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Molecular characteristic and pathogenicity of Indonesian H5N1 clade 2.3.2 viruses NLPI, Dharmayanti; R, Hartawan; DA, Hewajuli; ., Hardiman; H, Wibawa; ., Pudjiatmoko
Indonesian Journal of Animal and Veterinary Sciences Vol 18, No 2 (2013)
Publisher : Indonesian Animal Sciences Society

The outbreak of disease in late 2012 in Indonesia caused high duck mortality. The agent of the disease was identified as H5N1 clade 2.3.2. The disease caused economic loss to the Indonesian duck farmer. The clade 2.3.2 of H5N1 virus has not previously been identified, so this study was conducted to characterize 4 of H5N1 clade 2.3.2 viruses by DNA sequencing in eight genes segment virus namely HA, NA, NS, M, PB1, PB2, PA and NP.Â The pathogenicity test of clade 2.3.2 viruses in ducks was compared to clade 2.1.3 viruses which predominat circulating in Indonesia. Results of phylogenetic tree analysis showed that the four of clade 2.3.2 viruses isolated in 2012 was the new introduced virus from abroad. Further analysis showed eight genes were in one group with the clade 2.3.2 viruses, especially those from VietNam and did not belong to Indonesia viruses group. The pathogenicity test in ducks showed that virus H5N1 clade 2.3.2 and clade 2.1.3 have similar clinical symptoms and pathogenicity and cause death in 75% of ducks on days 3-6 after infection. Key Words: H5N1 Virus, Clade 2.3.2, Clade 2.1.3, Phylogenetic Tree, Pathogenecity

Screening test for detection of Newcastle Disease, Avian Influenza and Infectious Bronchitis viruses using multiplex reverse transcriptase polymerase chain reaction approach R, Hartawan; NLPI, DharmayantI
Indonesian Journal of Animal and Veterinary Sciences Vol 18, No 3 (2013)
Publisher : Indonesian Animal Sciences Society

Numerous viral pathogens are circulated in the environment of commercial chicken farms that causing the difficulty in the confirmation of diagnosis. A breakthrough on the diagnosis technique is required in order to deal with multiple viral infections. Ideally, the approach should have not only high accuracy but also economical and straightforward. The objective of this research is to develop a rapid multiplex reverse transcriptase polymerase chain reaction (mRT-PCR) diagnostic method for three common infectious viral diseases in poultry, Newcastle Disease (ND), Avian Influenza (AI) subtype H5N1 and Infectious Bronchitis (IB). This study was successful in developing and optimizing the mRT-PCR for these three pathogens in a single reaction test. Testing 67 field samples from Sukabumi district revealed the presence of several targeted viruses. Key Words: Screening Test, Multiplex RT-PCR, Newcastle Disease, Avian Influenza, Infectious Bronchitis

Identification of Mardivirus Serotypes Circulating in Poultry Farms in Sukabumi and Cianjur District, West Java, 2011 using Multiplex Polymerase Chain Reaction (mPCR) Approach Hartawan, Risza; NLPI, DharmayantI
Indonesian Journal of Animal and Veterinary Sciences Vol 18, No 4 (2013)
Publisher : Indonesian Animal Sciences Society

Three serotypes of Mardivirus had been circulating in the farm environments, these being Marekâs disease virus serotype 1 (MDV-1), Gallid hepesvirus 3 (GaHV3) and herpesvirus of turkey (HVT). However, only MDV-1 poses a significant hazard to the poultry farm. The virus causes a neoplastic syndrome that inflicting severe economic loss to the affected farms. Although vaccination has successfully reduced the frequency and severity of outbreaks, the threat does not disappear since several more pathogenic strains have evolved, and these can overcome protection by vaccination. The aim of this study was to investigate the circulation of three Mardivirus serotypes in commercial poultry farms in Sukabumi and Cianjur district using mPCR approach for the feather samples. A low prevalence of these three serotypes was detected. However, the practice of vaccinating using live attenuated MDV-1 caused difficulty in the investigation. Differentation between virulent field strains and CVI988 vaccine strain using the 132 bp repeat motif attenuation marker within the terminal and inverted repeats flanking the unique long region generated an ambiguous result. Thus, other approaches are required to address this issue, such as selection of other markers, restriction fragment length polymorphism (RFLP), high-resolution melt curve analysis (HRM) and gene sequencing. Key Words: Mardivirus serotype, MDV-1, GaHV3, HVT, multiplex PCR

Efficacy of application of vaccine AI H5N1 clade 2.1.3 on Mojosari ducks challenge against AI H5N1 clade 2.3.2 in laboratory conditions R, Indriani; NLPI, Dharmayanti; R.M.A., Adjid
Indonesian Journal of Animal and Veterinary Sciences Vol 19, No 1 (2014)
Publisher : Indonesian Animal Sciences Society

