potential sources for a return of smallpox

INTRODUCTION

The eradication of smallpox was defined bysuccessive WHO expert groups (WHOScientific Group on Smallpox Eradication,1968 ; WHO Expert Committee on SmallpoxEradication, 1972) as "the elimination ofclinical illness caused by variola virus". Animportant corollary of this definition was thatit did not involve the extinction of variolavirus, as some experts had urged . Theprocedures for the certification of smallpoxeradication described in Chapter 24 weretherefore designed to ensure that no humancases had occurred in the countries concernedfor at least 2 years-i .e., that the human-to-human chains of transmission of smallpoxhad been interrupted. Careful follow-up of

CHAPTER 30

POTENTIAL SOURCES FOR ARETURN OF SMALLPOX

Contents

1321

rumours of suspected smallpox by nationalauthorities and by WHO, described inChapter 28, has failed to confirm a single caseof smallpox in a field situation since the lastcase was recognized in Somalia in October1977, although there were 2 laboratory-associated cases in Birmingham, England, inAugust-September 1978 . The correctness ofthe conclusions that the Global Commissionfor the Certification of Smallpox Eradicationarrived at in its deliberations in December1979 (World Health Organization, 1980) hasbeen borne out by 10 additional years offreedom from the disease.

As the expert groups which defined eradi-cation recognized, the absence of cases ofsmallpox is not synonymous with theextinction of variola virus, and if the virus is

PageIntroduction 1321Is there an animal reservoir of variola virus? 1322

The example of yellow fever 1322Smallpox in apes and monkeys 1323Monkeypox 1325"Whitepox" virus isolations 1326Conclusion : There is no animal reservoir of variola

virus 1333Material stored by variolators 1333Laboratory stocks of variola virus 1334

Changes in attitude towards work with variola virus 1334Safety regulations in laboratories holding and

handling variola virus 1334Justification for the retention and use of variola

virus 1337Reduction in the number of laboratories retaining

variola virus 1338Deliberate release 1341Reactivation and excretion of virus in humans 1343Viral persistence in the environment 1343"Transformation" as a source of variola virus 1343General conclusions 1344

1 32 2

SMALLPOX AND ITS ERADICATION

not extinct it is possible that further cases ofsmallpox could occur. This chapter is con-cerned with the known and hypotheticalways in which variola virus could be pre-served and enter again into chains of human-to-human transmission. By far the mostimportant, because it would be the mostdifficult to eliminate or circumvent, would bethe continuing transmission of the virus insome wildlife reservoir or reservoirs . Indeed,such a situation would have made the perma-nent global eradication of smallpox animpossibility. This was therefore a matter thatgreatly exercised the staff of the WHOSmallpox Eradication unit from the outset ofthe global eradication programme (see Chap-ters 10 and 29). The lack of evidence ofinfection of humans from an animal host inEurope, North America and Australia, inwhich the disease no longer occurred, did notexclude this possibility for parts of Africa andAsia in which smallpox was still endemicuntil the 1970s. Although there was nodocumented evidence that this had takenplace, it was conceivable that infection of manfrom an animal source might have occurred inthe past without having been detected, asindeed proved to be the case with humanmonkeypox. Because of the reportedisolations in laboratories in Bilthoven (Neth-erlands) and Moscow (USSR) of variola-likeviruses ("whitepox" viruses) from animaltissues (see later in this chapter), investigationof this possibility remained a major concern ofvirologists involved in research associatedwith the eradication programme throughoutthe 1970s and into the early 1980s .

Material stored by variolators was anotherpotential source for the recurrence of cases ofsmallpox which concerned the WHO Sec-retariat and members of the GlobalCommission, especially in countries such asAfghanistan in which low temperatures pre-vailed for much of the year . However, themost obvious source of virus that might causehuman infection at some time in the futureconsists of laboratory stocks of variola virus,from which, indeed, the last known outbreakin the world originated (see Chapter 23) .These could be stocks of virus known byWHO to be held in microbiologically securelaboratories, or they could be specimens in thedeep-freeze storage facilities of any laboratorythat had ever worked with the virus . Finally,stocks of virus may be held secretly, forpossible use in microbiological warfare .

Other hypothetical sources of smallpox

could be from virus released by reactivation ina human subject who had had the infection, orrelease of viable virus long preserved in scabs,on old clothes, or even in coffins . For the sakeof completeness the possibility that anotherspecies of Orthopoxvirus might be "trans-formed" into variola virus (already discussedin Chapter 2) needs to be briefly reconsideredhere, as a possible source for the return ofsmallpox.

IS THERE AN ANIMAL RESERVOIROF VARIOLA VIRUS?

The presence of an animal reservoir ofvariola virus is in theory the most importantpotential source for a return of smallpox . Indiscussing the origin of variola virus inChapter 2, we suggested that conditionssuitable for its perpetuation as a specificallyhuman pathogen have existed for a fewthousand years at the most. Variola virus wastherefore probably derived from some closelyrelated orthopoxvirus that survived in natureby circulation in an animal that occurred inlarge numbers at the time of early man andhad a much shorter life-span. In this chaptertwo possibilities will be discussed : that thereis an animal reservoir of variola virus as weknow it, or that some other animalorthopoxvirus could be "transformed" intovariola virus by a few mutational steps orperhaps by chance recombination withanother animal orthopoxvirus . The greatimportance of this question, from the pointof view of smallpox eradication, can beillustrated by reconsidering briefly thehistory of the first deliberate attempt toeradicate a human disease, yellow fever,which has already been mentioned in anothercontext in Chapter 9 .

The Example of Yellow Fever

In 1915 the International Health Com-mission of the newly established RockefellerFoundation agreed to help with the globaleradication of yellow fever, an undertakingthat General W . C. Gorgas, Surgeon-Generalof the United States Army and the hero ofdisease control during the construction of thePanama Canal, considered eminently feasible .Yellow fever was regarded as an urbandisease, and its global eradication was basedon a simple epidemiological concept :

30. POTENTIAL SOURCES FOR A RETURN OF SMALLPOX

1323

" . . . that the disease could be acquired onlythrough the bite of an A. aegypti mosquito that hadbecome infected by feeding on a human being sickwith yellow fever ; that there were certain endemiccenters of the disease that served as seedbeds ; thatthese foci of infection were few in number, andthat if they were destroyed, yellow fever woulddisappear forever ." (Warren, 1951 .)

It is a matter of history that this conceptwas too simple. As early as 1907 Franco et al .(1911) had recognized the existence inColombia of another epidemiological situ-ation-the contracting of yellow fever in theforest, rather than in urban areas. Anotheroutbreak of yellow fever occurred in the samelocality in 1916, but since Aedes aegypti couldnot be found there, it was assumed by theGorgas Commission that the diagnosis waserroneous (Gorgas, 1917). Eventually Soper(1935, 1936) drew attention again to thepaper of Franco et al . and described what hecalled "jungle yellow fever", which hepostulated could be due to maintenance of thevirus in another vertebrate host or perhapslong persistence in an invertebrate vector. Atabout the same time surveys of forestmonkeys of many species in both Africa andSouth America revealed the presence ofneutralizing antibodies in their sera . With thediscovery of a vertebrate reservoir other thanman, it was clear that the global eradication ofyellow fever was impossible, although urbanyellow fever could be eliminated by riddingtowns and cities of Aedes aegypti.

The significance of the yellow feverexperience was not lost on those whocontemplated the global eradication ofsmallpox and investigations into the possibil-ity that there might be an animal reservoir ofvariola virus in Africa or Asia were initiatedsoon after the Intensified Smallpox Eradica-tion Programme was launched (see Chapter10).

a Varlola virus Isolated .bSerological and epidemiological evidence suggesting Infection with variola virus .

Smallpox in Apes and Monkeys

Experimental observations with variolavirus had demonstrated that, unlike vacciniaand cowpox viruses, both of which have awide host range, few laboratory animals couldbe infected with variola virus, except underunusual conditions (see Chapter 2) . However,it had been demonstrated during the latterpart of the 19th century that monkeys(probably Macaca mulatta) could be infectedwith variola virus (Zuelzer, 1874 ; Copeman,1894), and later studies (Hahon, 1961)showed that many species of monkeys andapes were susceptible . Noble (1970) foundthat 3 species of New World monkeys that hetested were insusceptible to variola minorvirus, although they reacted serologically butwithout symptoms to experimental infectionwith variola major virus .

Reported infections of primates in nature

Arita & Henderson (1968) reviewed thepublished accounts of supposed smallpox inprimates as well as other naturally occurringepidemics of "pox" infections among monkeypopulations. Only 8 such episodes are knownand only 4 of these occurred during thepresent century (Table 30.1). In only 2instances was laboratory confirmation avail-able ; in each of these there had been closeassociation between the primate concernedand cases of human smallpox .

