Tag Archives: Aedes aegypti

Mikhail Balayan and the Bizarre Discovery of Hepatitis E Virus

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.

Hepatitis E Virus
Hepatitis E Virus

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.”

References:

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 Virology 1:1-9.

 

 

 

 

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Zika Virus: Background, Politics, and Prospects

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.

The Aedes aegypti mosquito, the Zika virus vector
The Aedes aegypti mosquito, the Zika virus vector

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.

References:

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.

4. Norkin, L.C., Virology: Molecular Biology and Pathogenesis, ASM Press, 2010.

The Struggle Against Yellow Fever: Featuring Walter Reed and Max Theiler

The first part of this posting tells how a U.S. Army medical board, headed by Walter Reed, confirmed that the transmission of yellow fever requires a mosquito vector. The second part tells the story of the yellow fever vaccine developed by Max Theiler.

Bearing in mind the enormous benefit to mankind of the polio vaccines developed by Jonas Salk and Albert Sabin (1), and that Maurice Hilleman developed nearly 40 vaccines, including those for measles, mumps, and rubella (2), it would appear remarkable that Theiler was the only one of these four individuals to be recognized by the Nobel committee. In fact, Theiler’s 1951 Nobel award was the only one ever given for a vaccine! In any case, while Theiler’s vaccine was a major step forward in the fight against yellow fever, it came after a perhaps more dramatic episode in the struggle against that malady. But first, we begin with some background.

Yellow fever was another of mankind’s great scourges. Indeed, it was once the most feared infectious disease in the United States. And, while we might want to say that science has “conquered” yellow fever, that statement would not be entirely accurate. Unlike polio and measles, which have nearly been eradicated by the vaccines against them, that is not so for yellow fever. The reason is as follows. Humans are the only host for polio and measles viruses. Consequently, those viruses might be completely eradicated if a sufficient percentage of humans were to comply with vaccination regimens. In contrast, the yellow fever virus infects monkeys that range over thousands of square miles in Africa and the Amazon jungle. Thus, even with massive vaccination of humans, it would be impossible to eliminate the yellow fever virus from the world.

According to the World Health Organization’s estimates, there are still about 200,000 cases of yellow fever per year, resulting in about 30,000 deaths, about 90% of which occur in Africa. The yellow fever virus itself is the prototype virus of the flavivirus family of single-stranded RNA viruses, which also includes dengue hemorrhagic fever virus, Japanese encephalitis virus, and West Nile encephalitis virus, among others.

yellow fever map

Yellow fever is somewhat unique among the viral hemorrhagic fevers in that the liver is the major target organ. Consequently, the severe form of yellow fever infection is characterized by hemorrhage of the liver and severe jaundice. But, as in infections caused by other virulent viruses, most cases of yellow fever are mild.

Interestingly, the name “yellow fever” does not have its origin in the yellowing of the skin and eyes that is characteristic of severe disease. Instead, it has its origin in the term “yellow jack,” which refers to the yellow flag that was flown in port to warn approaching ships of the presence of infectious disease.

Yellow fever originated in Africa. It is believed to have been brought to the New World by slave ships in the year 1596. As noted above (and discussed below), yellow fever transmission, from an infected individual or primate to an uninfected one, requires a specific vector, the Aedes aegypti mosquito. The sailing ships of the day inadvertently transported the disease across oceans via the mosquito larvae in their water casks.

Before getting to our stories proper, we note a pair of intriguing instances in which yellow fever profoundly affected New World history. In the first of these, yellow fever was a key factor that led Napoleon to sell the Louisiana Territory to the United States in 1803; an act that doubled the size of the United States. It happened as follows. After Napoleon seized power in France, he reinstated slavery in the French colony of Saint Domingue (now Haiti); doing so for the benefit of the French plantation owners there. In response, the rather remarkable Toussaint Breda (later called Toussaint L’Ouverture, and sometimes the “black Napoleon”) led a slave revolt against the plantation owners. In turn, in February 1802, Napoleon dispatched an expeditionary force of about 65,000 men to Haiti to put down the revolt. The rebellious slaves, many fewer in number than the French, cleverly retreated to the hills, believing that the upcoming yellow fever season would wreak havoc on the French force. And, they were correct. By November 1803, the French lost 50,000 of the original 65,000 men to yellow fever and malaria. Thus, in 1804, Napoleon had to allow Haiti to proclaim its independence, and then become the second republic in the Western Hemisphere. Moreover, there is evidence suggesting that Napoleon’s actual purpose in dispatching the expeditionary force was to secure control of France’s North American holdings. With his expeditionary force decimated by yellow fever and malaria, that was no longer possible and, consequently, Napoleon sold France’s North American holdings (the Louisiana Purchase) to the United States.

louisiana purchaseThe Louisiana Purchase, in green.

