Muhammad Khaliim Jati Kusala1,2, Arif Nur Muhammad Ansori1,2, Reviany V. Nidom2,3, Setyarina Indrasari2,3, Anis F. Astutik2, Irine Normalina2, Mohammad Y. Alamudi2, Siti Rukmana2, Kadek Rachmawati2,4, Kuncoro P. Santoso2,4, Chairul A. Nidom2,4*
1Doctoral Program in Veterinary Science, Faculty of Veterinary Medicine,
Universitas Airlangga, Surabaya, 60115, Indonesia.
2Professor Nidom Foundation, Surabaya, 60115, Indonesia.
3PT. Riset AIRC Indonesia, Surabaya, 60115, Indonesia.
4Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, 60115, Indonesia.
In recent decades, zebrafish (Danio rerio) has been widely used as a model of biological experiments not only in the immunology and oncology aspects but also experimental animals for infectious diseases in human and fishes. The virus used in this study was the H5N1 avian influenza reverse genetic virus obtained from the Professor Nidom Foundation, Surabaya, Indonesia. We revealed that TCID50 was 3.1623×107/mL, then the virus was developed by inoculating into the primary cell culture and we performed HA test. Samples that showed positive results on the HA test then performed RNA extraction and confirmed by one-step RT-PCR. It can be seen from the presence of cytopathogenic effects (CPE) in primary cell cultures and also can be seen from HA titers in viral harvest samples from the primary cells of zebrafish (Danio rerio) that shows the same result of HA titers is 28. In conclusion, the present study demonstrated that there is an alternative development of new primary cell sources which leads to halal aspects of the production of avian influenza H5N1 vaccines.
Vaccination is one of the efforts to control an infectious disease, both human and animal which is preventive and efficient. However, the concept of vaccination has recently also been used to control non-infectious diseases such as cancer prevention. The vaccination process begins with the determination of the appropriate antigen for prevention followed by the formulation at the next stage. Determination of vaccine antigen will be adjusted to the character of the antigen and modulation of the antibody system that will be caused. The process from upstream to downstream, must follow the provisions of national and international regulations, including provisions on halal processes so that Muslims can use the vaccines1,2.
In addition, the level of lawfulness has become a concern, given that one third of the world's population are Muslim. Viral vaccines are generally multiplied using embryonic chicken eggs. However, this widely used influenza vaccine production platform has several disadvantages3. Production of vaccine based on mammalian cell culture has different advantages compared to the production of egg-based vaccines that are common today. Cell culture-based vaccines are easier to improve and have reduced risk for mutations that produce antigenic changes4.
Eissa et al. revealed that the H5N1 subtype was detected in the hemolymph of the red swamp cryfish (Procambrus clarkia) in three different waters in Egypt and was also found in the cone shell (Conus meditteraneus)5. Gabor et al. stated that zebrafish (Danio rerio) infection with seasonal influenza viruses showed that zebrafish (Danio rerio) were positively affected by seasonal influenza viruses with an increase in virus titers to cause death over time6. This study aimed to make an alternative development of the avian influenza H5N1 reverse genetic virus vaccine using zebrafish (Danio rerio) primary cells.
MATERIAL AND METHODS:
Reverse genetic H5N1 virus obtained from the laboratory of Professor Nidom Foundation, Indonesia, zebrafish (Danio rerio) primary cells, phosphate buffer saline (Sigma-Aldrich, USA), trypsin (non-animal origin) (Sigma-Aldrich, USA), Dulbecco's Modified Eagle's Medium (Sigma-Aldrich, USA), penicillin-streptomycin (Sigma-Aldrich, USA), aquadest, fetal bovine serum (Aldrich, USA), 70% ethanol, maintenance media (MM), bovine serum albumin (Sigma-Aldrich, USA), LL buffer, MgSO4 (Sigma-Aldrich, USA), forward primer, reverse primer, SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase (Invitrogen), distilled water, ethidium bromide (Sigma-Aldrich, USA), TrackItTM 1 Kb Plus DNA Ladder (Invitrogen), crystal violet (Sigma-Aldrich, USA), agarose (Sigma-Aldrich, USA).