Influenza virus HPAI H5N1 clade 2.3.2 outbreaks since September 2012 caused high mortality in ducks. Vaccination is one of strategies recommended by government. However, AI H5N1 clade 2.3.2 vaccine not yet available during this research, while AI H5N1 clade 2.1.3 vaccines available in markets. Therefore it was important to do study on efficay of HPAI H5N1 clade 2.1.3. vaccines on duck at laboratory scale. Three groups of Mojosari duck were used in this study, they were 1 group vaccinated with A Vaccine, 1 group vaccinated with B Vaccine, and 1 group as control (not vaccinated). Vacination groups consisted of 9 DOD and control group was consisted of 6 DOD. Vaccination was conducted when the duck at three weeks old of age using single dose recommended by producer. At three weeks later (ducks at 6 weeks old of age) all Groups of ducks were challenged with virus HPAI H5N1 clade 2.3.2 at dose 106 EID50/ml by drops intranasaly. Result showed that Group 1 (vaccinated with A Vaccine) produced 67% protection (3 out of 9 ducks died), Group 2 (vaccinated with B Vaccine) produced 100% protection (non out of 9 ducks died), and Group 3 (control, not vaccinated) produce 0% protection (all of 9 ducks died). This study give an alternative of choise to use AI H5N1 Clade 2.1.3 vaccine with high protection when AI H5N1 Clade 2.3.2 vaccine not available in markets to controll high mortality in ducks caused by HPAI H5N1 clade 2.3.2 outbreaks. Key Words: Duck, HPAI, AI, Avian Influenza, Vaccine

Protection level of AI H5N1 vaccine clade 2.1.3 commercial against AI H5N1 clade 2.3.2 virus from Ducks to SPF chicken in laboratory conditions Indriani R; Dharmayanti NLPI
Jurnal Ilmu Ternak dan Veteriner Vol 20, No 1 (2015): MARCH 2015
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Highly Pathogenic Avian Influenza (HPAI) subtype H5N1 clade 2.3.2 has infected chickens in farms, causing mortality and a decrease in egg production. Vaccination is one of the strategies to control disease of AI subtype H5N1. AI H5N1 clade 2.1.3 vaccine is available commercially. The effectiveness of two vaccines of AI H5N1 clade 2.1.3 (product A and B), and AI H5N1 clade 2.3.2 (Sukoharjo) against AI H5N1 clade 2.3.2 (Sukoharjo) virus SPF chickens was tested in laboratory. Four groups of SPF chickens were used in this study, there were (1) vaccinated with H5N1 clade 2.1.3 (product A), (2) vaccinated with H5N1 clade 2.1.3 (product B), (3) vaccinated with AI H5N1 clade 2.3.2 and (4) unvaccinated (as a control). Each vaccinated group consisted of 10 chicken except 8 chicken for control group. SPF chicken were vaccinated with 1 dose of vaccine at 3 weeks olds, and then after 3 weeks post vaccination (at 6 weeks olds). All group of chicken were challenged with 106 EID50 per 0.1 ml via intranasal. The results showed, chicken vaccinated with H5N1 clade 2.1.3 product A and B gave 100 and 80% protection respectively, but showed challenged virus shedding, whereas vaccine of H5N1 clade 2.3.2 gave 100% protection from mortality and without virus shedding. Vaccines of AI H5N1 clade 2.1.3 product A was better than vaccine product B, and when chicken vaccinated against H5N1 clade 2.3.2, H5N1 clade 2.3.2 vaccine was the best to be used. In order to protect chicken from AI subtype H5N1 clade 2.1.3 and 2.3.2 in the field, a bivalent vaccine of H5N1 clade 2.1.3 and 2.3.2 subtypes should be developed.

Screening test for detection of Newcastle Disease, Avian Influenza and Infectious Bronchitis viruses using multiplex reverse transcriptase polymerase chain reaction approach Hartawan R; DharmayantI NLPI
Jurnal Ilmu Ternak dan Veteriner Vol 18, No 3 (2013): SEPTEMBER 2013
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Numerous viral pathogens are circulated in the environment of commercial chicken farms that causing the difficulty in the confirmation of diagnosis. A breakthrough on the diagnosis technique is required in order to deal with multiple viral infections. Ideally, the approach should have not only high accuracy but also economical and straightforward. The objective of this research is to develop a rapid multiplex reverse transcriptase polymerase chain reaction (mRT-PCR) diagnostic method for three common infectious viral diseases in poultry, Newcastle Disease (ND), Avian Influenza (AI) subtype H5N1 and Infectious Bronchitis (IB). This study was successful in developing and optimizing the mRT-PCR for these three pathogens in a single reaction test. Testing 67 field samples from Sukabumi district revealed the presence of several targeted viruses. Key Words: Screening Test, Multiplex RT-PCR, Newcastle Disease, Avian Influenza, Infectious Bronchitis

