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 Health
Children’s Hospital Boston
Clinical Virology Symposium,
April, 2009
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.
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
• I prefer to think that “Virology” began a few
years before this, with a Scotsman named
John Brown Buist.
Clinical Virology Symposium,
April, 2009
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
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(diagnostic)
• John Brown Buist’s studies:
• He devised a method for staining the material from
a cowpox (vaccinia) vesicle
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(diagnostic)
• 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
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(diagnostic)
• 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.
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(diagnostic)
• 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
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(diagnostic)
• Inclusion Bodies
• Another early example was in rabies, from
impression smears made from rabid dogs,
by Negri in 1903
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
• 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.
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
• 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.
Clinical Virology Symposium,
April, 2009
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 SpanishAmerican 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.
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(public health and patient care)
• 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.
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(public health and patient care)
• 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.
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(public health and patient care)
• 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.
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(public health and patient care)
• 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.
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(public health and patient care)
• 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.
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(public health and patient care)
• Yellow fever (tissue culture and vaccine
development)
• Max Theiler
• The chick embryo tissue culture methods that
Theiler used were developed first by Alexis
Carrel.
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(public health and patient care)
• Yellow fever (tissue culture and vaccine
development)
• Max Theiler
• 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.
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(public health and patient care)
• Yellow fever (tissue culture and vaccine
development)
• Max Theiler
• 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:
Measles
Rubella
Mumps
Clinical Virology Symposium,
April, 2009
Polio
Varicella
The Evolution of Virology
• 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 nonsterile sites before the advent of antibiotics.
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(public health and patient care)
• 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.
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(public health and patient care)
• 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.
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(public health and patient care)
• 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.
Clinical Virology Symposium,
April, 2009
Smith, Andrewes, Laidlaw. Lancet 1933;2:66-68.
The Evolution of Virology
(diagnosis)
• 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
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(diagnosis)
• Growth of Virus
• Tissue culture
• 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.
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(diagnosis, public health and patient care)
• Growth of Virus
• Tissue culture
• 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)
Clinical Virology Symposium,
April, 2009
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.
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(diagnosis, public health and patient care)
• 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.
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(diagnosis, public health and patient care)
• 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.
Clinical Virology Symposium,
April, 2009
The Evolution of Virology (diagnosis,
public health and patient care)
• This preliminary experiment was followed by others
and published in a series of papers by Weller,
Enders, and Robbins.
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(diagnosis, public health and patient care)
• 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.
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(diagnosis)
• 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.
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(Parker and Muckenfuss, PSEBM 1932;29;483-5)
Disease
No. of
samples
Timing
No.
positive
Vaccinia
7
7
Variola
16
6-10 days
after
primary
vaccination
1-14 days
after
eruption
Control
10
Clinical Virology Symposium,
April, 2009
Remarks
14
Negative on 12th and
14th days.
0
Varicella, impetigo,
pemphigus, exfoliative
dermatitis
The Evolution of Virology
(diagnosis, patient care)
• 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.
Clinical Virology Symposium,
April, 2009
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.
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(diagnosis, patient care)
• 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.
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
(diagnosis, patient care)
• 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
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
• 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)
Clinical Virology Symposium,
April, 2009
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
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
• 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 BarréSinoussi, Luc Montagnier
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
• The Impact on Medicine (review)
• Public Health
• Diagnosis
• Patient Care
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
• 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
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
• 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
Clinical Virology Symposium,
April, 2009
The Evolution of Virology
• 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.
Clinical Virology Symposium,
April, 2009
Mortality Rates (% per year) among HIV
infected subjects enrolled in
PACTG 219 prior to Jan 1, 1996
6
5
4
3
2
1
0
1996
1997
1998
1999
Mortality (%)
5.25
2.07
0.94
0.7
Logrank test for trend significant P<0.0001
Clinical Virology Symposium,
April, 2009
Clinical Virology Symposium,
April, 2009
Cooper ER et al: JAIDS 2002;29:484-94
U.S. Is Close to Eliminating AIDS in Infants,
Officials Say
By 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.
Clinical Virology Symposium,
April, 2009
Thank you!
Clinical Virology Symposium,
April, 2009