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 When our father passed away on 8 March 2003, he left behind an unpublished autobiography.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 21, 2008

4:5 Su Hui and Sing Yuen

For a year or so before I became a Professor, my family had been living in University quarters in Ross Avenue, on the other side of Bukit Timah Road from the main University campus. It was while we were at Ross Avenue that my family was enlarged in 1960 by the arrival of a son who was named Su Hui (thinking with perception) by Grandma Yin. There was a twelve year gap between Su Hui and Su Chong, so we were in effect starting another family. By coincidence Carleton was in Singapore at that time and on visiting mother and child at Gleneagles Hospital, swiftly examined the new arrival and pronounced him free from any obvious defects. We did not exactly plan to have another child, but I told Rosie that it would be wise to have another so that Su Hui could have a companion, and so we did in due course, and Sing Yuen (Star Cloud) was born in 1962 when we were living in College Road.

Note: Chinese name their children with variations on a theme so that siblings recognize the cohort they belonged to. Soon Hock. Soon Lock and Soon Siew were three brothers well known in Raffles College. Long strings of name variations ordered by generations are available. My grandfather named his children unconventionally and I did likewise.

4:4 Master
During our absence in England the old FMS Hostel on the hill behind the College (now Faculty) of Medicine had been torn down and replaced by two four-storey blocks of student accommodation plus a two-storey administrative block where the dining facilities were located. The new hostel was named King Edward Hall of Residence in remembrance of the former name of the King Edward VII College of Medicine that was built in 1927. The main function of K.E. Hall was to accommodate medical students, both men and women, in their clinical years so that they could be near their work-place when on night duty, or to study their cases in after-class hours. Thus, the top floors of the residential blocks were reserved for women students. Priority of accommodation was given to medical students but dental students were also admitted when there were rooms to spare.

The previous Master (administrator) of K.E.Hall was Professor Sandosham who had resigned to take up a WHO appointment in Malaria control. I was appointed to succeed him. The Master’s duty was to be a father-figure for the students in his charge, to discipline them if necessary, and to direct the work of the cooks, cleaners, and gardeners, with the assistance of a Domestic Manager. To help him mind the students, a number of Fellows were appointed from the university staff, including a woman staff member whose job it was to mind the students on the D floors. For the responsibility for the welfare of over 200 students the Master was paid the token sum of $100, but of more practical value was the free use of a large house at the upper end of College Road past the Faculty building.

To live near my work-place as Carleton would have loved to do was something I much appreciated. When we had settled down in the official Master’s residence at 18 College Road, I asked Amak to move in with us. Eu Jin, Rosie’s brother, and his wife Francis had been living in Oberon with Amak, but Eu Jin had built his own house in Newton and was about to move into it. 18 College Road was a large colonial type of compound house with three large bedrooms and a verandah upstairs, a large dining room and study downstairs, with kitchen and servant quarters in an annex.

Editor’s note: Actually Uncle Eu Jin moved into his new house in Newton in the mid fifties. Grandma lived alone in Oberon for a few years, then in a terrace house in Cairnhill Road for a few years, before joining the family in College Road.

The Master’s duties were not onerous and I cast myself not so much as a father-figure, but as an older brother. I had liberal ideas and I believed that I should treat my charges as adults and I tried to improve facilities for their extra-curricular activities. I improved the tennis-courts; for the high- brows I acquired a baby grand piano for the main lounge over the dining hall; for the low-brows, we had downstairs in the Games Room Iwo billiards tables. I also partitioned off one corner of the Games Room as a card room which was used for mahjong, not bridge as I had hoped, and which engendered some criticism that I was teaching the students how to gamble. I did not have to, they already knew how. My intention was to provide an alternative to mahjong in the bedrooms, which was prohibited, not because gambling in the bedrooms was prohibited, but because the all-night sessions disturbed the occupants of adjacent rooms. There were suites in each block for occupancy by Fellows and I assigned a vacant suite as the “Senior Lounge” for use by Final Year students to entertain their friends privately. My idea was that the Hall should be the students’ home from home and they should be able to entertain their friends in a private area other than their bedrooms where visitors were not allowed in the evenings.

I invited all students to visit me at Chinese New Year and to entertain them I set up a roulette table as I had heard Amak did in Oberon in the old days. This was probably not such a good idea, but we played for small stakes and I had the notion that if the students had any itch for gambling, they should have it eased when they did not have so much money to lose! I was Master of K.E. Hall for two three-year terms and I was succeeded by Professor Khoo Oon Teik who converted the card room into a prayer room, and guided the students along more sober paths than I had. In those days Oon Teik and I held almost diametrically opposing views on religion! It was possibly a good thing for students that their teachers had different ways of thinking.
How much Oon Teik and I differed was shown in an incident that took place when I was still a young lecturer. The students had organized a debate with the challenging proposition “Religion is an unnecessary institution” or some such theme. Being a free-thinker in those days, I made so free as to declare in favour of the proposition since the existence of God cannot be proven. I was astonished, when the debate was over, to be confronted by an agitated female who told me, “Dr. Lim, you are wrong, you are wrong!” The good lady was Mrs. Khoo Oon Teik who was troubled by the fact that an university lecturer was teaching young people such heresy.

