E. H. Chowdhury

New Introduction of Clade 2.3.2.1 Avian Influenza Virus (H5N1) into Bangladesh

Since the first outbreak of highly pathogenic H5N1 avian inafluenza (HPAI) in Bangladesh in February 2007, a total of 519 disease events have been reported till 22 October 2011. Partial HA gene sequences of 11 selected H5N1 HPAI isolates of 2007 to 2011 were determined and subjected to phylogenetic analysis. The study revealed a recent introduction of clade 2.3.2 and 2.3.4 viruses into Bangladesh in 2011 in addition to clade 2.2 viruses that had been in circulation since 2007. Clade 2.3.2 virus isolates from Bangladesh are phylogenetically related to the newly designated clade 2.3.2.1 viruses, reported recently from Asia and Eastern Europe.

Full-genome analysis of avian influenza virus H9N2 from Bangladesh reveals internal gene reassortments with two distinct highly pathogenic avian influenza viruses

Low-pathogenic avian influenza viruses (LPAIVs) of subtype H9N2 have become widespread in poultry in many Asian countries with relevance to respiratory diseases of multifactorial origin. In Bangladesh, LPAIVs of subtype H9N2 co-circulate simultaneously with highly pathogenic avian influenza viruses (HPAIVs) of subtype H5N1 in commercial and backyard poultry. The aim of this study was to characterize LPAIVs of subtype H9N2 currently circulating in Bangladesh. The selected isolate A/Chicken/Bangladesh/VP01/2006 (H9N2) was propagated in chicken embryos. All eight gene segments were amplified by RT-PCR, cloned, and subjected to full-length sequencing. The sequence data obtained were compared with reference strains available in GenBank. Phylogenetic analysis of LPAIV H9N2 from Bangladesh revealed a close relationship to Indian, Pakistani and Middle Eastern isolates and identified an ancestor relationship to LPAIV H9N2 Quail/HK/G1/1997. The internal genes M and NP belong to lineage G1, whereas NS, PA, PB1 and PB2 belong to the prototype virus A/Chicken/Korea/38349-p96323/96. The internal genes showed high sequence homology to an HPAIV of subtype H7N3 from Pakistan, whereas the PB1 gene showed similarly high nucleotide homologies to recently circulating HPAIV H5N1 from Bangladesh, revealing two independent reassortment events. Examination of the hemagglutinin cleavage site of LPAIV H9N2 confirmed its low pathogenicity. The receptor-binding sites indicated a binding preference for human-type receptors. Several mutations in internal proteins are associated with increased virulence and altered host range, while other amino acids were found to be highly conserved among LPAIV H9N2 isolates.

Molecular evolution of H5N1 highly pathogenic avian influenza viruses in Bangladesh between 2007 and 2012

In Bangladesh, highly pathogenic avian influenza (HPAI) virus subtype H5N1 was first detected in February 2007. Since then the virus has become entrenched in poultry farms of Bangladesh. There have so far been seven human cases of H5N1 HPAI infection in Bangladesh with one death. The objective of the present study was to investigate the molecular evolution of H5N1 HPAI viruses during 2007 to 2012. Partial or complete nucleotide sequences of all eight gene segments of two chicken isolates, five gene segments of a duck isolate and the haemagglutinin gene segment of 18 isolates from Bangladesh were established in the present study and subjected to molecular analysis. 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.

Natural peste des petits ruminants virus infection in Black Bengal goats: virological, pathological and immunohistochemical investigation.

