BACTERIOLOGICAL REVEWS, Mar. 1973, p. 1-18Copyright ( 1973 American Society for Microbiology

Vol. 37, No. 1Printed in U.S.A.

Monkeypox VirusCHENG T. CHO AND HERBERT A. WENNER

Department of Pediatrics, University of Kansas Medical Center, Kansas City, Kansas 66103, and Children'sMercy Hospital of the University of Missouri School of Medicine, Kansas City, Missouri 64108

INTRODUCTION ......... ..................................... IHISTORICAL NOTE ........... ................................... 2Recognition of Monkeypox in Nonhuman Primates ....... ..................... 2Human Infection with Monkeypox Virus ..................................... 3

CLASSIFICATION .......... .................................... 3MONKEYPOX VIRUS AS AN INFECTIOUS AGENT ...... ................... 4Physical and Chemical Properties .............................................. 4Growth Characteristics in Cultured Cells ..................................... 5Cytopathic Effect ............ .................................. 5Plaque Formation ............. .................................. 5Viral Multiplication .............. ................................ 5

Growth on Chorioallantoic Membrane .......................................... 6Hemagglutinin Production .............................................. 6Antigenic Composition .............. ................................ 7

INFECTIONS IN ANIMAL HOSTS ............................................ 7Host Range .............................................. 7Routes of Transmission .............................................. 7Clinical Manifestations .............................................. 7Pathogenesis .............................................. 9Viremia .............................................. 10Histopathology ........ ...................................... 10Antibody Responses ........... ................................... 10Cross Immunity ........ ...................................... 10

SPECIAL ASPECTS ..............................................11

Antiviral Compound, Methisazone ............................................. 11Infection in Immunosuppressed Hosts .......................................... 12

EPIZOOTIOLOGY ......... ..................................... 12CLINICAL AND EPIDEMIOLOGICAL IMPLICATIONS ...... ................. 14Monkeys as a Reservoir for Smallpox? ........................................ 14

Monkeypox in Man .............................................. 14

CONCLUSIONS 14

ADDENDUM 15

LITERATURE CITED 15

INTRODUCTION

At this time in the history of smallpox amajor effort is being made to eradicate thedisease from people in geographic areas of highprevalence (2). Variola is considered a diseasetransmissible from person-to-person; no otherreservoir of the virus responsible for smallpoxhas been identified. The recent discovery of aclosely related pox disease in monkeys (70),called "monkeypox" has provoked inquiriesregarding clinical and epidemiological relation-ships of these two diseases. In its clinicalaspects monkeypox is analogous to smallpox,or to generalized vaccinia developing in humanbeings.

In relation to the epizootiology of monkeypoxseveral important questions arise regarding itsorigin. Did it originate accidentally in captive

monkeys from variola virus harbored by ahuman associate? Is it a variant of variola virusadapted long ago to simians, and now enzooticin certain species? Possibly the same questionscould be asked with respect to vaccinia virus.Finally, address is made to the query whetheror not monkeypox might be caused by anotherpreviously undefined member of the variola-vaccinia complex. Additionally, epidemiologi-cal implications must per force be brought intofocus, for natural infection from monkeypoxvirus (MPV) has been reported in humanbeings (3, 4).

Fourteen years have elapsed since the recog-nition of MPV. From a nosographic point ofview, this disease in a nonhuman primate hasproved to be a very satisfactory model for astudy of the variable characteristics of infec-tion and immunity of a poxvirus infection. The

on March 9, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

CHO AND WENNER

purpose of this review is to bring to the foreavailable information concerning (i) MPV and(ii) the evolution of the disease in simianspecies, and (iii) to reexamine the epidemiolog-ical potential with respect to an extrahumanreservoir of another poxvirus capable of causinghuman illness.

HISTORICAL NOTE

Recognition of Monkeypox in NonhumanPrimates

Smallpox has been a disease of major impor-tance to man for many centuries (25). Natu-rally occurring epidemics of pox diseases amongnonhuman primates have occasionally beenreported. In their survey in 1968, Arita andHenderson found seven recorded episodes ofpox diseases in nonhuman primates; one ofthese was confirmed by virus isolation (6).

In 1958, von Magnus et al. (7) observed twooutbreaks of a nonfatal poxlike disease in twoshipments of cynomolgus (Macacacynomolgus) monkeys arriving in Copenhagenafter shipment from Singapore. A poxlike skineruption developed among the animals be-tween 51 and 62 days after arrival in Copenha-gen. Approximately 20 to 30% of the animalsdeveloped clinical illness. The epizootics inCopenhagen suggested slow recruitment of sus-ceptible monkeys in a cycle of inapparentinfections with subsequent intensification andthe emergent clinical expression of monkeypox.Whether the risk of disease was dependentupon intensified replication of MPV in vivo orenhanced invasiveness, or both, is unknown.We assume that the primary source of MPVcame from a companion monkey; whetherMPV came from a recent (nasopharyngealcolonization) or remote (latent carrier of virusin tissues) infection is also unknown. The latterkind of origin is possible since a virus analogousto MPV has been recovered from the kidneys ofapparently healthy monkeys (quoted in 5, 6,31).

Viruses isolated from the dermal lesions ofaffected monkeys produced pock lesions on thechorioallantoic membrane (CAM) of develop-ing chicken embryos, cytopathic effects (CPE)in mammalian cell cultures, encephalitis inmice, and dermal lesions, as well as keratitis, inrabbits. Morphologically, the prototype virushad the rectangular shape and size typical ofother known poxviruses (200 by 250 nm);antigenically, it was closely related to thevaccinia-variola subgroup of poxviruses (Table1). Since it appeared to differ from variola andother known poxviruses, it was named mon-

keypox virus and given recognition as a specificmember of the group.

In 1959, an outbreak of monkeypox occurredin the animal quarters of Merck, Sharp, andDohme in Philadelphia (63, 64, 67). Within acolony of 2,000 monkeys (56% Macaca mulatta,41% Macaca philippinensis, and 3% Cercopi-thecus aethiops var. subaeus) at least 10% ofcompanion monkeys were considered to havebeen infected. Less than 0.5% of those affecteddied. The affected monkeys were predomi-nantly Macaca philippinensis, although Ma-

TABLE 1. Classification of the poxvirus groupa

Infections in natureSubgroup

Man Animals

I. Variola-vaccinia virusesVariola major (small-

pox) ................Variola minor (alas-

trim) ................Vaccinia ..............Cowpox ...............Monkeypox ...........Ectromelia (mousepox)Rabbitpox ............Buffalopox ............

II. Orf-like virusesBovine papular stomati-

tis ..................Contagious pustular

dermatitis (orf) .....Milker's nodules .......

III. Avian poxvirusesCanarypox ............

Fowlpox ..............Pigeonpox .............

Turkeypox ............

IV. Myxoma-fibroma virusesRabbit myxoma .......

Rabbit fibroma ........

Squirrel fibroma .......

Hare fibroma ..........

V. Unclassified poxvirusesMolluscom contagi-osum ...............

Yaba tumor virus.Goatpox ..............

Sheeppox .............

Swinepox .............

Entomopox (insect viruses;Horsepox ..............

Camelpox .............

Tanapox ..............

+

a See references: 9, 26, 54, 65.

Monkeys (?)

