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Detection of avian influenza virus H5N1 subtype in organs of chicken affected by higly pathogenic avian infuenza in East and West Java by using immunohistochemical technique Damayanti, R; Dharmayanti, N.L.P.I; Indriani, R; Wiyono, A; ., Darminto
Indonesian Journal of Animal and Veterinary Sciences Vol 9, No 3 (2004)
Publisher : Indonesian Animal Sciences Society

The study was conducted to detect antigen H5N1 of highly pathogenic Avian Influenza (HPAI) virus in various farms in East and West Java. The immunohistochemical technique was applied due to Hematoxilin-eosin (H&E) staining was impossible to visualize the antigen in tissue. Immunohistochemical staining was applied for some visceral organs collected from the areas where the outbreaks occurred in September-October 2003. The specimens were processed as histopathological paraffin blocks using standard method. The blocks that were suspected to have antigen H5N1 were cut and rabbit antisera to H5N1 produced from the local isolate was applied as the primary antibody. Biotinylated secondary antibody and avidin biotin peroxidase from a commercial kit were administered. The antigen present in the tissues were visualized by adding a substrate called Amino Ethyl Carbazole (AEC) resulting in reddish brown colour. This immunostaining proved to be accurate and reliably quick method to detect H5N1 antigen present in the avian tissues. In conclusion, the outbreak of bird flu was caused by H5N1 strain and the antigen could be found in wattles, combs, brain, trachea, lungs, heart, proventriculus, liver, spleen, kidney and ovary. Â Key words: Highly pathogenic Avian Influenza (HPAI), chicken, H5N1, outbreak, immunohistochemistry

The clinico-pathological effects of chicken infected with highly pathogenic avian influenza in some farms located in East Java and West Java Damayanti, R; Dharmayanti, NLP.I; Indriani, R; Wiyono, Achmad Selamet; ., Darminto
Indonesian Journal of Animal and Veterinary Sciences Vol 9, No 2 (2004)
Publisher : Indonesian Animal Sciences Society

The study was conducted to investigate the clinico-pathological features of highly contagious disease occurred in chicken located in East and West Java during the outbreak in September-October 2003. Six farms located in Districts of Surabaya, Malang and Blitar of East Java had been visited. They were mainly commercial layer, breeder layer and breeder broiler, which the population was between 14.000, 80.000, and aged 17-70 weeks. Where as five farms in West Java (Districts of Bogor, Bekasi and their surrounding areas) were visited and consisted of commercial layer and breeder broiler, having population of 3000-16.000 and aged 11-53 weeks. Observation was made according to clinical, gross pathological and histopathological changes. Clinically, most of them had cyanotic wattle and comb and subcutaneous petechiation of non-feathered part of the legs. These were also seen in necropsy, accompanied by general circulatory disturbances in most organs: namely pectoral and thigh muscle, trachea, lungs, epicard, myocard, proventriculus, liver, kidney and ovary. In addition, the liver was congested, friable and necrotic in some parts. Histologically, hemorrhage and non suppurative inflammatory reaction were observed in the brain, skin (comb, wattle and non feathered leg), skeletal muscle, trachea, lung, heart, proventriculus, liver, kidney and ovary whereas vasculitis was found especially in the skin of the wattle and comb, brain and kidney. It is concluded that based on the clinicopathological findings the outbreak of poultry disease in East and West Java were attributed to highly pathogenic avian influenza. Â Key words: Highly pathogenic avian influenza (HPAI), chicken, clinico-pathology, outbreak, East Java, West Java

Identification of avian influenza virus of Indonesian isolates by reverse transcriptase polymerase chain reaction (RT-PCR) method Dharmayanti, N.L.P.I.; Damayanti, R; Wiyono, A; Indriani, R; ., Darminto
Indonesian Journal of Animal and Veterinary Sciences Vol 9, No 2 (2004)
Publisher : Indonesian Animal Sciences Society

