Influenza Genetics Background

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

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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

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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

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Summary:

Neither of these papers is strictly necessary to graspthe 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 virulence[vii]. 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 questioned[viii],[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.)

1. 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.

1. 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?

[i] Crosby, Alfred W. (1989) America’s Forgotten Pandemic: The Influenza of 1918. CambridgeUniversity Press. Cambridge, UK

[ii] Steinhauer DA and JJ Skehel. (2002) Genetics of Influenza Viruses. Annual Review of Genetics. 36:305-32.

[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 Microbiology2:279-288.

[xiii]Ibid.