Calves, sheepCattleMonkeysMiceRabbitsBuffalo

Cattle

SheepCattle

CanaryChickensPigeonsTurkeys

RabbitsRabbitsSquirrelsHares

MonkeysGoatsSheepSwine

HorsesCamels

2 BACTERIOL. REV.

on March 9, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

MONKEYPOX VIRUS

caca mulatta also developed clinical evidenceof disease.During 1962, monkeypox was recognized in

the primate colony of the Walter Reed ArmyInstitute of Research, Washington, D.C. (49).The disease was observed initially in a cyno-molgus monkey which had been given totalbody irradiation. Subsequently, similar lesionsdeveloped in two other cynomolgus monkeys;of the two, one had been irradiated; the other,an apparently healthy animal, was untreated.Both irradiated monkeys died of the disease,whereas the healthy animal survived. Monkeysin the colony were studied serologically in orderto determine rates of past infection. Among 27cynomolgus monkeys exposed to MPV, 25(93%) had specific antibodies, whereas, inanother group of 45 cynomolgi wherein expo-sure had been unrecognized, only 5 (11%) hadspecific antibody. Fifty-two of 67 rhesus, and 6of 14 African green monkeys were also seroposi-tive. While it is likely that clinically inappar-ent infection occurred widely among exposedmonkeys, no pre-enzootic sera were tested;hence rates of seroconversion remain unknown.

In 1964, Peters reported an outbreak ofmonkeypox in the Zoological Garden, Rotter-dam, Netherlands. The affected animals in-cluded giant anteaters (Myrmecophagatridactyla), orangutans (Pongo pygmaeus),chimpanzees (Pan troglodytes), gorillas (Go-rilla gorilla), guenons (Cercopithecus sp.),squirrel monkeys (Siamiri sciurea), macaques(Macaca sp.), gibbons (Hylobates lar), andmarmosets (Hapale jacchus) (31, 60, 61).Eleven of the 23 affected animals died, includ-ing 6 of 9 orangutans, 3 squirrel monkeys, 1gibbon, and 1 marmoset. MPV was isolatedfrom 1 of the anteaters, 7 orangutans, and 3monkeys of various species (31). During 1964and 1965, three silent MPV infections wererecognized in colonies of cynomolgus monkeysat Utrecht, Netherlands. These infections wererecognized by recovery and identification of avirus obtained from kidney cells cultured fromapparently healthy monkeys (31).

In 1967, the World Health Organization(WHO) surveyed 26 major biological institutesin which large numbers of monkeys were usedto study outbreaks of monkeypox. Besides thefive outbreaks confirmed by virus isolationmentioned above, there were in the U.S.A.,between 1965 and 1967, four other instances ofpoxlike disease compatible with monkeypox;none of the diseases was confirmed virologically(6).Marennikova et al. (48) subsequently com-

pared the properties of five strains of MPV;

four of the five MPV strains tested were similarin biological properties and could be readilydistinguished from both variola and vacciniaviruses. A fifth strain differed from the otherfour in several characteristics and had proper-ties indistinguishable from variola virus.

Human Infection with Monkeypox VirusSix human infections from MPV were re-

ported in 1970 from the Democratic Republic ofthe Congo, Liberia, and Sierra Leone (3, 4). Allsix patients were unvaccinated; each illnesswas diagnosed as smallpox based on clinicalfeatures of infection. The agents recoveredfrom these patients were studied intensively byseveral reference laboratories and each wasidentified as MPV. The first case occurred in a9-month-old infant residing in the DemocraticRepublic of the Congo. The four affected chil-dren in Bouduo, Liberia, ranged from 4 to 9years of age. Three children who were play-mates developed rashes on successive days,thus suggesting common exposure. The fourthchild resided 12 miles distant from the others.The sixth case occurred in Sierra Leone; thepatient, a 24-year-old male, had removed thestomach and intestines from a "red monkey" 3to 4 weeks preceding onset of illness. None ofthe patients died of monkeypox. Moreover, noother member of the households of these pa-tients developed the disease. According toHenderson, another case had been discoveredsince the last report (41).

Large numbers of monkeys and apes inhabitthe affected areas; they are frequently killedand eaten by people, and their skins are used inhouseholds. Five of 18 monkeys (species un-stated) captured near Bouduo yielded seracontaining low titers of hemagglutination inhi-bition (HI) antibody against MPV. Four of 16chimpanzees captured in Sierra Leone werealso seropositive. In striking contrast, seraobtained from 55 monkeys (species unknown)in Nigeria where human infection is unrecog-nized were seronegative. Additionally, sera ob-tained from several thousand African andAsian monkeys have apparently failed to neu-tralize MPV.

CLASSIFICATIONAt the present time there is no generally

accepted overall classification of the pox-viruses. The commonly used classifications arelisted in Table 1. The available evidence indi-cates that MPV belongs to the subgroup ofvariola-vaccinia viruses. An important featureof this subgroup of viruses is that recovery from

3VOL. 37, 1973

on March 9, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

CHO AND WENNER

infection with one member confers immunityto another. Because of the lack of apparentdifferences in physical and chemical propertiesamong members of the variola-vaccinia sub-groups, biological properties have frequentlybeen used to distinguish one from the other (25,29). The major distinguishing features of MPVand other poxviruses are listed in Table 2. Onthe basis of these data, MPV has properties.placing it in an intermediate position betweenvariola and vaccinia viruses. However, studiesof MPV in monkeys in the CAM of developingchicken embryos and of its ceiling temperaturesuggest that MPV is more closely related tovariola than to vaccinia virus (11, 18). Failureof pock formation at 40.5 C has been used todifferentiate MPV and variola from vaccinia,cowpox, and rabbitpox viruses (11).

In addition to MPV, other poxviruses alsoproduce poxlike diseases in nonhuman pri-mates. These include smallpox (6, 13, 36, 58,59), Yaba tumor virus (10), Yaba-related virus(15, 22, 38, 53, 57), and molluscum contagi-osum (27). Most species of monkeys are notvery susceptible to experimental infection withsmallpox virus (5, 14, 36, 58). Natural infectionis also infrequent (6). Clinical distinction be-tween smallpox and monkeypox, both in mon-keys and in man is not possible (3, 75). Someoutbreaks of poxlike diseases in monkeys, as-

cribed to but unverified as smallpox, may

relate to infections caused by other viruses,e.g., monkeypox or herpesviruses (24). Naturalinfection with Yaba monkey tumor virus hasbeen reported only once in Yaba, Nigeria, in1957 (10). Other encounters with this tumorvirus have primarily related to experimentalinfection in monkeys and accidental or experi-mental infections in human beings (1, 5, 32, 33,43; Mira and Sheek, personal communication).

In 1966, three separate outbreaks of infectionfrom a Yaba-like virus occurred at about the

same time in primate colonies located in Cali-fornia, Oregon, and Texas (15, 38, 57). In eachinstance the affected monkeys had been re-

ceived from one distributor. Animal caretakers,particularly those handling monkeys, devel-oped cutaneous lesions similar to those encoun-

tered in affected animals (22, 53). The cutane-ous expression was characterized by the devel-opment of a solitary lesion in contrast to thedisseminated eruption of monkeypox. Similarsolitary lesions have been encountered among

human beings during outbreaks of Tanapox(26). Molluscum contagiosum has been recog-

nized in chimpanzees (27).

MONKEYPOX VIRUS AS ANINFECTIOUS AGENT

Physical and Chemical PropertiesPublished information concerning physical

and chemical properties of MPV is limited inscope. MPV has the morphological characteris-tics of other poxviruses. It is brick-shaped witha size of about 200 by 250 nm (55, 63, 70). Thevirus is resistant to ether and relatively resist-ant to dessication both in heat and cold. Heatstability tests indicated that 20 min of heatingat 40 C caused no significant loss of infectivity,whereas 20 min of heating at 50 or 56 C resultedeither in almost complete (92.3%) or completeloss of infectivity (66). Repeated freezing andthawing up to 12 times produced a loss of only0.2 to 0.6 log10 of infectious virus. The stabilityof virus stocks stored at different temperatureswas studied over a 15-month period. At the endof 6 months, the infectivity titer of stocks heldat 4 C remained unchanged from the original;however, at -20 C there was a 2.2 log10, and at-70 C a 1.5 log10 loss of infectious virus. By theend of 15 months of storage, the loss was 2.5log10 at 4 C, 3 log10 at -70 C, and more than 4log10 at -20 C (66). Various chemicals such as

TABLE 2. Biological properties of variola, vaccinia, and monkeypox viruses (16)

Properties Variola Vaccinia Monkeypox References

Lesion on CAMa Small Large Small 31, 64, 70Maximum temperature for growth 38.5 C 41.0 C 39 C 11on CAM

Dermal lesion in monkey Generalized Local Generalized 37, 75Rabbit skin passage No Yes Yes 31, 49, 64, 70Lesion in rabbit skin Not hemorrhagic Not hemorrhagic Hemorrhagic 31Pathogenicity in 3-week-old mice No Fatal Fatal 49, 64, 70by i.c. route

Plaque formation in chicken em- No Yes Yes 51, 56bryo cell culture

a CAM, Chorioallantoic membrane of chicken embryo.