"Smallpox" infection in a monkeypopulation in the forests of southern Brazilwas reported by Bleyer (1922), who notedthat carcasses of Mycetes seniculus and Cebuscapucinus were found under the trees, the deadanimals having fallen from the tree-tops . Sickas well as dead monkeys were covered withnumerous pustules like those seen in humansmallpox and the mortality was extremely

Table 30.1 Episodes of presumed or proved naturally occurring "pox" infection in non-human primates

Country Year Species Author

France 1767 ? Barrier, quoted by Schmidt (1870)Panama 1841 r Anderson (1861)France 1842 ? Rayer, quoted by Schmidt (1870)Trinidad 1858 ? Furlong, quoted by Schmidt (1870)Brazil 1922 Mycetes seniculus Bleyer (1922)

India 1936Cebus capucinusMacaca mulatta Rahman (quoted by Arita & Henderson, 1968)

Indonesia 1949 Orang-utana Gispen (1949)India 1966 Macaca mulattab Mack & Noble (1970)

1 32 4


high in certain districts . Anderson (1861)reported that a "smallpox" outbreak observedin 1841 in monkeys was followed by asmallpox outbreak in a human population inPanama. Two monkeys that he examinedwere covered with "perfectly formed"pustules. Schmidt (1870) mentioned 3 otherepisodes, 2 in France and 1 in Trinidad .

An outbreak of a vesiculo-pustular diseasewas observed by M . A. Rahman (quoted byArita & Henderson, 1968) in rhesus monkeysin Bengal, India, in 1936 . Many deaths wereobserved among monkeys which lived inmango groves near the town and visited thetown frequently in search of food and water .The sick monkeys were quiet and lethargicand had pustular lesions, particularly on theface, palms and soles . When they died theyfell from the trees and roof tops, and the car-casses were disposed of without any particularprecautions being taken. Despite the poorvaccinial immunity status of the population,no human pox-like illnesses were observed .

All these episodes must be regarded withcaution so far as their significance as evidenceof an animal reservoir of variola virus isconcerned. They may have been instances ofthe infection of primates from human cases ofsmallpox or they may have not been due tovariola virus or indeed to any kind ofpoxvirus. However, 2 episodes have been welldocumented which demonstrate that pri-mates in close contact with smallpox-infectedhumans may have become infected withvariola virus. Gispen (1949) observed 2orang-utans in the Jakarta Zoo whichcontracted a pox infection at the time of asmallpox epidemic in the area . Both animalswere in the same cage and demonstratedtypical lesions on the face, hands, and soles ofthe feet ; Bras (1952a) performed an autopsyon the one that died (Plate 30.1). Variolavirus was isolated on the chorioallantoicmembrane of the chick embryo from speci-mens taken from both affected animals . Othermonkeys in the zoo, none of which had beenpreviously vaccinated, remained unaffected .The other episode occurred in India, whereMack & Noble (1970) recorded an unusualsituation in which 1 or possibly 2 performingmonkeys (Macaca mulatta) appear to havebeen infected with variola virus by intimatehousehold contact with human cases ofsmallpox.

Throughout the global smallpox eradica-tion campaign attempts were made to iden-tify the source of infection in all outbreaks,

Plate 30.1 . Orang-utan (Pongo pygmaeus) in theJakarta Zoo, which was infected during the 1949epidemic of smallpox and died .

and as the incidence of smallpox fell to lowlevels in each endemic country, this became amatter of high priority, so that chains oftransmission could be traced . Thousands ofsuch outbreaks in Africa and south-easternAsia were investigated by skilled epidemi-ologists. In the vast majority, infectioncould be traced to a case of smallpox. Veryrarely fomites (e.g., laundry items or burialshrouds) were implicated . In a few instancesit was not possible to determine the sourceof the outbreaks. Some of them may havebeen due to infection acquired from mildunrecognized cases, or the source remainedundetected through faulty or incompleteinvestigation. But the possibility of infectionfrom an animal source was always present inthe minds of epidemiologists in the rareinstances in which no human source could beclearly identified . Never during the globalsmallpox eradication campaign could small-pox be traced back to an animal source .Finally, although monkeys are common inmany of the countries of Africa and Asia inwhich smallpox was once endemic, some-times living in close contact with man or

captured by hunters, no case of smallpox hasbeen found in any of these countries sinceeradication .

Experimental transmission of smallpox betweenprimates

More significant than isolated examples ofsmallpox in monkeys or the infection ofanimals by inoculation are 2 sets of observa-tions on the natural transmission of variolavirus from one primate to another. Noble &Rich (1969) showed that serial infectioncould be maintained for as many as 6successive passages in Macaca irus (cyno-molgus) monkeys placed in contact withother monkeys during the period of rash,but transmission then failed (Fig . 30.1) .Among chimpanzees, Kalter et al . (1979)observed that 2 animals situated in cages neara chimpanzee experimentally inoculated withvariola virus contracted smallpox, one suffer-ing a severe illness . One other animal in thegroup escaped infection. Clearly, primates ofseveral species are susceptible to variola virus,get a rash when infected, and can transmitthe disease to other primates in contact withthem. However, in Macaca irus, the onlyprimate in which adequate studies were con-ducted, the infection persisted with somedifficulty and then died out.

As was discussed in Chapter 2, smallpoxcould not be maintained in isolated human

1

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populations numbering under 200 000. Un-less smallpox in non-human primates pro-duced a situation in which recurrent infec-tivity could occur years after the primaryinfection, it is likely that populations of anyparticular species of monkey would be toosmall to maintain the disease. This is certainlytrue of chimpanzees and orang-utans, inwhich infection with variola virus causes asevere disease not unlike smallpox in man .

Monkeypox

It had been known since 1958 thatmonkeys in laboratory colonies occasionallysuffered epizootics due to another ortho-poxvirus-monkeypox virus (Magnus et al .,1959 ; see Chapter 29) . Known outbreaksof monkeypox were reviewed by Arita &Henderson (1968), and early in 1969 theWHO Smallpox Eradication unit convenedan Informal Group on Monkeypox andRelated Viruses (see Chapter 29) to advise iton matters relating to the problem of ananimal reservoir of variola virus . The fol-lowing year one of the members of the In-formal Group (Dr Svetlana Marennikova)recognized that the virus recovered from asuspected case of smallpox that had occurredin Zaire 2 years after the last outbreak ofsmallpox in the area (Ladnyj et al., 1972) wasindeed monkeypox virus (Marennikova et al .,

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Fig . 30 . I . Five successive contact infections of Macaca irus philippinensis before failure in transmission . Thefirst monkey was infected by intranasal inoculation and after 7 days (indicated in bar) developed many lesions,from which variola virus was recovered (first "+" symbol) and subsequently showed serological conversion(second " + " symbol) . The second monkey was placed in the same cage 3 days after the inoculation and developed54 lesions II days after exposure ; it was then placed in contact with the third monkey, and so on . The penultimatemonkey in the series developed 13 lesions, but virus was not recovered from the crusts, although antibodiesdeveloped. The last monkey failed to develop lesions or convert serologically . (Based on Noble & Rich, 1969 .)

1 326


1972b). Subsequently, human monkeypox hascome to be recognized as a rare zoonosisapparently confined to villages in tropicalrain forests in central and western Africa (seeChapter 29) . The significance of humanmonkeypox in the present context lies inobservations made in the course of ecologicalsurveys in Zaire designed to elucidate thenatural history of monkeypox virus . Theseresulted in a series of reports on what came tobe called "wild whitepox" and ultimately"whitepox" viruses .

"Whitepox" Virus Isolations

Three sets of isolations of "whitepox"viruses have been made which are best dealtwith separately and chronologically . The firstset ("Netherlands isolates") comprises 2strains, designated 64-7255 and 64-7275,apparently recovered from normal cyno-molgus monkey kidney cell cultures at theNational Institute of Public Health atBilthoven (Gispen & Kapsenberg,' 1966 ;Gispen & Brand-Saathof, 1972) . The second

White Pock Mutants, "Whitepox" Viruses and White Clones (Variants)of Monkeypox Virus

The appearance of pocks produced on the chorioallantoic membrane by differentspecies of Orthopoxvirus varies from bright red (cowpox virus), through a greyish ulceratedappearance with a haemorrhagic centre (monkeypox virus and some strains of vacciniavirus including rabbitpox virus) to a dense white pock with no sign of ulceration (variolavirus, some strains of vaccinia virus and camelpox virus) . All viruses that producehaemorrhagic pocks also yield a small proportion of non-haemorrhagic pocks ; these arethe "white pock mutants" of cowpox, rabbitpox, neurovaccinia or monkeypox virus,which have been used for genetic studies of orthopoxviruses .

In 1966 Gispen & Kapsenberg reported the recovery of orthopoxviruses fromapparently normal cynomolgus monkey kidney cells used for viral diagnostic work in theNational Institute of Public Health, Bilthoven. One of them was vaccinia virus ; the other3 were regarded as monkeypox virus. However, when studying these "monkeypox" virusisolates, Marennikova et al. (1971) showed that one of them resembled variola virus andwas clearly distinguishable from monkeypox virus . Gispen & Brand-Saathof (1972)confirmed this result with 2 of the 3 "monkeypox" virus isolates and in the same paperobserved that a white pock mutant that they recovered from monkeypox virus resembledthe parental virus in most properties and was clearly distinguishable from variola virus . Todiscriminate between them, the variola-virus-like strains were called "wild white"poxvirus, or "whitepox" virus .

