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

TWiV 49: Viral genomes

twiv-200Hosts: Vincent Racaniello and Dick Despommier

On episode 49 of the podcast ‘This Week in Virology”, Vincent and Dick continue Virology 101 with a discussion of the seven different types of viral genomes, and how to use the pathway to mRNA to understand viral replication.

Click the arrow above to play, or right-click to download TWiV #49 (45 MB .mp3, 62 minutes)

Subscribe to TWiV in iTunes, by the RSS feed, or by email

Links for this episode:
Dick talks about hookworm on Radio Lab
Dick’s video page at BigThink
The seven types of viral genome
Animation of HIV replication (thanks axiomatically atypical!)
Changes in transcript abundance relating to colony collapse disorder in honey bee (thanks Judi!)

Weekly Science Picks
Dick Discovery Channel: Planet Green
Vincent Influenza videos at BigThink: one, two, three, four, five, six

Send your virology questions and comments (email or mp3 file) to twiv@microbe.tv or leave voicemail at Skype: twivpodcast. You can also send articles that you would like us to discuss to delicious and tagging them with to:twivpodcast.

Pandemic H1N1 influenza virus outcompetes seasonal strains in ferrets

ferret-h1n1-coinfectionWhen more than one influenza A virus subtype is circulating in humans, as has been the case since 1977, there are several possible outcomes. The viruses might co-circulate, one virus might out-compete another, or co-infection of cells with two viruses can lead to the production of genetically distinct viruses by the process of reassortment of viral RNAs. Experiments have been done in ferrets to determine how the 2009 pandemic H1N1 strain interacts with seasonal H3N2 and H1N1 viruses.

Ferrets were intranasally co-infected with an H1N1 pandemic strain [Ca/04] and either a seasonal H1N1 virus [BR/59] or a seasonal H3N2 virus [BR/10].  One uninfected ferret was placed in the same cage (to allow contact transmission) and a second in another cage separated by a wire mesh (to allow aerosol transmission). They determined whether the viruses replicated in the animals by detecting viral RNA in nasal washes taken 1 day after infection using polymerase chain reaction (PCR), or by hemagglutination-inhibition assays to measure serum antibodies.

The results are striking. Both viruses replicate well in co-infected ferrets – look at panel C of the image above. The figure is a photograph of the DNA products of the PCR, separated by gel electrophoresis. Each lane shows the DNA corresponding to the individual influenza viral RNA. Panels A and B show the amplified DNAs from the nasal washes of two animals who were infected by contact. In these animals, the pandemic CA/04 virus replicates well (right half) while the seasonal H1N1 strain [BR/59] does not (left half).

When the nasal washes from the respiratory droplet contact ferrets were used to infect a new set of ferrets, only the pandemic CA/04 virus was detected; there was no evidence of the seasonal BR/59 or BR/10 viruses.

The pattern of the DNAs are distinct for each virus. For example, the PB2 DNA of BR/59 migrates more slowly on the gel than the PB2 DNA of CA/04 (panel C). Given these differences in migration of the DNAs, it would be easy to determine if there were reassortants in the co-infected ferrets. None can be detected by this analysis.

If these results were directly applicable to humans, we would predict that the 2009 H1N1 pandemic strain is not likely to reassort with the seasonal strains; and that it will out-compete those strains, which will eventually disappear. But we are not ferrets, and we don’t know whether these findings apply to humans. Nevertheless, the authors are allowed to speculate:

Although we must be cautious interpreting studies in the ferret model, it is reasonable to speculate that this prototypical pandemic strain, Ca/04, has all the makings of a virus fully adapted to humans.

In this case ‘fully adapted’ means that the pandemic strain replicates better than the seasonal strains or any reassortants that might arise in co-infections.

What did the press learn from this work? Reuters concluded:

And while a new study in ferrets suggested the virus spreads more quickly and causes more severe disease than seasonal flu, the good news is that it does not appear likely to mutate into a “superbug” as some researchers had feared.

Virologists consider mutation and reassortment to be two distinct phenomena. The mutation rate of the virus is determined by error-prone RNA synthesis. The host applies the selection pressure that enriches for a particular phenotype. The results of these studies reveal nothing about the ability of the virus to mutate.

Perez, D., Sorrell, E., Angel, M., Ye, J., Hickman, D., Pena, L., Ramirez-Nieto, G,, Kimble, B., & Araya, Y. (2009). Fitness of Pandemic H1N1 and Seasonal influenza A viruses during Co-infection PLoS Currents RRN1011.2.

