Our last posting, Vaccine Research using Children (July 7, 2016), addressed the history and ethics of testing vaccines in children. For a rather different take on the issue of children in biomedical research, see the appended Nature editorial, More support for clinical trials in children (Nature 535:465-466, 2016), which considers the use of children in cancer research. It raises issues that are similar to those on the earlier blog post (e.g., the problem of informed consent). More importantly, it raises dissimilar ones, which arise from the unique dilemma of cancer in children.
More support for clinical trials in children
US lawmakers should give drug firms the confidence to test pediatric cancer therapies.
27 July 2016
A cancer diagnosis is a shock, but adults with the disease can take some comfort in the numerous treatments available to them — both through clinical trials and as drugs that are already on the market. Children cannot. Because they make up only 1% of US patients with cancer, children are a low priority for pharmaceutical companies that want to launch an effective drug quickly. The hassle of a pediatric clinical trial may not seem worth it until after the drug has proved to be safe and effective in adults. This process can take decades, leaving children with therapies that are sometimes almost obsolete.
To access therapies early, parents of these children can turn to compassionate-use programs, in which companies give experimental drugs to people who are in desperate need. In the United States, firms that agree to provide medicines in this way will ask the Food and Drug Authority for emergency permission, which is almost always granted.
This system, although helpful for some, is rife with complications. Patients and their families report difficulties in applying for such programs, and say that they rarely receive responses. Companies that withhold a drug — because it is in short supply or not right for a patient — can find themselves on the receiving end of critical social-media campaigns highlighting individual patients. And firms worry that if a person dies or is harmed while taking a drug, it could hurt the drug’s chances of being approved. No one knows how many requests parents make and how often companies approve them, but anecdotally, firms often deny drugs on the grounds that they have not been tested in children.
Proper clinical trials for childhood cancer drugs are scarce. Designing a clinical trial is never simple, but adding children to the picture complicates the process immensely. Children are not just ‘small adults’ — they metabolize drugs in very different ways. It is difficult to predict from adult or animal studies whether a chemotherapy drug will be more or less toxic in a child, and at what dose. The process of obtaining informed consent for children participating in a trial can also be more complicated. And companies fear that the death of a child — even if unrelated to the treatment — could bring bad publicity for a new drug.
“Legal loopholes often prevent children with cancer from accessing new drugs.”
Recent years have seen attempts to make more drugs available to treat children. In the United States, a 2003 law known as the Pediatric Research Equity Act (PREA) requires that companies develop a plan for how they will test experimental drugs in children, although many trials are exempted. A second law, called the Best Pharmaceuticals for Children Act, motivates companies to perform pediatric clinical trials by granting an extra six months of market exclusivity for the adult drug.
Overall, these laws have been successful, leading to hundreds of drug labels being updated with information for use in children. But legal loopholes often prevent children with cancer from accessing new drugs. For instance, therapies for conditions that do not affect children — such as Alzheimer’s disease — are exempt from the PREA. And exemptions intended for such diseases have been broadly applied to cancer. For example, therapies that are being trialed in adults with breast cancer are exempted because children do not get that cancer, even if the drug could treat a childhood cancer in a different organ.
Also exempted are drugs for ‘orphan’ diseases that affect fewer than 200,000 people in the United States. The number of orphan designations has skyrocketed in recent years — the improved ability to define the molecular basis of an individual’s cancer means that diagnoses have become increasingly subdivided, and the majority of approved cancer drugs now carry this orphan designation.
Legislation is now attempting to close those loopholes. The Research to Accelerate Cures and Equity (RACE) for Children Act, introduced to the US Congress on 14 July, would require companies to apply the PREA to any therapy with a molecular target that is relevant to both an adult and a childhood disease. It would also end the exemption for orphan diseases. Last July, the European Medicines Agency passed similar rules to make it more difficult for companies to avoid testing drugs in children. This applies when the disease has a common mechanism in adults and children, unless the drug is likely to be unsafe in children.
With Congress now out of session and focused on the upcoming US election, the RACE for Children Act is unlikely to advance before next year. But when lawmakers pick it up, they should also address problems with compassionate-use programs — and ensure a transparent and useful process for people to gain access to unapproved drugs. They should also encourage companies to make more drugs available through market incentives, and provide increased protection should something go wrong.
Children have been used in vaccine research since its very beginning, usually said to have been in 1796, when Edward Jenner inoculated 8-year-old James Phipps with cowpox, and then challenged young James with actual smallpox (1). However, earlier, in 1789, Jenner inoculated his own 10-month-old son, Edward Jr., with swinepox. Edward Jr. then came down with a pox disease, which he fortunately recovered from. His father then challenged him with smallpox.
Edward Jr. survived his exposure to smallpox. But, since Edward Sr. wanted to determine the duration of young Edward’s protection, he again challenged his son with smallpox in 1791, when the boy was two. Edward Sr. inoculated his son yet again with smallpox when the boy was three. Fortunately, young Edward was resistant to each of the smallpox challenges his father subjected him to.
Jenner used several other young children in his experiments, including his second son, Robert, who was 11-months-old at the time. One of the children in Jenner’s experiments died from a fever; possibly caused by a microbial contaminant in an inoculum. [Microbes were not known in the late 18th century.]
We have no record of how Jenner (or his wife) felt about his use of his own children. However, there is reason to believe that Jenner felt some remorse over his use of James Phipps, who he referred to as “poor James.” Jenner looked after Phipps in later years, eventually building a cottage for him; even planting flowers in front of it himself.
By the 20th century, some of the most esteemed medical researchers were using children—in institutions for the mentally deficient—to test new drugs, vaccines, and even surgical procedures. These institutions were typically underfunded and understaffed. Several of them were cited for neglecting and abusing their residents. Moreover, their young patients were usually from poor families, or were orphans, or were abandoned. Thus, many of the children had no one to look out for their interests. In addition, research at these institutions was hidden from the public. [The goings-on at these institutions were, in general, hidden from the public, and most of the public likely preferred it that way.] Federal regulations that might have protected the children were not yet in existence, and federal approval was not even required to test vaccines and drugs.
In the early 1940s, Werner Henle, of the University of Pennsylvania, used children at Pennhurst—a Pennsylvania facility for the mentally deficient—in his research to develop an influenza vaccine. [Pennhurst was eventually infamous for its inadequate staffing, and for neglecting and abusing its patients (2). It was closed in 1987, after two decades of federal legal actions.] Henle would inoculate his subjects with the vaccine, and then expose them to influenza, using an oxygen mask fitted to their faces.
Henle’s vaccine did not protect all of his subjects. Moreover, it frequently caused side effects. Additionally, Henle maintained (correctly?) that a proper test of a vaccine must include a control group (i.e., a group exposed to the virus, but not to the vaccine). Thus, he deliberately exposed unvaccinated children to influenza. Children who contracted influenza had fevers as high as 104o F, as well as typical flu-like aches and pains.
Despite Henle’s investigations at Pennhuerst, he was a highly renowned virologist, best known for his later research on Epstein Barr virus. See Aside 1.
[Aside 1: While Henle was researching his influenza vaccine at Pennhurst, Jonas Salk concurrently worked on an influenza vaccine, using adult residents (ranging in age from 20 to 70 years) at the Ypsilanti State School in Michigan.]
Next, consider Hilary Koprowski, an early competitor of Jonas Salk and Albert Sabin in the race to develop a polio vaccine (3). By 1950, Koprowski was ready to test his live polio vaccine in people. [That was four years before Sabin would be ready to do the same with his live polio vaccine.] Koprowski had already found that his vaccine protected chimpanzees against polio virus. And, he also tested his vaccine on himself. Since neither he nor the chimpanzees suffered any ill effects, Koprowski proceeded to test his vaccine on 20 children at Letchworth Village for mentally disabled children, in Rockland County, NY. [Like Pennhurst, Letchworth Village too was cited for inadequately caring for its residents.] Seventeen of Koprowski’s inoculated children developed antibodies to the virus, and none developed complications.
Koprowski did not initiate his association with Letchworth. Actually, Letchworth administrators, fearing an outbreak of polio at the facility, approached Koprowski, requesting that he vaccinate the children. Koprowski gave each child “a tablespoon of infectious material” in half a glass of chocolate milk (4). Koprowski never deliberately infected the Letchworth children with virulent virus.
Koprowski reported the results of his Letchworth studies at a 1951 conference of major polio researchers, attended by both Salk and Sabin. When Koprowski announced that he actually had tested a live vaccine in children, many conferees were stunned, even horrified. Sabin shouted out: “Why did you do it? Why? Why (4)?” See Aside 2.
[Aside 2: In the 1930s, Canadian scientist Maurice Brodie tested a killed polio vaccine in twelve children, who supposedly had been “volunteered by their parents (4).” For a short time Brodie was hailed as a hero. However, too little was known at the time for Brodie to ensure that his formaldehyde treatment had sufficiently inactivated the live polio virus. Consequently, Brodie’s vaccine actually caused polio in several of the children. After this incident, most polio researchers could not conceive of ever again testing a polio vaccine, much less a live one, in children.]
Neither Koprowski nor Letchworth Village administrators notified New York State officials about the tests. Approval from the state would seem to have been required, since Koprowski later admitted that he was certain he would have been turned down. And, it is not clear whether Koprowski or the school ever got consent from the parents to use their children. However, recall there were not yet any federal regulations that required them to do so.
Koprowski was untroubled by the uproar over his use of the Letchworth children, arguing that his experiments were necessary. Yet he later acknowledged: “if we did such a thing now we’d be put on jail…” But, he added, “If Jenner or Pasteur or Theiler (see Aside 2) or myself had to repeat and test our discoveries [today], there would be no smallpox vaccine, no rabies vaccine, no yellow fever vaccine, and no live oral polio vaccine.” Moreover, he maintained that, secret or not, his use of the Letchworth children fit well within the boundaries of accepted scientific practice.
[Aside 2: Nobel laureate Max Theiler developed a vaccine against yellow fever in 1937; the first successful live vaccine of any kind (5). Theiler formulated a test for the efficacy of his vaccine, which did not involve exposing humans to virulent virus. Sera from vaccinated human subjects were injected into mice, which were then challenged with the Yellow Fever virus.]
Koprowski referred to the Letchworth children as “volunteers (6).” This prompted the British journal The Lancet to write: “One of the reasons for the richness of the English language is that the meaning of some words is continually changing. Such a word is “volunteer.” We may yet read in a scientific journal that an experiment was carried out with twenty volunteer mice, and that twenty other mice volunteered as controls.” See Aside 3.
[Aside 3: Koprowski was a relatively unknown scientist when he carried out his polio research at Letchworth. He later became a renowned virologist, having overseen the development of a rabies vaccine that is still used today, and having pioneered the use of therapeutic monoclonal antibodies. Yet, he is best remembered for developing the world’s first effective polio vaccine; several years before Salk and Sabin brought out their vaccines.
Most readers of the blog are aware that the Salk and Sabin vaccines are credited with having made the world virtually polio-free. What then became of Koprowski’s vaccine? Although it was used on four continents, it was never licensed in the United States. A small field trial of Koprowski’s vaccine in 1956, in Belfast, showed that its attenuated virus could revert to a virulent form after inoculation into humans. Yet a 1958 test, in nearly a quarter million people in the Belgian Congo, showed that the vaccine was safe and effective. Regardless, the vaccine’s fate was sealed in 1960, when the U.S. Surgeon General rejected it on safety grounds, while approving the safer Sabin vaccine. Personalities and politics may well have played a role in that decision (3, 4).
Interestingly, Sabin developed his vaccine from a partially attenuated polio virus stock that he received from Koprowski. It happened as follows. In the early 1950s, when Koprowski’s polio research was further along than Sabin’s, Sabin approached Koprowski with the suggestion that they might exchange virus samples. Koprowski generously sent Sabin his samples, but Sabin never reciprocated.
Koprowski liked to say: “I introduce myself as the developer of the Sabin poliomyelitis vaccine (7).” He and Sabin had a sometimes heated adversarial relationship during the time when their vaccines were in competition. But they later became friends.]
Sabin was at last ready to test his polio vaccine in people during the winter of 1954-1955. Thirty adult prisoners, at a federal prison in Chillicothe, Ohio, were the subjects for that first test in humans. [The use of prisoners also raises ethical concerns.]
Recall Sabin’s public outcry in 1951 when Koprowski announced that he used institutionalized children to test his polio vaccine. In 1954, Sabin sought permission to do the very same himself; asserting to New York state officials: “Mentally defective children, who are under constant observation in an institution over long periods of time, offer the best opportunity for the careful and prolonged follow-up studies…”
Although Sabin had already tested his attenuated viruses in adult humans (prisoners), as well as in monkeys and chimpanzees, the National Foundation for Infantile Paralysis, which funded polio research in the pre-NIH days of the 1950s, blocked his proposal to use institutionalized children. Thus, Sabin again used adult prisoners at the federal prison in Ohio. With the concurrence of prison officials, virtually every inmate over 21 years-old “volunteered,” in exchange for $25 each, and a possible reduction in sentence. None of the prisoners in the study became ill, while all developed antibodies against polio virus.