Between 1971 and 1975 Marennikova and her colleagues at the Moscow ResearchInstitute for Viral Preparations reported that they had recovered white-pock-producingorthopoxviruses from the tissues of 4 species of animals shot in Zaire (chimpanzee,monkey, sun squirrel and multimammate rat) . All isolates resembled Gispen's "wild white"poxvirus (and thus variola virus) in all the biological properties that they could test . Forpurposes of reference, the term "whitepox" virus was used to include all six of these viruses(Arita & Henderson, 1976) .

In 1978-1979 Marennikova and her colleagues reported that they had obtained"whitepox" by inoculating monkeypox virus on the chorioallantoic membrane and inhamsters. They called these isolates "stable white clones" or "white variants" ofmonkeypox virus.

In this way two terms came to be used by virologists associated with the smallpoxeradication programme

(1) White pock mutants-of rabbitpox, cowpox and monkeypox viruses ; and(2) "Whitepox" viruses-from normal cynomolgus monkey kidney cells, from

apparently normal wild animals shot in Zaire, or found as "stable white clones" or"white variants" of monkeypox virus .


set ("Zaire isolates") comprises 4 strains ofvirus reported by workers at the MoscowResearch Institute for Viral Preparations tohave been recovered from the tissues of 4different species of wild animal captured inZaire between 1971 and 1975 (Table 30 .2).The third set ("white clones") comprisesseveral isolates made from preparations ofmonkeypox virus maintained at the Moscow

1327

Research Institute for Viral Preparations(Marennikova & Shelukhina, 1978 ; Maren-nikova et al., 1979) .

Netherlands isolatesDuring 1964 and 1965 4 orthopoxvirus

isolations were made from cynomolgus mon-key kidney cells that were being used for the

Laboratory Contamination with Viruses

Bacteriologists are accustomed to the idea that unwanted organisms may occasionallyfind their way into culture media and multiply there . Indeed, the discovery of penicillinresulted from one such episode (Fleming, 1929) . Similar accidents may occur with viruses,especially if manipulations are of the kind that produce aerosols . Only a few cases of thecontamination of cultures have been reported (e.g., Andrewes et al ., 1944) . Another kindof environmental contamination is the accidental infection of laboratory workers (see, forexample, Pike, 1979 ; Wedum et al., 1972), which is an indicator of the possibility ofinfection of laboratory cultures.H. Mahnel (personal communication, 1984), concerned about the occasional occurrence

of vaccinia virus plaques on "uninfected" cell monolayers, carried out tests with vacciniaand monkeypox viruses under conditions simulating those in the laboratory . Theinfectivity of tissue culture fluid dried on coverslips and stored in Petri dishes in the shadein the laboratory (temperature 20-23 °C) dropped from 10 5 .8 plaque-forming units per mlto 10 2 .8 plaque-forming units per ml in 12 days, but the dried material did not completelylose infectivity until the 6th week . He believes that the principal source of laboratorycontamination of cultured cells arises from droplets of infected fluid drying on bench topsand in the dust of the laboratory .

Although the risk of cross-infection can be reduced by good microbiological technique,there is an ever-present possibility of the contamination of tissue cultures or eggs . Almostevery laboratory manipulation is associated with some risk of producing aerosol particlesthat could contain virus. Dimmick et al. (1973) have drawn up a table listing the "sprayfactors" of various laboratory operations and accidents . Assuming that a viral suspensioncontains 10 7 pock-forming units per ml, it is possible to calculate the number of infectiousaerosol particles released as a result of normal laboratory operations or minor accidents .

The only certain way of avoiding cross-infection with poxviruses is to use only oneisolate at a time, within a glovebox facility or a biocontainment hood, and thoroughlysterilize the working area before handling another strain of virus .

Without special markers and laborious experiments, it was very difficult to "prove" thatlaboratory contamination had occurred, as Dumbell & Kapsenberg (1982) were able to do,but every experienced laboratory scientist who has worked with poxviruses can recallinstances for which he or she believed that contamination was the most likely cause of anunexpected result.

Operation Spray factorVirions per m3of working area

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1 328

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isolation of viruses from human material inthe diagnostic laboratory at the NationalInstitute of Public Health, Bilthoven (Gis-pen & Kapsenberg, 1966) . One strain (64-9411) was eventually diagnosed asmonkeypox virus (see Chapter 29) and onestrain (65-3993) was vaccinia virus, recoveredtogether with herpes simplex virus fromvesicle fluid (] . G. Kapsenberg, personalcommunication, 1981) . The other 2 strains(64-7255 and 64-7275) were first regarded as

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Although in a comment at the Inter-national Symposium on Smallpox Vaccine,Bilthoven, in 1972 Gispen (1973) stated,correctly, that "there was no known possiblecontact ofthese animals [from which the kidneycell cultures infected with whitepox virus hadbeen derived ; our italics] with pox viruses", itsubsequently transpired that on 2 occasions inSeptember 1964, material from smallpoxpatients from Vellore, India, had beenhandled in the diagnostic laboratory in whichthe cynomolgus kidney cells were being used .Skilful detective work by Dr Kapsenbergidentified the passage transfer of cell culturesduring which viral contamination could haveoccurred ; there was a temporal coincidencewith manipulation of the cynomolgus cells inquestion and cells infected with materialfrom the smallpox cases. That laboratorycontamination was the explanation was

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other strains of variola virus (Dumbell &Kapsenberg, 1982) .

Zaire isolatesFive strains of poxvirus were recovered

from the kidneys of wild animals captured inZaire and processed in the Moscow ResearchInstitute for Viral Preparations . Four wereidentified as "whitepox" viruses (Table 30 .2),and the fifth (MK-10-73) as vaccinia virus .

Pocks were always scanty on the mem-branes on which they were first observed . Allsubsequent investigations, including detailedDNA mapping carried out independently in2 laboratories (St Mary's Hospital, Lon-don, England, and the Centers for DiseaseControl, Atlanta, USA), have confirmedthat the 4 strains of "whitepox" virus areindistinguishable from variola virus .

A priori it is difficult to believe that a viruswhich has such a narrow host range as variolavirus should be recovered from the tissues of2 species of healthy rodents that are commonin and near tropical forest villages in CentralAfrica, the rat Mastomys natalensis (syn . coucha)and the sun squirrel Heliosciurus rufobrachium .This view is reinforced by the observationthat variola virus and "whitepox" virus strainRZ-38-75, said to have been recovered fromMastomys natalensis, multiplied very poorlyafter intraperitoneal inoculation in that ani-mal (T. Kitamura, personal communication,1978).

Since the Moscow Research Institute forViral Preparations functioned as one of thetwo WHO collaborating centres that carriedout laboratory diagnosis of smallpox for theIntensified Smallpox Eradication Pro-gramme (see Chapter 10), specimens ofvariola virus were constantly being handledin the laboratories in which the "whitepox"virus isolates were made. The universalexperience of laboratories which havehandled orthopoxviruses, including theexperience of the National Institute of PublicHealth in Bilthoven, just described, attests tothe difficulty of avoiding occasional cases oflaboratory contamination .

Two features caution against too ready anacceptance of this explanation for all theMoscow "whitepox" virus isolates : the posi-tive orthopoxvirus antibody titres found in 3of the 4 animals whose tissues apparentlycontained virus, and the reported reisolationof virus from part of the stored kidney ofChimp 9 (from a chimpanzee) and from a

1329

Plate 30.2 . Farida Huq (b . 1942) undertookgraduate studies with Dr K .R. Dumbell in Londonbefore assuming responsibility for the smallpoxlaboratory in Dhaka, Bangladesh . Besides carryingout diagnostic work she studied the viability of variolavirus in scabs under tropical conditions .

ground-up suspension of kidney tissue of RZ-38-75 (from a sun squirrel) . However, inves-tigations of the ecology of monkeypox inZaire, reported in detail in Chapter 29,suggest that there may be several orthopox-viruses circulating among wild animals inthe forests of Zaire ; the antibody resultssignify prior infection with an orthopox-virus, not necessarily variola virus or evenmonkeypox virus . Reisolation is the standardprocedure for confirming the validity ofsuspicious isolates, but to be acceptable itmust be carried out with material that hadnot been processed in the laboratory at thetime the first isolation was made. Thissituation did not apply to the material fromHeliosciurus, but it did apply to the kidney ofthe chimpanzee (S . S. Marennikova, personalcommunication, 1983), a species which isknown to be susceptible to infection withvariola virus (Kalter et al ., 1979) . However,Chimp 9 was one of the two "whitepox"viruses tested by Dumbell & Huq (1986), whoshowed that it resembled Asian rather thanAfrican strains of variola virus in itsbiological characteristics (see below) .