4:3 Sabin Vaccine

WHO appointed me a member of the International Panel of Experts on Virus Diseases, and designated our department a Regional Enterovirus Centre. In 1962, the Minister of Health appointed me a member of its Poliomyelitis Committee that was tasked with the problem of preventing a recurrence of the polio outbreak of 1958 when there were more than 400 cases. The possible use of the Salk vaccine was soon ruled out; apart from the cost of the vaccine, the prospect of organizing an immunization programme involving the injection of over 300,000 children, our target population, was forbidding. We could be causing more cases of hepatitis through faulty sterilization of syringes (disposable syringes were not yet available then) than the polio cases we were hoping to prevent.

Note: Salk vaccine, pioneered by Jonas Salk, was made by treating poliovirus with heat and formalin until it was not able to cause disease. Because the inactivated virus did not grow in the body, a large amount of virus had to be inoculated to stimulate antibody production. Sabin vaccine was a live virus unable to cause paralysis, but because it grew in the intestines of vaccinees, a much smaller amount of virus needed be used to give protection.

The use of the Sabin oral vaccine had to be considered. This was made of polioviruses; one of each of the three types, that had been passaged many times in tissue culture and had lost their ability to cause paralysis in monkeys when inoculated by the most sensitive route. WHO had not yet declared the vaccine “safe”; although hundreds of thousands of children had been given the oral vaccine in field trials without getting polio, there lingered a suspicion that the vaccine virus could revert to pathogenicity as it was propagated to make vaccine, and actually cause poliomyelitis in the children it was meant to protect Many of the field trials had been done in the Soviet Union and the Soviets were waiting for the blessing of the WHO Poliomyelitis Committee of which I was a member (representing Asian countries!) before recommending to their government the general use of the vaccine.

“So far the vaccine has caused no polio cases,” complained the senior Russian at one meeting, “how many more trials do you want me to do before saying the vaccine is safe?” He was not going to take the blame if the child of a Party cadre got polio from the vaccine he was going to dish out. In Singapore, similar doubts of vaccine safety were raised and as mentioned earlier, it was suggested that we should wait until the vaccine was shown to be safe, rather than use Singapore children as guinea pigs in the first “mass use” of oral polio vaccine.

I relayed to our Committee the advice that Melnick had given me and which was mentioned above. There was no question in my mind that if we did not start soon to immunise the children that had been born since the 1958 outbreak, another polio outbreak would take place, engendered in the same way as the previous outbreak had been. I gave as the “considered opinion” of the Professor of Bacteriology that the Sabin vaccine should be used, and said that our department would monitor all children who developed polio-like symptoms to determine whether it was possible the oral vaccine had caused these symptoms, taking into consideration the time factors. It was a necessary spur to get theMinistry of Health going, and I believed that putting my head on the chopping block if anything went seriously wrong in the use of the vaccine was part of my job.

The vaccine was obtained through WHO initially and later from commercial sources under licence by Dr. Sabin who oversaw by correspondence the early use of oral poliovaccinc. Lee Liang Hin diluted the concentrated vaccine stocks which we mixed and dispensed in eye-dropper bottles so that one drop was the required dose for each administration of vaccine. The government polioimmunization programme was earned out in two phases. In phase 1, children between six months and two years of age were given two doses of polio vaccine at one month’s interval, reaching about 70%of the target population. In the second phase, the “routine programme”, children three months old would be given three doses of polio vaccine at monthly intervals, then two doses at four years of age and finally two doses 7 to 8 years of age on entering primary school. We joined the teams of Ministry of Health nurses who visited Maternity and Infant Welfare clinics to immunise young for the purpose, and took careful notes of the names and ages of children to whom the vaccine was given, and other useful details.

This vaccination regime so effectively reduced the incidence of polio in Singapore that the surgeons whose specialty was tendon transplantation to remedy muscle weakness had to turn their hands to other skills and hospital swimming pools for treatment and rehabilitation of polio patients were closed. Meanwhile, all cases of suspected polio were closely followed by Ministry of Health doctors who took stool specimens and blood samples for us. If we isolated a poliovirus, we tried to find out if it was a vaccine virus or not (very often it was) though the technique was difficult. From the date of onset of symptoms we back-tracked and determined if it was likely that the symptoms were vaccine-related, supposing the child had been given oral poliovaccine. Our fmding was that every child that actually developed polio had not been givenpoliovaccine recently, and as a corollary, we found that children who had received the full course of three doses of poliovaccine never developed poliomyelitis. This result eminently justified the great effort and expense the Ministry of Health invested in the polio immunization programme. In my Diploma of Public Health class, the doctors from neighbouring countries would ask when their country should start to use oral poliovaccine. My advice to them was to find out what their government was spending on treatment and rehabilitation of polio patients. I said, “When the cost of treating polio becomes greater than the estimated cost of buying and distributing the vaccine it will be time for you to adopt a polio immunization programme.”