Aquarium with a size of 20×30cm, 600mL plastic, biosafety cabinet class 2 (Biobase®, China), six well-plate, 1000µL micropipette, 200µL, and 50µL, 5mL, and 10mL serological pipettes, pipettor, conical tube 15mL, conical tube 50mL, T75 flask, T25 flask, Eppendorf 1.5 µL, flask, screwtube (Eppendorf), centrifuge (myFuge™), microcentrifuge (Thermo Fisher Scientific, USA), blue tip, yellow tip tip (Tarsons Product Pvt. Ltd., India), inverted microscope (Olympus CKX31, CKX41), CO2 incubator (Thermo Fisher Scientific, USA), Corning® 96 Well TC-Treated Microplates (Sigma-Aldrich, USA), microwave (Electrolux, Sweden), water bath, microcentrifuge tube, QIAamp mini spin column (Qiagen, Germany), SimpliAmp Thermal Cycler (Applied Biosystems™, USA), electrophoresis device (Mupid®-One Electrophoresis System), 3130 xL Applied Biosystems® Genetic Analyzers (Applied Biosystems™, USA), vortex (Vortex-Genie™ 2), refrigerator (Modena), scales (Krisbow).
Zebrafish (Danio rerio) Preparations:
Zebrafish (Danio rerio) are used an average of 3-5cm in size in the juvenile stage with healthy morphological features of the fish then packaged and taken to the laboratory. After that the fish is acclimatized to the aquarium for 5 days. Fish were fed with TetraMin (Tetra Werke Co.) once a day. All experimental procedures and maintenance of fish were conducted in accordance with the Guide for Care and Use of Laboratory Animals published by the US National Institutes of Health.
Avian Influenza Virus Preparations:
The avian influenza virus used in this study was the reverse genetic H5N1 virus obtained from the Poultry Disease Laboratory, Professor Nidom Foundation, Surabaya, Indonesia. The virus used has been tested TCID50 and has a yield of 3.1623×107/mL.
Zebrafish (Danio rerio) Primary Cells Culture:
Weighing zebrafish (Danio rerio) to be used is ±5.1 grams (equal to 20 fish). Then euthanasia of adult zebrafish (Danio rerio) by inserting it into cold water. After the fish fainted, then the fish was cut into two parts, namely the head (eyes removed) and body parts, then put it in petridisk and rinsed with sterile PBS (13 mM Na2HPO4, 1.5mM KH2PO4, 135mM NaCl, 2.5mM KCl; pH 7.0) three times. Then separate the two parts and each part of the insert into the syringe and press into the glass container that has a stirrer in it. Add 10 mL of trypsin (non-animal origin) solution. Tripsination with a medium stirer in a glass container containing an aquadest at 37°C for 50 minutes. Add 1 mL of FBS and the stirrer back for 10 minutes. Pour the results of trypsination into conical using a filter. Pour the cell suspension filtering results into the centrifuge tube and centrifuge at 3000 rpm for 10 minutes. Then discard the supernatant, resuspending the cell with DMEM so that the cells separate one by one. Insert the cell into the flask container. Then incubate 5% CO2 at 37°C, then observe the development and growth of zebrafish (Danio rerio) primary cell cultures every day.
Zebrafish (Danio rerio) Primary Cells Passages:
Passing cells of zebrafish (Danio rerio) primary culture begins by removing all the medium in the culture flask. Then, wash the cell using a PBS solution. Cell washing was done in three times and each cell was washed by inserting 5mL PBS into a culture flask (25cm2 size flask). After the washing process is complete, then add 1 mL of trypsin (non-animal origin) solution to the flask of each culture flask (25cm2 size flask). Cells that have been given a trypsin solution (non-animal origin) are then re-incubated in a CO2 incubator with 5% CO2 at 37 °C overnight (24 hours). After the incubation process is complete then do the tapping and observation under the microscope to see if the attached cells have been released or not. If the cell has escaped from the base of the flask, then add enough DMEM medium to the culture flask. Then transfer the entire liquid in the flask to the 15 mL cone. Perform 1500rpm centrifuge for 5 minutes. After the centrifugation process, the next step is to remove the supernatant, then add the DMEM medium and pipetting until the cells are evenly mixed. After the cell appears to be well mixed, then reinsert it into the culture flask container. Observe under the microscope to make sure the cells are separated one by one and mixed evenly. After that, re-incubation in CO2 incubator with 5% CO2 content at 37°C until the cell is confluent.
