S-Space College of Veterinary Medicine (수의과대학) Dept. of Veterinary Medicine (수의학과) Theses (Ph.D. / Sc.D._수의학과)
Novel molecular determinants related with mammalian pathogenicity in influenza A virus
인플루엔자 A 바이러스의 포유류 병원성 관련 신규 유전자 표지 분석
- 수의과대학 수의학과
- Issue Date
- 서울대학교 대학원
- 학위논문 (박사)-- 서울대학교 대학원 : 수의과대학 수의학과, 2018. 8. 김재홍.
- Influenza A virus (IAV) is an RNA virus belonging to family Orthomyxoviridae, which contains negative sense, single-stranded, segmented RNA genomes. IAV is further categorized into subtypes by surface glycoproteins, HA and NA proteins. Considering variety in subtypes of IAVs, migratory waterfowl has been suspected to be served as reservoirs for IAVs. The avian-origin influenza A virus (AIV) have been occasionally transmitted to human, and some of these viruses were successfully adapted, causing severe influenza pandemics. Nonetheless, the understanding of AIV transmission to mammals has not been well elucidated. In this study, I have generated several genetic reassortants of AIVs using reverse genetics techniques and found the novel pathogenic determinants in AIVs contributing to adapt AIVs to mammals and their role in viral transmission and pathogenicity in mammals.
The polymerase complex of IAV is a heterotrimer composed of PB2, PB1, and PA proteins. Given their role in the viral replication and transcription, the polymerase complex, especially PB2 protein, have been considered as one of the important host determinant factors. The polymerase complex of the low-pathogenic avian influenza viruses [A/chicken/Korea/KBNP-0028/2000 (H9N2)] (0028) and [A/chicken/Korea/01310/2001 (H9N2)] (01310) has previously been characterized, and novel amino acid residues present in PB2 and PA proteins that likely contribute to pathogenicity toward mammals have been identified. For the analysis of these novel molecular determinants in PB2 protein, I firstly generated the recombinant PR8 virus carrying the mutated 01310 PB2, and found that key amino acid mutations (I66M, I109V and I133V, collectively referred to as MVV) of 01310 PB2 increase the replication efficiency of recombinant PR8 virus carrying the mutated PB2 in both avian and mammalian hosts. The MVV mutations caused no weight loss in mice, but they did allow replication in infected lungs, and the viruses acquired fatal mammalian pathogenic mutations such as Q591R/K, E627K, or D701N in the infected lungs. The MVV mutations are located at the interfaces of the trimer and are predicted to increase the strength of this structure. These findings suggest that gaining MVV mutations might be the first step for AIV to acquire mammalian pathogenicity. Next, for evaluating the novel pathogenic determinants in PA proteins, I generated recombinant PR8 viruses containing the 0028-PA gene with a single amino acid substitution and test their pathogenicity and replication ability. A substitution from glutamate to glycine at position 684 (E684G) significantly increased viral replication in mammalian cells and mortality in mice, with significantly increased interferon β expression. Thus, the E684G mutation in the PA gene may play an important role in viral pathogenicity in mice by increasing viral replication and the host immune responses. In addition to the coding sequences of the viral genome, the noncoding sequences could also affect the viral pathogenicity. Hoffmanns 8-plasmid reverse genetics vector system introduced mutations at position 4, C4 to U4, of the 3′ ends of NA and M vRNAs of wild-type A/PR/8/34. For evaluating the effect of C4 to U4 mutation on viral pathogenicity, I generated 4 recombinant viruses with C4 in the NA and/or M vRNAs and rgPR8 by using reverse genetics and compared their pathobiological traits. The mutant viruses showed lower replication efficiency than rgPR8 due to the low transcription levels of NA and/or M genes. Furthermore, C4 in the NA and/or M vRNAs induced lower PR8 virus pathogenicity in BALB/c mice. The results suggest that the constellation of C4 and U4 among vRNAs may be one of the multigenic determinants of IAV pathogenicity. These novel pathogenic determinants found in this study will broaden the understanding of host jumping mechanism of AIVs, and these determinants may contribute to the surveillance and prevention of next pandemic threats.
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