4:3 Sabin Vaccine

WHO appointed me a member of the International Panel of Experts on Virus Diseases, and designated our department a Regional Enterovirus Centre. In 1962, the Minister of Health appointed me a member of its Poliomyelitis Committee that was tasked with the problem of preventing a recurrence of the polio outbreak of 1958 when there were more than 400 cases. The possible use of the Salk vaccine was soon ruled out; apart from the cost of the vaccine, the prospect of organizing an immunization programme involving the injection of over 300,000 children, our target population, was forbidding. We could be causing more cases of hepatitis through faulty sterilization of syringes (disposable syringes were not yet available then) than the polio cases we were hoping to prevent.

Note: Salk vaccine, pioneered by Jonas Salk, was made by treating poliovirus with heat and formalin until it was not able to cause disease. Because the inactivated virus did not grow in the body, a large amount of virus had to be inoculated to stimulate antibody production. Sabin vaccine was a live virus unable to cause paralysis, but because it grew in the intestines of vaccinees, a much smaller amount of virus needed be used to give protection.

The use of the Sabin oral vaccine had to be considered. This was made of polioviruses; one of each of the three types, that had been passaged many times in tissue culture and had lost their ability to cause paralysis in monkeys when inoculated by the most sensitive route. WHO had not yet declared the vaccine “safe”; although hundreds of thousands of children had been given the oral vaccine in field trials without getting polio, there lingered a suspicion that the vaccine virus could revert to pathogenicity as it was propagated to make vaccine, and actually cause poliomyelitis in the children it was meant to protect Many of the field trials had been done in the Soviet Union and the Soviets were waiting for the blessing of the WHO Poliomyelitis Committee of which I was a member (representing Asian countries!) before recommending to their government the general use of the vaccine.

“So far the vaccine has caused no polio cases,” complained the senior Russian at one meeting, “how many more trials do you want me to do before saying the vaccine is safe?” He was not going to take the blame if the child of a Party cadre got polio from the vaccine he was going to dish out. In Singapore, similar doubts of vaccine safety were raised and as mentioned earlier, it was suggested that we should wait until the vaccine was shown to be safe, rather than use Singapore children as guinea pigs in the first “mass use” of oral polio vaccine.

I relayed to our Committee the advice that Melnick had given me and which was mentioned above. There was no question in my mind that if we did not start soon to immunise the children that had been born since the 1958 outbreak, another polio outbreak would take place, engendered in the same way as the previous outbreak had been. I gave as the “considered opinion” of the Professor of Bacteriology that the Sabin vaccine should be used, and said that our department would monitor all children who developed polio-like symptoms to determine whether it was possible the oral vaccine had caused these symptoms, taking into consideration the time factors. It was a necessary spur to get theMinistry of Health going, and I believed that putting my head on the chopping block if anything went seriously wrong in the use of the vaccine was part of my job.

The vaccine was obtained through WHO initially and later from commercial sources under licence by Dr. Sabin who oversaw by correspondence the early use of oral poliovaccinc. Lee Liang Hin diluted the concentrated vaccine stocks which we mixed and dispensed in eye-dropper bottles so that one drop was the required dose for each administration of vaccine. The government polioimmunization programme was earned out in two phases. In phase 1, children between six months and two years of age were given two doses of polio vaccine at one month’s interval, reaching about 70%of the target population. In the second phase, the “routine programme”, children three months old would be given three doses of polio vaccine at monthly intervals, then two doses at four years of age and finally two doses 7 to 8 years of age on entering primary school. We joined the teams of Ministry of Health nurses who visited Maternity and Infant Welfare clinics to immunise young for the purpose, and took careful notes of the names and ages of children to whom the vaccine was given, and other useful details.

