Download Biology Lab Summary Final

Brittany Crockett
Article Summary
Results in Developing a Dengue Vaccine thus far
Dengue is a disease native to tropical areas, such as Central Africa and Guam, and is
known as breakbone fever for its characteristic fever and joint pain. Symptoms also include a
complete skin rash that resembles that of the measles, muscle pain, and headache, and in some
cases internal hemorrhaging resulting in death. The US Armed Forces Epidemiology Board has
been working since 1971 to come up with a vaccine, recognizing its worldwide spread and
effects. This procedure outlines tests done on known cultures of the virus, primary dog kidney
cells (PDK cells), and some monkey cell virulence results. These are based off of previous
experiments done in the early 80’s. Currently, the vaccine is still in development, and there is no
antiviral care available.
Dengue (DEN) virus, has four known types, numbered 1-4, and indicated as DEN-1, -2, 3, and DEN-4. Type DEN-3 is still in prototype strains. It is transmitted through mosquito bites,
with humans being the primary host of the virus. Previously, as discussed in the summarized
article, it has been inoculated onto primary cells that had been used to prepare vaccines. Some of
these include WI-38 fibroblasts, primary chick and duck embryos, and kidney cells from rabbit,
dog, and African green monkeys kidney (GMK). All four strains grew only in PDK and GMK
cells, and small amounts of DEN-1 and -2 were observed in WI-38 cells. Then they were
passaged into these cells for their use in human subjects, which was selected by the Walter Reed
Army Institute of Research for “phase II” human trials.
The present experiments re-attenuated wild DEN 1-4 strains as pure as they could
possibly be. They had no predictions which marker would match the virulence observed in
humans. Their only goal was to discover which marker would produce at the same levels that the
virus presents itself in humans.
What is attenuation and passaging? Why does that matter for discovering a cure?
Attenuation is to filter something to be less potent, such as sun through sun glasses, and
passaging is to split the cell and insert it into a new host. Human immunogenicity happens at
different virus passages, and in this test was tested from the highest passage possible down to the
lowest, as was the case with other vaccines.
Next, various plaque assays are performed. Assays are when passaged cells are coated in
a nutrient that forms a gel, and is incubated. The infected cells produce viral progeny, or
offspring. These progeny are restricted to neighboring areas because of the restrictive gel
formation, and thus the progeny can be seen in gel cultures as thick circles in the cell. In this
instance, the gel was a mixture of concentrations, and incubated at 37 degrees C for 60 minutes,
then after the gel overlay, incubated at the same temperature for 7 days, showing the sharpness of
the new plaque circles made of viral progeny.
PDKs and GMKs were taken from young animals free from viruses and in healthy,
monitored conditions. The strains of DEN 1-4 were passaged onto these kidneys and inoculated
at 32 degrees C, showing a maximal number of the virus around the sixth day after inoculation,
showing between 100 and 10,000 plaque forming units (PFU (plaque being the substance
observed in the Assays).
Finally, biological marker studies were done, showing how the antibody is produced in
animals. The major test was done on both male and female monkeys, in accordance to the
Animal Welfare Act. They were inoculated with the virus under the skin, then bled for 12 days to
study changes in viremia (where a virus gets into the bloodstream) and development of
antibodies.
The results show that no viremia was detected, although both monkeys developed some
antibodies for HI and DEN-4 strains. No viremia was observed in passage PDK 30 (passaged 30
times). What does this mean for the vaccine? After being infected with the original passaged
virus, the rhesus monkeys showed that the vaccinated virus was less infectious than the parental,
or original non-passaged, strain, but is still not a dormant virus. In order to be an effective
vaccine, the injected virus needs to be almost completely un-infectious, resulting in no symptoms
or affects but in the development of antibodies in the system. Thus, the vaccine is in need of
further development and study before it can be tested and passed in humans.