Real-time RT-qPCR assay for the analysis of human influenza A virus transcription and replication dynamics

2010 ◽  
Vol 168 (1-2) ◽  
pp. 63-71 ◽  
Author(s):  
Diana Vester ◽  
Antje Lagoda ◽  
Diana Hoffmann ◽  
Claudius Seitz ◽  
Stefan Heldt ◽  
...  
Keyword(s):  
Real Time ◽  
Influenza A Virus ◽  
Influenza A ◽  
Qpcr Assay ◽  
Human Influenza ◽  
Virus Transcription ◽  
Transcription And Replication

scholarly journals Functional Analysis of PA Binding by Influenza A Virus PB1: Effects on Polymerase Activity and Viral Infectivity

2001 ◽  
Vol 75 (17) ◽  
pp. 8127-8136 ◽  
Author(s):  
Daniel R. Perez ◽  
Ruben O. Donis
Keyword(s):  
Amino Acids ◽  
Influenza Virus ◽  
Amino Acid ◽  
Viral Replication ◽  
Influenza A Virus ◽  
Influenza A ◽  
Amino Acid Sequences ◽  
Influenza Viruses ◽  
Virus Growth ◽  
Transcription And Replication

ABSTRACT Influenza A virus expresses three viral polymerase (P) subunits—PB1, PB2, and PA—all of which are essential for RNA and viral replication. The functions of P proteins in transcription and replication have been partially elucidated, yet some of these functions seem to be dependent on the formation of a heterotrimer for optimal viral RNA transcription and replication. Although it is conceivable that heterotrimer subunit interactions may allow a more efficient catalysis, direct evidence of their essentiality for viral replication is lacking. Biochemical studies addressing the molecular anatomy of the P complexes have revealed direct interactions between PB1 and PB2 as well as between PB1 and PA. Previous studies have shown that the N-terminal 48 amino acids of PB1, termed domain α, contain the residues required for binding PA. We report here the refined mapping of the amino acid sequences within this small region of PB1 that are indispensable for binding PA by deletion mutagenesis of PB1 in a two-hybrid assay. Subsequently, we used site-directed mutagenesis to identify the critical amino acid residues of PB1 for interaction with PA in vivo. The first 12 amino acids of PB1 were found to constitute the core of the interaction interface, thus narrowing the previous boundaries of domain α. The role of the minimal PB1 domain α in influenza virus gene expression and genome replication was subsequently analyzed by evaluating the activity of a set of PB1 mutants in a model reporter minigenome system. A strong correlation was observed between a functional PA binding site on PB1 and P activity. Influenza viruses bearing mutant PB1 genes were recovered using a plasmid-based influenza virus reverse genetics system. Interestingly, mutations that rendered PB1 unable to bind PA were either nonviable or severely growth impaired. These data are consistent with an essential role for the N terminus of PB1 in binding PA, P activity, and virus growth.

scholarly journals Clinical Investigations. Comparison of Directigen Flu A+B with Real Time PCR in the Diagnosis of Influenza / Сравнение Иммунохроматографического Метода (Directigen Flu A+B) И Теста RT-PCR При Инфекциях Гриппа

Folia Medica ◽  
2015 ◽  
Vol 57 (2) ◽  
pp. 104-110 ◽  
Author(s):  
Golubinka Bosevska ◽  
Nikola Panovski ◽  
Elizabeta Janceska ◽  
Vladimir Mikik ◽  
Irena Kondova Topuzovska ◽  
...  
Keyword(s):  
Real Time ◽  
Influenza A Virus ◽  
Real Time Pcr ◽  
Influenza A ◽  
Age Groups ◽  
Influenza Viruses ◽  
Influenza B Virus ◽  
Influenza B ◽  
B Virus ◽  
Nose And Throat

AbstractEarly diagnosis and treatment of patients with influenza is the reason why physicians need rapid high-sensitivity influenza diagnostic tests that require no complex lab equipment and can be performed and interpreted within 15 min. The Aim of this study was to compare the rapid Directigen Flu A+B test with real time PCR for detection of influenza viruses in the Republic of Macedonia. MATERIALS AND METHODS: One-hundred-eight respiratory samples (combined nose and throat swabs) were routinely collected for detection of influenza virus during influenza seasons. Forty-one patients were pediatric cases and 59 were adult. Their mean age was 23 years. The patients were allocated into 6 age groups: 0 - 4 yrs, 5 - 9 yrs, 10 - 14 yrs, 15 - 19 yrs, 20-64 yrs and > 65 yrs. Each sample was tested with Directigen Flu A+B and CDC real time PCR kit for detection and typisation/subtypisation of influenza according to the lab diagnostic protocol. RESULTS: Directigen Flu A+B identified influenza A virus in 20 (18.5%) samples and influenza B virus in two 2 (1.9%) samples. The high specificity (100%) and PPV of Directigen Flu A+B we found in our study shows that the positive results do not need to be confirmed. The overall sensitivity of Directigen Flu A+B is 35.1% for influenza A virus and 33.0% for influenza B virus. The sensitivity for influenza A is higher among children hospitalized (45.0%) and outpatients (40.0%) versus adults. CONCLUSION: Directigen Flu A+B has relatively low sensitivity for detection of influenza viruses in combined nose and throat swabs. Negative results must be confirmed.

