Pilot Projects

Characterizing ABC Transporters that prevent the elimination of zoonotic toxocariasis

Dr. Jeba Jesudoss Chelladuri, College of Veterinary Medicine

Human toxocariasis is a zoonotic infection caused by the eukaryotic pathogen Toxocara canis, for which dogs are the final hosts. Eggs of the parasite in dog feces are the source of human infection. Major reservoirs that prevent the eradication of infection are muscle and tissue-dwelling larval stages in infected dogs. These tissue-dwelling stages reactivate and pass on to developing pups in the uterus, propagating the infection to the next generation of dogs and thus to humans in contact with the infected animals.

The reservoir larval stages cannot be killed by approved anthelmintics such as the macrocyclic lactone drugs (ivermectin, selamectin), despite good drug bioavailability. Involvement of efflux transporter proteins in the removal of macrocyclic lactones from nematode sites of action have been hypothesized as a mechanism of drug tolerance.

In Aim 1 of this pilot proposal, we propose to profile the transcription and polymorphisms of ATP-binding cassette (ABC) transporter genes as candidate targets for future studies.

In Aim 2 of this application, we propose to assess the tissue-specific distribution of three highly expressed P-glycoprotein (Pgp) genes in T. canis in relation to a molecular target of macrocyclic lactone drugs using in-situ hybridization. This will help understand the site-specific role of Pgps for rational drug design in future studies.

Together, results from this project are expected to be important for identifying novel drug targets, understanding drug tolerance mechanisms, and ultimately will provide new opportunities for chemotherapeutic development to eliminate human toxocariasis.

The project’s long-term objectives are to identify and characterize druggable targets to help develop clinical treatments for tissue-dwelling larvae in dogs to mitigate human toxocariasis using a “One Health” approach.

 

Dissecting the role of NSs and NSm in Rift Valley Fever reassortment

Dr. Natasha Gaudreault, College of Veterinary Medicine

The mechanisms of RVFV reassortment are poorly characterized. We have previously investigated reassortment between two virulent wild-type (wt) field strains of RVFV from Saudi Arabia and Kenya (SA01-1322 and KEN128B-15) as well as a virulent wt strain (KEN128B-15) and a vaccine strain (MP-12) using in vitro and in vivo systems. Those studies showed that reassortment was less efficient in Culex tarsalis mosquito derived cells than in mammalian (sheep) cells; however, we observed the opposite effect in vivo in the vertebrate host (sheep) versus the mosquito vector (Cx. tarsalis). Interestingly, we observed high rates of RVFV reassortment in orally exposed Cx. tarsalis mosquitoes, and the majority of virus plaques recovered from midgut and salivary gland tissues of RVFV co-infections comprised reassortant genotypes, with evidence for preferential inclusion of specific genome segments from different RVFV strains. Specifically, we saw the S segment of wt KEN128B-15 and the M segment of MP-12 preferentially represented among the reassortant viruses. The S and M segments of RVFV, in addition to structural proteins, encode non-structural proteins NSs and NSm, respectively. The current understanding is that NSs and NSm have critical roles in modulating both vertebrate as well as invertebrate infection dynamics. Therefore, our overarching hypothesis is that RVFV strain reassortment is also facilitated by NSs and NSm. Based on our preliminary data, we predict that swapping the MP-12 NSs into the KEN128B-15 backbone (KEN128B-15-NSs’) and the KEN128B-15 NSm into the MP-12 backbone (MP-12-NSm’) will impair reassortment and reduce the number of KEN128B-15 S and MP-12 M reassortant viruses.

The goals of this project are to (i) construct chimeric viruses that will (ii) be used to co-infect mosquito and mammalian derived cells to better understand the roles of the viral genes NSs and NSm in strain reassortment. Ultimately, these studies will contribute towards the goal of determining the molecular and immunologic mechanisms that drive reassortment in both vertebrate and invertebrate systems. The proposed work will lay the foundation and generate preliminary data for future applications that investigate the vector and host genes modulated by NSs and NSm that may govern reassortment success.

Aim 1. Generation of RVFV NSm and NSs chimeric virus strains. The S segment of wt KEN128B-15 and the M segment of MP-12 were preferentially represented among the majority of reassortant genotypes from our previous work. Moreover, well-characterized mutations in the NSs and NSm genes between these strains offer a possible genetic basis for these observed phenotypes. We propose to generate chimeric viruses from existing infectious clones by exchanging complete or partial NSs of KEN128B-15 and NSm of MP-12. Replication kinetics will be determined for the generated chimeric virus strains.

Aim 2. Dissect the roles of strain-specific RVFV NSm and NSs in reassortment in vitro. The chimeric viruses generated in Aim 1 will be used to co-infect mosquito and mammalian derived cell cultures. The viruses from infected cell supernatants will be plaque isolated and subsequent genotyping analysis performed to determine the frequency of reassortants. The results of this work will guide subsequent investigations into reassortment mechanisms in the arthropod vector and mammalian host.

