An amino acid substitution (V3I) in the Japanese encephalitis virus NS4A protein increases its virulence in mice, but not its growth rate in vitro

Abstract

Japanese encephalitis virus (JEV), which is transmitted to humans by mosquitoes, causes the serious neurological disorder Japanese encephalitis (JE). The fatality rate of JE is approximately 30 % and each year 30 000-50 000 clinical cases of JE and 10 000 deaths are reported, mainly in China, South East Asian countries, and India (Tsai, 2000). Most cases of JE occur in South, East and South East Asia; however, JE cases have been reported in northern areas of Australia in recent decades (Hanna et al., 1996). JEV belongs to the genus Flavivirus within the family Flaviviridae and is now classified into five genotypes (genotypes I-V) based on genomic RNA homology (Uchil & Satchidanandam, 2001; Solomon et al., 2003). JEV has a single-stranded, positive-sense RNA genome. The approximately 11 kb genome contains one ORF encoding three structural proteins [capsid (C), premembrane (prM) and envelope (E)] and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5), as well as 59 and 39 NTRs (Lindenbach et al., 2007). The E protein, the main structural protein, has a putative receptor-binding domain and neutralization epitopes. The E protein is thought to play major roles in determining viral pathogenicity by defining cell tropism and mediating entry into susceptible cells (Lindenbach et al., 2007;Gubler et al., 2007). Since the early 1990s, various attempts have been made to clarify the molecular basis of JEV virulence by comparing the nucleotide sequences of virus strains with different degrees of virulence (Nitayaphan et al., 1990; Aihara et al., 1991;Cecilia & Gould, 1991; Hasegawa et al., 1992;Sumiyoshi et al., 1995;Chen et al., 1996;Ni & Barrett, 1996, 1998Arroyo et al., 2001;Wu et al., 2003;Lee et al., 2004;Chambers et al., 2007; Pujhari et al., 2011;Wu et al. 2011). Some of these reports have suggested that nucleotide substitutions in the E protein may alter the virulence of JEV. Studies using an infectious clone of JEV demonstrated that a single glutamic acid-to-lysine substitution at position 138 of the E protein attenuated the JEV strain in which it was made (Sumiyoshi et al., 1995;Zhao et al., 2005;Liang et al., 2009). It has also been reported that a single methionine-to-lysine substitution at position 279 of S467N and rJEV-Mie41-NS4A V3I , which have, respectively, a serine-to-asparagine mutation at position 467 (S467N) in the NS3 region of Mie/41/2002 and a valine-to-isoleucine mutation at position three (V3I) in the NS4A region of Mie/41/2002 (Fig. 1a). Groups of mice (n510) were inoculated with 1610 3 p.f.u. of recombinant virus ( V3I in cultured cells. We first compared the plaque sizes of the strains as previously described (Tajima et al., 2010). The plaque morphologies of all three viruses were similar (Fig. 1b), indicating that the V3I mutation does not affect plaque formation and that the difference in virulence between Mie/41/2002 and Mie/40/2004 strains does not correlate with their plaque sizes in Vero cells. We next compared the growth rates of the three strains in four cell l...

In vitro growth, pathogenicity and serological characteristics of the Japanese encephalitis virus genotype V Muar strain

“…Other reports have also highlighted the importance of the E protein as a determinant of pathogenicity (Monath et al, 2002;Sumiyoshi et al, 1995;Zhao et al, 2005). In contrast, our group determined that an amino acid substitution in the JEV NS4A protein alters virulence in mice (Yamaguchi et al, 2011). To clarify the regions responsible for increased pathogenicity in the Muar genome, various GI-GV intertypic and point mutant viruses may be required.…”

Surveillance of Japanese Encephalitis Virus Infection in Mosquitoes in Vietnam from 2006 to 2008

“…29 Alignment of JEV E gene nucleotide sequences in the GenBank database showed that the great majority of JEV strains had Ser, not Arg, at residue 123. 29 The five Vietnamese JEV isolates in this study had Further data are needed to explain the function and mechanism of the E protein in JEV infection, and on other structural and non-structural JEV proteins (e.g., prM and NS4A [52][53][54] ) and on the JEV 5 and 3 NTRs. 29,55 This study has identified multiple JEV populations in Vietnam, and the nucleotide sequences of these JEVs were similar to those isolated in other area of Asia.…”

Flavivirus reverse genetic systems, construction techniques and applications: A historical perspective

“…Notably, a correlation between growth properties in cellulo and virulence in vivo was not always observed. Indeed, Yamaguchi et al (2011) revealed that an amino acid substitution NS4A-V3I of the JEV strain Mie/40/2004 increased its virulence in mice without influencing the growth rate in cell culture (Yamaguchi et al, 2011).…”