Seroepidemiology of Epstein-Barr virus and herpes simplex virus-1 in Taiwan

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1 Seroepidemiology of Epstein-Barr virus and herpes simplex virus-1 in Taiwan 1,3 Chao-Yu Chen, 1, 3 Jen-Hsiang Shen, 2 Yu-Chen Huang MD 1 College of Medical, Chang Gung University, Taoyuan, Taiwan 2 Pediatric Infection Department, Chang Gung Memorial Hospital, Taoyuan, Taiwan 3 Chang Gung Memorial Hospital, Taoyuan, Taiwan *CY Chen and JH Shen have contributed equally. Address correspondence to: Yu-Chen Huang MD, ychuang@cgmh.org.tw Running title: Seroepidemiology of EBV and HSV1 in Taiwan Funding: Funding and support were received from Dr. Yu-Chen Huang, Chang Gung Memorial Hospital and Medical Foundation in Memory of Dr. Deh-Lin Cheng. No other financial support had been received. Keyword: Epstein-Barr virus, herpes simplex virus type 1, seroprevalence, seroepidemiology Abstract Background: Both Epstein-Barr virus (EBV) and herpes simplex virus type 1 (HSV-1) are common pathogens associated with many human diseases. There have been no reports on the seroepidemiology of whole population for both viruses in Taiwan. Method: Residual sera from a pooled serum samples collected from 3552 subjects during a public health survey in 2007 were selected for this study. We used ELISA methods to detect EBV viral capsid antigen (VCA) IgG and HSV-1 IgG. Demographics and personal health data obtained originally were used for analysis. Result: A total of 1411 serum samples were included. Overall seroprevalence for EBV was 82%, and for HSV-1 was 59%. People in Taiwan had EBV infection in early life, with a seropositive rate of >50% at the age of 2 years, >80% at the age of 5-9 years and >90% for those beyond 10 years of age. Increasing age, more education, living in non-rural and seropositive for HSV-1 were the independent predictors of EBV seropositivity. In contrast, people in Taiwan had HSV-1 infection later in life and the rate increased slowly and steadily. The seropositive rate was up to around 30% for those less than 5 years of age, >50% for those aged >15 years and > 90% for those aged > 40 years. The indepenedent predictors of HSV-1 seropisitivity included incraesing age, less education and seropositive for EBV. Conclusion: In Taiwan, people had EBV infection early in life and the rate incraesed quickly; in contrast, people had HSV-1 infection later in life and the rate increased slowly and steadily. Introduction Epstein Barr Virus is known as the most common pathogen, and related to lot s of diseases: infectious

2 mononucleosis, nasopharyngeal carcinoma, hematological malignancies, etc.[1-4] Many also believe there is a relationship between EBV, SLE and multiple sclerosis.[5, 6] Seroprevalence of EBV is an old field in epidemiological studies, but it remains important in current Taiwan. Some research indicated that there are differences of epidemiological pattern between developing and developed country.[7] There also studies in Japan and Sweden showed changing in Seroprevalence pattern in harpers virus due to society and economical status differ from year by year.[8, 9] The last epidemiologic study of EBV was done in 1984[10] and Taiwan had already passed through many social changes. In old 1984, Seroprevalence of EBV in Taiwan is still like those in developing country. Due to most people had their primary infection younger than 4 years old, symptomatic infection cause by EBV like infectious mononucleosis is not that common. But Taiwan had gradually become a developed country, it may result in epidemiological changes of those common diseases. Herpes simplex type 1 had similar infection route as EBV, and it is capable of infecting most visceral organs and mucocutaneous areas. First symptomatic episode of HSV-1 infection usually presents as gingivostomatitis or pharyngitis. It can remain latent in human body without symptoms, and can stay infectious during patients lifetime. [11] However, we still had no epidemiological data in Taiwan until now. Seroepidemiological study is an important way to find out age-specific prevalence of infectious disease and may also reflect social economic status. Therefore, we aim to find out current epidemiological data, risk factors, offering references for clinical physician and publish health researchers. We will also compare Seroprevalence of EBV and HSV1 in Taiwan with other countries in the world in this study. Material and method Study subject and Ethic statement Our study enrolled 1411 serum samples from residual serum of one large public health survey in The original plasma sample was collected during August and October of 2007 under a Taiwan CDC-funded survey for investigating the seroprevalence of several vaccine preventable diseases. Total 3554 blood sample was collected from over 10 different hospitals and public health centers in Taiwan. Original inform consent is complete, and Serum from those who accept residual serum using for future studies was stored at -80 laboratory refrigerator. Our study was approved by original program conductor in 2007 with written form consent, and also reviewed in ethical committee of Chang Gung Memorial Hospital. We also obtained original questionnaire for demographic data and personal history. The demographic data of our study group list below: gender distribution is female(58.8%) and male(41.2%); Rural area(73.9%) and Urban(26.0%); living in North Taiwan(62.2%), South Taiwan(33.7%), Others(4.2%), age below 5 years old(38.5%). According to past epidemiologic study experience, it is known that age<5 is a peak age period of primary infection of EBV and HSV1, so we set each 1 year as an age group for under 5 years old children, and 5-9, 10-14, 15-19, 5 years a group, than each 10 year was pooling to a group after 20 years old.

