- -Adrian J Gibbs, John S Armstrong & Jean C Downie in Virology Journal
- 2) The "laboratory error" theory. We note that influenza viruses survive well in virus laboratories, that
- laboratories are not subject to routine surveillance, and that there are probably many laboratories in the world that share and propagate a range of swine influenza viruses from different sources and continents, and also share and use immortalized lines of cultured cells. The viruses are used for research, diagnostic tests and for making vaccines, and the cells are used for propagating the viruses. Thus if S-OIV had been generated by laboratory activity, when one host was simultaneously infected with strains from the different parental lineages, this would explain most simply why S-OIV's genes had escaped surveillance for over a decade, and how viruses last sampled in North America, Europe and Asia became assembled in one place and generated a reassortant.
So what sort of laboratory event might produce mixed infections with different strains of influenza, and thereby generate S-OIV? The simplest is that S-OIV is a reassortant produced during research. There is also the possibility that it was generated during the production of multivalent vaccines. Multivalent 'killed' vaccines are mixtures of virions that have been grown in hen's eggs and then chemically sterilized. Thus a reassortant might be produced if insufficient sterilant, usually formaldehyde or propiolactone, is added to the virion mixture. The live mixture could then infect pigs 'vaccinated' with it, and the growing viruses could reassort, infect piggery staff and hence spread to the broader human population. Finally, it is possible that serially passaged cells, such as the Madin-Darby canine kidney (MDCK) cells now widely used in influenza laboratories, became latently and serially infected with different strains of influenza as a result of lax laboratory practices. This process could generate reassortants and infect staff.
Circumstantial Evidence
There are clear historical precedents for most of the events described in the above scenarios. Viruses do 'escape' from laboratories, even high security facilities. The H1N1 influenza lineage that circulated in the human population for four decades after the 1918 Spanish influenza epidemic disappeared during the 1957 Asian influenza pandemic, was absent for two decades but then reappeared in 1977. Gene sequences of the 1977 isolate and others collected in the 1950s were almost identical, indicating that the virus had not replicated and evolved in the interim and had probably been held in a laboratory freezer between 1950 and 1977 and 'escaped' during passaging. The suggestion that persistently infected cells might be involved is also not outlandish; influenza virus can persistently and latently infect MDCK cells [27], and viruses do travel between laboratories in cells [28].
Multivalent 'killed' vaccines are widely used to control swine influenzas, particularly in North American piggeries [29], indeed one of the viruses identified by us and others (e.g. [30]) as closest to S-OIV, A/swine/Indiana/P12439/2000 (H1N2), seems to be the "2000 Indiana strain" used in commercial vaccines in North America [31]. We also note that isolates selected from the three clusters of viruses we find to be closest to S-OIV would probably make a useful trivalent vaccine for international use as they would provide a mixture of haemagglutinins of the swine H3, H1 'classical swine' and H1 'Eurasian avian-like' lineages.
The patchy occurrence of S-OIV infections in piggeries over the past six months is interesting and may be significant. Pigs have been shown to be fully susceptible to S-OIV. They shed the virus and readily transmit it between themselves, but whereas S-OIV has been reported in humans worldwide, it has not yet been reported from a pig farm in the USA (October 2009). By contrast it has been found in two piggeries each in Australia, Canada and Ireland, and one each in Argentina, Indonesia and Japan. In the outbreaks in Argentina, Australia and Canada, at least, the pigs had not been vaccinated (Jorge H. Dillon, J. Keenliside and Alain Laperle, personal communication), and became infected from infected farm staff. The apparent immunity to S-OIV of pigs in the USA and Mexico, but not elsewhere, may indicate that the swine influenza vaccines currently used in the USA and Mexico contain an immunogen that either protects against S-OIV infection or mitigates its symptoms.
Circumstantial evidence must always be treated with caution. One major uncertainty in trying to determine the origin of S-OIV is that one cannot predict which characters of the parental viruses have remained or changed during the reassortment process that produced S-OIV. If, for example, the significant infectiousness of S-OIV is an 'emergent' property of S-OIV, and not shown by its parents, then one could conclude that the final reassortment probably occurred at about the time it emerged in early 2009. However it is not yet known whether S-OIV's infectiousness is novel; the reassortment may have occurred a decade ago, and a recent mutation may have enhanced its infectiousness. Another widely reported feature of S-OIV is that it replicates poorly in embryonated eggs, but again this may be merely a specific feature of S-OIV and not its immediate parents. Similarly the fact that the evolutionary rate of all of the genes of S-OIV seem to be 'normal' during their unsampled pre-emergent period [8, 11]] does not prove that the virus or its parents have been maintained in "unsampled" pig herds and precluded the possibility of human involvement, as viruses grown for vaccines evolve, and indeed might be expected to show an increased evolutionary rate [32, 33] while adapting to eggs, a new host, although such an increase may have been offset by the practice of storing 'seed stocks' for use in several 'production cycles' in vaccine production, so that the evolutionary age of a vaccine virus may be less than its sidereal age, and the average could then appear to be 'normal'. Finally there is the report that the first human S-OIV infections were in Perote, a small Mexican town with a very large number of large piggeries, although it was also reported that none of the pigs showed signs of influenza. Among the earliest cases were some in Oaxaca, 290 kms to the south [34]. Perote is an unlikely place for an infected migratory pig to arrive from an intercontinental trip, as the town is in a remote high valley surrounded by mountains, 200 kms to the east of Mexico City where there is the nearest major airport, and 130 kms from the nearest port at Vera Cruz. The four month difference between 'The Most Recent Common Ancestor' date for S-OIV estimated from its phylogeny [8, 11], and its earliest detection in the human population makes it more difficult to make specific conclusions about its provenance. https://virologyj.biomedcentral.com/articles/10.1186/1743-422X-6-207
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