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The efficient extraction and purification of viral RNA are critical for downstream molecular applications, whether it is the sensitive and specific detection of virus in clinical samples, virus gene cloning and expression, or quantification of the avian influenza (AI) virus by molecular methods from experimentally infected birds. Samples can generally be divided into two types: enriched (e.g., virus stocks) and clinical. Clinical type samples, which may be tissues or swab material, are the most difficult to process due to the complex sample composition and possibly low virus titers. In this chapter, two well-established procedures for the isolation of AI virus RNA from common clinical specimen types and enriched virus stocks for further molecular applications will be presented.
Methods Mol Biol 2008
PMID:Avian influenza virus RNA extraction from tissue and swab material. 1837 36

Real-time RT-PCR (rRT-PCR) is a relatively new technology that has been used for avian influenza (AI) virus detection since the early 2000s for routine surveillance, during outbreaks, and for research. Some of the advantages of rRT-PCR are high sensitivity, high specificity, rapid time-to-result, scalability, cost, and quantitative nature. Furthermore, rRT-PCR can be used with numerous sample types, is less expensive than virus isolation in chicken embryos, and since infectious virus is inactivated early during processing, biosafety and biosecurity are also easier to maintain. This chapter will provide an overview of the USDA-validated rRT-PCR procedure for the detection of type A influenza.
Methods Mol Biol 2008
PMID:Type A influenza virus detection and quantitation by real-time RT-PCR. 1837 37

Serological methods, gene sequencing, and RT-PCR-based methods have all been used for the identification of influenza virus hemagglutinin (HA) subtypes. Compared to serological methods and gene sequencing, RT-PCR is fast, sensitive, and relatively inexpensive. However, since RT-PCR generally lacks the specificity of sequencing or serology, the most practical application of RT-PCR methods for subtype identification is either to target a few of the most important subtypes such as H5 and H7 or to use it in situations where a specific strain is being targeted, such as during an outbreak or with experimental samples. Since identification of viruses from the Asian-origin H5N1 highly pathogenic avian influenza virus lineage is a high priority worldwide, the procedure for real-time RT-PCR (rRT-PCR) identification of the H5 H subtype is presented here. This assay can identify the H5 hemagglutinin of any genetic lineage (North American or Asian) and either pathotype (highly pathogenic or low pathogenic), but does not differentiate between subtypes or pathotypes.
Methods Mol Biol 2008
PMID:Detection and identification of the H5 hemagglutinin subtype by real-time RT-PCR. 1837 38

The avian influenza (AI) virus is usually isolated and propagated by inoculating either swab or tissue samples from infected birds into the chorioallantoic sac of embryonating chicken eggs. This is the accepted method, but occasionally an isolation may only be successful when inoculated either into the yolk sac or onto the chorioallantoic membrane of embryonating chicken eggs. Chorioallantoic fluid is harvested from eggs with dead or dying embryos and is tested for the presence of hemagglutinating antigen. If hemagglutination-positive, this indicates that the isolate may be the AI virus. The presence of the AI virus may be confirmed by either an agar gel immunodiffusion (AGID) assay, RT-PCR specific for AI virus, or a commercially available immunoassay kit specific for type A influenza. Instructions for AI virus primary isolation and propagation, preparing antigen for an AGID test, setting up an AGID test, and interpreting results are given.
Methods Mol Biol 2008
PMID:Avian influenza virus isolation and propagation in chicken eggs. 1837 39

The hemagglutination-inhibition (HI) assay is a classical laboratory procedure for the classification or subtyping of hemagglutinating viruses. For the avian influenza (AI) virus, the HI assay is used to identify the hemagglutinin (H) subtype of an unknown AI virus isolate or the HA subtype specificity of antibodies to AI virus. Since the HI assay is quantitative, it is frequently applied to evaluate the antigenic relationships between different AI virus isolates of the same subtype. The basis of the HI test is inhibition of hemagglutination with subtype-specific antibodies. The HI assay is a relatively inexpensive procedure utilizing standard laboratory equipment, is less technical than molecular tests, and is easily completed within several hours. However, when working with uncharacterized viruses or antibody subtypes, the library of reference reagents required for identifying antigentically distinct AI viruses and/or antibody specificities from multiple lineages of a single hemagglutinin subtype requires extensive laboratory support for the production and optimization of reagents.
Methods Mol Biol 2008
PMID:Hemagglutination-inhibition test for avian influenza virus subtype identification and the detection and quantitation of serum antibodies to the avian influenza virus. 1837 41

