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Monitoring of Highly Pathogenic Avian Influenza Virus Circulating in Bangladesh and in Vitro Expression of Viral Proteins

M. Giasuddin
Animal Health Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka.

M. H. Rahman
Animal Health Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka.

M. Hasan
Animal Health Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka.

M.R. Islam
Proceedings of the Annual Research Review Workshop 2013-2014, BLRI, Savar, Dhaka. Publication No.261, pp: 270-278, October-2015.

E. H. Chowdhury
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensigh, Bangladesh.

M. E. Haque
Proceedings of the Annual Research Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensigh, Bangladesh.

M.J.F.A. Taimur
Proceedings of the Annual Research Review Workshop 2013-2014, BLRI, Savar, Dhaka. Publication No.261, pp: 270-278, October-2015.Animal Health Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka.

Abstract/ Executive Summary:

Highly pathogenic avian influenza (HPAI) causes enormous losses to the poultry industry of Bangladesh every year since the detection of first outbreak in 2007. In this study molecular characterization and phylogenetic analysis of circulating HPAI viruses from field outbreaks were determined as well as virological surveillance for avian influenza viruses in migratory birds and domestic poultry, including cloning and in vitro expression of structural proteins of HPAI were performed. A total 21 isolates from different species were sequenced which were collected from different areas of Bangladesh since 2007. Sequencing was done on HA gene. Topology of the phylogenetic tree shows that the all H5N1 viruses under these studies belong to the 2.2, 2.3.4 and 2.3.2.1 clade and similarity ranged between 97.4% and 100%. It was observed that from 2011, two new clades 2.3.2.1 and 2.3.4 viruses introduced in Bangladesh in addition to clade 2.2 viruses that have been in circulation since 2007. For AI monitoring purpose, a total of 1135 samples including 280 from migratory birds, 680 from live bird market (LBM) chickens, 118 from ducks and 57 from pigeons were collected from different locations of Bangladesh during the period from July 2013 to June 2014. All the samples were tested by real time R T - PCR for Type A influenza virus matrix gene. Out of 1135 samples, 69 were found positive for influenza A, of which 11 were from migratory birds (11/280; 3.93%), 48 from LBM chickens (48/680; 7.06%), 7 from ducks (7/118; 6.0%) and 3 from pigeons (3/57; 5.26% ). In addition, 741 Influenza A positive samples collected by different institutes under the FAO-led surveillance program were also received at BLRl for further analysis. The real time RT-PCR positive samples were inoculated in 10-day-old embryonated chicken eggs. Out of 810 (741+ 69) influenza A positive samples, 110 were selected on the basis of species and region to cover the whole country and were sent to the OlE reference laboratory for detailed study. The bio-molecular results revealed that in case of migratory bird samples one was H5 (HP AI), two were H9 and eight were of undetermined subtype. Among the LBM chicken samples, seven were H5 (HPAI), 51 were H9, six were mixed infection with H5 & H9 and three were of undermined subtype. In case of duck samples, one was H2, six were H4, six were H5 (HP AI), one with mixed infection with H5 & H2, one with both H5 & H4, two were H9 and one was H7N5, though H7 was genetically distinct from that of the novel H7N9 , virus of China. Among quail samples, one was H5 (HPAI) and one was H9. In case of pigeon samples, all three contained H9. From this study it was revealed that HPAI (H5N1) is still circulating in our poultry population. For cloning and in vitro expression study the full length HA gene of selected HPAI isolates has been amplified by RT-PCR and cloned in plasma vector by TA cloning method. The full length sequence of the cloned insert has been established. For in vitro expression of HA protein, specific primers have been designed and synthesized. The protocol of cloning cDNA corresponding to HA coding region in expression vector is now in the process of optimization.

Keywords: Avian influenza, H5Nl, Surveillance, Phylogenetic analysis, Bangladesh

Location: Department of Pathology of Bangladesh Agricultural University, Mymensingh.