Testing in children was still a necessary step before a polio vaccine could be administered to children on a widespread basis. But, Sabin’s vaccine could not be tested in children in the United States. Millions of American children had already received the killed Salk vaccine, and the National Foundation for Infantile Paralysis was not about to support another massive field trial of a vaccine, in children, in the United States (3).
Then, in 1959, after a succession of improbable events, 10 million children in the Soviet Union were vaccinated with Sabin’s vaccine (3). The Soviets were so pleased with the results of that massive trial that they next vaccinated all seventy-seven million Soviet citizens under 20 years-of-age with the Sabin vaccine. That figure vastly exceeded the number of individuals in the United States, who were vaccinated with the rival Salk vaccine during its field trials.
Next up, we have Nobel laureate John Enders who, in the 1950’s, oversaw the development of the first measles vaccine. Enders and co-workers carried out several trials of their attenuated measles vaccine; first in monkeys and then in themselves. Since the vaccine induced an increase in measles antibody titers, while causing no ill effects, they next tested it in severely handicapped children at the Walter E. Fernald State School near Waltham, Massachusetts.
Enders seemed somewhat more sensitive than either Henle or Koprowski to the ethics of using institutionalized children. Samuel L. Katz, the physician on Enders’ team, personally explained the trial to every Fernald parent, and no child was given the vaccine without written parental consent. [Federal guidelines requiring that step still did not exist.] Also, no child was deliberately infected with virulent measles virus.
Katz personally examined each of the inoculated Fernald children every day. None of these children produced measles virus, while all of them developed elevated levels of anti-measles antibodies. Also, the Fernald School had been experiencing severe measles outbreaks before the Enders team vaccinated any of its children. But, when the next measles outbreak struck the school, all of the vaccinated children were totally protected.
In 1963, the Enders vaccine became the first measles vaccine to be licensed in the United States. Several years later it was further attenuated by Maurice Hilleman (8) and colleagues at Merck. In 1971, it was incorporated into the Merck MMR (measles, mumps, and rubella) vaccine. See Aside 4.
[Aside 4: Before Enders carried out his measles investigations he pioneered the growth of viruses in tissue culture. In 1949, Enders, and collaborators Thomas Weller and Frederick Robbins, showed that poliovirus could be cultivated in the laboratory. This development was crucial, allowing Salk and Sabin to grow a virtually unlimited amount of polio virus and, consequently, to develop their polio vaccines. In 1954, Enders, Weller, and Robbins were awarded the Nobel Prize for Physiology or Medicine for their polio virus work.]
It may surprise some readers that before the mid 1960s the so-called Nuremburg Code of 1947 comprised the only internationally recognized ethical guidelines for experimentation on human subjects. The Nuremburg Code was drawn up by an American military tribunal during the trial of 23 Nazi physicians and scientists for atrocities they committed while carrying out so-called “medical” experiments during World War II. [Sixteen of the 23 Nazis on trial at Nuremburg were convicted, and 7 of these were executed (see Note 1)].
The Nuremberg Code’s Directives for Human Experimentation contained strongly stated guidelines. Its tenets included the need to obtain informed consent (interpreted by some to prohibit research using children), the need to minimize the risks to human subjects, and the need to insure that any risks are offset by potential benefits to society.
But, despite the well-articulated principles of the Nuremberg Code, it had little effect on research conduct in the United States. Federal rules, with the authority to regulate research conduct, would be needed for that. So, how did our current federal oversight of research come to be?
A 1996 paper in the The New England Journal of Medicine, “Ethics and Clinical Research,” by physician Henry Beecher, brought to the fore the need for rules to protect human subjects in biomedical research (9). Beecher was roused to write the paper in part by the early 1960s experiments of Saul Krugman, an infectious disease expert at NYU. Krugman used mentally deficient children at the Willowbrook State School in Staten Island, New York, to show that hepatitis A and hepatitis B are distinct diseases (9). Also, before a hepatitis vaccine was available, Krugman inoculated the children with serum from convalescing individuals, to ask whether that serum might protect the children against hepatitis. Krugman exposed the children to live virus either by injection, or via milkshakes seeded with feces from children with hepatitis.
Krugman found that convalescent sera indeed conferred passive immunity to hepatitis. Next, he discovered that by infecting passively protected patients with live hepatitis virus he could produce active immunity. Krugman had, in fact, developed the world’s first vaccine against hepatitis B virus (HBV) (see Aside 4). [Although Krugman used mentally deficient institutionalized children in his experiments, his investigations were nonetheless funded in part by a federal agency; the Armed Forces Epidemiology Section of the U.S. Surgeon General’s Office.]
[Aside 4: The first hepatitis B vaccine licensed for widespread use was developed at Merck, based on principles put forward by Nobel Laureate Baruch Blumberg, (10).]
Beecher was particularly troubled by two aspects of Krugman’s experiments. First, Krugman infected healthy children with live virulent virus. Beecher maintained that it is morally unacceptable to deliberately infect any individual with an infectious agent, irrespective of the potential benefits to society. [See reference 11 for an alternative view. “The ethical issue is the harm done by the infection, not the mere fact of infection itself.”]
Second, Beecher charged that the Willowbrook School’s administrators coerced parents into allowing their children to be used in Krugman’s research. The circumstances were as follows. Because of overcrowding at the school, Willowbrook administrators closed admission via the usual route. However, space was still available in a separate hepatitis research building, thereby enabling admission of additional children who might be used in the research.
Were the Willowbrook parents coerced into allowing their children to be used in the research there? Consider that the parents were poor and in desperate need of a means of providing care for their mentally impaired children. Making admission of the children contingent on allowing them to be used in the research might well be viewed as coercion. Yet even today, with federal guidelines now in place to protect human subjects, institutions such as the NIH Clinical Center admit patients who agree to participate in research programs. Is that coercion?
Beecher’s 1966 paper cited a total of 22 instances of medical research that Beecher claimed were unethical (9). Four examples involved research using children. Krugman’s work at Willowbrook was the only one of these four examples that involved vaccine research. Beecher’s other examples involved research using pregnant women, fetuses, and prisoners. But it was Beecher’s condemnation of Krugman’s hepatitis research at Willowbrook that is mainly credited with stirring debate over the ethics of using children in research.
Did Krugman deserve Beecher’s condemnation? Before Krugman began his investigations at Willowbrook, he plainly laid out his intentions in a 1958 paper in the New England Journal of Medicine (12). Importantly, Krugman listed a number of ethical considerations, which show that he did not undertake his Willowbrook investigations lightly. In fact, Krugman’s ethical considerations, together with his plans to minimize risks to the children, were not unlike the assurances one might now submit to an institutional review board (11).
Many (but not all) knowledgeable biomedical researchers claimed that Beecher misunderstood Krugman’s research and, thus, unjustly vilified him. Krugman was never officially censored for his Willowbrook investigations. Moreover, condemnation of Krugman did not prevent his election in 1972 to the presidency of the American Pediatric Society, or to his 1983 Lasker Public Service Award.
To Beecher’s credit, his 1966 paper was instrumental in raising awareness of the need to regulate research using human subjects. Beecher was especially concerned with the protection of children and, apropos that, the nature of informed consent.
In 1974, the National Research Act was signed into law, creating the National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research. The basic ethical principles identified by the Commission are summarized in its so-called Belmont Report, issued in 1978. Its tenets include minimizing harm to all patients, and the need to especially protect those with “diminished autonomy” or who are incapable of “self-determination.” In addition, federal guidelines now require universities and other research institutions to have Institutional Review Boards to protect human subjects of biomedical research. [Reference 13 (available on line) contains a detailed history of the establishment of these policies.] See Aside 6.
[Aside 6: The infamous U.S. Public Health Service Tuskegee syphilis research program, conducted between 1932 and 1972, in which several hundred impoverished black men were improperly advised and never given appropriate treatment for their syphilis, also raised public awareness of the need to protect human subjects. More recently, research involving embryonic stem cells and fetuses has stoked an ongoing and heated public debate. Policies regarding this research are still not settled, with stem-cell research being legal in some states, and a crime in others. Other recent technological advances, such as DNA identification and shared databases, have been raising new concerns, such as the need to protect patient privacy. In response to these new developments, in June 2016, the US National Academies of Sciences,Engineering and Medicine released a report proposing new rules (indeed a complete overhaul of the 1978 Belmont Report) to deal with these circumstances. The Academy’s report has stirred debate in the biomedical community]
Note 1: The use of children in medical research makes many of us profoundly uneasy. We may be particularly troubled by accounts of the exploitation of institutionalized children, who comprised a uniquely defenseless part of society. Indeed, it was the very vulnerability of those children that made it possible for them to be exploited by researchers. Consequently, some readers may well be asking whether the activities of vaccine researchers Krugman, Koprowski, Sabin, Henle and others might have been comparable to that of the Nazis on trial at Nuremberg. So, I offer this cautionary interjection. While in no way condoning the vaccine researchers using institutionalized children, their work was carried out for the sole purpose of saving human lives. As Koprowski suggested above, if not for that work, we might not have vaccines against smallpox, rabies, yellow fever, and polio. Now, consider Josef Mengele, a Nazi medical officer at Auschwitz, and the most infamous of the Nazi physicians. [Mengele was discussed several times at Nuremberg, but was never actually tried. Allied forces were convinced at the time that he was dead, but he had escaped to South America.] At Auschwitz, Mengele conducted germ warfare “research” in which he would infect one twin with a disease such as typhus, and then transfuse that twin’s blood into the other twin. The first twin would be allowed to die, while the second twin would be killed so that the organs of the two children might then be compared. Mengele reputedly killed fourteen twin children in a single night via a chloroform injection to the heart. Moreover, he unnecessarily amputated limbs and he experimented on pregnant women before sending them to the Auschwitz gas chambers.
Edward Jenner and the Smallpox Vaccine, Posted on the blog September 16, 2014.
Pennhurst Asylum: The Shame of Pennsylvania, weirnj.com/stories/pennhurst-asylum/
Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science, Posed on the blog March 27, 2014.
Oshinsky D, Polio: An American Story, Oxford University Press, 2005.
The Struggle Against Yellow Fever: Featuring Walter Reed and Max Theiler, Posted on the blog May 13, 2014.
Koprowski H, Jervis GA, and Norton TW. Immune response in human volunteers upon oral administration of a rodent-adapted strain of poliomyelitis virus. American Journal of Hygiene, 1952, 55:108-126.
Fox M, Hilary Koprowski, Who Developed First Live-Virus Polio Vaccine Dies at 96, N.Y. Times, April 20, 2013.
Hepatitis B virus (HBV), one of mankind’s most important pathogens, infects about 2 billion people worldwide, and more than 500 million individuals are life-long carriers of the virus; with most in Asia. HBV causes acute and chronic cirrhosis, as well as hepatocellular carcinoma. In point of fact, HBV is the 10th leading cause of death in the world! The serendipitous discovery of HBV, and the development of the first HBV vaccine, happened as follows. [See Note 1 for a brief review of the remarkable HBV replication strategy].
In the early 1940’s, during World War II, British doctor, F. O. MacCallum, was the first to suggest that an infectious agent might cause hepatitis. MacCallum was assigned to produce a yellow fever vaccine for British soldiers. That was how he happened to notice that soldiers tended to come down with hepatitis a few months after receiving the yellow fever vaccine.
It was fortunate that MacCallum also knew of hepatitis cases in children who received inoculations of serum from patients convalescing from measles and mumps (a means of protection against those viruses before vaccines were available), and of hepatitis cases in blood transfusion recipients, and of cases following treatments with unsterilized reused syringes.
To explain these coincidences, MacCallum hypothesized that hepatitis might be transmitted by a factor in human blood. And, since hepatitis could be transmitted by inoculation with serum that had been filtered, MacCallum proposed that the hepatitis factor might be a virus. [In 1947 MacCallum reported that hepatitis could be spread by food and water that had been contaminated with fecal material, as well as by blood. He coined the term hepatitis A for the form of the disease spread by food and water, and hepatitis B for the form transmitted via blood.] See Aside 1.
[Aside 1: The following episode, described in MacCallum’s own words (1), occurred in England during World War II: “One day in 1942, I received a message to go to Whitehall to see one of the senior medical advisers and when I arrived I was asked ‘What is this yellow fever vaccine and how dangerous is it?’ After explaining its constitution and the possibility of a mild reaction four to five days after inoculation, I was told that the Cabinet was at that moment debating whether or not Mr. Churchill should be allowed to go to Moscow, which he wished to do in a few days’ time. The yellow fever vaccine was theoretically essential before he could fly through the Middle East, but I explained that no antibody would be produced before seven to ten days so that there would be little point in giving the vaccine. It was finally decided that the vaccine would not be used, and the administrators would take care of the situation. Several months later I received an irate call from the Director of Medical Services of the RAF, who had been inoculated with the same batch of vaccine which would have been used for Mr. Churchill, and was informed that the D. G. had spent a very mouldy Christmas with hepatitis about 66 days after his inoculation…I will leave it to you to speculate on what might possibly have been the effect on the liver of our most famous statesman and our ultimate fate if he had received the icterogenic vaccine.”]
