Transovarial Transmission and Dengue Virus Serotypes in Aedes Aegypti in Kupang

Dengue is an endemic disease in almost all provinces in Indonesia, including NTT pro-vince and Kupang City. DHF prevention and control efforts need to be accompanied by understanding of the epidemiological and entomological aspects. This study proves the presence of transovarial transmission and serotypes of Dengue virus in Ae. aegypti in areas with different endemics in Kupang City. The study was conducted in endemic, sporadic and Dengue-free villages. Samples from each location were 30 head squash Ae. aegypti females for examination of Dengue virus by IHC methods and 10 Ae. aegypti females for serotypes check of Dengue virus with RTPCR. Data presented in tables and images then analyzed descriptively. This study finds that transovarial infection rates 9.2% with serotype Dengue virus-1. As many 97.5% of respondents have heard about Dengue by most cadres resources (24.5%) and health workers (24%). Transovarial infection is found in endemic, sporadic and free areas so that the Dengue vector control needs to be done in the three regions with different endemicity.


Introduction
Dengue Hemorrhagic Fever (DHF) is found in tropical and subtropical regions. Data from around the world shows that Asia ranks first in the number of Dengue fever patients annually. Meanwhile, starting from 1968Meanwhile, starting from until 2009, WHO notes that Indonesia as the country with the highest Dengue cases in Southeast Asia. Dengue case was first reported in Indonesia in 1968 in Surabaya and Jakarta with 58 cases and Case Fatality Rate (CFR) reaching 41%. Since then, cases of Dengue fever was spread throughout Indonesia, from 2 provinces and 2 cities in 1968 to 32 provinces (97%) and 382 cities / counties (77%) in 2009. The cases of Dengue also increased from 58 cases in in 1968 to 158 912 in 2009(Pusat Data Surveilans Epidemiologi, 2010. The incidence of Dengue fever continues to rise and is now an endemic disease in almost all provinces.
Dengue case in East Nusa Tenggara province always exists and occupies the lowest the city of Kupang.

