Former Year 4

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.