The Co-Evolution of Virology, Diagnostic Virology, and Clinical Medicine: A Century of Discovery

Clinical Virology Symposium, April, 2009

The Co-Evolution of Virology, Diagnostic Virology, and

Clinical Medicine: A Century of Discovery

Kenneth McIntosh, M.D.Harvard Medical School

Harvard School of Public HealthChildren’s Hospital Boston


The Evolution of Virology

• There is some question of when “Virology” can be said to have begun.• Many diseases we now know as viral were

recognized (smallpox, measles, polio), and even immunized against (smallpox), all of this before viruses were first described.

• Dmitry Ivanovsky was the first to show that the infectious agent that caused a disease, in this case tobacco mosaic disease, could be filtered through porcelain filters that would block bacteria. This was in 1892.



• I prefer to think that “Virology” began a few years before this, with a Scotsman named John Brown Buist.


The Evolution of Virology (diagnostic)

• The Beginning• John Brown Buist was a Scottish

pathologist and medical practitioner who devoted much of his professional life to vaccination.

• Published Vaccinia and Variola in 1887


• John Brown Buist’s studies:• He devised a method for staining the material from

a cowpox (vaccinia) vesicle



• Buist’s conclusions:• The particles he saw were considered to

be the spores of micrococci, evolving into the particles seen in pustules during the course of the disease

• Smallest particles were associated with most effective smallpox vaccination



• Buist’s contribution:• This was the first recorded detection of virus

directly in material obtained from a lesion in a disease caused by a virus (even though, since viruses had not been described, the particles were called spores).

• This was also the first rapid laboratory diagnosis of a viral disease.

• Buist also associated the small (viral) particles with infectivity.

• The work was all done 6 years before viruses as filterable, infectious particles, were described.



• Inclusion Bodies• Several physicians associated intracellular

inclusion bodies with the pathology of diseases that, usually later, were shown to be caused by viruses.

• An early example was in smallpox, where histologic studies were done of the base of the lesions, by Guarnieri in 1892



• Inclusion Bodies• Another early example was in rabies, from

impression smears made from rabid dogs, by Negri in 1903



• Growth of Virus• Animal inoculation

• Animal (or human) inoculation was essentially the only way to grow viruses in the early days (e.g. rabies virus in the CNS of rabbits, by Pasteur in the 1890’s; or maintenance of smallpox vaccine in orphans for transatlantic shipment in the early 19th century).

• Embryonated eggs• First successful passage of a virus in eggs was Rous

Sarcoma Virus by Rous and Murphy in 1911.



• Growth of Virus• Embryonated eggs

• The next use of embryonated eggs for virus passage was 20 years later, when smallpox and vaccinia were grown by Woodruff and Goodpasture in 1931. They also showed that the virus could be titrated by counting pox in the allantoic membrane.

• Burnet used chick embryos to grow influenza virus for the first time in 1940. This system has been used since that time for production of vaccine.



The Evolution of Virology (public health and patient care)

• Yellow fever (a flavivirus, transmitted by the bite of several species of mosquito)• Originated in Africa, brought to the Americas by

the slave trade.• Devastated the military during the Spanish-

American War (1898).• Thought to be spread by miasmas, or

contaminated fomites.• Killed many important people, including a number

of those investigating it, as well as volunteers who were inoculated with infected blood.



• Yellow fever (earliest studies)• Walter Reed

• Undertook experiments during the summer of 1900 in Cuba to prove that mosquito bites were, in fact, the mode of infection.

• At the time of these experiments, yellow fever was not known to occur in any animal species, so even animal experiments were not possible.



• Yellow fever (earliest studies)• Walter Reed

• In a facility built specifically for the purpose, Reed subjected consenting young male volunteers to bites by C. fasciatus (now known as Aedes aegyptae) mosquitoes that had, about 2 weeks previously, bitten patients early in the course of natural (often fatal) yellow fever.



• Yellow fever (earliest studies)• Walter Reed.

• The volunteers came down with clinical yellow fever 2-4 days later. Fortunately, all these volunteers survived.

• Reed was also able to show that fomites were not responsible for spreading the disease, by having volunteers sleep in grossly contaminated bed linens.

• In related experiments, Reed and his team showed that the responsible agent passed through porcelain filters and still

produced disease in volunteers, and was therefore a virus.



• Yellow fever • Walter Reed.

• Because of these scrupulously performed experiments, the incidence of Yellow Fever in the military, with proper mosquito control, dropped precipitously.

• Reed’s colleagues, Jefferson Kean and William Gorgas, instituted the control measures that allowed the digging of the Panama Canal just a few years later.



