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Background: Variant high pathogenicity avian influenza (HPAI) H5 viruses have recently emerged as a result of reassortment of the H5 haemagglutinin (HA) gene with different neuraminidase (NA) genes, including NA1, NA2, NA5, NA6 and NA8. These viruses form a newly proposed HA clade 2.3.4.4 (previously provisionally referred to as clade 2.3.4.6), and have been implicated in disease outbreaks in poultry in China, South Korea, Laos, Japan and Vietnam and a human fatality in China. There is real concern that this new clade may be wide spread and not readily identified using existing diagnostic algorithms., Findings: Fluorescent probe based reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) assays were developed to facilitate the identification of novel clade 2.3.4.4 viruses of H5N6 subtype emerging in Asia. Assays were aimed at the haemagglutinin (HA) gene for clade identification and at the NA gene to identify N6. The HA assay employing a minor groove binder (MGB) probe was able to detect and differentiate A/duck/Laos/XBY004/2014(H5N6) and related influenza A(H5N6) virus isolates belonging to the proposed clade 2.3.4.4 from other H5 HPAI viruses. In addition, an Eurasian N6 assay was able to differentiate N6 from other NA subtypes., Conclusions: Laos influenza A(H5N6) virus representative of proposed clade 2.3.4.4, was detected and differentiated from viruses in other H5N1 clades using a clade-specific HA RT-qPCR assay whereas the N6-NA subtype was determined by an Eurasian N6 RT-qPCR assay. Such a clade-specific assay would be of particular value for surveillance and in diagnostic laboratories where sequencing is not readily available.

The occurrence of highly pathogenic (HP) avian influenza (AI) H5N1 in Asia and its spread to Africa and Europe prompted costly monitoring programs of wild birds and domestic poultry. AI virus excretion is tested by examining avian swab samples by real-time reverse transcription PCR (RT-qPCR). In this study, pools of swab samples and a reagents volume reduction per RT-qPCR were evaluated as measures of economization. Viral transport medium and faecal matrices were spiked with different low pathogenic AI virus strains and tested for loss of target RNA during all processing steps as individual rayon swabs or in sample pools of 5, 10 and 15 swabs. Fresh faeces from Mallard ducks and other aquatic bird species as sample matrix resulted in loss of AIV RNA of about 90% compared to transport medium. Due to sample RNA dilution in pools the likelihood of detection of single positive samples is decreasing with increasing size of sample pools. However, pools of five samples containing only one positive sample consistently gave positive results. Similarly, no differences in detection rates were obtained when analyzing 1030 wild bird swab samples either individually or in pools of five. Reducing the reaction volume of influenza A virus generic as well as of subtype-specific RT-qPCRs to 12.5 μl (2.5 μl template) instead of 25 μl did not adversely affect the limit of detection of these RT-qPCRs. A significant economic benefit without impeding detection efficacy can be achieved when sample pools of five samples are analyzed by RT-qPCR using a reduction of the reaction mix to the half of the original volume., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Avian influenza (AI) caused by H7 AI viruses (AIVs) of both low pathogenicity (LP) and high pathogenicity (HP) are notifiable poultry diseases., Objectives: Design and validate two RealTime reverse transcriptase polymerase chain reactions (RRT PCRs) for Eurasian H7 AIV detection and pathotyping., Methods: The H7 RRT PCRs amplified within the (i) HA2 and (ii) cleavage site CS regions of the haemagglutinin gene. Both were validated against 65 H7 AIVs, 57 non-H7 AIVs and 259 poultry swabs in comparison to M gene (AI generic) RRT PCR and virus isolation (VI). An additional 38 swabs and 20 tissue specimens extended validation against M gene RRT PCR., Results: Both H7 RRT PCRs amplified all 61 Eurasian lineage H7 AIVs and none of 57 non-H7 AIVs. A total of 297 poultry swabs were used to determine diagnostic sensitivity and specificity relative to M gene RRT PCR, sensitivity was 95.4% and 64.6% for the HA2 and CS RRT PCRs respectively, and specificity 97.9% and 99.6% respectively. The H7 HA2 RRT PCR was more sensitive than VI. This was emphasized by analysis of 37 swabs from turkeys infected experimentally with HPAI H7N1 virus sampled at 24 hours post-inoculation and LPAI H7N1 chicken infections sampled at 40-64 hours. Although less sensitive, usefulness of the H7 CS RRT PCR was confirmed by the correct molecular pathotyping for all 61 Eurasian lineage H7 AIVs tested., Conclusions: The high sensitivity of H7 HA2 RRT PCR confirms its suitability for use in poultry surveillance and disease diagnosis. H7 CS RRT PCR provides an opportunity for rapid pathotyping of H7 AIVs.