This vaccination regime so effectively reduced the incidence of polio in Singapore that the surgeons whose specialty was tendon transplantation to remedy muscle weakness had to turn their hands to other skills and hospital swimming pools for treatment and rehabilitation of polio patients were closed. Meanwhile, all cases of suspected polio were closely followed by Ministry of Health doctors who took stool specimens and blood samples for us. If we isolated a poliovirus, we tried to find out if it was a vaccine virus or not (very often it was) though the technique was difficult. From the date of onset of symptoms we back-tracked and determined if it was likely that the symptoms were vaccine-related, supposing the child had been given oral poliovaccine. Our fmding was that every child that actually developed polio had not been givenpoliovaccine recently, and as a corollary, we found that children who had received the full course of three doses of poliovaccine never developed poliomyelitis. This result eminently justified the great effort and expense the Ministry of Health invested in the polio immunization programme. In my Diploma of Public Health class, the doctors from neighbouring countries would ask when their country should start to use oral poliovaccine. My advice to them was to find out what their government was spending on treatment and rehabilitation of polio patients. I said, “When the cost of treating polio becomes greater than the estimated cost of buying and distributing the vaccine it will be time for you to adopt a polio immunization programme.”

Saturday, December 13, 2008

4:2 Dengue Haemorrhagic Fever

In 1960, soon after I got over my operation for perforated duodenal ulcer, dengue haemorrhagic fever appeared for the first time in Singapore and in neighbouring countries, the vector (spreading agent) for which were the mosquitoes Aedes egypti and Aedes albopictus. Dengue was a disease that had been long known, causing fever, headache and severe body pains for which reason it was also known as “Break-bone fever”.
Dengue haemorrhagic fever, however, was almost a different disease although the causative viruses were related. In dengue haemorrhagic fever the patients occasionally go into severe shock because of rapidly falling blood pressure that results in death. Such patients often had bleeding into the skin that caused skin blotches or tiny little spots. It turned out that there were at least four variants of dengue virus, some more apt to cause haemorrhagic fever and shock, but it was not possible by tests on a patient’s blood to determine which variant was causing the illness. There seemed to be no animal reservoir for dengue viruses so the disease had to be spread from human to human. How an epidemic started was not easy to determine, but that Aedes mosquitoes spread the disease was clear.

After many attempts we isolated dengue viruses from mosquitoes caught in the open. The control of mosquito breeding places near human habitations brought down the number of cases of dengue, but cases continue to occur though the years and early recognition of patients likely to go into shock enabled prompt treatment and reduced fatalities.

Editor’s note: The researchers went out to trap mosquitoes at various places in the countryside. For this purpose there was a blue van belonging to the Department of Microbiology. It was called the “mosquito van” and parked at our home in College Road, sometimes occasionally used for the Prof’s own affairs.

CHAPTER FOUR: COLLEGE ROAD


4:1 Professor

On July 1, 1959, I was appointed Hale’s successor as Professor of Bacteriology. in my Inaugural Lecture I addressed the theme that was my main work, “Encephalitis in Singapore.” I said that in the same way as we take a lot of trouble to rehabilitate children physically affected by poliomyelitis, we should also take the trouble to rehabilitate children mentally affected by Japanese B encephalitis and who often had psychiatric problems that were little understood. I do not think anyone heard my plea and I doubt if anything much was done for the latter problem.

My assistants were Lee Liang Hin who was soon promoted Senior Lecturer, and Chan Yow Cheong, a Lecturer, who had a PhD in Zoology. Liang Hin took the brunt of our work in poliomyelitis and enteroviruses and was appointed Director of the WHO Polionyelitis Centre in Singapore (later the Enterovirus Centre) while Yow Cheong concentrated on arboviruses (an acronym for arthopod-borne viruses, which included mosquito-borne viruses). I have sometimes considered my position in the light of the observation of Northcote Parkinson who said of “empire-builders” within institutions, “Work expands to fill the time available.” Parkinson quoted the remark of a departmental head who proved that he had so much to do that he was given two assistants between whom he divided his work load. When asked what his own work was, the worthy said, “Why, I am fully occupied making sure my assistants do their work properly!”

Note: Northcote C.Parkinson, Professor of History, University of Singapore, 1960s. Most well known for his book, The Law and the Profits, the first line of which read, ‘Expenditure rises to meet income.

In practice, I undertook as my personal project the detection of polioviruses in Singapore waters. The procedures involved detection of as few as 100 particles of poliovirus in one cubic metre of water and involved filtration of water and clarified sewage through micro fillers and subsequent recovery of the entrapped virus in tissue cultures. The question I was trying to answer was if the Singapore sewage purification process successfully eliminated polioviruses that might have been excreted by poliovirus carriers, and if not, where surviving polioviruses ended up. If in the sea, where? I fear that I never got to the bottom of this question, being enmeshed on the way by technical problems that I had not foreseen. It was unfortunately a waste of my time and energy in Singapore, though Melnick’s group were more successful in Houston.