3 Laboratory and statistic methods We use commercial ELISA kit (EUROIMMUN(Lübeck, Germany)) for our serology test to detect seropostivity of anti-ebv VCA IgG and anti-hsv1 VCA IgG. 100μg serum was first incubated for 30 minutes in room temperature. After three times wash cycles, enzyme conjugate was added and incubated for another 30 minutes. Then, we went through another three times wash cycles and add substrate. Finish 15 minutes incubation, stop solution was added and photometric measurement was made at 450nm. Due to two standard known kit, we can do quantitative analysis for each serum. Serum that concentration higher than 22 IU/ml is consider as positive. Then we use SPSS statistic software (SPSS Statistics 17.0; 2008 SPSS Inc, Chicago, IL) for statistics analysis, Chi-square or Mann-Whitney test was performed when we compare difference in seroprevalence. We have also done backward logical regression for multiple risk factor analysis. In our studies, urban and rural classfication was according to original administrative divisions in 2007(Two special municipality include Taipei city and Kaoshiung city plus five provincial city include Keelung city, Hsiuchu city, Taichung city, Chiayi city and Tainan city were defined as urban, and others as rural). Drinking history devide into three grade: less than twelve times drinking in lifetime, less than twelve times drinking in one year, and more than twelve times in one years. Educatinal status from under elementary school, elementary school, junior high, high school, university or college devided into five degrees. Smoking and marriage history only defined ever-smoked, ever-marriaged or not. Factors that above mentioned and other medical cormobilities(dm, hepatitis, chronic cough) was input into our logic regression. Only significant factors will discuss in our result. Result The seroprevalence of EBV and HSV1 in Tawan, 2007 is shown in Table 1. Total seroprevalence of EBV in our study is 82.28%, whereas that of HSV-1 is only 59.25%.There s over 90% people in Taiwan have anti-ebv VCA IgG over years old. However, there s also over 90% seropostivity to anti-hsv-1 IgG after ageband years olds. It showed most peoples in Tawan have ever infected to HSV-1 in their life time. The age-specific seroprevalence curve of EBV, comparing 2007 and 1984 in figure 1 showed The EBV infection period is slowly delayed. In 1984, the curve is quickiy reach a peak and over 80% children had alreay infected by EBV in 4-5 years old. Comparing to the curve of 1984, the curve of 2007 is mild shifted to right. However, there s no signigicance(p>0.05) between each age group. Figute 2 is age-specific seroprevalenc curve of HSV1, It s much smoother than EBV. Unlike most EBV infection period is concentrated in young age, the main infection period is not that clear; the most steep part of slope may happen in adolescent. Besides, we can see a decrease seroprevalence in 0-2 years old period, the same situation may see in EBV but in 0-1 years period. Result of logic regression for risk factor evaluation is shown in figure 2, 3. Age is obvious a main factor in seroprevalence rate. However, there s other factor that determine who will get infection or not, early or late.