With the advent of cell culture in the 1950s, researchers hoped that a hepatitis agent might soon be cultivated in vitro. Nonetheless, HBV was not discovered until 1966. What’s more, the discovery did not involve growing the virus in cell culture. And, reminiscent of the case of MacCallum above, the discovery was made by a researcher, Baruch S. Blumberg, who was not even working on hepatitis. Rather, Blumberg was interested in why individuals varied in their susceptibilities to various illnesses.
Blumberg sought to answer that question by identifying possibly relevant genetic differences between population groups, which, in the pre-molecular biology era, might be revealed by differences in their blood proteins. Thus, in the early 1950s, Blumberg, then working at the NIH, began collecting a panel of blood samples from diverse populations throughout the world.
Blumberg looked for serum protein variations (i.e., serum protein polymorphisms) by asking if sera from multiply-transfused individuals (defined by Blumberg as persons who received 25 units of blood or more) might contain antibodies that reacted with proteins in the serum samples of his panel. His rational, in his own words, was as follows: “We decided to test the hypothesis that patients who received large numbers of transfusions might develop antibodies against one or more of the polymorphic serum proteins (either known or unknown) which they themselves had not inherited, but which the blood donors had (2).” In other words, patients who received multiple transfusions were more likely than others to have antibodies against polymorphic serum proteins in donor blood, and those antibodies might also react with polymorphic serum proteins in the samples from his panel. See Aside 2.
[Aside 2: Blumberg used the Ouchterlony double-diffusion agar gel technique in these experiments. Serum samples to be tested against each other were placed in opposite wells of a gel. The proteins they contained could then diffuse through the gel. Antigen-antibody complexes that formed between the two samples appeared as white lines in the gel.]
Hemophilia and leukemia patients were well-represented in Blumberg’s collection of serum samples from multiply-transfused individuals. And, a serendipitous aspect of Blumberg’s experimental approach was that he used these samples to probe for serum protein polymorphisms in samples from geographically diverse populations. Thus it happened that Blumberg detected a cross-reaction between a New York hemophilia patient’s serum and a serum sample from an Australian aborigine. But what could these two individuals have had in common that might have triggered the cross-reaction?
His curiosity thus aroused, Blumberg and collaborator, Harvey Alter, of the NIH Blood Bank, tested the hemophilia patient’s serum against thousands of other serum samples. Blumberg and Alter may have been surprised to find that whatever the antigen in the Aborigine’s serum was that reacted with the hemophilia patient’s serum, reactivity against that antigen was common (one in ten) in leukemia patients, but rare (one in 1,000) in normal individuals. In any case, because the antigen was first identified in an Australian aborigine, it was termed the Australia antigen.
Bear in mind that Blumberg’s original purpose was to explain why individuals differed in their susceptibilities to various illnesses. Thus, Blumberg at first believed that he detected an inherited blood-protein that predisposes people to leukemia. However, additional experiments showed that the antigen was more common in older individuals than in younger ones; a finding more consistent with the possibility that the antigen might be associated with an infectious agent.
Blumberg’s first clue that the Australia antigen might be associated with hepatitis came to light when he tested serum samples from a 12-year old boy with Down syndrome. The first time that the boy was tested for the Australia antigen, he was negative. However, several months later, when retested, the boy was positive. Moreover, sometime during that interim, the boy also developed hepatitis.
Blumberg, and other researchers, carried out additional experiments, which confirmed that the Australia antigen indeed associated with hepatitis. In addition, the antigen was more frequently detected in hepatitis sufferers than in individuals with other liver diseases. Thus, the Australia antigen was a marker of hepatitis in particular and not of liver pathology in general. See Aside 3.
[Aside 3: Blumberg had a personal reason motivating him to identify the cause of hepatitis. His technician (later Dr. Barbara Werner) became ill with hepatitis, which she almost certainly acquired in the laboratory. Fortunately, she underwent a complete recovery.]
In 1970, British pathologist David Dane and colleagues at Middlesex Hospital in London, and K. E. Anderson and colleagues in New York, provided corroborating evidence that hepatitis is an infectious disease. Using electron microscopy, they observed 42-nm “virus-like particles” in the sera of patients who were positive for the Australia antigen. In addition, they saw these same particles in liver cells of patients with hepatitis.
What then is the Australia antigen? Actually, it is the surface protein of the 42-nm HBV particles; now known as the hepatitis B surface antigen (HBsAg). Since HBV particles per se were described for the first time by David Dane, they are sometimes referred to as Dane particles.
Now we can explain Blumberg’s early finding, that individuals who received multiple transfusions (e.g., leukemia and hemophilia patients) were more likely than the general population to have antibodies against the Australia antigen. Those individuals were more likely than the general population to have received donated blood and, thus, were more likely to have been recipients of blood contaminated with HBV. At that time, a large percentage of the blood supply came from paid donors, at least some of whom were syringe-sharing, intravenous drug abusers and, consequently, more likely than most to be HBV carriers. In 1972 it became law in the United States that all donated blood be screened for HBV. See Note 2.
But it was important to protect all people from HBV; not just transfusion recipients. In 1968, Blumberg, now at the Fox Chase Cancer Center in Philadelphia, and collaborator Irving Millman, hypothesized that HBsAg might provoke an immune response that would protect people against HBV and, consequently, that a vaccine could be made using HBsAg purified from the blood of HBV carriers. In Blumberg’s own words: “Irving Millman and I applied separation techniques for isolating and purifying the surface antigen and proposed using this material as a vaccine. To our knowledge, this was a unique approach to the production of a vaccine; that is, obtaining the immunizing antigen directly from the blood of human carriers of the virus (1).” The Fox Chase Cancer Center filed a patent for the process in 1969.
Blumberg was willing to share his method and the patent with any pharmaceutical company willing to develop an HBV vaccine for widespread use. Nonetheless, the scientific establishment was somewhat slow to accept his experimental findings and his proposal for making the vaccine. Then, in 1971, Merck accepted a license from Fox Chase to develop the vaccine. In 1982, after more years of research and testing, Maurice Hillman (3) and colleagues at Merck turned out the first commercial HBV vaccine (“Heptavax”). Producing an HBV vaccine, without having to cultivate the virus in vitro, was considered one of the major medical achievements of the day. See Notes 3 and 4.
The consequences of Blumberg’s vaccine were immediate and striking. For instance, in China the rate of chronic HBV infection among children fell from 15% to around 1% in less than a decade. And, in the United States, and in many other countries, post-transfusion hepatitis B was nearly eradicated.
Moreover, Blumberg’s HBV vaccine was, in a real sense, the world’s first anti-cancer vaccine since it prevented HBV-induced hepatocellular carcinoma, which accounts for 80% of all liver cancer; the 9th leading cause of death. Jonathan Chernoff (the scientific director of the Fox Chase Cancer Center, where Blumberg spent most of his professional life) stated: “I think it’s fair to say that Barry (Blumberg) prevented more cancer deaths than any person who’s ever lived (4).”
In 1976 Blumberg was awarded the Nobel Prize in Physiology or Medicine for “discoveries concerning new mechanisms for the origin and dissemination of infectious diseases.” He shared the award with Carlton Gajdusek, who won his portion for discoveries regarding the epidemiology of kuru (5). See Note 5.
Blumberg claimed that saving lives was the whole point of his career. “This is what drew me to medicine. There is, in Jewish thought, this idea that if you save a single life, you save the whole world, and that affected me (7).” See Aside 4.
[Aside 4: Blumberg received his elementary school education at an orthodox yeshiva in Brooklyn, and he attended weekly Talmud discussion classes until his death. Interestingly, Blumberg graduated from Far Rockaway High School in Queens, N.Y.; also the alma mater of fellow Nobel laureates, physicists Burton Richter and Richard Feynman.]
As we’ve seen, Blumberg’s landmark discovery of HBV sprang from a basic study of human genetic polymorphisms. In Blumberg’s own words, “… it is clear that I could not have planned the investigation at its beginning to find the cause of hepatitis B. This experience does not encourage an approach to basic research which is based exclusively on specific-goal-directed programs for the solution of biological problems (1).”
Saul Krugman (Note 4) had this to say about Blumberg’s discovery: “It is well known that Blumberg’s study that led to the discovery of Australia antigen was not designed to discover the causative agent of type B hepatitis. If he had included this objective in his grant application, the study section would have considered him either naïve or out of his mind. Yet the chance inclusion of one serum specimen from an Australian aborigine in a panel of 24 sera that was used in his study of polymorphisms in serum proteins…led to detection of an antigen that subsequently proved to be the hepatitis B surface antigen (1).” See Note 6.
In 1999, Blumberg’s scientific career took a rather curious turn when he accepted an appointment by NASA administrator, Dan Goldin, to head the NASA Astrobiology Institute. There, Blumberg helped to establish NASA’s search for extraterrestrial life. Blumberg also served on the board of the SETI Institute in Mountain View, Calif.
Blumberg passed away on April 5, 2011, at 85 years of age.
[Note 1: HBV is the prototype virus for the hepadnavirus family, which displays the most remarkable, and perhaps bizarre, viral replication strategy known. In brief, in the cell nucleus, the cellular RNA polymerase II enzyme transcribes the hepadnavirus circular, double-stranded DNA genome, thereby generating several distinct species of viral RNA transcripts, all of which are exported to the cytoplasm. The largest of these viral transcripts is the pregenomic RNA; a transcript of the entire circular viral DNA genome, as well as an additional terminal redundant sequence. Remarkably, the pregenomic RNA is then packaged in nascent virus capsids, within which it is reverse transcribed by a virus-encoded reverse transcriptase activity, thereby becoming an encapsulated progeny hepadnavirus double-stranded DNA genome. Thus, reverse transcription is a crucial step in the replication cycle of the hepadnaviruses, as it is in the case of the retroviruses. But, while the retroviruses replicate their RNA genomes via a DNA intermediate, the hepadnaviruses replicate their DNA genomes via an RNA intermediate.]
[Note 2: The highly sensitive radioimmunoassay (RIA) technique, developed by Rosalyn Yallow and Solomon Berson, is the basis for the test that screens the blood supply for the Australia antigen. The story behind this assay is worthy of note here because it is yet another example of serendipity in the progress of science. In brief, Yallow and Berson sought to develop an assay to measure insulin levels in diabetics. Towards that end, they happened to find that radioactively-labeled insulin disappeared more slowly from the blood samples of people previously given an injection of insulin than from the blood samples of untreated patients. That observation led them to conclude that the treated patients had earlier generated an insulin-binding antibody. And, from that premise they hit upon the RIA procedure. Using their insulin test as an example, they would add increasing amounts of an unlabelled insulin sample to a known amount of antibody bound to radioactively labeled insulin. They would then measure the amount label displaced from the antibody, from which they could calculate the amount of unlabelled insulin in the test sample. Their procedure has since been applied to hundreds of other substances. RIA is simpler to carry out and also about 1,000-fold more sensitive than the double-diffusion agar gel procedure that Blumberg used to identify the Australia antigen. Yallow and Berson refused to patent their RIA procedure, despite its huge commercial value. Yallow received a share of the 1977 Nobel Prize in Physiology or Medicine for her role in its development. Berson, died in 1972 and did not share in the award.]
[Note 3: Making Heptavax directly from the blood of human HBV carriers was somewhat hindered because it required a continuing and uncertain supply of suitable donor blood. Moreover, there was concern that even after purifying the HBsAg, and treating it with formalin to inactivate any infectivity, the vaccine might yet contain other live dangerous viruses. Concern increased in the early 1980s with the emergence of HIV/AIDS, since much of the HBV-infected serum came from donors who later developed AIDS. Thus, in 1990 Heptavax was replaced in the United States by a safer genetically engineered (i.e., DNA recombinant) HBV vaccine, which contained no virus whatsoever. That vaccine was the first to be made using recombinant DNA technology. Moreover, it was yet another instance in which Hilleman played a key role in the development of a vaccine (3).]
[Note 4: In 1971, Saul Krugman, working at NYU, was actually the first researcher to make a “vaccine” against HBV. Krugman’s accomplishment began as a straightforward inquiry into whether heat (boiling) might kill HAV (see Note 5). Finding that it did, Krugman repeated his experiments; this time to determine whether boiling might likewise kill HBV in the serum of HBV carriers. As Krugman expected, boiling indeed destroyed HBV infectivity. But, to his surprise, while the heated serum was no longer infectious, it did induce incomplete, but statistically significant protection against challenge with live HBV. Krugman considers his “vaccine” discovery, like Blumberg’s discovery of HBV, to have resulted from “pure serendipity” (1).
Krugman could not answer whether HBsAg per se in his crude vaccine induced immunity. However, Hilleman, in 1975, using purified HBsAg, as per Blumberg’s concept, showed that HBsAg indeed induced immunity against an intravenous challenge with HBV.