Molecular characteristic and pathogenicity of Indonesian H5N1 clade 2.3.2 viruses Dharmayanti NLPI; Hartawan R; Hewajuli DA; Hardiman .; Wibawa H; Pudjiatmoko .
Jurnal Ilmu Ternak dan Veteriner Vol 18, No 2 (2013): JUNE 2013
Publisher : Indonesian Center for Animal Research and Development (ICARD)

The outbreak of disease in late 2012 in Indonesia caused high duck mortality. The agent of the disease was identified as H5N1 clade 2.3.2. The disease caused economic loss to the Indonesian duck farmer. The clade 2.3.2 of H5N1 virus has not previously been identified, so this study was conducted to characterize 4 of H5N1 clade 2.3.2 viruses by DNA sequencing in eight genes segment virus namely HA, NA, NS, M, PB1, PB2, PA and NP. The pathogenicity test of clade 2.3.2 viruses in ducks was compared to clade 2.1.3 viruses which predominat circulating in Indonesia. Results of phylogenetic tree analysis showed that the four of clade 2.3.2 viruses isolated in 2012 was the new introduced virus from abroad. Further analysis showed eight genes were in one group with the clade 2.3.2 viruses, especially those from VietNam and did not belong to Indonesia viruses group. The pathogenicity test in ducks showed that virus H5N1 clade 2.3.2 and clade 2.1.3 have similar clinical symptoms and pathogenicity and cause death in 75% of ducks on days 3-6 after infection. Key Words: H5N1 Virus, Clade 2.3.2, Clade 2.1.3, Phylogenetic Tree, Pathogenecity

Efficacy of application of vaccine AI H5N1 clade 2.1.3 on Mojosari ducks challenge against AI H5N1 clade 2.3.2 in laboratory conditions Indriani R; Dharmayanti NLPI; Adjid R.M.A.
Jurnal Ilmu Ternak dan Veteriner Vol 19, No 1 (2014): MARCH 2014
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Influenza virus HPAI H5N1 clade 2.3.2 outbreaks since September 2012 caused high mortality in ducks. Vaccination is one of strategies recommended by government. However, AI H5N1 clade 2.3.2 vaccine not yet available during this research, while AI H5N1 clade 2.1.3 vaccines available in markets. Therefore it was important to do study on efficay of HPAI H5N1 clade 2.1.3. vaccines on duck at laboratory scale. Three groups of Mojosari duck were used in this study, they were 1 group vaccinated with A Vaccine, 1 group vaccinated with B Vaccine, and 1 group as control (not vaccinated). Vacination groups consisted of 9 DOD and control group was consisted of 6 DOD. Vaccination was conducted when the duck at three weeks old of age using single dose recommended by producer. At three weeks later (ducks at 6 weeks old of age) all Groups of ducks were challenged with virus HPAI H5N1 clade 2.3.2 at dose 106 EID50/ml by drops intranasaly. Result showed that Group 1 (vaccinated with A Vaccine) produced 67% protection (3 out of 9 ducks died), Group 2 (vaccinated with B Vaccine) produced 100% protection (non out of 9 ducks died), and Group 3 (control, not vaccinated) produce 0% protection (all of 9 ducks died). This study give an alternative of choise to use AI H5N1 Clade 2.1.3 vaccine with high protection when AI H5N1 Clade 2.3.2 vaccine not available in markets to controll high mortality in ducks caused by HPAI H5N1 clade 2.3.2 outbreaks. Key Words: Duck, HPAI, AI, Avian Influenza, Vaccine

Identification of Mardivirus Serotypes Circulating in Poultry Farms in Sukabumi and Cianjur District, West Java, 2011 using Multiplex Polymerase Chain Reaction (mPCR) Approach Risza Hartawan; DharmayantI NLPI
Jurnal Ilmu Ternak dan Veteriner Vol 18, No 4 (2013): DECEMBER 2013
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Three serotypes of Mardivirus had been circulating in the farm environments, these being Marek’s disease virus serotype 1 (MDV-1), Gallid hepesvirus 3 (GaHV3) and herpesvirus of turkey (HVT). However, only MDV-1 poses a significant hazard to the poultry farm. The virus causes a neoplastic syndrome that inflicting severe economic loss to the affected farms. Although vaccination has successfully reduced the frequency and severity of outbreaks, the threat does not disappear since several more pathogenic strains have evolved, and these can overcome protection by vaccination. The aim of this study was to investigate the circulation of three Mardivirus serotypes in commercial poultry farms in Sukabumi and Cianjur district using mPCR approach for the feather samples. A low prevalence of these three serotypes was detected. However, the practice of vaccinating using live attenuated MDV-1 caused difficulty in the investigation. Differentation between virulent field strains and CVI988 vaccine strain using the 132 bp repeat motif attenuation marker within the terminal and inverted repeats flanking the unique long region generated an ambiguous result. Thus, other approaches are required to address this issue, such as selection of other markers, restriction fragment length polymorphism (RFLP), high-resolution melt curve analysis (HRM) and gene sequencing. Key Words: Mardivirus serotype, MDV-1, GaHV3, HVT, multiplex PCR

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