3:18 Joe Melnick and Sabin

The great thing I learned from Joe Melnick was objectivity. He never took anything for granted and weighed carefully any scientific judgement even from the highest authorities. “Show me,” he would say, “don’t just tell me.” He had a confrontation with Dr. Albert Sabin, the inventor of live oral polio vaccine, when Sabin claimed that the vaccine viruses did not attack nerve cells and were not able to cause paralysis in monkeys even when injected directly into the spinal cord, a procedure considered the most testing for neuropathogenicity.
Melnick repeated Sabin’s experiments and confirmed that the monkeys did not develop paralysis, but he found that their spinal cord and brain cells showed signs of virus invasion. To Melnick this meant that the vaccine virus still retained its affinity for nerve cells even if the monkeys did not have paralysis. Perhaps, he argued, the search for a vaccine virus without affinity for nerve cells should be continued. Sabin had to admit that the vaccine virus retained some neuropathogenicity, but the health authorities continued with small trials of oral poliovaccine in the hope that failure of the vaccine virus to cause paralysis in monkeys really indicated that the vaccine virus was not neuropathogenic in humans.

Melnick was not against this, for he accepted that practical tests of the vaccine where it was going to be used was the final arbiter. When the Singapore government debated in 1962 whether Singapore should adopt the Sabin oral vaccine to protect all children against polio, some doctors questioned whether Singapore children should be used as experimental guinea-pigs, so to speak, when the vaccine was not of proven safety. Melnick said to me,“Ann, Singapore is already in the oral vaccine safety experiment. You can choose to be in the group of guinea-pigs that are given the vaccine or you can chose to be in the group of guinea-pigs that are not. After a few years we shall compare the number of polio cases in the first group of guinea-pigs and in the second group. If polio cases in the first group are significantly fewer than in the second, then the vaccine will have been proven to be safe and effective. Otherwise, not.”

In the event, the Singapore experiment proved to be successful in reducing the incidence of paralytic polio in Singapore to less than two annually and no child given the full initial course of Sabin vaccine ever contracted polio.
In Houston I shared an apartment with two of Melnick’s PhD students. One was Dan Turshack from Philadelphia and the other Arwind Diwan from India. Dan gave me an insight into the American way of studying. He had a reading list as long as his arm; once a week the PhD students had to present a seminar on a virology topic in which one of them had to review a journal article and discuss the significance of the new knowledge reported. Everyone was expected to join in the discussion and draw upon their own reading of the literature. “I have time to read everything once only,” Dan told me, “so I have to read for retention.” This was a new concept for me. I had taken for granted that one remembered what one has read, but Dan’s notion of retention was to be able to cite the authors and the date and journal reference of the report and to quote passages verbatim. It was something that I did not know how to do.

Sunday, December 07, 2008

3:17 Invention of LBM antisera pools
Melnick’s laboratory was appointed by WHO as the International Enterovirus Centre that collaborated with national enterovirus centres in the identification of viruses isolated in polio outbreaks to determine what types of poliovirus were involved and if there were other enteroviruses that could cause polio-like symptoms (this was rarely the case).

(For convenience though not strictly correct, in the following account, the term enterovirus included those coxsackieviruses that grew in tissue culture. )

One important bit of collaborative work between different laboratories was the preparation and standardization of neutralizing or typing antisera. These were usually prepared by vaccinating rabbits or other animals with purified virus grown in tissue culture. It was most important that the virus used to make an antiserum should be of one virus type only, and complications sometimes resulted when a laboratory used a vaccine “virus” that had two different types of viruses in it. High quality typing antisera was as precious as gold because a rabbit does not yield much serum.

To match a virus isolate with one of 20 or more enterovirus (or coxsackievirus) types was no easy task. To avoid doing over twenty tests of which all but one was unnecessary, the identification test was done in steps. Three serum-pools were made, each containing a mixture of about one third of the typing sera that had to be typed against. If one of the serum-pools neutralized the virus isolate we proceeded to the next step, and that was to type the isolate against each component of the neutralizing serum-pool. This was relatively a simple procedure and I identified a number of virus isolates in this way.
One day it occurred to me that three serum-pools were used in step 1 because there were three poliovirus types, the commonest of which was poliovirus type 1. Serum-pool A contained neutralising antiserum for poliovirus type 1 and the antisera for a number of enteroviruses that were thought to be most common. Serum-pool B neutralized poliovirus type 2 and some other enteroviruses; Serum-pool C neutralized poliovirus type 3 and the remaining enteroviruses.