Krugman also carried out key studies on the epidemiology of hepatitis, demonstrating that “infectious” (type A) hepatitis is transmitted by the fecal-oral route, while the more serious “serum” (type B) hepatitis is transmitted by blood and sexual contact.
Krugman reputation was somewhat tarnished because he used institutionalized disabled children as test subjects in the experiments that led to his vaccine. While that practice astonishes us today, it was not unheard-of in the day. In any event, it did not prevent Krugman’s election in 1972 as president of the American Pediatric Society, or his 1983 Lasker Public Service Award.]
[Note 5: Gajdusek’s reputation was later sullied when he was convicted of child molestation (5).]
[Note 6: In 1973 and 1974, research groups led by Stephen Feinstone and Maurice Hilleman (3) discovered hepatitis A virus (HAV), a picornavirus.
After the discoveries of HAV and HBV, it became clear that blood samples cleared of HAV and HBV could still transmit hepatitis. In 1983 Mikhail Balayan identified a virus, now known a hepatitis E virus (the prototype of a new family of RNA viruses), as the cause of a non-A, non-B infectious hepatitis (6).
In 1989, a mysterious non-A, non-B hepatitis agent, now known as hepatitis C virus (a flavivirus), was identified by a team of molecular biologists using the cutting-edge molecular biology techniques of the day (8).]
Krugman, S. 1976. Viral Hepatitis: Overview and Historical Perspectives. The Yale Journal of Biology and Medicine 49:199-203.
Blumberg, B, Australia Antigen and the Biology of Hepatitis B, Nobel Lecture, December 13, 1976.
There have been several instances in which medical researchers, for the sake of mankind, allowed themselves to be infected with a potentially deadly pathogen. A well known example involved the discovery that the Aedes aegypti mosquito is the vector for yellow fever (1). Here we consider a less known and slightly bizarre example in which Mikhail S. Balayan, of the Russian Academy of Medical Sciences in Moscow, discovered the hepatitis E virus.
But first, hepatitis refers to an inflammatory disease involving the liver. Four unrelated viruses, hepatitis A, hepatitis B, hepatitis C, and hepatitis E viruses cause epidemic viral hepatitis (see Aside 1). Hepatitis E was initially identified in 1980 as a non-A, non-B infectious hepatitis. The differences between hepatitis A, B, and E virus infections are as follows. Hepatitis A and hepatitis E are similar, insofar as the etiologic agent of each usually gives rise to an acute (i.e., self-limiting) infection and illness. In contrast, hepatitis B and hepatitis C viruses usually give rise to persistent infections that may lead to chronic hepatitis, cirrhosis, and liver cancer. The mortality rate for hepatitis E is generally “only” about 1% to 2%. Yet, hepatitis E is unusual among hepatitis viruses for its severity in pregnant woman, in whom the fatality rate may reach 20%.
[Aside 1: For aficionados, hepatitis A is a picornavirus, hepatitis B is a hepadnavirus (a DNA retrovirus), and hepatitis C is a flavivirus. Hepatitis E-like viruses were originally classified as calciviruses. However, sequencing of their RNA genomes revealed that they are more similar to rubella virus, a togavirus, than to the calciviruses. Yet they are different enough from togaviruses to merit their own family. The prototype is the hepatitis E virus, discovered by Balayan. Like hepatitis A virus, it is spread by the fecal-oral route. Hepatitis E virus is found worldwide, but it is most problematic in developing countries.]
Here then is Balayan’s tale. In 1983 Balayan was investigating an outbreak of non-A, non-B hepatitis in Tashkent; now the capital city of Uzbekistan. Balayan wanted to bring patient samples back to Moscow to study. However, he had no means for refrigerating the samples. Moreover, he may not have had permission from his supervisors to return with the samples. So, he solved his dilemma by a rather extreme form of self sacrifice—he drank a pooled filtrate of patient stool samples. He is said to have made his private inoculum more palatable by first mixing it with yogurt.
Belayan’s efforts were not for naught since, after returning to Moscow, he indeed came down with hepatitis, as he presumably desired. In fact, he became seriously ill. He then began to collect his own stool samples, in which he detected, by electron microscopy, 32 nm virus particles that produced a hepatitis-like illness when inoculated into monkeys. Balayan then observed a virus in the stool of these monkeys that appeared to be identical to the virus in the original patient samples, which he transported in, and recovered from himself.
Belayan’s virus looked like hepatitis A virus in electron micrographs. But, he could show that it was not hepatitis A virus. He already had antibodies against the hepatitis A virus, and these did not react with the new virus.
Balayan mentions himself in his original report (2), as follows: “Hepatitis E virus (HEV) was first identified in the excreta of an experimentally infected human volunteer and further confirmed by similar findings in clinical specimens from patients with acute jaundice disease different from hepatitis A and B.”
1. The Struggle Against Yellow fever: Featuring Walter Reed and Max Theiler, Posted on the blog May 13, 2014.
2. Balayan, M.S., 1983. Hepatitis E virus infection in Europe: Regional situation regarding laboratory diagnosis and epidemiology. Clinical and Diagnostic Virology1:1-9.
In 1911 Peyton Rous, at the Rockefeller Institute, discovered the Rous sarcoma virus; the first virus known to cause solid tumors (1). Although Rous’ eponymous virus also would be known as the prototype retrovirus, his discovery generated only scant interest at the time, and would not be recognized by the Nobel Committed until 65 years later! [Nobel prizes are not awarded posthumously. Fortunately, Rous had longevity on his side. He died 4 years after receiving the prize, at age 87.]
In 1976 Harold Varmus and J. Michael Bishop, then at the University of California San Francisco, discovered that the Rous sarcoma virus oncogene, v-src, as well as the oncogenes of several other tumorgenic retroviruses, actually were derived from cellular genes that normally play an important role in controlling cell division and differentiation (2). Moreover, Varmus and Bishop showed that these cellular “proto-oncogenes” can be altered by mutation, to become “oncogenes” that contribute to cancer. [Varmus and Bishop received the 1989 Nobel Prize in Physiology or Medicine for their discovery of proto-oncogenes.]
But what is the actual activity of the protein coded for by the normal cellular c-src, and by v-src as well? The story of that discovery is rather delightful and begins as follows.
In 1978, Raymond Erikson and coworker Marc Collette, then at the University of Colorado Medical Center, were the first researchers to isolate the Src protein. They accomplished this by first preparing lysates from avian and mammalian cells, which had been transformed in culture into tumor cells by Rous sarcoma virus. Next, they precipitated those lysates with antisera from rabbits that bore Rous sarcoma virus-induced tumors. The premise of their strategy was that antibodies from the tumor-bearing rabbits would recognize and precipitate proteins that were specific to cells transformed by the virus .
With the Src protein now in hand, Ericson and Collette next sought its function. They initially asked whether Src might have protein kinase activity (i.e., an activity that adds a phosphate group to a protein.). This was a reasonable possibility because protein phosphorylation was already known to play a role in regulating various cellular processes, including cell growth and differentiation.
Ericson and Collette tested their premise by incubating their Src immunoprecipitates with [γ-32P] ATP (i.e. 32P-labelled adenosine triphosphate). In agreement with their proposal, they found that the antibody molecules in the Src immunoprecipitates had been phosphorylated. [Note that Src’s protein kinase activity was simultaneously and independently discovered by Varmus and Bishop.]
Ericson and Collette also carried out control experiments that were particularly revealing. When the same rabbit antisera was used to immunoprecipitate extracts from normal cells, or extracts from cells infected with a transformation-defective mutant of Rous sarcoma virus, no signs of protein kinase activity were seen in those immunoprecipitates. What’s more, the protein kinase activity was found to be temperature sensitive in immunoprecipitates from cells infected with a mutant Rous sarcoma virus that was temperature-sensitive for transformation.
These control experiments confirmed that the protein kinase activity in the immunoprecipitates was coded for by the virus. What’s more, they confirmed that the kinase activity of the retroviral Src protein plays an essential role in transformation. Furthermore, when taken with the earlier findings of Varmus and Bishop, they implied that the kinase activity of the cellular Src protein plays a key role in the control of normal cell proliferation.
While Erickson and coworkers were carrying out the above experiments in Denver, Walter Eckhart and TonyHunter, at the Salk Institute, were looking into the basis for the transforming activity of the mouse polyomavirus middle T (MT) protein. [Unlike Rous sarcoma virus, which is a retrovirus, the mouse polyomavirus is a member of the Polyomavirus family of small DNA tumor viruses. SV40 is the prototype Polyomavirus.]
Since Erickson’s group was finding that Src expresses protein kinase activity, Eckhart and Hunter asked whether the polyomavirus MT protein might likewise be a protein kinase. Thus, as Erickson and Collette had done in the case of Src, Eckhart and Hunter examined immunoprecipitates of MT to see if they too might express a protein kinase activity, and found that indeed they did.
Interestingly, it was not known at the time of these experiments that MT actually does not express any intrinsic enzymatic activity of its own. Instead, MT interacts with the cellular Src protein to activate its protein kinase activity. See Aside 1.
[Aside 1: For aficionados, MT is a membrane-associated protein that interacts with several cellular proteins. Importantly, the phosphorylation events carried out by MT-activated Src cause a variety of signal adaptor molecules [e.g., Shc, Grb2, and Sos] and other signal mediators [e.g., PI3K and PLCγ] to bind to the complex, thereby triggering a variety of mitogenic signaling pathways. These facts were not yet known when Eckhart and Hunter were doing their experiments.]
At the time of these experiments, serine and threonine were the only amino acids known to be phosphorylated by protein kinases. In fact, Erikson and Collette, as well as Varmus and Bishop, believed that threonine was the amino acid phosphorylated by the Src kinase (see below). Consequently, Hunter asked whether the polyomavirus MT protein likewise would phosphorylate threonine. [Recall that MT actually does not express any intrinsic enzymatic activity of its own.]
Hunter’s experimental procedure was relatively straightforward and reminiscent of Erikson’s and Collette’s. It involved incubating immunoprecipitates of MT with [γ-32P]ATP, hydrolyzing the immunoglobulin, and then separating the amino acids in the hydrolysate by electrophoresis. But, to Hunter’s surprise, the position of the labeled amino acid in his electropherogram did not correspond to that of either threonine or serine.
Hunter was well aware that tyrosine is the only other amino acid with a free hydroxyl group that might be a target for the MT kinase activity. And, while there was no precedent for a tyrosine-specific protein kinase, Hunter proceeded to ask whether the polyomavirus MT protein indeed might phosphorylate tyrosine.
Hunter began by synthesizing a phosphotyrosine molecule that could be used as a standard marker against which to compare the labeled amino acid in a repeat of his earlier experiment. And, to his pleasure, Hunter found that the amino acid that was phosphorylated by the MT kinase activity ran precisely with the phosphotyrosine standard marker in his new electropherograms.
But why had other researchers not detected tyrosine phosphorylation earlier? It was partly because phosphotyrosine accounts for only about 0.03% of phosphorylated amino acids in normal cells. The remaining 99.97% are phosphoserine and phosphothreonine. But, again, that is not the entire explanation. The rest is truly precious.
In Hunter’s own words, he was “too lazy to make up fresh buffer” before doing his experiments. Had the buffer been fresh, its pH would have been the usual 1.9; a pH that, unbeknownst to all at the time, does not separate phosphotyrosine from phosphothreonine during the electrophoresis procedure. The pH of the old buffer that Hunter used in his experiment had inadvertently dropped to 1.7; a pH at which phosphotyrosine is resolved from phosphothreonine. That fact enabled Hunter to discriminate phosphotyrosine from phosphothreonine for the first time. Thus, Hunter attributes his hugely important discovery to his laziness.
The finding that tyrosine is the amino acid phosophorylated by the polyomavirus MT protein kinase activity led Hunter and his Salk Institute-colleague Bart Sefton to ask whether Src too might phosphorylate tyrosine, rather than serine or threonine (4). Indeed, they found that the retroviral Src protein, as well the normal cellular Src protein, function as tyrosine-specific protein kinases. [Recall that it became clear only later that MT actually has no intrinsic enzyme activity of its own and that it acts through Src.] Moreover, the levels of phosphotyrosine were 10-fold higher in cells infected with wild-type Rous sarcoma virus than in control cells, consistent with the premise that Src’s protein tyrosine kinase activity accounts for the altered growth potential of those cells.
Subsequently, Stanley Cohen, at Vanderbilt University, discovered that the epidermal growth factor (EGF) receptor contains an intrinsic protein-tyrosine kinase activity, further underscoring the importance of protein-tyrosine kinases in the normal control of cell proliferation. [Cohen shared the 1986 Nobel Prize in Physiology or Medicine with Rita Levi-Montalcini for their discoveries of growth factors, including EGF.] Subsequent studies identified additional receptor protein-tyrosine kinases, such as the fetal growth factor (FGF) receptor, and non-receptor protein-tyrosine kinases, such as Abl, each of which activates a mitogenic intracellular signaling pathway.