4 Living in rural is seem like a protective factor, odd ratio is 0.557(compared to urban). Besides, high educational status(odd ratio:1.706) and those who have ever infected by HSV1 (odd ratio: 2.742) is much easier get infected by EBV. There is no sexual difference in seroprevalence of EBV. For HSV-1, education status is no more a risk factor, but a protective factor(odd ratio: 0.818). Drinking is a risk facor(odd ratio: 1.793), and anti-ebv igg seropositivity have also effect on HSV1 infection(odd ratio: 2.917). Marriage status is at the border line of 95% confidence interval(95% CI of odd ratio is [0.993,4.212], and p-value= 0.052). There s no sexul or rural/urban difference in seroprevalence of HSV1. Besides, residence(living in North or south of Taiwan), smoking history, other medical comorbidity (DM, hepatitis, chronic cough) show no influence on both EBV and HSV1 infection. Discussion This study is fisrt seroepidemiological study focus on EBV and HSV-1 of whole age-group survey in entire Taiwan. In 1984 study of EBV[10], they only enrolled children in Taipei, so it may lead to some bias. The serum sapmle of our study is from residual serum from a big public health program conducted by CDC TAIWAN. Althogh age distribution can t reflect toal population and gender distribution. However, age-specifc seroepidemiological curve may reflect each age group seroantibody status and we can know how many people have ever infected by EBV and HSV-1 before. According to our result, EBV and HSV-1 infection are still very common in Taiwan. Most people had ever infected by EBV, HSV-1 in their lifetime. There s over 90% seroprevalence in EBV after years old. Current peak age-group of primary infected by EBV may be pre-school to early elemtary years, and it is delayed if comaring to those in This finding is compatable with Japan which showed significant decrease prevalence of EBV in early life.[9] However, the decreasing rate of early infection to EBV is still far away from Japan. The current seroepidemiological pattern in Taiwan is similar to other asia countries except Japan. The seroepidemiological difference may due to differnet public health policy, hygeine behavior or other socialeconomical activity. Comparing seroepidemiology between asia and europe was organized in table 3. [7-9, 12-18]In Asia, most countrys(except Japan) had early infected to EBV, unlike people in western europe had their first infection in adolescent age. There are differences between them, and lead to a hypothesis that socialeconomical behavior may do more than race or region. Cecilia study in Brazil [12]also support that EBV infection may more fequent in younger children from families with low socialeconomical status. However, our multiple regression found living in rural is relative protective method(odd ratio:0.557) comparing to people lived in urban. The reason may be due to that most children living in urban was sent to kindergarden or other childcare earlier in Taiwan. Therefore, country differences about peak age period of primary infection to EBV should be multifactorial induced rather than a single factor problem. On the other hand, country difference also exists in seroepidemiology of HSV-1. We compare age specific

