmune sera in foals against equine G3 and G14 viruses.The above foal infection experiment will enable us to generate genotype-specific antibodies (G3 and G14) that can be used to antigenically characterize equine rotaviruses. We will obtain a panel of strongly positive, weekly positive and negative serum from infected foals from this objective. 2.2. Identification of the genetic basis of antigenic changes and the level of cross-neutralization of equine G3 and G14 viruses.With the sequence and structure analysis, we have identified a cluster of variable amino acid residues distributed through the antigenic sites between equine G3 and G14 viruses. We will utilize the genotype-specific antibodies in combination of the RGSs of equine G3 and G14 viruses to identify critical amino acid changes responsible for the limited cross-neutralization observed between equine G3 and G14 viruses. Specifically, we will exchange variable amino acid residues in equivalent positions between G3 and 14 viruses, which are in antigenic sites, and then rescue these variants through the reverse genetics approach. G3- and G14- specific variants will be further analyzed in replication fitness experiment and virus-neutralizing assays to assess the impact of genetic changes on viral antigenic properties. We anticipate that completion of these experiments will allow us to elucidate the genetic basis of antigenic variations between ERVA G3 and G14 viruses. This work will also offer novel insights about the levels of cross-neutralization of equine G3 and G14 viruses.Research Objective 3. To further characterize genetic and antigenic landscape of circulating equine rotaviruses.3.1. Isolation, full-genome sequence determination, and phylogenetic analysis of equine rotaviruses.We will utilize an innovative set of assays (virus isolation, virus neutralization, and full-genome sequencing) to identify equine rotavirus positive fecal samples, determine the full-genome sequences, and isolate equine G3 and G14 rotaviruses. We expect to obtain 60-90 equine rotavirus strains of different geographic regions over a 3-year period of the project, which should be sufficient for our proposed studies in this objective.3.2. Replication and antigenic characterization.Next, we will examine the replication fitness of these field isolates in a range of cell lines including equine primary intestinal epithelial cells. Cells will be infected with viruses over a range of MOIs (0.001, 0.01, 0.1, 1.0, and 10) and culture supernatants will be collected every 12 h over a 5-day period followed by determining viral titers. Upon the completion of viral replication experiment, we will employ the virus neutralization assay to determine the antigenic distance among the field strains and our reference G3 and G14 strains. The assay will be performed, and mean titers and standard deviations will be calculated from triplicate tests. All the data here and elsewhere will be presented as mean ± SEMs of triplicate samples from at least three independent experiments. GraphPad Prism5 software will be used for statistical analyses to determine statistical differences between different groups, which will include one-way analysis of variance (ANOVA) and two-way ANOVA. P values of less than 0.05 will be considered statistically significant.We anticipate that the successful completion of these studies will offer novel epidemiological insights into equine rotavirus genotypes or variants that are currently circulating in foals, which will help the U.S. equine industry in understanding the current infection landscape of equine rotaviruses. This new knowledge can significantly aid in development of the next-generation vaccines to protect foals from infection by various strains or genotypes of equine rotavirus.