Virons le virus (Institut Merieux Benelux, 1991)
“Virons le virus” (Institut Merieux Benelux, 1991)

One of the important drivers of influenza virus evolution is mixed infection: Infection of the same individual with two different strains of virus, which can then reassort to generate brand-new viral genomes. This presumably what happened, for example, with the recent swine-origin influenza virus (SOIV): some pig was simultaneously infected with North American swine flu and a Eurasian swine flu, the two reassorted so that two of the Eurasian virus’s segments joined with 6 of the North American segments, and the new virus thus produced turned out, just by chance, to be good at infecting humans.

Reassortment, notoriously, can generate rapid large changes in the personality of the virus. Pandemic influenzas have been reassortants, unrecognized by the population’s immune systems. But that’s not the only possible outcome; reassortants between closely-related viruses can lead to small changes, reassortants between two circulating strains would still be recognized by the immune response, and so on. Reassortment per se isn’t inevitably devastating, the big concern is reassortment between widely-differing viruses — human and avians strains being the major issue today.

I’ve tended to think of multiple infection and reassortment as quite a rare phenomenon. Reassorted influenza viruses appear and circulate relatively often, but not, you know, daily;1 and those are the product of millions upon millions of infected individuals. On the other hand, most reassortments are probably either dead on arrival (their different segments are simply not compatible) or at best very unfit (their different segments make them easily outcompeted by the wild flu that’s already well adapted to the individuals in question). That means we don’t know the frequency of reassortants, because most of them would be invisible to us.

I’ve also tended to think, perhaps naively, that multiple infections would be a little unusual, because the timing would have to be fairly precise. Viruses generally rely on a couple of days of relative peace (immunologically speaking) to quickly replicate and bank a virus load that then keeps pace with the increasing immune response. If Virus B tries to infect you a couple of days after Virus A is already present, Virus B is going to run right into the thick of the immune response to Virus A, never have that chance to bank its progeny virus, and you’d expect it to be quickly overwhelmed. So you probably need nearly simultaneous infections to get a real multiple infection.

Chicago influenza poster 1918
“Influenza is prevalent” (Chicago, 1918)

But this is all speculation. A recent paper2 went out and actually looked for evidence of mixed infection in humans.3 They used previously-collected samples, and this is going to greatly underestimate the extent of mixed infection,4 but they did detect evidence of several mixed infections in their collection of over 1000 influenza samples. A plausible number they offer is about 0.5% of their samples — half a dozen individuals — were potentially mixed infections.5

(Later they suggest, as unpublished data, that the number may be as high as 3%. An important caution, that they don’t mention here, is that the 3% number is from influenza database analysis, and we know that these databases are not high quality — see On the accuracy of the influenza databases and the paper referenced therein6 — in fact, about 3% of the samples in the database are contaminated, so I don’t know if the present authors took this into account when interpreting evidence for mixed infections.)

However, sticking with the 0.5% figure — which is still remarkably high, and would represent tens of thousands of cases per year — they were able to look more closely at several of these samples and confirmed that they did, in fact, represent true mixed infections. This is another spinoff of the rapid, high-throughput sequencing that’s now becoming widely available. One patient, from New Zealand, was simultaneously infected with two viruses:

…one closely related to viruses cocirculating in New Zealand during 2004 and a second lineage that clustered with A/H3N2 viruses that became dominant in the following (2005) influenza season in the southern hemisphere 2

Another, in New York, was infected with two different influenza strains that are antigenically distinct — that is, viruses that would require different vaccines for protection. Remember that influenza vaccines are customized, year by year, to match up against the dominant circulating virus of that particular year. This patient would have needed two distinct vaccines to get adequate protection from his two infections.

A third, “even more dramatic” example was another New Yorker who was infected with two viruses that were not merely antigenically different, but that came from two distinct, broad groups — influenza A and influenza B viruses. I don’t think A and B can reassort, or at least the progeny would be very unlikely to be fit, but it illustrates that very mixed infection is quite possible.

It’s important to note that they were looking for mixed infection, not reassortment. Reassortment woud be much less common than mixed infection — you need mixed infectio nfor reassortment, but it’s not inevitable following mixed infection. Still, the background of mixed infection seems to be rather higher than I thought it would be.

In sum, we propose that mixed infection of diverse influenza viruses, a necessary precursor to reassortment, is a common occurrence during seasonal influenza in humans and will in turn accelerate the rate of adaptive evolution in this virus. In addition, intrahost populations of influenza virus will harbor genetic diversity generated by de novo mutation, which we have not assessed in the current study. As a consequence, we urge that intrahost sequencing be more routinely employed to assess the degree of genotypic and phenotypic diversity in populations of acute RNA viruses. With the advent of high-throughput next-generation sequencing platforms, viral variants are being much more explicitly revealed within specimens, and this type of data can be made available on a routine basis.2

  1. Offhand, actually, I don’t know how often reassortants have been identified. I’ll try to find that[↩]
  2. Ghedin, E., Fitch, A., Boyne, A., Griesemer, S., DePasse, J., Bera, J., Zhang, X., Halpin, R., Smit, M., Jennings, L., St. George, K., Holmes, E., & Spiro, D. (2009). Mixed Infection and the Genesis of Influenza Virus Diversity Journal of Virology, 83 (17), 8832-8841 DOI: 10.1128/JVI.00773-09[↩][↩][↩]
  3. It would probably be more interesting to look for mixed infection in swine, or wild ducks, but it’s only humans that have enough close attention to detect these relatively rare events.[↩]
  4. Most samples of a mixed infection are simply going to pick up the more abundant of the viruses present[↩]
  5. This comes with a large helping of caveats; it could over- or under-estimate the frequency. But it’s a reasonable starting point and they did confirm some of them.[↩]
  6. Krasnitz, M., Levine, A., & Rabadan, R. (2008). Anomalies in the Influenza Virus Genome Database: New Biology or Laboratory Errors? Journal of Virology, 82 (17), 8947-8950 DOI: 10.1128/JVI.00101-08[↩]