Inoculation of the Virus in Primary Cells:
Confluent culture cells were washed three times with 5 mL PBS, added the virus that had been tested TCID50 and had a yield of 3.1623×107/mL of 2mL in each flask (25mL flask) then incubated 5% CO2 at 37°C for 60 minutes. After cells incubation in the 25mL flask media 5mL maintenance medium (DMEM + BSA 1% + Penstrep 0.1%) was then cultured in the culture flask to be incubated for 3 days in a 5% CO2 incubator at 37°C. Observation of the developments cells every day. Cells harvesting was carried out 3 days after inoculated virus samples in the cell. Cells harvesting was carried out by taking each liquid contained in each flask and then placed in an microcentrifuge tube 1.5µL to be centrifuged at a speed of 3000rpm at 4°C for 10 minutes. The results of the supernatant from the centrifuge process were then stored at 4°C before further testing.
Staining of Primary Cells:
After the harvesting process, the cells that are still attached to the base of the flask are stained starting with giving a 10% formalin solution to a 2mL flask (covering all the base surfaces of the flask). Then incubate for 30 minutes for the cell fixation process. After that, remove formalin and add the crystal violet dye solution to the flask to cover the entire base of the flask. Then incubate for 5 minutes and rinse with distilled water.
HA Test :
The HA test in this study used a "V" microplate. First filling 50µL of microplate with PBS starting from numbers 1-12 on lines A to C. Microplate wells on line C are used as erythrocyte control (without antigen). The number 1 line A-B microplate well is filled with 50µL cell culture supernatant liquid, given cell culture supernatant liquid on row A1 (head cell) and B1 (body cell) then dilution. Dilution is done by taking 50µL at the A1 well and inserting and blending it until it flattens the A2 well, 50µL is taken in A2 well and inserted into A3 well as in the previous step until well A12, then 50µL is removed. Dilution was also carried out on the well in row B. The next step was to fill all well lines A-C on the microplate with 0.5% chicken RBC as much as 50µL, then incubate at room temperature for 30 minutes. Line C is used as a control containing RBC 0.5% and PBS. Finally, HA titre readings from the samples examined7.
Samples that show positive results on the HA test are then extracted from RNA. The viral RNA obtained from culture fluid was extracted using the QIAamp viral RNA Mini kit (Qiagen, Germany) following the manufacturer’s guidelines. Labels in accordance with the name until then can be continued with the PCR test.
Reverse Transcription-Polymerase Chain Reaction:
HA forward (5’-AGCAAAAGCAGGGGTTC AATCTGTCTAAAATGG-3’)
HA reverse (5’-AGTAGAAACAAGGGT GTTTTTTAACTAATCTG-3’)
PCR amplification was carried out by making a master mix on the microcentrifuge tube 0.5 mL sterile with components 12.5µL 2× RT Mix, 2.5µL 5 mM MgSO4, 1 µL forward primer, 1 µL reverse primer, 0.5µL SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase, 2.5µL distilated water and 5µL RNA so that a total of 25µL was obtained. Tubes that already contain a mixture of cDNA and master mix are incubated in a thermal cycler with a program temperature of RT 55°C for 1 hour, predenaturation of 94 °C, for 4 minutes; denaturation of 94°C, for 20 seconds; annealing 58°C, for 1 minute (30 cycles); extention 68°C, for 10 minutes; and keep at 4 °C.
After the gel is ready, make a sample mixture (loading dye 1µL plus 3µL samples) and for the mixture marker (loading dye 1µL plus 3µL DNA ladder) as a reference standard for DNA fragments. Insert the print and gel into the Electrophoresis tool. Put the sample into a well in the gel. Then running turn on the 100 V device for 30 minutes. After that, place the gel on the UV-transilluminator media to see the results.
RESULTS AND DISCUSSION:
Results of Growth of Zebrafish (Danio rerio) Primary Cell Culture:
Maintenance of zebrafish (Danio rerio) primary cell cultures was carried out using a 5% CO2 at 37°C. The process of replacing the primary cell culture medium is done every two days with a new medium. On the second day, some cells have begun to form colonies and stick to the bottom of the flask. For body cells there tends to be more giant cells compared to head cells which appear more uniform. The single cell form initially looks round with the cell nucleus looking like a point, then when forming a colony the shape becomes different, looks elongated with a variety of short lengths that vary. The cell nucleus resembles the formation of neurons that connect between one cell and another when forming colonies.
