Virus isolation and identification steps!
Although animal inoculation, chicken embryo culture, organ culture (such as tracheal cartilage rings), and cell culture can all be used for the isolation and culture of respiratory infection viruses, almost all virus laboratories use cell culture to grow respiratory viruses, while chicken embryo culture is used in vaccines. It is more used in production, and laboratory testing is rarely used.
1. Steps
The basic steps of virus isolation and identification can be divided into the following types according to the different types of viruses:
(1) Virus isolation
1. Selection of Cell Lines
For different respiratory viruses, sensitive cell lines/strains should be selected for culture. In order to adapt the cultured cells to the isolation and culture of various viruses, a mixed culture cell line can be established, such as the R-mix cell line combined with mink lung cells (MV-1-Lu) and human adenocarcinoma cells (A549). For the isolation and cultivation of influenza A and B viruses, respiratory syncytial virus, parainfluenza virus and adenovirus.
2. special culture conditions
(1) Influenza virus: The prerequisite for the virus to penetrate into the host cell is that its hemagglutinin is cleaved by trypsin or trypsin-like enzymes. Passage cells MDCK do not produce such enzymes. Therefore, in order to help influenza virus enter MDCK cells, it is necessary to Add a low concentration of trypsin to the culture medium. In addition, the calf serum in the culture medium will inactivate trypsin, so the cells should be washed with serum-free culture medium or other balanced salt solutions before inoculating the cells with virus samples.
(2) Coronavirus: For different coronaviruses, select different cell lines for culture (Table 6-6). It is worth noting that the temperature for cultivating coronavirus is slightly lower, which is 33-35°C
3. cytopathic features
(1) Influenza virus: The lesions that cause MDCK cells are characterized by cell swelling and rounding, increased intercellular space, nuclear pyknosis or rupture, and in severe cases, some or all of the cells fall off.
(2) Parainfluenza virus: Different parainfluenza viruses have different characteristics of cell lesions, for example, cells infected with hPIV-1 become smaller and round; cells infected with hPIV-2 are prone to cell fusion; cells infected with hPIV-3 Cells are prone to bridge-like structures; cells infected with hPIV-4 are prone to changes in the entire monolayer of cells, with swelling and rounding of cells, increased gaps, and even shedding.
(3) Respiratory syncytial virus: Cytopathies are characterized by rounded cells and cell fusion.
(4) Human metapneumovirus: Cytopathic effects appeared 3 to 23 days after inoculation (average 17.3 days). Most of the lesions were characterized by the appearance of small, round, granular refractory cells, and syncytial lesions were occasionally seen.
(5) Coronavirus: The cytopathy caused by SARS-CoV is obvious, and the cells become round and fall off. The cytopathy caused by MERS-CoV manifests as cell fusion under acidic conditions, and as cell rounding and shedding under neutral or slightly alkaline conditions. The lesions of HCoV-229E, OC43, and NL63 are mild, and the cells are filamentous, with increased granules, rounded, scattered on the cell layer, and the cells are rarely completely destroyed.
(6) Adenovirus: The characteristic of cell lesions is that the cells first become round and then spherical, with enhanced refraction, and many diseased cells gather into clusters of grapes.
(2) Virus identification and typing
1. Immunological methods include classic neutralization tests, hemagglutination inhibition tests, and various immunolabeling techniques that are still widely used. The premise of this type of detection method is to have the corresponding antibody to the virus, such as using the antibody against the nucleoprotein of influenza virus or the fusion protein of respiratory syncytial virus to determine whether it is the corresponding virus, and then use specific antiserotype or determine the specific antigen type .
2. Methods based on nucleic acid detection
(1) Reverse transcription PCR: It is increasingly used for identification and typing of viruses, which can have several forms:
① Real-time fluorescent RT-PCR, using different primers and probes, and performing typing and identification according to the presence or absence of specific amplification products.
② Conventional RT-PCR, using a combination of common primers and complementary typing primers, electrophoresis after amplification, and different types of viruses can be distinguished according to the size of the amplified product fragments, such as the G protein gene of respiratory syncytial virus in different subtypes. According to the characteristics of high variation between types, three primers were designed, P1, P2 and P3.
P1 is designed according to the nucleotide sequence of the 5' end of the G protein gene. The nucleic acid sequence in this region is highly conserved between A and B subtypes, and it is a general primer for A and B subtypes. P2 and P3 are located at the 3' end of the nucleotide respectively. , are complementary to subtype A and subtype B respectively. If it is subtype A, P2 and P1 will amplify a fragment of 277bp together. If it is subtype B, a fragment of 863bp will be amplified by P3 and P1. The PCR product is in the agar Electrophoresis is carried out in sugar gel, and A and B subtypes can be determined according to the size of the bands.
(2) Sequence determination: identify and type by sequence comparison, and study the kinship of different viruses according to the phylogenetic tree. With the development of sequencing technology and the reduction of cost, this technology will be more widely used.
2. Matters needing attention
(1) Identification and disposal of specimen toxicity:
If the cells senescence rapidly within 1 to 2 days of inoculation, it may be due to the non-specific toxic reaction caused by the toxic substances contained in the specimen. Take 0.2ml of the virus (this is the second generation of the virus) and inoculate it on a new monolayer of cells; ②If the toxic reaction occurs again, the original sample should be diluted with PBS and inoculated into the same type of cells again.
(2) Pollution and countermeasures:
Bacterial contamination can easily lead to turbidity of the culture medium or cell death, making the infected cells unable to exhibit cytopathic effect (CPE), or fungal contamination, the culture medium becomes sour, and the cells cannot grow normally. Improper handling of bacteria, or operational contamination, or insufficient/invalid concentration of antibiotics in the culture medium. Mycoplasma contamination, the cell shape does not change significantly, and the culture medium is not turbid, but the growth characteristics of the cells, cell membrane composition or chromosomes will be abnormal. Therefore, it is necessary to regularly detect whether the cells are contaminated by mycoplasma. Once contaminated, the contaminated cells need to be discarded.
(3) Avoid virus cross-contamination:
When pouring the cell culture medium inoculated with virus, the liquid should be removed with a pipette, and a new pipette should be replaced at each step to avoid aerosols caused by violent vibration.
(4) Blind pass:
When there is still no cytopathic disease after 7 to 10 days of culture, or other techniques do not show the presence of the corresponding virus in the culture, the cultured cells are also harvested, and the cells are inoculated again. After 3 generations of blind passage, there is still no cytopathic disease, or other tests When both are negative, the virus isolation can be considered negative.
