Prompt You have isolated what appears to be an intracellular

Prompt: You have isolated what appears to be an intracellular parasite from a blood culture from a human case of chronic fatigue syndrome. Electron microscopic studies indicated that the isolate looks like a virus, but the results are not conclusive. Your virology/bacteriology training makes you believe that this isolate is either a new virus or a protozoan parasite. Your virology professor tells you that your isolate is most likely a virus.

Assume that you have many eggs, and you can grow this pathogen in large quantity and purify it with ease. How would you go about to establish conclusively that your new isolate is a virus? Please explain in detail.

Solution

Chronic fatigue syndrome (CFS),which is caused by the infection with herpes human virus or cytomegalovirus. It may also be caused by the infection with protozoans like brucella,mycoplasma,chlamydia,or coxiella as confirmed by the virology/bacteriology electron microscopy training experiments in the laboratory by students.

PROTOCOL--Blood cells were collected along with the addition of EDTA. The samples were centrifuged at 13,000rpm for 5-10min and the supernatant was discarded. The remaining centrifuged precipitated sample was mixed with chelex solution and dissolved by heating at 60 degree centigrade and 100 degree centigrade.

Protozoan mycoplasma analysis--The sample were then mixed with Tris-HCl and triton-X-100 and PCR analysis was done to by designing primers which specifically analyse particular genes present in the genome of mycoplasma, or chlamydia.Further agarose gel electrophoresis and UV-analysis was done for the presence of amplification of DNA bands and further 11-dUTP probe is designed for specific genes in protozoan which is used for southern hybridization to confirm the infection by the protozoan. If the bands are not present, then it is confirmed that the infection is not caused by protozoan like chlamydia or mycoplasma but may be due to a virus like human herpes virus-6.

ELECTRON MICROSCOPY OF HUMAN HERPES VIRUS INFECTION LEADING TO CHRONIC FATIGUE SYNDROME--the infected sample of blood cells along with the virus are grown in the culture medium along with carbon dioxide to maintain sufficient growth of virus cells inside the infected blood cells. The cells are again washed with culture medium like DMEM. The electron microscopy experiments is done further to confirm the presence of virus particles. In the starting, the virus particle just starts developing after about 2.5 hrs of infection. The virus consists of outer covering of capsid and envelope proteins and genetic material inside. After about 48 hrs , the capsid develop in the form of crystal leading further to the formation of dense granules inside the infected cells. The virus cells then fuse with the membranes of other organelles inside the blood cell and lead to infection. Further caesium chloride ultracentrifugation is done to purify and separate the viral particles from the blood cells by spinning at high speeds which separates the capsomere and capsid of viral particles from the genetic material of virus and further separates the infected blood cells which are degraded and lysed after spinning and are discarded.