The number of strains of variola viruswhose DNAs have been mapped by restric-tion endonuclease cleavage is too small toallow any conclusions to be drawn about the

1 330

Table 30.3. The distribution of 4 independentbiological properties characteristic ofAsian variola major virus according tothe geographical origin of the strainstesteda

a Based on Dumbell & Huq (1986).b The characters were: chick embryo virulence; ceiling tempera-

ture; haemadsorption on human embryo fibroblast monolayers;and haemagglutinin production in HEp 2 cells.

CChimp 9 and MK-7-73 (see Table 30 .2) .

relationship of these "whitepox" isolates toother strains of variola virus from western orcentral Africa. However, comparison of 4biological characteristics of 32 variola virusstrains originating from the Indian subcon-tinent, western Africa or Zaire with those ofthe "whitepox" strains Chimp 9 and MK-7-73 (purportedly recovered from primatescaptured in Zaire) showed that these"whitepox" isolates had the same group ofbiological characteristics as the great majorityof strains of Asian variola major and werequite unlike 12 strains of variola virusderived from western Africa or Zaire (Table30.3). Dumbell & Huq (1986) concluded thatthese two "whitepox" viruses (Chimp 9 andMK-7-73) "did not enter into the chains oftransmission which maintained humanvariola in West Africa" .

In summary, and in view of the results setout in the next section, it seems likely that the"whitepox" virus isolates made in Moscowwere due to laboratory contamination withAsian strains of variola virus.

"White clones" ("variants") of monkeypox virusSeeking an explanation for the recovery of

whitepox virus strains from the tissues ofseveral species of wild animals from Zaire, DrMarennikova postulated that they may havearisen as "white clones" of monkeypox virus,which was known to infect non-human pri-mates in that country (see Chapter 29) . Sev-eral investigators (Bedson, 1964 ; Gispen &Brand-Saathof, 1972) had previously isolatedwhite pock mutants of monkeypox virus, but


found them to resemble the parental virusrather than variola virus in properties such asspecies-specific antigenicity (Gispen &Brand-Saathof, 1974) and the pattern of theirintracellular polypeptides (Harper et al .,1979). In this section attention will befocused on two reports of what Marennikovaand her colleagues call "stable white clones"recovered from stocks of monkeypox virusthat had been maintained for several years inthe Moscow laboratory . All these "whiteclones", recovered either as pocks on thechorioallantoic membrane (Marennikova etal., 1979) or from organs of hamsters that hadbeen inoculated some weeks earlier with themonkeypox virus material (Marennikova &Shelukhina, 1978), were identical ; they wereindistinguishable by biological tests from theZaire and Bilthoven "whitepox" virus iso-lates, which have now been shown to bevariola virus .

In view of the importance of this claim asto the origin of "whitepox" viruses, from thepoint of view of the feasibility of smallpoxeradication, the WHO Smallpox Eradicationunit organized a series of studies to determinewhether these results could be independentlyconfirmed. The studies in question wereconducted in the WHO collaborating centresin London and Atlanta . Dumbell & Archard(1980) reported on 17 white pock mutantsrecovered on the chorioallantoic membranefrom their own stocks of the Copenhagenstrain of monkeypox virus and comparedtheir biological characteristics and DNAmaps with those of wild-type monkeypoxvirus, the Harvey strain of variola virus,the 2 Netherlands "whitepox" isolates, 2Zaire "whitepox" viruses and 2 "whiteclones" recovered from monkeypox virus bypassage through hamsters (Marennikova &Shelukhina, 1978) . All the "whitepox"viruses and the 2 "white clones" had DNAsthat were virtually identical with that ofvariola virus (Table 30.4). The monkeypoxwhite pock mutants differed amongthemselves in both genome maps andbiological properties (Table 30.5). Some,which had been recovered after serial passageof the monkeypox virus stock at highconcentrations, showed complex genomechanges, with deletions and transpositions ;some showed minor differences from wild-type monkeypox virus, others were indistin-guishable from it. In all cases the DNA mapswere readily distinguishable from those ofvariola and "whitepox" viruses (Fig . 30 .2) .

Source of variola Number of biological propertiesbvirus strains 0 I 2 3 4

Indian subcontinent 0 0 0 5 13Western Africa 4 2 I 0 0Zaire 4 I 0 0 0Whitepox virusesc 0 0 0 0 2


Table 30.4 . The derivations of various viral strains and the nature of their genomes as determined byrestriction endonuclease analysisa

Strain

Recovered :

DNA analysis

a Based on Dumbell & Archard (1980) and Esposito et al . (1985).b var = DNA pattern like that of variola virus ; mp = DNA pattern like that of monkeypox virus ; mp,del = DNA pattern like that of

monkeypox virus, with terminal deletion; mp,mod = DNA pattern like that of monkeypox virus but with complex modifications .

Table 30.5 . Phenotypic characteristics of white pock mutants of monkeypox virus

Studies at the Centers for Disease Control(CDC), in Atlanta, confirmed the resultsobtained in London. Four white pockmutants-2 derived in Atlanta from CDCstocks of a monkey strain of monkeypox virus(Copenhagen) and 2 from their stocks of ahuman strain (Congo)-resembled monkey-

1331

a Mutants described by Dumbell & Archard (1980) .b Numbers of mutants in each group. There were differences (not shown) within groups in the appearance of the white pocks, the size of

the lesions produced In rabbit skin and the extent of growth in pig embryo kidney cells .C Mutants described by Esposito et al . (1985) .d Not tested .

pox virus in both their DNA maps (Table30.4) and their biological characteris-tics (Table 30 .5). On the other hand, Espositoet al . (1985) reported that isolations of whitepock mutants made by a member of the staffof the Moscow laboratory working in theCDC, but not using the glovebox facility, had

Virus

Pocks on chorloallantoicmembrane Lesions In

Growth atrabbit skin

35.5 °C inafter Intradermal

pig embryo kidneyInoculatlona

cellsAppearance Growth at39 °C

Monkeypox, wild typeVarlola (Including "whitepox")

Grey, haemorrhagic centreOpaque white

+ Large, haemorrhagicSlight transient erythema

+

Mutants:a 5b6

Mutants:c 3

Opaque white„„ d

dSmall nodule

-Small nodule

±

Characterization Number In from Patternb Laboratory

"Whitepox" 64-7255 Netherlands Cynomolgus monkey kidney cells var Atlanta, London64-7275Chimp 9

..Moscow

11

Chimpanzee, Zairevarvar

Atlanta, LondonAtlanta, London

MK-7-73 .. Cercopithecus, Zaire var LondonRZ-10-74 .. Mastomys, Zaire var LondonRZ-38-75 He!losclurus, Zaire var London

"White clones" Ham 8 Moscow Passage of Moscow/Copenhagen var LondonHam 9 monkeypox virus In hamstersCpMW Moscow/Copenhagen monkeypox

virus on CA membranevar Atlanta

CnMW ~. Moscow/Congo-8 monkeypoxvirus on CA membrane

var Atlanta

White pock CPCWN I Atlanta CDC-pock-purified Copenhagen mp Atlantamutants ofmonkeypox virus

monkeypox on CA membrane

11 CPCWN2'

mp,del Atlanta11 CpCnNI CDC pock-purified Congo-8

monkeypox on CA membranemp Atlanta

11 CpCnN2 11 P, mp AtlantaWhite pock h16 London London pock-purified Copen- mp London

mutants of hagen monkeypox on CAmonkeypox virus membrane

11 h2,15 mp,del Londonh3,4,5,6 1h7,8,10 I mp,mod London

1332

3


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biological properties and DNA maps identi-cal with those of variola virus . On the basis ofa detailed analysis of all the data, theseauthors concluded that "the spontaneousproduction of whitepox from monkeypoxvirus would be genetically impossible", andthat "the whitepox viruses recovered frommonkeypox virus stocks had originatedexogenously" .

I

Variola major HarveyWhitepox Netherlands

Whitepox Mastomys

Monkeypox h7

Monkeypox h 15

Monkeypox Copenhagen

0Index of dissimilarity

B

Fig. 30 .2. A: Physical map locations of Hindlll, Xhol and Smal cleavage sites in DNA from variola, "whitepox"and monkeypox viruses : Harvey strain of variola major, Netherlands "whitepox" 64-7255, "whitepox"Mastomys (= Moscow RZ-10-74), Monkeypox Copenhagen, and 4 white pock mutants derived from Monkey-pox Copenhagen (Monkeypox h2, h7, h8 and h IS . (Based on Dumbell & Archard, 1980 .) B: Dendrogramillustrating the similarities and differences between these DNAs . Presence, absence or impossibility (becauseDNA molecules were too small) of coincidence of the cleavage sites illustrated was determined after aligning allmaps on a common Hindlll cleavage site about 60 kilobase pairs from the left-hand end of the molecule ; theresults were then analysed as described by Gibbs & Fenner (1984), using the squared Euclidean metric (numberof attributes = 60) . The "index of dissimilarity" has no absolute value, but illustrates the close resemblancesbetween the DNAs of variola virus and the 2 "whitepox" viruses, and between the DNAs of monkeypox virusand the 4 mutants derived from it .