Second, in 1882, France began its attempt to build a canal across the Isthmus of Panama. However, thousands of French workers succumbed to yellow fever, causing France to abandon the project. The United States was able to successfully take up the task in 1904; thanks to the deeds of the individuals in part I of our story, which now begins.

In May 1900, neither the cause of yellow fever, nor its mode of transmission was known. At that time, U.S. Army surgeon, Major Walter Reed, was appointed president of a U.S. Army medical board assigned to study infectious diseases in Cuba, with particular emphasis on yellow fever. Cuba was then thought to be a major source of yellow fever epidemics in the United States; a belief that was said to have been a factor in the American annexation of Cuba.

ReedMajor Walter Reed

When Reed’s board began its inquiry, a prevailing hypothesis was that yellow fever might be caused by the bacterium Bacillus icteroides. However the board was unable to find any evidence in support of that notion.

Another hypothesis, which was advanced by Cuban physician Dr. Carlos Juan Finlay, suggested that whatever the infectious yellow fever agent might be, transmission to humans requires a vector; specifically, the mosquito now known as Aedes aegypti. Reed was sympathetic to this idea because he noticed that people who ministered to yellow fever patients had no increased risk of contracting the disease, which indicated to Reed that people did not pass yellow fever directly from one to another.

Reed, as president of the medical board, is generally given major credit for unraveling the epidemiology of yellow fever. Yet there were other heroes in this story as well. Finlay, whose advice and experience were invaluable to Reed’s board, was one. He was the object of much ridicule for championing the mosquito hypothesis, at a time when there little evidence that might support it. In any case, Reed, in his journal articles and personal correspondences, gave full credit to Finlay for the mosquito hypothesis.

Acting Assistant Surgeon Major James Carroll was another hero. As a member of Reed’s board, Carroll volunteered to be bitten and, promptly, developed yellow fever. Major Jesse Lazear, also a board member, asked Private William Dean if he might be willing to be bitten. Dean consented, and he too contracted yellow fever. Fortunately, Dean and Carroll each recovered. Not so for Lazear. After allowing himself to be bitten, he died after several days of delirium.

Lazear’s contribution to gaining recognition of the mosquito hypothesis went significantly beyond his tragic martyrdom. When Reed examined Lazear’s notebook after his death, Reed found that it contained several key observations. First, Lazear had carefully documented that in order for a mosquito to be infected; it had to bite a yellow fever patient within the first three days of the patient’s illness. Second, twelve days then had to elapse before the virus could reach high enough levels in the insect’s salivary glands to be transmitted to a new victim.

The observations of the board, up to then, convinced Reed and the others that the mosquito hypothesis indeed was correct. Yet Reed knew that more extensive controlled experiments would be needed to convince the medical community. So, he directly supervised those experiments, which involved twenty-four more volunteers, each of whom may rightly be considered a hero.

Just as Reed benefited from Finlay’s insights, William C. Gorgas, Surgeon General of the U.S. Army, applied the findings of Reed’s board to develop vector control measures to combat urban yellow fever; first in Florida, then in Havana, Cuba, and next in Panama, where those measures enabled the United States to complete the canal in 1914. The last urban yellow fever outbreak in the United States occurred in New Orleans in 1905, and the last in the New World occurred in 1999 in Bolivia.

The vector control strategy works for urban yellow fever because the Aedes aegypti mosquitoes have a very short flight range and, consequently, the female mosquito does not stray far from the source of her blood meal before laying her eggs. Thus, it is only necessary to control the vector population in the immediate vicinity of human habitation. In practice, this is accomplished by draining potential mosquito breeding sites such as swamps and ditches, and destroying water-collecting objects such as discarded tires.

After Reed’s board was disbanded, he made yet another key contribution to the wiping out of yellow fever. The focus of the board had been on the means of yellow fever transmission; not with the infectious agent itself. In 1901, at the suggestion of William Welch, an eminent Johns Hopkins pathologist, Reed and James Carroll (who nearly died of yellow fever after being experimentally infected while in Cuba), asked whether yellow fever might be caused by a filterable virus. Indeed, they found that they could infect volunteers by inoculating them with filtered serum taken from yellow fever patients. What’s more, theirs was the very first demonstration of a human illness being caused by a filterable agent. That is, yellow fever was the first human illness shown to be caused by a virus. [Pasteur developed an attenuated rabies vaccine in 1885, more than a decade before the discovery of viruses. Remarkably, this most brilliant of experimentalists did not recognize that he was dealing with a previously unknown, fundamentally distinct type of infectious agent; the topic of a future posting.]