An outbreak of avian influenza in Indonesia was reported at the first time at the beginning of September 2003 causing high mortality among poultry population especially commercial layer chicken farms in Java, Sumatra and Bali islands. From the outbreaks highly pathogenic avian infuenza viruses have been isolated and characterized by rapid, HA, HI and AGP tests. However, these isolates are still needed to be further molecularly characterized. The aim of this study is to identify by further subtyping the avian viruses by means of RT-PCR using Matrix, H7 and H5 primers. The study reveals that the RT-PCR using Matrix primer amplified a 200-300 basepairs (bp) Jawa Timur isolates were collected from East Java, while Jawa Barat isolates were from West Java. The RT-PCR using H7 primers did not amplify any product, while H5 primer amplified a 500-600 bp product from the isolates. It is concluded that the outbreak of poultry disease in East and West Java was caused by an avian influenza H5 subtype. Â Key words: Identification, avian influenza virus, RT-PCR, H5 subtype

Indonesian avian influenza viruses character in second wave epidemic Dharmayanti, N.L.P.I.; Indriani, R; Damayanti, R; Wiyono, A; Adjid, R.M.A.
Indonesian Journal of Animal and Veterinary Sciences Vol 10, No 3 (2005)
Publisher : Indonesian Animal Sciences Society

Second wave of epidemic avian influenza occurred from December 2004 until April 2005. In March 2005, the disease had infected some districts in South Sulawesi such as Wajo and Sopeng. More than 21 field isolates have been collected and identified as avian influenza virus subtype H5N1. In this study further characterized was undertaken for 14 isolates of avian influenza using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and sequencing in region of HA1 gene. It was then followed by genetic analysis to identify the mutation and phylogenetic relationship of the isolates. The study indicates that the Indonesia isolates collected in second wave epidemic are generally having a different group to the isolates group in 2003 and 2004. There is point mutation in the nucleotide sequence of the isolate collected at August 2004-March 2005, that is the replacement of adenine by guanine in the position of 195. Â Â Key Words: Avian Influenza Virus, Second Epidemic Wave, Mutation

Development inactivated vaccine prototype of avian influenza (AI) H5N1 local isolate and its application at laboratory level Indriani, R; Dharmayanti, N.L.P.I; Syafriati, T; Wiyono, A; Adjid, R.M.A
Indonesian Journal of Animal and Veterinary Sciences Vol 10, No 4 (2005)
Publisher : Indonesian Animal Sciences Society

A preliminary study related on vaccine safety and vaccination effectivity for controlling avian influenza (AI) subtype H5N1 was carried out at Virology Laboratorium, Indonesian Veteriner Institute, Bogor. A Prototype of inactivated vaccine was made using AI H5N1 local isolate (A/Chicken/West Java/67-2/2003). The vaccine was then tested for safety and protection in DOC of layers. Antibody response, protection and shedding virus challenge were observed in the experiment. Result showed that the vaccine was saved and protected against virulent viral challenge. Efective vaccination was achieved at 3 weeks chicken old started with low level of antibody. Antibody titre increased gradually and reached the top at 8 weeks post vaccination. Challenge test using AI virulent at the age of 4 and 8 weeks post vaccination showed that the vaccine gave high protection (90%). Viral shedding was not longer expressed than 7 days after challenge. It is concluded that this prototype is a satisfied AI vaccine in laboratory level. Â Â Key Words: Vaccine, Avian Influenza, H5N1, HPAI

Molecular characterization of Indonesia avian influenza virus Dharmayanti, N.L.P.I.; Damayanti, R; Indriani, R; Wiyono, A; Adjid, R.M.A.
Indonesian Journal of Animal and Veterinary Sciences Vol 10, No 2 (2005)
Publisher : Indonesian Animal Sciences Society

