Amino acid 159 of the envelope protein affects viral replication and T-cell infiltration by West Nile virus in intracranial infection

Abstract

West Nile virus (WNV) is an important cause of viral encephalitis in birds and animals, includinghumans. Amino acid 159 of the envelope (E) protein is reportedly implicated in the different levels of neurovirulence in mice infected with WNV NY99 or Eg101. We investigated the role of amino acid 159 of the E protein in the pathogenesis of WNV infection. We produced recombinant WNV with the structural proteins of the NY99 or Eg101 strain (NY-WT or EgCME-WT) and mutant viruses with substitutions of amino acid 159 of the E protein (NY-E-V159I or EgCME-E-I159V). The NY-WT and NY-E-V159I or EgCME-WT and EgCME-E-I159V titers in culture supernatant were similar. The mortality rate and viral titer in the brains of mice inoculated intraperitoneally with NY-WT or NY-E-V159I were also similar. In contrast, the mortality rate and viral titer in the brains of mice inoculated intracranially with EgCME-E-I159V were significantly higher than those of mice inoculated with EgCME-WT. The numbers of CD3positive and CD8-positive T cells were greater in brains inoculated with EgCME-E-I159V than in those inoculated with EgCME-WT. Therefore, amino acid 159 of the E protein modulates the pathogenicity of WNV by affecting viral replication and T-cell infiltration in the brain. Figure 4. Infiltration of T cells in mouse brains infected with recombinant WNV. C57BL/6 mice were inoculated intracranially with 10 pfu EgCME-WT or EgCME-E-I159V. (A) Images of haematoxylin and eosin-stained sections of the cerebral cortex, sections stained by TUNEL or immunostained for WNV antigen, CD3, or CD8 (n = 5). White arrowheads, degenerated neuronal cells; black arrowheads, TUNEL-positive cells, CD3-positive cells, or CD8-positive cells. Scale bar, 25 μm. (B) Number of CD3-or CD8-positive cells in the hippocampus (n = 5). Statistical significance was assessed by the two-tailed Student's t-test. *p < 0.05; **p < 0.01. (C) Relationship between CD3-positive T cells and WNV antigen-positive cells. Sections were stained with antibodies against CD3 (green) and WNV antigen (red). Nuclei were stained with DAPI (blue). White arrowheads indicate CD3-positive T cells adjacent to viral antigen-positive cells. Scale bar, 20 μm. (D) Expression levels of genes encoding immune factors in brains inoculated with EgCME-WT or EgCME-E-I159V. MethodsCells. Vero cells, obtained from the JCRB Cell Bank (JCRB0111), were cultured in an atmosphere containing 5% CO 2 at 37 °C in modified Eagles' medium (MEM; Wako, Osaka, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS). SH-SY 5Y cells, obtained from the European Collection of Authenticated Cell Cultures (94030304), were cultured in an atmosphere containing 5% CO 2 at 37 °C in Dulbecco's MEM (DMEM)/ Nutrient Mixture F-12 Ham (Wako) supplemented with 10% heat-inactivated FBS. HEK-293T cells, kindly provided by Dr. Matsuura (Osaka University), were cultured in an atmosphere containing 5% CO 2 at 37 °C in high-glucose DMEM (Wako) supplemented with 10% heat-inactivated FBS.

Molecular Determinants of West Nile Virus Virulence and Pathogenesis in Vertebrate and Invertebrate Hosts

“…A recent study by Kobayashi et al [111] supports this scenario. WNV recombinants between the highly virulent lineage 1 strain NY99 (V159) and the less virulent Eg101 (I159) strain were generated but did not show any significant differences in in vitro cultures or in C57BL/6 mice inoculated intraperitoneally.…”

“…In contrast, following the intracranial inoculation of mice, viral replication in the brain was higher for EgCME-E-I159V than for the parental strain EgCME. The mutation also increased levels of CD3+ and CD8+ T cells in the brain, suggesting that residue 159 of the E protein modulates WNV pathogenicity by influencing both viral replication and T-cell recruitment [111].…”

West Nile Virus Lineage 1 in Italy: Newly Introduced or a Re-Occurrence of a Previously Circulating Strain?

“…Goshawks and kestrels can in fact be considered either migrant or resident birds. Unfortunately, it was not possible to check the carcasses, and eventually make out the bird behavior from wing, claw, and feather characteristics [74][75][76]. In support of the re-introduction of WNV-L1 is the lack of detection for consecutive years by the national surveillance program, which has been in place since 2002, and has been drawn to detect virus circulation early.…”

“…A threonine residue was observed at the 249 position in the strain MW627239, as well as in JF719065 and MW835362, circulating in Italy in 2008 (jay) and 2011 (owl), and in JF719069 and MT863559, circulating in Spain (horse) and France (human) in 2010 and 2015, respectively. Furthermore, the amino acid valine at the residue 159 of the E protein is considered a determinant of WNV neurovirulence, influencing viral replication and pathogenesis, and being involved in WNV infection and T-cell infiltration in the brain [75]. This amino acid is observed in the isolate NC009942, circulating in the USA in 1999, but not in the Italian and European strains, all characterized by isoleucine at this residue position.…”

Different viral genes modulate virulence in model mammal hosts and Culex pipiens vector competence in Mediterranean basin lineage 1 West Nile virus strains

“…Comparison of WN-IS98 and WN-IT08 genomes revealed 22 non-synonymous mutations, including E-V159I, also found on IS98-NS1, and NS4B-E249D. Kobayashi et al (2020) , demonstrated that E-I159V increased virulence in a mouse model. Here, the attenuated phenotype of WN-IT08 in mammals could be due to the E-V159I or the NS4B-E249D mutations, as well as to other modifications discussed earlier.…”