5 seroprevalence of Taiwan with China, USA and Japan.[19-21] In figure 3, seroprevalence in China rose quickly in early life, then Taiwan, USA, and Japan. Both China and Taiwan had over 80% seroprevalence in years old, wheras there s only 60-70% in USA and japan. HSV1 had similar transmission way as EBV, and many study had already pointed that the seroepidemiological pattern is similar to EBV, too. The corelationship is also found in our study, people who had infected by HSV1 had higher odd ratio in seropositve of EBV(odd ratio:2.742), vice versa(odd ratio:2.917). In our multiple risk factor evaluation, we use backward logcal regression to offer more significant result and adjusted by age. For EBV, living in urban, higher educational status and anti-hsv1 IgG seropostive are risk factors. There s no gender difference in our study. Study in Bangladesh [13] also showed no gender difference while some studies indicate some difference to limited age group(10-14 years old)[14]. There is also no gender difference in HSV seroprevalence in our study. Others risk factors for HSV1 as low educational status, marriage status, drinking hostory, EBV seropositivity was found in Taiwan. Limtied study disscussed about this part, question like drinking history in HSV1 is a physiological problem or socicalogical behavior relaed may need further research to answer. Limitation This study is based on the past public health survey of CDC TAIWAN in Both serum sample and questionnaire come from that study. Therefore, we can t design our own questionnaire to find out if there s other factors influencing on seroprevalence of EBV and HSV1. Besides, personal choice to receive public health survey or not will affect age and gender distribution in our study. Although we do have gender and age difference from current distribution, the age specific seroprevalence curve can still offer enough information on epidemiology of Taiwan. Acknowledgement This study was support by Chang Gung Memorial Hospital and Medical Foundation in Memory of Dr. Deh-Lin Cheng. Greatly thank for their Funding. We sincerely thank to the original investigators of public health study in 2007, Dr. Lin TY and Chen CJ offer residual serum, original questionnaire and inform consent to our investigation. Also, special thanks to Miss. Yi-Chen Kuan, Miss. Yun-Hsuan Don, and Miss. Hsin-Yi Chang for their generous assistance in our study. References 1. Chan, C.W., et al., Epstein-Barr virus-associated infectious mononucleosis in Chinese children. Pediatr Infect Dis J, (11): p Hsu, W.L., et al., Familial tendency and risk of nasopharyngeal carcinoma in taiwan: effects of covariates on risk. Am J Epidemiol, (3): p

6 3. Chien, Y.C., et al., Serologic markers of Epstein-Barr virus infection and nasopharyngeal carcinoma in Taiwanese men. N Engl J Med, (26): p Kawa, K., Epstein-Barr virus--associated diseases in humans. Int J Hematol, (2): p Lu, J.J., et al., Association of Epstein-Barr virus infection with systemic lupus erythematosus in Taiwan. Lupus, (3): p Lucas, R.M., et al., Epstein-Barr virus and multiple sclerosis. J Neurol Neurosurg Psychiatry, (10): p Kangro, H.O., et al., Seroprevalence of antibodies to human herpesviruses in England and Hong Kong. J Med Virol, (1): p Svahn, A., et al., Changes in seroprevalence to four herpesviruses over 30 years in Swedish children aged 9-12 years. J Clin Virol, (2): p Takeuchi, K., et al., Prevalence of Epstein-Barr virus in Japan: trends and future prediction. Pathol Int, (3): p Tsai, W.S., et al., Seroepidemiological study of Epstein-Barr virus infection in children in Taipei. Zhonghua Min Guo Xiao Er Ke Yi Xue Hui Za Zhi, (2): p Nahmias, A.J. and B. Roizman, Infection with herpes-simplex viruses 1 and 2. II. N Engl J Med, (14): p Figueira-Silva, C.M. and F.E. Pereira, Prevalence of Epstein-Barr virus antibodies in healthy children and adolescents in Vitoria, State of Espirito Santo, Brazil. Rev Soc Bras Med Trop, (5): p Haque, T., et al., Seroepidemiological study of Epstein-Barr virus infection in Bangladesh. J Med Virol, (1): p Morris, M.C., et al., Sero-epidemiological patterns of Epstein-Barr and herpes simplex (HSV-1 and HSV-2) viruses in England and Wales. J Med Virol, (4): p Ozkan, A., et al., Seropositivity of Epstein-Barr virus in Eastern Anatolian Region of Turkey. Asian Pac J Allergy Immunol, (1): p Leogrande, G. and E. Jirillo, Studies on the epidemiology of child infections in the Bari area (south Italy). VII. Epidemiology of Epstein-Barr virus infections. Eur J Epidemiol, (4): p Ternak, G., et al., [Sero-epidemiologic study of Epstein-Barr virus markers in patients without mononucleosis at a department for infectious diseases]. Orv Hetil, (50): p Crowcroft, N.S., et al., Epidemiology of Epstein-Barr virus infection in pre-adolescent children: application of a new salivary method in Edinburgh, Scotland. J Epidemiol Community Health, (2): p Beydoun, H.A., et al., Socio-demographic and behavioral correlates of herpes simplex virus type 1 and 2 infections and co-infections among adults in the USA. Int J Infect Dis, Suppl 3: p. e Doi, Y., et al., Seroprevalence of herpes simplex virus 1 and 2 in a population-based cohort in Japan. J Epidemiol, (2): p Lin, H., et al., Herpes simplex virus infections among rural residents in eastern China. BMC Infect Dis, : p. 69. Seroprevalence of EBV Seroprevalence of HSV-1 Age Positive/Tested Seroprevalence(%) 95% CI of Seroprevalence Positive/Tested Seroprevalence(%) 95% CI of Seroprevalence <1 9/ [9.24, 36.91] 6/ [3.54, 27.23] 1 27/ [20.35, 39.65] 10/ [4.49, 17.73] 2 67/ [43.95, 61.56] 18/ [8.02, 20.32]