Observation of cell growth of primary zebrafish (Danio rerio) culture is done every day to see the development of cell growth until cell growth will appear denser and meet the base of the flask (confluent). To then need to pass the cell to the new growth place (flask) so as to provide an opportunity for cells to continue to develop by providing empty space for attachment cells and growing. The first phase is carried out on day 5 after the process of making primary cell cultures. In cells that have been confluent, many cell colonies appear that attach and grow to meet the base of the flask, while cells non-sticking will be removed together with media replacement.
Results of Passage of Zebrafish (Danio rerio) Primary Cells:
The process of zebrafish (Danio rerio) cell culture cell growth is repeated over cells that have been confluent before inoculation using a virus. To pass a confluent cell culture, a trypsination process is carried out. The trypsination process begins by taking all the mediums inside the flask and washing the cells using PBS three times. Only after that was given an additional 1 mL of trypsin (non-animal origin) per T25 flask, then continued according to the method described earlier.
The trypsination process is carried out overnight (24 hours) incubation. In the picture of cells undergoing the trypsination process there is a difference between the head cell and the body cell. After overnight incubation (24 hours) on body cells, the cells appear more rounded and many have been released from the base of the flask, whereas in the head cell the cell formation still appears tight and there are still many attached to the base of the flask, therefore cell passages process we do it with the bending method on the base of the flask. After the passage process, the cells are then re-incubated in a 5% CO2 incubator with a temperature of 37°C until the cells can grow and develop to reach confluence (growing attached to meet the base of the flask) before the inoculation process is carried out with the virus.
Virus Inoculation in Cell Culture:
The virus used for inoculation of zebrafish (Danio rerio) primary cell culture is the H5N1 avian influenza virus that has been made with reverse genetic technology obtained from the stock of laboratory of the Professor Nidom Foundation. Reverse genetic virus which has TCID50 value of 3.1623×107/mL, then inoculated into a T25 flask of 2mL/T25 flask and then maintenance medium is added and incubated in a CO2 incubator at 37 °C and CO2 content of 5%. Observations are made every day for 3 days.
When zebrafish (Danio rerio) primary cell culture was inoculated with reverse genetic avian influenza virus, cell damage was formed due to infection with avian influenza virus. Cell damage is called Cytopathogenic Effect (CPE). The existence of CPE indicates that avian influenza virus has replicated in zebrafish (Danio rerio) primary cell culture both in the head cell and body cells. At 24-hour observation, morphological changes in cells (CPE) have appeared, namely the cell nucleus appears to be lysis so that the cell will die and appear not to stick to the base of the flask. The CPE formation that appeared was more widespread in the 48-hour observation and also at the 72-hour observation. After 3 days of observation, the virus was harvested from zebrafish (Danio rerio) primary cell cultures in the head cell and body cells.
Staining of Post-Harvest Cell Culture:
Coloration of cell cultures using crystal violet staining. From the results of the coloring, we can see a picture of the difference between living cells and cells that have CPE as a result of the reverse genetic avian influenza virus infection. Cells that are still alive, the cell nucleus will be able to absorb the dye, so the cell will become colored. While cells that experience CPE/cells that have lysis do not have the ability to absorb dyes, so the cells will not be stained.
Hemagglutination Test (HA) Results:
The yield of the virus that has been inoculated on zebrafish (Danio rerio) cells is then tested for hemagglutination (HA). The avian influenza virus used has the structure of the hemaglutinin protein that can agglomerate erythrocytes. Hemagglutination testing is carried out using erythrocytes (red blood cells) derived from chicken. In HA testing using samples from head cells and bodies, both of which showed the ability to routine erythrocytes up to well number 8, whereas in wells 9 to 12 hemagglutination did not occur. Therefore, the results of HA titers for viruses in head cells are 28 and on body cells also show the same results as 28 HA titers. HA titers are the highest dilutions of a virus that can still drain erythrocytes. In the red blood cell control line erythrocyte agglutination did not occur, indicating that in sample C there was no presence of a virus that could advertise erythrocytes.
Test Results using One-Step RT-PCR (Reverse Transcription-Polymerase Chain Reaction):
Samples that show positive results on the HA test are then extracted from RNA. After extracting RNA the next stage is PCR amplification using HA forward and reverse HA primers. Then the results will appear using gel electrophoresis. To detect pieces of DNA in the form of bands-DNA in agarose gels using low concentration of fluorescent dyes, such as the intercalating agent ethidium bromide.
