Recipient Organization
UNIV OF PENNSYLVANIA
(N/A)
PHILADELPHIA,PA 19104
Performing Department
5805 - Clinical Studies-New Bolton Center
Non Technical Summary
Equine hepacivirus (EqHV) is a very common virus and we have recently observed that it can cause deadly hepatitis (liverdisease) in horses. This condition strongly resembles liver disease caused by the closely related hepatitis C virus (HCV) inhumans. Given this increased recognition of the health burden of EqHV infection in horses, our long-term objective is toreduce and/or prevent transmission and disease caused by EqHV. Our specific hypothesis is that EqHV is readily transmittedamong horses and can cause chronic hepatitis and liver failure during persistent infection. To test this, we will (1) model EqHVinfection and transmission by monitoring infection in horse herds over 4 years and testing the ability of mosquitoes to carryEqHV, (2) characterize the clinical condition of chronic EqHV hepatitis by serial testing of affected horses over time andexamination of diagnostic liver biopsies, and (3) identify risk factors of persistence and severe disease including age, sex, hostimmune response, and viral genetics by comparing horses that clear the infection without serious illness and those that developchronic infection and hepatitis. The significance of this work is to develop a foundation for EqHV disease diagnosis, prevention,and control. Determining the transmission and disease burden of the virus, and risk factors for severe disease, can drive futuredevelopment of vaccines, treatments, and management strategies, including possible governmental regulations on equinebiologic products, as has been done for the other recently discovered liver pathogen, equine parvovirus-hepatitis. As such, thiswork can contribute to an overall understanding of and reduction in cases of transmissible liver disease in horses with hithertounknown etiology.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
Our long-term goal is to identify the extent that Equine hepacivirus (EqHV) contributes to equine liver disease and to reduceand/or prevent those cases. EqHV is a recently discovered, highly prevalent equine virus, with 22-84% of horses worldwidehaving ongoing or previous infection. EqHV is closely related to hepatitis C virus (HCV), which causes persistent infection andchronic hepatitis in humans. Most horses clear EqHV infection, and such acute resolving infection has been the focus ofresearch so far. However, recent case reports and our own preliminary data indicate that persistent EqHV also occurs andcauses fatal chronic hepatitis in horses.In addition to reducing the welfare and economic burden of EqHV on the horse industry, it is important to study EqHV for threemajor reasons. First, EqHV can be transmitted in USDA-regulated equine biologic products, such as hyperimmune plasma, andin unregulated products such as whole blood transfusions. Second, equine serum-based products are also used in medicationsintended for human administration, such as antitoxins, although spread of EqHV to humans has not been documented. Third,horse sera are commonly used as cell culture additives for many purposes, including propagating viruses for infection studiesand for vaccine production for use in horses and other species. The presence of EqHV could affect the outcome of these studiesand could contaminate live attenuated vaccines.It is therefore essential to accurately determine the epidemiology of viral spread and disease burden. The studies proposed herewill be used to improve diagnostic criteria for EqHV-associated hepatitis and to determine risk factors for severe disease. Thiswill inform regulatory, prevention and management strategies. The specific hypothesis of this study is that EqHV is readilytransmitted among horses and can cause chronic hepatitis and liver failure during persistent infection. To test this, the followingaims are proposed:Aim 1: Epidemiologic modeling of EqHV infection kinetics and transmission in horses. To understand the risk of chronic hepatitisin the equine population, we first need to determine the proportion of exposed horses that develop persistent infection, definedas serum RT-qPCR-positive for longer than 6 months. Additionally, to prevent spread, we need to understand natural horizontaltransmission modes. To address this, we will 1) monitor viremia and serology quarterly over 5 years (collections ongoing for acomplementary project) in horse herds to develop a model of natural transmission dynamics and kinetics, 2) test for viralshedding in nasal, oral, and fecal secretions and semen, and 3) test for vector competence of mosquitoes. Results will elucidatehow the virus circulates in horse populations, which is essential for improved management strategies to reduce spread.Aim 2: Establish the clinical characteristics of EqHV-associated chronic hepatitis and develop diagnostic criteria. Our preliminarydata suggest persistent EqHV infection can cause chronic severe hepatitis. Because it can take many years of persistentinfection to develop disease, experimental infection studies will not be feasible. Therefore, we will use a retrospective review ofbanked liver biopsies with the specific histopathologic patterns of 1) normal liver, 2) non-neutrophilic cholangiohepatitis, 3)neutrophilic cholangiohepatitis, 4) lobular lymphocytic hepatitis, 5) individual hepatocyte necrosis, and 6) fibrosis, regardless offinal diagnosis. Liver samples will be tested by RT-qPCR and