What if, I asked, I made up a Serum-pool D that contained some of the antisera that made up Serum-pool A, excluding poliovirus type 1 with some of the antisera that made up Serum-pool B, excluding poliovirustype 2. If a virus isolate was neutralised by Serum-pool D but not by Serum-pools A, B or C, it showed that the virus was not a poliovirus, but one of the “other” enterovirus types common to Serum-pools A and B that I had put in Serum-pool D.
Further thinking along this line led me to the realization that by making six antiserum-pools I could further narrow down the identification of a virus by its “neutralizing pattern” that corresponded to the way I put together the typing antisera. For example, if antiserum for each of six virus types was incorporated in only one of the six serum-pools, say Serum-pools A to F, and if each of the antisera for the remaining types were distributed in more than one pool, then neutralization by one serum-pool only showed that the virus isolate belonged to the type that was put in the serum-pool and none other! Moreover, with six serum-pools, I had available 15 double neutralization patterns, A+B, B+C, C+D and so on, and 20 triple (!) neutralization patterns, A+B+C, B+C+D, and so on, more than the number of existing enteroviruses. The principle will be familiar to those who bet in football pools and, of course, to those who played with permutations and combinations in algebra.

To recapitulate: I had hit upon the principle of identifying a virus isolate in one test, using antiserum pools in which the typing antisera had been distributed uniquely. If a virus was neutralized only by one Serum-pool A, B, or C..., it would be identified as enterovirus No.1, No.2 or No.3, as the case may be. If the virus was neutralized by two Serum-pools A and B, and by none other, it could be identified as enterovirus No.7, and if neutralized by Serum-pools A+C, as enterovirus No.8, and so on. The possibilities were limitless!

Melnick was intrigued by the labour- and time - saving aspects of my invention. The existing method required setting up three tests in the first step and possibly eight other tests against the individual components of a serum pool. If the identification could be achieved in one step, it halved the time necessary to identify a virus isolate. Melnick told me to concentrate on my bright idea.
There was quite a lot of work to be done to show that the combination pools system was workable. First, I had to find out if any enterovirus was neutralized by antiserum for other enteroviruses and to what extent. There were indeed one or two enteroviruses that gave such cross reactions. Then I had to choose the components of my serum-pools to take such cross-reactions into account. Eventually I had a set of six serum-pools that enabled the typing of the 24 types of enteroviruses (or coxsackieviruses) then known in Houston and I was ready to publish.
Mati played a great part in the work for she had made most of the typing antisera herself and she double-checked the validity of my experiments. The report was sent to the Journal of Immunology under the authorship of K.A.Lim and M. Benyesh-Melnick and entitled, Typing virus by Combinations of antiserum pools. Application to Typing of enteroviruses (Coxsackie and ECHO).

Note: ECHO viruses. When cytopathogenic agents were first discovered that were not poliomviruses, and apparently not related to any disease, they were called ‘orphan viruses’ , that is, viruses looking for diseases to be named after. Thus, the acronym for them, ECHO viruses: Enteric Cytopathogenic Human Orphan viruses -- ‘Human’ because similar viruses were found in monkeys.

Based on this pilot scheme I drafted a scheme for combining antisera for 42 enterovirus types in eight combination antiserum pools, but had to leave the details to Mati as my time at Baylor was up. WHO found my invention of practical value as Melnick had foreseen and Melnick was givena grant to make enterovirus antisera in large quantity and to make combination antiserum pools for distribution to national enterovirus centres. Melnick found that horses responded well to vaccination with enterovirus vaccines and gave antisera in two litre lots as compared with 20 ml for a rabbits. He freeze-dried the antiserum pools and distributed them under the label of LBM enterovirus antiserumpools (LBM for Lim &Benyesh-Melnick).
“We shall have enough material here to last well into the 21st century,” he told me. Whenever a polio outbreak takes place, the investigators take faecal specimens from cases and their contacts to determine the type of polio virus responsible for the outbreak and to try to trace the source. Many non-polio CPE agents were isolated, the great majority of them very likely enteroviruses. Rather than throw them away, attempts were made to identify them, and as few laboratories had a full range of enterovirus antisera, the “unknowns” were sent to the national public health laboratories from where they found their way to the regional WHO enterovirus laboratory.

The LBM antiserum pools freed regional centres from having to identify the unknowns usually sent to them. Instead, WHO distributed LBM antiserum pools to all laboratories working on polioviruses on the premise that those who can isolate poliovirus can do neutralization tests and identify any CPE agent believed to be enterovirus. Without the LBM antiserum pools it would have been necessary to distribute typing antiserum for each of the antisera that the antiserum pools comprised. In 1992 Melnick reported that the first batch of LBM antiserum pools he had made were exhausted and reviewed reports he had received concerning their use. He further stated that he was about to make a second batch of LBM antiserum pools from the horse antisera from which he had made the first batch, about half of which had been stored in deep freeze. He thus fulfilled the prediction he had made about the LBM pools being used in the 21st century.

Editor’s Note: Recently my nephew Koi Jun demonstrated a card trick, in which he was able to identify one card I had selected. He laid out combinations of cards and had me state which combinations contained my card. He was then able to identify my card ... using the same principle as his grandfather has described above for the LBM anti-sera typing pools.

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.