Start Date: 00-07-2013

End Date: 00-06-2014

Program Area: Animal Health and Management

Commodity: Poultry

Objectives:

To conduct virological surveillance in migratory water fowls and their immediate contacts (domestic water fowls) for possible introduction of new viruses or possible re-combination.

Methodology:

Samples were collected from new outbreak site of different areas of the country by using standard protocol and brought in the laboratory for further characterization. HA genome segments of selected isolates of H5N1 AI viruses were amplified by RT-PCR. Primers were selected from published literature and synthesized commercially. RNA was extracted and RT-PCR was conducted following protocols described in the literature and using commercially available kits. The sequence of haemagglutinin gene of isolated viruses was determined by using standard protocol. RNA was extracted from HPAI/LPAI isolates, obtained from allantoic fluid of the inoculated embryos which was positive to HA test, using Qiagen RNA extraction kit. HA gene fragment was amplified by RT-PCR. The PCR product was purified with the EZ-10 Spin Column DNA Gel Extraction kit (BIO BASIC INC) and EZ-10 spin Column PCR purification kit (BIO BASIC INC). The cleaned RT-PCR product was sequenced directly from a commercial source using RT-PCR primers. Amplified cDNA was sequenced using the sequencing primers and Dye-Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA polymerase Cycle reaction products analyzed in a gene sequencer at a commercial laboratory. Determined sequences of each genome segments of Bangladeshi isolates were compared with the corresponding sequences of other HP AI strains of different countries using computer software. Such sequences were downloaded from the Gene Bank. Phylogenetic analysis was performed and based on this analysis the genotype of Bangladeshi isolates was determined. Amplified cDNA corresponding to each genome segments were cloned in plasmid vectors using TA cloning protocol. Trasformed Escherichia coli containing recombinant plasmids were grown overnight and plasmids were extracted. Plasmids DNA were purified and insert cDNA were sequenced from a commercial sequencing laboratory using appropriate primers. A total of 1135 samples including 280 environmental fecal samples from migratory birds, 680 swabs samples from chickens of live bird market (LBM), 118 swabs samples from native ducks and 57 swab samples from pigeons were collected randomly during the period from July 2013 to June 2014 from different locations of Bangladesh and transported to the NRL-AI. About 741 Influenza A positive samples were received by NRL-AI from a FAO program consisting 05 samples from the Department of Pathology of Bangladesh Agricultural University (BAU), 34 samples from CDIL, 656 from the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) and 46 from Chittagong Veterinary and Animal Sciences University (CVASU) for further analysis. These samples were collected from different species including chickens, ducks, pigeons and quails from different areas of Bangladesh under the FAO-Ied surveillance program in the year of 2013. Real time RT -PCR positive samples were inoculated in ten days old embryonated chicken eggs through allantoic route and were incubated at 37°C for 48-72 hours. Allantoic fluid was harvested from eggs after death of the embryos or at the end of the incubation period. Allantoic fluid was subjected to haemagglutination (HA) test. The HA positive sample was further tested by the RT-PCR for confirmation of AI virus using specific primer sets from published literature. One hundred ten (110) representative influenza A positive samples were selected on the basis of species and region to cover the whole country and were sent to the OlE reference laboratory, Padova, Italy for further detailed study.

Source of Information: Proceedings of the Annual Research Review Workshop 2013-2014, BLRI, Savar, Dhaka. Publication No.261, pp: 270-278, October-2015.

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

From 2007-2012 a total of 556 clinical outbreaks of HPAI H5N1 were confirmed, but the submi of suspected samples were remarkably declined in 2013-2014. The study revealed that introduction two new clades 2.3.2.1 and 2.3.4 viruses in to Bangladesh in addition to clade 2.2 viruses that has in circulation since 2007. It was observed that clade 2.2 viruses are being replaced mainly by c. 2.3.2.1 viruses. From virological surveillance it was revealed that HPAI (H5N1) is still circulating our poultry population and no novel avian influenza virus H7N9 was found during this study, results of our study emphasize the need of continuous surveillance activities in Bangladesh that WI allow detection of any newly emerged AIV with pandemic potential.

Knowledge Management: Report/Proceedings