Isolation and titration of dengue viruses by the mosquito inoculation technique

Mosquito inoculation is a highly sensitive technique for isolation and titration of dengue virus (DENV) from sera, human tissues, wild animals, or mosquitoes. It has been under utilized since it was described 40 years ago because most dengue laboratories do not have access to an insectary to rear mosquitoes. This technique requires good eye-hand coordination while doing manipulation under a stereoscopic microscope, and extensive practice is needed to become proficient at inoculating mosquitoes. Following inoculation, mosquitoes are held for 10 days to allow dengue virus to replicate and disseminate to tissues throughout the mosquitoes. They are then harvested and examined for the presence of viral antigens in head tissue by either immunofluorescence assay (IFA) or PCR (polymerase chain reaction). The mosquito infectious dose 50 (MID50) is calculated using the method of Reed and Muench to quantitate the virus. This method can be used for other arboviruses as well as for dengue.