Avian influenza outbreaks in poultry have been reported in Java island since August 2003. A total of 14 isolates of avian influenza virus has been isolated from October 2003 to October 2004. The viruses have been identified as HPAI H5N1 subtype. All of them were characterized further at genetic level and also for their pathogenicity. Phylogenetic analysis showed all of the avian influenza virus isolates were closely related to avian influenza virus from China (A/Duck/China/E319-2/03(H5N1). Molecular basis of pathogenicity in HA cleavage site indicated that the isolates of avian influenza virus have multiple basic amino acid (B-X-B-R) indicating that all of the isolates representing virulent avian influenza virus (highly pathogenic avian influenza virus). Â Â Key Words: Avian Influenza Virus, Molecular Characterization, Poultry, Indonesia

Genetic characteristic of protein membran of avian influenza viruses H5N1 subtype Dharmayanti, N.L.P Indi; Hewajuli, D.A; Ratnawati, A; Indriani, R; ., Darminto
Indonesian Journal of Animal and Veterinary Sciences Vol 15, No 3 (2010)
Publisher : Indonesian Animal Sciences Society

In 2006-2008 there were findings about the antigenic drift on AI virus due to vaccination and the AI H5N1 subtype viruses which was similar to H5N1 viruses in human. The findings indicated that the AI viruses continue and undergoing to mutate and try to adapt with their environment.Â The objective of this study was to characterize the mutation of recent AI viruses (2009) on the membran protein namely Hemagglutinin (HA), Neuraminidase (NA) and Matrix 2 (M2). In this study RT-PCR â sequencing methods and genetic analysis for the protein membran of AI viruses were used. Result revealed that there were specific mutation belong to AI 2009 viruses on HA and NA protein such as AI virus mutation in 2008 which was isolated from backyard chicken. The mutations were non synonimous and not caused by immunological pressure. Furthermore, M2 analysis indicated that the viruses were resistant to amantadine. Key Words: Mutation, AI Subtype H5N1 Viruses, Membran Protein

Purification and production of monospecific antibody to the hemagglutinin from Subtype H5N1 influenza virus Tarigan, Simson; Indriani, R; Hewajuli, D
Indonesian Journal of Animal and Veterinary Sciences Vol 15, No 4 (2010)
Publisher : Indonesian Animal Sciences Society

The purpose of this study was to purify the hemagglutinin from H5N1 virus and to generate monospecific antibody appropriate for production of sensitive and specific immunoassay for H5N1 avian influenza. For this purpose, a local isolate H5N1 virus (A/Ck/West Java/Hamd/2006) was propagated in chicken embryos. The viral pellet was dissolved in a Triton-X-100 solution, undissolved viral particles were pelleted by ultracentrifuge, and the supernatant containing viral surface glycoproteins (Hemagglutinin and neuraminidase) was collected. The neuraminidase in the supernatant was absorbed by passing the supernatant through an Oxamic-acid-superose column. After dialyzing extensively, the filtrate was further fractionated with an anion exchange chromatography (Q-sepharose) column. Proteins adsorbed by the column were eluted stepwisely with 0.10, 0.25, 0.25 and 0.75 M NaCl in 20 mM Tris, ph 8. Hemagglutinin (H5) was found to be eluted from the column with the 0.5 M NaCl elution buffer. The purified H5 was free from other viral proteins based on immunoassays using commercial antibodies to H5N1 nucleoprotein and neuraminidase. When used as ELISAâs coating antigen, the purified H5 proved to be sensitive and specific for hemagglutinin H5. Cross reactions with other type-A-influenza virus, H6, H7 dan H9, were negligibly low. For the production of monospecific antiserum, the purified H5 was separated with SDS-PAGE, the band containing the H5 monomer was cut out , homogenised and injected into rabbits. The antiserum was capable of detecting the presence of inactivated H5N1 virus in a very dilute suspension, with a detection limit of 0.04 heagglutination (HA) unit. The purified hemagglutinin and the serum raised against it should be useful for developing specific, sensitive and affordable immunoassay for H5N1 avian influenza. Key Words: H5N1, Hemagglutinin, Triton, Oxamic-acid Sepharose, Q sepharose, ELISA