Why don’t DNA based organisms discard error repair?

quasispecies1The recent series of posts on polymerase error rates and viral evolution has elicited many excellent and thought provoking comments from readers of virology blog. Here is one that I had not thought of before, and which I’ll use on an exam in my virology course:

Here’s a tough question. In the follow up blog to this, you say that the high mutation rates of RNA viruses is beneficial to survival in a complex environment. If this is true, why don’t DNA viruses evolve high mutation rates also? It would be simple for them to delete their proofreading domain.

There is no answer to this question, so I’ll speculate. I believe that DNA viruses have error correction mechanisms so that they can have very long genomes. RNA viral genomes are no longer than 27-31 kb. This limit is probably imposed by their high error rate: if RNA genomes were longer, they would likely sustain too many lethal mutations to survive. Error correction mechanisms allow for DNA viral genomes up to 1.2 million bases in length. Smaller DNA viruses don’t have their own DNA polymerases – they use those of the cell. Cellular DNA polymerases have error repair to avoid mutations that lead to diseases such as cancer. Both forms of reproduction are evolutionarily sustainable: shorter RNAs with lots of errors; longer DNAs with fewer errors.

The reader who posed the original question then came back with this retort:

If reduced fidelity is beneficial to RNA viruses, because of the complex environment they are in, why don’t DNA viruses do the same thing?

I think the same endpoint, in terms of surviving in complex environments, is achieved by both strategies. RNA viruses have high diversity; DNA genomes have many more gene products which allow them to survive in diverse situations. Both strategies appear to be evolutionarily sustainable.

It’s important to keep in mind that the goal of viral evolution is survival. Evolution does not move a viral genome from “simple” to “complex”, or along a trajectory aimed at “perfection”. Change is effected by elimination of the ill adapted of the moment, not on the prospect of building something better for the future.

While researching this subject I came across a series of papers on DNA synthesis by African swine fever virus, a virus with a DNA genome of 168-189 kb. The viral genome encodes a complete DNA replication apparatus, including DNA polymerase and DNA repair enzymes. Incredibly, the DNA repair pathway itself is error-prone, which is believed to contribute to the genetic variability of the virus. There is some controversy concerning the error rate for this virus, so we don’t know the consequence of this observation for fidelity of DNA replication. Nevertheless, these observations suggest that evolution has seen fit to tinker with, and perhaps increase, the error rates of certain DNA based organisms.

Lamarche, B., Kumar, S., & Tsai, M. (2006). ASFV DNA Polymerase X Is Extremely Error-Prone under Diverse Assay Conditions and within Multiple DNA Sequence Contexts. Biochemistry, 45 (49), 14826-14833 DOI: 10.1021/bi0613325

Influenza virus RNA: Translation into protein

influenza-rna-2

figure 1

Let’s resume our discussion of the influenza virus genome. Last time we established that there are eight negative-stranded RNAs within the influenza virion, each coding for one or two proteins. Now we’ll consider how proteins are made from these RNAs.

Figure 1 shows influenza RNA segment 2, which encodes two proteins: PB1 and PB1-F2. The (-) strand viral RNA is copied to form a (+) strand mRNA, which in turn is used as a template for protein synthesis. Figure 2 (below) shows the nucleotide sequence of the first 180 bases of this mRNA.

The top line, mostly in small letters, is the nucleotide sequence of the viral mRNA. During translation this sequence is read in triplets, each of which specifies an amino acid (the one-letter code for amino acids is used here). Translation usually begins with an ATG which specifies the amino acid methionine; the next triplet, gat, specifies aspartic acid, and so on. Only the first 60 amino acids of the PB1 protein are shown; the protein contains a total of 758 amino acids.

Most of the influenza viral RNAs code for only one protein. However, RNA 2 (and two other RNAs) code for two proteins. In the case of RNA 2, the second protein is made by translation of what is known as an overlapping reading frame.

On the second line of the RNA sequence in figure 2 is an atg highlighted in red. You can see that this atg is not in the reading frame of the PB1 protein. However, it is the start codon for the second protein encoded in RNA 2, the PB1-F2 protein (F2 stands for frame 2, because the protein is translated from the second open reading frame). Figure 3 shows how PB1-F2 is translated. The sequence of the viral RNA is shown from the beginning, except that reading frame 1, which begins at the first ATG, is not translated. Rather, we have begun translation with the internal atg, which is in the second reading frame. This open reading frame encodes the PB1-F2 protein which, in this case, is 90 amino acids in length (its length varies in different isolates). The protein is much shorter than PB1 because translation stops at a termination codon (tga) long before the end of the RNA. Because PB1-F2 is encoded in reading frame 2, its amino acid sequence is completely different from that of PB1.

figure 2

figure 2

figure 3

figure 3

The sequences used for this example are from the 1918 H1N1 strain of influenza. Notice the amino acid of PB1-F2 which is highlighted in blue. This amino acid has an important role in the biological function of the protein, which we will consider in a future post.

