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Molecular Evolution of H5n1 Highly Pathogenic Avian Influenza Viruses in Bangladesh Between 2007 and 2012

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

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

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

M. Giasuddin
Principal Scientific Officer
National Reference Laboratory for Avian Influenza, Bangladesh Livestock Research Institute, Savar, Bangladesh

Abstract/ Executive Summary:

In Bangladesh, highly pathogenic avian influenza (HPAI) virus subtype H5N1 was first detected in February 2007. The objective of the present study was to investigate the molecular evolution of H5N1 HPAI viruses during 2007 to 2012. In addition, full-length sequences of different gene segments of other Bangladeshi H5N1 isolates available in GenBank were included in the analysis. The analysis revealed that the first introduction of clade 2.2 virus in Bangladesh in 2007 was followed by the introduction of clade 2.3.2.1 and 2.3.4 viruses in 2011. However, only clade 2.3.2.1 viruses could be isolated in 2012, indicating progressive replacement of clade 2.2 and 2.3.4 viruses. There has been an event of segment re-assortment between H5N1 and H9N2 viruses in Bangladesh, where H5N1 virus acquired the PB1 gene from a H9N2 virus. Point mutations have accumulated in Bangladeshi isolates over the last 5 years with potential modification of receptor binding site and antigenic sites. Extensive and continuous molecular epidemiological studies are necessary to monitor the evolution of circulating avian influenza viruses in Bangladesh.

Keywords: H5N1, Avian influenza viruses, Bangladesh

Location: Bangladesh Livestock Research Institute, Savar, Dhaka

Start Date: 01-01-2007

End Date: 31-12-2012

Program Area: Animal Health and Management

Commodity: Chicken

Objectives:

To investigate the molecular evolution of H5N1 HPAI viruses during 2007 to 2012.

Methodology:

Virus isolates. A total of 21 H5N1 avian influenza virus isolates were subjected to molecular characterization in this study. They were propagated in embryonated chicken eggs. The allantoic fluid was harvested, stored in aliquots at −70°C and used in the subsequent study. Amplification of partial or full-length genome segments by reverse transcriptase-polymerase chain reaction. Partial H5 (545 base pairs) and N1 (343 base pairs) gene fragments were first amplified from all 21 HPAI virus isolates for reconfirmation of the virus identity. RNAwas extracted from the isolates using the Qiagen RNA extraction kit (Qiagen, Hilden, Germany). The partial H5 and N1 gene fragments were amplified with the Qiagen one-step reverse transcription-polymerase chain reaction (RT-PCR) kit using the primers H5-155F (5′-ACA CAT GCY CAR GAC ATA CT-3′) and H5-699R (5′-CTY TGR TTYAGT GTT GAT GT-3′) for the H5 gene fragment and N1-580-607F (5′-TGAAGT ACA ATG GCA TAA TAA CWG ACA C-3′) and N1-891-918R (5′-CAC TGC ATA TAT ATC CTA TTT GAT ACT CC-3′) for the N1 gene fragment (WHO, 2007), respectively. The RT-PCR products were analysed by electrophoresis on 1.5% agarose gel stained with ethidium bromide. For amplification of full-length genome segments from selected isolates, a two-step RT-PCR was followed. First-strand cDNA was synthesized from extracted RNA using Uni-12 primer and the ImProm II First Strand cDNA Synthesis Kit (Promega, Madison, WI, USA) as per the manufacturer’s instructions. Synthesized first-strand cDNA was used as the template for amplification of different full-length gene segments by PCR using gene-specific primers. PCR products were analysed by electrophoresis on 1% agarose gel stained with ethidium bromide. Cloning and sequencing. The PCR products were purified with the Wizard® SV Gel and PCR Clean-Up System (Promega) and were subjected to direct sequencing with respective PCR primers. Alternatively, the cleaned PCR products were cloned by the TA cloning method using the pGEM®-T Easy Vector System Cloning Kit and chemically competent E. coli JM109 (Promega). The plasmid DNA was isolated and purified with EZ Spin Column and the cloned cDNA was sequenced with universal primers. If needed, overlapping primers were used to sequence longer gene segments. Sequencing was carried out from a commercial laboratory. Molecular and phylogenetic analysis. All sequences have been deposited into GenBank. In addition to the sequences generated in the present study, full-length sequence data of other Bangladeshi H5N1 avian influenza virus isolates that are available in the GenBank were downloaded and used in the analysis. In the case of the HA gene, representative strains from neighbouring countries such as India, Nepal, Bhutan and Myanmar were also included in the phylogenetic analysis. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. A bootstrap resampling (1000 replications) was used to assess the robustness of individual nodes of the phylogeny, incorporating the maximum likelihood substitution model defined above. Any sequence appearing as a distinct outgroup was subjected to a BLAST (Basic Local alignment Search Tool) search in the GenBank database to look for its closest neighbours. The deduced amino acid alignment table for each gene segment was thoroughly examined for any substitution that might be of biological significance.

Source of Information: Avian Pathology, 2014; Vol. 43, No. 2, 183–194, http://dx.doi.org/10.1080/03079457.2014.898244

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The newly introduced clade 2.3.2.1 H5N1 viruses have apparently replaced previously circulating clade 2.2 viruses in Bangladesh. This needs to be confirmed by extensive and systematic surveillance. There has been an event of reassortment between H5N1 virus of clade 2.3.2.1 and H9N2 virus of Eurasian lineage (“Quail/HK/G1/97-like” sublineage) in Bangladesh, where H5N1 virus has acquired the PB1 gene of an H9N2 virus. Point mutations are accumulating in Bangladeshi isolates with potential modification of receptor binding site and antigenic sites. Extensive and continuous molecular epidemiological studies are necessary to monitor the evolution of circulating avian influenza viruses in Bangladesh, because such information is very important in assessing the pathogenic, zoonotic and pandemic potential of the viruses and selecting appropriate vaccine strains.

Knowledge Management: Journal