4 BACTERIOL. REV.

on March 9, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

MONKEYPOX VIRUS

formaldehyde, sodium dodecyl sulfate (SDS),chloroform, methanol, and phenol also inacti-vate MPV (66, 70).

Growth Characteristics in Cultured CellsCytopathic effect (CPE). A broad spec-

trum of cultured cells is permissive of growth ofMPV with an associated cytopathology. Thesecultured cells include primary and secondarylines of kidney cells derived from rhesus, cyno-molgus, and African green monkeys (20, 49, 66,70); cells from rabbit, bovine (49, 66) andguinea pig kidneys (48); mouse liver cells (66);and some of human origin, i.e., amnionic andhuman lung fibroblasts (31, 48, 66, 70). CPEhas not been observed in our laboratory inHep-2 cells, certain lines of HeLa cells, andchicken embryo cell cultures (66), althoughothers (48, 70) have reported CPE in these cellcultures. Marennikova et al. (48) reported thata strain of MPV "64-7275" had all the proper-ties of variola virus and was unable to replicatein a continuous line of pig embryonic kidneycells which supported four other strains ofMPV.The CPE of MPV in most cell cultures is

characterized by rounding up, granulation, andcondensation of cells and then final detach-ment from the side of the glass, leaving micro-scopic visible "holes" in the residual cell mono-layer (20, 70). Affected cells in monkey kidneyand human amnion cell cultures are intercon-nected by threadlike syncytial elongations, butsuch cellular bridges are not apparent in HeLacells (70). The time of appearance of CPE inMPV-infected cell cultures is a function ofmultiplicity of infection. Depending upon thesize of inoculum, CPE- of MPV-infected CV-1cells (a continuous line of African green mon-key kidney cells) may be observed as early as 8h or as late as 10 days or more (20). When thesuspension of pustular material from infectedmonkeys is inoculated into such tissue cul-tures, the CPE usually develops in 2 to 3 days.Complete destruction occurs after 5 days ofincubation (70). In tissue cultures the infectiv-ity titers of most of the passage fluids varybetween 10-4 and 10-6 50% tissue cultureinfective doses (TCID)5Jml (20, 70).The physical characteristics of CPE pro-

duced by MPV in monkey kidney-cultured cellscannot be distinguished from those of variola(31) and vaccinia viruses (20).Plaque formation. MPV regularly forms

plaques in a variety of cultured cells (20, 51, 56,66). Generally, tissue culture cells that give riseto CPE also form discrete plaques (66). When

MPV-infected monolayers of monkey kidneycells grown in petri dishes are overlayed withagar and stained with neutral red, well-definedplaques 2 to 3 mm in diameter can be regularlydemonstrated. Thus, the plaque methods canbe used for relatively precise quantitative stud-ies of MPV (17,20,66).

It has been suggested that MPV may bedifferentiated from variola virus by the smallersize of the plaques (51) and by an ability toMPV to form plaques in chicken embryo fi-broblasts (51, 56). Attempts to induce plaqueformation in chicken embryo fibroblasts withboth variola and alastrim have failed (56). Theinability of MPV to form plaques in chickenembryo fibroblasts has also been reported byothers (66).Viral multiplication. Studies on the course

of MPV infection in tissue culture cells indicatethat the kinetics of intracellular replication-namely synthesis of viral antigens, changesin cell morphology, formation of inclusion bod-ies, and release of virus from cells-resemblethose of vaccinia (23, 30, 68, 69) and variolaviruses (34, 62). The minor variations notedbetween these viruses may be attributable totype of cell culture, multiplicities of infection,and conditions of cell growth, among otherfactors.

After inoculation of an appropriate quantityof virus into primary rhesus monkey kidneycells or kappa cells (a continuous line of rhesusmonkey kidney cells), the majority (75 to 85%)of the virions are attached to cell receptorswithin 2 h and all (100%) are similarly attachedwithin 4 h (66). The uncoating process beginsat 2 h in monkey kidney cells, and synthesis ofmessenger ribonucleic acid is necessary for theprimary uncoating (79). The detailed biochem-ical events associated with MPV replication areunknown; presumably, they are similar tothose of vaccinia virus (44, 78).A one-step growth curve of MPV in CV-1

cells (Fig. 1) using a multiplicity of infection of2 plaque-forming units (PFU)/cell revealed a6-h period of partial eclipse, presumably repre-senting the period of attachment, uncoating,and synthesis of the earliest virions. There-after, virus replication occurred at an incre-mental rate until exhaustion of available sub-strate by the 14th hour. The pattern of increaseof "cell-free virus" followed closely that of cell-associated virus, with a lag of 3 or 4 h betweenintracellular maturation and release into ex-tracellular milieu. The time required for MPVto reach maximal levels of infectivity variedwith multiplicity of infection and type of cell

5VOL. 37, 1973

on March 9, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

CHO AND WENNER

culture used in tests (20, 66). Release of virusfrom infected cells into culture fluid is approxi-mately 1% for kappa cells (66) and 10% forCV-1 cells (20).By using the immunofluorescent method,

MPV antigens can be revealed in infected CV-1cells just before detectable increases of infec-tious virus (Fig. 1). Cytoplasmic immuno-fluorescence is the general rule, but, not infre-quently, antigens may be present also in thenuclear area or in long cellular bridges ofinfected cells (20). Inclusion bodies can bedemonstrated readily in MPV-infected cell cul-tures (20, 31, 63). The development of cytoplas-mic inclusions in MPV-infected monkey kid-ney cells coincides with the kinetic aspects ofviral replication (Fig. 1). The inclusions appearto contain both deoxyribonucleic acid and otherviral antigens as shown by histochemical andimmunofluorescent staining (20).

Growth on the Chorioallantoic MembraneMPV produces pocks on the CAM of de-

veloping chicken embryos which are quite simi-lar to those of variola virus; the pocks are con-sistently smaller in size than those of vacciniavirus (18, 31, 70). Inoculation on the CAM ofdermal pustular material, or blood obtainedfrom monkeys infected with MPV, was fol-lowed initially by edematous clouding of themembrane and development of small discretepocks (18, 70). On serial passage of the CAM-adapted virus, the edematous reaction sub-sided and small opaque dome-shaped pockswere formed. The titer on CAM of the originalpustular materials may reach levels as high as108 PFU (70). In general, pocks on the CAM arenonhemorrhagic (18, 31, 70); however, pockswith hemorrhage in the center have been en-countered. Four strains of MPV studied byMarennikova et al. (48) ("Copenhagen""65-31," "65-32," and "7-61") produced pockswith hemorrhagic centers, whereas one strain("64-7275") was consistently nonhemorrhagic.MPV forms pocks on the CAM when in-

cubated between 33 C and 39 C (11, 18). Themaxiumum temperature permissive of pockformation on CAM varies for members of thepoxvirus group (11); the ceiling temperaturerange has been reported as follows: alastrim37.5 C, variola 38.5 C, monkeypox 39.0 C,ectromelia 39.0 C, cowpox 40.0 C, vaccinia 41.0C, and rabbitpox 41.0 C. These ceiling temper-atures have been used as one of the means ofdifferentiating strains of poxviruses within thevaccinia-variola subgroups.

