Jembrana Disease Research Articles

Article Review: The Role of Blood-sucking Insect Vectors in the Spread of Jembrana Disease in Bali Cattle

ABSTRACT. Jembrana disease is a contagious disease in cattle and causes enormous economic losses for farmers. This disease, known as bovine lentivirus, was first reported in 1964 in Jembrana District. Jembrana disease specifically attacks Bali cattle at various ages. The cause of this disease is a lentivirus from the Retoviridae family. Jembrana disease transmission occurs by direct contact and is mediated by blood-sucking insect vectors. Not all blood-sucking insects can be vectors for Jembrana disease. Some blood-sucking insects capable of spreading Jembrana disease are Tabanus rubidus flies Culicoides sp mosquitoes, and Aedes lineatopennis, which act as mechanical vectors. As for the Boophilus microplus tick, it is suspected that it acts as a biological vector. Even after re-testing the tick, it could not prove its role as a biological vector for JDV. Prevention and control of this disease can be carried out by vaccination, biosecurity, biosafety, administration of vitamins, isolation of infected livestock, and control of blood-sucking insect vectors by administering insecticides to cages.

Development of hyperimmune serum for diagnosis and therapy against Jembrana disease in Bali cattle

Bali cattle are indigenous Bali that has been widely distributed throughout Indonesia, form about 27% of the total cattle population, and they make the highest contribution to beef production in Indonesia. The animals possess many superiorities compared to other breeds but they are very susceptible to Jembrana disease virus (JDV) infection which is classified as one of the strategic-infectious animal diseases. The purpose of this study was to provide hyperimmune serum appropriate for the treatment of JDV infection and routinely serological diagnosis. The hyperimmune serum against JDV infection was provided using the method of Mhoma method and hyperimmune was developed using a different types of vaccines. firstly immunized intramuscularly with vaccine containing the CFA adjuvants and the second were prepare with the commercial vaccine twice at one monthly interval. Prophylaxis with these hyperimmune after infection was failed in challenge experiments.

IDENTIFIKASI VIRUS PENYAKIT JEMBRANA PADA SAPI BALI MENGGUNAKAN POLYMERASE CHAIN REACTION (PCR) DI PROVINSI KEPULAUAN BANGKA BELITUNG

Bali cattle is one of the typical Indonesian livestock that is widely cultivated because of its high reproductive and adaptation capabilities to the environment. However, behind these advantages, bali cattle have a weakness, namely they are susceptible to the jembrana virus which can cause jembrana disease. This disease will cause a decrease in the immune system of bali cattle so that they are susceptible to other diseases that will lead to the death of the cattle. The method used in the identification of the Jembrana disease virus is the Polymerase Chain Reaction-Reverse Transcription (PCR-RT) technique. Identification do 10 Bali cows in Panca Tunggal Village, South Bangka Regency and 11 Bali cattle in Temberan Village, Pangkalpinang City. The bali cattle serum used for virus identification was centrifuged to obtain buffycoat. The PCR-RT technique is carried out in several stages, namely sampling, buffycoat making, extraction, master mix making, amplification, agar manufacture, electrophoresis and visualization. Based on the test, it was found that A1 and A2 bali cattle from Panca Tunggal Village, South Bangka Regency indicated the presence of the Jembrana virus. So that this Polymerase Chain Reaction Technique can help detect the presence of the Jembrana virus in bali cattles.

Design of the env-su Gene Coding for Surface Unit Protein as a Vaccine Candidate for Jembrana Disease Virus in In Silico

Bali cattle are superior meat producers, but they are susceptible to Jembrana disease. The injection of the crude vaccine was considered ineffective, so the env-su gene was selected to express the Jembrana Surface Unit (JSU) protein as a candidate for the Jembrana vaccine. This study aimed to analyze the potential of the JSU protein as a candidate for the Jembrana vaccine and analyze the increase in the env-su gene expression which codon has been optimized in silico. Vaccine design was carried out through in silico including the selection of the SU protein sequences of the genus Lentivirus and sequence alignment of UniProt. The construction phylogeny tree of SU protein using MEGA-X program, optimization of env-su gene codon with preference codon Esherichia coli str. K-12 substr. MG1655 using Optimizer. The optimized env-su gene was inserted into the plasmid pET-21a (+) using GenScript. The result of sequence alignment showed that there is no SU protein that has a percent identity value of more than 30% with JSU protein. The SU JDV and BIV proteins are monophyletic groups and have a percent identity of 20.57%. Codon optimization showed an increase in CAI by 1,000 and GC 54.5%, and a decrease in ENc to 22 and AT 45.5%. EcoR1 and HindIII can recognize the gene target and MCS cut regions on the plasmid so that the env-su gene can be inserted into the pET-21a (+) plasmid. The JSU protein has the potential to be a candidate for the Jembrana vaccine, but it needs further research in vitro and in vivo.

