Abstract
In Bangladesh, highly pathogenic avian influenza (HPAI) virus of subtype H5N1 was first detected in February 2007. Since then the virus has become entrenched in poultry farms of Bangladesh. So far, there have been 549 poultry outbreaks reported to OIE and seven human cases of H5N1 HPAI infection in Bangladesh with one death. The present study aimed at pathological investigation of natural outbreaks of HPAI in poultry of Bangladesh as well as molecular characterization and evolutionary analyses of HPAI viruses circulating in Bangladesh. For pathological study, formalin fixed tissue samples were processed, sectioned and stained with hematoxylin and eosin. Tissue sections from ducks and quails were subjected to streptavidin-peroxidase immunohistochemical staining for the localization of avian influenza virus antigen. To investigate molecular evolution of H5N1 HPAI viruses during 2007 to 2012, a 545 bp partial HA gene fragment of 21 Bangladeshi isolates were amplified, sequenced directly and subjected to phylogenetic analysis for HA clade determination. Subsequently, full-length genes of all the eight segments of two selected chicken isolates and five segments of a duck isolate were amplified by RT-PCR. The amplified cDNA were sequenced either directly or after cloning in pGEM-T Easy plasmid vector and subjected to molecular analysis. In addition, full-length sequences of different gene segments of other Bangladeshi H5N1 isolates were also downloaded from the GenBank and included in the analysis. The gross and histopathological lesions in chickens and quails were primarily characterized by haemorrhages and congestion in the subcutis and visceral organs, and necrotic changes in the parenchymatous organs like liver, kidney and pancreas. Histopathological lesions were also observed in the brain, which were characterized by neuronal degeneration and gliosis. In ducks, no obvious gross lesion was seen but histological lesions similar to that in chickens were also found. Viral antigen was detected in various tissues, such as degenerating neurons and proliferating glial cells in the brain, in the mucosa of trachea, interstitial cells in the lungs, hepatocytes in the liver and necrosed acini in the pancreas. The molecular analysis revealed that the first introduction of clade 2.2 virus of genotype Z in Bangladesh in 2007 was followed by the re-introduction of clade 2.3.2.1 and 2.3.4 viruses of genotype V 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 by clade 2.3.2.1 viruses. There also has been an event of segment re-assortment between H5N1 and H9N2 viruses in Bangladesh, where a H5N1 virus acquired PB1 gene from a H9N2 virus. Point mutations were found to have accumulated in Bangladeshi isolates over the last five years with potential modification of the receptor binding site and antigenic sites. Detailed pathological investigation on avian influenza in chickens and aquatic birds on natural and experimental infections are necessary for better understanding of the pathobiology of HPAI viruses. Extensive and continuous molecular epidemiological studies are needed to monitor the evolution of circulating avian influenza viruses in Bangladesh with regard to virulence, adaptation to human and antiviral drug resistance.