This work was carried out in 1978 and1979, and in an assessment of the situation in1980, on the eve of the declaration ofsmallpox eradication by the Thirty-thirdWorld Health Assembly, Fenner et al .(SE/80.154) concluded that "the variety ofmutants derivable from monkeypox in thelaboratory does not present any greater threatto the success of smallpox eradication than

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does the existence in nature of orthopoxvirusspecies other than variola" .

Conclusion : There is No Animal Reservoirof Variola Virus

The evidence reviewed in the foregoingpages suggests that smallpox was indeed aspecifically human disease . Variola virus is adistinctive species ; DNA analysis of strains ofwidely differing virulence for man (variolamajor and variola minor viruses) shows thattheir DNAs are very similar, but quitedifferent from those of any other species oforthopoxvirus or from those of any viralmutants that have been recovered underconditions that precluded the possibility oflaboratory contamination. Variola virus has anarrow host range ; besides man the onlyanimals in which serially transmissible infec-tion is known to have occurred are chim-panzees, orang-utans and one species ofmonkey. The "whitepox" viruses isolatedfrom a variety of animals between 1964 and1975 are strains of variola virus . The mostacceptable explanation is that all of themwere laboratory contaminants.

The laboratory cvidence must be seen inboth epidemiological and evolutionary per-spectives. Apart from known contaminants(the Bilthoven viruses), the 4 "whitepox"viruses were recovered from areas of Zaire inwhich human monkeypox occurred andcontinues to occur more frequently thananywhere else in the world . The isolation ratein Moscow for the recovery of a virus fromwild animals was high (4 out of 61 animalsfrom the areas that were tested, comparedwith none of 930 animals from the area testedby the Centers for Disease Control, Atlanta,primarily in a search for monkeypox virus) .Since human monkeypox is a zoonosis andintensive surveillance in selected areas ofZaire between January 1982 and December1984 revealed 210 cases of humanmonkeypox, of which 144 were thought tohave an animal source (see Chapter 29), thereis clearly the kind of contact between humansand animals in these tropical rain forests thatwould make infection of humans with variolavirus likely, if indeed it occurred in the ani-mals there. However, all cases of "suspectedsmallpox" in western and central Africa since1970 that were due to orthopoxvirus infec-tion have been shown by epidemiological andlaboratory investigation to have been causedby monkeypox virus.

MATERIAL STORED BYVARIOLATORS

Variolation was a matter of concern as asource of outbreaks of smallpox during theoperations of the Intensified Smallpox Eradi-cation Programme in Afghanistan, Pakistanand Ethiopia (see Chapters 14 and 21) . Inaddition, it was known that an outbreak innorthern Yunnan Province in China in 1958was due to variolation, 126 cases havingoccurred a year after the last known case inthat subregion . Late in 1984 it was learnedfrom Dr Jiang Yutu, in a personalcommunication, that there had been otheroutbreaks of smallpox in North China (NeiMonggol Autonomous Region (Inner Mon-golia) and 2 nearby counties in ShanxiProvince) which were ascribed to theactivities of variolators and the first of whichoccurred some 6 years after the last reportedcases of smallpox in these regions (seeChapter 8, Table 8 .13 ; and Chapter 27) . Theexplanation for these outbreaks (Jiang Yutu,1985 ; and personal communications, 1984,1987) appears to be that variolators in Shanxiand adjacent parts of Inner Mongolia contin-ued to keep variolation material, stored withhoney in sealed jars, and maintained itspotency by annual passage in susceptiblefamily members and by the addition of freshmaterial to the jars . The 1962-1964 and 1965outbreaks were initiated by such variolatedpersons but the chains of transmission havenot been elucidated. The occurrence of theseoutbreaks long after smallpox had beeneradicated from China led to measures thateliminated this source of the disease and nocase of smallpox has been recorded in thecountry since 1965 .

Apart from these episodes in China, whichdid not come to the attention of WHO until1984, major concern with the danger ofvariolators' material causing outbreaks ofsmallpox after the interruption of transmis-sion centred on Afghanistan . Tests for viablevirus in material collected from variolatorsthere gave positive results in one sample 9months after the material had been collectedfrom a patient ; all the others were negativelong before this (see Chapter 14, Table 14 .15).All the variolators who were questionedabout their mode of operation said that, if itwas available, they preferred to use freshmaterial (from a recent case), and as smallpoxbecame less common they usually added fresh

1333

1 334

scabs or pustule fluid to their stored materialwhenever possible . One sample testedcontained herpesvirus particles, doubtlessderived from a misguided attempt to keepviable stocks for variolation .

Within scabs, out of sunlight and in coolsurroundings, viable variola virus can survivefor several years. However, variolators' ma-terial was never held under such "ideal"conditions, and tests showed that it rarelycontained viable virus for as long as one year.The major factor that now further reduces therisk presented by variolation material as asource for the recurrence of smallpox is thepassage of time. The last cases of smallpox incountries in which variolation was widelypractised during the 20th century occurred in1965 (China), 1973 (Afghanistan), 1974(Pakistan) and 1976 (Ethiopia) . In Ethiopia,in which smallpox was the most recentlyendemic, variolation was usually carried outwith fresh pustular material when an epi-demic threatened, and material was rarelystored (see Chapter 21). The lapse of 10-20years since the last cases of smallpox in theother countries has reduced the incentive topractice variolation, and even under the beststorage conditions (short of refrigeration) theviability of variola virus gradually falls off.The absence of any recurrence of smallpoxdue to variolation for 10 years or more givesreason to believe that this practice will neveragain initiate an outbreak of smallpox .

LABORATORY STOCKS OF VARIOLAVIRUS

While the existence of an animal reservoirwas without doubt the most serious risk tothe achievement of the permanent eradica-tion of smallpox, the existence of stocks ofvariola virus in laboratories constituted and,while such stocks of variola virus are retained,will continue to constitute a real thoughremote risk that further cases of humansmallpox could occur .

Changes in Attitude towards Work withVariola Virus

The handling of variola virus has beensubject to progressively stricter control overthe years. In Jenner's day variolation wasextensively practised, with no control orsupervision of the way in which the virus was


handled or administered and usually little orno check on the movements of the inoculatedsubject . Jenner, and vaccinators after him, didnot hesitate to test the efficacy of vaccinationby challenge inoculation with variola virus .Variolation, which involved the uncon-trolled distribution of variola virus, was notmade illegal until 1840 in Great Britain and1870 in British India, and, as has beennoted above, it was widely practised inAfghanistan up to the early 1970s and inEthiopia until as late as 1976.

Pathologists who were interested insmallpox and vaccinia experimented withvariola virus as a matter of course and withfew precautions, secure in the protectionafforded by a previous attack of smallpox orby repeated vaccination . The attitude ofvirologists towards handling the virus in the1950s is revealed in the report on experimentson viability by Wolff & Croon (1968), whostored scabs from a patient in an envelope in acupboard in their laboratory in 1954, andtested one scab annually for the presence ofvariola virus . At that time reliance was placedon vaccination and revaccination of alllaboratory workers, good laboratory tech-nique, and careful disposal and sterilization ofinfected material and glassware . Biohazardhoods had not then been invented and workwith variola virus was carried out on openbenches, although special cubicles were some-times used when the virus was handled on alarge scale. In countries in which smallpoxwas still endemic-for example, in India,Africa and South America-material forsmallpox diagnosis was regarded, with reason,as being less hazardous to handle in thelaboratory than, for example, material con-taining the tubercle bacillus .

Safety Regulations in LaboratoriesHolding and Handling Variola Virus

Recommendations made in 1969 and 1974The first comment by WHO on precau-

tions to be taken when handling variola virusin the laboratory appeared in the publicationGuide to the Laboratory Diagnosis of Smallpox forSmallpox Eradication Programmes (WorldHealth Organization, 1969a), which wasprepared by 2 virologists with extensiveexperience in handling variola virus (DrA. W. Downie and Dr J. Noble) and 2epidemiologists (Dr I. Arita and Dr A . S .Benenson). The guide suggested that methods


that ensured high standards of microbiologi-cal safety should be employed and that every-one who might have occasion to enter thelaboratories should be vaccinated annually .However, recommendations on physical con-tainment were relatively simple (Fig. 30.3A)-a separate room entered via a vestibule,but no provision for sterilization of theambient air, for biosafety hoods or for 2-wayautoclaves .

Following the accidental infection of alaboratory worker in London in 1973 (seeChapter 23), virologists in the UnitedKingdom interested in smallpox drew up acode of safety practice (Cox, 1974). This codeformalized arrangements for vaccination andrevaccination and for the first time suggestedthe use of safety cabinets and emphasized therisk of aerosol production during laboratorymanipulations. It recommended that animalinoculation should be performed only in"institutes with specially designed facilities" .During the middle and late 1970s

laboratory safety in general became a muchmore prominent issue. There were severalreasons for this, including deaths from Lassaand Marburg fevers contracted in thelaboratory, the invention and developmentof sophisticated methods for the physicalcontainment of infectious agents, and thewidespread concern for laboratory safetyengendered by the introduction of recom-binant DNA technology.