Entrapment Formulation for env-Tm Gene Based on Chitosan Low Molecular Weight as a Jembrana Disease Virus Vaccine Candidate

Jembrana disease (JD) caused by Jembrana Disease Virus (JDV) becomes an obstacle in Bali cattle (Bos javanicus). The development of JD vaccines has a critical meaning to prevent losses in the B. javanicus in Indonesia and is one of the models in the development of the Human Immunodeficiency Virus (HIV) vaccine. The development of vaccines for JDV has carried out DNA vaccines that are expected to provide better immunological effects. This study aimed to determine the low molecular weight chitosan (LC) entrapment towards pEGFP-C1-env-Tm in the formation of Chitosan Nanoparticles Low/pEGFP-C1-env-Tm complex. The env-Tm gene was inserted in pEGFP-C1 into the pEGFPC1/ env-Tm construct transformed on the E. coli DH5α host. The construct was formulated into LC/pEGFP-C1/env-Tm complex with a low molecular weight chitosan concentration of 0.06 % and the ratio of pEGFP-C1/env-Tm: LC (wt/wt) was 1:0.5-1:3. The complexes were then analyzed by gel retardation assay agarose 1 %. The results of this study indicated that the best entrapment results of low molecular weight chitosan to pEGFPC1/ env-Tm was in the mass ratio of pEGFP-C1/env-Tm: LC was 1:2. The best formulation entrapment for env-Tm by low molecular weight chitosan 0.06 % is in the rate 1:2.

Potential of polylactic-co-glycolic acid (PLGA) for delivery Jembrana disease DNA vaccine Model (pEGFP-C1-tat)

BackgroundThe development of a vaccine for Jembrana disease is needed to prevent losses in Indonesia's Bali cattle industry. A DNA vaccine model (pEGFP-C1-tat) that requires a functional delivery system will be developed. Polylactic-co-glycolic acid (PLGA) may have potential as a delivery system for the vaccine model.ObjectivesThis study aims to evaluate the in vitro potential of PLGA as a delivery system for pEGFP-C1-tat.MethodsConsensus and codon optimization for the tat gene was completed using a bioinformatic method, and the product was inserted into a pEGFP-C1 vector. Cloning of the pEGFP-C1-tat was successfully performed, and polymerase chain reaction (PCR) and restriction analysis confirmed DNA isolation. PLGA-pEGFP-C1-tat solutions were prepared for encapsulated formulation testing, physicochemical characterization, stability testing with DNase I, and cytotoxicity testing. The PLGA-pEGFP-C1-tat solutions were transfected in HeLa cells, and gene expression was observed by fluorescent microscopy and real-time PCR.ResultsThe successful acquisition of transformant bacteria was confirmed by PCR. The PLGA:DNA:polyvinyl alcohol ratio formulation with optimal encapsulation was 4%:0.5%:2%, physicochemical characterization of PLGA revealed a polydispersity index value of 0.246, a particle size of 925 nm, and a zeta potential value of −2.31 mV. PLGA succeeded in protecting pEGFP-C1-tat from enzymatic degradation, and the percentage viability from the cytotoxicity test of PLGA-pEGFP-C1-tat was 98.03%. The PLGA-pEGFP-C1-tat demonstrated luminescence of the EGFP-tat fusion protein and mRNA transcription was detected.ConclusionsPLGA has good potential as a delivery system for pEGFP-C1-tat.