7 3 64/ [59.98, 79.15] 29/ [21.85, 41.20] 4 58/ [58.95, 79.14] 20/ [14.51, 33.11] / [76.82, 90.75] 52/ [37.42, 56.28] / [85.44, 97.17] 43/ [36.35, 57.13] / [90.66, 98.91] 61/ [43.78, 62.30] / [96.11, 100] 86/ [61.70, 78.14] / [97.69, 100] 109/ [78.17, 90.83] / [95.21, 100] 94/ [89.26, 98.74] / [91.31, 99.90] 90/ [96.72, 100] / [96.08, 100] 122/ >70 96/ / Total 1161/ [80.29, 84.28] 836/ [56.68, 61.82] Table 1. Seroprevalence of EBV and HSV1 on 2007 in Taiwan Figure 1. Age specific seroprevalence curve of EBV: comparing 1984 and 2007 Figure 2. Age specific seroprevalence curve of HSV1 in 2007 EBV multiple risk factor analysis HSV1 multiple risk factor analysis Significant factors Sig.* Odd ratio 95% CI of Odd ratio Significant factors Sig.* Odd ratio 95% CI of Odd ratio Age 0.000** [1.017,1.061] Age 0.000** [1.049,1.110] Rural/Urban 0.046* [0.313,0.990] Educational status 0.029* [0.683,0.980]

8 Educational status 0.000** [1.361,2.139] Drinking history 0.004** [1.207,2.662] Anti-HSV1 IgG 0.001** [1.503,5.003] Marriage status [0.993,4.212] seropositivity Anti-EBV IgG seropositivity 0.001** [1.554,5.475] Table 2. Multiple risk factor analysis of EBV (only significance factors was shown) Area Country(study year) Seroprevalence(age band) Europe UK(1993) 61%( y/o) 80% ( y/o) UK( ) 54%(10-14 y/o) 72%(15-19 y/o) Turkey(2003) 96.3%(0-9 y/o) 100%(10-19 y/o) Sweden(1997) 62%(9-12 y/o) Hungary(1995) 80%(5-10 y/o) Italy(1993) 80.2%(5-7 y/o) 81.9%(8-10 y/o) Scotland(1998) 56%(6-8 y/o) Asia Taiwan(2007) 83.8%(5-10 y/o) 91.3%(10-15 y/o) 94.8%(15-20 y/o) Hongkong(1993) 95%( y/o) 100%( y/o) Tailand(2001) 92.3%(6-8 y/o) 96.6%(9-12 y/o) 97.6%(12-14 y/o) Japan( ) 59%(5-7 y/o) 86%(10-12 y/o) Table 3. A comparison on seroprevalence of EBV between Asia and Europe

9 Figure 3. Age specific seroprevalence curve of HSV1 comparison in four different country: Taiwan, China, USA and Japan (*age group for first spot of Japan is years old)