博士(獣医学)山口大学Yamaguchi Universit

SHELLY WULANDARI

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(Appended form No. 3) (About 800 words in English) Abstract of Doctoral Thesis Name SHELLY WULANDARI Title: Establishment of a reverse genetic system and determination of the responsible gene for the  enhanced virulence of trisegmented bunyaviruses.  (3) Abstract of thesis:  Trisegmented bunyaviruses constitute a category of viruses within the  order, distinguished by their segmented, negative-sense RNA genomes that are encoded in an orientation opposite to that of messenger RNA. Their members infect broad ranges of hosts, including several significant human pathogens. Reverse genetic systems have been essential in the investigation of trisegmented bunyaviruses. The process of introducing a precise genetic change into a viral genome, known as viral reverse genetics, has revolutionized our understanding of negative-sense, segmented RNA viruses. While the specific characteristics of reverse genetic systems vary among different viruses, the fundamental principles of biology remain the same. Here, we examine the development of reverse genetic systems as applied to these virus members, emphasizing conserved approaches illustrated by some of the prominent members that cause significant human disease. We also describe the utility of their genetic systems in the determination of the responsible gene for the enhanced virulence of trisegmented bunyaviruses.    The genus Orthonairovirus includes highly pathogenic tick-borne viruses, such as the Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV). A reverse genetics system is an indispensable tool for determining the viral factors related to pathogenicity. Tofla orthonairovirus (TFLV) is a recently identified virus isolated from ticks in Japan, and our research has suggested that TFLV is a useful model for studying pathogenic orthonairoviruses. In this study, we successfully established a reverse genetics system for TFLV using T7 RNA polymerase. Recombinant TFLV was generated by transfecting cloned complementary DNAs encoding the TFLV genome into BSR T7/5 cells expressing T7 RNA polymerase. We were able to rescue infectious recombinant TFLV mutant (rTFLVmt) and wild-type TFLV (rTFLVpt) viruses, which exhibited indistinguishable growth kinetics in mammalian cells and pathogenicity in A129 mice compared with the authentic virus. Our approach provides a valuable method for establishing a reverse genetic system for orthonairoviruses.   Severe fever with thrombocytopenia syndrome (SFTS) is a significant public health concern, with a high fatality rate in humans and cats. In this study, we explored the genetic determinants that contribute to the different virulence of SFTS virus (SFTSV) based on Tk-F123 and Ng-F264 strains isolated from cats. Tk-F123 was 100% lethal in type I interferon receptor-knockout mice, whereas Ng-F264 exhibited no fatality. We identified a pair of amino acid residues in the Gc protein, glycine and serine, at residues 581 (Appended form No. 3) (About 800 words in English) and 934, respectively, derived from Tk-F123, leading to a fatal infection. Those in Ng-F264 were arginine and asparagine. These results suggest that this pair of residues affects the Gc protein function and regulates SFTSV virulence. Our findings provide useful clues for the elucidation of viral pathogenicity and the development of effective live-attenuated vaccines and antiviral strategies.  In conclusion, we were able to rescue recombinant TFLV and SFTSV from cDNA clones, allowing detailed studies on viral genetics and pathogenesis. We also describe the molecular determinants of virulence in SFTSV, which is crucial for developing effective vaccines and therapeutics. Finally, we highlight that reverse genetics continues to be a powerful tool in elucidating the molecular mechanisms of virulence in trisegmented bunyaviruses, which will contribute to our understanding of viral biology, pathogenesis, and intervention strategies.

3分節ゲノムを有するブニヤウイルスを対象としたリバースジェネティクス系の確立および病原性に関与するウイルス遺伝子の特定

https://petit.lib.yamaguchi-u.ac.jp/30178/files/168307

3分節ゲノムを有するブニヤウイルスを対象としたリバースジェネティクス系の確立および病原性に関与するウイルス遺伝子の特定

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