Method
This study was an observational study using a descriptive study through crosssectional design where the study variables were studied in the same period. The study was conducted in endemic areas (Oebobo village Oebobo District and Alak Village, Alak District), sporadic area (South Oesapa South Village, Kelapa Lima District) and free area (Fatukoa village, Maulafa District). Therefore, four villages in four districts in Kupang were involved in this study. Every survey was conducted in 30 houses per village.
Fieldwork was conducted to survey the knowledge and attitudes about Dengue and its prevention as well as for the collection of eggs of Aedes sp. The egg collection was done by mounting ovitrap which was contained 200ml glass painted black on the outside, filled with water 2/3, then mounted filter paper (ovitsrip) with a size of 5cm x 20cm full circle in the glass limited to surface water so ovistrip became moist. Coarse filter paper was mounted with hard section facing inward as mosquito breeding ground. Ovitrap was set inside or outside the house in a dark place that was expected to be potential spawning grounds for Aedes sp. Each house was fitted two pieces of ovitrap inside and outside the house. After three endemic areas of Dengue disease. Therefore, epidemiological aspects including mechanisms of transmission of Dengue virus (Dengue virus) is important aspect to learn. Transmission of Dengue virus can occur horizontally i.e. from transmission of Dengue virus from sick patients to mosquitoes and vice versa, as well as vertically (transovarial) i.e. the transmission of Dengue virus from female mosquitoes into the ovum in the uterus or transmission from male mosquitoes infected with Dengue virus to female mosquitoes which occurs during the venereal activities (Mardihusodo, 2007). This vertical transmission of Dengue virus causes the transmission of the Dengue virus will never stop because once mosquito is infected with Dengue virus, then the virus remains in the animal for the rest of its life and it is also found throughout the season (Angel, 2008;Rohani, 2008).
Until now there is no cure or right vaccine for Dengue disease. Therefore, in the prevention and control efforts incidence of DHF, it is necessary to understand the epidemiological aspects including climatic conditions, the vector density and the presence of transovarial transmission and serotypes of Dengue virus in Ae. Aegypti. This study aims to prove the presence of transovarial transmission and serotypes of Dengue virus in Ae. aegypti in endemic, sporadic and free of Dengue areas in  (2013) days, the water of ovitrap was checked and was taken after 1 week. Each place was done for 3 weeks or 3 repetitions.
The dried ovistrip with eggs was then separated according to location and was sent to a parasitology laboratory of Medical Faculty, Gadjah mada University to do rearing until the first offspring (F1) and the examination of the Dengue virus in Ae. Aegypti. The eggs became adult mosquitoes colonized up to expectations when there was a virus in eggs then they were replicated during the development of the egg to adult mosquitoes, making it easier to detect the presence of Dengue virus. Positive eggs Ovistrips were laid in the tray. After they were hatched into larvae, they were fed with meat of dried liver. Pupae emerged later were transferred to mosquito breeding nest and waited 1-2 days to become adult mosquitoes. Adult mosquitoes that had appeared were separated by location in a mosquito cage sized 20x20x20cm and then were maintained until the age of 7 days to be only given sugar water 10%. The samples for examination of transovarial transmission with IHC method were 30 Ae.Aegypti female mosquitoes. Therefore, 120 Ae. aegypti females were involved in four locations. While samples for examination serotype of Dengue virus in mosquitoes Ae.Aegypti method RTPCR were 10 mosquitoes Ae. aegypti females coming from 4 locations.
Source: Umniyati (2004) Figure 2. ISBC Method Scheme to Detect Antigen Virus in body tissue/cell The detection of Dengue virus in mosquitoes used Immunohistochemistry Streptovidin Biotin Peroxidase Complex (ISBPC) method by setting preparation of head squash which had been developed and standardized by Umniyati (2004). The steps were: preparations of head squash were made by pressing the head of Ae. aegypti aged > 7 days that had been killed and separated from the body on an object glass coated with Poly L Lysine and made per location; preparations were fixed with cold methanol -20 o C for 3-5 minutes, then stored at -20 o C if not immediately checked; washing the preparations under distilled water and then with Phosfat Buffer Saline (PBS) until it was stagnant; activities of Peroxidase Blocking Solution in a room temperature for 10 minutes or under tap water for 5 minutes; preparations were incubated in prediluted Horse Serum for 10 minutes at a temperature of 25 o C or the tray of damp then wrapped; Primary antibody commercial monoclonal antibody 1:10 that had been prepared was added as much as 100μl per preparation (until stagnant) and were then incubated in a moist tray at room temperature (25 o C) for 60 minutes or overnight in the refrigerator; further, the preparations were put in fresh PBS for 5 minutes; Biotinylated Universal Secondary Antibody (BUSA) as much as 100μl per slide was added, and then incubated at a temperature of 25 o C for 10 minutes or in a tray of moist, then the preparations were washed for 5 minutes with fresh PBS; the preparations were incubated in ready-to-use streptovidin/peroxidase complex reagent as much as 100μl for 10 minutes in a tray of moist, after the preparations were washed with PBS for 5 minutes; preparations incubated in 100μl DAB/preparations for 2-10 minutes (the thicker the longer the incubation preparation) at room temperature (25 o C); preparations were washed using tap water, then Mayer hematoxylin (counterstain) of 100μl was added and incubated for 5 minutes, then washed under tap water; The slide was dipped into 95% alcohol, then dipped into xylol and was cleaned for contrasting colors; The slide was dropped using mounting media and was then covered with a cover slip, then a dry slide was ready to be examined under a microscope at 400x and 1.000x distribution; brown preparation meant that it was positive with Dengue virus antigen, whereas pale blue preparation meant that it did not contain the Dengue virus antigen.
Dengue virus examination was done separately for each location and conducted only for female mosquitoes. Examination of Dengue virus with RTPCR method began with the isolation of viral RNA from the head of females Ae. aegypti using the High Pure Viral nucleid Acid Kit from Roche. Viral RNA obtained was stored in a temperature (-20 o C) prior to RTPCR.
Primary data included data on transovarial transmission rates of Dengue virus in Ae. aegypti females which were obtained by observation/direct examination in the laboratory with ISBPC method. Mosquitoes implied transovarial transmission when the virus antigen was detected by the formation of brown color on inspection method of ISBPC. No transovarial transmission in mosquito was shown when brown color was not formed; instead, the preparations slide became blue on the examination by the ISBPC method. Dengue virus serotype data was obtained by observation / inspection using RTPCR. The primary data was about people's behavior in elimination of DHF mosquito breeding sites was directly obtained through interviews. The collected data was processed and presented in the form of tables and pictures, and then analyzed descriptively.

