Source: CORNELL UNIVERSITY submitted to NRP
MECHANISMS UNDERLYING SENECAVIRUS A PERSISTENT INFECTIONS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1032589
Grant No.
2024-67015-42737
Cumulative Award Amt.
$650,000.00
Proposal No.
2023-07924
Multistate No.
(N/A)
Project Start Date
Jul 1, 2024
Project End Date
Jun 30, 2027
Grant Year
2024
Program Code
[A1221]- Animal Health and Production and Animal Products: Animal Health and Disease
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
(N/A)
Non Technical Summary
Senecavirus A (SVA) has recently emerged in several of the major swine producing countries worldwide, causing VD outbreaks in swine that are clinically indistinguishable from FMD and other high consequence FADs of livestock. Notably, since the emergence of the virus in the US in 2015 over 3000 outbreaks of SVA-induced VD have been confirmed. Vesicular diseases are among the most important diseases of livestock. The annual cost of FMD, for example, has been estimated to amount between $6.5 and $21 billion per year in endemic regions, whereas FMD outbreaks in disease free countries (like the US) could cost up to $84 billion. The similarity of SVA with such high consequence FADs and the fact that the virus is capable of establishing persistence and a carrier state in infected animals which transmit the virus to other susceptible animalshighlight the significance of the disease. Understanding the mechanisms underlying establishment and maintenance of SVA persistence is critical to implement effective prevention and control measures to mitigate the impact that circulation of the virus causes to the US livestock industry.The main goals of the study proposed here are to elucidate the mechanisms and host cell functions exploited by SVA during persistence as well as to identify viral determinants underlying SVA's ability to establish and maintain long-term persistent infections.The outcomes of this study will significantly improve our understanding of SVA infection biology and persistence and are likely to shed light on the pathways and molecular mechanisms that may have enabled the maintenance and transmission of the virus in the swine population for the last three decades.A direct application of the basic knowledge generated here through the identification of molecular determinants of SVA persistence could lead to the development of safer and highly efficient vaccines devoid of their ability to establish persistence.
Animal Health Component
30%
Research Effort Categories
Basic
60%
Applied
30%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3114099110170%
3117010103030%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
4099 - Microorganisms, general/other; 7010 - Biological Cell Systems;

Field Of Science
1101 - Virology; 1030 - Cellular biology;
Goals / Objectives
Senecavirus A (SVA) is an emerging picornavirus of swine that causes foot-and-mouth- (FMD)-like vesicular disease (VD) in affected animals and is known to establish persistent infections in pigs. Most importantly, carrier animals can serve as source of infection to other susceptible pigs. Currently, however, little is known about the mechanisms underlying SVA persistence. In the present project we will test the hypothesis that modulation of host survival and antiviral pathways by SNA determines the fate of infected cells, which cycle between lytic and persistent infection. We propose to dissect the mechanisms underlying SVA persistence using in vivo and in vitro and model systems of persistent infection. The objectives of this study are:To define virus-host cell interactions underlying establishment and maintenance of persistent SVA infection in vivo.To characterize molecular determinants of SVA persistence.These objectives are related to the program area of Diseases of Agricultural Animals (A1221). The work is directly aligned with the priority areas of "Cellular aspects of animal health, maintenance and homeostasis" and the sub priority area of "disease prevention and control". The work proposed here will significantly contribute to the improvement and sustainability of the US livestock industry by generating foundational knowledge regarding SVA persistence in swine. Such knowledge may be translated to safe live attenuated SVA vaccines and to improved strategies for prevention and control of SVA and other highly relevant VDs of swine.
Project Methods
The overall goal of the proposed study is to dissect the mechanisms underlying establishment and maintenance of SVA persistence in the swine host. To achieve this goal we proposed two objectives and a series of experimental studies as outlines below:Aim 1: To define virus-host cell interactions underlying establishment and maintenance of persistent SVA infection. In Aim 1 we propose: (ii) to determine the host cell responses at different stages of infection (acute, transitional and persistence) in the tonsil in vivo (Aim 1.1); and (ii) to dissect the role and function of host factors modulated during persistence on SVA life cycle and on its ability to establish and maintain persistence (Aim 1.2). For this, we will use cutting edge single-cell RNA sequencing (scRNA-seq) coupled with spatial transcriptomics (ST) to unveil SVA host cell interactions as they take place during establishment and maintenance of the persistent infection in the tonsil in vivo. Additionally, porcine tonsillar cell culture libraries stably expressing- or containing CRISPR/Cas9 gene knockouts will be used to dissect the function of identified host factors on SVA infection, replication and persistence.Aim 2: To identify and characterize molecular determinants of SVA persistence. In Aim 2 we plan to identify viral determinants of persistence and characterize their role on SVA persistence. Mutations that we identified in specific SVA proteins during persistence will provide the foundation for these studies. They will be coupled with our SVA reverse genetics system to determine their contribution to virus persistence using in vitro and in vivo model systems.