5

4

-3 3

2

0 8

NOVI S

16 24

FIG. 1. One-step growth curves of MPV in CV-1cells (20). The data were derived from multiplicity ofinfection of 2 PFU/cell. Infectivity titer is expressedin log ,PFU/0.1 ml (la, cell-associated virus; lb,cell-free virus). Hemagglutinin titer is expressed as

reciprocals of dilution of infected culture (Ha, cell-associated fraction; Hb, cell-free fraction); (top) thetemporal course of development of cytoplasmic inclu-sions (IC) and of cellular immunofluorescence (IF).Reproduced by permission (Proc. Soc. Exp. Biol.Med. 139:1206-1212, 1972).

Hemagglutinin Production

MPV, like some other poxviruses, has an

associated hemagglutinin (HA; see references20, 21, 29, 44, 48, and 70). The (HA) of MPVagglutinates erythrocytes obtained from chick-ens, but not from mice (70). Among five strainsof MPV tested, four strains produced HA titersbetween 1:64 and 1:128 (48). Another strainproduced only a low titer of HA (1: 4) similar tothat of variola virus. Production of HA islargely dependent upon the type of cell cultureemployed for infection. During the initial stud-ies of the "Copenhagen strain" by von Magnuset al. (70), no HA was demonstrable; however,in later studies of other cell cultures, HA was

...................... IC-__________----IF

la

.0lb

l l

l l

I*I*Ha 16

I I /

oh\/ 1 /

0 \* ,/ /

/ / // / / Hb

/ //

IV/,1,

MAn AO -Ace

6 BACTERIOL. REV.

on March 9, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

MONKEYPOX VIRUS

detected (20, 48). The titer of HA is generallyhigher in extracts from infected CAM thanfrom tissue culture fluids (48, 70). Finding thatthe amount of HA produced by the same virusstrain varies in different tissue systems used forviral growth suggests that, although the viralgenome contains information to cause synthe-sis of HA, the information necessary for itssynthesis resides in the genome of the host cell(44).HA of MPV is present in both "cell-free" and

"cell-associated" fractions of MPV-infectedcells and increases in titer value during the latephase of virus multiplication (Fig. 1). HA ofMPV resembles that of vaccinia virus (46)where HA becomes detectable only after ma-ture virus has accumulated. As with otherpoxviruses (44), the HA ofMPV appears to be anonessential part of the virion, since dissocia-tion of infectivity and HA activity can beclearly demonstrated by sucrose density cen-trifugation (Cho, unpublished data). Viral par-ticles separated in the sediment containedlittle HA and high infectivity, whereas the"top-band" of the supernatant fluid containedabundant HA and little infectivity.

Antigenic CompositionMPV has a complex antigenic structure.

This virus shares with vaccinia and variolaviruses common structural and soluble anti-gens (31, 70, 77). Studies on antigenic relation-ships between vaccinia, variola, and mon-keypox viruses by HI, complement-fixation(CF; see reference 70), and by diffusion-in-gelprecipitin tests (31) have not revealed readilyrecognizable differences between them (70).Vaccinia virus and MPV are neutralized to thesame extent by MPV antisera (40). Cross-reac-tivity between vaccinia virus and MPV has alsobeen observed by immunofluorescent antibodylabeling using either conjugated vaccinia orMPV antibody (Cho et al., unpublished data).On the other hand, Prier et al. (63) in

cross-CF tests of vaccinia and monkeypox serafound that homologous exceeded heterologoustiters, suggesting differences in their solubleantigens. von Magnus et al. (70) also reportedthat when monkeypox and vaccinia viruseswere tested against vaccinia antisera by agarprecipitin methods, five precipitin bands de-veloped for vaccinia virus, whereas only foursuch bands developed for MPV. Although theseviruses share many common antigens, minorantigenic differences may exist between MPVand vaccinia virus; these differences requirefurther elucidation.

INFECTIONS IN ANIMAL HOSTS

Host RangeStudies on the host spectrum of MPV in

tissue culture cells reveal that both primaryand secondary cell lines derived from manyspecies (i.e., mouse, rabbit, dog, bovine, mon-key, and human) support the growth of thisvirus (66). In this respect, MPV resemblesvariola (25, 34, 62) qnd vaccinia (25) virusesstudied in tissue culture systems. AlthoughMPV produced CPE in a variety of mam-malian cell cultures, the virus is not pathogenicfor many of the animal species from whichthese cells are derived. Similarly, variola virusmultiplies in many types of cultured cells, andlike MPV has a very limited host range withrespect to naturally occurring, or experimental,infections (34, 36).MPV appears to have a wider host range

than variola virus (5). Among primates thesusceptible monkeys include cynomolgus (49,63, 70, 75), rhesus (49, 75), African green (49,60), marmoset, and squirrel monkeys (60);apes, orangutans, gibbons, gorillas (40, 60),and chimpanzees (52, 60) may also be involvedby disease. Susceptible nonprimate animalsinclude the giant anteater (60), rabbit (31, 48,63, 70), mouse (63, 70), chicken embryo (17, 18,31, 48), and guinea pig (63). As noted earlier,human infections have been recognized (3, 4,41).

Routes of TransmissionMonkeypox has developed in monkeys fol-

lowing intradermal, subcutaneous, intramus-cular, and intravenous inoculation of MPV (31,37, 58). Clinical and subclinical infectionsregularly develop among uninoculated com-panion monkeys separately caged among ex-perimentally infected animals (58, 73, 75). Thenatural route of these latter infections is pre-sumably by the respiratory pathway (37), al-though autoinoculation or ingestion of viralparticles, or both, are possible portals of entry.

Clinical ManifestationsThe descriptive clinical features of mon-

keypox have been recorded during naturallyoccurring and experimental infections (49, 60,63, 70, 73, 75). As a rule, the disease isgeneralized with the development of a rash ofvarying severity in different species of pri-mates. In almost all respects it resemblesnaturally occurring variola in man (25, 65) andexperimental variola in monkeys (14, 35, 37, 42,47, 59, 76).

7VOL. 37, 1973

on March 9, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

CHO AND WENNER

The incubation period of the disease inexperimentally infected animals has variedfrom 7 to 14 days in cynomolgus and rhesusmonkeys, and in baboons (40, 73, 75). Incynomolgus monkeys fever is the first clinicalsign, usually of abrupt onset on the 3rd dayafter virus inoculation (Fig. 2). Shortly there-after, a generalized lymphadenopathy developsand the mucocutaneous lesions generally ap-pear between the 7th and 14th day. Theeruptions are usually seen on the face, trunk,extremities (particularly on palms and soles),tail, and oral mucosae. The lesions on the skinrapidly pass through the stages of papule,vesicle, pustule, and crust. The papular andvesicular stages are brief, lasting only 1 or 2days. The pustular stage lasts about 2 days;crusting ensues and persists for about a week,or longer if pyoderma intervenes.Cynomolgus monkeys readily develop clini-

cal disease; they express disease much more

CYNOMOLGUS D 388

41-

D 40

W 39t

38

37

DAYS 2 4 6 8 ID 12 14 16

EXANTHEMA + + +

ENANTHEMA + +

VIREMIA 0000+00000+

HI 80ANTIBODY <10 8

CYNOMOLGUS MONKEYS

-J40 C]~~~~~~ONSETI 0 ~~~~~~~~~HEIGHT

cr38 [gMENANTHEMARASH *--,~ DECLINE

37

2 4 6 8 10 12 14 1618 20 22 42 60

EXANTHEMA + K m VESICULATIONENANTHEMA +VIREMIA ++++++.++. CRUST

HI '10 1380 390 175Z 0 PR c4 48 140 1700

intensely than rhesus monkeys, both in natu-rally occurring outbreaks (49, 63) and in experi-mental infections (75). The baboon seems torank between the rhesus and cynomolgus mon-keys with respect to clinical susceptibility (40).Subclinical infections probably occur duringepizootics more commonly than is currentlyrecognized, for, as noted above, antibodies maydevelop in the absence of clinical disease (40,63, 70, 75). As noted earlier, MPV has beenrecovered from the kidneys of apparentlyhealthy monkeys (5, 6, 31).The mortality rate of monkeypox varies from

less than 3% (40, 63, 70, 75) to 48% (60) and isdependent upon species susceptibility, the bio-logical pressures applied in experimental infec-tions and in some instances, on interaction andintervention of other microorganisms leading tobacterial septicemia. The mortality rate isgreatly increased in animals receiving im-munosuppressive treatment either after ir-

