Influenza Genetics Background
Paul Nagami
Bi 226 – Current Topics in Genetics
The presentation material:
Paper to be presented: Kobasa et al (2004) Enhanced virulence of influenza A viruses
with the haemagglutinin of the 1918 pandemic virus. Nature, 431:703-707
URL: http://www.nature.com/cgitaf/DynaPage.taf?file=/nature/journal/v431/n7009/full/nature02951_fs.html
Summary:
Using the known hemagglutanin (HA) and neuraminidase (NA) sequences of the
influenza A virus that caused the 1918 Spanish Flu pandemic, Kobasa et al recreated
those segments of the viral genome, placed them in flu viruses that are normally
nonpathogenic flu in mice, and tested them on mice. The resulting symptoms resembled
those of the 1918 flu epidemic in humans. In addition, they performed immunological
tests on the 1918 HA and NA-carrying viruses, and found that those of us who were not
around for the 1918 outbreak could be susceptible.
Suggested background paper: Neumann et al (1999) Generation of influenza A viruses
entirely from cloned cDNAs. PNAS. 96:9345-9350
URL:
http://www.pnas.org/cgi/content/full/96/16/9345?maxtoshow=&HITS=10&hits=10&RE
SULTFORMAT=&fulltext=influenza+cloned&searchid=1114403141275_6818&stored_
search=&FIRSTINDEX=0&journalcode=pnas
Second suggested background paper: Reid, Fanning, et al (1999) Origin and evolution of
the 1918 “Spanish” influenza virus hemagglutinin gene. PNAS. 96(4):1651-1656
URL:
http://www.pnas.org/cgi/content/full/96/4/1651?ijkey=73aeefaabda86ee2283999b6f4c4d
478eb776c59
Summary:
Neither of these papers is strictly necessary to grasp the first, but they’re both
worth reading. The first describes the methods of reverse genetics employed in the
presented paper, and the second is one of the intriguing and slightly frightening papers by
Reid and Fanning, who used tissue samples from autopsy collections and Alaskan grave
sites to sequence the genes of the 1918 Spanish flu. This work provided the genetic
sequences used in the presented paper.
Another optional background paper: Steinhauer and Skehel, 2002. Genetics of Influenza
Viruses. Annual Review of Genetics. 36:305-32.
[In the following background, when I refer to influenza, I mean influenza A. Influenza B
and C are closely related, as all belong to the Orthomyxoviridae, but not nearly as heavily
studied.]
The influenza pandemic of 1918 (or, a good reason to know about influenza):
The influenza pandemic of 1918 struck the young, spread quickly, and killed
more people than World War I. It came in waves, seemingly dying off before reemerging
with renewed virulence, likely due to novel mutations. By the time effective quarantine
measures were in place, resources mustered, and an ineffective vaccine prepared, the
brunt of the pandemic had past, leaving over 20 million dead.i
Influenza Genetics:
Influenza’s negative-strand RNA genome is divided into eight segments. Each
segment encodes one protein, with the exception of a segment that encodes two proteins
by alternative splicing. This setup presents a problem for the reproducing virus, for a
viable progeny virion must carry all eight segments to survive. We know that flu viruses
may carry more than eight segments at a time, so it is possible that newborn virions
simply take up several segments at random and only survive if they get a full genome.
However, there is some evidence that the segments bear some kind of packaging signal.
Either way, if two influenza viruses infect the same cell, they may freely trade genomic
segments, a process of reassortment that leads to rapid adaptation. ii
From an epidemiological point of view, the most interesting influenza genes are
those coding for the glycoproteins hemagglutinin (HA) and neuraminidase (NA). HA
binds to sialic acid residues and is needed for viral entry into cells; it provides host
specificity. NA serves the opposite function, cutting new virions free from their sialic
acid tethers and allowing them to escape the cell. Due to their opposite functions,
mutations in hemagglutinin and neuraminidase can suppress each other.iii
(In spite of the seeming simplicity of the flu genome, we can still have suppressor
mutants, temperature-sensitive mutants, and other phenomena familiar to geneticists
working in more advanced organisms. That influenza has eight genomic segments was
originally ascertained by complementation testing; mutations in the different segments
fell into eight complementation groups.) iv
Normally, hemagglutinin must be cleaved outside the cell by host proteases,
which limits influenza infection to cells where those proteases are present. However,
mutations in hemagglutinin that make it cleavable before it has been shipped to the cell
surface remove this requirement and allow the virus to infect a wider variety of cells,
causing increased virulence. HA and NA mutations can also produce antigenic drift and
shift, as these glycoproteins are the virus’s primary points of recognition for the immune
system; this is the reason for the continual need for updated influenza vaccines.v
This is not to say that only mutations in HA and NA are responsible for virulence;
the Hong Kong H5N1 virus, which caused 6 deaths in 18 cases, had virulence-inducing
mutations in both HA and the polymerase subunit PB2.vi
Recombination, both homologous and nonhomologous, is less prevalent than
reassortment, but does happen. An outbreak of highly pathogenic avian influenza H7N3
in British Colombia was likely caused by a virus in which the genes for hemagglutinin
and structural proteins recombined. This recombination produced a small insertion in the
HA gene that made it more readily cleavable, which, as explained earlier, can lead to
increased virulencevii. It has also been theorized that the original 1918 epidemic was
caused by a recombination event between swine and human influenza, but this analysis
has been questionedviii,ix.
