Technician Deborah Clouser
prepares purified plasmids
containing individual swine
influenza A virus gene
segments for constructing a
live swine influenza A virus.
ARS scientists studying
a strain of swine influenza new to this part of the world have found
that taking one step backward can lead to many steps forward.
Veterinary medical officers Jürgen Richt and Kelly
Lager of the ARS National Animal Disease Center (NADC) in Ames, Iowa,
are using a process called reverse genetics to gain insight into an
alarming development: rapid spread throughout North America of a swine
flu type that contains gene segments from birds and humans as well as
The researchers, who work in NADC's Virus and Prion Diseases
of Livestock Research Unit, are using reverse genetics to create new
flu viruses in efforts to explore individual components of the virus.
The hope is that these components can in turn be exploited by vaccines.
In studies to construct a
swine flu vaccine, cells
are observed for signs of
change that indicate a
live swine influenza A
virus was generated by
Reverse genetics has been developed over the past decade
for virus studies. "The technology has now advanced to where one
can confidently generate influenza viruses entirely from cloned DNA
resulting from the process," Lager says. Richt says this work is
unique because, while the process has been used on human flu strains,
it has never been used on swine flu.
Swine influenza presents a special challenge in genetics
because its genome comprises eight segments of ribonucleic acid, better
known as RNA. RNA viruses such as swine flu store their genetic information
in RNA, which is more susceptible to mutation than DNA. This allows
RNA viruses to evolve far more rapidly than DNA viruses and sometimes
makes it hard for an infected host to develop lasting immunity.
What's "Reverse" Got To Do With It?
What made reverse genetics attractive for exploring the
new swine flu strains is that manipulations commonly done on DNA cannot
be performed with RNA.
Veterinary medical officer
Jürgen Richt removes
embryonated chicken eggs
from an incubator. These
eggs are used to propagate
swine influenza A viruses.
| That's where the "reverse"
in reverse genetics comes in. "Scientists can convert RNA viruses'
genetic material into a DNA state," says Richt. "This is called
reverse transcription. At this point, mutations can be introduced into
the resulting cloned DNA. Once the DNA is converted back into RNA, the
introduced mutations will occur in the genome of the RNA virus. Through
this approach, we use cloned DNA to generate swine influenza viruses,"
Richt and Lagercollaborating with veterinary pathologist
Bruce H. Janke of Iowa State University and virologist Richard J. Webby
of St. Jude Children's Research Hospital in Memphis, Tennesseegenerated
the A/Swine/Texas/4199-2/98 virus, or TX/98 for short.
"When tested in experimentally infected pigs, this
generated virus showed characteristics similar to its parental wild
type," says Richt. In addition, pigs infected with TX/98 viruses
that were genetically altered through mutation or deletion showed significantly
less evidence of flu infection, says Lager. "This makes these viruses
potential candidates for modified-live vaccines."
Jürgen Richt (left) and
veterinary medical officer
Kelly Lager use a laryngoscope
to inoculate an anesthetized
pig, while Deborah Clouser
| Swine influenza is an acute respiratory
disease of swine whose symptoms include anorexia, fever, depression, coughing,
and troubled breathing. It is among the type A influenza viruses, which
can affect humans as well as chickens, ducks, horses, seals, whales, and
New Flu Strain Changes Everything
Specialists in North America used to diagnose almost exclusively
only one type of flu virus in pigs: H1N1. That changed in 1998, when
pigs started to be diagnosed with H3N2, a strain that up to that time
was rarely seen here.
Since then, Lager says, these H3N2 viruses have combined,
or reassorted, further with the classical H1N1 viruses, resulting in
new H1N2 and H1N1 swine influenza viruses. The increased virulence represented
by this new strain also raised concerns, he adds.
The H3N2 virus appeared in two types: a double reassortant
(DR), labeled as such because it contains gene segments from both human
and swine flu; and a triple reassortant (TR) that also contains gene
segments from avian viruses. Richt says it's the TR viruses that are
causing most of the trouble. "By the end of 1999, these had spread
throughout the United States, whereas the DR viruses had not,"
Birds play an important role in the flu dynamic, providing
a global reservoir of A-type viruses. It is believed flu resides harmlessly
in birds, where viruses are genetically stable. When a virus from birds
infects pigs that are already infected with a swine influenza virus,
gene segments from each virus can be mixed, and a new influenza virus
can arise. This reassortment likely produced the TR H3N2 virus.
Richt and Lager believe reverse genetics can greatly benefit
the study of influenza in humans as well as in pigs.
"Current human and swine vaccines are inactivated
vaccines that vary in efficacy, depending on the match of the vaccine
with influenza virus strains circulating in the susceptible population,"
says Richt. "Modified-live-virus vaccines generated through reverse
genetics can stimulate a better, broader immune response than killed-virus
There is a trade-off, however, in that modified-live-virus
vaccines may not be as safe as killed-virus vaccines. "There's
always a chance a modified-live-virus vaccine may gain virulence as
it replicates in the vaccinated host," Richt says.
"But if a modified-live-influenza-virus vaccine can
be developed, it may be an important tool in preventing flu in pigs
and humans. We also believe reverse genetics can contribute to our understanding
of how influenza virus causes disease in various host species."By
Agricultural Research Service Information Staff.
This research is part of Animal Health, an ARS National
Program (#103) described on the World Wide Web at www.nps.ars.usda.gov.
A. Richt and Kelly
M. Lager are in the USDA-ARS Virus
and Prion Diseases of Livestock Research Unit, National Animal Disease
Center, 2300 Dayton Ave., Ames, IA 50010; phone (515) 663-7366 [Richt],
(515) 663-7371 [Lager], fax (515) 663-7458.
"Reverse Process May Be Key To Developing Swine Flu Vaccine"
was published in the February
2004 issue of Agricultural Research magazine.