immunohistochemistry for EqHV. Prevalence of EqHV infectionwill be compared between groups. Due to the high prevalence of EqHV infection in horses, these controlled studies will beessential to establish an association of infection with specific histopathologic patterns. Second, we will perform a prospectivecase series evaluating horses with EqHV infection and chronic hepatitis, to determine diagnostic markers of hepaciviral hepatitisand to characterize disease progression.Aim 3: Determine risk factors for persistence and severe disease. To examine risk factors for persistence and chronic hepatitis,we will perform a case-control analysis to compare the following groups: i) EqHV-infected horses that clear the virus from Aim 1,ii) horses with persistent infection without hepatitis from Aim 1, and iii) horses with persistent infection with hepatitis from Aim 2.Based partially on known risk factors for HCV-infected people, we will focus on age, sex, breed, EqHV sequence, immuneresponse, and co-morbidities. Factors will be screened for significant effect by bivariate analyses and additive multiple logisticregression models will be built to assess risk for persistence and risk for severe disease.The significance of this work is to develop a foundation for EqHV disease diagnosis, prevention, and control. We expectthe aims to lead to a better understanding of the epidemiology and transmission of EqHV, which will guide prevention strategies.Characterizing deleterious immune responses and the risk factors for severe disease can guide vaccine design and treatmentdecisions. Finally, characterizing the overall health burden of EqHV is important to guide possible governmental regulations onequine biologic products, as has been done for the recently discovered EqPV-H.
Project Methods
Aim 1: Epidemiologic modeling of equine hepacivirus (EqHV) infection kinetics and transmission in horses. We will monitor the spread of EqHV and the frequency of persistent infection by quarterly serum RT-qPCR and luciferase immunoprecipitation system (LIPS) serology in ~270 horses over four years, and monthly testing in 200 horses for one year. Full genome deep sequencing of the virus will be performed on each positive horse, to determine if there are multiple strains circulating, or multiple introduction events occurring on these farms. We will develop models of viremia dynamics within a horse host following a compartmental form, and representing basic feedbacks between host cell infection and depletion and viral replication using a Hamiltonian Monte Carlo approach. The estimated parameters for within host viral dynamics will then be used to infer the generation time distribution andtransmission rate of the infection. Both frequency-dependent and density-dependent models of between host transmission will be assessed. These models will describe the transmissibility between hosts and will delineate seasonality of transmission.We will assess EqHV shedding in infected horses by performing RT-qPCR on oral, nasal, and fecal swabs collected weekly over 3 weeks from at least 20 EqHV positive horses. This will define whether shedding is a common feature of infection, and therefore whether inhalation or ingestion are likely routes of transmission.To assess mosquito vector competence to transmit EqHV, we will perform targeted collection of mosquitoes on horse farms with infected horses. Gravid traps, which attract mosquitoes that have fed and are ready to lay eggs, will be employed, thus increasing the likelihood of detecting infected insects. Mosquitoes will be speciated and then tested by RT-qPCR for EqHV. Replication will be confirmed by 3' end tailing assay to detect negative strand RNA, and RT-qPCR on dissected salivary glands. AdditionallyAedes albopictus mosquitoes will be fed onblood from 2 positive horses and one negative control donor. Mosquitoes will be sacrificed at 3, 7, 14, and 21 days post infection. RT-qPCR will be performed on bodies for infection rate and legs to measure dissemination rate.Aim 2: Establish the clinical characteristics of EqHV-associated chronic hepatitis and develop diagnostic criteria. Standardized histopathologic analysis, EqHV immunohistochemistry, and EqHV RT-qPCR will be performed on diagnostic submissions ofequine liver biospies. The proportion of positive samples will be compared between groups based on histopathologic patterns (e.g. fibrosis, necrosis, etc.) to see if any particular pattern is associated with EqHV infection.Thirty horses with evidence of chronic hepatitis which are serum or liver EqHV RT-qPCR positive will be enrolled in a case series. Liver tissue will be tested by liver culture, heavy metal analysis, and histopathology with hematoxylin and eosin, Masson's trichrome, reticulin, and IHC for EqHV. Serum liver biomarkers, serum RT-qPCR, and serum cytokine profiles will be performed monthly for 6-12 months to assess disease progression.Aim 3: Determine risk factors for persistence and severe disease. To examine host risk factors for persistence and chronic hepatitis, we will use a prospective case-control study to compare the following groups: i) EqHV-infected horses that clear the virus, ii) persistently infected horses without hepatitis, and iii) persistently infected horses with hepatitis. Serum and liver cytokine concentrations, liver RNA expression patterns, case metadata (age, breed, sex, co-morbidities), and viral sequences will be compared between groups using multivariate generalized logistic regression.