Safety measures recommended by WHO in 1977Because of this change in attitude towards

laboratory safety, and the imminent achieve-ment of the global eradication of smallpox,the WHO Smallpox Eradication unit inAugust 1977 organized a meeting of expertsto discuss safety measures in laboratoriesretaining variola virus. The expert groupincluded senior staff from the WHOcollaborating centres for smallpox research inLondon, Tokyo, Moscow and Atlanta, andDr J. H. Richardson of the Office of Biosafetyat the Centers for Disease Control, Atlanta .Recommendations for physical containmentand the packaging and shipping of specimenswere drawn up (SME/77 .2). Although small-pox laboratories in the WHO collaboratingcentres had to a greater or lesser degreealready installed the physical facilities out-lined in this report, it was the first officialstatement by WHO prescribing the kinds ofphysical containment necessary for holding

1335

and handling dangerous pathogens, withstrict control of entry, facilities for air andbiowaste sterilization, gloveboxes (see Plate30 .3), 2-way autoclaves and biologicalsafety cabinets (see Fig. 30.3 B), as well as theusual stipulations about vaccination andregular revaccination.

The laboratory-associated outbreak inBirmingham, England, in August-September1978 (see Chapter 23) caused serious concernamong the general public and the healthauthorities of many countries . Reacting tothis concern, the WHO Smallpox Eradicationunit arranged a meeting in Geneva in April1979 to study the safety measures adopted inlaboratories then retaining stocks of variolavirus and to discuss the destruction ortransfer of the virus if the laboratory con-cerned was not using it for research .Government officials from the 7 countriesand scientists from the 8 laboratories thatthen retained variola virus attended themeeting and reviewed the situation (WHO/SE/79.137), with special reference to the roleof national authorities in maintaining safetystandards. They agreed on modifications tothe WHO recommendations for physicalcontainment in accordance with furtherexperience with such facilities ; these wereissued as WHO document SME/77 .2 Rev . 1and later reproduced in Annex 9 to the finalreport of the Global Commission (WorldHealth Organization, 1980) . Eventually, inMarch 1983, the WHO Committee onOrthopoxvirus Infections, noting that theOrganization, through its Special Pro-gramme on Safety Measures in Microbiology,had published a book entitled LaboratoryBiosafety Manual (World Health Organization,1983) which provided a standard referencefor a maximum containment laboratory,recommended that WHO documentSME/77.2 Rev. 1 should be withdrawn .

Over the several years after the 1977meeting steps were taken to ensure that thefacilities in the WHO collaborating centres inAtlanta and Moscow were upgraded, and newlaboratories were built in each centre . It is notwidely appreciated how difficult it is to builda highly secure biocontainment laboratoryand maintain it properly, particularly inrelation to airflow . For various reasons it tookmore than 3 years, to construct and test thenew facilities in both centres, and all researchwork with variola virus ceased during thisperiod, although studies with variola virusDNA, which is not infectious, continued in

1 33 6

300z

7 qN V

RefrigeratorFreezer

Microscope 0

-------------

CENTRIFUGEROOM

iFreezer

TOILET

Eggrack

Stool

Cupboard for supplies

Glovebox

Refrig .

Stool

Incubator


WINDOW

Towel

Glovebox

Sink forstain

shelves

O Disinfectantbucket

Hooks for lab gowns r T -r T

r---r--~-9-~--r -

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ANIMALROOM

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VESTIBULE

DIRTYDRESS .ROOM

sterilizer

Stool

q

0N

0o t.

LIQUIDDECONTAMINATION

ROOM

CLEANDRESSINGROOM

A

B

Fig . 30 .3 . A : Suggested layout for a smallpox diagnostic laboratory as conceived in 1969 . (From WorldHealth Organization, 1969a.) B : Plans of part of the smallpox laboratory at the Centers for Disease Control,Atlanta, GA, USA, drawn in 1979 . Note the much more stringent physical containment, with access and exit ofpersonnel through a shower and access of materials through a decontamination box and exit via a two-wayautoclave . Effluent air is passed through a HEPA (high-efficiency particulate air) filter and liquid and solid wastesare sterilized by heat treatment. Within the laboratory, virus is handled within a glovebox or under a laminarflow hood and special precautions are taken with centrifugation and other laboratory manipulations .

Discard Egg

T 0Boiling pan

candler

a c bucketTest tube rack n


Atlanta. Between August 1981 and March1983 urgent diagnostic work on specimensfrom cases of suspected smallpox ormonkeypox was carried out in the SpecialViral Pathogens Laboratory at the Centersfor Disease Control in Atlanta . Work withvariola virus in other laboratories that heldthe virus in 1979 (see Table 30 .6) ceased atvarious times between 1979 and 1982, whentheir stocks were transferred to a variola virusstorage facility in the Centers for DiseaseControl .

Two of the 19 recommendations of theGlobal Commission (World Health Orga-nization, 1980), all of which were accepted bythe Thirty-third World Health Assembly inMay 1980, dealt with the inspection of WHOcollaborating centres approved to hold andhandle variola virus. It was recommendedthat each laboratory that still held variolavirus (whether or not a WHO collaboratingcentre) should report annually to WHO on itssafety measures and should be inspectedperiodically by WHO. The implementationof these recommendations has been describedin Chapter 28 .

1337

Plate 30 .3 . Glovebox facility (Class III biological safety cabinet) in use at the WHO collaborating centre inthe Centers for Disease Control, Atlanta, GA, USA . To eliminate the risk of laboratory contamination, only asingle specimen was handled at a time and the cabinet was sterilized with peracetic acid before another specimenwas introduced .

Justification for the Retention and Useof Variola Virus

The potential danger of experimentationwith variola virus in a laboratory notequipped with full microbiological securityfacilities was dramatically demonstrated bythe Birmingham outbreak, discussed inChapter 23, some 10 months after the lastknown case of naturally transmitted smallpoxhad been reported in Somalia . In response tothe concern expressed by many WHO Mem-ber States, a consultation of public health andvirological experts who were not themselvesinvolved in laboratory work with variolavirus was convened in Geneva in February1979. Its report (WHO/SE/79 .135) was sub-sequently used by the Global Commission forthe Certification of Smallpox Eradication informulating its final report (World HealthOrganization, 1980) . In essence, the consulta-tion recommended that research with variolavirus was justified, at least for the next 3 years,because of the problem then posed by reportsof the isolation of "whitepox" viruses and thework on DNA mapping that provided a

1 33 8

The Maximum Containment Laboratory

The 1977 report and the Laboratory Biosafety Manual recommended the use of amaximum containment laboratory for the handling of dangerous pathogens. In essence,this is a room or a few rooms (or, for veterinary laboratories handling exotic viruses, acomplete laboratory building) in which special measures are taken for the physicalcontainment of dangerous pathogens, to minimize the risk of their escape outside thelaboratory. The principal features of a maximum containment laboratory are :

(1) Controlled access . The entry and exit of personnel and supplies are through airlocksystems . On entering, personnel put on a complete change of clothing, and they shower onexit before changing back into their street clothes.

(2) Controlled air ystem . Negative pressure is maintained by an individual supply andexhaust air mechanical ventilation system with HEPA (high-efficiency particulate air)filters in the exhaust (and in the intake when necessary) .

(3) Decontamination of effluents . All effluents from the maximum containment laboratoryare rendered safe, including the shower water .

(4) Sterilization of waste and materials . A double-door pass-through autoclave is provided .

Properly installed, operated and maintained, these arrangements should prevent theescape of dangerous pathogens from such a laboratory . To protect personnel frominfection and to minimize the risk of contamination of apparatus and cultures, anadditional facility can be operated within the maximum containment laboratory. This iscalled the primary containment facility, of which there are 3 forms

(1) a fixed "Class III" biosafety cabinet (Plate 30.3), in which material is handled viaglove ports and all effluent air is passed through a HEPA filter,

(2) a flexible-film plastic equivalent of a Class III biosafety cabinet, or(3) a positive-pressure ventilated suit, operated only in a special laboratory which

provides a decontamination shower for personnel leaving the risk area .The WHO collaborating centre in Atlanta handled all specimens of known or suspected

variola virus within a Class III biosafety cabinet . One specimen was handled at a timeand the cabinet sterilized with peracetic acid before another specimen was introduced .This practice enabled that laboratory to avoid completely any cases of laboratorycontamination of cultures by its personnel, throughout its operations in smallpoxdiagnosis and research .

method of at least partially solving thisproblem. In January 1979, the WHO Execu-tive Board had asked the Secretariat torecount what measures were being taken inresponse to the Birmingham outbreak. TheSmallpox Eradication unit was able to informthe Board of the proposed consultation andthe anticipated meeting on laboratory safetymeasures, mentioned in the previous section,which were designed to deal with the essenceof the problem-namely, whether furtherresearch with variola virus was necessary and,if so, whether the laboratories in which suchwork was undertaken were microbiologicallysecure.