RHESUS D 419

40 -X\

,

3

37 _

DAYS 2 4 6 8 10 12 14 16

EXANTHEMA ++

ENANTHEMA + + +

VIREMIA 00000+00000

ANTIBODY <10 160

RHESUS MONKEYS

,40 *' -. .ONSET

~~~~-~~~~~~~HEIGHT*_ 3ENANTHEMA SHIH

38 RASH M * DECLINEU 37_

DAYS 2 4 6 8 10 12 14 1618 20 22 42 60I I

EXANTHEMA ++ urnS VESICULATIONENANTHEMA + + + CRUSTVIREMIA +++

,_ S Hi'IO 800 200 140

o PR '4 100 200 200

FIG. 2. Clinical and laboratory features of cynomolgus and rhesus monkeys inoculated intramuscularlywith MPV (75). Top panels pertain to typical findings for a cynomolgus and rhesus monkey. Bottom panelspertain to the composite picture obtained from both species. The temperature values are expressed as thearithmetic values for at least nine monkeys on the stated days; antibody (geometric mean values) expressed asthe reciprocal of the serum dilution end point. Reproduced by permission (Amer. J. Epidemiol. 87:551-566,1968).

8 BACTERIOL. REV.

on March 9, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

MONKEYPOX VIRUS

radiation (49) or after the use of antilym-phocytic sera (ALS) (72). Experimentally in-fected animals were given ALS, a principalcause of death related to bacterial infection,intervening at about the 3rd week duringpost-eruptive phase of monkeypox. Apparently,during this phase bacteria within dermal orintestinal lesions penetrate the blood streamwhich leads to septicemia and death. In theRotterdam Zoo (60) an outbreak among mon-keys and apes was also complicated by fatalbacterial infections. A variety of bacteria havebeen recovered including several species ofstaphylococci, streptococci, enterobacteria,pseudomonas, proteus, and enterococci (60, 71,72). Not all animals necessarily die of interven-ing bacterial infection; Heberling et al. (39)observed that germ-free baboons had an ac-celerated clinical course with leukopenia anddied of monkeypox, whereas conventionallyreared baboons with leukocytosis survived theinfection. In both sets of baboons HI antibodydeveloped by the 10th day after infection.

PathogenesisIn monkeys, monkeypox is analogous to vari-

ola and generalized vaccinia in man. Providedwith the right experimental conditions, wehave shown that MPV regularly produces dis-

ease in susceptible cynomolgus monkeys.When inoculated intramuscularly, one TCID5Ois sufficient to produce infection (73).Pathogenetic studies using cynomolgus mon-

keys infected intramuscularly (71, 73) indicatethat the earliest multiplication of MPV takesplace in local cellular components, probablyfixed or wandering cells of connective tissue.The sequential events of pathogenesis of mon-keypox appear to develop in an orderly way(Fig. 3). MPV is first detected at the local siteof infection and is associated with an intenseinflammatory response characterized by cellnecrosis, phagocytosis, vasculitis, and localreplication of MPV. These early cellular reac-tions appear to be the forerunners of transportof virus to other cellular loci through regionallymphatics and vascular channels (primaryviremia). MPV is transported in lymph toregional lymph nodes and very likely in bloodto spleen, tonsils, and bone marrow. Theseorgans comprise, among others, secondary sitesof virus multiplication, and with further releaseof virus there is a consistently measurable levelof viremia; presumably at this stage, the virusis transported to tertiary target organs (skin,testes, etc.) resulting in clinically recognizabledisease. In most respects the pathogenesis ofmonkeypox generated either experimentally orin sentinel animals follows similar patterns to

PATHOGENESIS OF MONKEYPOX

- 10

- 11

Clinicaldisease

Associated Responses

Primary site of infection

Local virus multiplication

/ \Lymphatics Blood stream

(l viremic phase)

Draining lymph nodes Spleen, tonsils

Systemic lymph nodes /

\ 4\ //\ /d

Blood stream(29viremic phase)

Skin and other organs(3 target organs)

I Local inflammation

Virus release

Febrile response

Lymphoid hyperplasia

Mucocutaneous lesions

Systemic tissue injury

Antibody formation

FIG. 3. Model for the pathogenesis of monkeypox (16, 73). Model is based on data derived from MPVinfected intramuscularly in cynomolgus monkeys.

Days

Infection 0

r 1

Incubation .5

VOL. 37, 1973 9

on March 9, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

CHO AND WENNER

those described for ectromelia (28), rabbitpox(12, 19), and variola (35).The evidence supporting the association of

the inflammatory lesions of the skin and otherorgans with MPV is based on several docu-mented facts; among the first of these is thehigh concentrations of virus recovered fromaffected tissues (71, 73). Moreover, foci clearlylabeled by fluorescent antibody (indicating thepresence of viral antigen) when restained withhematoxylin and eosin, or other stains, revealsnot only an inflammatory response, but in-tracytoplasmic inclusions as well (71, 72). Al-though a strong stand can be taken in defenseof a direct attack by MPV on selected cells, wemust point out that antibodies appear at aboutthe same time as detectable cell injury, sug-gesting (remotely to us) that a part of the in-flammatory response may relate to antigen-antibody interactions.

ViremiaIn cynomolgus monkeys viremia is a constant

feature of infection (73, 74, 75; Fig. 2 and 4) andappears to be responsible for dissemination ofthe MPV to some of the secondary and proba-bly all tertiary target organs (Fig. 3). Viremiaoccurs between the 3rd and 14th day (73, 74).There is a considerable variability in the inten-sity and in the duration of viremia; someanimals have high and others have relativelylow concentrations of virus; in some monkeysviremia appears to last only several days, andin others 7 to 10 days. When clean separation ofblood components (red blood cells, buffy coat,and plasma) is accomplished, MPV appears tobe mainly cell-associated, particularly with theleukocytes of the buffy coat. This associationcan be demonstrated both by virus isolationand by the immunofluorescent method (72).Another finding worthy of note relates to thepersistence of viremia beyond the time ofappearance of humoral antibodies (Fig. 4). Thepersistence of virus in blood (and also intissues) in the presence of antibodies suggeststhat MPV, because of intracellular location,may be "protected" from antibody action, orthe early formed antibody molecules are in-competent for complete neutralization of theseearly virions.

HistopathologyHistopathological aspects of monkeypox

have been reported for both naturally occurringand experimental infections (31, 67, 71, 75).Experimentally, the inoculation of MPV in-tramuscularly is followed within 24 to 48 h byan intense local inflammatory response (71)

characterized by pronounced focal cell destruc-tion, particularly of interstitial connective tis-sue components. Excepting this site, furthercellular injury from MPV is usually not foundduring the preeruptive period. Foci of cellnecrosis may develop in the spleen as early asthe 7th day; thereafter, such foci are noted withincreasing fequency in tonsils, lymph nodes,testes, ovaries, kidneys (71, 75), and in liverand lungs (31). Lesions encountered in theseorgans are characterized by inflammation, cel-lular proliferation, degeneration, and focal ne-crosis. Lesions involving the skin and mucousmembranes are characterized by epithelial de-generation, reticulation, endothelial prolifera-tion, inflammatory cell infiltration, necrosis,and granulation. Cytoplasmic inclusion bodiescan be found; they may or may not be numer-ous (63, 71); intranuclear inclusions have beenobserved also (63). The lesions accompanyinginfection in monkeys from MPV are entirelylike those described in human beings frominfection with variola virus (25, 36, 65, 71).

Antibody ResponsesHI antibodies may be detectable as early as

