We identified a unique amino acid of NS2A113, 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

Daisuke Hayasaka

Kouichi Morita

T. S. Dermody

Yuki Takamatsu

PubMed

Takamatsu, Yuki

Morita, Kouichi

Hayasaka, Daisuke

Institutional Repositories DataBase (IRDB)

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

Nagasaki university's Academic Output SITE

ABSTRACT
          
            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
            .
          </jats:p

Crossref

Journal of Virology

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

Takamatsu Yuki

Morita Kouichi

Hayasaka Daisuke

Nagasaki University's Academic Output SITE: NAOSITE

1  Title 1 A unique amino acid substitution in NS2A protein of Japanese encephalitis virus affects 2 virus propagation in vitro but not in vivo 3  4 Running title 5 A unique amino acid substitution in NS2A of JEV 6  7 Authors 8 Yuki Takamatsu, Kouichi Morita and Daisuke Hayasaka* 9  10 Affiliations 11 Department of Virology, Institute of Tropical Medicine, GCOE program, Leading 12 Graduate School Program, Nagasaki University, Nagasaki, Nagasaki, Japan 13  14 *Corresponding author: Daisuke Hayasaka 15 Address: 1-12-4, Sakamoto, Nagasaki, Nagasaki 852-8524, Japan 16 Phone: +95-819-7828, Fax; +95-819-7830 17 E-mail: hayasaka@nagasaki-u.ac.jp  18 2  Abstract 19 We identified a unique amino acid of NS2A113 phenylalanine that affects the 20 efficient propagation of two Japanese encephalitis virus strains, JaTH160 and 21 JaOArS982 in neuroblastoma Neuro-2a cells but not in cell lines of extraneural origin. 22 This amino acid did not affect viral loads in the brain nor survival curves in mice. These 23 findings suggest that virus propagation in vitro may not reflect the level of virus 24 neuroinvasiveness in vivo. 25   26 3  Japanese encephalitis (JE) virus (JEV) causes approximately 30,000 to 50,000 cases 27 and 10,000-15,000 deaths in Asian countries annually (1, 2). JEV belongs to the family 28 Flaviviridae, genus Flavivirus (3, 4), whose genomic RNA encodes one polyprotein, 29 cleaved into three structural (C, prM, and E) and seven nonstructural (NS1, NS2A, 30 NS2B, NS3, NS4A, NS4B, and NS5) proteins (5). The clinical symptoms of JE vary 31 from mild to severe and include a non-specific febrile illness, meningitis, encephalitis 32 and meningoencephalitis (6, 7). The mechanism of severe central nervous system (CNS) 33 disease is not fully understood. 34 To evaluate disease pathogenesis and virulence, mice have been employed as an 35 infection model. Several viral and host factors affect disease severity during JEV 36 infection. We previously suggested that the host response, resulting in 37 immunopathological effects, contributes to fatal infections (8). Furthermore, we also 38 demonstrated that the JaOArS982 and JaTH160 strains of JEV exhibited different 39 virulence in mice (8). Therefore, a genetic-based comparison between these strains may 40 provide an effective approach to identify viral factors contributing to severe disease. 41 Our previous results showed that following subcutaneous infection with 104 PFU of 42 JaTH160, mice showed 100% mortality, whereas JaOArS982 caused approximately 43 30% mortality in mice (8). We first constructed infectious cDNA clones harboring full 44 4  length genes of JaOArS982 and JaTH160, and produced infectious viruses of S982-IC 45 and JaTH-IC from each cDNA, respectively (9). In the present study, subcutaneous 46 infection with 104 PFU of S982-IC and JaTH-IC viruses caused 40% and 100% 47 mortality, respectively, in C57BL/6j (B6) mice (Japan SLC Cooperation, Japan CLEA 48 Cooperation) (Figure 1A), indicating that both JaTH-IC and S982-IC viruses possessed 49 virulence potentials similar to their parent JaOArS982 and JaTH160 viruses. Our animal 50 experimental protocols were approved by the Animal Care and Use Committee, 51 Nagasaki University (approval number: 091130-2-7 /0912080807-9, 100723-1-3 / 52 1008050873-3). 