Virology question of the week: why a segmented viral genome?

influenza-reassortmentThis week’s virology question comes from Eric, who writes:

I’m working on an MPH and in one of my classes we are currently studying the influenza virus. I’d forgotten that the genome is in 8 separate parts. Curious, I’ve been searching but can’t find any information as to why that is?

What evolutionary advantage is conferred by having a segmented genome?

Terrific question! Here is my reply:

It’s always hard to have answers to ‘why’ questions such as yours. We answer these questions from a human-centric view of what viruses ‘need’. We might not be right. But I’d guess there are at least two important advantages of having a segmented RNA genome.

Mutation is an important source of RNA virus diversity that is made possible by the error-prone nature of RNA synthesis. Viruses with segmented genome have another mechanism for generating diversity: reassortment (illustrated).

An example of the evolutionary importance of reassortment is the exchange of RNA segments between mammalian and avian influenza viruses that give rise to pandemic influenza. The 2009 H1N1 pandemic strain is a reassortant of avian, human, and swine influenza viruses.

Having a segmented genome is another way to get around the limitation that eukaryotic mRNAs can only encode one protein. Viruses with segmented RNA genomes can produce at least one protein per segment, sometimes more. There are other ways to overcome this limitation – for example by encoding a polyprotein (picornaviruses), or producing subgenomic RNAs (paramyxoviruses).

Other segmented viral genomes include those of reoviruses, arenaviruses, and bunyaviruses.

There are various ways to achieve genetic variation and gene expression, and viruses explore all aspects of this space.

TWiV 89: Where do viruses vacation?

Hosts: Vincent Racaniello and Alan Dove

On episode #89 of the podcast This Week in Virology, Vincent and Alan review recent findings on the association of the retrovirus XMRV with ME/CFS, reassortment of 2009 pandemic H1N1 influenza virus in swine, and where influenza viruses travel in the off-season.

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Click the arrow above to play, or right-click to download TWiV #89 (56 MB .mp3, 78 minutes)

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What if influenza virus did not reassort?

rewiring-influenzaWould influenza virus be the same pathogen if it could not undergo reassortment of its segmented RNA genome? This is the question being asked in the wake of the development of a method to prevent the free assortment of influenza viral RNAs.

The process of influenza virus replication includes the incorporation of eight viral RNAs into each newly synthesized virion. This process, called packaging, depends upon specific RNA sequences in each genome segment. By swapping the packaging sequences for the nonstructural protein (NS) and HA genes, a virus was produced which replicated but lost the ability to independently reassort the HA or NS gene. The authors note that the other influenza A virus RNA segments could be modified in a similar way to reduce or eliminate their ability to form reassortant viruses.

Some have speculated that replacement of all the influenza viruses on the globe with reassortment-defective influenza viruses would eliminate pandemics. Reassortment is a major driver of influenza virus evolution, and the mechanism by which pandemic strains arise. In a world full of reassortment-defective influenza viruses, antigenic drift would still occur, which means that seasonal influenza would not be eliminated. But that could probably be accepted in exchange for eliminating the ability, for example, of H5N1 viruses to reassort with human strains.

Unfortunately, this scenario is highly improbable. Influenza viruses infect so many animal species that it would be virtually impossible to replace all viruses with a reassortment-defective strain. To predominate, this strain would require a strong selective advantage over wild strains, an unlikely scenario. The fact that the strain is engineered by humans virtually guarantees that it will be less fit than wild viruses, and hence unable to replace them.

Even in the unlikely event that we could devise ways to replace all the influenza virus strains in the world with one that cannot undergo reassortment, it is unlikely that this phenotype would endure. Influenza viruses, like all RNA viruses, undergo high mutation rates as a consequence of error-prone RNA replication. Long before the laboratory-engineered strain circled the globe, mutations would likely occur that restore its ability to undergo reassortment.

There is at least one practical use for a reassortment-defective influenza virus. The infectious, attenuated influenza virus vaccines currently in use could in theory reassort with circulating wild type viruses, leading to the the production of a pathogenic strain. This possibility would be reduced if the attenuated vaccines were engineered so they could not undergo reassortment.

[Technical note: As shown in the figure, influenza reassortment was examined by gel electrophoresis, not PCR. The approach is similar to my method from 1979, with the exception that viral RNAs are detected by silver staining, not autoradiography.]

Gao, Q., & Palese, P. (2009). Rewiring the RNAs of influenza virus to prevent reassortment Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.0908897106

Influenza virus reassortment, then and now

influenza-rna-gelIn a recent study of influenza virus reassortment in ferrets, the authors used polymerase chain reaction (PCR) to search for viruses with RNA segments from the 2009 pandemic H1N1 strain and seasonal H1N1 and H3N2 strains. I thought you might like to see how I did a similar experiment in 1979 – a very different era for laboratory techniques.