the 8th day after infection, almost coincidingwith and occasionally preceding the earliestappearance of the skin eruption. HI antibodiesreach their peak during the 3rd week, approach-ing titers of approximately 1:640 to 1:1,280(Fig. 5). HI antibodies decline between the 3rdand 6th week and plateau between the 2nd and3rd month. At 6 months, titer values rangebetween 1: 20 to 1: 30. Serum-neutralizing (SN)antibodies appear concomitantly with orclosely follow the development of HI antibody.SN antibody may reach peak titers by the 3rdweek, or even later; the peak values are similarto HI titers and remain constant for longer than3 months (our longest period of observation).CF antibodies appear approximately 1 weekafter HI antibody and decline slowly over aperiod of 90 to 230 days (40, 73).Monkeys inoculated with small doses of

MPV (.600 TCID50) showed development oflower titers of HI antibody, and also earlierpeak and initial decline, than animals givenlarge doses (<60,000 TCID50). This phenome-non remains unexplained, but may be due to amodulation of intracellular virus replicationand switch-off of antibody formation whenenough is formed to prevent further replicationof infectious virus (73).

Cross ImmunityAs indicated earlier, MPV shares common

antigens with vaccinia and variola viruses,

10 BACTERIOL. REV.

on March 9, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

MONKEYPOX VIRUS 11

DAYS

FIG. 4. Titration data on monkeypox: some clinical and laboratory findings (16, 73). Cynomolgus monkeyswere infected with various dilutions (10-1 to 10-') of MPV. Eight monkeys were selected for the purpose ofcomparison of their clinical responses (temperature and onset of skin rash) and laboratory findings (HIantibodies and viremia). See test for discussion. Monkey 584 and 582 developed hypothermia with a bodytemperature of less than 35 C. D, died; K, killed. Note the delayed incubation in monkey 610. The monkey was

a sentinel control and acquired monkeypox while exposed to experimentally infected monkeys housed inadjacent cages. The same may apply to monkey 607 inoculated with 10- " dilution of virus, a value very close to<1 ID50.

1280640 -

c 320 -

260 -

A80 -

40 -

20-4 B

2 4 6 8 10 12 14 21 28 35 42 49 56 60

DAYS

FIG. 5. Rise and fall of HI antibodies (73). CurveA (open circles) was derived from the geometric meanvalues obtained on six monkeys inoculated with 104.8and 102.6 ID,. of MPV; curve B (solid triangles) wasderived similarly from values on seven monkeysinoculated with 102.8 and 101.8 ID50. Reproduced bypermission (Arch. Gesamte Virusforsch. 27:166-178,1969).

hence there ought to be cross-resistance on

challenge. McConnell et al. (50, 52) have dem-onstrated that monkeypox is preventable in

rhesus monkeys and chimpanzees by intrader-mal inoculation of vaccinia virus. Similar cross

protection for cynomolgus monkeys has alsobeen observed by Gispen et al. (31). In ourlaboratory, monkeys recovering from mon-

keypox also resisted intradermal challengewith vaccinia virus (74, 75). In contrast, mon-

keys recovered from infection from monkeypoxor vaccinia virus, or both, were not immune tochallenge with Yaba tumor virus (74).

SPECIAL ASPECTS

Antiviral Compound, Methisazone

Isatin-fl-thiosemicarbazone has been usedextensively to study the mechanism of action ofits antiviral activity among poxviruses (44).Methisazone (1-methyl-isatin-3-thiosemicar-bazone or marboran), one of the more effectivederivatives, has been used in prophylacticcontrol of smallpox (7, 8).

VOL. 37, 1973

41

40

C 39

38

37

*40

I 6 0

'10

VI RENUA

41so37

'40

1..1 684 se'gmRRASH RAW RAWHA...~~~~~ / - :

0 4 K

................. ............... ...............

10s 693 *la 604 r" b 607 _ontrol *1RASH RASH RASH RASH

, Il

..................... ....................... ....... ............................. ..............4

0 S 10 IS 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20

on March 9, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

CHO AND WENNER

Methisazone has been used by us to studythe cellular expression of MPV (17). In vitrostudies revealed that methisazone suppressedplaque development in CV-1 cells infected withMPV. However, in vivo experiments with me-thisazone showed only a slight prolongation ofsurvival time among MPV-infected chickenembryos. A temporary prolongation of survivaltime was also observed among treated miceinoculated intraperitoneally with MPV. Noprotection was afforded in mice inoculatedintracerebrally. Methisazone failed to preventthe development of monkeypox in MPV-infected monkeys; although the illness seemedto be mild in intensity, the pathogenetic andimmunologic features of infection were indistin-guishable from those of infected control mon-keys. Methisazone was used also during anoutbreak of monkeypox in the Rotterdam Zooand apparently failed to curtail the spread ofthe disease (60).

Infection in Immunosuppressed HostsMonkeypox provides an ideal model for stud-

ies of infection and immunity. During theoutbreak of monkeypox at the Walter ReedArmy Institute of Research in 1962, pre-expo-sure irradiated monkeys died while non-irradiated animals survived the disease (49).Studies on the effects of ALS on monkeypoxrevealed that ALS adversely enhanced morbid-ity and mortality of infected monkeys (72).Azathioprine used either singly or in combina-tion with ALS intensified the clinical andpathological responses of cynomolgus monkeysto MPV (Wenner et al., unpublished data).Among 14 inculated monkeys (5 control ani-mals) fever occurred in all at similar intervals(8 i 1.5 days), as did the appearance of pocks(9 ± 0.8 days). The major differences relate tothe late course of infection, where impairedhealing, cutaneous gangrene of the inoculatedlimb, and risks of dying during a shocklikesyndrome were notably greater in monkeysgiven either ALS or azathioprine than in theuntreated controls. The significant pathologi-cal features of the dermal lesions in all treatedmonkeys related to modulation of the inflam-matory response and delay in tissue reparativeprocesses. Adrenal hemorrhage occurred in fourtreated monkeys and in one control animal.Viremia developed and persisted until deathresulted in ALS-treated, but not in azathio-prine-treated, monkeys. Despite this sustainedviremia, concentrations of MPV in tissuescorresponded with those obtained in untreatedanimals; moreover, the histological studies sug-

gested that cellular injury was not a majorfactor contributing to death. ALS temporarilydelayed development of HI and CF antibodies;similar delays were not observed in azathio-prine-treated animals. These data suggest thatthe principal area of defense vulnerability re-lates to suppression of cellular immunity anddelayed repair, rather than humoral antibodysynthetic events.

EPIZOOTIOLOGYRecently, attention has been paid to the

possible existence of a reservoir of smallpox innonhuman primates (5, 6, 58). Serologicalsurveys have been made to determine theextent of specific antibodies to poxviruses indifferent monkey populations and their varioushabitats (5, 45, 58).

Several serological tests have been usedproviding evidence of poxvirus infection. TheCF test is not entirely optimal because offrequent anticomplementary activity of mon-key sera (5). The HI test is reasonably reliable,but temporal persistence of HI antibody is stilllargely unknown. Such antibodies may notpersist longer than 1 year. SN antibody persistsfor a longer period (<1 year), but this rathertedious test is not practical for large-scaleepidemiological studies.We have presented data suggesting that