Tony Hunter and coworkers went on to demonstrate that protein-tyrosine kinases play key roles in additional crucial cellular processes, including cellular adhesion, vesicle trafficking, cell communication, the control of gene expression, protein degradation, and immune responses. Moreover, discoveries regarding the role of protein-tyrosine kinases in cell transformation and cancer gave rise to a promising new rational approach to cancer therapy; i.e., the targeting of protein-tyrosine kinases. For example, the drug Gleevec, which inhibits activation of the Abl and platelet-derived growth factor (PDGF) tyrosine kinases, was approved by the U.S. Food and Drug Administration for the treatment of chronic myelogenous leukemia and several types of gastrointestinal tumors.
Much has happened since our lasting posting on the Brazilian Zika outbreak (1). In particular, the major topic of our last posting was the uncertainty regarding whether Zika virus causes congenital birth defects. Recent findings may be settling the issue.
One reason for the earlier uncertainty was that although Zika virus has spread to more than a dozen countries since its discovery in Uganda more than 50 years ago, Brazil remained the world’s only country in which the virus was associated with microcephaly. However, in February 2016, Brazil’s neighbor, Colombia, now the world’s second-most Zika-affected country, reported its first cases of birth defects linked to Zika.
More direct and compelling evidence for Zika as an agent of microcephaly was reported early this March in the New England Journal of Medicine (2). Ultrasound examination of Zika-infected pregnant woman revealed that 29 percent of them carried fetuses suffering “grave outcomes, including fetal death, placental insufficiency, fetal growth restriction, and CNS injury.” Zika infection of the mothers was confirmed by reverse-transcriptase–polymerase-chain-reaction assays of blood and urine specimens. “To date, 8 of the 42 women in whom fetal ultrasonography was performed have delivered their babies, and the ultrasonographic findings have been confirmed.”
Although the above study examined only 88 women, all at one clinic in Rio de Janeiro, an article in the New York Times (March 5, 2016) quotes Anthony Fauci, the director of the National Institute of Allergy and Infectious Diseases as saying, “Now there’s almost no doubt that Zika is the cause.”
Another notable report described a case of a pregnant woman who, while living in Brazil, came down with a Zika-like feverish illness at the end of the first trimester of her pregnancy (3). The mother opted to abort her 29-week-old fetus after it showed signs (by ultrasonography) of microcephaly—subsequently confirmed by autopsy of the fetus. Importantly, a flavivirus was visualized in the fetal brain by electron microscopy, and the entire Zika genome (unambiguously identified by reverse-transcriptase–polymerase-chain-reaction assay) was recovered from it.
Next, we consider a new finding that Zika can be present in breast milk. Whereas Zika is an arthropod-borne virus that is transmitted primarily by its mosquito vector, our first posting on the Brazilian Zika outbreak noted at least one instance in which Zika was transmitted via a blood transfusion (4). In addition, there were reports of Zika being sexually transmitted (5). Now there is a report of Zika virus in the breast milk of a mother in New Caledonia (6).
The woman was feverish in July 2015 when she arrived at the hospital to give birth. Nevertheless, she breast fed her apparently healthy baby immediately after delivering it. Samples of the mother’s serum and breast milk then tested positive for Zika virus by reverse-transcriptase–polymerase-chain-reaction assay, while a test of a serum sample from the 3-day–old baby was ambiguous. The mother’s fever, now accompanied by a characteristic Zika rash, persisted for the next several days. Nonetheless, she and her baby were each healthy when they left the hospital.
This report would appear to raise considerable concern that a Zika-infected mother might transmit the virus to her baby via her breast milk. All the same, the US Centers for Disease Control and Prevention (CDC) maintains that the benefits of breastfeeding outweigh the theoretical risks of Zika virus infection via breast milk, and recommends that infected women should breastfeed.
Next, we consider some recent history. On February 1, 2016 the WHO declared that Brazil’s Zika outbreak is as an international public-health emergency. But, uncharacteristically, the WHO put forth this pronouncement despite the fact that the scientific community was still not sure of the threat that Zika poses to humans. In point of fact, this was the first instance in which the WHO proclaimed its highest level of alarm for an agent of uncertain danger. [The CDC likewise elevated its Zika virus surveillance program to its highest priority level.]
Why did the WHO make its frightening declaration when the threat posed by Zika was still not clear? Obviously, a failure to take immediate action might allow the Zika outbreak to get well out of hand, with possibly devastating consequences.
In contrast to the hurried response by the WHO to the Zika outbreak, that agency responded more leisurely to the 2013/2014 West African Ebola outbreak, which did get out of control, and which persists even to this day. So, perhaps the more urgent response of the WHO to the Zika outbreak reflects a lesson learned from the Ebola affair.
But, why did the WHO wait longer before responding to the West African Ebola outbreak? One reason is because it was strongly criticized for “overreacting” to the risk posed by the 2009 H1N1 influenza epidemic—which turned out to be far less threatening than originally feared.
While the WHO may have learned a lesson from its somewhat unhurried response to the Ebola outbreak, its more urgent February 1, 2016 Zika declaration did not go far enough for some observers, since it stopped short of advising pregnant women not to travel to Zika-affected regions. For that reason, the WHO has been accused of taking political considerations into account, to the detriment of good public health policy. Any travel ban—even one aimed only at pregnant women—would be embarrassing and costly to Brazil, which has been moving ahead with its plans to host the Olympic Games this summer. Still, hundreds of thousands of people from around the world, including female spectators and participants, some of whom may be pregnant, are expected to attend.
Lastly, we note that the Zika outbreak has been stirring up a fierce religious debate in Latin America; a debate that is actually challenging the very authority of the Catholic Church in the hemisphere. But first, an earlier posting on the blog recounted how in 2002 Colin Powell, at the time Secretary of State in the George W. Bush administration, advocated that sexually active young people should use condoms to protect themselves against HIV/AIDS (7). Powell’s advocacy of condom usage was contrary to the Bush administration’s strongly held abstinence-only approach for preventing sexual transmission of HIV. Moreover, then as now, Powell’s stance was contrary to the official position of the Catholic Church on artificial contraceptives. Nevertheless, Powell asserted, “I certainly respect the position of the Holy Father and the Catholic Church. In my own judgment, condoms are a way to prevent infection. Therefore, I not only support their use, I encourage their use among people who are sexually active and need to protect themselves.”
Now, presumably in response to reports that Zika virus might be transmitted sexually, Pope Francis declared on February 18, 2016—during a mid-air news conference on his flight from Mexico back to Rome—that contraceptives could be used to block the spread of Zika virus. That same day, the WHO advised the sexual partners of pregnant women to use condoms, or to abstain from sex, if they live in a Zika-affected area, or if they are returning from one of those areas. Also, several Latin American governments asked their female citizens to delay getting pregnant.
Those suggestions, whether from Latin American governments, or from the WHO, offended many Latin American women, in part because of the strict anti-abortion laws, and laws that restrict access to contraceptives in some of those countries. Moreover, the situation is compounded in some regions of the hemisphere by rampant sexual violence against women. In any event, the Pope’s pronouncement intensified an angry debate over contraception, and abortion as well, that was already underway in Latin America.
Pope Francis did not condone abortion, which he referred to as an “absolute evil.” But, he did make a point of justifying his statement condoning contraception by citing as a precedent a 1960 judgment by Pope Paul VI, which permitted nuns in the Belgian Congo, who were in danger of being raped, to use contraceptives.
Pope Francis’ remarks, such as “avoiding pregnancy is not an absolute evil,” has encouraged Latin American opponents of the church’s longstanding ban on the use of artificial contraceptives to campaign harder against those policies. In any case, the Pope’s pronouncement, and the heated response it is provoking, shows that the Zika outbreak is now impacting religious institutions. And, as noted by Ana Ayala, the director of the Global Health Law Program at Georgetown University, “The pope’s positioning on this subject can significantly shift how governments see access to contraception.” See Aside 1.
[Aside 1: Ayala’s comment can be found in a February 18, 2016 article in the New York Times, entitled “Francis Says Contraception Can Be Used to Slow Zika”, by Simon Romero and Jim Yardley. This piece offers an extensive account of the response in Latin America, and elsewhere, to the Pope’s comments. “While international researchers are still trying to prove definitely a link between Zika and microcephaly, the pope’s comments on contraception seemed to catch up to the reality in parts of the hemisphere where many Catholics pay little heed to the church’s teachings on birth control.”]
Zika Virus, Part 2: The Link to Birth Defects, Is It Real?, Posted on the blog February 23, 2016.
Brasil, P., Pereira, J.P., Gabaglia, C.J., et al., Zika Virus Infection in Pregnant Women in Rio de Janeiro — Preliminary Report, N. Engl. J. Med., March 4, 2016DOI: 10.1056/NEJMoa1602412
Mlakar, J., Korva, M., Tul, M., et al., Zika Virus Associated with Microcephaly, N. Engl. J. Med. 2016; 374:951-958 March 10, 2016 DOI:10.1056/NEJMoa1600651
Zika Virus: Background, Politics, and Prospects, Posted on the blog February 4, 2016.
Foy, B.D., K. C. Kobylinski, J.L. Foy, et al., 2011. Probable Non–Vector-borne Transmission of Zika Virus, Colorado, USA, Emerg Infect Dis.17: 880–882.6.
Gravitational waves were detected for the first time this past February by the Laser Interferometer Gravitational-Wave Observatory (LIGO), which consists of two widely separated installations within the United States — one in Livingston, Louisiana and one in Hanford, Washington. Before LIGO, there was no technology able to detect these vanishingly weak waves. Consequently, LIGO’s accomplishment has generated considerable excitement in the physics and astronomy communities. First, it confirmed the existence of gravity waves; a key prediction of Einstein’s 1915 theory of general relativity. Second, and remarkably, LIGO detected gravitational waves that were emitted during the final fraction of a second of the merger of two black holes, which were over a billion light-years away (one light-year is about 5.88 trillion miles)! What’s more, LIGO’s findings in fact proved the existence of black holes. Prior to LIGO’s achievement, the existence of black holes was widely accepted, but based only on indirect evidence.
Physicists and astronomers are also excited by the potential of gravitational-wave detectors to shed light on other basic concerns, such as determining whether gravitational waves travel at the speed of light—an important issue since it would answer whether gravity is transmitted by particles having no mass. These detectors may also enable astronomers to measure the rate at which the universe is expanding, and perhaps observe the effect of dark energy on space.
Most exciting perhaps, gravitational wave detectors may enable astronomers to see almost all the way back to the big bang. Until now, astronomers could only see as far back as 380,000 years after the big bang, when the universe became transparent to light and other electromagnetic radiation. However, gravitational waves would have traveled unhindered through the newborn universe. By scrutinizing gravitational waves from the infant universe, cosmologists hope to learn more about its beginning and, perhaps, even uncover evidence for the existence of other universes. Moreover, gravitational wave detectors might even lead to a “theory of everything (1).”
Scientists from other disciplines, as well as lay people, might very well marvel at the sheer ingenuity and persistence of the physicists and engineers who designed LIGO; a result of decades of instrument research and development. But first, here is a very brief account of gravity waves.
Einstein’s theory of general relativity predicts that matter emits gravity waves. These waves disturb the fabric of space, in fact causing the distances between objects to ebb and flow in an oscillatory manner. However, these oscillations are far too small to have been detected prior to LIGO.
Here is Lawrence M. Krauss’ account in the New York Times of the LIGO technical achievement (2). “To see these waves, the experimenters built two mammoth detectors, one in Washington State, the other in Louisiana, each consisting of two tunnels about 2.5 miles in length at right angles to each other. By shooting a laser beam down the length of each tunnel and timing how long it took for each to be reflected off a mirror at the far end, the experimenters could precisely measure the tunnels’ length. If a gravitational wave from a distant galaxy traverses the detectors at both locations roughly simultaneously, then at each location, the length of one arm would get smaller, while the length of the other arm would get longer, alternating back and forth …To detect the signal they observed they had to be able to measure a periodic difference in the length between the two tunnels by a distance of less than one ten-thousandth the size of a single proton. It is equivalent to measuring the distance between the earth and the nearest star with an accuracy of the width of a human hair….If the fact that this is possible doesn’t astonish, then read these statements again. This difference is so small that even the minuscule motion in the position of each mirror at the end of each tunnel because of quantum mechanical vibrations of the atoms in the mirror could have overwhelmed the signal. But scientists were able to resort to the most modern techniques in quantum optics to overcome this.” See Asides 1 and 2.
[Aside 1: Lawrence M. Krauss is a theoretical physicist and director of the Origins Project at Arizona State University. He is the author of “A Universe from Nothing: Why There is Something Rather than Nothing.”]
[Aside 2: Interestingly, the LIGO detectors had just been turned on for their first observing run when they discovered a clear signal emanating from the colliding black holes. Also, recall that these black holes were over a billion light-years away.]