For my Ph.D. thesis project, I wanted to isolate reassortants of two influenza B virus strains, B/Lee and B/Maryland. The goal was to obtain viruses with a genome consisting of one RNA segment from one parent, and 7 RNA segments from the other parent. These viruses would then be used to identify the protein product of each viral RNA.

To isolate these reassortants, I co-infected cells in culture with both viruses, allowed them to replicate, and then harvested the newly synthesized viruses. I then did a plaque assay with the viruses produced by the co-infected cells, and isolated individual clones by plaque purification. I prepared virus stocks from each plaque-purified clone, and then asked if any of these viruses were reassortants.

In 1979, we identified viral reassortants by a tedious method. Each virus to be examined was used to infect cultured cells in the presence of radioactive phosphate. The viruses were purified by centrifugation, and the viral RNA was extracted, concentrated, and fractionated by gel electrophoresis. The gel was dried and exposed to X-ray film. Because the viruses were propagated in the presence of radioactive phosphate, which was incorporated into the viral RNAs, it was possible to visualize each viral segment as a band on the film. The X-ray is shown above.

The RNAs of three different viruses are included: B/Lee on the left, B/Maryland in the middle, and a plaque-purified virus called R3. You can see that each lane contains 8 RNAs, as expected. It is also evident that the migration pattern of the RNAs is different for B/Lee and B/Maryland. This property allowed us to determine that the plaque purified virus R3 is a reassortant that inherits RNA 2 from B/Lee, and the remaining 7 RNAs from B/Maryland. Just what I wanted!

In fact, in just one infection, I isolated all the different reassortant viruses that I needed. I remember showing the gel to my thesis advisor: he told me I didn’t deserve such luck.

The whole procedure – from infecting cells in the presence of radioactive phosphate, to producing the X-ray, took about a week. And to get all the right viral reassortants required a great deal of luck.

Today, the process of identifying influenza viral reassortants is far simpler and faster. The process begins in a similar way – co-infect cells (or animals) with two different viruses. Once the infection is complete, a small sample of the cell culture medium is taken, heated to disrupt the virions, and the viral RNA is converted to DNA using reverse transcriptase. The DNA is amplified by PCR, in eight separate reactions, using primer pairs specific for the individuals segments. The products are then fractionated by gel electrophoresis, as shown here.

Total time to identify reassortants by PCR – less than a day. That’s progress.

In a few years, we’ll skip the gel electrophoresis and simply determine the sequence of the RNAs using a small, inexpensive machine that will be on most laboratory benches. And who knows what will be next? That’s one of the beauties of science: it is driven forward by technological innovation.

Racaniello VR, & Palese P (1979). Influenza B virus genome: assignment of viral polypeptides to RNA segments. Journal of virology, 29 (1), 361-73 PMID: 430594

Reassortment of the influenza virus genome

Mutation is an important source of RNA virus diversity that is made possible by the error-prone nature of RNA synthesis. Viruses with segmented genomes, such as influenza virus, have another mechanism for generating diversity: reassortment.

When an influenza virus infects a cell, the individual RNA segments enter the nucleus. There they are copied many times to form RNA genomes for new infectious virions. The new RNA segments are exported to the cytoplasm, and then are incorporated into new virus particles which bud from the cell.

If a cell is infected with two different influenza viruses, the RNAs of both viruses are copied in the nucleus. When new virus particles are assembled at the plasma membrane, each of the 8 RNA segments may originate from either infecting virus. The progeny that inherit RNAs from both parents are called reassortants. This process is illustrated in the diagram below, which shows a cell that is co-infected with two influenza viruses L and M. The infected cell produces both parental viruses as well as a reassortant R3 which inherits one RNA segment from strain L and the remainder from strain M.


One example of the evolutionary importance of reassortment is the exchange of RNA segments between mammalian and avian influenza viruses that give rise to pandemic influenza. For example, the 2009 H1N1 pandemic strain is a reassortant of avian, human, and swine influenza viruses, as illustrated.


Reassortment can only occur between influenza viruses of the same type. Why influenza A viruses never exchange RNA segments with influenza B or C viruses is not understood. However, the reason is probably linked to the packaging mechanism that ensures that each influenza virion contains at least one copy of each RNA segment.