53 Our previous data showed that viral loads in the CNS of JaTH160-infected mice 54 were significantly higher than those of JaOArS982-infected mice (8). This raised the 55 possibility that virus propagation in neuronal cells is different between JaTH160 and 56 JaOArS982. Thus, we next compared virus propagations in murine neuroblastoma 57 Neuro-2a (N2a) cell lines. 58 N2a cells were infected with each JEV strain at an m.o.i. of 0.1 and supernatants 59 were harvested at 0, 24, 48 and 72 hours post-infection (pi). Virus titers were 60 determined by plaque forming assays on Baby Hamster Kidney (BHK) cells (10). In 61 N2a cells, JaTH-IC and the parent JaTH160 viruses exhibited significantly higher virus 62 5  yields compared with S982-IC and the parent JaOArS982 viruses (Figure 1B). However, 63 there were no significant differences of virus yields between the four viruses in BHK 64 cells (Figure 1C), Vero (rhesus monkey kidney), PS (porcine kidney) and HeLa (human 65 epithelial) cells (data not shown). These results suggest that replication in neuronal cells 66 is different between JaTH160 and JaOArS982 viruses. 67 To determine the specific region of the viral gene affecting virus propagation in N2a 68 cells, we constructed four chimeric JEV clones, S982_J1-IC, S982_J2-IC, S982_J3-IC 69 and S982_J4-IC, as shown in Figure 2A. S982_J2-IC exhibited a similar growth curve 70 to JaTH-IC and produced significantly higher virus titers than S982-IC, S982_J1-IC, 71 S982_J3-IC and S982_J4-IC (Figure 3A). Thus, the viral genome sequence in the region 72 of NS1322 to NS335 affects virus propagation in N2a cells. 73 There are three amino acid differences in this region between JaOArS982 and 74 JaTH160. Thus, we inserted amino acid substitutes into S982-IC and JaTH-IC, as 75 shown in Figure 2B. Site-directed mutagenesis was used, as described previously (11). 76 S982_IA23FA113DB81 showed significantly higher virus production in N2a cells compared 77 with S982-IC, S982_VA23LA113DB81 and S982_IA23LA113EB81 (Figure 3B). Conversely, 78 JaTH_VA23LA113EB81 exhibited significantly lower virus yields than JaTH-IC, 79 JaTH_IA23FA113EB81 and JaTH_VA23FA113DB81 (Figure 3C). These results indicate that an 80 6  amino acid substitution in NS2A113, F in JaTH-IC and L in S982-IC, is responsible for 81 the difference in propagation in N2a cells. 82 To examine whether the amino acid substitution in NS2A113 contributes to the 83 virulence and virus propagation in vivo, B6 mice were subcutaneously inoculated with 84 S982_IA23FA113DB81 and JaTH_VA23LA113EB81, and their mortality was observed. Viral 85 loads in the brain were also compared as previously shown (8, 12). Unexpectedly, 86 S982_IA23FA113DB81 showed similar survival curves to the parent S982-IC virus (Figure 87 4A) and there was no significant difference in viral loads in the brain between S982-IC- 88 and S982_IA23FA113DB81-infected mice (Figure 4B). JaTH_VA23LA113EB81 also showed 89 similar survival curves and similar viral loads in the brain to the parent JaTH-IC virus 90 (Figure 4B). Thus, an amino acid substitution in NS2A113 did not explain the different 91 viral loads and virulence in the brain between S982-IC and JaTH-IC viruses. 92 Flavivirus NS2A protein is a 22-kDa hydrophobic protein (13). Previous studies have 93 shown that NS2A protein is involved in viral assembly/release, viral RNA synthesis, 94 regulation of NS1’ expression and inhibition of type-I interferon response (14-20). 95 These functions are affected by amino acid substitutions within NS2A, such as NS2A 96 -G11A, -E20A, -P30A, -T33I, -L46H, -I59N, -D73H, -R84S/A/E, -E100A, M108K, 97 D125A, -Q187A, -K188A, -K190S, and -G200A (14, 16-25). 