 

Comparative transcriptomic analysis of IAV infection in porcine organoids

Dr. Laura Miller, College of Veterinary Medicine

There has been a re/emergence of zoonotic pathogens driven by multiple and complex factors, with livestock animals frequently in the interface of the disease transmission between wild animals and humans. These constant interactions have significantly increased the chances of potential spillover of viruses from wild animals to domestic animals and/or to humans, as evidenced by recent influenza and coronavirus outbreaks. Mammalian innate and adaptive immune responses are complex, interconnected and crucial for host defense against infectious disease. However, in some situations, some of these responses may lead to deleterious consequences. This highlights the need for alternative and adjunctive therapeutic options that target host-responses. Organoids can be used to document species-specific differences and are useful to assess the pandemic threat of animal influenza viruses. We have established an ex vivo three-dimensional (3D) organotypic air-liquid interface primary porcine respiratory epithelial cell culture system (ALI-PRECs) recreating a cell culture environment morphologically and functionally more representative of the epithelial lining of the swine trachea than traditional culture systems. ALI-PRECs are proposed for ex vivo comparison of influenza A virus infection. Specific Aim 1 will be to harness the ALI-PRECs for comparative influenza A virus studies. Intensive molecular and cellular characterization will be performed in parallel to virus infection tests. Specific Aim 2 will be to perform genomic analysis of ALI-PRECs following human-, swine-, avian-lineage influenza A virus infection(from Specific Aim 1) to evaluate the virus-host relationship. Transcriptomic
annotation will allow clustering of shared expression patterns. Moreover, genome-wide profiling will delineate the epigenetic regulation of both viral and host factors, thus sequentially determining productive infection and clinical disease (pathogenesis) outcome. Understanding pathogenesis will help in the development of novel therapeutic options that minimize immunopathology without impairing beneficial host defenses.

 

Target Genes of Global regulators SinRI in Clostridioides difficile

Dr. Revathi Govind , College of Arts & Sciences

Clostridioides difficile is an important nosocomial pathogen that has been classified as an “urgent threat” by CDC.Antibiotic use is the primary risk factor for the development of C. difficile associated disease because it disrupts healthy protective gut flora and enables C. difficile to colonize the colon. C. difficile damage host tissue by secreting toxins and disseminates by forming spores. Biofilm formation further aids in successful colonization and persistence of C. difficile in the host gut. We discovered that a new set of global regulators (SinR and SinI) to regulate toxin production, sporulation, and biofilm formation in C. difficile. In this grant proposal, we hypothesize that Sin regulators controls the regulatory networks of all three pathogenic traits
by controlling the regulators in these pathways. Main objective of this proposal is to identify the genes that are directly under the control of Sin regulators.

 

The Role of Conserved ORF3a Protein Domains in SARS-CoV-2 Replication and Pathogenesis

Dr. Edward Stephens, Professor, Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center

Coronaviruses infect numerous animal species including pigs, dogs, cats, cattle, horses, llamas, camels, civets, bats, and pangolins. In the last two decades, three highly pathogenic coronaviruses (MERS-CoV, SARS-CoV, and SARS-CoV-2) have evolved to cross the species barrier to cause morbidity and mortality in humans. As an example, SARS-CoV-2, the causative agent of COVID-19, caused a pandemic with over 6 million deaths worldwide and over 1 million deaths in the United States alone (as of April 3, 2023). With the concurrent global expansion of human and domestic animal populations, it is likely that novel coronaviruses will emerge because of cross-species transmission among humans and domestic and wild animals. The immunity generated from current vaccines is transitory and requires periodic boosters. Concomitant with the monitoring of species for new variants or distinct new coronavirus species, additional studies on the pathogenesis of SARS-CoV-2 is required to develop better vaccines in the future that generate long-term immunity. Using SARS-CoV-2 as the best-studied member of the β-coronaviruses, which is highly related to SARS-CoV, we propose to generate recombinant viruses with select mutations within the ORF3a protein of SARS-CoV-2. The ORF3a protein causes ER stress, activates the NLRP3 inflammasome, induces apoptosis, causes incomplete autophagy, and may facilitate in virus release through the endosome/lysosome pathway of virion assembly and release. Orf3a also has a PDZ-binding domain (PBM), which is known to interact with host cell proteins through its PDZ domain. The Orf3a is transported to the cell plasma membrane and co-localizes with the LAMP-1 marker for lysosomes. Proteins containing small linear motifs such as NPXY, YxxΦ, and dileucine sorting signals can be targeted to different compartments of the cell including the plasma membrane, recycling endosomes, late endosomes, lysosomes, and the trans-Golgi network (TGN). While the Orf3a has no dileucine or NPXY signals, it has three potential tyrosine sorting motifs in its cytoplasmic domain. Additionally, ORF3a has a PBM at its C-terminus. We removed the tyrosine-based sorting signals (Orf3a-∆YxxФ) and showed they were not significantly transported to the cell surface or associated with lysosomes. In Aim 1, we will analyze Orf3a-∆YxxФ and ORF-mutPBM proteins for the functions discussed above. In Aim 2, we propose to construct viruses expressing ORF3a-∆YxxΦ, ORF3a-mutPBM, Orf3a-[∆YxxФ/mut-PBM]
and analyze these viruses for replication in cell lines (VeroE6, A549) or primary human small airway epithelial cells to determine if these viruses have reduced/decreased pathogenicity in cell cultures. We will also analyze these viruses for the functions described above. Finally, we propose to analyze the pathogenicity of these viruses in the K18 ACE-2 mouse model. Overall, the results of these studies will further our knowledge of the Orf3a transport and the PBM in the SARS-CoV-2 replication cycle and pathogenicity, may aid in the development of more effective vaccines with long-term immunity against this disease, and provide important preliminary data used to secure NIH funding.