1 Cells for culturing influenza virus 1?1 Selection of cell lines Chicken embryos are used when initially cultivating influenza viruses, but isolation of influenza viruses from chicken embryos can easily cause virus mutation and allergic reactions, and can also cause There is a problem of contamination of exogenous viruses during large-scale production. The use of Vero and MDCK cells to culture influenza viruses not only solves the above problems, but also makes the virus's immunogen more stable. Qi Fengchun [1] et al. found that under the same experimental conditions, the HA titer of viruses cultured on MDCK cells was significantly higher than that on Vero cells. Therefore, when we culture influenza virus cell lines, we choose MDCK cells, whose passage number is preferably less than 35 generations. , too old cells will make it difficult for viruses to load. 1?2 Cells are best loaded with viruses. MDCK cells of a cell age of two days are very vigorous in their reproduction. It is necessary to inoculate the cells in the cell culture flask into a six-well plate one day in advance. If MDCK cells with a cell age of two days are used to load the virus, then The positive rate of virus culture is almost 0, while the positive rate of virus load in MDCK cells with a cell age of 12-24 hours has little difference [2]. 1?3 The optimal density of cells to load viruses. First of all, the density of MDCK cells should not be too thin. If the density of MDCK cells is too thin, they will not be able to grow, and the cells should not be packed densely. This will also greatly reduce the positivity of virus inoculation. Rate. It is best to make the cells just cover the well, when the HA titer of the virus harvested after loading the virus is the highest. When workers inoculate cells in a 6-well plate one day in advance, the growth rate of the cells should be considered to ensure that the cell density is appropriate when loading the virus the next day. Secondly, when MDCK cells are inoculated into a six-well plate, the density must be uniform. It is easy to happen that the cells gather at the edge of the well, and the density in the middle is too thin, which will also affect the positive rate of virus load. To avoid this situation, the staff must be skilled. If this situation occurs, gently shaking the culture plate (to avoid liquid splashing) can effectively improve the distribution of cells. 2. Reagents for culturing influenza viruses. Different reagents used to culture influenza viruses will also greatly affect the positive rate of virus culture. First of all, do not use expired reagents; secondly, it is best to use reagents from well-known manufacturers, such as GIBCO; thirdly, take the reagents out of the refrigerator for a period of time before use, and it is best to wait until the temperature of the reagents has equilibrated to room temperature before use. If the cells are replaced, super-cold reagents will affect the growth status of the cells; if the cells are washed before loading the virus, super-cold reagents will cause the cells to suddenly shrink and reduce the positive rate of virus culture. 3 Samples 3.1 Storage of samples The storage time of samples directly affects the positive rate of virus load. The fresher the specimen, the higher the positive rate of virus culture. If the sample cannot load cells in time, the sample should be placed in a -70°C refrigerator to avoid repeated freezing and thawing. The more freezing and thawing times, the lower the positive rate of virus culture. Note that samples cannot be placed in a refrigerator at -20°C. Influenza viruses are very unstable at -20°C. If the sample of the day cannot be inoculated until the next day, the sample should be placed in a refrigerator at -20°C. 3.2 Aseptic processing of samples Because the sampling conditions of the samples cannot be sterile, although there are antibiotics in the sampling solution, it cannot guarantee that all bacteria will be killed, so it is best to filter the positive samples into a sterile 1.5ml centrifuge tube. spare. Some workers add antibiotics to cell culture plates to avoid contamination. Although this saves trouble, it will damage the cells and reduce the positive rate of virus culture. 4 Virus loading steps 4?1 Cell washing Pour out the cell culture medium in the six-well plate and wash each well with HBSS more than three times. Its function is to wash away the residual FBS. FBS inhibits influenza by inhibiting trypsin. Replication of viruses. After pouring out the washing solution, add the washing solution or virus solution immediately. Exposing the cells to the air for too long will greatly reduce the positive rate of virus culture. It is best to shorten the time the cells are exposed to the air to 5 seconds. Within. 4?2 The inoculation amount of virus liquid The inoculation of virus liquid is very particular. The virus liquid loaded on the cells should be greater than 300u, otherwise it will not be enough to cover the entire well of cells.
After 1 to 2 hours of incubation with 37!, no matter how much volume of virus liquid is initially loaded, 300ul of virus liquid should be left in each well before adding virus culture medium. If all the virus liquid is discarded or too little is left, the virus culture will be compromised. The positive rate is greatly reduced; if too much virus fluid is retained, for example, more than 400u, the positive rate will also be greatly reduced, because retaining too much defective virus will seriously affect the replication of normal viruses. 4.3 Incubation time of virus liquid Theoretically, the virus liquid can be incubated at 37°C for 1 to 2 hours, but we found that 1 hour is still too short. It is best to add the virus culture medium at 1 hour and 15 minutes. Do not take too long, which will also cause the virus to be incubated. The decrease in the positive rate of culture may be because the pH value of the sampling solution is different from that of the virus culture medium, and the slightly longer incubation time may cause damage to the cells. In blind transfer, the incubation time can be more than 2 hours, because the virus liquid used at this time is also a virus culture medium, not a sampling liquid. 4?4 Preparation of virus culture medium The virus culture medium is prepared by adding DMEM, HEPES and trypsin. The function of HEPES is to enhance the buffering capacity of the system. The function of trypsin is to convert the non-cleavable HA0 of the virus particles into cleavage. Types of HA1 and HA2, thereby improving the ability of influenza viruses to replicate in cells, because influenza viruses are infectious only if the hemagglutinin (H) is cleaved. However, storing HEPES and trypsin in DMEM for too long at 4!*** will cause the pH value to rise, so we generally prepare the virus culture medium for immediate use. 4.5 Determination of virus HA titer The emergence time of CPE varies with the type and strain of the virus and the size of the infection. We will observe whether CPE occurs in the specimen 3 days after inoculation. If CPE occurs, the HA titer will be measured. If the titer is greater than 8, the virus will be harvested. The virus subtype will be determined by HI experiment. If the virus titer is less than 8, blind transmission will be carried out. Then HA will be measured on the 3rd day. Titer. If CPE does not appear within 7 days of loading the virus, and no HA activity can be detected in the maintenance solution, the specimen can be considered negative and discarded. The highest peak of virus titer appeared on 3d, and both 4d and 2d were lower than the virus titer on 3d. Before harvesting the virus strains, freeze and thaw the cells once at -80°C/37°C to help increase the HA titer of the virus.