Potential of Nanoparticles Chitosan for Delivery pcDNA3.1-tat

The development of Jembrana disease vaccine is importance to prevent the loss of Bali cattle industry in Indonesia. This study aims to prepare a Jembrana DNA vaccine. The data Tat protein sequences gained from NCBI and the consensus process has been finished by the MultAlign program, and then Cloning of the pcDNA3.1-tat has been successfully performed on E. coli DH5α and confirmed by PCR, restriction analysis and sequencing. The propagated plasmids were prepared as DNA-chitosan complex and physiochemical characterized using Particle Size Analyzer. Complex with a 1:2 (wt/wt) ratio of DNA and chitosan have a mean diameter of 268.5 nm and zeta potential +25.1 mV and the value of Cytotoxicity Assay 80-90% as compared to the untreated cells that used as negative control, so it can be concluded that nanoparticles chitosan has good potential as a carrier agent for pcDNA3.1-tat.

POLYCLONAL ANTIBODY UTILIZATION FOR DETECTION OF JEMBRANA ANTIGEN AT BALI CATTLE IN WEST SUMATRA PROVINCE

The aims of this study were to detect Jembrana antigen with polyclonal antibody and to describe antigen distribution in the Bali cattle organ that positively infected with Jembrana disease. Spleens, lungs, and livers were harvested from 10 naturally infected Bali cattle whose infection was confirmed through positive Polymerase Chain Reaction (PCR) result for Jembrana virus. Immunohistochemistry test was performed using polyclonal antibodies produced in rabbits. The results showed that cells infected by Jembrana viruses displayed positive reaction with a reddish brown color. Immunohistochemistry methods using polyclonal antibodies can detect Jembrana antigens in the spleen, liver, and lung with the highest average detection score (P0.05) was found in spleen, followed by liver and lungs. There was significant difference in the distribution of Jembrana antigens between the spleen, liver, and lungs with spleen having the highest antigen density.

Recombinant Vaccine - A Vaccine Development and Antigen for Controlling Jembrana Disease in Bali Cattle

Most vaccines in Indonesia were initially made up from relying on attenuation or inactivation of pathogens. However, there have been developed several recombinant vaccines for poultry diseases. There have not been noticed recombinant vaccines for ruminant animals. Advances in molecular biology, genomics and proteomics have facilitated in creating of vaccine development. Nowadays, vaccine can be developed by approach of recombinant protein technology. Jembrana disease is a viral and infectious disease and only attacks in Bali cattle. Up to present the vaccination of Jembrana disease has been conducting by using a crude vaccine which is made up from infected lymph origin and processed by ultra-centrifuge and emulsified with adjuvant. This crude vaccine has a short immunity for about 3 months. Breakthrough in molecular biology with approach of protein recombinant technology, Jembrana vaccine was developed in our laboratory of Animal Molecular Genetics of the Research Center for Biotechnology – LIPI. Jembrana disease virus (JDV) belongs to Lentivirus, it has three major genes termed as gag, pol and env. These genes encode proteins needed for replication of the virus. The env gene encodes TM and SU proteins located on surface of the virus and induce a protective immunity against lentivirus infections. Another protein is TAT which encoded by tat gene as one of small accessory genes located between pol and env genes. Two genes of tat and env su were originated from infected lymph by Jembrana virus and constructed into vector plasmid pET. Lately the two of tat and env su genes were synthetically made up and constructed into vector plasmid of pBT-7-His-C. Both nature and synthetic gene original constructions were transformed through Escherichia coli strain BL21. Both nature and synthetic tat gene original were already expressed in a small scale production of protein recombinant. The protein recombinant JTAT was first used in field trial for vaccine candidate and was examined by ELISA for jembrana disease detecting as diagnostic candidate. The result of both field trial and detecting Jembrana disease will be discussed for vaccine and antigen candidates, respectively.