scholarly journals Cooperation between the Hemagglutinin of Avian Viruses and the Matrix Protein of Human Influenza A Viruses

2002 ◽  
Vol 76 (4) ◽  
pp. 1781-1786 ◽  
Author(s):  
Christoph Scholtissek ◽  
Jürgen Stech ◽  
Scott Krauss ◽  
Robert G. Webster
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Matrix Protein ◽  
Gene Products ◽  
Human Influenza ◽  
Influenza A Viruses ◽  
M Gene ◽  
Human Virus ◽  
Ha Gene ◽  
M Proteins

ABSTRACT To analyze the compatibility of avian influenza A virus hemagglutinins (HAs) and human influenza A virus matrix (M) proteins M1 and M2, we doubly infected Madin-Darby canine kidney cells with amantadine (1-aminoadamantane hydrochloride)-resistant human viruses and amantadine-sensitive avian strains. By using antisera against the human virus HAs and amantadine, we selected reassortants containing the human virus M gene and the avian virus HA gene. In our system, high virus yields and large, well-defined plaques indicated that the avian HAs and the human M gene products could cooperate effectively; low virus yields and small, turbid plaques indicated that cooperation was poor. The M gene products are among the primary components that determine the species specificities of influenza A viruses. Therefore, our system also indicated whether the avian HA genes effectively reassorted into the genome and replaced the HA gene of the prevailing human influenza A viruses. Most of the avian HAs that we tested efficiently cooperated with the M gene products of the early human A/PR/8/34 (H1N1) virus; however, the avian HAs did not effectively cooperate with the most recently isolated human virus that we tested, A/Nanchang/933/95 (H3N2). Cooperation between the avian HAs and the M proteins of the human A/Singapore/57 (H2N2) virus was moderate. These results suggest that the currently prevailing human influenza A viruses might have lost their ability to undergo antigenic shift and therefore are unable to form new pandemic viruses that contain an avian HA, a finding that is of great interest for pandemic planning.

scholarly journals Objective pathogen monitoring in nursery and finisher pigs by monthly laboratory diagnostic testing

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Nicole B. Goecke ◽  
Maja Kobberø ◽  
Thomas K. Kusk ◽  
Charlotte K. Hjulsager ◽  
Ken Steen Pedersen ◽  
...  
Keyword(s):  
Real Time ◽  
San Francisco ◽  
Influenza A Virus ◽  
High Throughput ◽  
Real Time Pcr ◽  
Influenza A ◽  
Clinical Signs ◽  
Swine Influenza ◽  
Porcine Cytomegalovirus ◽  
Swine Influenza A Virus

Abstract Background Infectious diseases are of great economic importance in commercial pig production, causing both clinical and subclinical disease, with influence on welfare, productivity, and antibiotic use. The causes of these diseases are often multifactorial and laboratory diagnostics are seldom routinely performed. The aim of the present study was to explore the benefits of monthly pathogen monitoring in nursery and finisher herds and to examine association between laboratory results and observed clinical signs, including coughing and diarrhoea. Three monthly samplings were conducted in three different age groups in six nursery and four finisher production units. For each unit, two pens were randomly selected in each age group and evaluated for coughing and diarrhoea events. Furthermore, faecal sock and oral fluid samples were collected in the selected pens and analysed for 18 respiratory and enteric viral and bacterial pathogens using the high-throughput real-time PCR BioMark HD platform (Fluidigm, South San Francisco, USA). Results In total, 174 pens were sampled in which eight coughing events and 77 diarrhoeic events were observed. The overall findings showed that swine influenza A virus, porcine circovirus 2, porcine cytomegalovirus, Brachyspira pilosicoli, Lawsonia intracellularis, Escherichia coli fimbria types F4 and F18 were found to be prevalent in several of the herds. Association between coughing events and the presence of swine influenza A virus, porcine cytomegalovirus (Cq ≤ 20) or a combination of these were found. Furthermore, an association between diarrhoeic events and the presence of L. intracellularis (Cq ≤ 24) or B. pilosicoli (Cq ≤ 26) was found. Conclusions The use of high-throughput real-time PCR analysis for continuous monitoring of pathogens and thereby dynamics of infections in a pig herd, provided the veterinarian and farmer with an objective knowledge on the distribution of pathogens in the herd. In addition, the use of a high-throughput method in combination with information about clinical signs, productivity, health status and antibiotic consumption, presents a new and innovative way of diagnosing and monitoring pig herds and even to a lower cost than the traditional method.

Sign in / Sign up

Export Citation Format

Share Document