Influenza

Influenza virus

Influenza virus is the major cause of acute upper and lower respiratory tract infections in humans and is commonly known as the flu virus. Influenza viruses are classified into three types A, B and C, within the family Orthomyxoviridae. They contain eight negative-sense single-stranded RNA segments which encode 11 proteins (HA, NA, NP, M1, M2, NS1, NEP, PA, PB1, PB1-F2, PB2). The best-characterized of these viral proteins are the two major envelope glycoproteins, hemagglutinin (H) and neuraminidase (N). They are the immunodominant antigens recognized by the human immune system. Type A influenza viruses are further divided into subtypes according to the specific variety and combinations of the H and N proteins. Currently, influenza A(H1N1)pdm09 and influenza A(H3N2) are the circulating seasonal influenza A virus subtypes. In addition, there are two type B viruses that are also circulating as seasonal influenza viruses, which are named after the areas where they were first identified, Victoria lineage and Yamagata lineage. Type C influenza causes milder infections and is associated with sporadic cases and minor localized outbreaks. As influenza C poses much less of a disease burden than influenza A and B, only the latter two are included in seasonal influenza vaccines.

DDL offers the following influenza analyses:

• Detection and quantification of influenza A and B by qRT-PCR, aimed at the matrix gene
• Subtyping of influenza A(H1N1)pdm09, influenza A(H1N1) and influenza A(H3N2) by real time RT-PCR
• Segment-specific sequencing of influenza A and B by next generation sequencing
• Whole genome sequencing of influenza A by next generation sequencing
• Broad viral screening can be performed using e.g., the Luminex xTAG RVP FAST v2 assay, detecting: RSVA/B, influenza A (H1, H1pdm2009, H3), influenza B, parainfluenza (1-4), metapneumovirus, adenovirus, entero-rhinovirus, coronavirus (NL63, HKU1, 229E, OC43), bocavirus.

Samples

• Nasal, nasopharyngeal or oropharyngeal
• Sputum
• Cultured strains.

Influenza A whole genome amplification and sequencing assay

The influenza A whole genome amplification and sequencing assay can be applied to study:

• structural changes in the genome (gaps, insertions, deletions and mutations)
• minority variant composition in the sample down to 1% of read coverage
• influenza evolution (surveillance programs, outbreak analysis)

DDL’s influenza A whole genome sequencing and reporting pipeline consists of several steps:

Step 1. Universal influenza A single-tube whole genome amplification (RT-PCR)

Step 2. High-throughput next-generation sequencing

Step 3. Data analysis through our next-generation sequencing analysis and reporting pipeline

Step 4. Project-specific custom analysis

Step 1. Universal influenza A single-tube whole genome amplification (RT-PCR)

The influenza A RT-PCR amplifies all influenza RNA segments in one tube. The RNA segments are amplified with relatively equal efficiency down to low viral loads. The assay was optimized for minimal sample dilution in order to ensure a rich representation of minor variants in the sequencing data.

Step 2. High-throughput next-generation sequencing

Next-generation sequencing is performed on the Illumina platform which has a very low error rate and yields highly reliable data. Next-generation sequencing, in contrast to Sanger sequencing, allows for sensitive detection of minor variant populations and structural changes in genome in relation to the reference sequence.

Step 3. Data analysis through our next-generation sequencing analysis and reporting pipeline

DDL’s Athena virology pipeline is used to process the FASTQ files to a set of reports that are suitable for submission to health authorities. The Athena software is an amino acid and nucleotide sample composition analysis pipeline. It has been developed and validated following the requirements from GxP guidelines allowing for traceability, digital signing of records and database handling. For more information see Athena virology pipeline.

In the case of influenza A whole genome analysis, the raw FASTQ files are filtered based on the read quality, then the appropriate reference for mapping is chosen, the mapped sample is approved or rejected based on the requirements and relevant analysis can be exported (e.g. coverage, amino acid and nucleotide minor variant composition, consensus sequences).

Step 4. Project-specific custom analysis

Should you need any additional data analysis, DDL can perform it or assist with advice. Custom analysis such as, phylogenetic tree preparation, in-depth minor variant analysis or visualization of the mapping to the reference can be performed. For information on other possibilities please see custom pipeline development.