[Aside: Walter Reed spent the early years of his Army career at different posts in the American west. The Mount Vernon Barracks in Alabama, which served as a prison for captured Apache Native Americans, including Geronimo, was a particularly interesting stop for Reed. Captain Walter Reed, serving as post surgeon in the 1880s, looked after Geronimo and his followers.]

Part II of this posting concerns the development of Max Theiler’s yellow fever vaccine. But first, here is a bit more background.

Vector control measures ended yellow fever epidemics in most, but not all urban centers worldwide. Outbreaks have not occurred in the United States for more than a century. However, jungle yellow fever still persists in areas of Sub-Saharan Africa and, to a lesser extent, in tropical South America. Individuals who are infected in the jungle by wild mosquitoes can then carry the virus to densely populated urban areas, where Aedes aegypti mosquitoes can transmit the virus from one individual to another. [Vector-mediated, human-to-human transmission happens because the level of yellow fever virus in the blood of an infected person becomes high enough for the infected person to transmit the virus to a biting mosquito. In this regard, the yellow fever virus is an exception to the generalization that humans are a “dead end” host for arthropod-borne (arbo) viruses.]

Fortunately, people who live in high risk areas for yellow fever can be protected by vaccination. Indeed, the World Health Organization’s strategy for preventing yellow fever epidemics in high risk areas is, first, to mass immunize the population, and then to routinely immunize infants. [Vaccinated American or European visitors to West Africa or the Amazon need not be concerned about yellow fever. However, the risk to an unvaccinated person of acquiring yellow fever during a two-week stay at the height of the transmission season (July through October), is estimated to be 5%. Individuals wanting to enter or return from countries where yellow fever is endemic may need to show a valid certificate of vaccination. ]

Part II of our story, concerning Max Theiler and the development of the yellow fever vaccine now begins.

Even as late as the 1920s, some reputable bacteriologists remained unconvinced by the earlier findings of Reed and Carroll that yellow fever is caused by a filterable agent. Instead, they persisted in the belief that the illness is caused by a bacterium. The notion of a bacterial etiology for yellow fever was finally put to rest after A. H. Mahaffy in 1927 discovered that the yellow fever agent could be propagated and cause illness in Asian rhesus monkeys. With an experimental animal now at hand, yellow fever workers were able to prove conclusively that the disease is caused by a virus. [Mahaffy drew the virus he used in his experiments from a 28-year-old African man named Asibi, who was mildly sick with yellow fever. That isolate, referred to as the Asibi strain, will play an important role later in this anecdote.]

Regardless of the significance of the discovery that the yellow fever virus could be propagated in rhesus monkeys, Max Theiler had to contend with the fact that these monkeys were quite expensive; especially for a not yet established young investigator. [They cost the then princely sum of about $7.00 apiece.] As for mice, while they could be bred for pennies apiece, other researchers were not able infect them via the usual practice of inoculating them under the skin or in the abdomen. However, Theiler took a cue from Pasteur’s inability to propagate the rabies virus in laboratory rabbits until he put the virus directly into their brains. Thus, in 1929 Theiler attempted to do the same with yellow fever virus in mice.

TheilerlMax Theiler

Theiler’s attempts to infect the mice by intracranial injection were a success. All of the inoculated mice died within several days. Surprisingly, the dead mice did not display the liver or renal pathology characteristic of yellow fever. Instead, the mice appeared to have succumbed to inflammation of their brains. Thus, the yellow fever virus appeared to be neurotropic in mice. Also, Theiler himself contracted yellow fever from one of his inoculated mice. He was fortunate to survive.

A fortuitous result of Theiler’s perilous bout with yellow fever was that he had become immune to the virus, as revealed by the presence of antiviral antibodies in his blood. Importantly, Theiler’s acquired immunity to the virus validated the possibility of developing an attenuated yellow fever vaccine. And, in a sense, Theiler was inadvertently the first recipient of the nascent vaccine he soon would be developing.

Theiler also determined that the virus could be passed from one mouse to another. And, while the virus continued to cause encephalitis in mice, it caused yellow fever when inoculated back into monkeys; quite a unique and striking set of findings. But, and crucially significant, while continued passage of the virus in mice led to its increased virulence in those animals, the virus was concurrently losing its virulence in monkeys. [In 1930, Theiler moved from the Harvard University School of Tropical Medicine to the Rockefeller Foundation’s Division of Biological and Medical Research. The Rockefeller Foundation shared facilities with the Rockefeller Institute (now University); although it was otherwise administratively separate from it.]