subclinical poxvirus infection may be a com-mon event in captive monkeys. McConnell etal. (49) apparently described another suchoccurrence. After the appearance of mon-keypox in two animals, he subsequently found25 out of 27 (93%) cynomolgus, 52 out of 67(78%) rhesus, and 6 out of 14 (43%) Africangreen monkeys quartered in the same com-pound to have HI antibodies. On the otherhand, only 5 out of 45 (11%) cynomolgusmonkeys housed in a separate compound hadsuch antibody. Within this separate compoundmonkeypox had not been recognized.At this time (5, 45; see Addendum) more

than 2,000 sera from 20 different species ofmonkeys originating in Africa, India, Pakistan,Philippines, Japan, and South America havebeen tested for MPV antibodies, primarilyusing the HI and for some the SN test. Theresults of positive HI tests are difficult tointerpret, however; only a few unequivocallypositive results were obtained. Two out of 250sera (mostly from cynomolgus, rhesus, andfuscata monkeys) showed weakly positive SNtests; both sera were obtained from cynomolgusmonkeys. Additional serological surveys havebeen conducted by Noble (58) and Kalter and

12 BACTERIOL. REV.

on March 9, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

MONKEYPOX VIRUS

Heberling (45). The results are summarized inTable 3. Since the source of animals and thetime held in captivity are often unknown, thedata are difficult to interpret also. Neverthe-less, there appears to be a great deal of varia-tion in positivity among various species of Oldand New World monkeys. In the series ofKalter and Heberling, approximately 3.5% (46out of 1,281) of the monkeys are positive for HIantibody to vaccinia virus, and 12.2% (149 outof 1,219) were positive to MPV. Noble found a

comparable result: approximately 5% (26 out of535) were positive for HI antibody to vacciniavirus. Among the monkeys we have tested (98young cynomolgus and 13 rhesus monkeysoriginating from India or the Philippines), none

had HI antibody to MPV upon arrival in our

laboratory (17, 71-75).As noted earlier in this review, monkeypox

and vaccinia viruses share major common anti-gens thereby making it virtually impossible todistinguish them in conventional serologictests. The data summarized in Table 3 revealin general that a larger proportion of sera

procured from human beings and nonhumanprimates was reactive with monkeypox thanvaccinia HA. There were two exceptions in-volving rhesus and vervet monkeys. Assumingthat the HI antibodies were engendered as a

result of previous infection from a poxvirus,then the reactions in human beings presuma-

bly apply to vaccinia-variola, and those innonhuman primates to monkeypox. While thedata may be ascribed to a greater sensitivity ofMPV HA, the possibility also exists that cer-

tain sera representing infections acquired more

recently than others may have differing avidi-ties that are hidden in a screening test whereinfull serum dilution end points were not deter-mined. Nevertheless, these data point up thevariable but appreciable risks of infection by a

poxvirus among primates; presumably thisvirus is MPV. Neither of the rates of HIantibodies determined for human beings or

species of nonhuman primates can be consid-ered absolute in view of the known decline ofHI antibodies within several months post-infection.

TABLE 3. Antibody to vaccinia and monkeypox viruses in human and nonhuman primatesa

Screening of sera for HI antibody to

Species Vaccinia virus Monkeypox virus

No. positive/no. tested Positive (%) No. positive/no. Positive(%)tested

Humanb 13/109 11.9 31/115 26.9Old World monkeys

Gorilla ......................... 0/39 0.0 0/38 0.0Chimpanzee ................... 4/267 1.5 34/261 13.0Orangutan ..................... 0/89 0.0 1/74 1.4Gibbon ........................ 0/12 0.0 0.8 0.0Baboon ........................ 0/145 (3/74) 0.0 (4.0) 17/147 11.6Rhesus ........................ 12/210 5.7 11/160 6.9Cynomolgus .................... 1/77 (0/64) 1.3 (0) 4/80 5.0Japanese macaques ........ ..... 0/44 0.0 3/44 6.9Vervet ......................... 4/77 (2/21) 5.2 (9.5) 3/83 3.6Patas .......................... 0/55 (20/317) 0.0 (6.3) 2/52 3.5Talapoin ....................... 0/21 0.0 4/22 18.2

New World monkeysMarmosets ..................... 2/43 4.7 6/43 13.9Squirrel ........................ 20/63 (0/5) 31.7 (0) 37/63 58.7Capuchin ...................... 0/14 (1/4) 0.0 (25.0) 1/2 50.0Woolly ......................... 0/1 (0/11) 0.0 (0) 4/12 33.3Owl .......................... 0/8 0.0 0/8 0.0Spider ......................... 3/7 (0/11) 42.9 (0) 7/7 100.0

a These data are compiled from Kalter and Herling (45); data in parentheses are obtained from Noble (58).These represent results of sera obtained from various parts of the world, some having been tested in variedlaboratories. The majority of the sera were tested for the presence or absence of antibody to poxviruses byusing both vaccinia and monkeypox antigens.

b Sources of human sera are from Kenya, Tex., U. S. Army recruits, etc. The vaccination status of these menis unknown.

13VOL. 37, 1973

on March 9, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

CHO AND WENNER

CLINICAL AND EPIDEMIOLOGICALIMPLICATIONS

Monkeys as a Reservoir for Smallpox?The absence of an animal reservoir of variola

virus is essential for the success of world-widesmallpox eradication. Smallpox appears to behighly host-specific; besides human beingsonly a few primate species are known to besusceptible to infection (5, 14, 36, 58).

Arita and Henderson (6) recently reviewedthe problems of smallpox and monkeypox innonhuman primates. They, along with Noble(58), conclude that a natural reservoir forsmallpox in nonhuman primates is unlikely.Supportive evidences used in arriving at thisconclusion are: (i) epidemiological surveillanceindicates that outbreaks of smallpox in mon-keys are rare phenomena; only 7 such episodesare reported, and no outbreak has been recog-nized since 1936 (6); (ii) human smallpox hasvirtually disappeared from several geograph-ical areas containing large monkey popula-tions, e.g., Panama and the Philippines; (iii)serological surveys of monkeys for evidence ofpoxvirus infection reveal that usually only a

fraction of the animals exhibit HI antibody topoxviruses (5, 36, 45, 58); and (iv) althoughcynomolgus monkeys are susceptible to small-pox, the disease is not readily transmissible inserial passages (58). However, the susceptibil-ity of certain species of monkeys to smallpox(35, 36, 58, 76) and the recent recognition of theclosely related virus of monkeypox (70) warrantfurther studies and continued field observa-tions (5, 6) particularly with respect to (i)circumscribed, exogenous (nonhuman) reser-voirs of variola virus and (ii) the epizootiologyof monkeypox.

Monkeypox in ManThere appeared to be virtually no risk of

human infection among personnel exposed dur-ing the various outbreaks of monkeypox (seeHistorical Note). The initial impression was

that human beings may be comparatively in-susceptible to MPV (6, 65). However, most ifnot all such persons had been vaccinatedagainst smallpox. Now it has become clear thatnatural infection with MPV may occur inhuman beings (3, 4). Although the primarysource of infection in the small outbreaksoccurring in South Africa was not identified,the information available suggests primarytransmission of MPV from monkeys to humanbeings. Since the clinical features of thesehuman infections resembled smallpox, difficul-

ties in differential diagnosis can be anticipatedand discrete outbreaks of monkeypox mayremain unrecognized. At this time there is nosolid evidence for the transmission of MPVfrom person to person; however, opportunityfor critical study of clusters of human infectionhave been too few to establish the epidemiolog-ical pattern (see Addendum). Having at handinformation on the relative ease of cross-infec-tion between captive monkeys, and recognizingas yet no biological reason why similar eventsshould not prevail for susceptible (unvacci-nated) members of the human population, welook forward to the further development ofcritical studies relating MPV to infectionsamong children and adults residing in enzooticareas.