Effect of storage time and temperatures on growth rate of Eschericia coli BL21 containing JSU construct for Jembrana disease candidate vaccine

The viability of Eschericia coli BL21 (DE3) bearing JSU (Jembrana Surface Unit) recombinant protein depend on several factors, such as temperatures and time of storage. JSU act as a vaccine candidate for the Jembrana disease in Bali cattle, therefore, it is important to keep the quality and quantity the yield of JSU recombinant protein, the E. coli need to maintenance. The study was designed to examine the viability of Escherichia coli at different temperatures and time of cultivation. The E. coli BL21 bearing JSU was cultured in LB broth (+Amph 100µg mL−1) overnight and aliquot it in 1.5mL sterile tube (3 tube every temperatures). The tubes were incubated in three storage temperatures (Room Temperatures/RT, 4°C and -20°C. The viabilities of the cells were observed in 24, 48 and 120 hours by measuring the Optical density by spectrophotometer, and then inoculated it in LB plate (+Amp 100µg mL−1). The result showed that statistically, there were significant effect (P<0.05) in temperature -20°C at 24h and 120h observation on viability of E. coli BL21. The viability of E. coli at -20°C and storage time observed at 120h are more stabile, compared the other temperatures (RT and 4°C). There was no significant effect on both temperatures RT and 4°C in OD600 at 24 and 48h and the growth was decreased until 120h observation. The observation at 24h with 4°C cultured has a higher OD600 (1.098) followed by RT (1.070) and -20°C (0.773). The viability of E. coli was decreased at 48h with RT (1.011) and 4°C (1.0611), meanwhile, at -20°C the cells were increased (0.958). Meanwhile, the observation at 120h, the cells was more stabile at -20°C (0.961) compared with cultures from 4°C (0.981) and RT (0.719). Based on the OD600, the viability at the -20°C intended more stabile compared to RT and 4°C, but in the 24h the growth at -20°C are very low because no protectant added in the culture, it will shock the cells and dead. This finding showed that viability of E. coli BL21 bearing JSU recombinant protein better to keep in -20°C for longer period storage time with an addition of protectant agent in the culture media to keep the cells alive and more stabile.

The Jembrana disease virus Rev protein: Identification of nuclear and novel lentiviral nucleolar localization and nuclear export signals.

The lentiviral Rev protein, which is a regulatory protein essential for virus replication, has been first studied in the human immunodeficiency virus type 1 (HIV-1). The main function of Rev is to mediate the nuclear exportation of viral RNAs. To fulfill its function, Rev shuttles between the cytoplasm and the nucleus. The Jembrana disease virus (JDV), a lentivirus, is the etiologic agent of the Jembrana disease which was first described in Bali cattle in Indonesia in 1964. Despite the high mortality rate associated with JDV, this virus remains poorly studied. Herein the subcellular distribution of JDV Rev, the nuclear and nucleolar localization signals (NLS and NoLS, respectively) and the nuclear export signal (NES) of the protein were examined. JDV Rev fused to the enhanced green fluorescent protein (EGFP) predominantly localized to the cytoplasm and nucleolus of transfected cells, as determined by fluorescence microscopy analyses. Through transfection of a series of deletion mutants of JDV Rev, it was possible to localize the NLS/NoLS region between amino acids (aa) 74 to 105. By substituting basic residues with alanine within this sequence, we demonstrated that the JDV Rev NLS encompasses aa 76 to 86, and is exclusively composed of arginine residues, whereas a bipartite NoLS was observed for the first time in any retroviral Rev/Rev-like proteins. Finally, a NES was identified downstream of the NLS/NoLS and encompasses aa 116 to 128 of the JDV Rev protein. The JDV Rev NES was found to be of the protein kinase A inhibitor (PKI) class instead of the HIV-1 Rev class. It also corresponds to the most optimal consensus sequence of PKI NES and, as such, is novel among lentiviral Rev NES.

In Vitro Evaluation of Chitosan-DNA Plasmid Complex Encoding Jembrana Disease Virus Env-TM Protein as a Vaccine Candidate.

IntroductionThe development of Jembrana disease vaccine is an important effort to prevent losses in the Bali cattle industry in Indonesia. This study aims to prepare a Jembrana DNA vaccine encoding the transmembrane portion of the envelope protein in pEGFP-C1 and test the success of its delivery in culture cells using a chitosan-DNA complex.Material and MethodsCloning of the DNA vaccine was successfully performed on E. coli DH5α and confirmed by colony PCR, restriction analysis and sequencing. The plasmids were prepared as a chitosan complex using the complex coacervation method and physicochemically characterised using a particle size analyser. A transfection assay was performed in HeLa cells with 4 h exposure, and mRNA expression was assessed at 24 h post transfection.ResultsWith a 1:2 (wt./wt.) ratio of DNA and chitosan, the complexes have a mean diameter of 236 nm, zeta potential value of + 17.9 mV, and showed no high toxicity potential in the HeLa cells. This complex successfully delivered the DNA into cells, as shown by the presence of a specific RT-PCR product (336 bp). However, the real-time PCR analysis showed that the delivery with chitosan complex resulted in lower target mRNA expression when compared with a commercial transfecting agent.ConclusionpEGFP-env-tm JDV as a candidate vaccine can be delivered as the chitosan-DNA complex and be expressed at the transcription level in vitro. This initial study will be used for further improvement and evaluation in vivo.

