After reports of unusually high mortality rates among ducks on farms in Java Island, Indonesia, in September 2012, influenza A(H5N1) viruses were detected and characterized. Sequence analyses revealed all genes clustered with contemporary clade 2.3.2.1 viruses, rather than enzootic clade 2.1.3 viruses, indicating the introduction of an exotic H5N1 clade into Indonesia.
Available avian influenza (AIV) serological diagnostic tests cannot distinguish vaccinated from naturally infected birds. Differentiation of vaccinated from infected animals (DIVA) is currently advocated as a means of achieving the full control of H5N1. In this study, for the first time, recombinant ectodomain of M2 protein (M2e) of avian influenza virus (H5N1 strain) was used for the DIVA serology test. M2e was cloned into pMAL-P4X vector and expressed in E. coli cells. We used Western blot to recognize the expressed M2e-MBP protein by chicken antisera produced against live H5N1 virus. Also, the specificity of M2e-MBP protein was compared to the M2e synthetic peptide via ELISA. In M2e-MBP ELISA, all sera raised against the live avian influenza viruses were positive for M2e antibodies, whereas sera from killed virus vaccination were negative. Furthermore, M2e-MBP ELISA of the field sera obtained from vaccinated and non-vaccinated chickens showed negative results, while challenged vaccinated chickens demonstrated strong positive reactions. H5N1-originated recombinant M2e protein induced broad-spectrum response and successfully reacted with antibodies against other AIV strains such as H5N2, H9N2, H7N7, and H11N6. The application of the recombinant protein instead of synthetic peptide has the advantages of continues access to an inexpensive reagent for performing a large scale screening. Moreover, recombinant proteins provide the possibility of testing the DIVA results with an additional technique such a Western blotting which is not possible in the case of synthetic proteins. All together, the results of the present investigation show that recombinant M2e-MBP can be used as a robust and inexpensive solution for DIVA test.
Avian influenza H5N1 infection in humans is typically associated with close contact with infected poultry or other infected avian species. We report on human cases of H5N1 infection in Indonesia where exposure to H5N1-infected animals could not be established, but where the investigation found chicken faeces contaminated with viable H5N1 virus in the garden fertilizer. Human cases of avian influenza H5N1 warrant extensive investigations to determine likely sources of illness and to minimize risk to others. Authorities should regulate the sale and transportation of chicken faeces as fertilizer from areas where H5N1 outbreaks are reported.
The highly pathogenic avian influenza (HPAI) A(H5N1) virus is endemic in Indonesian poultry and has caused sporadic human infection in Indonesia since 2005. Surveillance of H5N1 viruses in live bird markets (LBMs) during 2012 and 2013 was carried out to provide epidemiologic and virologic information regarding viral circulation and the risk of human exposure. Real-time RT-PCR of avian cloacal swabs and environmental samples revealed influenza A-positive specimens, which were then subjected to virus isolation and genomic sequencing. Genetic analysis of specimens collected at multiple LBMs in Indonesia identified both low pathogenicity avian influenza (LPAI) A(H3N8) and HPAI A(H5N1) viruses belonging to clade 2.1.3.2a. Comparison of internal gene segments among the LPAI and HPAI viruses revealed that the latter had acquired the PB2, PB1, and NS genes from LPAI progenitors and other viruses containing a wild type (wt) genomic constellation. Comparison of murine infectivity of the LPAI A(H3N8), wt HPAI A(H5N1) and reassortant HPAI A(H5N1) viruses showed that the acquisition of LPAI internal genes attenuated the reassortant HPAI virus, producing a mouse infectivity/virulence phenotype comparable to that of the LPAI virus. Comparison of molecular markers in each viral gene segment suggested that mutations in PB2 and NS1 may facilitate attenuation. The discovery of an attenuated HPAI A(H5N1) virus in mice that resulted from reassortment may have implications for the capability of these viruses to transmit and cause disease. In addition, surveillance suggests that LBMs in Indonesia may play a role in the generation of reassortant A(H5) viruses and should be monitored.
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