From the results of the PCR test, both viral yield samples from head cells and body parts showed positive results. There were bands in the same two samples with positive control samples (reverse genetic H5N1 virus) at a molecular weight of 1778 bp which indicated the reverse genetic H5N1 HA virus avian influenza virus gene. This shows that viral harvest samples from zebrafish (Danio rerio) primary cell cultures from the head and body also contain the reverse genetic H5N1 virus that successfully grows and replicates in cell culture after inoculation.
The results of the study entitled zebrafish (Danio rerio) primary cell culture as a development material for avian influenza vaccine provided an alternative development of new cell sources that could be used as a basis for developing aspects leading to aspects of halal H5N1 bird flu vaccine production processes. This research has succeeded in making primary cell cultures from zebrafish (Danio rerio) both from the head and cell parts of the body.
Zebrafish (Danio rerio) primary cell culture in this study was carried out using DMEM is in accordance with what has been done in developing primary culture cells from adult zebrafish (Danio rerio) with endogenous oligodendrocyte progenitor cells (OPCs) at 28°C and CO2 levels of 5% and replacing the medium every two days8. The Dulbecco's modified eagle's medium (DMEM) media, supplemented with additional antibiotics, salts, and fetal bovine serum (FBS), was stated to be sufficient to develop primary cell cultures from marine fish muscles and showed good growth results for 20 days9.
Zebrafish (Danio rerio) primary cell cultures in this study were carried out in an incubator at 37°C and CO2 levels of 5%. Other studies suggest that zebrafish (Danio rerio) cells can grow at room temperature and can survive for long periods of time when confluent, and do not even need to use CO2-enriched atmospheres, which greatly simplifies the process of cell culture. The growth of primary cell cultures from adult zebrafish (Danio rerio) cell gills was carried out using a variety of temperatures namely 25, 28, 32, 37 and 40°C and the results of zebra gill cells showed different growth in different temperature. Cells can grow at temperatures between 25 and 32°C10.
The cell form obtained in this study is that the single cell form initially looks round with the cell nucleus looks like a point, then when forming colonies the shape becomes different, looks elongated with a variety of short lengths that vary. The cell nucleus resembles the formation of neurons that connect between one cell and another when forming colonies. This is in accordance with research by Meade et al. which conducted research using primary culture cells from adult zebras in the morphology of glial cells characterized by small cell bodies, then undergoing a process of tapering and enlargement and in some cases elongated cellular extensions11.
Meanwhile, in this study, primary cell cultures from the head and body of zebrafish (Danio rerio) have been produced which can grow and develop well at 37°C with CO2 levels of 5%. This condition has resulted in confluent cells and passages can be carried out for inoculation using the H5N1 reverse genetic influenza virus. Preliminary research conducted by Nidom et al. using the H5N1 reverse genetic influenza virus can grow and develop well in madine darby canine kidney (MDCK) cell culture as well as vero cells incubated at 37°C with CO2 levels of 5% which is the optimum temperature for the development of influenza viruses12.
The use of reverse genetic as a method to produce influenza vaccines originating from HPAI is a relatively new method. This method is very relevant to address concerns about the safety and immunogenicity of this vaccine, as well as the dosages and regimens needed to protect immunologically naive individuals from serious infections. Another concern is the suitability of antigens used to protect against the threat of infection, considering that pathogens that will cause an outbreak cannot be known with certainty, so a vaccine antigen needs to be prepared that can cause cross reactions. Cross protection in HA subtypes will allow the use of vaccines that until there are special vaccines with special strains available13.
The use of reverse genetic in preparing vaccine reference strains offers several advantages over traditional methods. First, the reverse genetic approach is a rational approach directly compared to the traditional approach that still has the potential not on target. Second, reverse genetic will decontaminate wild types of viruses that may be derived in unvalidated systems, such as cell lines that are not validated for vaccine purposes, or which may contain additional pathogens. Third, at the plasmid stage, HA can be engineered to eliminate pathogenic properties14.
