Virulence of Japanese Encephalitis Virus Genotypes I and III, Taiwan.
In Japan, the confirmed case incidence of JEV suddenly decreased from 20-50 cases each year during 1980-1990 to <10 cases after 1992 (2). This decrease may be related to implementation of JEV vaccine in Japan but also to less virulent GI JE viruses circulating there (3-6). A similar decrease was not seen in the other countries where genotype replacement had also occurred in recent years (7). In Taiwan, JEV GI was first detected in 2008 and became the island-wide dominant circulating genotype within a year (8,9), which provided an excellent opportunity to study the transmission dynamics and pathogenicity of these 2 JEV genotypes.
A mouse model showed that the pathogenic potential is similar among different JEV genotypes (10). However, the pathogenic difference between GI and GIII virus infections among humans remains unclear. Endy et al. reported that the proportion of asymptomatic infected persons among total infected persons (asymptomatic ratio) is an excellent indicator for estimating virulence or pathogenicity of dengue virus infections among humans (11). We used the asymptomatic ratio method for a study to determine if GI JEV is associated with lower virulence than GIII JEV among humans in Taiwan.
JEVs were identified in 6 locations in Taiwan during 1994-2012 (online Technical Appendix Figure, panel A, https:// wwwnc.cdc.gov/EI D/arti cl e/23/11/16-1443-Techapp1. pdf). GIII viruses were the only known circulating JEVs in Taiwan before 2009 (online 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 period (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 [greater than or equal to] 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 [less than or equal to] 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 (online 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).
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 [greater than or equal to] 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 [+ or -] 1.25:2.82 [+ or -] 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).
We thank the study subjects and the nurses of the Mennonite Christian Hospital and the Tungs' Taichung MetroHarbor Hospital for their assistance with the study subjects and specimen processing.
This work was supported by the Ministry of Science and Technology, R.O.C. (research grant NSC 102-2321-B-005-019).
Dr. Fan is a postdoctoral researcher at Graduate Institute of Microbiology and Public Health, National Chung Hsing University, Taiwan. Her research interests focus on the host tropism of flavivirus and the underlying mechanism(s) of genotype replacement of Japanese encephalitis virus.
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Address for correspondence: Shyan-Song Chiou, Graduate Institute of Microbiology and Public Health, National Chung Hsing University, 250 Kuo Kuang Rd, Taichung 40227, Taiwan; email: email@example.com
Author affiliations: National Chung Hsing University, Taichung, Taiwan (Y.-C. Fan, J.-W. Lin, S.-Y. Liao, J.-M. Chen, Y.-Y. Chen, C.-M. Chen, Y.-T. Ko, C.-C. Chang, S.-S. Chiou); National Defense Medical Center, Taipei, Taiwan (H.-C. Chiu); Tri-Service General Hospital (H.-C. Chiu); Taiwan Mennonite Christian Hospital, Hualien, Taiwan (C.-C. Shih); Tungs' Taichung MetroHarbor Hospital, Taichung (C.-M. Chen); National Dong Hwa University, Hualien (R.-Y. Chang); National Taiwan University, Taipei (C.-C. King); Chang Gung University, Taoyuan, Taiwan (W.-J. Chen)
 These authors contributed equally to this article.
Caption: 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 calculate JEV genotype-specific asymptomatic ratios. Horizontal lines indicate mean; error bars indicate SEM. JEV, Japanese encephalitis virus.