Reduction in the Number of LaboratoriesRetaining Variola Virus

The possibility of escape of variola virusfrom either of the two high-security labora-tories that now retain it is extremely remote .But in 1976, as global smallpox eradicationappeared imminent, stocks of the virus wereheld by many more than these two labora-tories ; in fact, no one knew how many more .It was reasonable to argue that the smaller thenumber of laboratories holding variola virus,the lower would be the risk of the virusescaping. The Twenty-ninth World HealthAssembly, in May 1976, requested "all

Philosophical Considerations regarding the Destruction of Variola Virus

From time to time commentators in scientific journals have questioned the moralrectitude of destroying all known stocks of variola virus, on the grounds that man shouldnot knowingly cause the extinction of any living thing. With the cloning of variola virusDNA this raises some interesting philosophical problems . Are variola virions, in anampoule, "living things"? In fact they are inert until their genetic potential can beexpressed, and the only "natural" form of living variola virus would be that found in ahuman being, since man was the only known host of the virus . Presumably, the periodicoccurrence of cases of smallpox is not what conservationists have in mind when they arguethat variola virus stocks should never be totally destroyed .

The other problem is whether the cloned fragments of variola virus can be regardedphilosophically as still constituting the essence of life of that virus . Certainly, molecularbiologists are, or soon will be, able to produce all the known viral products from suchcloned fragments. They could even, with considerable (albeit misguided) effort, recon-stitute the intact variola virus DNA molecule and thus by laboratory manipulations, thevirus itself.

When viewed against the regrettable but who, .le extinction of species that resultsfrom human interventions in natural ecosystems, concern about the preservation ofvariola virus seems to us to be misplaced . The only criterion by which to judge thenecessity for the preservation of the virus, we believe, is whether it is necessary forscientific work .

governments and laboratories to cooperatefully in preparing an international registryof laboratories retaining stocks of variolavirus" but, at the same time, urged all lab-oratories which did not require such stocksof variola virus to destroy them (resolutionWHA29.54). This resolution followed in-quiries that the WHO Smallpox Eradicationunit had addressed in 1975 both to gov-ernmental authorities and to the directors of823 laboratories included in the WHO WorldList of Virus Laboratories. The unit had alsoscanned the Index medicus in search ofreferences to papers on variola virus pub-lished from laboratories during the previousfew decades and had written to the directorsof these laboratories to find out whether theyheld stocks of the virus . This informationhaving been obtained, laboratories other thanthe WHO collaborating centres in Atlantaand Moscow and the WHO laboratories forpoxvirus research were asked to destroy theirstocks or transfer them to one of the twoWHO collaborating centres . Governmentswere asked to inform WHO of the response tothis request. Prior to this, the Director of theNational Smallpox Eradication Programmein India had carried out a similar survey in

30. POTENTIAL SOURCES FOR A RETURN OF SMALLPOX 1339

that country and reported that by the end of1976 all stocks of variola virus in laboratoriesin India had been destroyed (Basu et al .,1979). The situation in the 6 WHO regions in1975 and in July 1977 is shown in Table 30.6.

Some noteworthy inferences can be drawnfrom this table . First, the response from bothnational authorities and laboratory directorswas remarkably comprehensive. Apart fromChina, the countries that failed to respondwere small in size and population and wereknown not to have a laboratory that had everhandled variola virus. Secondly, there wereno fewer than 75 laboratories, several in eachWHO region, that were then holding variolavirus. Indeed, the real figure was somewhathigher, for it is known that virologists inseveral laboratories discovered that they didstill possess variola virus, after having, ingood faith, declared to the contrary ; in suchinstances, the stocks were privately destroyedor taken to the nearest WHO collaboratingcentre for destruction. This widespreadpossession of stocks of variola virus is notsurprising. Smallpox was endemic in severalpopulous countries and importations intoEurope, especially, had occurred in the recentpast. In order to have "controls" for the

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Table 30 .6 . Laboratories holding variola virus stocks in 1975 and the reduction in this number by July 1977,in response to requests from WHO

laboratory diagnosis of agents that they rarelyhandle, such as variola virus, virologistsusually keep strains of these agents in theirdeep-freeze cabinets, for comparison with asuspicious isolate. Provided that their staffwere regularly vaccinated and used goodmicrobiological techniques, laboratory direc-tors rightly regarded variola virus as lessdangerous to handle than other agentssometimes encountered in diagnostic labora-tories, such as various rickettsias . Further,even though smallpox might no longer beendemic in their country, it did not occur tothem to destroy their laboratory stocks ofvariola virus. However, in response to therequest from WHO, and bearing in mind theimminent world-wide eradication of small-pox, the directors of 57 of the 74 laboratoriesagreed to destroy or transfer their stocks ofthe virus .

Further reduction in the number of laboratoriesretaining variola virus, 1977-1983

With strong support from the Consulta-tion on the Worldwide Certification ofSmallpox Eradication, convened in Genevain October 1977, and subsequently from theGlobal Commission, WHO continued toexert pressure to try to reduce further thenumber of laboratories holding variola virusstocks. In its final report in December 1979(World Health Organization, 1980) the Glo-bal Commission recommended that : "Nomore than four WHO collaborating centresshould be approved as suitable to hold andhandle stocks of variola virus . A collaboratingcentre would be approved only if it hadadequate containment facilities ." Biocontain-ment standards had been laid down for


a Although China did not respond to the WHO letter of request, stocks of variola virus were then held at the Institute for the Control ofDrugs and Biological Products, Beijing .

such laboratories (SME/77 .2 Rev. 1) and itwas stipulated that each laboratory thatheld variola virus (whether it was aWHO collaborating centre or not) shouldbe periodically inspected by WHO.

The Birmingham outbreak had a dramaticeffect on the attitudes of the directors ofseveral European laboratories, because ofProfessor Bedson's suicide and the extensivepress coverage of the event (see Chapter 23) .By the end of 1979, 5 of the remaining 8European laboratories holding variola virushad disposed of their stocks (Table 30 .7).Every year, at the World Health Assembly,Member States continued to express concernabout variola virus stocks in laboratories.Gradually, the remaining laboratoriesholding such stocks responded to the requestsfrom WHO to destroy or transfer theirholdings, since with the eradication ofsmallpox the rationale for retaining stocks ofthe virus in any laboratory except the WHOcollaborating centres in Atlanta and Moscowhad disappeared . Scientific developments,notably the cloning of restriction fragmentsrepresenting the total genome of variola virus(see Chapter 2), removed the last objections,

Table 30 .7. Laboratories holding variola virusstocks at the end of calendar years1977-1983

a WHO collaborating centre at the Centers for Disease Control,Atlanta, USA.

b WHO collaborating centre at the Moscow Research Institutefor Viral Preparations, Moscow, USSR.

Number of countriesNumber of laboratories By July 1977

WHO regionInformation

soughtResponded

Respondedto WHO

Holdingvariolavirus in1975

Destroyed ortransferredvariola virus

Retainedvariolavirus

Africa 46 43 is 5 4 1

Americas 34 34 506 18 13 5

South-East Asia II II 57 13 13 0

Europe 36 36 185 29 19 10

Eastern Mediterranean 23 23 25 3 3 0

Western Pacific 31 29 35 6(7)a 5 1(2)3

Total 181 176 823 74(75) 57 17(18)

Continent 1977 1978 1979 1980 1981 1982 1983

Africa I I I I I I 0America 5 3 2 I I I I aAsia 2 I I I 0 0 0bEurope 10 8 3 3 2 I 1


voiced by the Ministry of Health of SouthAfrica (see Chapter 28, Plate 28 .4), and by theend of 1983 only 2 laboratories in the world,both with newly completed biocontainmentfacilities, still held stocks of variola virus : theWHO collaborating centres at the Centers forDisease Control, Atlanta, and the MoscowResearch Institute for Viral Preparations . Ata meeting in March 1986, the Committee onOrthopoxvirus Infections recommended thatall stocks of variola virus should be destroyedin October 1987, subject to approval by anad hoc committee to be convened by WHO.

There remains the possibility that am-poules containing variola virus may still bestored in deep-freeze cabinets in laboratories,unknown to anyone in the laboratory orindeed in the country concerned. In mostlaboratories deep-freeze cabinets are rarelythoroughly cleaned out, though sections ofthem may be. As staff replacements occur andsmallpox recedes into the past, all memoriesof working with variola virus will be lost .Two such incidents have come to the noticeof the WHO Smallpox Eradication unit-thediscovery of variola virus stocks in alaboratory in California in 1979 (Emmons,1979), and in a laboratory in the UnitedRepublic of Tanzania during the visit of aWHO international commission in 1979(J. G. Breman, personal communication,1979). Another possible but unconfirmedexample was described in London in Decem-ber 1985 . The dangers posed by such forgot-ten material are very small provided thelaboratory carries out the recommendedsafety measures-namely, the destruction byautoclaving of any unlabelled ampoules orany ampoules labelled "variola virus" or"smallpox virus". This risk is hypotheticaland in any case beyond the control of WHOor any other organization .