Trifonov, V., Khiabanian, H., & Rabadan, R. (2009). Geographic Dependence, Surveillance, and Origins of the 2009 Influenza A (H1N1) Virus New England Journal of Medicine DOI: 10.1056/NEJMp0904572

Packaging of the segmented influenza RNA genome

The RNA genome of influenza viruses is segmented . The virions of influenza A and B viruses contain 8 different RNAs, while those of influenza C viruses contain 7. How is the correct number of RNA segments inserted into newly synthesized virus particles?

During influenza virus assembly, viral RNAs and viral proteins – called a ribonucleoprotein complex or RNP –  travels to the plasma membrane. There the virion forms by a process called budding, during which the membrane bulges from the cell and is eventually pinched off to form a free particle.


Production of an infectious virus particle requires incorporation of at least one copy of each of the eight RNA segments. Two different mechanisms – random and selective packaging – have been proposed to explain how each virion receives a full complement of genomic RNA.

If the 8 influenza viral RNA segments were randomly packaged into new particles, we would expect to observe 1 infectious particle for every 400 particles assembled (8!/88). This ratio falls within the range of infectious to noninfectious particles that occur in virus stocks. If more than 8 RNA segments could be packaged into each virion, then the fraction of infectious particles would be significantly increased. For example, if 12 RNA molecules could fit into each virion, then 10% of the particles would have the complete viral genome. In support of this mechanism, influenza viruses with more than 8 RNA segments have been observed.

In the selective packaging mechanism, each of the eight genomic RNAs has a different signal that allows incorporation into virus particles. These signals are believed to be within the noncoding and coding sequences at the 5′- and 3′-ends of the viral RNAs. The sequences interact and form structures that are unique to each segment, and which have been shown to be essential for incorporation of each segment into virions. Consistent with this hypothesis, electron microscopy reveals that during budding, the viral RNPs are organized in a distinct pattern, as shown in the image.


This observation argues that RNPs are not randomly incorporated into virions, and is consistent with the presence of specific signals in each RNA segment that enable the RNPs to be packaged as a complete set. The mechanisms by which these signals are recognized, and how they ensure incorporation of one copy of each RNA segment into the particle, are not known.

There is clear evidence for a selective mechanism during the packaging of the bacteriophage ψ6 genome. Viral particles contain one copy each of a S, M, and L dsRNA segment. All particles contain a complete complement of genome segments, as indicated by the fact that every virus particle is infectious. Only the S RNA segment can enter newly formed particles; once that segment is packaged, then the M RNA can enter. The L RNA can only enter particles that contain both the S and M segments. Precise packaging is therefore the result of a serial dependence of packaging of the RNA segments.

Muramoto, Y., Takada, A., Fujii, K., Noda, T., Iwatsuki-Horimoto, K., Watanabe, S., Horimoto, T., Kida, H., & Kawaoka, Y. (2006). Hierarchy among Viral RNA (vRNA) Segments in Their Role in vRNA Incorporation into Influenza A Virions Journal of Virology, 80 (5), 2318-2325 DOI: 10.1128/JVI.80.5.2318-2325.2006

Noda, T., Sagara, H., Yen, A., Takada, A., Kida, H., Cheng, R., & Kawaoka, Y. (2006). Architecture of ribonucleoprotein complexes in influenza A virus particles Nature, 439 (7075), 490-492 DOI: 10.1038/nature04378

Frilander, M. (1995). In Vitro Packaging of the Single-stranded RNA Genomic Precursors of the Segmented Double-stranded RNA Bacteriophage ψ6: The Three Segments Modulate Each Other’s Packaging Efficiency Journal of Molecular Biology, 246 (3), 418-428 DOI: 10.1006/jmbi.1994.0096

The trajectory of evolution

quasispecies-selectionScientists and philosophers have long debated the trajectory of evolution. Some of the questions they consider include: is there a predictable direction for evolution, and if there is, what is the pathway? Are there evolutionary dead ends?

Viruses are excellent subjects for the study of evolution: they have short generation times, high yields of offspring, and prodigious levels of mutation, recombination, and reassortment. Furthermore, selection pressures can be readily applied in the laboratory, and may be often be identified in nature.

When studying evolution of viruses, it is important to avoid judging outcomes as ‘good’ or ‘bad’. Anthropormorphic assessments of virus evolution come naturally to humans, but concluding that viruses become ‘better adapted’ to their hosts, for example, fails to recognize the main goal of evolution: survival. Or, in the case of the non-living viruses, existence.

Evolution does not move a viral genome from simple to complex, or along a trajectory aimed at perfection. Change comes about by eliminating those viruses that are not well adapted for the current conditions, not by building something that will fare better tomorrow.