98 7  The influence of the NS2A-L113F substitution identified here in JEV infection has 99 not been reported previously. NS2A has eight predicted transmembrane segments 100 (pTMS), and NS2A113 appears to localize to pTMS4 (14). However, how NS2A113 101 substitution affects virus propagation in N2a cells remains unclear. Further investigation 102 may provide information on the unknown function of NS2A. 103 Although a single amino acid substitution in NS2A113 alters viral propagation in N2a 104 cells, this substitution did not affect viral loads in the brain nor survival curves in mice. 105 These findings suggest that virus propagation in vitro does not necessarily reflect virus 106 replication in vivo. Further, other amino acid and/or nucleotide substitutions may affect 107 host responses such as antiviral activity. In this regard, this study helps to elucidate the 108 mechanism of pathogenesis due to JEV infection in a mouse model. 109 Interestingly, our preliminary experiments showed that there were no significant 110 differences of mortality following intracerebral inoculation between JaOArS982 and 111 JaTH160. In our previous study of tick-borne encephalitis viruses, we suggested that the 112 mechanism of fatal infection is fundamentally different between intracerebral and 113 peripheral infection (10, 26). We further showed that immune responses were different 114 between JaOArS982- and JaTH160-infected mice (8). From these observations, we 115 assumed that different viral replications in the brains between JaOArS982 and JaTH160 116 8  attributes to the peripherally induced host immune responses and those immune cells 117 infiltrating in the brains. In addition, it appears that most of the volume of inoculum 118 leaked from the brain due to intracranial pressure following intracerebral inoculation. 119 Thus, we consider that intracerebral inoculation does not simply reflect virus infection 120 and replication in neurons, and it appears that it is difficult to examine the different 121 virulence mechanism between JaOArS982 and JaTH160. 122  We propose that actual virus propagation in the brain in vivo reflects a combined 123 mechanism of viral replication properties in neuronal cells and the host antiviral 124 immune response. Furthermore, we believe that the disease mechanisms of JEV in vivo 125 involve a complex mechanism that includes the host immune response and neuronal 126 infection in the CNS. Further investigations in a step-by-step fashion will provide clues 127 to elucidate the precise pathogenic mechanisms of JEV infection and enable the 128 development of effective treatment strategies for JE. 129  130 Acknowledgments 131 We acknowledge Dr. Tomohiko Takasaki from National Institute of Infectious Diseases, 132 Tokyo, Japan, for providing the JaTH160 strain. We also thank Jun Iriki and Toshiki 133 Nakamura of the faculty of medicine, Nagasaki University for technical support and 134 9  Corazon C. Buerano from Department of Virology, Institute of Tropical Medicine, 135 Nagasaki University for the editing of the paper. This work was supported by JSPS 136 KAKENHI Grant Numbers 25304045, 25660229, 23658243 and from the Japan Society 137 for the Promotion of Science; Health and Labour Sciences Research Grant on Emerging 138 and Re-emerging Infectious Diseases from the Japanese Ministry of Health, Labour and 139 Welfare; Research on International Cooperation in Medical Science (Japan-US 140 Cooperative Program), Health and Labour Sciences Research Grants; the Cooperative 141 Research Grant(s) of NEKKEN, 2014 and the Japan Initiative for Global Research 142 Network on Infectious Diseases. 143  144 References 145 1. Ghosh D,  Basu A. 2009. Japanese encephalitis-a pathological and clinical 146 perspective. PLoS Negl Trop Dis 3:e437. 147 2. Solomon T. 2004. Flavivirus encephalitis. N Engl J Med 351:370-378. 148 3. Westaway EG, Brinton MA, Gaidamovich S, Horzinek MC, Igarashi A, Kaariainen L, 149 Lvov DK, Porterfield JS, Russell PK,  Trent DW. 1985. Flaviviridae. Intervirology 150 24:183-192. 