DELIBERATE RELEASE

The United Nations (1970) and a WHOgroup of consultants (World Health Orga-nization; 1970) both included the smallpoxvirus among some 20 infectious agents thatcould be used in biological warfare . However,it ranked low in suitability compared withseveral other viruses, rickettsias and bacteria .As the WHO consultants commented :

"Because of the effectiveness of the vaccine andthe relative ease with which it can be produced

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and administered, it is unlikely that smallpox viruswould be used as an agent for a large-scalebiological attack against countries systematicallypractising periodic vaccination .

"The variola virus, however, can easily beemployed for acts of sabotage . Such acts, at selectedpoints within a country, could have serious socio-economic effects unless efficiently dealt with bythe public service."

That was written in 1969 . At present nocountry is "systematically practising periodicvaccination" and as time passes the immunityof those who have been vaccinated will waneand populations will become completelysusceptible.

In 1972 many countries of the world signeda convention outlawing the use of biologicalweapons in warfare (United Nations, 1984) .Unfortunately, as international conventionsare subject to infringement, this does notcompletely exclude the possibility that variolavirus might be deliberately released as a meansof warfare . However, the risk of any such actleading to the re-establishment of endemicsmallpox should not be exaggerated . As hasalready been mentioned, smallpox spreadcomparatively slowly, by face-to-face contact .Unless the public health services had comple-tely broken down, the existence of reservestocks of vaccine (see Chapter 28) and thecapacity for the production of vaccine to berapidly reactivated would ensure the contain-ment of any outbreak that followed a deliber-ate release of variola virus. With the cessationof vaccination and vaccine production, it willbecome increasingly difficult for any personor group contemplating the release of variolavirus to assure themselves and their colleaguesof protection against smallpox . A country'sresumption of vaccination against smallpoxwould now be interpreted as a sign that itmight be considering the use of variola virusfor aggressive purposes.

Deliberate release, or the threat of it, by anindividual or a group, as an act of sabotage orterrorism, is remote because access to thevirus is so restricted . A document madepublic in December 1984 (Young & Lenarcic,1984) describes tests made in 1964 and 1965with aerosols containing Bacillus subtilis as amarker. The aerosols were experimentally butsecretly produced in a crowded airport inWashington, DC, USA, to test the possibilitythat variola virus might be released in thisway and thus cause "inexplicable" outbreaksof smallpox a few weeks later in the diverse

1 342 SMALLPOX AND ITS ERADICATION

Plate 30 .4 . Only 2 laboratories in the world retain variola virus. A: Entrance to the maximum containmentlaboratory at the Moscow Research Institute for Viral Preparations, USSR . The control panel for biowastedisinfection and disposal is on the right and on the door is a list of persons authorized to enter . The door is keptlocked and the key retained by the director. B : Variola virus is stored in two nitrogen vapour phase freezers ina maximum containment laboratory at the Centers for Disease Control, Atlanta, GA, USA .


places to which infected passengers travelled .The existence of such a possibility emphasizesthe need for "military" as well asmicrobiological security in laboratoriesholding variola virus stocks. It is melancholybut perhaps realistic to suggest that thepossibilities of biological warfare or terrorismnow constitute the chief reason for holdingreserves of vaccine and for maintainingepidemiological and laboratory expertise forthe diagnosis and control of smallpox .

REACTIVATION AND EXCRETIONOF VIRUS IN HUMANS

Viruses of many viral families persist forlong periods in their hosts and viruses insome families are persistently (Arenaviridae)or intermittently (Herpesviridae) releasedinto the environment . In addition, there isnow a vast experience of the reactivation oflatent herpesvirus infections (especially cyto-megalovirus) in immunosuppressed persons,both those suffering from lymphoreticulardiseases and those in whom chemical suppres-sion of cell-mediated immunity is employedto prevent transplant rejection . In addition,steroid therapy has immunosuppressiveeffects and is associated with the reactiva-tion of latent infections . Such therapies havenow been practised for many years, in manycountries, but no evidence has ever come tolight of the reactivation of variola or vacciniavirus in immunosuppressed persons or thoseon steroid therapy. This supports the viewelaborated in Chapter 3 that the poxviruses ingeneral and variola and vaccinia virusesin particular do not produce persistent infec-tions. This hypothetical possibility can bedismissed .

VIRAL PERSISTENCE IN THEENVIRONMENT

Although it does not cause persistentinfection in man, variola virus in scabs is,for a virus, very resistant to inactivation,especially at moderate temperatures and outof sunlight . Clothing and bedding fromsmallpox cases, which were often heavilycontaminated with virus from salivary excre-tions and skin lesions, were an occasionalsource of outbreaks of smallpox amonglaundry workers (England and Wales, Min-istry of Health, 1928b), and virus on im-ported raw cotton was suspected (without

1343

good reason, according to Dixon, 1962) ofcausing some outbreaks in England. How-ever, the periods for which the virus survivedto cause such infections were measured indays or weeks-not years .

Interred corpses have also been suggestedas a possible source of infectious virus (e.g .,Razzell, 1976), but the only evidence isanecdotal and comes from situations in whichthe possibility of infection by direct contactwith cases of smallpox could not be excluded .The chance of viable variola virus survivingin corpses or coffins for the length of timethat has elapsed since there were numeroussmallpox deaths in temperate climates seemsto us to be extremely remote, and the topicdoes not lend itself to scientific investigation .

A more bizarre possibility has been raisedby Ewart (1983). Noting that bacteria can bepreserved in permafrost for years (Boyd &Boyd, 1964), he suggested that active variolavirus might still be preserved in interredbodies at York Factory on the shores ofHudson Bay in northern Canada . Six Indianswere reported to have died of smallpox therein 1782, and it was the practice to inter thebodies of the dead in wooden coffins, whichwere placed in graves hacked out of the rock-hard earth of the permafrost . Most of thisgraveyard has now been eroded by the nearbyHayes river, but Ewart's contention is thatvariola virus could be preserved for longperiods in some such way, somewhere in theArctic. Whether viable variola virus could bereleased from a thawed corpse in such a way asto infect a susceptible person is a matter ofconjecture . There is no scientific evidence onwhich to base a firm reply to speculations ofthis kind ; one can merely say that thecircumstances that would give rise to infec-tion from such a source are exceedinglyremote and impossible to study .

In whatever form the virus may persist inthe environment, it is gradually inactivated,probably even at subzero temperatures (seeChapter 2). As the intervals since the lastcases of smallpox in various parts of the worldhave extended from months to years and nowto decades, this danger has lessened until itmust now (1987) be considered to be verysmall indeed .

"TRANSFORMATION" AS A SOURCEOF VARIOLA VIRUS

The term "transformation" belongs to anera predating the development of microbial

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Plate 30.5 . Albert Herrlich (1902-1970), aleading German virologist and public health expertwho was Director of the Institute for ComparativeTropical Medicine at the University of Munich . Hewas principal author of major books on the poxvirusesand on vaccination and carried out a definitive studywhich showed that variola virus could not be "trans-formed" into vaccinia virus by passage in laboratoryanimals or cattle .

genetics . It now has a specific meaning : thealteration of the genome of a bacterium oreukaryotic cell by the incorporation of DNAfrom another source. As it used to be appliedto the orthopoxviruses, the term was mostfrequently used to describe what was re-garded as the conversion of variola virus intovaccinia virus, usually by passage throughcows. It is impossible to achieve any suchchange when experiments are carried outunder conditions that rigidly exclude thepossibility of contamination (Herrlich et al .,1963) .

The "white clones" of monkeypox virus,described above, were viewed by Maren-nikova and her colleagues as examples ofthe "transformation" of monkeypox intovariola virus, but the evidence presented


earlier argues strongly that these isolatesarose because of the contamination ofmonkeypox virus stocks with variola virus .Likewise, there is no likelihood that eithervaccinia or cowpox virus, of which suitablestrains yield a great variety of white pockmutants, could give rise to variola virus byone or a few mutational steps . The clear-cutdistinctions in the DNA maps of differentspecies of Orthopoxvirus (see Chapter 2)preclude such a possibility .

GENERAL CONCLUSIONS

At this time, 10 years after the last knowncase of endemic smallpox, there is only onecredible source from which another case ofsmallpox might arise-namely, the infectionof a susceptible person with variola virus heldin storage, either in a known location (stocksheld in the WHO collaborating centres inAtlanta or Moscow) or in an unknown place .The latter could be either a stock maintainedfor possible use in microbiological warfare ora forgotten and possibly unlabelled specimenlying in a deep-freeze cabinet in a laboratoryin which smallpox diagnosis or research wasonce performed . The likelihood of variolavirus still surviving on scabs or associatedwith corpses or coffins in a form that mightgive rise to new cases of smallpox is nowextremely remote.

Unless there were a complete breakdown ofhealth services, so that countermeasurescould not be mounted, the occurrence of anaccidental case of smallpox could be readilycontained, as was apparent in the Birming-ham outbreak. Even microbiological warfarewith variola virus should not present asignificant hazard, in terms of the re-establishment of smallpox as an endemicdisease. Its nature would be quickly recog-nized and countermeasures could be taken,calling if necessary on the personnel andmaterials provided by WHO as part of the"insurance policy" (see Chapter 28) .

potential sources for a return of smallpox

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