NS2B/NS3 mutations enhance the infectivity of genotype I Japanese encephalitis virus in amplifying hosts

Abstract

Genotype I (GI) virus has replaced genotype III (GIII) virus as the dominant Japanese encephalitis virus (JEV) in the epidemic area of Asia. The mechanism underlying the genotype replacement remains unclear. Therefore, we focused our current study on investigating the roles of mosquito vector and amplifying host(s) in JEV genotype replacement by comparing the replication ability of GI and GIII viruses. GI and GIII viruses had similar infection rates and replicated to similar viral titers after blood meal feedings in Culex tritaeniorhynchus. However, GI virus yielded a higher viral titer in amplifying host-derived cells, especially at an elevated temperature, and produced an earlier and higher viremia in experimentally inoculated pigs, ducklings, and young chickens. Subsequently we identified the amplification advantage of viral genetic determinants from GI viruses by utilizing chimeric and recombinant JEVs (rJEVs). Compared to the recombinant GIII virus (rGIII virus), we observed that both the recombinant GI virus and the chimeric rJEVs encoding GI virus-derived NS1-3 genes supported higher replication ability in amplifying hosts. The replication advantage of the chimeric rJEVs was lost after introduction of a single substitution from a GIII viral mutation (NS2B-L99V, NS3-S78A, or NS3-D177E). In addition, the gain-of-function assay further elucidated that rGIII virus encoding GI virus NS2B-V99L/NS3-A78S/E177E substitutions re-gained the enhanced replication ability. Thus, we conclude that the replication advantage of GI virus in pigs and poultry is the result of three critical NS2B/NS3 substitutions. This may lead to more efficient transmission of GI virus than GIII virus in the amplifying host-mosquito cycle.

Fig 1. Growth curves of GIII and GI JEVs in Aedes albopictus and Culex tritaeniorhynchus mosquito-derived cell lines.

(A-C) GIII CH1392 virus (), GIII T1P1 virus (), GI YL2009-4 virus (), or GI TC2009-1 virus ()-infected mosquito-derived cells at 0.5 multiplicity of infection (MOI). Virus was amplified in C6/36 cells at 28°C (A) and in CTR209 cells at 28°C (B) or 34°C (C). The viral titer (focus-forming unit [ffu] per ml) was determined in the supernatant at 12, 24, 36, 48, and 60 hours post infection (HPI) by micro-antigen focus assay. Mean, with standard error of the mean (SEM), for triplicates is shown. A dotted line indicates the detection limit. The difference in viral titer was calculated for each time point using one-way ANOVA followed by Turkey’s Multiple Comparison Test.

Fig 2. The indistinguishable infectivity of GIII and GI JEVs in Culex tritaeniorhynchus.

Female Culex tritaeniorhynchus mosquitoes (n = 12 per group) were fed with the pig blood meal mixed with 8×10⁶ ffu/ml of JEVs (CH1392, T1P1, YL2009-4, or TC2009-1). (A) The viral infection rate was calculated by the number of JEV-positive mosquitoes divided by the number of feeding mosquitoes. (B) The viral titer (ffu) for each whole mosquito body was detected by micro-antigen focus assay at 14 days post-feeding. Each dot represents an individual mosquito. The mean value is shown as a horizontal line. Statistical analysis was performed by one-way ANOVA followed by Turkey’s Multiple Comparison Test.

Fig 3. The replication advantage of GI JEVs is due to the amplifying host and/or the replication temperature.

(A-F) GIII CH1392 virus (), GIII T1P1 virus (), GI YL2009-4 virus (), and GI TC2009-1 virus () were inoculated onto VERO (A and D), PK-15 (B and E), and DF-1 cells (C and F) at 0.5 MOI and incubated at 37°C (A-C) or 41°C (D-F). The viral titer (ffu/ml) was determined in the supernatant at 12, 24, 36, 48, 60 HPI by micro-antigen focus assay. Mean with SEM for the triplicates is shown. The difference in viral titer was analyzed with one-way ANOVA followed by Turkey’s Multiple Comparison Test. A significantly genotype-specific difference is indicated by an asterisk (P< 0.05).

Fig 4. Comparison of viremia in experimentally-infected pigs and poultry using GI and GIII JEVs.

(A and B) Nine specific pathogen free (SPF) pigs (n = 3 per group) were subcutaneously inoculated with 10⁵ ffu of GIII CH1392 virus () or GI YL2009-4 virus (), or PBS (). (A) The change of each pig’s body temperature before and after viral infection is shown. (B) The viral titer in pig plasma was detected by micro-antigen focus assay at 1, 2, and 4 days post infection (DPI). (C and D) One-day old chickens (n = 4 per group) and two-day old ducklings (n = 4 per group) were subcutaneously injected with 10⁴ ffu of GIII CH1392 (), GIII T1P1 (), GI YL2009-4 (), and GI TC2009-1 viruses (). The viral titer in plasma recovered from JEV-infected chickens (C) or ducklings (D) at 1, 2, 4, and 6 DPI (n = 4 per group) was determined by micro-antigen focus assay. The detection limit is indicated as a dotted line. (A-D) A dot plus a horizontal line represent an individual animal and a mean viral titer (C, D) of the group, respectively. The statistics comparing either two or four viruses were determined by a Student’s Two-Tailed t-test or one-way ANOVA followed by Turkey’s Multiple Comparison Test. The statistical difference was noted by an asterisk (P< 0.05).

Fig 5. NS1-3 genes determine the major replication advantage of rGI virus in amplifying host-derived cell lines.

