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 When our father passed away on 8 March 2003, he left behind an unpublished autobiography.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

Monday, September 22, 2008

3:3 Japanese B Encephalitis.

Hale had been Senior Lecturer in the London University College Hospital Medical School where he had been doing research on influenza. He intended to continue working on viruses and immediately found a good line when he visited the Institute of Medical Research (IMR) in Kuala Lumpur shortly after he came to Singapore. The US Army maintained a research unit in the IMR studying matters of interest to the US Army which covers just about everything. At that time the US Army was interested in Japanese B encephalitis* because its headquarters was in Tokyo and the Japanese health authorities were concerned by epidemics of the disease in Japanese children.

* Encephalitis means inflammation of the brain and is also known as brain fever. It is a disease due to destruction of brain cells resulting in loss of muscular co—ordination and mental ability, and occasionally causing coma and death. The condition was first described in adults and occurred all the year round. When cases were identified in Japan they were found to occur mainly in summer, and in children. At first described as summer encephalitis, the disease was named Japanese encephalitis B to distinguish it from Japanese encephalitis A, the other form which had also been recognized. Japanese scientists did not like the association of the disease with Japan because they had found evidence of the same disease in other Asian countries but they were out-voted in the scientific committee that named viruses. Indeed, while the virus of Japanese B encephalitis has been recovered from animals and from humans, the causative agent of encephalitis A has not been discovered.

The start given us was indirect. Some race-horses recently imported from Australia at great cost had taken ill with symptoms of encephalitis, fever and muscular in coordination (they were staggering a bit). Blood tests showed that the horses had developed antibodies to Japanese B virus and it was assumed that the horses had got infected when they arrived in Malaya for the disease was not prevalent in Australia. It was known that pigs in Japan could be infected by Japanese Boon Keng encephalitis so it was possible for horses in Malaya to be infected, if the virus was present there. A horrible thought: if horses can be infected, why not humans? Hale decided to pursue this question.

An economical way of growing viruses was to use chick embryos or rather, fertilized hen eggs, as is done with influenza virus, but Jap B virus (for convenience we shall use this abbreviation for “Japanese encephaltis B virus”) could not be grown in eggs, we had to use mice. Hale got some mice from Kuala Lumpur and very soon we were breeding white mice, not only in the laboratory, but also at home where my children made pets of them, especially when we stained them different colours. In the laboratory, the mice were kept in rooms that were mosquito screened, to prevent stray mosquitoes infecting our mice, or to prevent them biting our infected mice and getting out again. We grew Jap B virus from seed virus given us and confirmed that the serum of horses that had recovered from encephalitis was able to neutralize (prevent) the infectivity of Jap B virus in mice. A control test with normal horse serum (from newly arrived horses), showed that this was unable to neutralize the virus. We were in the business!

The question whether the disease was prevalent in Singapore was easily answered. Tests on blood samples from Singapore children showing signs of brain fever showed that they often developed antibodies against Jap B virus, a sure sign that they had the disease. -Though we had no fatal cases at first, some patients had severe brain damage and were unable to do anything for themselves after the acute stage. Now, how did the children get infected with a virus last of whichwe heard of was infecting race-horses? How Japanese children get Japanese encephalitis gave us a clue. Japanese encephalitis in Japan was spread by mosquitoes that got the virus from pigs that got the virus from mosquitoes that had bitten other infected animals or humans. The mosquito responsible was named Culex tritaeniorrhyncus (meaning, mosquito with three horns, markings on the head of the mosquito). As is well-known, the female of the species is the deadlier. Female Culicine mosquitoes need a blood meal before they lay eggs. When they bite an animal with virusin the blood they also pick up the virus which then multiplies in the mosquito and finally finds its way to its salivary glands. A blood-sucking mosquito feeds by inserting its needle-like feeding tube into the skin to find a capillary. Before use, the needle is lubricated with some saliva thus passing on the virus. All this had been worked out before in Japan; the question was how to make use of the knowledge. With the help of the veterinary department we found some cattle-sheds in Pasir Panjang not far from where the Tiger BaIrn Gardens are now located. We could have looked at some piggeries elsewhere on the island, but Hale thought we should test our methods first at a convenient site, just off the West Coast Road. We were lucky.