Isolation and characterization of virus of highly pathogenic avian influenza H5 subtype of chicken from outbreaks in Indonesia Wiyono, Agus; Indriani, R; Dhamaryanti, N.L.P.I; Damayanti, R; Parede, L; Syafriati, T; ., Darminto
Indonesian Journal of Animal and Veterinary Sciences Vol 9, No 1 (2004)
Publisher : Indonesian Animal Sciences Society

A study on the isolation and characterization of Highly Pathogenic Avian Influenza of chicken from outbreaks in Indonesia was conducted at Indonesian Research Institute for Veterinary Science. Outbreaks of avian disease had been reported in Indonesia since August 2003 affecting commercial layer, broiler, quail, and ostrich and also native chicken with showing clinical signs such as cyanosis of wattle and comb, nasal discharges and hypersalivation, subcutaneous ptechiae on foot and leg, diarre and sudden high mortality. The aim of this study is to isolate and characterize the causal agent of the disease. Samples of serum, feather follicle, tracheal swab, as well as organs of proventriculus, intestine, caecal tonsil, trachea and lungs were collected from infected animals. Serum samples were tested haemaglutination/haemaglutination inhibition to Newcastle Disease and Egg Drop Syndrome viruses. Isolation of virus of the causal agent of the outbreak was conducted from samples of feather follicle, tracheal swab, and organs using 11 days old specific pathogen free (SPF) embryonated eggs. The isolated viruses were then characterised by agar gel precipitation test using swine influenza reference antisera, by haemaglutination inhibition using H1 to H15 reference antisera, and by electron microscope examination. The pathogenicity of the viruses was confirmed by intravenous pathogenicity index test and its culture in Chicken Embryo Fibroblast primary cell culture without addition of trypsin. The study revealed that the causative agent of the outbreaks of avian disease in Indonesia was avian influenza H5 subtype virus based upon serological tests, virus isolation and characterization using swine influenza reference antisera, and electron microscope examination. While subtyping of the viruses using H1 to H15 reference antisera suggested that the virus is very likely to be an avian influenza H5N1 subtype virus. The pathogenicity test confirmed that the viruses are highly pathogenic to experimental animals. It is concluded that the causative agent of the outbreaks of avian disease in Indonesia was avian influenza H5 subtype virus. The result has been the basis of further study such as development serological tests and vaccine production. The decission of Indonesian Government to conduct vaccination program using homolog vaccine in order to control the disease is regarded as the correct choice. However, it should be accompanied by conducting surveillance and monitoring of the disease as well as the possibility of mutation of virus. The program should be coordinated nationally. Â Key words: Virus isolation, characterization, chicken, outbreak, highly pathogenic avian influenza (HPAI), H5 subtype, Indonesia

Indonesian avian influenza viruses character in second wave epidemic N.L.P.I. Dharmayanti; R Indriani; R Damayanti; A Wiyono; R.M.A. Adjid
Jurnal Ilmu Ternak dan Veteriner Vol 10, No 3 (2005): SEPTEMBER 2005
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Second wave of epidemic avian influenza occurred from December 2004 until April 2005. In March 2005, the disease had infected some districts in South Sulawesi such as Wajo and Sopeng. More than 21 field isolates have been collected and identified as avian influenza virus subtype H5N1. In this study further characterized was undertaken for 14 isolates of avian influenza using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and sequencing in region of HA1 gene. It was then followed by genetic analysis to identify the mutation and phylogenetic relationship of the isolates. The study indicates that the Indonesia isolates collected in second wave epidemic are generally having a different group to the isolates group in 2003 and 2004. There is point mutation in the nucleotide sequence of the isolate collected at August 2004-March 2005, that is the replacement of adenine by guanine in the position of 195. Key Words: Avian Influenza Virus, Second Epidemic Wave, Mutation

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