Present status of avian influenza virus in Bangladesh Md. Giasuddin, PhD Director National Reference Laboratory for Avian Influenza & Head Animal Health Research Division Bangladesh Livestock Research Institute (BLRI) Savar, Dhaka, Bangladesh

Introduction Influenza viruses are enveloped, negative sense, single stranded RNA viruses. They have 2 Pathotypes :HPAI and LPAI. HPAI epidemic started in late 2003 from south east . The disease has spread in Asia to all over the world including 63 countries of 5 continents. The persistence of the virus in poultry may increase the risk to mutant and evolve to initiate a human pandemic. Mutation LPAI HPAI

Sample Collection Clinical samples: Tracheal tissue collected by FDIL & CDIL and send to NRL-AI. Only 05 samples submissions from suspected HPAI outbreaks were received during the period from July 2013 to up to June 2014. Surveillance samples: Cloacal, Oropharyngeal & fecal samples collected from ducks, chickens and migratory birds. Wild birds Native birds Species Sample Type Sample No. Migratory Bird Environmental feces 280 LBM Chicken Swab 680 Duck 118 Pigeon 57 Total 1135

Influenza A positive samples from other laboratories NRL-AI were received influenza A positive samples from other laboratories under the FAO lead program. Sample Sources Sample Types Sample No. Department of Pathology, BAU Influenza A positive 03 Department of Microbiology, BAU 02 CVASU, Chittagong 46 CDIL, Dhaka 34 icddr,b 656 Total 741

Processing of Samples Clinical samples: The tracheal tissues were processed using sterile PBS and centrifuged @3000 rpm for 8 min. Then supernatant fluid used for RNA extraction Surveillance samples: Samples were pooled (1:5 ratio) for initial screening. Then positive pool were then tested individually for identifying the individual positive samples.

Automated NA Extraction RNA Extraction Extraction of viral RNA was performed by- Automated nucleic acid extraction method (Thermo Scientific Kingfisher ML). Manual extraction method with Qiagen RNeasy One-step Mini Kit. Reagents for RNA extraction (Qiagen) Manual RNA Extraction Automated NA Extraction

Molecular Detection of AI Master mix Preparation Master mix was prepared by Ambion AgPath_IDTM One-step RT-PCR Kit or Qiagen one step RT- PCR kit. Real Time PCR (RRT-PCR) The samples were directly tested by RRT-PCR with 7500 Real Time Fast Machine. Master mix Preparation Template mixing RRT-PCR Results

Culture of AI virus The RRT-PCR positive samples were inoculated into 10 days old chicken embryos. Haemagglutination test Virus Inoculation HA positive sample were then subjected to Real time RT-PCR for confirmation of AI virus. HA Test

Results (Clinical) From 2007-2012 a total of 556 In 2013, only three cases June 2014, two clinical cases of HPAI H5N1 were diagnosed in samples submitted to BLRI.

Results (Surveillance) For virological surveillance, out of 1135 samples from migratory birds and domestic poultry. The RRT-PCR results are : Species Sample No. Influenza A Positive Positive (%) Migratory Bird 200 11 5.5 LBM Chicken 680 48 7.06 Duck 118 07 6.0 Pigeon 57 03 5.26 Total 1135 69 6.19 NRL-AI selected 110 influenza A positive samples from 810 (741+69) influenza A positive samples on the basis of species and region. Finally sent to the OIE Reference Laboratory, Padova, Italy for detail study with the help of FAO.

Contd. Bio-molecular results of OIE Reference Laboratory, Padova, Italy for 110 representative influenza A positive samples: Species Test Results Influenza A H2 H4 H5 H5& H2 H5 & H4 H5 & H9 H7N5 H9 Total Migratory bird 05 - 01 02 08 LBM chickens 03 07 06 51 67 Duck 12 30 Quail Pigeon 20 15 59 110 One sample from duck tested positive for H7N5 (LPAI) which is genetically distinct from H7N9 of China.

Conclusion From our study it was revealed that: HPAI (H5N1) is still circulating in our poultry population. The high prevalence of H9N2 in poultry market may provide the opportunity of human infections by reassortment with the existing HPAI H5N1. No novel avian influenza virus H7N9 was found during this study. This study emphasize the need of continuous surveillance activities that would allow detection of any newly emerged AIV with pandemic potential.