This protocol of a disease model you see dengue virus infection in the brain become an in vivo platform for screening viral and host vectors associated with neuropathogenesis. Unlike previous models, our models use tools of the infection with a low viral titer, to infect the immunocompetent Institute of Cancer Research suckling mice for encephalitic-like disease procreation induction. Demonstrating the procedure will be Ting-Jing Shen, a PhD candidate from my laboratory.
Before initiating the infection procedure, dilute non-adapted dengue virus two stock, to a one times 10 to the fifth plaque forming units per 40 microliters of RPMI 1640 medium concentration. Next, load one 0.3 milliliter syringe equipped with a 30 gauge needle with 10 microliters of diluted virus. And one 0.3 milliliter syringe equipped with a 30 gauge needle, with 30 microliters of virus per animal.
For intercerebral delivery of the virus, press the oracle between the thumb and index finger to gently restrain a seven day old suckling Institute of Cancer Research mouse in a prone position and inject the 10 microliter volume of diluted virus into the lambda area, at the intersection of the sagittal and lambdoid suture. After the intercerebral injection, use the thumb and index finger to hold the mouse in a supine position, and gently intraperitoneally, inject the 30 microliter volume of diluted virus, into the murine abdomen. For scoring of the disease progression, assign a score of zero to five to each inoculated mouse daily, according to the grade of acute viral encephalitis-like illnesses, and plot the scores for each day as a curve based figure, using the mean plus and minus the standard deviation of the daily test scores in each group.
Compared with the body weight changes measured in mock, medium only inoculated mice, dengue virus inoculated animals exhibit a decrease in body weight at every time point during the trial. In addition, dengue virus infected animals demonstrate a significant increase in hunch back postures, limbic seizures, limbic weakness, paralysis, and death compared to the symptoms displayed by mock inoculated animals. Indeed, dengue infected mice exhibit a time dependent reduction in survival rates that is not observed in mock inoculated animals, confirming the establishment of an infectious model of dengue virus within the central nervous system that demonstrates the progression of acute viral encephalitis-like illnesses in mice.
Puncture of the wrong site may result in needle blockage by the score and the overflow of the virus solution outside the head, resulting in failed injection and a failed infection. Following the procedure, brain samples can be harvested for pathological damage, immune cell infiltration, and viral replication analysis by hematoxylin and eosin staining flow cytometry, and plaque formation assay, respectively. Our immunocompetent murine model, provides a platform for exploring the pathogenic mechanisms of dengue virus infection, for the development of dengue virus vaccines, or anti-dengue virus drugs.
Dengue virus is a biosafety level two pathogen, and, as such, all experiment with this virus should be completed in a qualified animal biosafety level two laboratory.
