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The primary goals of this chapter are to discuss common viral RNA isolation and purification methods that are routinely used by various diagnostic laboratories and to highlight the advantages and drawbacks of each method and to identify the most suitable and reliable method to increase the sensitivity and specificity of RT-PCR assays for the detection of equine influenza virus (EIV) in clinical specimens. Our experiences and review of literature show that magnetic bead-based nucleic extraction methods (manual and automatic) work well for isolation and purification of EIV RNA from nasal swab specimens. Furthermore, most of the information presented in this chapter could be directly applicable to isolation and purification of nucleic acids (both DNA and RNA) from other equine clinical samples.In horses, presumptive diagnosis of equine influenza is commonly made on the basis of clinical signs. This alone is insufficient for confirmation of equine influenza, because other equine infectious respiratory diseases can in some degree have similar clinical presentations. Surveillance and control of equine influenza also necessitate detection of subclinical cases. Effective diagnosis of equine influenza virus infection is critically dependent on obtaining adequate specimens of virus-containing respiratory secretions for testing. These specimens are also valuable as sources for isolation of virus strains for antigenic characterization and potential inclusion in vaccines. Both nasal swabs and nasopharyngeal swabs are employed with horses. These differ little in their invasiveness, but nasopharyngeal swabs typically yield more virus than nasal swabs and are superior diagnostic specimens. Methods for obtaining nasopharyngeal swab specimens are described.Equine influenza virus (EIV) is a common respiratory pathogen of horses and other equids in most parts of the world. EIV are Type A influenza viruses and two subtypes are known H3N8 and H7N7. Both are believed to have evolved from avian influenza virus ancestors. The H3N8 subtype circulates widely, but the H7N7 subtype is thought to be extinct. The clinical disease in horses, caused by either subtype, is an upper respiratory infection of varying severity depending upon the immune status of the individual animal. It is not normally life-threatening in itself except in very young foals; however it predisposes infected equids to secondary infections capable of producing life-threatening pneumonias. Vaccines are available and widely used in some horse populations, but their effectiveness is limited by antigenic drift and other factors, and vaccinated animals with subclinical infections have been responsible for introduction of EIV into susceptible populations. EIV has spread into canines.Swine influenza is a disease of the respiratory tract caused by influenza A virus (IAV). Experimental inoculation of pigs involves either aerosolization of virus and inhalation or the direct introduction of virus into the upper or lower respiratory tract. This chapter covers methods for experimental IAV infection of pigs and collection of specific samples to study the pathogenesis of swine influenza and vaccine efficacy.The neuraminidase (NA) of influenza A viruses (IAV) is a structurally and antigenically important envelope glycoprotein. There are eleven known subtypes of NA of which two, N1 and N2, circulate in swine. The sialidase activity of NA is required for the release of nascent virus particles from infected cell membranes and inhibition of NA enzymatic activity can significantly reduce virus titers and duration of infection. Efforts to improve IAV vaccine technology in humans have focused on the generation of neuraminidase inhibiting (NAI) antibodies and should be considered in swine as well. The enzyme-linked lectin assay (ELLA) conducted in 96-well plates has enabled high-throughput analysis of serum samples for NAI antibody titers. Through the use of reverse genetics, custom antigen panels and antisera can be generated to encompass the antigenically diverse population of NA that circulate in swine. The ELLA is a robust method to assess NAI antibody titers and characterize the antigenic difference between NA antigens.The serum virus neutralization (SVN) assay is a serological test used to detect the presence and magnitude of functional systemic antibodies that prevent infectivity of a virus. The SVN assay is a highly sensitive and specific test that may be applied to influenza A viruses (IAV) in swine to measure the titer of neutralizing antibodies postexposure, postvaccination, or after passive transfer of maternally derived antibody (MDA). Conventional SVN methods performed in vitro are based on inhibition of virus infectivity in cell culture in the presence of neutralizing antibodies in serum. Titer determination may be based on the presence or absence of cytopathic effect or evidence of viral infection using an immunoreactive technique. The SVN assay is relatively inexpensive using standard laboratory equipment, although it requires cell culture, more time and labor, and technical skill to conduct the assay compared to other serological methods. The SVN test may be used to evaluate the level of serological cross-reactivity between IAV exposure or vaccine antisera and heterologous influenza viruses that may correlate with cross-protection in the host.Enzyme-linked immunosorbent assays can be used to detect isotype-specific anti-influenza antibodies in biological samples to characterize the porcine immune response to influenza A virus (IAV). The isotype antibody assay is based on an indirect ELISA using whole influenza virus as antigen and commercial antibodies directed against porcine IgG and IgA. Samples such as serum, nasal wash, and bronchoalveolar lavage fluid allow for evaluation of systemic, upper, and lower respiratory tract mucosal antibody responses, respectively. The isotype ELISA assay is performed in a 96-well format using IAV test antigen and anti-swine IgG or IgA detection antibodies conjugated to an enzyme that catalyze a color change reaction. selleck inhibitor The optical density of the sample is measured using an automated plate reader. The assay is useful to characterize the IgG or IgA immune response to challenge or vaccination against specific IAV isolates in different compartments of the immune system.