BackgroundPeste des Petits Ruminants (PPR), also known as Goat Plague, occurs in goats, sheep and related species. It is caused by a morbillivirus in the family Paramyxoviridae. In Bangladesh PPR is endemic and it causes serious economic losses. Pathology of PPR has been reported in different goat and sheep breeds from natural and experimental infections. Field results are better indicators of pathogenicity of the circulating virus. The severity of the disease varies with species, breed and immune status of the host. Pathological investigations of natural outbreaks of PPR in Balck Bengal goats are very limited. The current investigation was aimed at describing pathology and antigen localization in natural PPR infections in Black Bengal goats.ResultsA total of 28 outbreaks were investigated clinically and virologically. Average flock morbidity and mortality were 75% and 59%, respectively, with case fatality rate of 74%. Necropsy was conducted on 21 goats from 15 outbreaks. The major gross lesions were congestion of gastrointestinal tract, pneumonia, engorged spleen, and oedematous lymphnodes. Histopathological examination revealed severe enteritis with denudation of intestinal epithelium, severe broncho-interstitial pneumonia with macrophages within lung alveoli and extensive haemorrhages with depletion of lymphoid cells and infiltration of macrophages in the sinuses of spleen. In lymph nodes, the cortical nodules were replaced by wide sinusoids with severe depletion of lymphocytes, infiltration of mononuclear cells and some giant cells in sub-capsular areas and medullary sinuses. PPR virus antigen was found in pneumocytes and alveolar macrophages in lungs. Viral RNA could be detected by RT-PCR in 69 out of 84 nasal swab, 59 out of 84 blood and 21 out of 21 lymph node samples. Sequence analyses revealed closeness of Bangladeshi strains with other recent Asian isolates.ConclusionNatural outbreaks of PPR in Black Bengal goats in Bangladesh resulted in 75% and 59% flock morbidity and mortality, respectively, with a case fatality rate of 74%. The striking histo-morphologic diagnosis of PPR was acute pneumonia and severe gastro-enteritis. A detailed experimental pathological study on Black Bengal goats infected with recent isolates is required.

Genetic characterization of highly pathogenic H5N1 avian influenza virus from live migratory birds in Bangladesh

Since the first outbreak of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 in Bangladesh in 2007, the virus has been circulating among domestic poultry causing severe economic losses. To investigate the presence of HPAIV H5N1 in migratory birds and their potential role in virus spread, 205 pools of fecal samples from live migratory birds were analyzed. Here, the first virus isolation and genome characterization of two HPAIV H5N1 isolates from migratory birds (A/migratory bird/Bangladesh/P18/2010 and A/migratory bird/Bangladesh/P29/2010)are described. Full-length amplification, sequencing, and a comprehensive phylogenetic analysis were performed for HA, NA, M, NS, NP, PA, PB1, and PB2 gene segments. The selected migratory bird isolates belong to clade 2.3.2.1 along with recent Bangladeshi isolates from chickens, ducks, and crows which grouped in the same cluster with contemporary South and South-East Asian isolates. The studied isolates were genetically similar to other H5N1 isolates from different species within the respective clade although some unique amino acid substitutions were observed among them. Migratory birds remain a real threat for spreading pathogenic avian influenza viruses across the continent and introduction of new strains into Bangladesh.

Differentiation of infectious bursal disease virus (IBDV) genome segment B of very virulent and classical lineage by RT-PCR amplification and restriction enzyme analysis.

Molecular characterization of IBDV usually relies on the analysis of segment A of the bi-segmented, double-stranded RNA genome. Although segments B of classical and very virulent IBDVs differ significantly, re-assortment of genome segments does occur, and molecular epidemiological studies demand the analysis of both segments. An RT-PCR and restriction enzyme analysis for molecular discrimination between genome segment B of classical and very virulent IBDVs is described. Tested on eight IBDV strains/isolates, the protocol successfully identified very virulent and classical IBDVs as well as a segment reassortant. This approach is a valuable tool for molecular epidemiological studies on IBDV.

Dried fluid spots for peste des petits ruminants virus load evaluation allowing for non-invasive diagnosis and genotyping

BackgroundActive surveillance of peste des petits ruminants (PPR) should ease prevention and control of this disease widely present across Africa, Middle East, central and southern Asia. PPR is now present in Turkey at the gateway to the European Union. In Bangladesh, the diagnosis and genotyping of PPR virus (PPRV) may be hampered by inadequate infrastructures and by lack of proper clinical material, which is often not preserved under cold chain up to laboratories. It has been shown previously that Whatman® 3MM filter paper (GE Healthcare, France) preserves the nucleic acid of PPRV for at least 3 months at 32°C.ResultsIn this study, we demonstrate the performances of filter papers for archiving RNA from local PPRV field isolates for further molecular detection and genotyping of PPRV, at -70°C combined with ambient temperature, for periods up to 16 months. PPR-suspected live animals were sampled and their blood and nasal swabs were applied on filter papers then air dried. Immediately after field sampling, RT-PCR amplifying a 448-bp fragment of the F gene appeared positive for both blood and nasal swabs when animals were in febrile stage and only nasal swabs were detected positive in non-febrile stage. Those tested positive were monitored by RT-PCR up to 10 months by storage at -70°C. At 16 months, using real time RT-PCR adapted to amplify the N gene from filter paper, high viral loads could still be detected (~2 x 107 copy numbers), essentially from nasal samples. The material was successfully sequenced and a Bayesian phylogenetic reconstruction achieved adequate resolution to establish temporal relationships within or between the geographical clusters of the PPRV strains.ConclusionsThis clearly reveals the excellent capacity of filter papers to store genetic material that can be sampled using a non-invasive approach.