Gabor et al. stated that zebrafish (Danio rerio) infected with seasonal influenza viruses, showed the presence of viral infections and caused death in fish, this suggests that aquatic biota can be contaminated by seasonal influenza virus infection. The results of this study illustrate that the H5N1 influenza reverse genetic virus has succeeded in growing and developing in zebrafish (Danio rerio) primary cell cultures in the cells of the head and also cells in the body. This can be seen from the presence of CPE in primary cell cultures of the head and body which indicate that the virus can grow and replicate in the primary cell culture. These results are in accordance with the preliminary study, which showed that zebrafish (Danio rerio) could be infected with the H5N1 Avian Flu virus which was infected through soaking or dipping6. A study by Nidom et al. also states that zebrafish (Danio rerio) infection with the reverse genetic H5N1 virus has been carried out and shows that the H5N1 virus can infect zebrafish (Danio rerio) in almost all organs15. Usually positive or negative CPE is evaluated manually under an inverted microscope in three to five days. To avoid the subjective errors of manual CPE evaluation, media is often tested for the presence of viruses by hemagglutination16. Therefore, in this study the virus yields from head and cell cells from zebrafish (Danio rerio) which were obtained were then tested for hemagglutination (HA test).
Molecular analysis of the reverse genetic bird flu virus was carried out using the Polymerase Chain Reaction (PCR) method using universal HA and NA primers. The nucleotide length of the HA gene is 1778 basepairs (bp) while the NA nucleotide length gene is 1400 basepairs (bp). The results of molecular analysis obtained showed that the HA gene both from the multiplication of TAB, and culture cells (MDCK cells and Vero cells) had the same DNA band with positive controls. This shows that the results of the inoculation of the reverse genetic bird flu virus have developed well in each of the inoculation media.
Current techniques such as Reverse Transcriptase - Polymerase Chain Reaction (RT-PCR) are widely used to detect influenza viruses. Avian Influenza is a single-stranded RNA virus so that in the PCR reaction a stage of DNA copy (cDNA) synthesis is required. This stage requires a reverse transcriptase enzyme. Some of the reverse transcriptase enzymes that can be used include Taq DNA Polymerase, mesophilic viral reverse transcriptase (RTase) and Tth DNA Polymerase. Taq DNA Polymerase is an enzyme that is resistant to high temperatures and has a high polymerase rate and high ability to continuously combine nucleotides with a primer without dissociating from the primary-DNA complex complex. RTases encoded by avian mycoblastosis (AMV) or M-MuLV viruses are highly processive and are capable of synthesizing cDNA up to 10 kb, while DNA polymerase is capable of synthesizing cDNA up to 1-2 kb. Based on the above reasons, this test is called RT-PCR17.
Viral yields from zebrafish (Danio rerio) primary cell cultures were then confirmed by using a RT-PCR. PCR is carried out using HA forward primers and reverse HA primers. The results of molecular analysis obtained showed that both HA genes derived from harvested zebrafish (Danio rerio) head cells and zebrafish (Danio rerio) body cells had the same DNA band as positive control at 1778 bp molecular weight indicating the reverse genetic virus H5N1 avian influenza HA gene. The results of this study are in accordance with previous studies that have carried out molecular analysis of the HA gene from the reverse genetic H5N1 virus. This shows that the result of inoculation of the reverse genetic H5N1 virus has developed and grown well in zebrafish (Danio rerio) primary cell cultures in the head cells and also in the body cells.
In sum, we found tissue or organ parts of the head and body of zebrafish (Danio rerio) can be used as primary cell culture and grow and develop well until it reaches confluence and passages can be carried out on cell culture. The H5N1 bird flu virus reverse genetic can infect zebrafish (Danio rerio) primary cell cultures in head cells and body cells and cause CPE. H5N1 avian influenza virus reverse genetic can grow and develop in zebrafish (Danio rerio) primary cell cultures in head cells and body cells as indicated by the presence of HA titers and confirmed by PCR test.
In conclusion, the present study demonstrated that there is an alternative development of new primary cell sources which leads to halal aspects of the production of avian influenza H5N1 vaccines.
This study was supported by PMDSU Grant with the reference number 1341/UN3.14/LT/2018 from the Ministry of Research, Technology and Higher Education (KEMENRISTEKDIKTI) of the Republic of Indonesia. This study was also supported a part by PT. Riset AIRC Indonesia, Surabaya. We thank Prof. Yoshihiro Kawaoka, DVM., Ph.D. and Dr. Shinya Yamada from the Institute of Medical Science, the University of Tokyo, Japan, for helping dan providing plasmids for reverse genetic technology.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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Received on 29.11.2019 Modified on 10.02.2020
Accepted on 28.03.2020 © RJPT All right reserved