3:15 US Tour 1958 - Joseph L. Melnick, Baylor Medical College

Hale had with great energy obtained grants from the WHO, Geneva, and from the Rockefeller Foundation. New York. In the summer of 1958 he obtained a travel grant to visit US laboratories but learned before he left Singapore that Professor Wilson Smith was about to retire. Hale decided to resign from the University of Singapore so that he could be in England when Wilson Smith vacated his chair for it was Hale’s ambition to occupy it. In the circumstances, he felt obliged to cancel his American tour and recommended that I should use the funds provided for a six-months visit to the US; meanwhile, he would hold the fort until his six-month notice of resignation took effect. I was given only two weeks to prepare for my departure, but it was an offer that I could scarcely refuse though I was still in service bond on account of the leave I had taken in 1953-54. The University rule was that for each month of study leave you were given you had to work two months for the University, else repay the salary you were given while you were away. I had little intention of leaving the University, but the bond was an irritating reminder that I was a paid servant, a condition that my father had warned me against.

Rosie was exhilarated beyond words when she heard that she would accompany me to the US, well, not exactly accompany me, for she scoffed when she heard my travel plans: Singapore / London, non-stop, take the next convenient plane for New York and the great beyond. “Fly over all the interesting places in between?” she asked in derision, “I don’t want to fly when for the same money, or even less. I could travel by boat.” So Rosie made her own travel plans, and insisted on paying her own expenses from her savings. Her routing was Singapore to Rangoon by plane to see friends Amak had there, then take plane to Athens for a tour of the Grecian Isles, then take the Orient Express train to Brussels and London where we could meet briefly, then take ship from London to New York where we would rendezvous at Gajdusek’s house in New York, after which, “Que sera, sera,” we shall see what we shall see. Rosie’s plans were not unreasonable, so she left a two week before I did; we met in London for a couple of days before she took ship and I took plane for New York. Carleton Gajdusek came from Washington to meet me in New York and we visited his home in Bethesda, Maryland where he lived, near his work place, the National Institutes of Health, nominally in Washington, but actually located in Maryland and not in Washington, D.C. We went back to New York to meet Rosie off her boat and stayed with Carleton’s mother in New York before I set off for my final destination in America.

The first thing I did in New York was actually to report to the Rockefeller Foundation to see what plans they had for me because I had left Singapore at such short notice that I had no time to arrange where I should spend my six months in the US. When the Rockfeller Foundation learned that I was interested in poliomyelitis virus and tissue cultures they said that I should go to Dr. Joseph L. Melnick who had recently been appointed Chairman of the Department of Epidemiology in Baylor Medical College in Houston, and had a grant for work on poliomyelitis. I was a trifle put out when I discovered that Houston was in Texas, for I thought the best science centres were either in Eastern USA or in California, not in the deep south. I was mistaken. Houston had the NASA, the Space Centre, and was leading in open heart surgery, in the Methodist Hospital next to Baylor, amongst other things, and of course, had its share of the oil wealth of Texas. Melnick had been invited to Baylor Medical College to start a Department of Virology, and was meanwhile Chairman of the Department of Epidemiology and was responsible for teaching medical microbiology. Melnick had recently published a students’ text-book on Medical Microbiology. The arrangement in American universities was that they provided space and salary for the teaching staff one of whom was designated Chairman. For research expenses including the cost of major equipment, research assistants and technicians, each staff member was on his own and had to seek the aid of private foundations or government institutions interested in extending the scope of their work. Melnick was well funded in this respect, having received grants from the National Institutes of Health for work on polio, as well as from WHO and elsewhere. Instead of accompanying me to Houston, Rosie decided to take a course in the Visual Arts at Columbia University that covered the cinema and the stage. She stayed with Carleton’s mother in White Plains, a north New York suburb, and commuted to her classes by sub-way which in those days was not as dangerous as now. She had a wonderful 3-4 months in New York going to matinees on Broadway and visiting museums between her classes before she took off again to join me in Houston.


3:14 Flu Fighter.
On the first Monday that I returned to the laboratory I got a call from Dr. Huang who had attended the course we had been giving for Diploma in Public Health students. He asked me if I would like to visit Pulau Bukom with him to see some patients suspected of having got influenza from the Hong Kong outbreak. “What outbreak?” I asked, “Hong Kong?”. It had been in the papers, I was told, but I had not caught up with the news yet. Hale confirmed that an outbreak of influenza had been reported in Hong Kong and very likely the patients in Pulau Bukom, where Shell Oil Company had a large installation and where some port workers lived, had caught influenza from passengers off ships they had visited. It was an opportunity to perform our duties as a WHO Influenza Observer, appointed by WHO in various parts of the world to detect new variants of influenza as early as possible.

