Influenza virus is the pathogen of influenza, belonging to Orthomyxoviridae, and its nucleic acid is composed of Segmented Negative-Sense RNA virus(sNSV). Influenza virus is spherical with a diameter of 80~120 nm, and it has an envelope. Its structure can be divided into three parts from outside to inside: the envelope, the matrix protein and the core.
1. Influenza A virus, IAV
In addition to seasonal influenza, several pandemics caused by IAV in history have imposed serious disease burden on human beings. IAV has a wide range of hosts, which is prone to antigenic drift and shift. Based on antigenicities of the hemagglutinin (HA) and neuraminidase (NA), IAV can divide the HA into 16 subtypes (H1-H16) and the NA into 9 subtypes (N1-N9). Therefore, influenza virus can be classified into various subtypes by different combinations of HA and NA.
IBV differs from IAV in gene structure and encoded protein, mainly in the following aspects:
① Segment 6 of IBV is a bicistronic structure, encoding NA and NB proteins, while IAV lacks NB protein.
② IBV is a bicistronic structure, with Fragment 7 of the two viruses both encoding M1 and M2 (BM2) proteins, and IAV is mainly obtained by splicing the transcribed mRNA.
③ PB1-F2 protein and PA-X protein encoded by IAV are unique to iteself, and haven’t been discovered in IBV.
Figure1. Comparison of gene structure between IAV and IBV
1. IAV continues to circulate in hosts like humans, pigs, dogs, horses, marine mammals, poultry, etc.
2. IBV almost exclusively infects humans with only a few reported cases in seals, horses, dogs and pigs. Due to the limitation of the host, the influenza B viruses have never been known to be associated with any pandemic.
3. ICV mainly infects children, pigs, feral dogs and dromedary camels. The relatively low prevalence of ICV and mild clinical outcomes have discouraged its inclusion in the routine virological screenings.
4. A novel influenza virus, recently classified as the influenza D virus (IDV), has been identified in several animals, such as swine, cattle and sheep from across the globe.
Figure 2. Hosts of influenza viruses
It is particularly important to detect some highly pathogenic influenza viruses early, and hence here are some detection methods.
Several mature technology platforms for influenza viruses detection both at home and abroad
Molecular diagnostics has become an indispensable and important technique for virus diagnosis and research for its highly sensitive and specific, faster and simple diagnosis.
RT-PCR is one of the most commonly used techniques for influenza viruses detection, which has become a gold standard assay for influenza diagnosis by replacing the role of viral isolation and culture.
2.Real-time PCR(quantitative PCR, qPCR)
qPCR is often used for the detection and typing of various viruses including influenza. This method adds fluorophores into the PCR reaction system, and then monitors the whole PCR process in real time by the accumulation of fluorescence signals. Finally, the unknown template is quantitatively analyzed by standard curve, including SYBR Green method and TaqMan Probe method. The high specificity of probe technology, sensitivity and quantification of spectral technology, which has the advantages of intuition, good repeatability, strong specificity, high sensitivity and easy operation. In particular, Probe method features high specificity, and spectroscopic techniques have sensitive and quantitative characteristics. Both of them enjoys advantages of intuition, good repeatability, strong specificity, high sensitivity and easy operation, etc.
Figure 4. Basic principles of qPCR
3. Loop mediated isothermal amplification (LAMP)
LAMP is a novel isothermal nucleic acid amplification method, which can be used to detect viruses such as SARS, adenovirus, rhinovirus, etc. By designing four specific primers for specific regions of target genes, it uses a strand displacement DNA polymerase to keep the temperature at isothermal conditions (about 65℃) for several tens of minutes, and then the nucleic acid amplification reaction can be completed. There is no need for thermal denaturation of template, long-time temperature cycle, tedious electrophoresis, ultraviolet observation and other processes. The sensitivity of this method is similar to that of RT-PCR.
Figure 5. Basic principles of LAMP
Microarray is an effective tool for influenza virus detection and typing. In recent years, researchers have more interests in using nanoparticles and silver staining to replace the fluorescent dyes to improve the sensitivity. It has become a new method for accurate and rapid diagnosis of seasonal influenza or pandemic.
Influenza virus antigen detection technique contains the direct flourescent antibody (DFA) test. Rapid influenza diagnostic tests (RIDTs) can provide results within 30 minutes. Influenza virus antibody detection technique includes the colloidal gold-based immunochromatographic and virus neutralization test.
Based on the detection of influenza viruses, Vazyme has introduced relevant diagnostic materials and solutions:
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