Monoclonal antibodies (MAbs) against the recently emerged Asian H5N1 virus (A/crow/Kyoto/53/2004) were generated. From five anti-hemagglutinin (HA) MAbs, four antibodies (3C11, 4C12, 3H12, and 3H4) broadly in vitro recognized and neutralized H5 subtypes, including H5N1. By contrast, the 4G6 MAb specifically reacted with H5N1-HA and not with H5N2- or H5N3-HAs from previous epidemics. The 4G6 MAb was useful for immunofluorescence assays but not for immunoblotting, suggesting that this antibody recognizes a conformational epitope of the H5N1-HA protein. An intensive epitope-mapping analysis demonstrated that the 4G6 MAb recognizes Asp59, which is highly conserved among currently circulating H5N1 lineages. Further, a 4G6-based antigen capture enzyme-linked immunosorbent assay detected H5N1 even that derived from clade 2.2 (A/chicken/Egypt/CL-61/2007) from infected chicken lung before virus isolation. Taken together, these results suggest that the established MAbs, especially 4G6, are useful for rapid and specific detection of Asian H5N1 viruses.

Bird flu or H5N1 infection is a new emerging zoonosis. With the pandemic in avian species in Asia, it is now under surveillance for a possible new public health threat to human. There are many present researches focusing on several aspects of bird flu. There are some recent patents and patent applications published within a few years. In this article, the recent patients relating to bird flu infection covering the diagnostic and treatment aspects for both avian species and human are reviewed and discussed.

A rapidly spreading, highly pathogenic avian influenza virus A H5N1 in the domestic poultry population has crossed the species barrier to humans and other mammalian species, thus, posing an increasing pandemic threat. The World Health Organization, other agencies, and countries worldwide are closely monitoring the prevalent influenza viruses and their related illnesses to detect any increased virulence or transmissibility that might signal the beginnings of any future pandemic. So far, the H5N1 virus has infected birds in more than 30 countries in Asia, Europe and Africa, while further geographical spread remains likely. Human infections are still rare and the virus does not spread easily from birds to humans or readily from person to person. Although antiviral drugs and vaccination are among the most important measures to be used in case of an influenza pandemic, a timely supply of sufficient quantities will not be possible. This review describes various aspects of avian influenza in birds and in humans; epidemiology, transmission, diagnosis and clinical manifestations. Also presented are the global preparedness, the anti-influenza drugs and vaccines.

In December 2003, the largest outbreak of highly pathogenic avian influenza H5N1 occurred among poultry in 8 Asian countries. A limited number of human H5N1 infections have been reported from Vietnam and Thailand, with a mortality rate approaching 70%. Deaths have occurred in otherwise healthy young individuals, which is reminiscent of the 1918 Spanish influenza pandemic. The main presenting features were fever, pneumonitis, lymphopenia, and diarrhea. Notably, sore throat, conjunctivitis, and coryza were absent. The H5N1 strains are resistant to amantadine and rimantadine but are susceptible to neuraminidase inhibitors, which can be used for treatment and prophylaxis. The widespread epidemic of avian influenza in domestic birds increases the likelihood for mutational events and genetic reassortment. The threat of a future pandemic from avian influenza is real. Adequate surveillance, development of vaccines, outbreak preparedness, and pandemic influenza planning are important. This article summarizes the current knowledge on avian influenza, including the virology, epidemiology, diagnosis, and management of this emerging disease.