Since the mouse-passed virus was becoming attenuated in monkeys, Theiler’s belief in the possibility of generating an attenuated yellow fever vaccine was bearing out. However, because the mouse-passed virus remained neurovirulent in mice, Theiler was reluctant to inoculate that virus into humans. In an attempt to solve this problem, Theiler turned from passing the virus in the brains of live mice and, instead, began passing the virus in mouse tissue cultures.

Theiler carried out seventeen different sets of trials to further attenuate the virus. In the 17th of these, Theiler used the wild Asibi strain, isolated earlier by Mahaffy. Initially, this virus was extremely virulent in monkeys, in which it caused severe liver damage. But, after passing the virus from culture to culture several hundred times, over a period of three years, a flask labeled 17D yielded the virus that was to become the famous 17D yellow fever vaccine.

Theiler never gave a satisfactory accounting for the “D” in the “17D” designation, and for what, if anything became of A, B, and C. Regardless, the genesis of 17D was as follows. Theiler initially took an Asibi sample that had been multiplying in mouse embryo tissue and continued passing it in three separate types of minced chicken embryo cultures. One of these sets contained whole minced chicken embryos, and was designated 17D (WC). A second set contained chick embryo brain only, and was designated 17D (CEB). In the third set, the brains and spinal cords were removed from the otherwise whole chick embryo tissue cultures. This set, alone among all the sets, generated an attenuated virus that did not induce encephalitis when injected directly into monkey brains. Indeed, Theiler removed the central nervous systems from the chicken tissue in this set of cultures, in the express hope of generating just such an attenuated virus. And, by hook or by crook, the virus emerging from that particular set of passages became the vaccine that is now known simply as 17D.

Field tests of Theiler’s yellow fever vaccine were underway in 1937 in Brazil, and were successfully completed by 1940. In 1951 Theiler was awarded the Nobel Prize in Physiology or Medicine for developing the vaccine.

Next, we return to a point noted above, and discussed in two earlier postings. Neither Jonas Salk nor Albert Sabin were awarded Nobel prizes for developing their polio vaccines (1). And, Maurice Hilleman was never awarded a Nobel Prize, despite having developed nearly 40 vaccines, including those for measles, mumps, and rubella (2). Indeed, Max Theiler’s Nobel Prize for the yellow fever vaccine was the only Nobel Prize ever awarded for a vaccine! Why was that so?

Alfred Nobel, in his will, specified that the award for Physiology or Medicine shall be for a discovery per se; not for applied research, irrespective of its benefits to humanity. With that criterion in mind, the Nobel committee may have viewed the contributions of Salk and Sabin as derivative, requiring no additional discovery. [Hilleman’s basic discoveries regarding interferon should have been sufficient to earn him the award (2). The slight to him may have been because the Nobel committee was reluctant to give the award to an “industrial” scientist. Hilleman spent the major part of his career at Merck & Co.]

So, what was there about Theiler’s yellow fever vaccine that might be considered a discovery? Hadn’t Pasteur similarly developed an attenuated Rabies vaccine in 1885?

Perhaps the “discovery” was Theiler’s finding that passage of the Asibi strain of yellow fever virus in chick embryo cultures, which were devoid of nervous system tissue, generated attenuated yellow fever virus that was no longer neurovirulent in mice and monkeys. But, consider the following.

Theiler indeed believed that removing the brains and spinal cords from the chick embryo cultures in which 17D had been serially passed was the reason why the virus lost its neurovirulence. Nevertheless, as a serious scientist he needed to confirm this for himself. So, he repeated the long series of viral passages under the same conditions as before. But, this time, there was no loss of neurovirulence. Thus, a cause and effect relationship, between the absence of the brains and spinal cords from the tissue cultures and the emergence of non-neurovirulent virus, was not confirmed.

So, perhaps the Nobel committee merely paid lip service to the directives in Alfred Nobel’s will. In any case, Theiler’s 17D yellow fever vaccine has had a virtually unblemished safety record, and is regarded as one of the safest and most effective live-attenuated viral vaccines ever developed.

Theiler’s unshared 1951 Nobel award paid him $32,000. At the time, he resided in Hastings-on-Hudson; a village in Westchester County, NY, from which he commuted to the Rockefeller labs. Theiler’s next door neighbor in Hastings-on-Hudson was Alvin Dark, the star shortstop of the New York Giants. Nobel laureate Max Theiler was known to fellow commuters from Hastings-on-Hudson as the man who lives next door to Alvin Dark.

Virus Hunters, by Greer Williams (Alfred A, Knoff, 1960) was my major source for the material on Max Theiler.

1. Jonas Salk and Albert Sabin: One of the Great Rivalries of Medical Science. On the blog.

2. Maurice Hilleman: Unsung Giant of Vaccinology. On the blog.