151 4. Gubler DJ, Kuno G,  Markoff L. 2007. Field's Virology, vol. fifth edition. Wolters 152 Kluwer Lippincott Williams and Wilkins, Philadelphia. 153 5. Sumiyoshi H, Hoke CH,  Trent DW. 1992. Infectious Japanese encephalitis virus 154 RNA can be synthesized from in vitro-ligated cDNA templates. J Virol 66:5425-5431. 155 6. Tsai TF. 2000. New initiatives for the control of Japanese encephalitis by 156 vaccination: minutes of a WHO/CVI meeting, Bangkok, Thailand, 13-15 October 157 1998. Vaccine 18 Suppl 2:1-25. 158 7. Misra UK,  Kalita J. 2010. Overview: Japanese encephalitis. Prog Neurobiol 159 10  91:108-120. 160 8. Hayasaka D, Shirai K, Aoki K, Nagata N, Simantini DS, Kitaura K, Takamatsu Y, 161 Gould E, Suzuki R,  Morita K. 2013. TNF-alpha acts as an immunoregulator in the 162 mouse brain by reducing the incidence of severe disease following Japanese 163 encephalitis virus infection. PLoS One 8:e71643. 164 9. 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Molecular and functional analyses of 216 Kunjin virus infectious cDNA clones demonstrate the essential roles for NS2A in 217 virus assembly and for a nonconservative residue in NS3 in RNA replication. J Virol 218 77:7804-7813. 219 26. Hayasaka D, Nagata N, Hasegawa H, Sata T, Takashima I,  Koike S. 2010. Early 220 mortality following intracerebral infection with the Oshima strain of tick-borne 221 encephalitis virus in a mouse model. J Vet Med Sci 72:391-396. 222  223  224 Figure Legends 225 FIG 1  Virulence in mice and viral yields in cultured cells infected with the S982-IC 226 and JaTH-IC viruses. (A) Survival curves and (B) Weight ratios of mice subcutaneously 227 12  infected with 104 PFU of each virus (n=10). P: Log-rank (Mantel-Cox) Test. 228 Propagation of JaOArS982 (original virus) and S982-IC (derived from infectious cDNA 229 clone of JaOArS982), JaTH160 (original virus) and JaTH-IC (derived from infectious 230 cDNA clone of JaTH160) viruses in N2a (C) and BHK cells (D) at 0, 24, 48 and 72 231 hours post-infection. Error bars represent standard deviations. p: One-way analysis of 232 variance. 233  234 FIG 2  Schematic representation of full-length chimeric and amino acid 235 substituted-viruses derived from S982-IC and JaTH-IC. (A) A genome representations 236 of S982_J1-IC, S982_J2-IC, S982_J3-IC and S982_J4-IC showing the replacement of  237 5’UTR-NS1322, NS1323-NS335, NS336-NS5566, NS5567-3’UTR of S982-IC, respectively, 238 with the corresponding region of JaTH-IC. (B) A genome representation of a single 239 amino acid substituted-S982-IC and JaTH-IC. The white and black arrowheads indicate 240 amino acids derived from S982-IC and JaTH-IC, respectively. S982-IC and JaTH-IC 241 are also named S982_IA23LA113DB81 and JaTH_VA23FA113EB81 , respectively. 242  243 FIG 3  Growth curves for virus propagation of the chimeric and amino acid 244 substituted-viruses in N2a cells at 0, 24, 48 and 72 hours post-infection. (A) Viral yields 245 13  of S982_J1-IC, S982_J2-IC, S982_J3-IC and S982_J4-IC compared with S982-IC and 246 JaTH-IC viruses. Viral yields of JaTH-IC and S982-IC are the same data as FIG 1C. 247 Viral yields of amino acid substituted-S982-IC (B) and JaTH-IC (C) viruses. p: 248 One-way analysis of variance. The white and black arrowheads indicate amino acids 249 derived from S982-IC and JaTH-IC, respectively. 250  251 FIG 4  Virulence in mice and viral loads in the brains of mice following subcutaneous 252 infection with 104 PFU of S982_IA23LA113DB81 (S982-IC), S982_IA23FA113DB81, 253 JaTH_VA23FA113EB81 (JaTH-IC), and JaTH_VA23LA113EB81. (A) Survival curves (n=10) 254 P: Gehan-Breslow-Wilcoxon Test. (B) Viral loads in the brain (n=6). P: Kruskal-Wallis 255 test, *: p<0.05 by Dunn’s Multiple Comparison Test. 256 

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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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