The neuraminidase-inhibition (NI) assay is a laboratory procedure for the identification of the neuraminidase (NA) glycoprotein subtype in influenza viruses or the NA subtype specificity of antibodies to influenza virus. A serological procedure for subtyping the NA glycoprotein is critical for the identification and classification of avian influenza (AI) viruses. The macro-procedure was first described in 1961 by D. Aminoff et al. [2] and was later modified to a microtiter plate procedure (micro-NI) by Van Deusen et al. [4]. The micro-NI procedure reduces the quantity of reagents required, permits the antigenic classification of many isolates simultaneously, and eliminates the spectrophotometric interpretation of results. Although the macro-NI has been shown to be more sensitive than the micro-NI, the micro-NI test is very suitable for testing sera for the presence of NA antibodies and has proven to be a practical and rapid method for virus classification. This chapter will provide an overview of the USDA-validated micro-NI procedure for the identification of subtype-specific NA in AIV and antibodies.
Methods Mol Biol 2008
PMID:Neuraminidase-inhibition assay for the identification of influenza A virus neuraminidase subtype or neuraminidase antibody specificity. 1837 42

Immunohistochemical methods are commonly used for studying the pathogenesis of the avian influenza (AI) virus by allowing the identification of sites of replication of the virus in infected tissues and the correlation with the histopathological changes observed. In this chapter, the materials and methods for performing immunohistochemical detection of AI virus antigens in tissues are provided. The technique involves the following steps: heat-induced antigen retrieval; binding of a primary antibody to a virus type-specific antigen; antibody-antigen complex binding by a biotinylated secondary antibody; and binding of an enzyme-streptavidin conjugate. The enzyme is then visualized by application of the substrate chromogen solution to produce a colorimetric end product. Demonstration of AI virus antigen in tissues is based on chromogen deposition in the nucleus and/or cytoplasm of infected cells.
Methods Mol Biol 2008
PMID:Immunohistochemical staining for the detection of the avian influenza virus in tissues. 1837 43

Avian influenza (AI) viruses have been isolated from a wide diversity of free-living avian species representing several orders. Isolations are most frequently reported from aquatic birds in the Orders Anseriformes and Charadriiformes, which are believed to be the reservoirs for all AI viruses. Since their first recognition in the late 1800 s, AI viruses have been an important agent of disease in poultry and, occasionally, of nongallinaceous birds and humans. However, the recent highly pathogenic avian influenza (HPAI) H5N1 virus epidemics have increased the awareness of AI viruses and their potential implications among the scientific community, politicians, and the general public. In response to the spread of HPAI H5N1 viruses to Europe and Africa in 2005-2006, many countries developed surveillance plans to detect AI viruses; a large portion of these sampling efforts was targeted at migratory avian species. This chapter is intended to give general concepts and guidelines for surveillance of the AI virus in wild birds. Separate sections are included for low pathogenicity avian influenza (LPAI) and HPAI H5N1 viruses because the unique biological characteristics of HPAI H5N1 require a modified surveillance plan tailored to these viruses.
Methods Mol Biol 2008
PMID:Wild bird surveillance for the avian influenza virus. 1837 44

The measurement of avian cellular immunity is critical to understanding the role and regulation of avian lymphocytes following avian influenza (AI) virus infection. Although the ability to measure avian T cell responses has steadily increased over the last few years, few studies have examined the role of cell-mediated immunity in avian species against the AI virus. Because of the structural and functional differences between mammalian and avian immune systems-including MHC architecture, different modes of somatic recombination for antibody production, and the absence of lymph nodes in birds-the extent to which birds and mammals regulate similar immune responses against the AI virus is currently under investigation. The increasing availability of monoclonal antibodies recognizing avian T cell-associated antigens as well as a number of inbred lines of chickens with genetically defined MHC haplotypes make this an important field of research for the future.
Methods Mol Biol 2008
PMID:Evaluating the cell-mediated immune response of avian species to avian influenza viruses. 1837 46

During the latter half of 2005 a widespread outbreak caused by influenza highly pathogenic H5N1 virus among wild and domestic birds occurred in Russia. As pathogenicity level is a polygenic feature and majority of individual genes of influenza A viruses contribute to pathogenicity of influenza viruses to birds, animals and humans. Nucleotide sequencing of the entire genome of influenza H5N1 virus isolates obtained in Kurgan region (Western Siberia) was performed. Structure of viral proteins was analyzed according to the predicted amino acid sequences. HA receptor-binding site of A/chicken/Kurgan/05/2005 and A/duck/Kurgan/08/2005 strains was typical for avian influenza viruses and contained Glu and Gly at positions 226 and 228, respectively. Structure of the cluster of positively charged amino acid residues at the cleavage site was identical for all isolates: QGERRRKKR. According to the data of neuraminidase structure analysis NA of the H5N1 isolates tested was suggested to belong to Z genotype. Amino acid residues typical for birds were revealed in 30 out of 32 positions of M1, M2, NP, PA and PB2 proteins determining host range specificity. One strain isolated in Kurgan contained lysine in position 627 of PB2 protein. Kurgan isolates was shown to have remantadine-sensitive genotype. Glutamic acid was found at position 92 of NS1 protein in both strains indicating virus resistance to interferon. Phylogenetic analyses allowed relating Kurgan isolates to subclade II of clade II of highly pathogenic H5N1 influenza viruses.
Mol Biol (Mosk)
PMID:[Molecular characteristic of influenza virus A H5N1 Strains isolated from poultry in Kurgan Region in 2005]. 1838 23


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