Abstract
In early July 2014, the National Institutes of Health in the USA discovered a few vials containing smallpox virus in their Bethesda, Maryland facility. The subsequent investigation, performed by US CDC, documented viable virus in two of the discovered vials that were subjected to tissue culture testing.
Genetic testing of the recovered virus verified the virus to be smallpox and a review of office records documented that the original specimen was likely to have been placed into the facility sometime around 1950. This discovery brings up the likelihood that similar forgotten variola virus specimens may exist in other laboratories elsewhere in the world. In this regard, the finding highlights the need to establish a standard protocol of how to handle such circumstances in the future.
But first, let’s put this issue into its historical context. During final stages of smallpox eradication, between 1977 and 1980, the WHO made a special effort to uncover all smallpox virus remaining in laboratories throughout the world. To do that, first, a list of all smallpox-related publications over the previous 20 years was developed. Then, a list of all laboratories that performed diagnostic work for detection of smallpox during smallpox eradication program (1967–1980) was constructed. And finally, WHO sent a special letter to the responsible health authorities of each member state requesting each country to report the presence of smallpox virus in laboratories in their country. Based on the results of these investigations, a special follow-up letter was sent to all of the national governments reporting virus stocks requesting them to destroy any remaining stocks.
Two laboratories were exempt from this destruction – the US CDC laboratory in Atlanta and the Russian laboratory which was renamed Vector and moved to Novosibirsk. These laboratories were authorized by WHO to keep viable variola virus and, under special safety and security precautions to ensure that the variola virus could not escape, undertake important research.
If, for a special reason, any government did not want to destroy their virus stocks, they were asked to remove the stocks from their laboratory and dispatch them to one of the official laboratories (Atlanta or Novosibirsk). Such requests were submitted to the WHO Smallpox Certification Commission and approved by World Health Assembly in 1980. The transfer was done under special security measures by responsible laboratories.
Not surprisingly, everything did not go exactly as planned. During the smallpox eradication end game, a laboratory diagnosis of suspect smallpox patients was encouraged by WHO. But, given the large number of cases, WHO decided that during the initial phase of the global program, containment vaccination activities should be launched with a clinical diagnosis alone without laboratory confirmation of smallpox virus, and when during the latter years, the laboratory diagnosis became important, WHO did set up special measures, namely if smallpox suspect is found, its specimens should be collected by national program and such specimens with WHO supplied special dispatch containment kits be sent to WHO HQ which send one of the WHO collaborating centers as mentioned above for smallpox diagnosis. At that time the procedures were conducted under shortest time possible, use of reserved ready flight, readiness of preparation at testing laboratories and so on. During such emergency surveillance activities, many national laboratories were interested to undertake laboratory research on smallpox and monkey pox – a virus which produced a clinical picture similar to smallpox – both in nonhuman primates and in humans.
In this regard, during the final variola surveillance activities a rather peculiar experience occurred. A staff member of one of the WHO collaborating centers for pox virus, called the WHO office one day, having said that their monkey pox serial tissue culture work produced a mutant-like smallpox virus on tissue culture. The author advised them why not they tried to reproduce the same finding at some other WHO collaborating centre. So the arrangement was made and the trial started. While the study was continuing, WHO office had a call from the chief of the laboratory where such a reproduction study was being done. He was asked by the trial team to provide them smallpox virus so that they could compare the lesions with suspected monkey pox mutants during the trial. WHO office agreed immediately to provide the smallpox virus for the study. Then, one day, we received the call that the team confirmed the appearance of smallpox virus like lesions, confirmed that the reproduction study succeeded. This virus initially considered to be a mutated monkey pox virus was smallpox virus. Here, some parenthetic comments on this: namely at that time the diagnosis of smallpox infection was not difficult. First clinically, smallpox infection in man was typical with type of rash with history of contact with either smallpox patients or virus-contaminated materials. As for the laboratory test, the specimen was inoculated into chick embryo chorioallantoic membrane which produced, if the specimen contains variola virus, opaque white, small elevated lesions on the chorioallantoic membrane. Such phenotype was special of variola virus. It differed from other orthopox virus such as caw pox, vaccinia and so on. The lesion by monkey pox virus showed usually gray pock with hemorrhagic center. There were some additional tests like rabbit skin inoculation, but this chorioallantoic membrane test was most common. Frankly, it was never thought that the study team deceived the test using the given smallpox virus material. But at the same time it was hard to believe the mutation, and in the circumstance, obviously cross contamination had to be cautioned a priori. It was a great puzzle. Some years later pertinent studies by Dr. Keith Dumbell et al. revealed that the monkey pox genome was very distinct from the smallpox genome Citation[1]. This indicated that monkey pox virus could not have mutated to smallpox virus. In fact, these ‘mutated monkey pox viruses’ were actually smallpox virus that had contaminated the tissue cultures during their laboratory work.