Serological and virological surveillance of avian influenza virus in domestic ducks of the north-east region of Bangladesh

BackgroundWild waterfowl are considered as the natural reservoir for avian influenza (AI) viruses. Bangladesh has been experiencing highly pathogenic avian influenza (HPAI) outbreaks since 2007, mostly in chickens and occasionally in ducks. Ducks play an important role in the persistence and genetic recombination of AI viruses. This paper presents the results of serological and virological monitoring of AI in domestic ducks in 2013 in the north-east region of Bangladesh.ResultsA total of 871 and 662 serum samples and 909 and 302 pairs of cloacal and oropharyngeal swabs from domestic ducks of Mymensingh and Sylhet division, respectively, were analysed. Antibodies to type A influenza virus were detected by blocking ELISA in 60.73 and 47.73% serum samples of Mymensingh and Sylhet division, respectively. On haemagglutination-inhibition (HI) test 17.5% of ELISA positive serum samples were found to be seropositive to H5 avian influenza virus. Five cloacal swabs and one oropharyngeal swab were positive for M gene of type A influenza virus by real time RT-PCR (rRT-PCR), but all of them were negative for H5 influenza virus. Three of the six viruses were successfully characterized as H1N5, H2N5 and H7N5 subtype of AI virus, the other three remained uncharacterized. On sequencing and phylogenetic analysis the HA and NA genes were found to be of Eurasian avian lineage. The H7 virus had cleavage site motif of low pathogenic virus.ConclusionsLow pathogenic avian influenza viruses were detected from apparently healthy domestic ducks. A small proportion of domestic ducks were found seropositive to H5 AI virus.

Histomorphological and morphometric studies of the pancreatic islet cells of diabetic rats treated with aqueous extracts of Momordica charantia (karela) fruits

Abstract Objective To investigate the effect of aqueous extract of Momordica charantia (karela) (M. charantia) fruits on blood glucose level, pancreatic weight changes and histopathology of pancreatic changes in the streptozotocin (STZ) induced diabetic rats. Methods Thirty-six albino rats were used in the experiment; diabetes mellitus was induced in 30 adult albino rats, using intraperitoneal injection of 55 mg/kg STZ. Six non diabetic rats remained as control (T1). The diabetic rats were randomly assigned into five equal groups: diabetic control (T2) without any treatment, groups T3, T4, T5 and T6 were treated with aqueous extract of karela fruits daily at a doses of 250, 500 and 750 mg/kg and glibenclamide (5 mg/kg) up to 90 d, respectively. At Day 90, all rats were sacrificed, the pancreases of the rats were excised and processed. Results The results of this study indicate that aqueous extract of M. charantia fruits was able to reduce blood glucose level significantly compared with the diabetic control group (P Conclusions The present study suggests that oral feeding of M. charantia fruit juice has a significant anti-hyperglycemic effect and may have a role in the regeneration of the β cells in STZ diabetic rats.

Further evidence for the association of distinct amino acid residues with in vitro and in vivo growth of infectious bursal disease virus

A cell-culture-adapted reverse genetics strain of very virulent infectious bursal disease virus (IBDV) of chickens, designated as BD-3tcC, having four amino acid substitutions (Gln253His, Asp279Asn, Ala284Thr and Ser330Arg) in the capsid protein VP2 was tested for its genetic stability during serial passage in chickens and chicken embryo fibroblast (CEF) cell culture. Results of in vitro and in vivo experiments demonstrated that all four introduced mutations in BD-3tcC remained stable during serial passage in CEF cell culture, but during passage in chickens, amino acid residues at position 253 and 284 reverted from histidine to glutamine and threonine to alanine, respectively. In a parallel experiment, the same substitutions also occurred in a conventionally attenuated vaccine strain D-78 on serial passage in chickens. However, no reversion or substitution took place at positions 279 and 330 during in vivo passage of the mutant virus BD-3tcC or vaccine virus D-78. The findings provide conclusive evidence that while IBDV requires histidine and threonine at positions 253 and 284 for cell culture adaptation, glutamine and alanine at these positions are selected preferentially during in vivo replication.