Krauss later says, “Too often people ask, what’s the use of science like this, if it doesn’t produce faster cars or better toasters. But people rarely ask the same question about a Picasso painting or a Mozart symphony. Such pinnacles of human creativity change our perspective of our place in the universe. Science, like art, music and literature, has the capacity to amaze and excite, dazzle and bewilder. I would argue that it is that aspect of science — its cultural contribution, its humanity — that is perhaps its most important feature (2).”
Also, consider the following from an editorial in the New York Times. “The curiosity of our species knows no bounds; more remarkably, neither does our capacity for satisfying it. And that is truly wonderful in itself, even if it doesn’t lead to a better toaster (3).” See Aside 3.
[Aside 3: The development of LIGO was made possible by support from the National Science Foundation. “By coincidence, at about the same time that the LIGO discovery was announced, the U.S. House of Representatives passed a bill requiring that National Science Foundation grants be justified ‘in the national interest.’ It is doubtful that LIGO would have survived such political meddling (3).”]
“The Theory of Everything,” Posted on the blog September 15, 2015.
Lawrence M. Krauss, Finding Beauty in the Darkness, Opinion in Sunday Review, New York Times, February 14, 2016.
The Editorial Board, New York Times, February 17, 2016
Zika virus was discovered in the Zika Forest of Uganda in 1947 and, despite its current prominence in the media, until this past year it was thought to be relatively benign. However, matters changed dramatically in 2015 when Zika virus emerged in Brazil, where it has since been associated with a striking surge in the incidence of infants born with microcephaly (abnormally small heads and brains). Astonishingly, the number of Brazilian children born with microcephaly allegedly jumped from 147 in 2014 to 4,783 cases as of February 2, 2016.
The link between Brazil’s cases of microcephaly and in-utero infection with Zika virus was first implied by the geographic and temporal correspondence between the emergence of the virus in Brazil and the remarkable rise in the number of Brazilian cases of microcephaly (1). Subsequently, Brazilian health officials investigated 1,113 of their microcephaly cases and confirmed that 404 of them could be linked to Zika infection. In addition, actual Zika virus was detected in the amniotic fluid of several microcephalic fetuses, and anti-Zika virus antibodies were detected in the amniotic fluid of others—evidence that the virus indeed might cross the placenta and possibly infect the fetus (2). With those sorts of findings at hand, why do public health authorities still refer to the Zika/microcephaly link as merely suggested?
Some health officials and researchers claim that the surge in the Brazilian incidence of microcephaly, which allegedly occurred after the Zika outbreak, was merely an artifact, accounted for by a significant under-reporting of cases before the Zika outbreak. To that point, 21 Brazilian medical centers recently collaborated to reassess the head circumferences of 16,208 Brazilian neonates from Northeast Brazil (which contained the epicenter of the 2015 Zika epidemic) from late 2012 until the entry of the Zika virus into Brazil in mid-2014. As reported earlier this month (3), they found an astonishing incidence of microcephaly during that pre-Zika period; ranging from 2% to 8%. Moreover, and importantly, the number of affected babies actually peaked in 2014, before Zika virus had even been seen in Brazil! See Aside 1.
[Aside 1: These researchers also found an increased incidence of the most extreme cases of microcephaly in the last quarter of 2015, after Zika virus emerged in Brazil; a finding consistent with the possibility that something new was occurring after the Zika outbreak. See Note below.]
What might explain the under-reporting of microcephaly cases in Brazil, before its Zika outbreak? The authors of the collaborative study claim that it was mainly because there are no standardized criteria for diagnosing microcephaly—loosely defined as a condition, not an illness, characterized by an occipital-frontal head circumference smaller than expected for gestational age and gender. The absence of standardized diagnostic criteria was said to account for the discrepancies between the incidence of microcephaly found by the authors of the collaborative study and that recorded earlier in official sites. [A lack of consensus over the defining limits of microcephaly also accounts for the wide range in incidence of cases, of from 2% to 8%, in the collaborative study’s report.] See Note 1, below.
Interestingly, when these investigators narrowed the criteria for microcephaly to include only the most extreme cases (i.e., those neonates who fell into the lower third of all three criteria enumerated in Note 1; see below) the rates (0.04 percent to 1.9 percent) were still high, although now within the ranges reported elsewhere in the world. Consequently, the authors conclude: “It is possible that a high incidence of milder forms microcephaly has been occurring well before the current outbreak, but that only those extreme cases, with classical phenotypes, were being notified. And as the number of extreme cases increased over these past three or four months so did the awareness of health professionals who started to notify milder forms (3).”
The authors call attention to the fact that the clinical significance of the milder forms of microcephaly, which comprise the vast majority of reported cases, remains to be determined. They then assert that, “These observations highlight the need to review the situation carefully. Many questions need to be answered prior to concluding what problem we are facing, how it came about and which consequences it is likely to bring to the Brazilian population in years to come… We can only conclude that we are facing a new and challenging public health problem and that limited epidemiological and clinical data hinders conclusions at this early stage.”
In February 2016 another team of Brazilian researchers claimed that the lack of standardized diagnostic criteria for microcephaly led to an overestimate of the incidence of microcephaly after the Zika outbreak, rather than to an underestimatebefore the outbreak (5). That is, an increased incidence of microcephaly was reported after the outbreak because normal children, whose heads were small, actually comprised the majority of alleged cases.
Here is another reason for questioning the link between Zinka infection and microcephaly. Whereas Zika virus was discovered in Uganda more than 60 years ago, and has since spread to more than a dozen countries, the recent surge of microcephaly in Brazil is the only case-in-point, anywhere in the world, in which microcephaly has been associated with Zika virus. Brazil’s neighbor, Colombia, is the world’s second-most Zika-affected country, with around 20,000 confirmed Zika infections. Yet while more than 2,000 of the Columbian Zika infections were of pregnant women, none of their fetuses were diagnosed with microcephaly.
Moreover, and in contrast to the lack of an association between Zika and microcephaly outside of Brazil, the Zika outbreak has been associated with a surge in Guillain-Barré syndrome (a temporary paralysis) in Colombia, El Salvador, Suriname, Venezuela, and French Polynesia, as well as in Brazil. The apparent universal association of Zika with Guillain-Barré syndrome, but not with microcephaly, might be taken as an argument against an etiologic role for Zika virus in microcephaly.
Yet, even in the case of Guillain-Barré syndrome, the World Health Organization considers the link to Zika to be tenuous; in part because other arthropod-borne viruses, including dengue, chikungunya and Zika viruses, have all been circulating simultaneously in the Americas (5). [Guillain-Barré syndrome occurs after infection by a variety of pathogens, including dengue and chikungunya viruses, which are related to Zika.] Likewise, the circulation of these other arthropod-borne viruses in areas hard hit by Zika raises the possibility that they might be involved in microcephaly.
So, where do we stand? Uncertainty regarding the connection between Zika and microcephaly underscores the need for clinicians to come to a consensus regarding the criteria that define that condition. Moreover, since most of the mothers who participated in earlier epidemiologic studies were not tested for Zika (even if they might have been infected), and since Zika causes a relatively mild illness that often goes undetected, and since other pathogenic arthropod-borne viruses also circulate in areas in which Zika is prevalent, there is a crucial need for a convenient and unambiguous molecular diagnostic test to identify these infections. See Aside 2.
[Aside 2: A convenient diagnostic test for Zika virus is also needed to protect the blood supply in countries were the virus is spreading by local transmission. Many of these countries are poverty stricken and already suffer from low donation rates and dwindling blood supplies. They can not long depend on outside sources of blood for their transfusions.]
But even with standardized diagnostic criteria for microcephaly, as well as accurate tests for infection, important gaps still remain in our knowledge of Zika virus—gaps that must be filled before the role of the virus in microcephaly can be known with certainty.
But isn’t the presence of Zika virus in the amniotic fluid of microcephalic fetuses proof enough? It isn’t because the handful of other viruses that are able to cross the human placenta (e.g. rubella, cytomegalovirus) are not known to cause microcephaly at the extraordinary rates currently being reported in Brazil. Thus, it is necessary to establish with certainty that Zika virus does, in fact, target and harm the fetal brain. [If it were ascertained that Zika virus does target the fetal brain, then it will be important to know how much time elapses after the mother is infected, before the virus can strike the fetus. Moreover, it will be important to determine whether the fetal brain is at risk to Zika during all, or during only some stages of its development.] See Aside 3.
[Aside 3: The MMR vaccine largely protects American children against congenital rubella. However, worldwide, more than 100,000 children continue to be born each year with this condition. Cytomegalovirus, for which there is no vaccine, causes at least 5,000 cases of birth defects each year in the United States alone.]
Importantly, despite the reservations noted above, many, if not most researchers believe that Zika virus indeed is the agent behind a very real surge in the incidence of microcephaly in Brazil. Moreover, the World Health Organization declared that the rise in microcephaly constitutes a global health emergency. Thus, while we await more rigorous proof of the Zika/microcephaly connection, it remains essential to act as though it were real.
“In this study (3), classification of microcephaly was based on three different criteria, as follows:
1. Brazilian Health Ministry proposed criteria, where microcephaly equals an occipital-frontal head circumference (OFC) smaller than 32 cm for term neonates.
2. Fenton curves, where microcephaly equals an OFC less than -3 standard deviation (SD) for age and gender.
3. Proportionality criteria, where microcephaly equals an OFC less than ((height/2) + 10) ± 2.
Neonates were classified with microcephaly according to each one of the three criteria.
A separate group was created for those who fulfilled all three criteria. Finally, those who fell into the lower third in each criterion were grouped as extreme cases of microcephaly.”
1. Zika Virus: Background, Politics, and Prospects, Posted on the blog February 4, 2016.
2. Oliveira Melo AS, Malinger G, Ximenes R, Szejnfeld PO, Alves Sampaio S, Bispo de Filippis AM. Zika virus intrauterine infection causes fetal brain abnormality and microcephaly: tip of the iceberg? 2016 Ultrasound Obstet Gynecol. 47: 6–7.
3. Soares de Araújo JS, Regis CT, Gomes RGS, Tavares T R, Rocha dos Santos C,
Assunção PM, et al. Microcephaly in northeast Brazil: a review of 16 208 births
between 2012 and 2015 [Submitted]. Bull World Health Organ E-pub: 4 Feb 2016. doi: http://dx.doi.org/10.2471/BLT.16.17063
4. Victora CG, Schuler-Faccini L, Matijasevich A, Erlane Ribeiro A, Pessoa A,
Fernando Celso Barros FC. Microcephaly in Brazil: how to interpret reported numbers? The Lancet, Published Online February 5, 2016 http://dx.doi.org/10.1016/
Ebola, MERS, and Hepatitis C viruses dominated virology news during the past year (2015). Now, early in 2016, Zika virus has taken center stage. The reasons are clear. This once seemingly innocuous virus, initially restricted to Equatorial Africa, has of late spread to the Western Hemisphere, and is now suspected (but not proven) to cause microcephaly—an otherwise rare condition in which babies have unusually small heads and incomplete brain development—in transplacentally infected fetuses of infected pregnant woman. Moreover there is evidence which links Zika virus to Guillain–Barré syndrome—a potentially severe autoimmune attack on peripheral nerves that may occur after signs of a viral infection. We begin with some background.
Zika virus is a member of the flavivirus family of plus-strand RNA viruses. The family also includes several notable human pathogens, including yellow fever, dengue, hepatitis C, and West Nile viruses. Like most other flaviviruses, Zika virus too is spread by an arthropod vector; in this instance Aedes mosquitoes. 80% of Zika virus infections are asymptomatic and, prior to recent developments, symptomatic infections were seen as mild, acute febrile illnesses, similar to dengue.
Zika virus was discovered by accident in the Zika Forest of Uganda in 1947. The discovery was made by scientists who had been studying yellow fever. They isolated Zika virus from one of their rhesus macaques, which was suffering from an unknown fever. The following year the same virus was found in Aedes mosquitos from the same Ugandan forest, thus identifying the mosquito as a vector for Zika virus. Zika virus was detected for the first time in humans in 1954, in Nigeria.
Until recently, Zika virus infections were rare and were reported only within equatorial Africa and Southeast Asia. Then, in 2007, an outbreak occurred in Yap Island, Micronesia. The Yap Island Zika outbreak was the first one outside of Africa and Asia. None of the Yap Island cases, which included 49 in which Zinka virus was confirmed by the presence of Zinka RNA, resulted in either hospitalization or death.
The Yap Island outbreak was followed by epidemics in Polynesia, Easter Island, the Cook Islands and New Caledonia. The Polynesian outbreak was notable for being the first in which Zika infection was associated with Guillain–Barré syndrome.
Concern over Zika virus was heightened, particularly in the Americas, when, in April 2015, a large and still ongoing outbreak of Zika virus occurred in Brazil. The Brazilian outbreak marked the first appearance of Zika virus in the Western Hemisphere. It is not clear how Zika virus made its way to Brazil, but it is widely believed that the virus made the leap from Polynesia to Brazil during the 2014 World Cup soccer tournament.