Kombinasi One-step Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) dan Nucleic Acid Lateral Flow (NALF) sebagai Metode Deteksi Gen env-tm Virus Jembrana Strain Tabanan 1987

Jembrana disease is an acute viral disease in Bali cattle that have short incubation period, high mortality and morbidity rate. Early diagnostic methods are needed to prevent the further spreading of this disease. In this study, we combined One-step RT-PCR and NALF methods to detect env-tm gene of Jembrana virus Tabanan 1987 strain. Viral RNA isolated from a spleen of the infected cattle was used as the template. One-step RT-PCR procedure was performed using oligoprobes labeled digoxigenin and right primer sequence that were designed using the primer3plus program based on the conserves region of env-tm gene from NCBI database. The product of one-step RT-PCR was tested using NALF method instead of electrophoresis. Positive result was shown by the appearance of dark lines on the test line of digoxigenin in NALF dipstick device.

Expression of gag-CA Gene of Jembrana Disease Virus with Cationic Liposomal and Chitosan Nanoparticle Delivery Systems as DNA Vaccine Candidates

Expression of gag-CA Gene of Jembrana Disease Virus with Cationic Liposomal and Chitosan Nanoparticle Delivery Systems as DNA Vaccine Candidates

Jembrana disease virus Vif antagonizes the inhibition of bovine APOBEC3 proteins through ubiquitin-mediate protein degradation

Viral infectivity factor (Vif) encoded by lentiviruses is essential for viral replication and escaping from antiviral activity of host defensive factors APOBEC3. Jembrana disease virus (JDV) causes an acute disease syndrome with approximately 20% case fatality rate in Bali cattle. However, the interplay mechanism between JDV Vif and Bos taurus APOBEC3 (btA3) is poorly understood. In this study, we determined that JDV Vif recruits ElonginB, ElonginC(ELOB/C), Cul2 and RBX1 but without the need of CBF-β to form E3 ubiquitin ligase and induces the degradation of btA3 proteins. Further investigation identified BC-box (T149LQ151) motif required for ELOB/C binding, Cul2 box (Y167xxxxV/X172) and a zinc-binding motif (H95-C113-H115-C133) required for Cul2 binding in JDV Vif. The precise mechanism of JDV Vif overcoming the antiviral activity of btA3 proteins is helpful for the application of the broad spectrum antiviral drug targeting conserved functional domains of various species Vif proteins in the future.

A naturally occurring bovine APOBEC3 confers resistance to bovine lentiviruses: implication for the co-evolution of bovids and their lentiviruses.

Mammals have co-evolved with lentiviruses for a long time. As evidence, viral infectivity factor (Vif), encoded by lentiviruses, antagonizes the anti-viral action of cellular APOBEC3 of their hosts. Here, we address the co-evolutionary dynamics of bovine APOBEC3 and the following two bovine lentiviruses: bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV). We determined the sequences of three APOBEC3 genes of bovids belonging to the genera Bos and Bison and showed that bovine APOBEC3Z3 is under a strong positive selection. We found that APOBEC3Z3 of gaur, a bovid in the genus Bos, acquired resistance to JDV Vif-mediated degradation after diverging from the other bovids through conversion of the structural composition of the loop 1 domain. Interestingly, the resistance of gaur APOBEC3Z3 can be attributed to the positive selection of residue 62. This study provides the first evidence, suggesting that a co-evolutionary arms race between bovids and lentiviruses occurred in Asia.