• Yellow fever (tissue culture and vaccine development)• Max Theiler

• Theiler was a South African, born in 1899 to Swiss parents, who began work at the Rockefeller Institute in New York City in 1930.

• Theiler used a combination of animal inoculation and chick embryo tissue culture to study the dual pathogenicity of yellow fever virus in the liver and the brain.




• The chick embryo tissue culture methods that Theiler used were developed first by Alexis Carrel.




• During these pathogenicity studies, and after over 200 passages in chick embryo tissue culture, Theiler gradually attenuated the Yellow Fever virus and developed the 17D vaccine that is used today.




• The attenuation methods that Theiler pioneered (passage in tissue culture) were the basis for the development of vaccines for essentially all attenuated human viral vaccines before the molecular era:

MeaslesRubellaMumps

PolioVaricella



• The studies described so far were all of severe viral diseases where infectious material from patients could be removed from sterile sites (brain, skin pustules, blood, liver).

• Influenza is an example of the difficulties inherent in trying to work with viruses in non-sterile sites before the advent of antibiotics.



• Influenza• From the huge pandemic of 1918 until the early

1930’s controversy raged about what was the cause of the “great influenza.”

• The battle came down to two camps:• Haemophilus influenzae

• A filterable virus

• There were innumerable “demonstrations” of disease, induced in human volunteers or various animal models, from both camps. None of them was convincing.



• Influenza• Order began to appear in 1931

• Shope, at the Rockefeller Institute, developed an elegant model in pigs: Swine influenza, where mild respiratory disease was produced with the filterable agent, and severe disease with a combination of the virus and H. influenzae suis.

• Then in 1933 Smith, Andrewes, and Laidlaw in England transferred a filterable agent from clinical cases to ferrets.

• This agent proved serologically close to Shope’s swine influenza.



• Influenza: Consistency• In 1934, from a separate outbreak of influenza,

Thomas Rivers at the Rockefeller Institute duplicated exactly the ferret experiments of Smith, Andrewes and Laidlaw.

• In 1940, Macfarlane Burnet in Australia isolated influenza virus in embryonated eggs using samples from an earlier local outbreak.

• Suddenly, everything became much clearer.

Smith, Andrewes, Laidlaw. Lancet 1933;2:66-68.


• Growth of Virus• Tissue culture

• Early efforts: vaccinia was grown in 1913 in explants of rabbit cornea.

• First recognition of cytologic changes in culture was in 1929 (early concepts of cytopathic effect, or CPE):

• Andrewes in England, with Virus III (a herpesvirus of rabbits)

• Rivers and colleagues, with vaccinia

The Evolution of Virology (diagnosis)



• Rolling of cultures• Alexis Carrel, 1913, suggested the idea• Gey, 1933, actually did it, on a large scale, at the

Johns Hopkins Hospital, and then, with Frederick Bang, grew and studied LGV, including demonstration of the inclusion bodies in unstained monolayers.




• After World War II, antibiotics began to be used in tissue culture. This made it possible to inoculate clinical specimens directly into culture.

• Advances came at a rapid pace:• Vaccinia (Feller, Enders and Weller, 1940)• Poliovirus (Enders, Weller and Robbins, 1949)

The Evolution of Virology (diagnosis, public health and patient care)


• Growth of Poliovirus in Tissue Culture• The situation before 1949:

• Poliovirus was grown dependably only in monkeys, passaging by inoculation of neural tissue into the CNS.

• Simon Flexner, in 1910, had tried and failed to grow poliovirus in non-neural tissue culture.

• Sabin had tried the same thing and failed in the 1930’s.



• Growth of Poliovirus in Tissue Culture• Thomas Weller’s first tissue culture experiments:

• The Enders lab had a strain of poliovirus (the Lansing strain – a Type 2 strain) that had been adapted to growth in mouse central nervous system.

• Weller was trying to grow varicella virus in “Maitland” tissue cultures: mixtures of human embryonic skin and muscle cells kept alive in 25 ml Erlenmeyer flasks with frequent changes of the fluid medium.



• Growth of Poliovirus in Tissue Culture• Weller’s first tissue culture experiments:

• During an experiment on varicella in April, 1948, there were four unused flasks. On a whim Weller inoculated them with mouse brain containing the Lansing strain of poliovirus. Nine days later, attempts to find residual varicella failed. However,when mice were inoculated intracerebrally with the tissue culture fluid from the polio cultures, they became paralyzed.




• This preliminary experiment was followed by others and published in a series of papers by Weller, Enders, and Robbins.


• Growth of Poliovirus in Tissue Culture• With the work on poliovirus, the word

“cytopathogenic effect” was first used by Enders.

