JEVs were identified in 6 locations in Taiwan during 1994–2012 (Technical Appendix Figure, panel A). GIII viruses were the only known circulating JEVs in Taiwan before 2009 (Technical Appendix Figure, panel B). A genotype shift was complete by 2009, and since then, all JEV isolates in Taiwan have evolved from GI viruses (8). To investigate differences in the virulence of GIII and GI viruses in human infections, we conducted a subcohort and cross-sectional combined study to determine the JEV asymptomatic infection ratio. We used serum panels collected during the GIII JEV endemic period (1994–2000 [12,13]) and during the GI JEV endemic pe-riod (2010–2012).

The institutional review boards of the Mennonite Christian Hospital and the Tungs’ Taichung Metroharbor Hospital reviewed and approved clinical protocols for the serum sample collection. Approximately 10% of total specimens were paired serum samples. We used the plaque reduction neutralization test (PRNT) and IgM antibody-capture ELISA (MAC-ELISA) to determine the infection status of each serum specimen (14). We tested serum panels collected before and after 2009 by using MAC-ELISA and PRNT and used viral antigens and viruses derived from the GIII-T1P1 and GI-YL2009–4 strains, respectively.

We further tested all neutralizing and IgM antibody-positive specimens by using GI YL2009–4, GIII T1P1, and dengue virus 2 (DENV-2) viral antigens to determine the genotype-specific infection status and to exclude false-positive results possibly caused by DENV infection. We used SAS version 9.4 (SAS Institute Inc., Cary, NC, USA) for statistical analyses.

We collected 5,557 specimens from 4,617 participants (Table 1). The average participant age by year ranged from 40.9 to 52.0 years (p<0.05); overall, 49.6% of participants were male and 50.4% female (p>0.05). The proportion of persons receiving JEV vaccinations increased over time from 33% to 49% (p<0.05).

The positivity rate for neutralizing antibody, elicited by natural infection or immunization in the past, differed significantly, ranging from 58% to 75% among the 8 serum panels (p<0.05) (Table 2). A JEV infection that had been acquired recently was indicated by the presence of IgM among the seronegative population, seroconversion of the neutralizing antibody, and a ≥4-fold increase in the neutralizing antibody titer among the seropositive population. For the 8 serum panels, the IgM positivity rate ranged from 0.90% to 4.91% (p<0.05), and the proportion of seroconversion and a ≥4-fold increase in the neutralizing antibody titer ranged from 0% to 1.37% (p>0.05). The incidence rate of JEV infection ranged from 1.83% to 5.49% (p<0.05) in the 8 serum panels.

We calculated the asymptomatic ratio by dividing the number of confirmed JE cases by the number of JEV infection cases. The number of confirmed JE cases (Table 2) was obtained from the CDC National Infectious Disease Statistics System, Taiwan (https://nidss.cdc.gov.tw/en/Default.aspx), which reported 1–4 cases/year in the regions from which the 8 serum panels were collected (Table 2). The number of JEV infection cases, calculated by multiplying the total population by the incidence of JEV infection, ranged from 10,243 to 53,657 in the 8 serum panels. The asymptomatic ratios (95% CI) for GIII virus infection were 1:6,640 (1:5,102–1:8,000) for Changhua County in 1994; 1:17,886 (1:15,408–1:21,322) for Taipei City in 1995; 1:8,779 (1:8,000–1:9,709) for Pingtung County in 1999; and 1:10,243 (1:8,065–1:13,947), for Miaoli County in 2000. The asymptomatic ratios for GI virus infection were 1:7,029 (1:6,329–1:7,874) for Taichung City and 1:6,063 (1:5,348–1:6,993) for Hualien County in 2010; ratios were 1:12,410 (1:10,846–1:14,493) for Taichung City and 1:8,435 (1:7,229–1:10,010) for Hualien County in 2012.

We applied the log-linear Poisson regression model to adjust the asymptomatic ratio by the number of patients who had encephalitis and were infected by other pathogens, and also by age, sex, and vaccination status (Technical Appendix Table) (15). The results revealed that non–JEV-specific encephalitis symptoms (fever, headache, convulsion, and seizure) (intercept, p<0.05) influenced the calculation of asymptomatic ratios but not age, sex, or vaccination status (p>0.05).

Figure

Figure. JEV genotype-specific asymptomatic ratios for 8 serum panels collected during the GIII- and GI-endemic periods (1994–2000 and 2010–2012) in Taiwan. The adjusted asymptomatic ratios estimated from genotype-representing populations were included to...

We calculated the overall GI JEV–­ and GIII JEV–specific asymptomatic ratios by using the adjusted asymptomatic ratios of the 8 serum panels collected during the GI JEV– and GIII JEV–endemic periods (Figure). The GI-specific asymptomatic ratio was 1:15,378 (1:6,168–1:24,588) and the GIII-specific ratio was 1:18,842 (1:6,624–1:30,260); these ratios were not significantly different (p>0.05).

The possible effects of JEV genotype replacement remain unclear, particularly in terms of disease burden, virulence, vaccine efficacy, and policy decisions. In this study, we combined the IgM seropositivity rate, the proportion of seroconversion, and paired samples displaying a ≥4-fold increase in the neutralizing antibody titer to calculate incidence of the infection (Table 2). The results showed that the incidence of JEV infection fluctuated over years and in different regions in Taiwan. Nevertheless, genotype replacement had no significant effect on this fluctuation (GI:GIII = 3.78 ± 1.25:2.82 ± 3.18; p>0.05).

The dramatic decline observed in the number of clinical JE cases after the genotype replacement from GIII to GI in Japan suggested that GI viruses were less virulent than GIII viruses (3–6). However, we found that the asymptomatic ratio of GI and GIII JEV infections was similar, indicating equal virulence of GIII and GI JEVs. These results were also supported by the mouse virulence and neurovirulence experiments and disease burden estimation (10).