1. 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

2. 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

3. 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

4. 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

5. Processing of Samples Clinical samples:
The tracheal tissues were processed using sterile PBS and 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.

6. 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

7. 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

8. 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

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

10. 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.

11. 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.

12. 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.