In another instance, one high level WHO collaborating center reported that, during its preparation of healthy monkey kidney cell tissue culture, they by chance found that one tissue culture specimen carried smallpox virus. With these findings, it was hypothesized that monkeys may be a natural reservoir of smallpox virus. At that time the scientific community indicated the absence of natural reservoir of smallpox should be critical research to be completed for successful smallpox eradication. The laboratory also stressed that there was no chance of any cross-contamination in that laboratory. Later, through, it was known, that at that time of this tissue culture work, smallpox virus was present, not only in the same room, but actually on the same laboratory bench on which this tissue culture work had been conducted.
Thus, cross contamination was certainly possible although the frequency varies tremendously as I have personally seen the related episodes, and feel even now that in biological studies or control measures in general we should be extremely careful about a chance of cross contamination. The late Frank Fenner, who chaired the WHO Commission for Certification of Smallpox Eradication accurately predicted personally that the white pox virus or monkey pox mutant virus will turn out to be smallpox virus contaminants – ‘no doubt about it’. Frank Fenner was the most respected virologist and his word was based on his long laboratory work, so his statement made a solid impact on all who heard it.
If such cross contamination event occurred, although it occurred rare, in high level collaborating centers working for WHO, who did play great role for these surveillance as well as research during smallpox eradication work. Then now a difficult question would be what may have happened in other laboratories around the world? The WHO laboratories followed strict security practices. If cross contamination took place in these laboratories, could there have been similar cross contaminations in other laboratories that were never reported? If this had occurred, how would it have been treated? Would the specimens have been placed in some laboratory waste and been destroyed? Or would they, by chance, be left in some freezer and forgotten?
The discovery of smallpox virus in a forgotten NIH freezer is a case in point. Although not officially recorded by WHO, other instances of the discovery of forgotten variola specimens have certainly occurred in the past. At this stage, it is impossible to determine exactly what happened to each and every smallpox specimen/stock in laboratories that operated 30 to 40 years ago. It would not be surprising that forgotten smallpox virus specimens remain today in one or more of these laboratories. The question is: should we attempt to actively search out such laboratories or should we develop a strategy to cope with the ‘unknown’ which has evolved from this somewhat unusual circumstance.
It is proposed that we take advantage of the rather embarrassing situation of finding variola virus at a distinguished American government research institution. While we should try some realistic strategy to find out further suspected laboratory keeping hidden or forgotten variola virus stocks or vials, through extensive consultation with experts who carried our extensive work for variola research there would be some more urgent preparations; in the author’s opinion, the destruction of variola virus stocks remaining at the two laboratories in the USA and Russia, would not add anything to global security. That being said, the current plan to strengthen the global security against a variola biohazard must be reinforced with the fully organized global smallpox vaccine stockpile being a top priority. It seems opportune for WHO to convene a meeting to review these important matters and make reasonable recommendations.
Acknowledgements
While preparing this report the author remembers fully devoted contribution by small pox eradication teams with thankfulness. This time the author was also grateful to K Hayashi, International Office Kumamoto Medical Center for her examining collected report material as well as repeated preparation of manuscripts.
Financial & competing interests disclosure
The author is the former director of Smallpox eradication at the WHO. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
Reference
- Dumbell KR, Archard LC. Comparison of white pox (h) mutant of monkey pox virus with parental monkey pox and variola like viruses isolated from animal. Nature 1980;286:29-32