Table 1. Descriptive characteristics of populations for study of virulence of JEV genotypes I and III, Taiwan * Region Characteristic Changhua Taipei Pingtung Miaoli Taichung Year 1994 1995 1999 2000 2010 Circulating JEV GIII GIII GIII GIII GI No. participants 795 886 571 274 510 No. (%) vaccinated 36 41 41 33 48 Average age, 42.8 40.9 47.1 51.3 49.6 y ([double dagger]) Male sex, % 45 52 47 54 50 Region p Characteristic Hualien Taichung Hualien value ([dagger]) Year 2010 2012 2012 ND Circulating JEV GI GI GI ND No. participants 754 527 300 ND No. (%) vaccinated 48 49 46 <0.05 Average age, 48.1 52.0 49.2 <0.05 y ([double dagger]) Male sex, % 48 52 49 >0.05 * JEV, Japanese encephalitis virus; ND, no data. ([dagger) Analyzed by using 1-way analysis of variance. ([double dagger]) Range 40.9-52.0 y. Table 2. Estimated incidence of infection and asymptomatic ratios of JEV genotypes I and III among study populations, Taiwan * Region Characteristic Changhua Taipei Pingtung Year 1994 1995 1999 No. specimens 966 (171) 974 (88) 638 (67) (incidence of infection) NT positive, % 62 69 60 IgM positive, % (A) 4.91 0.90 1.93 Seroconversion + [greater 0.58 1.13 0 than or equal to] 4-fold increase in NT titer, % ([double dagger)] (B) Incidence of infection, % 5.49 2.03 1.93 (C = A + B) Population ([section]) 483,766 2,643,221 909,778 (D) Predicted no. infections 26,559 53,657 17,559 (E = C x D) Confirmed cases (F) 4 3 2 ([paragraph)] Asymptomatic ratio (G = 1/6,640 1/17,886 1/8,779 F/E) Asymptomatic ratio, 95% 1/5,102- 1/15,408- 1/8,000- CI 1/8,000 1/21,322 1/9,709 Characteristic Miaoli Taichung Hualien Year 2000 2010 2010 No. specimens 411 (137) 656 (146) 905 (151) (incidence of infection) NT positive, % 58 65 75 IgM positive, % (A) 1.10 0.98 2.25 Seroconversion + [greater 0.73 1.37 1.32 than or equal to] 4-fold increase in NT titer, % ([double dagger)] (B) Incidence of infection, % 1.83 2.35 3.57 (C = A + B) Population ([section]) 559,703 598,186 339,659 (D) Predicted no. infections 10,243 14,057 12,126 (E = C x D) Confirmed cases (F) 1 2 2 ([paragraph)] Asymptomatic ratio (G = 1/10,243 1/7,029 1/6,063 F/E) Asymptomatic ratio, 95% 1/8,065- 1/6,329- 1/5,348- CI 1/13,947 1/7,874 1/6,993 Region Characteristic Taichung Hualien p value ([dagger]) Year 2012 2012 ND No. specimens 632 (105) 375 (75) ND (incidence of infection) NT positive, % 72 66 <0.05 IgM positive, % (A) 3.23 3.67 <0.05 Seroconversion + [greater 0.95 1.33 >0.05 than or equal to] 4-fold increase in NT titer, % ([double dagger)] (B) Incidence of infection, % 4.18 5.00 <0.05 (C = A + B) Population ([section]) 593,780 337,382 ND (D) Predicted no. infections 24,820 16,869 ND (E = C x D) Confirmed cases (F) 2 2 ND ([paragraph)] Asymptomatic ratio (G = 1/12,410 1/8,435 >0.05 F/E) Asymptomatic ratio, 95% 1/10,846- 1/7,299- ND CI 1/14,493 1/10,010 * Serum samples were collected after the Japanese encephalitis season (May-September); multiple serum samples were collected from some participants. JEV, Japanese encephalitis virus; ND, no data; NT, neutralizing antibody. ([dagger]) Analyzed by using one-way analysis of variance. ([double dagger]) Among the IgM-negative population, JEV infection was estimated by the observation of an increase in the NT titer, including seroconversion and [greater than or equal to] 4-fold increase in the NT titer among subjects with multiple serum samples. ([section]) In Taichung, only residents of the districts around the serum-collecting hospital were included. ([paragraph]) According to the official report of the Taiwan Centers for Disease Control.
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|Author:||Fan, Yi-Chin; Lin, Jen-Wei; Liao, Shu-Ying; Chen, Jo-Mei; Chen, Yi-Ying; Chiu, Hsien-Chung; Shih, Ch|
|Publication:||Emerging Infectious Diseases|
|Date:||Nov 1, 2017|
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