Our assistants went on field work in the mornings, using butterfly nets made with mosquito netting, beating about the bushes adjoining the cattle-shed and behind the door in the shed, anywhere dark where mosquitoes might be resting after their nightly feasting. The mosquitoes were sorted out by our colleagues in the Parasitology Department who picked out the C.tritaeniorrhyncus for us. My job was to grind up the mosquitoes and to inject the clarified suspension into baby mice. Ouch! You might say, and indeed it was a brutal thing to do, but have you ever seen a child lying immobile in a bed like a vegetable because his brain was destroyed? That, also, is not nice, and our work was to try to understand how it happened.

We watched the mice daily, morning and evening, turning them over gently to see if they wriggled normally. Soon I was able to recognize the twitching that was the early sign of encephalitis in the mice, and which was followed by paralysis and death. To prove that what had affected the mice was Jap B virus we made an extract of their brains and treated some of the suspension with anti-serum for Jap B virus. We then injected the treated and untreated brain suspensions into suckling mice.. The untreated brain suspension killed inoculated mice with typical signs of encephalitis whereas mice inoculated with treated (neutralized) brain suspension were spared. The neutralization test definitely identified the virus we had isolated. There was Jap B virus about the place and somehow Singapore children were getting infected by it.

It is axiomatic in science that one question leads to another, for truth lies at the bottom of a well the depth of which we do not know. The study of how a disease spreads is called epidemiology, the ultimate aim of which is prevention. We needed to know how many children get Japanese encephalitis each year and if there is any seasonal distribution. This was easily determined: the Paediatric wards in the General Hospital right next door was admitting encephalitis cases, proven by blood tests to be due to Jap B virus, all the year round. We needed to know where the mosquitoes get the virus from in the first place, for mosquitoes do not transmit the virus to each other. Quite often, a disease agent persists in humans or animals over long periods, perhaps without any sign of its presence, and by some means is transmitted to the patients with whom we are concerned. From our knowledge of the Japanese scene, we guessed that pigs, of which there were far more than cattle in Singapore at that time, should be the reservoir for Jap B virus. But this was hard to prove because the virus stays only a day or two in the blood of an infected pig, and how to catch a pig red-handed, so to speak, would be a problem, so we did the next best thing and that was to show that Singapore pigs could be infected by mosquitoes that carried Jap B virus and that mosquitoes could be infected by feeding on infected pigs.

With great ingenuity Hale converted an outhouse in the medical faculty grounds to a piggery and made it mosquito proof, with screened double doors. The Veterinary Department helped us buy a pregnant sow and I became a pig farmer for a while. One technical problem had to be overcome: there were two grades of non-academic staff, technicians and servants; the latter did all the dirty work such as cleaning and looking after our experimental animals. I knew that our Malay servants could not handle pigs but I was a little surprised to learn that Indians (Hindus) also considered pigs unclean so it was left to our sole Chinese servant to feed our pig and clean the pig sty. To encourage him in his work I would visit the pig(s) often and help him hose down the place. Our problems were intensified when the piglings were born for they were our experimental subjects and my assistant had to hold the little beast while I injected it or took a blood sample. This part of the experiment was not without hazards for the procedure was to make a small cut in the pig’s ear and collect the blood in a test-tube. More often than not, the pig would twitch its ear out of my hand when it felt the knife and the blood that came out went all over the place. We did wear gloves and protective gowns, but it was no joking matter and all in the trade.

Our experiments showed that piglings at birth were protected by maternal antibodies, but after six months the antibodies had waned and they could be infected by injection of Jap B virus by allowing infected mosquitoes to bite them. The injected virus multiplied in the animal and could be detected in its blood, and antibodies for Jap B virus developed afterwards. This knowledge was purely academic, however, because it did not lead to any practical way of preventing children from getting Japanese encephalitis. It was useless to advise people to keep away from piggeries where infected mosquitoes might be found, for the wind might carry an infected mosquito far from its feeding site. Vaccination was not a solution because the vaccine developed by the Japanese was not without harmful side-effects and they used it only in anticipation of a summer out-break. Japanese encephalitis in Singapore was endemic, occurring all the year round.
Ultimately, the problem solved itself when for environmental reasons, the Singapore government phased out pig farms and removed a large potential animal reservoir for Jap B virus, and the disease is now scarce.


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