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Molecular Characterization of Avian Influenza Virus Strains Circulating in Commercial and Backyard Chickens in Bangladesh.

M. Giasuddin
Animal Health Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka 1341.

M. E. Haque
Department of Pathology, Faculty of Veterinary Science,Bangladesh Agricultural University, Mymensingh-2202.

R. Parvin
Department of Pathology, Faculty of Veterinary Science,Bangladesh Agricultural University, Mymensingh-2202.

A. R. Bhuiyan
Department of Pathology, Faculty of Veterinary Science,Bangladesh Agricultural University, Mymensingh-2202.

M. A. Samad
Animal Health Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka 1341.

J. Alam
Animal Health Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka 1341.

E. H. Chowdhury
Department of Pathology, Faculty of Veterinary Science,Bangladesh Agricultural University, Mymensingh-2202.

P. Monura
Animal Health Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka 1341.

M. J. F. A. Taimur
Animal Health Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka 1341.

M. R. Islam
Department of Pathology, Faculty of Veterinary Science,Bangladesh Agricultural University, Mymensingh-2202.

Abstract/ Executive Summary:

For molecular characterization and phylogenetic analysis, RNA extracted from four selected H5NI isolates of 2007, 2008, 2009, 2010 respectively. In the first step Bangladeshi sequences were compared with the sequences selective strains representing all clades and sub-clades of H5NI viruses. The results confirmed that all Bangladeshi strains belong to Clade 2.2. In the second step, sequences of Bangladeshi strains were aligned with that of clade 2.2 H5NI strains of other countries. All the four Bangladeshi isolated were found 100% identical and clustered together along with the viruses from West Bengal and Assam of India indicating that the same virus is in circulation since 2007.

Keywords: Molecular characterization, Avian influenza, Virus strains, Commercial and backyard chickens

Location: Animal Health Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka 1341.

Start Date: 01-07-2009

End Date: 30-06-2010

Program Area: Animal Health and Management

Commodity: Chicken

Objectives:

1. Epidemiological study Identification of potential reservoirs. 2. Molecular characterization, phylogenetic analyses and genotype determination of the Bangladeshi isolates of AI virus. 3. In vitro expression of haemagglutinin and neuraminidase proteins of Bangladeshi strains of HPAI.

Methodology:

1. Molecular characterization, phylogenetic analyses and genotype determination Amplification of cDNA: All eight genome segments of selected isolates of H5NI AI viruses will be amplified by RT-PCR Primers will be selected from published literature (Guan et al., 2002) and synthesized commercially. RNA will be extracted and RT-PCR will be conducted following protocols described in the literature and using commercially available kits. Cloning and sequences of cDNA: Amplified cDNA corresponding to each genome segments will be cloned in plasmid vectors using TA cloning protocol. Transformed Escherichia coli containing recombinant plasmid will be grown overnight and plasmids will be extracted. Plasmid DNA will be purified and insert cDNA will be sequenced from a commercial sequencing laboratory using appropriate primers. Determined sequences of each genome segments of Bangladeshi isolates will be compared with the corresponding sequences of other HPAI strains of different countries using computer software. Such sequences will be downloaded from the Gene Bank. Phylogenetic analysis will be performed and based on this analysis the genotype of Bangladeshi isolates will be determined. 2. Epidemiological study Identification of potential reservoirs: Blood samples will be collected from duck and pig of three epicenter of avian influenza. Samples will be tested by ELISA kit. Virological samples also collect and tested by RT PCR to tress out the potential source of infection. 3. In vitro expression of haemagglutinin and neuraminidase proteins: (1) cDNA corresponding to the coding region of the haemagglutinin and neuraminidase gene will be transferred to an expression vector suitable for E. coli in vitro expression system. (2) Recombinant proteins will be expressed in E. coli and the protein will be extracted by column chromatography. (3) The proteins will be identified by western blotting using appropriate antibodies.

Source of Information: blri. Proceedings of the Annual Research Review Workshop 2009-2010, Bangladesh Livestock Research Institute, Savar, Dhaka 1341.

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

To find out the potential reservoir hosts, 1500 cloacal awab and 500 sera samples from native ducks and 250 fecal samples from wild migratory birds have been collected. Out of one thousand five hundred clocal swabs 17 samples showed haemagglutinin positive and three samples were confirmed influenza A positive by RT PCR. On the other hand 32% duck was found sero positive to influenza A virus. For molecular characterization and phylogenetic analysis, RNA extracted from four selected H5NI isolates of 2007, 2008, 2009, 2010 respectively. The extracted RNA was subjected to RT-PCR for H5 and NI gene using previously standardized protocols. In the first step Bangladeshi sequences were compared with the sequences selective strains representing all clades and sub-clades of H5NI viruses. The results confirmed that all Bangladeshi strains belong to Clade 2.2. In the second step, sequences of Bangladeshi strains were aligned with that of clade 2.2 H5NI strains of other countries. All the four Bangladeshi sequences clustered together along with the isolates from West Bengal and Assam of India indicating that the same virus is in circulation since 2007 in this Genetic plane. However, 2006 isolates from western India and 2007 isolate from Manipur of India clustered separately indicating at least three separate introduction of HPAI in India. For full-length cloning of all genome segments, the protocols have now been standardized, which included the procedure for amplification of all the eight full-length genome segments, ligation of RT-PCR products in pCR4 TOPO cloning vector, preparation of competent E. coli transformation of competent E. coli with recombinant plasmids and isolation and analysis of plasmids form transformed E. coli. The cloning of full-length genes is in progress.

Knowledge Management: Report/Proceedings