Apprehension over Zika virus increased in November 2015 when the virus was isolated from a Brazilian newborn with microcephaly. By December 2015 many more cases of this generally rare disorder were reported. The European Center for Disease Prevention and Control then warned of a possible association between Zika virus infection and congenital microcephaly, and with Guillain–Barré syndrome as well.
More than a million Brazilian people since been infected with Zika virus, and the number of Brazilian children born with microcephaly jumped from 147 in 2014 to nearly 4,000 in 2015. There is no anti-Zika vaccine, nor is there an effective therapy.
The first Zika virus-associated case of microcephaly in the United States occurred in early January 2016 in a baby born in Oahu, Hawaii. The baby and its mother each tested positive for a past Zinka infection; probably acquired in May 2015 when the mother, then pregnant, had been traveling in Brazil.
On January 24, 2016 the World Health Organization warned that Zika virus will likely spread to every nation in the Western Hemisphere (possibly excepting Canada and Chile), since its Aedes aegypti vector can thrive in tropical and sub-tropical climates here. The Aedes mosquito has long been present in the United States, ranging as far north as New York and west into Indiana and Illinois. [An earlier posting reported that Aedes aegypti may have been brought from Africa to the New World by slave ships in 1596 (1). Mosquito larvae, present in the water casks of the sailing ships of the day, also carried yellow fever to the New World.]
Global concern over the Brazilian Zika outbreak was heightened by the fact that Brazil is scheduled to host the Olympic Games this summer, and about 500,000 people are expected to attend from all over the world, including 200,000 Americans. Some of these attendees will, of course, be bringing the virus back to their home countries.
Brazilian officials no doubt are concerned that their Zika outbreak will affect attendance at the upcoming Olympic Games. Consequently, commercial considerations may be one of the motives behind Brazil’s extensive campaign to eradicate its mosquitoes. Unfortunately, standard approaches, such as using insecticides and removing standing water where mosquitoes breed, have not done the job. Thus, the Brazilian Zika outbreak may not be under control by the start of the Olympic Games. [Brazil also experienced more than 1.6 million cases of dengue during 2015, with 863 people dying from the disease, underscoring that the Aedes mosquito vector is not well contained in that country.]
The failure of Brazilian vector-control approaches suggests that new strategies may be needed to contain the outbreak. Apropos that, this past January Colombia began releasing mosquitoes treated with bacteria, which are hoped might limit the mosquitoes’ capacity to spread disease. Note that insecticides have limited effectiveness. Not only are they toxic to humans, but after decades of overexposure to them, many mosquitoes are now resistant.
Zika virus is now present in the continental United States. Thus, it is timely to consider how grave a threat Zika virus might impose here. To that point, consider that yellow fever, dengue and chikungunya viruses are dangerous pathogens that also are spread by Aedes mosquitoes. Yet these viruses are not regarded as important threats in the United States. That is so because our vector control measures have thus far been able to contain them. Those measures might likewise be expected to contain local transmission of Zika virus here.
But, what if Zika virus has a mode of transmission other than via its mosquito vector? To that point, there is a single reported case of Zika transmission via a blood transfusion. Also, it was suggested that Zika virus might have a sexual route of transmission, as per the finding of high levels of the virus in the semen of a man from French Polynesia. In addition, there is a report of an American scientist, Brian D. Foy, who contracted Zika virus while working in Senegal in 2008, and who transmitted the virus to his wife after returning home (2). Serologic analyses of the couple’s convalescent serum confirmed that they had been infected with Zika. Sexual transmission is implicated in this instance since neither Foy nor his wife passed the infection to their children or to other close relatives. Moreover, Foy and his wife observed signs of hematospermia (red–brown fluid in his ejaculate).
Foy notes in his scientific report (2), “If sexual transmission could be verified in subsequent studies, this would have major implications toward the epidemiology of Zika virus and possibly other arthropod-borne flaviviruses.” [Human sexual transmission of an arthropod-borne virus has not yet been documented.] Foy has been trying to get funds to investigate sexual transmission of Zika. However, according to a January 26, 2016 article in the N.Y. Times, the CDC says that the “theoretical risk” of sexual transmission in the above instances is insufficient to justify a warning (and funding?). But, see the following paragraph.
As I’m sitting at my computer on the evening of February 2, 2016, NPR, CNN, BBC News, the N.Y. Times, etc., are reporting a case of Zika virus infection in Texas that appears to have been sexually transmitted. According to the Dallas County Health and Human Services Department, a patient with the Zika virus was infected after having sex with someone who returned from Venezuela, where Zika is circulating. The CDC appears to give credence to the Texas report, since it quickly responded to it by advising men having sex after traveling to these areas to “consider” wearing condoms, and advised pregnant women to avoid “contact with semen” from men recently exposed to the virus.
Sexual transmission will probably account for only a very small fraction of Zika cases, but that isn’t known for certain. As in instances of mosquito-borne transmission, its contribution will depend in part on how long the virus might persist in infected individuals.
Since the vast majority of Zika virus infections are likely transmitted via its mosquito vector, and since Zika virus mainly threatens fetuses infected in utero, the most severe consequences of Zika virus infection can be largely avoided if pregnant women, or women planning to become pregnant, avoid traveling to places where Zika virus remains prevalent (a fact which doesn’t help individuals living in those regions). For that reason, on January 15, 2016, the United States Centers for Disease Control and Prevention (CDC) released a list of countries—Brazil, Colombia, El Salvador, French Guiana, Guatemala, Haiti, Honduras, Martinique, Mexico, Panama, Paraguay, Suriname, Venezuela, and Puerto Rico—where mosquitoes are spreading the Zika virus, and which pregnant women should avoid at this time. On February 1, 2016, the World Health Organization added Costa Rica and Jamaica.
Political and commercial considerations may have been behind the Brazilian minister of tourism taking exception to the CDC’s warning, claiming that measures adapted by Brazilian health authorities are bringing the Zika outbreak under control, and that Brazil is, in fact, a safe destination for pregnant women. The Brazilian health minister added, “Zika virus doesn’t worry us…,” calling it a “benign disease.” Those pronouncements were made despite the fact that Brazilian health authorities were at the same time investigating more than 3,500 cases of microcephaly. But at least some Brazilian health professionals did endorse the CDC announcement.
On February 1, 2016 the World Health Organization took the further step of declaring that Zika virus and its suspected link to birth defects constitute an international public health emergency. Yet the WHO stopped short of advising pregnant women not to travel to affected regions. Some public health experts claimed that the WHO’s silence on that point was more about politics than public health. Any travel ban—even one aimed only at pregnant women—would be embarrassing and costly to Brazil, which is moving ahead with its plans to host the Olympic Games this summer. And, while there have been calls to cancel, postpone, or move the Rio games, the International Olympic Committee (IOC) hasn’t expressed any concerns over the Games taking place as planned.
Meanwhile, the governments of Columbia, El Salvador, Ecuador, and Jamaica have taken the rather extraordinary step of recommending that women avoid getting pregnant until the Zika outbreak might be brought under control in their countries. This advisory was not well received by many El Salvadoran women, especially in view of the strict abortion laws and high levels of sexual violence against women in that country.
And, as I’m putting the final touches on this piece, an article in today’s (February 4, 2016) N.Y. Times reports that the Zika virus/microcephaly link is causing a fierce debate in Brazil over its strict abortion laws; under which abortion is illegal under most circumstances. [Remarkably, Brazil’s strict abortion laws are actually less restrictive than those in other Latin American countries.] Some Brazilian doctors are already seeing pregnant women who are seeking abortions because they fear microcephaly. Yet conservative Brazilian lawmakers actually want to make the restrictions against abortion more stringent than they already are. [The Times article says that their position reflects “the influence of Roman Catholic leaders and the increasingly powerful preachers at the helm of a growing evangelical Christian movement.”] Regardless, individuals on both sides of the debate might be troubled by the fact that microcephaly can not be detected by ultrasound scans until the end of the second trimester, when the “child” is already very much formed. Moreover, the criteria for diagnosing microcephaly are rather non-specific, and it is difficult to predict what its consequences might be.
A crucially important question regarding Zika virus concerns determining its true pathologic potential, particularly its role in microcephaly—a role that is strongly inferred (but not proven) by the geographic and temporal relationship between microcephaly and Zika infection. To that point, no increase in microcephaly has been linked to Zika virus outside of Brazil. For instance, Colombia is the second-most Zika-affected country, with around 20,000 confirmed cases. More than 2,000 of the Columbian cases have been pregnant women. Yet none of their fetuses have been diagnosed with microcephaly.
Did Zika virus become an etiologic agent for microcephaly only after reaching Brazil? If so, how did that happen? Was it because of the emergence of a new strain of the virus? Or, does Zika virus cause microcephaly only if the mother has had a previous infection, like dengue? Alternatively, was the link simply missed in the past because, until now, the virus has not invaded a country where there are a large enough number of non-immune individuals, who also are living under conditions that are ideal for the virus to spread? Or, were previous cases merely under-reported, such that the 147 Brazilian cases in 2014 were a vast underestimate?
The flip side is that the current extraordinarily high number of reported cases of microcephaly in Brazil might merely be due to a heightened awareness of that condition; a possibility that is favored by some Brazilian officials. A supporting argument is that the criteria for diagnosing microcephaly are relatively unspecific. However, others point out that physicians were reporting a rise in cases as early as November 2015, before the increased attention from health authorities and the media.
Another unexplained yet key factor is the unusually severe congenital deformities—extensive loss of brain tissue, unusually smooth, wrinkleless brains, many calcium deposits, and smaller cerebellums—seen in the Brazilian microcephaly cases. These features are not characteristic of microcephaly caused by other pathogens, such as toxoplasmosis, cytomegalovirus, or rubella.
And, presuming that Zika virus indeed causes microcephaly, how or why is it able to cross the human placenta and enter the fetal brain? [In December 2015, the Pan American Health Organization reported that Zika virus RNA was identified by reverse transcription-polymerase chain reaction (RT-PCR) in amniotic fluid samples from two pregnant women whose fetuses were found to have microcephaly by prenatal ultrasound. Moreover, Zika virus RNA was identified in multiple fetal body tissues, including the brain of an infant with microcephaly (3).] Remarkably, only a handful of viruses cross the human placenta and infect the fetus with any notable frequency (4). These include rubella virus, cytomegalovirus, and HIV; none of which is related to Zika virus. Yellow fever, dengue, and West Nile viruses, which are related to Zika virus, are not known to harm embryos.
Since most Zika virus infections are either asymptomatic, or present with flu-like symptoms that mimic other infections, a rapid diagnostic test for Zika infection is needed to accurately measure the prevalence of the virus in a population, and to measure its spread. Such a test might also help sort out whether the Brazilian microcephaly cases indeed have been due to Zika, rather than to another virus, such as the related dengue virus. Efforts are currently underway to develop Zika-specific immunological reagents for these purposes.
Vaccine researchers say that a vaccine against Zika virus may be available for testing by the end of 2016. But, even if the vaccine were effective, how long might it take for it to gain approval?
Meanwhile, an increasing, but still small number of Zika virus infections are being detected in the continental United States. With the exception of the Texas case noted above, all cases have thus far involved travelers who recently returned from overseas. Thus, with the exception of the Texas case, there is no evidence yet for local transmission here. But that well might change as summer approaches.
So, Zika now joins Lyme, West Nile, Chagas, dengue, and chikungunya on the list of recently emergent arthropod-borne diseases. Still, as we’ve noted, it is not yet clear how much of a threat Zika virus actually poses. Regardless, until that is known, it will be necessary to prepare for the worst. Even if the threat of Zika has been vastly overblown, progress towards its containment will pay important dividends in the containment of established threats, such as dengue and chikungunya.
And, if Zika is indeed a dangerous pathogen that is responsible for severe birth defects, then current conditions—global warming, more people traveling worldwide on jet airliners, cities in tropical countries becoming larger and ever more crowded—don’t portend well for the future. Stand by for new developments.
1. The Struggle Against Yellow Fever: Featuring Walter Reed and Max Theiler, Posted on the blog May 13, 2014.
2. Foy, B.D., K. C. Kobylinski, J.L. Foy, et al., 2011. Probable Non–Vector-borne Transmission of Zika Virus, Colorado, USA, Emerg Infect Dis.17: 880–882.
3. Pan American Health Organization. Neurological syndrome, congenital malformations, and Zika virus infection. Implications for public health in the Americas—epidemiological alert. Washington DC: World Health Organization, Pan American Health Organization; 2015. This paper is in Spanish.
Harold Varmus and J. Michael Bishop changed cancer research in a fundamental way in the 1970s, when they discovered proto-oncogenes at the University of California at San Francisco (UCSF). Proto-oncogenes are cellular genes that normally play an important role in controlling cell division and differentiation. However, Varmus and Bishop found that proto-oncogenes can be altered by mutation, to become oncogenes that contribute to cancer. When Varmus and Bishop first began their collaboration in 1970, cancer research was, for the most part, focused on epidemiology (e.g., studies linking smoking to lung cancer) and empirical approaches to therapy (e.g., radiation and chemotherapy).
