RNA Extraction from Swine Samples and Detection of Influenza A Virus in Swine by Real-Time RT-PCR

Real-time RT-PCR (rRT-PCR) assays are currently the method of choice in many laboratories for the detection and subtyping of influenza A virus (IAV) in swine. Traditionally, nasal swabs and lung tissues (sometimes broncho-alveolar lavage and tracheal tissues) are the primary specimens for IAV testing. However, oral fluids are becoming more common for IAV prognostic profiling. In this chapter, we describe (1) procedures of RNA extraction from the common clinical specimens, (2) two rRT-PCR assays for detection of IAV in swine, and (3) an rRT-PCR assay for subtyping swine IAV. RNA extraction procedures include a magnetic bead method optimized for extraction from nasal swabs and tissue homogenates and a magnetic bead method optimized for extraction from oral fluids. Two rRT-PCR assays for detection of swine IAV include a USDA-validated IAV rRT-PCR targeting the matrix gene and the USDA-licensed VetMAX™-Gold Swine Influenza Virus Detection rRT-PCR kit (Life Technologies) targeting the nucleoprotein and matrix genes. The swine IAV subtyping assays described here are multiplex SIV HA (H1 and H3) and NA (N1 and N2) subtyping rRT-PCR reagents from Life Technologies.
Source: Springer protocols feed by Infectious Diseases - Category: Infectious Diseases Source Type: news

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The dissemination of Influenza A virus (IAV) throughout the world has become one of the main concerns for the health of both animals and human beings. An efficient and sensitive diagnostic tool is thus needed for the early detection of IAV. Here, we developed a wash-free magnetic bioassay and further integrated it with a handheld platform based on giant-magnetoresistance (GMR) sensors. The wash-free magnetic bioassay significantly accelerates and simplifies the detection process. This brand-new system was successful in detecting both IAV nucleoprotein and IAV-contained nasal swab samples from pigs on the farm. The extrapol...
Source: Frontiers in Microbiology - Category: Microbiology Source Type: research
Avian influenza viruses (AIVs) are zoonotic pathogens that proliferate in a wide variety of host and undergo continuous genetic reassortment.1 Currently, 18 different haemagglutination (HA, H1-H18) and 11 different neuraminidase (NA, N1-N11) subtypes have been discovered, among which H1-H16 and N1-N9 were identified in avian species and had adapted to humans, equines, and swine.2 The continuous reassortment among AIV subtypes resulted in the emergence of novel AIVs and poised significant zoonotic risk to human.
Source: Journal of Infection - Category: Infectious Diseases Authors: Source Type: research
The emergence of novel avian influenza A viruses is frequently reported in this journal, 1,2 while other emergent diseases, e.g., African swine fever (ASF), which also cause great losses in recent years. African swine fever is caused by the African swine fever virus (ASFV). ASFV is the only member of the Asfarviridae family, and the genus Asfivirus.3 Infections of swine with ASFV show high morbidity and mortality (up to 100%). At present, there is no effective vaccine to prevent ASF and thus it is a great threat to global pig production.  Since its first described in Kenya in the 1920s, ASF has been recorded in most s...
Source: Journal of Infection - Category: Infectious Diseases Authors: Tags: Letter to the Editor Source Type: research
Human infection with swine flu viruses can occur. Here's how to diagnose and treat swine flu.CDC Expert Commentary
Source: Medscape Infectious Diseases Headlines - Category: Infectious Diseases Tags: Infectious Diseases Commentary Source Type: news
Conclusion and Future Perspectives The mechanisms which result in reduced viral replication and lack of disease in African wild suids after ASFV infection are largely unknown. The data so far indicate that this is not due to an intrinsic difference in the ability of the virus to replicate in macrophages from these hosts. A more likely explanation is that the innate immune system of these hosts is better able to control virus replication resulting in a reduced systemic infection and reduced pathogenesis. This may involve a balance between virus and host factors which has evolved over long term infections of these hosts. Se...
Source: Frontiers in Genetics - Category: Genetics & Stem Cells Source Type: research
We read with interest the recent communication by Guo et al. concerning avian influenza virus pathogenicity.1 Swine has been considered an intermediate host for avian influenza viruses to adapt to humans. Cross-species transmissions caused by novel reassortant swine-originate influenza A virus (S-OIV) are of particular concern after the 2009 pandemic caused by pdH1N1 virus and epidemic outbreaks caused by H3N2v.2,3 The genesis of these viruses shows that reassortant is the major driving force for producing highly infectious variants.
Source: Journal of Infection - Category: Infectious Diseases Authors: Tags: Letter to the Editor Source Type: research
Mark K. Slifka1* and Ian J. Amanna2 1Division of Neuroscience, Oregon National Primate Research Center, Oregon Health &Science University, Beaverton, OR, United States2Najít Technologies, Inc., Beaverton, OR, United States Vaccines play a vital role in protecting our communities against infectious disease. Unfortunately, some vaccines provide only partial protection or in some cases vaccine-mediated immunity may wane rapidly, resulting in either increased susceptibility to that disease or a requirement for more booster vaccinations in order to maintain immunity above a protective level. The durability of a...
Source: Frontiers in Immunology - Category: Allergy & Immunology Source Type: research
Conclusions and Perspectives It is interesting that watershed events, such as the loss of Neu5Gc from the glycocalyx of human cells have occurred numerous times in many mammalian and other vertebrate species. These cases of convergent evolution represent precious opportunities for increased understanding of evolutionary processes. In some respects, Neu5Gc is an ideal self-molecule as it is “private” to vertebrates and, based on current data, has yet to be successfully mimicked by microbes. Against the background of this benefit, the loss of Neu5Gc appears paradoxical and may implicate strong selective regimes,...
Source: Frontiers in Immunology - Category: Allergy & Immunology Source Type: research
Recently, a study in this journal suggested that the 2014 H1N1 pandemic 2009 (H1N1/pdm2009) had gene communication with 2016/2017 H3N2 1. The influenza A H1N1/pdm2009 virus, a novel swine-derived, triple reassortant virus, was rapidly transmitted between humans and spread to 168 countries, resulting in over 123,000 human deaths globally from March to December 2009 2, 3. Since then, it has replaced the previous seasonal H1N1 and circulated as a seasonal virus along with the H3N2 virus, posing substantial risks to human populations 4, creating an opportunity for coinfection and therefore recombination or reassortment between them.
Source: Journal of Infection - Category: Infectious Diseases Authors: Tags: Letter to the Editor Source Type: research
Recently, a study in this journal suggested that the 2014 H1N1 pandemic 2009 (H1N1/pdm2009) had gene communication with 2016/2017 H3N2.1 The influenza A H1N1/pdm2009 virus, a novel swine-derived, triple reassortant virus, was rapidly transmitted between humans and spread to 168 countries, resulting in over 123,000 human deaths globally from March to December 2009.2,3 Since then, it has replaced the previous seasonal H1N1 and circulated as a seasonal virus along with the H3N2 virus, posing substantial risks to human populations,4 creating an opportunity for coinfection and therefore recombination or reassortment between them.
Source: Journal of Infection - Category: Infectious Diseases Authors: Tags: Letter to the Editor Source Type: research
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