My apologies if the figures and text are not optimally aligned. A blog post is not the optimal format for such information, but in the interest of time I have not explored other options. Suggestions for improvement are welcome.

Send your questions to virology@virology.ws.

Influenza virus RNA genome

influenza_virus_rna

Within the influenza A virion are eight segments of viral RNA. These molecules carry the all the information needed to make new influenza virus particles. These eight RNAs are shown schematically as olive green lines at the top of the illustration. RNAs are chains of four different nucleotides, A, C, G, U. In the case of influenza virus, the eight RNAs are a total of about 14,000 nucleotides in length. The nucleotides make up the genetic code – it is read by the cell’s translation machinery in groups of three, with each triplet specifying an amino acid.

There are two important aspects of these viral RNA that we must consider. First, you can see that the ends of the RNAs are labeled 3′ and 5′. Nucleic acids have polarity, in that one end of the chain is chemically different from the other. Such polarity is represented by 5′ or 3′. The second point is that when a nucleic acid is copied, or duplicated, by enzymes called polymerases, a strand of the complementary polarity is produced. Influenza viral RNAs are called (-), or negative strand RNAs, because they are the opposite polarity of the RNA that is translated to make protein. The RNA molecules that are templates for the synthesis of proteins are defined as having having (+), or positive polarity. Upon entering the cell, the (-) strand influenza viral RNAs must be copied into complementary (+) strands, so that they can serve as templates for proteins. The viral RNAs are copied by an enzyme – called RNA polymerase – that is carried into the cell with the virus.

In the above scheme, the olive green lines are the (-) strand RNAs found in the influenza virion. Once the virion enters the cell, these 8 RNAs are copied into (+) strand mRNAs. Finally, the mRNAs can serve as templates for the synthesis of proteins. The specific viral proteins that are produced by each viral mRNA are shown at the bottom of the illustration. From this picture we see that, for example, RNA segment 4 codes for the viral HA protein, and RNA segment 6 codes for the viral NA protein. Note also that some RNA segments encode for more than one protein. Both influenza A and B viruses have 8 RNA segments, while the influenza C viruses have 7.

Influenza viruses are called (-) strand RNA viruses because of the polarity of the RNA that is carried in the virion. Other RNA viruses – such as poliovirus – are (+) strand RNA viruses, because their genomic RNA can be translated into protein immediately upon entering the cell.

Any questions before we proceed?

Structure of influenza virus

influenza-virion3In this week’s discussion of swine flu A/Mexico/09 (H1N1), we have considered many aspects of influenza virus biology that might not be familiar to some readers of virology blog. I thought it might be useful to explain how the virus multiplies, how it infects us, and how we combat infection. Today we’ll start with the basic structure of influenza virus, illustrated above.

The influenza virion (as the infectious particle is called) is roughly spherical. It is an enveloped virus – that is, the outer layer is a lipid membrane which is taken from the host cell in which the virus multiplies. Inserted into the lipid membrane are ‘spikes’, which are proteins – actually glycoproteins, because they consist of protein linked to sugars – known as HA (hemagglutinin) and NA (neuraminidase). These are the proteins that determine the subtype of influenza virus (A/H1N1, for example). We’ll discuss later how the HA and NA are given subtype numbers. The HA and NA are important in the immune response against the virus; antibodies (proteins made by us to combat infection) against these spikes may protect against infection. The NA protein is the target of the antiviral drugs Relenza and Tamiflu. Also embedded in the lipid membrane is the M2 protein, which is the target of the antiviral adamantanes – amantadine and rimantadine.

Beneath the lipid membrane is a viral protein called M1, or matrix protein. This protein, which forms a shell, gives strength and rigidity to the lipid envelope. Within the interior of the virion are the viral RNAs – 8 of them for influenza A viruses. These are the genetic material of the virus; they code for one or two proteins. Each RNA segment, as they are called, consists of RNA joined with several proteins shown in the diagram: B1, PB2, PA, NP. These RNA segments are the genes of influenza virus. The interior of the virion also contains another protein called NEP.

This week, when we discussed the nucleotide sequence of swine influenza RNAs, we were referring to these RNA molecules. Tomorrow I’ll show you how each RNA codes for protein. This way it will be easier to understand the meaning of the swine flu virus sequences that were released this week.

Let me know if this type of explanation is useful, and if you would like me to continue.