Pelacakan Gen Env-TM Virus Penyakit Jembrana Galur Tabanan 1995 dengan Metode Nucleic Acid Sequence Based Amplificaton

Jembrana disease is an infectious disease in Bali cattle cause by a member of lentivirus calledjembrana disease virus (JDV). It causes an acute and severe disease syndrome with short incubationperiod. As the disease has spread to several areas in Indonesia, a simple and rapid detection method isrequired. The objective of this study to apply rapid diagnostic method for JDVTabanan 1995 strain basedon Nucleic Acid Sequence Based Amplification (NASBA) methods targeting env-tm gene. The steps of thisresearch consisted of viral RNA isolation from organ and blood of cattle experimentaly infected withJDVTabanan 1995 strain . RNA amplification was conducted by NASBA using waterbath. The NASBAproducts were then separated on 2 % agarose gel. Using this technique JDV positive result was obtainedfrom organ samples such as spleen, liver, lung, prefemoralis lymph node, prescapularis lymph node andblood generating a RNA fragment of 207 bp. In this study, diagnosis method for env tm of JDV Tabanan1995 strain can be conducted by isothermal amplification NASBA.

Use of reverse transcription loop-mediated isothermal amplification combined with lateral flow dipstick for an easy and rapid detection of Jembrana disease virus

Jembrana disease virus (JDV) is a viral pathogen that causes Jembrana disease in Bali cattle (Bos javanicus) with high mortality rate. An easy and rapid diagnostic method is essential for further control this disease. We used a reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with lateral flow dipstick (LFD), based on conserved tm subunit of Jembrana disease virus env gene. The RT-LAMP conditions were optimized by varying the concentration of MgSO4, betaine, dNTP, and temperature as well as the time and duration of reaction. The primers sensitivity for JDV was confirmed. The method was able to detect env-tm gene dilution which contained 2×10(-15)g of template. Comparatively, the sensitivity of RT-LAMP/LFD was 100-fold more sensitive than reverse transcription-polymerase chain reaction. The primers specificity for JDV was also confirmed using positive and negative controls. This work also showed that virus detection could be done not only on total RNA extracted from blood but various organs could also be analyzed for the presence of JDV using RT-LAMP/LFD method. The whole process, including the LAMP reaction and the LFD hybridization step only lasts approximately 75min. Results of analysis can be easily observed with naked eyes without addition of any chemical or further analysis. The combination of RT-LAMP with LFD makes the method a more suitable diagnostic tool in conditions where sophisticated and expensive equipments are not available for field investigations on Jembrana disease in Bali cattle.

Gene Transfer Vector Derived from Jembrana Disease Virus: A Review

The etiological agent of Jembrana disease is a virus which was accordingly named Jembrana Disease Virus (JDV). JDV was a member of the family of Retroviridae, of the subfamily of lentiviruses. As a member of the lentivirus, JDV has general property to integrate their genome into the chromosomal DNA of the host-cells. It is one of their common properties which is not shared by simple retroviruses such as murine leukimia virus or avian sarcoma virus. Lentiviruses are highly species-specific and share the property of replicating in dividing and non-dividing terminally differentiated cells. JDV-based gene transfer vector which had been developed is appeared to possess a broad tropism and a unique capacity of mediating efficient gene transfer. This review summarize JDV-based viral vector system that has been developed. The main principles and components of JDV viral gene transfer system and its implication in the future research will be discussed.

Immunodiagnosis in Jembrana Disease: A Review

Jembrana disease is a bovine disease that affects Bali cattle (Bos javanicus). The causal agent is named Jembrana Disease Virus (JDV), a lentivirus member. The disease development of Jembrana disease in Bali cattle is unique for a lentivirus infection as it is related with severe clinical syndrome in an acute period. In experimentally JDV-infected Bali cattle, the death rate was about 21% and occurred within only 1 to 2 weeks post-infection. Indeed, the mortality of more than 60 000 cattles in a year was observed during the first outbreak and the disease is now endemic through-out parts of Indonesia. Early diagnosis constitutes a preventive method of further disease outbreaks. The Jembrana disease can be diagnosed by the clinical symptoms but more reliable diagnostic tools are available, based on either antigen (immunodiagnosis) or viral genome (molecular diagnosis). In this review, we summarize about immunodiagnostic tools of Jembrana disease which has been developed so far.