Construction of an infectious cDNA clone for Omsk hemorrhagic fever virus, and characterization of mutations in NS2A and NS5

Yoshii, Kentaro; Igarashi, Manabu; Ito, Kimihito; Kariwa, Hiroaki; Holbrook, Michael R.; Takashima, Ikuo

doi:10.1016/j.virusres.2010.08.023

Cited by 25 publications

(19 citation statements)

References 49 publications

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“…The four amino acids identified in the present study are located in the C-terminal region of the RdRp domain. Several studies have shown that amino acid changes in the RdRp domain affect flavivirus genome replication and disease development (10,37). However, the KFK-D motif described here did not affect viral multiplication either in vitro or in vivo.…”

Section: Discussionmentioning

confidence: 52%

“…Recombinant TBEV (Oshima 5-10 strain) and OHFV (Guriev strain) were recovered from infectious cDNA clones of the respective viruses (TBEV-pt and OHF-pt, respectively) as reported previously (10,11). To prepare the infectious cDNA clones of chimeric viruses ( Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, we constructed infectious cDNA clones of both TBEV and OHFV (10)(11)(12). In the present study, we used these cDNA clones to generate TBEV-OHFV chimeric viruses with the specific objective of identifying viral determinants critical for the development of neurological disease in mice.…”

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confidence: 99%

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A Critical Determinant of Neurological Disease Associated with Highly Pathogenic Tick-Borne Flavivirus in Mice

Yoshii

Sunden

Yokozawa

et al. 2014

J Virol

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Tick-borne encephalitis virus (TBEV) and Omsk hemorrhagic fever virus (OHFV) are highly pathogenic tick-borne flaviviruses; TBEV causes neurological disease in humans, while OHFV causes a disease typically identified with hemorrhagic fever. Although TBEV and OHFV are closely related genetically, the viral determinants responsible for these distinct disease phenotypes have not been identified. In this study, chimeric viruses incorporating components of TBEV and OHFV were generated using infectious clone technology, and their pathological characteristics were analyzed in a mouse model to identify virus-specific determinants of disease. We found that only four amino acids near the C terminus of the NS5 protein were primarily responsible for the development of neurological disease. Mutation of these four amino acids had no effect on viral replication or histopathological features, including inflammatory responses, in mice. These findings suggest a critical role for NS5 in stimulating neuronal dysfunction and degeneration following TBEV infection and provide new insights into the molecular mechanisms underlying the pathogenesis of tick-borne flaviviruses. IMPORTANCE Tick-borne encephalitis virus (TBEV) and Omsk hemorrhagic fever virus (OHFV) belong to the tick-borne encephalitis serocomplex, genusFlavivirus, family Flaviviridae. Although TBEV causes neurological disease in humans while OHFV causes a disease typically identified with hemorrhagic fever. In this study, we investigated the viral determinants responsible for the different disease phenotypes using reverse genetics technology. We identified a cluster of only four amino acids in nonstructural protein 5 primarily involved in the development of neurological disease in a mouse model. Moreover, the effect of these four amino acids was independent of viral replication property and did not affect the formation of virus-induced lesions in the brain directly. These data suggest that these amino acids may be involved in the induction of neuronal dysfunction and degeneration in virus-infected neurons, ultimately leading to the neurological disease phenotype. These findings provide new insight into the molecular mechanisms of tick-borne flavivirus pathogenesis.

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Section: Discussionmentioning

confidence: 52%

Section: Methodsmentioning

confidence: 99%

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A Critical Determinant of Neurological Disease Associated with Highly Pathogenic Tick-Borne Flavivirus in Mice

Yoshii

Sunden

Yokozawa

et al. 2014

J Virol

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“…Reverse genetics platforms have been developed for a wide range of flaviviruses including YFV [57,58], Dengue Types 1–4 [59,60,61,62,63,64,65,66], JEV [67], Kunjin virus [68], Tick-borne encephalitis virus (TBEV) [69,70,71], Murray Valley encephalitis virus [72], Langat virus [73], West Nile virus (WNV) [6,74], and Omsk hemorrhagic fever virus [75]. Advances in the recombinant flavivirus approaches have been instrumental in new vaccine design efforts.…”

Section: Positive-strand Rna Virus Biology Reverse Genetics and mentioning

confidence: 99%

RNA Virus Reverse Genetics and Vaccine Design

Stobart

Moore

2014

Viruses

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RNA viruses are capable of rapid spread and severe or potentially lethal disease in both animals and humans. The development of reverse genetics systems for manipulation and study of RNA virus genomes has provided platforms for designing and optimizing viral mutants for vaccine development. Here, we review the impact of RNA virus reverse genetics systems on past and current efforts to design effective and safe viral therapeutics and vaccines.

