Investigation Of C.Elegans Susceptibility To Vesicular Stomatitis Virus Infection And Its Antiviral Strategies

As a successful multicellular model organism,the nematode Caenorhabditis elegans(C.elegans)h as been widely used for half a century in diverse research fields including development,aging,metabolism,stress,and neurobiology.Recently,C.elegans has also been developed as models for microbial pathogenesis and innate immunity studies in a variety of human pathogens including bacteria and fungi.However,because nature has very few natural virus species that can infect C.elegans,and living nematodes are insusceptible to almost all human pathogenic viruses,the contribution of nematode models to virologically relevant research has always been quite limited.Although researchers have attempted to construct some virus-associated experimental models using nematodes,these models have generally suffered from problems such as technical difficulties,lack of in vitro and in vivo relevance or physiological significance,and eventually have not been generally accepted and widely used by academia.Therefore,the development of a simple and convenient large-scale C.elegans virus infection model and a better understanding of the complete antiviral mechanisms in C.elegans would greatly benefit virology-related research.In this thesis,using the nematode model organism Caenorhabditis elegans,we comprehensively and systematically examined the infection and transmission of vesicular stomatitis virus(VSV)at different interfaces and in different tissue organs of the nematode.By developing a large-scale viral infection model,we exploited the phenomenon of vertical transmission of VSV in the nematode and explored conserved and novel antiviral mechanisms in C.elegans.We first examined the susceptibility of different physical interfaces of live nematodes to the human pathogenic virus VSV.We infected RNAi-deficient nematode mutants using soaking,feeding,and microinjection of green fluorescent protein(GFP)-labeled VSV and found that only the interface of the inner basement membrane covering the nematode body lumen was susceptible to VSV virus infection,whereas no significant infection occurred after the outer surface of the epidermis,intestine,valve,and anus were exposed to equal titers of virus.We next used RNAi knockdown to disrupt the cuticle structure,the anchoring structure of the cuticle and epidermis,and the intestinal architecture.We found that VSV was unable to replicate and spread within the nematode,indicating that various physical barriers play an important role in defense against VSV invasion.In the process of constructing a nematode model of VSV infection,we found that VSV was able to transmit to nematodes across generations to three generations in the nematode body,and presented different rates of hereditary infection.In parallel,we also compared the characteristics of wild-type C.elegans and RNAi-deficient mutants upon virus infection and showed that both exhibited a clear lethal phenotype and that infection of C.elegans with VSV and the lethal phenotype it resulted in were dose-dependent.In examining the systemic spread of VSV within the nematodes,we found that regardless of the initial infected tissues,VSV was able to cross tissue barriers between tissues for cross tissue transmission,indicating that various different tissue organs within nematodes are generally susceptible to VSV.Through optimization of the VSV microinjection approach,we successfully constructed nematode strains with unlimited trans-generational transmission of VSV,a model that presents features of stable virus transgenerational transmission rate,constant viral load,and stable lethality of VSV-infected nematodes,and is able to yield large numbers of virus-infected nematode individuals.Our use of this model to discover that deletion of specific collagen molecules from the extracellular matrix of epidermal cells significantly increases viral load within the epidermal cells reveals a novel function for extracellular matrix collagens in resisting viral infection and exemplifies the advantage of this model in antiviral infection-related basic research.Finally,we found that the newly constructed nematode virus infection model could be used for the screening of antiviral drugs and detection of toxic side effects of the candidate drugs,indicating the potential application of this model in biomedical field.In summary,using C.elegans as a research object,we fully elucidated the susceptibility of different tissues and organs of C.elegans to VSV,and clarified the important role of various physical barriers in C.elegans against the invasion of external VSV.Through a modified experimental approach for microinjection of viruses,we have constructed a facile and efficient large-scale VSV trans-generational infection nematode model.This model will provide a large number of virus-infected individuals for the study of nematode antiviral mechanisms,which can be used for the screening of novel antiviral molecular drugs,large-scale unbiased genomic mutation screening,searching for new antiviral molecules and signaling pathways,and ultimately exploring novel antiviral mechanisms in organisms.The research results obtained from this study will not only provide a new model of viral infection,but will also shed new light on researches involving the host process of infection with multiple human pathogenic viruses.