Gong Gong says

This is a posthumous blog of our father's (Lim Kok Ann) life. When our father passed away on 8 March 2003, he left behind an unpublished autobiography. We'd like to celebrate his life by sharing his autobiography through this blog.

"I have dredged these anecdotes from memory just to pass the time; if they amuse my grandchildren their purpose will have been served; if they provide any instruction, it will be a happy coincidence; that they are disjointed is probably to be expected.

Aurora was the name of my grandfather’s house in Kulangsu.   Amoy, where I spent the first five or six years of my life.   I still have vivid memories of events that took place when I was barely three years old.

Lim Kok Ann
October 1996"

Sunday, December 07, 2008

3:16 Polioviruses
Melnick’s first question when I saw him in his office was what I wanted to do and I told him I wanted to learn about poliomyelitis (or polio). “You should have stayed home,” said Melnick who had a dry sense of humour. He then showed me a newspaper cutting with the headline, “Polio strikes Singapore.”
There was no turning back, of course; I would have to study polio in Houston. Melnick put me to work with his wife, Dr. Matilda Benyesh-Melnick. She was a doctor from Israel who had come to study virology in US and Melnick had married her recently. Melnick was not a medical doctor but a PhD, and was one of the top-flight virologists of America and who understood neuropathology better than most doctors with MD. He was not allowed to touch patients and in this respect, Matilda Melnick or Mati, was a valuable colleague who could go into the wards without eye-brows being raised, and whose clinical judgement Melnick greatly valued. Mati’s family, named Benyesh, were of Bulgarian stock and had not long ago emigrated to Israel.

Mati was studying an outbreak of polio in Mexico from which she had obtained numerous blood and (frozen) faecal samples, for virus isolations. My task was to help her test the blood samples and identify the viruses isolated from faecal specimens of patients and their contacts. There are three immunological types of polio viruses. Infection with one type of polio virus conferred immunity against that type but not against another type.
Until a few years back, it was thought that polioviruses would grow only in nerve tissue because it was a neurotropic (preferring nerves) virus and destroyed nerve cells, and was found in the spinal cord and brain of patients. Thus, the accepted way of isolating polioviruses from autopsy material was to inoculate brain or spinal cord extracts into monkeys by the intra-cranial route. Infected monkeys would develop typical signs of muscular weakness and paralysis and the virus could be recovered from their brains and spinal cords. The discovery of at least two immunological types of polioviruses was confirmed by an experiment that involved the inoculation of hundreds of monkeys, an expensive procedure. Then one day Dr.Tom Weller who was working with Dr. Enders on the use of tissue cultures for growing measles virus happened to have one tissue culture tube left over from an experiment. He also happened to have some poliovirus brain extract at hand from another experiment. “Let’s put the poliovirus in the tissue culture and see what happens,” he said. And what happened was a discovery waiting to be discovered; polio virus grew much better in tissue cultures than measles virus and with cytopathogenic (cell-destruction) effect easily recognized with a low power microscope.

At first human fore-skin was used as starting materials for tissue cultures; there being a ready source from circumcision of infants which yielded young epithelial cells that grew well in favourable conditions. A more convenient source used later was monkey kidneys and this was what we used in Houston. The kidney was cut up into small pieces and treated with the enzyme trypsin which digested away the protein that held kidney cells together. The liberated individual kidney cells were then seeded in test tubes in a nutrient solution and placed in an incubator. After two or three days, the kidney cells grew out on the glass surface as a monolayer carpet of cells. Virus growing in such a tissue culture causes the kidney cells to swell up and separate from the monolayer, a result described as cytopathogenic effect (CPE) that was easily observed by use of a low-power microscope. In a way analogous with the haemagglutination-inhibition test, the CPE caused by a virus could be neutralized by its specific antiserum and this test was used for typing or for identifying polioviruses.

For a long time it had been assumed that polio was spread by contamination by pharyngeal secretions of patients, there being an obvious association between the pharynx and the brain and possibly by faecal material containing swallowed polioviruses. That polioviruses were present in stools of patients was easily proved when tissue cultures became available for growing them. That polioviruses grew in intestinal cells was a surprising discovery and led to the conclusion that infection by poliovirus was initially an intestinal infection without obvious clinical manifestations and that neural involvement, especially of the nerves concerned with motor functions, was only a complication that occurred in a small minority of those infected with the virus.
Along with poliovirus that were isolated from polio patients in the Mexico outbreak, Mati had also a number of CPE agents isolated from stool specimens of patients and contacts and even from non-polio patients. Because the CPE they showed was similar and all were recovered from stool, and had, as well, other common properties, for example, size, they were called enteroviruses, of which the polioviruses were the most important.
It was soon discovered that some CPE agents found in faeces were not enteroviruses, but were viruses normally found in the upper respiratory tract and named coxsackieviruses. The coxsackieviruses, some of which did not grow in tissue culture, caused respiratory infections as well as a wide variety of diseases, and complicated the identification of enteroviruses because they both produce CPE similar in appearance and had to be distinguished by neutralization tests. The number of different CPE agents that were enterovirus and coxsackievirus types exceeded 70 within a few years. For convenience though not strictly correct, in the following account, the term enterovirus included those coxsackieviruses that grew in tissue culture.


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