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“…Previous studies have shown that NS2A protein is involved in viral assembly/release, viral RNA synthesis, regulation of NS1= expression, and inhibition of type-I interferon response (14)(15)(16)(17)(18)(19)(20). These functions are affected by amino acid substitutions within NS2A, such as NS2A-G11A, -E20A, -P30A, -T33I, -L46H, -I59N, -D73H, -R84S/A/E, -E100A, M108K, D125A, -Q187A, -K188A, -K190S, and -G200A (14,(16)(17)(18)(19)(20)(21)(22)(23)(24)(25).…”

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confidence: 99%

A Single Amino Acid Substitution in the NS2A Protein of Japanese Encephalitis Virus Affects Virus Propagation In Vitro but Not In Vivo

Takamatsu

Morita

Hayasaka

2015

J Virol

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We identified a unique amino acid of NS2A 113 , phenylalanine, that affects the efficient propagation of two Japanese encephalitis virus strains, JaTH160 and JaOArS982, in neuroblastoma Neuro-2a cells but not in cell lines of extraneural origin. This amino acid did not affect viral loads in the brain or survival curves in mice. These findings suggest that virus propagation in vitro may not reflect the level of virus neuroinvasiveness in vivo. J apanese encephalitis virus (JEV) causes approximately 30,000 to 50,000 cases and 10,000 to 15,000 deaths in Asian countries annually (1, 2). JEV belongs to the family Flaviviridae, genus Flavivirus (3, 4), whose genomic RNA encodes one polyprotein, cleaved into three structural (C, prM, and E) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins (5). The clinical symptoms of JE vary from mild to severe and include a nonspecific febrile illness, meningitis, encephalitis, and meningoencephalitis (6, 7). The mechanism of severe central nervous system (CNS) disease is not fully understood.To evaluate disease pathogenesis and virulence, mice have been employed as an infection model. Several viral and host factors affect disease severity during JEV infection. We previously suggested that the host response, resulting in immunopathological effects, contributes to fatal infections (8). Furthermore, we also demonstrated that the JaOArS982 and JaTH160 strains of JEV exhibited different virulence in mice (8). Therefore, a geneticsbased comparison between these strains may provide an effective approach to identify viral factors contributing to severe disease.Our previous results showed that following subcutaneous infection with 10 4 PFU of JaTH160, mice showed 100% mortality, whereas JaOArS982 caused approximately 30% mortality in mice (8). We first constructed infectious cDNA clones harboring fulllength genes of JaOArS982 and JaTH160 and produced infectious viruses S982-IC and JaTH-IC from the respective cDNAs (9). In the present study, subcutaneous infection with 10 4 PFU of S982-IC and JaTH-IC viruses caused 40% and 100% mortality, respectively, in C57BL/6j (B6) mice (Japan SLC Cooperation, Japan CLEA Cooperation) (Fig. 1A), indicating that both JaTH-IC and S982-IC viruses possessed virulence potentials similar to those of their parent JaOArS982 and JaTH160 viruses. Our animal experimental protocols were approved by the Animal Care and Use Committee, Nagasaki University (approval numbers 091130-2-7/0912080807-9 and 100723-1-3/1008050873-3).Our previous data showed that viral loads in the CNS of JaTH160-infected mice were significantly higher than those of JaOArS982-infected mice (8). This raised the possibility that virus propagation in neuronal cells is different between JaTH160 and

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Construction of an infectious cDNA clone for Omsk hemorrhagic fever virus, and characterization of mutations in NS2A and NS5

Cited by 25 publications

References 49 publications

A Critical Determinant of Neurological Disease Associated with Highly Pathogenic Tick-Borne Flavivirus in Mice

A Critical Determinant of Neurological Disease Associated with Highly Pathogenic Tick-Borne Flavivirus in Mice

RNA Virus Reverse Genetics and Vaccine Design

A Single Amino Acid Substitution in the NS2A Protein of Japanese Encephalitis Virus Affects Virus Propagation In Vitro but Not In Vivo

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