(A) The schematic diagram displays viral genes encoded in the infectious clones and rJEVs. GIII virus- and GI virus-derived genes are shown in dark gray and red, respectively. (B) The infectious clones were transfected into BHK-21 cells and incubated for 3 to 4 days. The replication of rJEVs was detected in the cells by immunofluorescence using mouse anti-JEV NS1 antibodies and Alexa Fluor 488-labeled goat anti-mouse IgG antibodies. The cellular nucleus was localized by DAPI staining. The secretion of rJEVs was analyzed by the plaque assay in BHK-21 cells. (C-F) The rJEVs were inoculated onto C6/36 (C), VERO (D), PK-15 (E), and DF-1 (F) cells at 0.5 MOI, and incubated at 28°C (C6/36) or at 41°C (VERO, PK-15, DF-1). Supernatants were collected from each rJEV-infected cell culture at 24 and 48 HPI, and viral titers determined by micro-antigen focus assay. Mean with SEM for triplicates is shown. A horizontal dotted line indicates the detection limit. The statistical analysis was conducted with one-way ANOVA followed by a post test using Dunnett’s Multiple Comparison Test using rGIII virus as a control. An asterisk indicates that rJEV has a significantly higher titer than rGIII virus (P< 0.05).

Fig 6. Enhancement of GI virus NS1-3 proteins in infected pigs and chickens.

10⁵ and 10⁴ ffu of rGIII (), rGI (), and rGIII/ GI NS1-3 viruses () were subcutaneously inoculated into SPF pigs (A) and one-day old chickens (B). The viral titer (A and B) in plasma collected from the infected pigs at days 0 to 8, and from the infected chickens at 60 HPI, were detected by micro-antigen focus assay. Each dot represents an individual animal. A horizontal dotted line indicates the detection limit. Mean with SEM is shown. The viremia in chicken plasma was statistically analyzed with one-way ANOVA followed by Turkey’s Multiple Comparison Test. A significant difference is indicated by an asterisk ([B], P< 0.05).

Fig 7. Effect of introduction of GIII virus-specific NS2B/NS3 substitutions into GI virus.

(A) The schematic diagram shows the location of sixteen GIII virus-specific substitutions on NS1-3 proteins. (B-E) The sixteen mutant rGIII/ GI NS1-3 viruses encoding single NS1, NS2A, NS2B, or NS3 substitutions are respectively colored with orange, purple, green, and blue. The rGIII and rGIII/ GI NS1-3 virus controls are shown colored with white and yellow. These rJEVs were infected into C6/36 (B), VERO (C), PK-15 (D), and DF-1 (E) cells at 0.5 MOI and replicated at 28°C (C6/36) or 41°C (VERO, PK-15, and DF-1). The viral titer in each supernatant was detected at 48 HPI by micro-antigen focus assay. Mean with SEM for the triplicate samples is shown. (F) 10⁴ ffu of the selected rJEVs were subcutaneously inoculated into one-day old chickens (n = 6 per group). The viral titer in collected plasma was detected at 60 HPI and measured by micro-antigen focus assay. A horizontal dot line represents an individual animal and mean of a group respectively. Error bars indicate SEM. A horizontal dot line indicates the detection limit. The statistical analysis was determined by one-way ANOVA followed by Dunnett’s Multiple Comparison Test utilizing rGIII/ GI NS1-3 virus as a control. A significant difference is indicated by an asterisk (B-E) or shown as a red dot (F) (P< 0.05).

Fig 8. The contribution of NS2B/NS3 substitutions on replication advantage of GI virus in amplifying hosts.

(A) The schematic diagram displayed the location of GI virus-specific NS2B/NS3 substitutions. (B-E) The single, double, or triple substitutions were introduced into NS2B/NS3 proteins of rGIII viruses. Single NS2B or NS3 substitutions with green or blue color. Double or triple substitutions with cyan or dark blue color. The rGIII, rGI, and rGIII/ GI NS1-3 viruses with white, red, and yellow color. The mutant rGIII viruses infected C6/36 (B), VERO (C), PK-15 (D), and DF-1 (E) cells at 0.5 of MOI and replicated at 28°C (C6/36) or 41°C (VERO, PK-15 and DF-1). The viral titer in the supernatant was detected at 48 HPI by the micro-antigen focus assay. Mean with SEM of the triplicates was displayed. (F) 10⁴ ffu of the rJEVs were subcutaneously inoculated into one-day old chickens (n = 6 per group). The viral titer in plasma was detected at 48 HPI by the micro-antigen focus assay. A dot and horizontal line represent an individual animal and mean of a group. Error bars indicate SEM. A dotted line indicates the detection limit. The statistical analysis was calculated with one-way ANOVA followed by Dunnett’s Multiple Comparison Test utilizing the control of rGIII virus. A significant difference was indicated as an asterisk (B-E) or shown as a red dot (F) (P< 0.05).

Fig 9. The conformational location of GI virus-specific substitutions on NS2B/NS3 proteins.

(A) The homologous modeling of GI virus NS2B/NS3 proteins was carried out with SWISS-MODEL [47] by utilizing the template structure of MVEV NS2B/NS3 proteins (PDB: 2WV9). The 49 to 131 residues of NS2B protein, protease domain of NS3 protein, and the helicase domain of NS3 protein are highlighted with pink, green, and blue. The linker domain connecting proteinase and helicase domains of NS3 protein is colored orange. All GI virus-specific substitutions on NS2B/NS3 proteins are highlighted as red spheres.