I went over to Pulau Bukom with the Public Health doctor and found typical cases of influenza - the patients had fever, running noses, red eyes, some cough,, and were miserable. I took throat swabs and blood specimens and returned to the laboratory. I got some 8-day-old and some 14 day-old chick embryos from Crawford Street where there were a number of hatcheries (who thought I was making some kind of medicine with the un-hatched chicks) and inoculated them with extracts of the throat swabs to which a mixture of penicillin and streptomycin was added. Before the days of these antibiotics, inoculation of bacteria from the throat would assuredly kill the chicks. Then I waited. On the second day - Wednesday - I opened a couple of the 14-day eggs that had been inoculated amniotically, removed the lungs from the baby chick and ground them up to make a suspension. I mixed the clarified suspension with some chick red blood cells and was elated to find that the red blood cells had clumped together as they would be by an influenza virus. I took the precaution of mixing the red blood cells with a suspension made from lungs of un-inoculated chicks to show that the “negative control” lung suspension did not clump chick cells.

Hale was just as excited as I was when I reported my findings. He watched me closely as I prepared my identification tests, using the typing influenza antiserum I had brought back from Melbourne. I mixed the chick embryo extract with the antiserum and added the mixture to chick red blood cells. As controls, I mixed normal serum with the chick embryo extract to the chick red blood cells as well as chick embryo extract alone to the red blood cells. To my consternation, the chick embryo extracts in all cases caused clumping of the red blood cells, whether mixed with influenza antiserum or not. If our assumption was correct that we had isolated an influenza virus, then the HI (anti-clumping) test that we had done should have prevented the chick embryo extract from clumping the red blood cells. That the chick embryo extract had a clumping agent was proven, but I could not prove that it was an influenza virus. Hale said that we had better send the virus isolate, if that was what we had, to the International Influenza Centre for identification, so I prepared sealed glass ampoules of the chick embryo abstract, and sealed ampoules of the patient’s serum taken when the throat specimen was taken and another serum taken three days later. I packed the ampoules in dry ice and sent them off by air-freight to the London International Influenza Centre with instructions for the dry ice to be refilled every 24 hours. Identical packages were sent to the American International Influenza Centre in Washington and other influenza virus laboratories including the Hall Institute in Melbourne. I cabled Dr. Eric French to tell him that I thought I had isolated an influenza virus but I could not type it with the serum he had given me; could he help? It was Friday morning.

The specimens arrived in London on Saturday afternoon and sat in the Airport icebox until the following Monday. French personally went to Melbourne Airport on Friday night to retrieve the specimens that I had sent him and immediately inoculated some chicks amniotically the way that he had taught me. On Sunday morning French went to his laboratory and opened up some inoculated eggs and found that there was, indeed, a red blood cell clumping agent present. He repeated the tests that I had done using the same influenza antisera that he had given me and confirmed my negative results. He then tested the chick embryo extract by another test called the complement-fixation test (CIFT) which proved that our virus isolate belonged to the Influenza A virus group (there were two other Influenza virus groups, B and C Influenza groups that behaved differently by the CFT) but was sufficiently different from other Influenza A viruses as not be neutralized by their antisera in HI tests. his implied also that a person who had experienced the old Influenza A viruses would not be protected against the new variant. French called Sir Mac who came to the laboratory to review his results then called the newspapers. On Monday morning the Singapore Straits Times reported my coup with front page headlines: “Brilliant Singapore scientist discovers new influenza virus”. In following write-ups they called me Flu-fighter. I did not think that they knew my Grand Uncle by marriage, Dr.Wu Lien-Teh, had published his memoirs under the title Plague Fighter.

The aftermath of my discovery of the new Influenza virus, subsequently named Influenza A2virus was something of an anti-climax. The main aim of the world-wide WHO Influenza Observer network was the early detection of new influenza virus variants. The A2 virus was present in Hong Kong at least two weeks before it came to Singapore where the virus was isolated about a week after cases had been recognized. It then took a further week before the virus was identified as a new type. WHO’s interest in new influenza variants was not purely academic; WHO had hoped that early detection and isolation of new influenza variants could lead to better control measures because vaccines could be prepared against the virus and populations elsewhere protected against the new virus before it arrives. This was a vain hope. It took about a month for vaccines to be made against the virus isolates sent to England and to the United States. First the virus had to be adapted for growth in large quantities, then tests for safety and efficacy would have to be made before the vaccine was actually used. By the time the health authorities had a vaccine ready, the new influenza had arrived on their shores and in most cases, second epidemic waves had already begun. Modern transportation enables large numbers of infected people, some not even showing signs of illness to be landed among susceptible populations well within 30 days, the minimum lead time for isolating a new virus and preparing a vaccine from it. Flu-Fighter was an empty label; I fingered the enemy, but nothing I did affected its progress round the world.