• It was also recognized that CPE could be used to quantitate virus

• In 1949, with the addition of penicillin and streptomycin to culture fluid, poliovirus was recovered in tissue culture from the stools of patients with poliomyelitis.



• Immunology in the service of viral diagnosis• Agglutination

• Early 20th Century, agglutination of smallpox particles from vesicular fluid with animal sera

• Development of complement fixation (Bedson and Bland, 1929)

• Application to the fluid of smallpox vesicles by Parker and Muckenfuss, 1932.



Disease No. ofsamples

Timing No.positive

Remarks

Vaccinia 7 6-10 daysafterprimaryvaccination

7

Variola 16 1-14 daysaftereruption

14 Negative on 12th and14th days.

Control 10 0 Varicella, impetigo,pemphigus, exfoliativedermatitis


(Parker and Muckenfuss, PSEBM 1932;29;483-5)


• Immunofluorescence• Coons and colleagues, in 1942, conjugated antibodies

with fluorescein for the first time, and used them to identify pneumococcal antigens in tissue.

• In conjunction with tissue culture, immunofluorescence became a potent tool:

• In 1953, Weller, after finally producing CPE with varicella virus in roller tube cultures, used the FA technique to prove that the virus reacted with convalescent serum, thereby demonstrating the antibody response, as well as the location of the virus intracellularly.

The Evolution of Virology (diagnosis, patient care)


• Immunofluorescence for rapid diagnosis• Ch’ien Liu (1956) identified influenza

directly in the secretions of Harvard undergraduates in the winter of 1952-53.



• Solid phase assays for viral diagnosis• RIA as a technique

• Yalow and Berson, 1960, developed RIA for measurement of insulin in serum

• Use of a “solid” support was first described by Jacob and Monod in 1961

• Plastic tubes, beads or wells were first used in 1967.




• The quality, specificity, speed of all immune-based tests designed to detect viral antigens in clinical materials was vastly improved with the development of monoclonal antibodies in the early 1970’s by Kohler and Milstein


• Nobel Prizes for Physiology and Medicine• Alexis Carrel (1912)• Max Theiler (1951)• Enders, Weller and Robbins (1954)• Macfarlane Burnet (1960)• Monod and Jacob (1965)• Peyton Rous (1966)• Rosalyn Yalow (1977)• Jerne, Kohler and Milstein (1984)



The Evolution of Virology(diagnosis, public health and patient care)

• The molecular virology era• Of course progress has not slowed in the last few

decades:• PCR and other molecular methods for diagnosis• Virus discovery and progress using purely molecular

methods (hepatitis C virus; human papillomavirus; coronaviruses, bocavirus, many others)

• Designer antiviral drugs (protease and integrase inhibitors for HIV)

• Molecular epidemiology



• The molecular virology era• Nor have Nobel Prizes stopped coming:

• 1975 - David Baltimore, Renato Dulbecco, Howard Temin

• 1989 - Michael Bishop, Harold Varmus • 1997 - Stanley Prusiner • 2008 - Harald zur Hausen, Françoise Barré-

Sinoussi, Luc Montagnier



• The Impact on Medicine (review)• Public Health• Diagnosis• Patient Care



• The Impact on Medicine• Public Health

• The ability to understand pathophysiology, and therefore control diseases (e.g. yellow fever)

• The ability to describe the epidemiology, and therefore control diseases (e.g. influenza)

• The development of vaccines



• The Impact on Medicine• Diagnosis

• We have gone from purely clinical diagnosis, through retrospective diagnosis by acute and convalescent antibody measurement, to rapid diagnosis.

• Buist• Immunofluorescence• Solid phase, point-of-care assays• PCR and other molecular methods



• The Impact on Medicine• Patient care

• More rapid, specific, and point-of-care diagnostics

• Antiviral treatment and prophylaxis• Amantadine, acyclovir• Ganciclovir, neuraminidase inhibitors• Drugs for HIV, including NRTI’s, NNRTI’s, Protease

Inhibitors, Integrase Inhibitors, and more.


Mortality Rates (% per year) among HIV infected subjects enrolled in

PACTG 219 prior to Jan 1, 1996

0

1

2

3

4

5

6

Mortality (%) 5.25 2.07 0.94 0.7

1996 1997 1998 1999

Logrank test for trend significant P<0.0001


Cooper ER et al: JAIDS 2002;29:484-94


U.S. Is Close to Eliminating AIDS in Infants, Officials SayBy MARC SANTORA

Published: January 30, 2005

AIDS among infants, which only a decade ago took the lives of hundreds of babies a year and left doctors in despair, may be on the verge of being eliminated in the United States, public health officials say.


Thank you!

The Co-Evolution of Virology, Diagnostic Virology, and Clinical Medicine: A Century of Discovery

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