The discovery of proto-oncogenes is a pertinent topic for our Virology blog because it depended crucially on Varmus and Bishop’s earlier finding that retroviral oncogenes are mutated versions of cellular genes that retroviruses “captured” from their host cells. Varmus and Bishop hypothesized and then demonstrated that since retroviral oncogenes are versions of genes that actually are part of a normal cell’s genetic makeup, mutations in those genes, or their inappropriate expression, can lead to cancer. The v-src gene of Rous sarcoma virus was the first retroviral oncogene that Varmus and Bishop showed is derived from a cellular genome (1).
Varmus and Bishop continued searching for proto-oncogenes in the 1980s. Varmus also began investigating HIV (also a retrovirus and the cause of AIDS). In 1989 Varmus and Bishop were awarded the Nobel Prize in Physiology or Medicine for their discovery of proto-oncogenes.
In the early 1990s Varmus stepped out from his role as a research scientist to take up the cause of public funding for biomedical research. In 1993 President Bill Clinton acknowledged Varmus’ efforts in that regard, as well as his stature as a scientist, by appointing him to serve as Director of the National Institutes of Health (NIH). Thus, Varmus became the first Nobel laureate to head the NIH.
In 2000 Varmus left the NIH to accept the presidency of the Memorial Sloan-Kettering Cancer Center in New York. In 2010 Varmus returned to the NIH, this time appointed by President Barak Obama to serve as director of the National Cancer Institute (NCI). In 2015 Varmus was back again in New York where he is the Lewis Thomas University Professor of Medicine at Weill-Cornell Medical College.
Varmus was featured in two earlier blog postings. The first of these described how he mediated the dispute between Robert Gallo and Luc Montagnier over the right to name the AIDS virus (2). The second posting covered some of the political and social dilemmas Varmus faced during his days leading the NIH (3).
Here, we relate first how Varmus opted for a career in biomedical science and, second, how his collaboration with Bishop came about. This is an interesting tale because Varmus’ remarkable career as a science researcher, administrator, and spokesperson happened despite his initial intention to become a teacher of English literature. Indeed, his career in science did not begin until after he earned an M.A. degree in English from Harvard University, and then spent four years in medical school preparing for a career in clinical medicine.
We begin our story in 1950 as Varmus recounts how, as a ten-year-old, he witnessed his physician father receive a call that conveyed shocking news: “one of my mother’s favorite cousins, a robust man in the middle of his life, had just been diagnosed with leukemia. Of course, I did not know very much about leukemia, but I did know immediately from my parents’ expressions–and within a few weeks, from our cousin’s death—that his disease was a veritable tidal wave.” [All quotations are from Varmus’ book The Art and Politics of Science (4), in which he reflects back on his entire career.]
His cousin’s leukemia actually resulted from a mutation in one of the genes that Varmus would discover more than two decades later. And Varmus notes just how far the science in general had progressed during that 25-year interim: “…when my father heard about our cousin’s leukemia, biologists were not even sure that genes were made of DNA, had no idea how genetic information could be encoded in genes, and, of course, had no way of knowing that cancers are driven by mutations.”
Varmus was urged by his father to prepare for a career in medicine. Nonetheless, when Varmus enrolled as a freshman at Amherst College he strongly favored studying the humanities. Thus, he “toyed with the idea of majoring in philosophy (ultimately too abstract), physics (ultimately too hard), and English literature (ultimately selected).”
Throughout his undergraduate days, Varmus envisioned preparing for an academic career teaching literature. Still, he dutifully fulfilled premed requirements to keep open the possibility of obliging his father’s wishes that he become a medical doctor. Yet he never considered majoring in biology. “I couldn’t understand how some of my close friends (among them, some now distinguished scientists) could spend long afternoons and evenings incarcerated in a laboratory, when they could be reading books in a soft library chair or reciting poetry on Amherst’s green hills.”
Varmus began having doubts about his career choice when his Amherst College classmate Art Landy (later a well-known molecular biologist at Brown University) won an Amherst biology prize that allowed him to attend a 1961 international biochemistry meeting in Moscow. Importantly, Landy invited Varmus to accompany him to the Moscow meeting, where Varmus learned that Marshal Nirenberg had deciphered the genetic code. “Even though I did not understand its meaning or its importance at the time, I was not oblivious to the excitement around me…Scientists seemed likely to discover new, deep, and useful things about the world, and other scientists would be excited by these discoveries and eager to build on them. Would this be true of literary critics and teachers?”
Notwithstanding these misgivings, Varmus continued on his path to a career in English literature after graduating from Amherst College in 1961, earning an M.A. in English from Harvard in 1962 (his focus was on Anglo-Saxon poetry). But his uncertainties about his future only grew stronger. “Despite outward signs that I had chosen a life of studying and teaching literature, soon after starting my graduate work at Harvard I began to suffer some further internal doubts about abandoning medicine. The graduate curriculum in English literature was not especially onerous, but it felt like a prolongation of college. Most of my courses were heavily populated with Harvard and Radcliff undergraduates.” Varmus leaves the impression that he looked upon much of his course work at Harvard as a tiresome chore.
Varmus was also aware of the enthusiasm of former Amherst College classmates who were then studying at Harvard Medical School. “Occasionally, on Saturday mornings, I traveled across the Charles River to join some Amherst classmates at Harvard Medical School, while they sat in the Ether Dome at Massachusetts General Hospital, entranced by diagnostic dilemmas discussed at the weekly pathology conference. These stories struck me as far more interesting than those I was reading, and my medical school friends expressed general excitement about their work. They also seemed to have formed a community of scholars, with shared interests in the human body and its diseases and common expectations that they would soon be able to do something about those diseases…These Saturday excursions probably account for an influential dream I had one night about my continuing indecision. In that dream, my future literature students were relieved when I didn’t turn up to teach a class, but my future patients were disappointed when I didn’t appear. It seemed I wanted to be wanted.”
So, Varmus finally came to grips with his qualms about a career teaching English literature, hastily preparing an application to Harvard Medical School and biking across the frozen Charles River to deliver it just in time to meet the deadline. But it was to no avail, since the dean of admissions thought Varmus was “too inconstant and immature” for medical school.
Varmus next sent off an application to Columbia University’s College of Physicians and Surgeons (P&S). His interviewer at Columbia was an esteemed physician named David Seegal, who also happened to be rather literate. Seegal asked Varmus if he might translate the Anglo Saxon phrase Ich ne wat. “This was easy; it simply means ‘I don’t know.’” Seegal used his question as a lead-in to discuss why a physician might admit fallibility to a patient. In any case: “By the fall of 1962, I was happily enrolled at P&S, helped for the first, but not the last, time by someone’s exaggerated appreciation of my competence in two cultures.”
Now ensconced at P&S, Varmus thought he might become a psychiatrist; an ambition stoked by an interest in Freud and by his winning of an essay prize at P&S in psychiatry. But, he found his “first hour alone in a room with a psychotic patient to be more difficult and less interesting than an hour reading Freud.” So, Varmus’ interests in medical school turned from the “elusive mind” to the physical brain and then, more generally, to diseases that might be explained by known physiology and biochemistry.
When graduation from medical school was impending, Varmus had to consider his career options more deliberately than he had in the past. A key factor was the Vietnam War, which was in progress, and which he and many others of that era vehemently opposed. “I was determined not to serve in it. Medical graduates were subject to the draft; however, we did have the more palatable option of two years training at one of the agencies of the Public Health Service. For most of my classmates with academic ambitions similar to my own, the NIH was the favored choice. As the largest biomedical research campus in the world, it offered unequaled opportunities to learn virtually any form of biomedical research…”
Varmus confesses that he had a “woeful lack of laboratory credentials.” Nonetheless, he entered the competition for one of the coveted research slots at the NIH. But, because of his lack of research experience, he was not encouraged by most of the NIH laboratory chiefs who interviewed him. However, one of them, endocrinologist Jack Robbins, suggested to Varmus that he speak to Ira Pastan; a young endocrinologist who, at that time, was interested in the production of thyroid hormones.
As Varmus relates, “The recommendation proved to be wise and fateful. My schooling in literature turned out to be more important than my interest in endocrinology, Ira’s field, because Ira’s wife Linda, a poet, had often complained that Ira’s colleagues seldom talked about books…When matches were announced I was told I would become Ira’s first clinical associate, having been passed over by the more senior investigators. This outcome could not have been more fortunate.”
But, before Varmus could take up his position at the NIH, he received a “shocking phone call” from Pastan, to the effect that he (Pastan) was giving up his work on thyroid hormones because he and colleague Bob Perlman “had made a shocking discovery about gene regulation in bacteria.” Pastan and Perlman found that cyclic AMP is a major regulator of bacterial gene activity, and that it plays a similar role in animal cells—findings which led Pastan to pioneer the field of receptor biology in animal cells.
The discovery by Pastan and Perlman had important consequences for Varmus. First, it immediately forced him to give up his plan to train in endocrinology. Instead, Pastan assigned Varmus to find out whether cyclic AMP augments bacterial gene expression by increasing transcription of mRNA. Second, as explained below, Pastan’s new research direction led to Varmus’ introduction to and fascination with virology.
So, Varmus was now a budding molecular biologist. But, since he had no prior research experience, his first days in the Pastan lab were a near disaster, leading Pastan to half jokingly ask, “Now remind me why I took you into the lab.”
In any case, Varmus worked closely with Pastan to develop a molecular hybridization assay to measure transcription of E. coli lac mRNA. [Their specific the goal was determine whether the mechanism by which cyclic AMP reverses catabolite repression of the E. coli lac operon is by enhancing transcription of lac mRNA.] And, they used an E. coli phage, which had incorporated the lac operon into its genome, as their source of isolated lac operon DNA. Thus, Varmus was introduced to virology. [Aficionados, note, “These experiments with the lac operon proved to be analogous in several ways to experiments that revealed the first proto-oncogene a few years later.”]
The satisfaction that Varmus derived from his research in Pastan’s lab caused him to reconsider his aspirations for a career in clinical medicine, and instead to think about a future in biomedical research. He thought he might next try his hand at cancer research, motivated in part, by his mother’s breast cancer, first diagnosed in 1968, to which she succumbed two years later. But there were other factors at work as well. In particular, Varmus’ use of the E. coli phage in Pastan’s lab led to his fascination with virology. And his interest in virology was relevant to his new plans because the DNA and RNA tumor viruses held immense potential for cancer research. 1970s technology could not identify which one of the tens of thousands of cellular genes might have mutated to result in a cancer. However, that technology was potentially able to identify which of the handful of a tumor virus’ genes might underlie its ability to transform a normal cell into a tumor cell.
That line of thought led Varmus to apply for a research position in Renato Dulbecco’s lab at the Salk Institute. [Dulbecco would win a share of the 1975 Nobel Prize in Physiology or Medicine for his pioneering studies of the DNA tumor viruses (5).] However, reminiscent of Varmus’ unsuccessful application to Harvard Medical School, he was “rebuffed by not one but two letters from his (Dulbecco’s) secretary.”
While the rejection from Dulbecco was a disappointment, it would be another of the seemingly providential happenings in Varmus’ career. In the summer of 1969 he chanced to visit Harry Rubin, an eminent Rous sarcoma virus researcher at U Cal Berkeley. Rubin, who had earlier introduced Howard Temin to virology (another auspicious happening; see reference 6), told Varmus about a new group at UCSF that had begun to study retroviruses. Importantly, the goal of the UCSF group was to discover cancer-causing genes. Thus, Varmus stopped over at UCSF, where he met members of the group, including a smart young virologist named Mike Bishop. Varmus reports, “we recognized from the first moments that we were destined to work together.”
Varmus came to Bishop’s lab in 1970 as a postdoctoral fellow. However, their relationship quickly evolved to one of equals, and they made all of their major discoveries in the 1970s and 1980s as a team, and they rose together through the UCSF academic ranks. Bishop relates that their bond formed not just by a shared fascination with cancer viruses but “by our mutual love of words and language.” Varmus, for his part, notes that “after many years of ambivalence and indecision…I appeared to be headed in a clear direction, even if not towards medicine or literature.”
1. Stehelin D, Varmus HE, Bishop JM, Vogt PK., 1976. DNA related to the transforming gene(s) of avian sarcoma viruses is present in normal avian DNA. Nature 260:170-173.
2. How the Human Immunodeficiency Deficiency Virus (HIV) Got Its Name, posted on the blog February 4, 2014.
3. The Politics of Science: Vignettes Featuring Nobel Laureate Harold Varmus during his Tenure as Director of the NIH, posted on the blog June 2, 2014.
4. Varmus, H. 2009. The Art and Politics of Science, (W. W. Norton & Company).
5. Renato Dulbecco and the Beginnings of Quantitative Animal Virology, posted on the blog December 3, 2013.
6. Howard Temin: “In from the Cold”, posted on the blog December 14, 2013.