Influenza Reverse Genetics:
Although methods for propagating influenza in culture are long-established,
creating transgenic influenza viruses has been difficult until recently. As the influenza
viruses contain negative strand RNA, transforming cells directly with the viral genome
has no effect; it cannot be translated into protein without the viral polymerase that usually
accompanies it in the virion.
Earlier researchers solved this problem by including purified influenza
ribonucleoprotein complexes (RNPs) to transcribe the added RNA. Although this allowed
the production of influenza viruses with transgenic RNA, the purification of the RNPs
was troublesome and ‘helper’ influenza viruses needed to be added to provide additional
protein machinery.x
In 1999, Neumann et al demonstrated a technique that eliminated the need for
purified RNPs and helper viruses. Instead of using genomic RNA, the researchers
transfected embryonic kidney cells with eight plasmids that each carried cDNA
corresponding to a viral genome segment, flanked by RNA polymerase I promoters, as
well as plasmids that expressed the influenza viral proteins needed for transcription. The
RNA polymerase I present in the cells produced vRNA copies of the viral segments, and
the viral proteins transcribed mRNA from them and provided other structural functions.
This method reliably produces recombinant influenza viruses carrying genome segments
from multiple strains.xi
Influenza Population Genetics:
Although this subject is not directly relevant to the papers covered in the talk, it is
both practically and scientifically important. In addition to making it possible to more
accurately predict the upcoming year’s flu strains, an understanding of influenza’s
population genetics could tell us much about the mechanisms of evolution. Due to their
high error rate of RNA transcription, the RNA viruses are the fastest mutating genetic
systems known, with mutation rates of “roughly one mutation per genome, per
replication.”xii
One method of using RNA viruses to study evolutionary change is to infect cell
monolayers with virus, then transfer infected samples to culture dishes at regular
intervals, counting plaque numbers over time as an indicator of viral titer. If inhibitors of
growth are added to the culture media, the relative fitness of wild-type and mutant viruses
in the inhibitory environment can be gauged by this method.xiii
Talking points:
1. How accurate an evolutionary model can RNA viruses be for anything other than
viral evolution?
a) Does the “bottleneck” population structure, consisting of large populations
within separated hosts, make them less amenable to traditional models of
population genetics? More amenable?
b) Should selection be assumed to act on individual viruses, or is it better to
treat all of the viruses in a single host as one selectable unit? (This is the
concept of quasispecies; see Moya et al 2004.)
2. Working in level 4 containment, Kobasa et al reconstituted viruses that could be
essentially equivalent to the 1918 flu. Was this advisable? What about the work of
Reid and Fanning in sequencing the virus in the first place? Should information
that could allow the production of a deadly flu be public?
Note: Bear in mind that the original Spanish flu HA and NA may very well
still exist in avian reservoirs, and that descendants of those genes ARE present in
the modern-day flu.
3. Influenza’s viral packaging system is mysterious; what sorts of experiments could
further explain how individual segments are packaged? Would there be any
advantages to a purely random system?
Crosby, Alfred W. (1989) America’s Forgotten Pandemic: The Influenza of 1918. Cambridge University
Press. Cambridge, UK
ii
Steinhauer DA and JJ Skehel. (2002) Genetics of Influenza Viruses. Annual Review of Genetics. 36:30532.
iii
ibid
iv
ibid
v
ibid
vi
Hatta, M et al (2001) Molecular Basis for High Virulence of Hong Kong H5N1 Influenza A Viruses.
Science. 293:1840-1842
vii
Hirst, M et al. (2004) Novel Avian Influenza H7N3 Strain Outbreak, British Columbia. Emerging
Infectious Diseases. 10(12):2192-2195
viii
Gibbs, MJ et al (2001) Recombination in the Hemagglutinin Gene of the 1918 “Spanish Flu.” Science.
293:1842-1845
ix
Worobey M, et al. (2002) Questioning the Evidence for Genetic Recombination in the 1918 “Spanish
Flu” Virus. Science 296:211a
x
García-Sastre, A and P Palese. (1993) Genetic Manipulation of Negative-Strand RNA Virus Genomes.
Annu. Rev. Microbiol. 47:765-90
xi
Neumann, G et al. (1999) Generation of influenza A viruses entirely from cloned cDNAs. PNAS.
96:9345-9350
xii
Moya, A et al (2004) The Population Genetics and Evolutionary Epidemiology of RNA Viruses. Nature
Reviews Microbiology 2:279-288.
xiii
Ibid.
i