Progress 07/01/24 to 06/30/25

Outputs
Target Audience:During the reporting period the audience reached by the project and its results include the animal health scientific community, scientists and graduate students at Cornell. We have presented the results in scientific meetings such as the American Society for Virology Meeting (ASV) and the CRWAD meeting. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided opportunity for training of 3 graduate students and 1 post-doctoral research associate. One of the PhD students has completed her PhD rsearch and is currently working on her thesis. She has started Vet School at Cornell, also as a result of her involvement with animals in this study. The other two PhD students are working on the characterization of host-factors on the persistence of SVA. How have the results been disseminated to communities of interest?We have presented the results from the project in national scientific conferences as well as in local graduate school presentations at Cornell. A manuscript was submitted for publication and is currently under review. A second manuscript is being drafted/edited and a PhD dissertation is being written and revised. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue the characterization of host factors on SVA persistence in vitro We also plan to conduct an in vivo study with the recombinant SVA virus that is no longer able to establish persistence to determine what is the role of the 2C function on 3C on the ability of the virus to persist.

Impacts
What was accomplished under these goals? During the reporting period we made important strides in understanding the mechanisms underlying SVA persistence, using in vitro models of SVA persistent infection: To define virus-host cell interactions underlying establishment and maintenance of persistent SVA infection in vivo. By using in vitro cell culture models of SVA persistence including H1299, Pk15 and porcine tonsil cells we have demonstrated that SVA is capable of persisting for several days (up to 280) in H1299 and Pk15 cells witth cells constantly shedding virus in the supernatant of affected cells. We have shown using TUNEL and western blots that during persistence SVA inhibits host cell apoptosis. Additionally, by using bul RNAseq and single cell RNA seq in pi culture cells we identified a set of innate immune and prosurvival genes that are modulated during indection. We initiated to asses the role of a set of these host factors on SVA infection and replication in vitro. We have showed that the ISGs control SVA replication leading to reduced replication in cell culture in vitro. We are now investigating the steps of the infection cycle that are affected by 9 of these genes identified during the transcriptomic experiments. To characterize molecular determinants of SVA persistence. We have also investigated potential viral determinants of persistence. We have identified a unique function of SVA 2C protein which binds to SVA 3C protease and induces its degradation. Notably, 3C is a major inducer of host-cell apoptosis, a process that is inhibited by SVA during persistence. Using site-directed mutagenesis we have identifid a specific motif in 2C that is responsible for its activity in the 3C protease. Using site-directed mutagenesis we were able to demonstrate that by introducing a mutation on this site, SVA was cleared from the tonsil of infected animals suggesting that this function of 2C is important for SVA persistence in vivo. We plan to conduct additional studies using RNAseq and scRNAseq coupled with Spatial Transcriptomics to further characterize this function in establishment and maintenance of persistent infection. .

Publications

  • Type: Other Journal Articles Status: Under Review Year Published: 2025 Citation: Diaz, A.N., Ribeiro, L.C., Butt, S.L, Diel, D.G#. Stem loop I mutations in Senecavirus A 5 UTR reduce replication and virulence in pigs. Submitted, Frontiers and Cellular and Molecular Microbiology, July 7, 2025.