sybr-green-i and Influenza--Human

Topics: Animals; Benzothiazoles; Diamines; Humans; Influenza A virus; Influenza A Virus, H1N1 Subtype; Influenza, Human; Orthomyxoviridae Infections; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Swine

Toll-like receptors (TLRs) play key roles in innate immune recognition of pathogen-associated molecular patterns of invading microbes. Among the 10 TLR family members identified in humans, TLR10 remains an orphan receptor without known agonist or function. TLR10 is a pseudogene in mice and mouse models are noninformative in this regard. Using influenza virus infection in primary human peripheral blood monocyte-derived macrophages and a human monocytic cell line, we now provide previously unidentified evidence that TLR10 plays a role in innate immune responses following viral infection. Influenza virus infection increased TLR10 expression and TLR10 contributed to innate immune sensing of viral infection leading to cytokine induction, including proinflammatory cytokines and interferons. TLR10 induction is more pronounced following infection with highly pathogenic avian influenza H5N1 virus compared with a low pathogenic H1N1 virus. Induction of TLR10 by virus infection requires active virus replication and de novo protein synthesis. Culture supernatants of virus-infected cells modestly up-regulate TLR10 expression in nonvirus-infected cells. Signaling via TLR10 was activated by the functional RNA-protein complex of influenza virus leading to robust induction of cytokine expression. Taken together, our findings identify TLR10 as an important innate immune sensor of viral infection and its role in innate immune defense and immunopathology following viral and bacterial pathogens deserves attention.

Topics: Animals; Benzothiazoles; Blotting, Western; Diamines; DNA Primers; Dogs; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Humans; Immunity, Innate; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H5N1 Subtype; Influenza, Human; Macrophages; Madin Darby Canine Kidney Cells; Mice; Organic Chemicals; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Toll-Like Receptor 10

Contemporary influenza B viruses are classified into two groups known as Yamagata and Victoria lineages. The co-circulation of two viral lineages in recent years urges for a robust and simple diagnostic test for detecting influenza B viruses and for lineage differentiation. In this study, a SYBR green-based asymmetric PCR assay has been developed for influenza B virus detection. Apart from identifying influenza B virus, the assay contains sequence-specific probes for lineage differentiation. This allows identifying influenza B virus and detecting influenza B viral lineage in a single reaction. The test has been evaluated by a panel of respiratory specimens. Of 108 influenza B virus-positive specimens, 105 (97%) were positive in this assay. None of the negative control respiratory specimens were positive in the test (N = 60). Viral lineages of all samples that are positive in the assay (N = 105) can also be classified correctly. These results suggest that this assay has a potential for routine influenza B virus surveillance.

Topics: Benzothiazoles; Diamines; Genotyping Techniques; Humans; Influenza B virus; Influenza, Human; Molecular Diagnostic Techniques; Organic Chemicals; Polymerase Chain Reaction; Quinolines; Retrospective Studies; Sensitivity and Specificity; Staining and Labeling

The emergence and rapid spread of the 2009 H1N1 pandemic influenza virus showed that many diagnostic tests were unsuitable for detecting the novel virus isolates. In most countries the probe-based TaqMan assay developed by the U.S. Centers for Disease Control and Prevention was used for diagnostic purposes. The substantial sequence data that became available during the course of the pandemic created the opportunity to utilize bioinformatics tools to evaluate the unique sequence properties of this virus for the development of diagnostic tests. We used a comprehensive computational approach to examine conserved 2009 H1N1 sequence signatures that are at least 20 nucleotides long and contain at least two mismatches compared to any other known H1N1 genome. We found that the hemagglutinin (HA) and neuraminidase (NA) genes contained sequence signatures that are highly conserved among 2009 H1N1 isolates. Based on the NA gene signatures, we used Visual-OMP to design primers with optimal hybridization affinity and we used ThermoBLAST to minimize amplification artifacts. This procedure resulted in a highly sensitive and discriminatory 2009 H1N1 detection assay. Importantly, we found that the primer set can be used reliably in both a conventional TaqMan and a SYBR green reverse transcriptase (RT)-PCR assay with no loss of specificity or sensitivity. We validated the diagnostic accuracy of the NA SYBR green assay with 125 clinical specimens obtained between May and August 2009 in Chile, and we showed diagnostic efficacy comparable to the CDC assay. Our approach highlights the use of systematic computational approaches to develop robust diagnostic tests during a viral pandemic.

Topics: Benzothiazoles; Chile; Diamines; DNA Primers; Hemagglutinin Glycoproteins, Influenza Virus; Humans; Influenza A Virus, H1N1 Subtype; Influenza, Human; Neuraminidase; Organic Chemicals; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Staining and Labeling; United States; Viral Proteins; Virology

Influenza A virus (IAV) epidemics are the result of human-to-human or poultry-to-human transmission. Tracking seasonal outbreaks of IAV and other avian influenza virus (AIV) subtypes that can infect humans, aquatic and migratory birds, poultry, and pigs is essential for epidemiological surveillance and outbreak alerts. In this study, we performed four real-time reverse transcription-PCR (rRT-PCR) assays for identification of the IAV M and hemagglutinin (HA) genes from six known AIVs infecting pigs, birds, and humans. IAV M1 gene-positive samples tested by single-step rRT-PCR and a fluorogenic Sybr green I detection system were further processed for H5 subtype identification by using two-primer-set multiplex and Sybr green I rRT-PCR assays. H5 subtype-negative samples were then tested with either a TaqMan assay for subtypes H1 and H3 or a TaqMan assay for subtypes H2, H7, and H9 and a beacon multiplex rRT-PCR identification assay. The four-tube strategy was able to detect 10 RNA copies of the HA genes of subtypes H1, H2, H3, H5, and H7 and 100 RNA copies of the HA gene of subtype H9. At least six H5 clades of H5N1 viruses isolated in Southeast Asia and China were detected by that test. Using rRT-PCR assays for the M1 and HA genes in 202 nasopharyngeal swab specimens from children with acute respiratory infections, we identified a total of 39 samples positive for the IAV M1 gene and subtypes H1 and H3. When performed with a portable SmartCycler instrument, the assays offer an efficient, flexible, and reliable platform for investigations of IAV and AIV in remote hospitals and in the field.

Topics: Adolescent; Benzothiazoles; Child; Child, Preschool; Diamines; DNA Primers; Hemagglutinin Glycoproteins, Influenza Virus; Humans; Infant; Influenza A virus; Influenza, Human; Molecular Sequence Data; Nasopharynx; Organic Chemicals; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; Staining and Labeling; Viral Matrix Proteins

A rapid SYBR green I real-time reverse transcription-PCR (RT-PCR) assay was developed to identify pandemic influenza H1N1 virus from clinical specimens in less than 1 h. Probe real-time RT-PCR influenza A/B, H1/H3, and swNP/swHA assays were modified into the same PCR program, which allows for rapid and simultaneous typing and subtyping of influenza viruses.

Topics: Benzothiazoles; Diamines; Humans; Influenza A Virus, H1N1 Subtype; Influenza, Human; Oligonucleotide Probes; Organic Chemicals; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Staining and Labeling; Time Factors; Viral Proteins