Group A rotavirus is the main pathogen causing diarrhea in infants and young children, and there is no specific drug yet. Only two reconstituted live attenuated vaccines are available on the market, but there are safety concerns such as limited spectrum of protection and recovery of viral virulence. It is important to study the pathogenesis of rotaviruses in order to develop effective vaccines and targeted drugs. Since the 1980s, people have been exploring animal models that are close to human rotaviruses in terms of clinical manifestations and histopathologic changes. In the early stage of research, large mammals such as neonatal cattle and sheep, pigs, etc. were used, but there are limitations of species, complex genetic background of animals. However, there are some drawbacks such as species restriction, complex genetic background, weak reproduction ability, sterility cannot be guaranteed, high experimental cost, and it is difficult to obtain specific animal antibodies from commercial channels, etc. Instead, people have turned their attention to small mammals with clear genetic background and strong reproduction ability, including rabbits, rats, mice and other commonly used experimental animals. These animals are easy to obtain inbred strains, and are also easy to use in the laboratory. These animals are easy to obtain inbred lines, SPF levels, and increase the number of samples, which increases the reliability of experimental data and greatly reduces the cost of experiments. However, it must be pointed out that due to the obvious species limitation of rotavirus, there is no truly ideal animal modlels of rotavirus-infection. In the future, it is expected that the ideal animal model can be established by transgenic animals, provided that the mechanism of virus-host interaction and the viral receptor are understood.
Principle
The pathogenesis of rotavirus diarrhea is still unclear. Early studies suggest that diarrhea is caused by destruction of the small intestinal mucosa. Rotavirus infection leads to shortening and flattening of the villi of the small intestine, loss of the columnar epithelial cells at the top of the villi, and replacement of the normal brush border by cuboidal cells in the crypts without a brush border.
Due to the low function of the new epithelial cells, the absorptive area becomes smaller, resulting in intestinal malabsorption. Secondly, there is a deficiency of lactase, and Holmes et al. showed that the epithelial cells of the small intestine are particularly sensitive to damage to the villi because the location of maximal expression of lactase is the apical mature absorptive cells of the villi, which are susceptible to secondary lactose malabsorption. This effect is exacerbated by lactose deficiency in newborn epithelial cells due to disruption of the intestinal mucosa. This effect is exacerbated by the lack of lactose in the neoplastic epithelium due to disruption of the intestinal mucosa, and the accumulation of products from the bacterial breakdown of lactose, which can lead to osmotic diarrhea due to increased luminal osmolality. However, diarrhea that occurs in the early stages when there is no damage to the intestinal mucosa or when there is no intestinal pathology on pathologic examination cannot be explained by this theory.
In recent years, it has been suggested that intestinal epithelial cell necrosis is not obvious, and the prominent pathological manifestation is the extensive vacuolike degeneration of small intestinal villi. The mechanism of rotavirus diarrhea is complex and unclear. Possible mechanisms include malabsorption due to intestinal mucosal damage, viral toxin action, stimulation of the enteric nervoussystem (ENS), and villi ischemia. A variety of cytokines are involved in the pathogenesis of rotavirus diarrhea.
Appliance
The common application areas of animal models of rotavirus infection are as follows: Animal models for the study of rotavirus infection can generally be divided into two categories, one is purely for the model of infection. Adult rats are commonly used; one is a diarrhea model, in which the animal develops diarrhea after infection. Because adult animals are less susceptible to rotavirus infection, most reported studies of rotavirus infection have been conducted in younger animals. Neonatal mice are internationally recognized as the best animal model for rotavirus infection.
Operation method
Animal models of rotavirus infection
Principle
The mechanism of rotavirus diarrhea is complex and unclear. Possible mechanisms include malabsorption due to intestinal mucosal damage, viral toxin action, stimulation of the enteric nervoussystem (ENS), and ischemia of the villi. A variety of cytokines are involved in the pathogenesis of rotavirus diarrhea.
Materials and Instruments
Equipment: Pigs and dogs Move The basic process of rotavirus infection can be divided into the following steps: For more product details, please visit Aladdin Scientific website.
Reagents: Hp bacterial solution (10)
6
-10
9
CFU/0.2 ml)
A. Viral culture: Monkey kidney MA104 cells (African green monkey fetal kidney cell line) were selected and grown in high glucose medium (IDMEM) containing 10% new bovine serum. Cells were washed three times with Hanks' solution after monolayer formation, and then rotavirus SA11 pretreated with 10 U/ml trypsin was added and adsorbed at 37 ℃ for 60 min. The supernatant was discarded, and 1 U/ml trypsin was added, and then the cells were incubated in bovine serum-free DMEM (37 ℃, 59% CO2). Collect the cell culture after the cell lesion is obvious.
B. Virus concentration:
Method 1, cell culture was frozen and thawed three times, centrifuged at 4 ℃ for 30 minutes at 3000 xg, discarded the precipitate, and the supernatant was dispensed and centrifuged at 100,000 xg for 3 hours, and then the precipitate was added with phosphate buffer solution (PBS), and then frozen at -70 ℃ for spare use. The titer of virus infection was determined by the plaque forming method, and animal experiments were carried out when the titer of virus reached 10' plaque forming unit (PFUYml).
After 48 hours of MA104 cell infection, collect the cells and supernatant, freeze and thaw the cells 3 times, then concentrate the virus by ultrafiltration, and take 10＇PFU virus to infect the animals orally.
C. Viral infection: Choose clean grade healthy pregnant mice, test their rotavirus antigen negative, wait for the pregnant mice to give birth, and then breastfeed them naturally, and then use a modified 1 ml syringe to instill 5x10＇PFU monkey rotavirus SA11 viral suspension orally on the 4th day. On the 4th day, 5x10＇PFU monkey rotavirus SA11 virus suspension was administered orally in a modified 1 ml syringe, followed by normal feeding.
D. Clinical Observation and Collection of Animal Fecal Samples: Dairy mice infected with SA11 were observed continuously and fecal samples were collected from the mice. The animals were observed once every 12 hours, and each time the abdomen was gently massaged with a finger to observe the production of diarrhea, which was classified into 6 grades according to the literature, Grade 1 is no stool, Grade 2 is yellow formed stool, Grade 3 is yellow paste-like stool, Grade 4 is yellow watery mucus stool, Grade 5 is yellow egg soup-like stool, and Grade 6 is completely yellow watery stool. Grade 3 (yellow paste-like stool) and above is diarrhea, Grade 4-5 is mild diarrhea, and Grade 6 is severe diarrhea. Grade 6 is severe diarrhea. The judgment of diarrhea is made by the same person.
E. RV antigen detection: Take appropriate amount of fecal samples into a container, add 9 times the volume of isotonic saline, stir and mix for 2 minutes, and then leave it to stand or centrifuge, and then use the Group A rotavirus diagnostic kit to detect the virus.
F. Small intestinal tissues for light microscopy and electron microscopy observation: Randomly execute 2 suckling mice in each group by cervical dislocation on the 4th day of the viral attack, respectively. About 1 cm of the jejunum was cut out to make samples for histopathological diagnosis and transmission electron microscopic observation, and photos were taken.
G. Measurement of the height of small intestinal villi: 2 mice were taken from each group, and 4 paraffin sections were taken from the jejunum of each mouse, with a total of 8 slices. After hematoxylin Ⅰ eosin (HE) staining, photos were taken under an optical microscope, and the height of all the villi was measured by professional image analysis software, and the reference length was the marking line of the counting pool of the blood cell counting board.