Results and Discussion
Based on education, the majority of This research probes at 120 mosquito Aedes aegypti. The transovarial infection rates (TIR) in Kupang is 9.2%. The highest TIR which is found in endemic areas is 13.3% and in the free areas of Dengue fever, transovarial transmission is still found although their TIR is lower than in most endemic and sporadic areas as shown in Table 2. This study detects the presence of Dengue virus in the head squash of Aedes aegypti mosquitos using Immunohistochemistry method. It is said to be positive when the brown color on the head quash is mosquito Aedes aegypti formed. The presence of the Dengue virus is said to be negative when it does not form a brown color but instead blue color, as shown in Figure 1.
This study also examines the serotypes of the Dengue virus in Ae. aegypti which comes from Kupang and was only found Dengue virus serotype 1 and it is found in Oesapa (sporadic area) and Alak (endemic area  (Ditjen PP & PL, 2013).
This study shows that the Dengue virus which causes the Dengue disease in Kupang is the virus Den-1, and this study proves also that the virus Den-1 can be transovarially transmitted, which is passed down from mosquitos parents to descendants (in this study, the first offspring (F1)). Previous research in Malaysia has also proven that the Dengue virus has been even descended to the Ae. aegypti F5, and it is proved in Den-2 virus (Spiritual et al., 2008). Minimum infection rate (MIR) in the study is approximately 30‰ -45 ‰ in generation F1 -F5, and in F6 and F7 there is no Dengue virus (Rohani et al., 2008). Den-4 viruses can also be transovarially transmitted in Ae. aegypti females and males with a MIR of 10.5 ‰ (Da Cruz, 2014). Research in Thailand and Bantul even find that transovarial transmission in Ae. aegypti could happen to the virus Den-1, Den-2, Den-3 and Den-4 (Thongrungkiat 2011;Satoto, 2014).
In this study, the transovarial transmission of Dengue virus is only demonstrated in Ae. aegypti females, whereas in previous studies, it is also found in Ae. aegypti males, even minimum infection rate (MIR) which is higher in males than in females (Thongrungkiat et al., 2011). The finding shows that Ae. aegypti males can be infected with the virus from their females through venereal activities or male mosquitoes transmit Dengue virus to female mosquitoes during the reproduction (Thongrungkiat, 2011).
In this study, not all regions which are positive IHC virus are also positive at the time of inspection with RTPCR. This is because the mosquitoes used in the examination of IHC with RTPCR are different mosquitos. Therefore, the mosquitos used for RTPCR examination could be the ones without the Dengue virus. Other possibility is the sensitivity of IHC examination which is higher as compared to RTPCR examination, i.e. at a low titer of Dengue virus can be read by the IHC method but cannot be read by RTPCR. Dengue virus with RTPCR examination in this study is found in two regions (50%) which contain transovarial transmission. This is different from the research of Satoto (2014) who finds the 70.58% area contained transovarial transmission of Dengue virus in Ae. Aegypti.
Den-2 virus in the previous experimental studies can also be descended to the offspring, where the number of transovarial transmission and hatchability of infected eggs of Ae. aegypti is lower than uninfected mosquitoes in both the F0 and F1 (Satoto et al, 2013). This study is different from experimental research. In this study, transovarial transmission rate is only 9.2% while in experimental research is 82.2% for Ae. aegypti first offspring (F1) (Satoto et al, 2013). This difference is due to a variety of possibilities, among others, not all of Ae. aegypti can be infected by the Dengue virus. This is because their immune system in the body which ends up being a good barrier in case of infection by viruses or other microbes in the body and is also a barrier in the middle of the stomach and salivary glands of mosquitoes (Luplertlop, 2014). The barrier causes not all Dengue virus can live and multiply in the mosquito's body. Besides, temperature and humidity also affect transovarial transmission. Besides the viral strain and strain mosquitoes geography also affect the amount of transovarial transmission (Rohani, 1997). This study only proves the ability of Ae. aegypti in transmitting the Dengue virus transovarially (vertically), whereas previous studies have demonstrated that Ae. albopictus is capable of transmitting Dengue virus vertically namely Den-2 virus can be found in the larvae or Ae. albopictus that have not suck human blood (Cecilio, 2009). This proves that both Ae. aegypti and Ae albopictus are susceptible to the transovarial transmission of Dengue virus.
This study finds that transovarial transmission does not only occur in endemic areas, but also in sporadic and free areas of Dengue infection although the percentage number of transovarial transmission is higher in endemic areas. This shows that all regions in this study become the risk in the spread of Dengue fever because Ae. aegypti containing Dengue virus. And it is ready to transmit the Dengue virus to humans. Transovarial transmission of Dengue virus plays a role in the sustainability of the transmission of Dengue both at the time of epidemic as well as between epidemic because the mosquitoes that have been infected with Dengue virus can transmit the virus throughout their life, although they never suck infected human blood (Ditjen PP & PL, 2013;Espinosa, 2014) , The information on the ability of Ae. aegypti to transovarially transmit Dengue virus in Kupang and elsewhere is very useful in combating Dengue and Dengue vector control, especially also because the transovarial transmission of Dengue virus is not only found in endemic areas, but also free and sporadic areas. This means that the area which is free from Dengue remains a risk for the occurrence of Dengue fever since the existing vector and virus. Therefore, when the supporting environmental factors, immune factors people declined, and vector control are not being encouraged in the free areas, it is not impossible that there will be also an increase in Dengue cases in the area. So it is expected to add awareness to the community and the government to intensify vector control in all areas including Dengue-free areas.
In addition, area-based management needs to be done, namely the concept of prioritizing, working or focusing on controlling the source of the disease which is done early to prevent the occurrence of extraordinary events and carried out simultaneously with the search and eradication of breeding places based on the characteristics of the RT, RW, or village (Achmadi, 2010). DHF conditions between regions can be different, including the determinant factors of incidence of Dengue, for example on research in Puskesmas Sekaran in Semarang, it is found that the incidence of Dengue is associated with the presence of larvae in shower tub and clothes hanging habit (Sukowinarsih, 2011), but this does not necessarily apply equally in elsewhere, for the area-based management in the eradication of Dengue here, it is very necessary.

Conclusion
This study proves that the Dengue virus transovarial infection can be found in all endemic, sporadic and free areas with the highest rate is in endemic areas (13.3%) and the average of Kupang is 9.2%. Den-1 virus can be found only in sporadic and endemic areas. It is expected that Dengue vector control is not only performed in endemic areas but also in areas of sporadic and free of Dengue.