Moreover, MPV has been recovered from thetissue cultures derived from the kidneys ofapparently healthy cynomolgus monkeys (31),and seroconversion has been defined amongmonkeys with inapparent infections; therefore,"silent" infections from MPV in nature may bemore common (5) than is generally appreci-ated! The demonstrable presence of MPV incaptive monkeys should alert those concernedwith usage of tissue cultures in the field ofbiological research to the potentiality of MPVas a risk to human beings. Hence, it appearsadvisable to protect persons against infectionfrom such poxviruses by vaccination, especiallythose who handle monkeys or work with biolog-ical materials involving tissue cultures of pri-mate species.

CONCLUSIONSMPV was isolated in 1958 during outbreaks

of a pox disease in laboratory colonies ofcynomolgus monkeys in Copenhagen. Sincethen, several outbreaks have occurred in differ-ent species of non-human primates housed inlaboratories in various parts of the world.Naturally occurring infections among monkeysin their native habitat is unknown; however,the appearance of infection by MPV in childrenresiding in West Africa suggests that wildmonkeys (or related species) are the likelyharborers of MPV.

Studies on the biological properties of MPVindicate that it is closely related to the vac-cinia-variola subgroup of poxviruses. MPV pro-duces pock lesions on the CAM of developingchicken embryos, encephalitis in mice, andcharacteristic dermal lesions and keratitis inrabbits. Morphologically, the virus is 200 by250 nm in size and has a rectangular shape.Many mammalian cells in culture support the

14 BACTERIOL. REv.

on March 9, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

MONKEYPOX VIRUS

growth of MPV. Cytopathic effects and plaqueformation can be easily produced in culturedcells.

Clinically, monkeypox as found in monkeysand in human beings cannot be differentiatedfrom variola. Serological data support the oc-currence of subclinical infection in monkeys;the ratio of subclinical-clinical infection isunknown. In its pathogenesis monkeypox fol-lows patterns described for variola, ectromelia,and rabbitpox. Histopathological features ofinfection are similar to those found in humaninfections from variola virus, to wit: inflamma-tion, cellular proliferation, degeneration, andfocal cellular necrosis in various organ systemsincluding the dermis. Specific antibodies areengendered in infected hosts and these may bedemonstrated by using HI, CF, and SN tests.MPV shares common antigens with vacciniaand variola. Therefore, there is a strong sero-logical cross-reactivity and clinical cross-immunity between them.

Serological surveys to determine the fre-quency of specific antibody to poxviruses indifferent monkey populations show wide rangesbetween species; on the average, less than 12%of monkeys originating from different parts ofthe world contain HI antibody. Whether ac-quisition of infection was in their native habi-tat, or followed captivity, remains unknown.Epidemiological surveillances suggest that anatural reservoir of smallpox in nonhumanprimates is unlikely; however, further observa-tions are needed, particularly with respect tomonkey populations with high infection ratesbased on existent HI antibodies.MPV is pathogenic to man; this feature

undoubtedly has clinical and epidemiologicalsignificance. Protection of human beingsagainst monkeypox by vaccination with vac-cinia virus is mandatory among those whohandle monkeys or tissue cultures of primatespecies.

Uniquivocal answers cannot be given toquestions asked in the introductory section. Ifmonkeypox is variola, the contagiousness isexceptional, for variola virus is not easilyacquired by sentinel companions quarteredamong infected monkeys. If infection relates tovaccinia virus, it is unique also, for to ourknowledge generalized vaccinia has never beenreported in healthy primates (immunologicallyand dermatologically intact). If it is a hybridvirus of recent or remote origin derived byreactivation in vivo of variola and anotherpoxvirus, it is yet to be described. Finally, if itis uniquely a primary poxvirus (MPV) of mon-keys, as it well might be, many of its principal

properties have been defined.

ADDENDUMAfter this review was written, a series of

papers appeared in the Bulletin of the WorldHealth Organization (vol. 46, p. 567-639, 1972).None of these reports significantly alters thedata presented herein, but they do amplifyseveral points of discussion. At least two morehuman infections have been recognized (Lad-nyj et al., p. 593-597; Bourke and Dumbell, p.621-653). The clinical characteristics of MPVinfection in humans is described by Foster et al.,p. 569-570. The epidemiological data point outthe low, and possibly negligible, risk of human-to-human transmission; some susceptibles atrisk were successfully vaccinated, suggestinglittle if any immunity to vaccinia virus. Studieson properties of MPV suggest that (i) the virusis a specific homogeneous poxvirus (Rondle andSayeed, p. 577-583) or (ii) heterogeneous, sincemutants closely related to variola have beenselected from strains isolated from healthymonkeys. Thus the most recent published dataleave unanswered most of those questionsposed in the introductory section of this review.

ACKNOWLEDGMENTSThis investigation was supported in part by Public

Health Service grants AI-06263 from the NationalInstitute of Allergy and Infectious Diseases and5S01RROS373. H.A.W. was a Research CareerAwardee (NIH no. K6-AI-13976).

LITERATURE CITED1. Ambrus, J. L., E. T. Feltz, J. T. Grace, and G.

Owens. 1963. A virus induced tumor in pri-mates. Nat. Cancer Inst. Monogr. 10:447-458.

2. Anonymous. 1968. The smallpox eradication pro-gramme. WHO Chronicle 22:354-362.

3. Anonymous. 1971. Human infection with mon-keypox virus. Morbidity and Mortality WeeklyReport, vol. 20, no. 8.

4. Anonymous. 1971. A rare smallpox-like disease.WHO Chronicle 25:370-372.

5. Arita, I., F. Fenner, R. Gispen (Chairman), S. B.Gurrich, S. S. Kalter, S. S. Marennikova, G.Meiklejohn, J. Noble, Jr., G. M. Sheluchina,V. D. Soloviev, and I. Tayaga. 1968. Report ofinformal discussion on monkeypox virus stud-ies. Draft to report to WHO.

6. Arita, I., and D. A. Henderson. 1969. Smallpoxand monkeypox in non-human primates. Bull.WHO 39:277-283.

7. Bauer, D., L. St. Vincent, C. H. Kempe, and A.W. Downie. 1963. Prophylactic treatment ofsmallpox contacts with N-methylisatin-beta-thiosemicarbazone (compound 33T57 marbo-ran). Lancet 2:494-496.

8. Bauer, D. J., L. St. Vincent, and H. Kempe.

15VOL. 37, 1973

on March 9, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

CHO AND WENNER

1969. Prophylaxis of smallpox with methisa-zone. Amer. J. Epidemiol. 90:130-145.

9. Baxby, D., and B. J. Hill. 1971. Characteristicsof a new poxvirus isolated from indian buf-faloes. Arch. Gesamte Virusforsch. 35:70-79.

10. Bearcroft, W. G. C., and M. F. Jamieson. 1958.An outbreak of subcutaneous tumours in rhe-sus monkeys. Nature (London) 182:195-196.

11. Bedson, H. S., and K. R. Dumbell. 1961. Theeffect of temperature on the growth of poxviruses in chick embryo. J. Hyg. (Camb.)59:457-469.

12. Bedson, H. S., and J. J. Duckworth. 1963.Rabbitpox: an experimental study of the path-ways of infection in rabbits. J. Pathol. Bacte-riol. 85:1-20.

13. Bleyer, J. C. 1922. Uber Auftreten von Variolaunter Affen der Genera Mycetes und Cebus beiVordringen einer Pockenepidemie im Urwald-gebiete an den Nebenflussen des Alto Uruguayin Sudbrasilien. Munch. Med. Wochschr.69:1009-1010.

14. Brinckerhoff, W. R., and E. E. Tyzzer. 1905-06.Studies on experimental variola and vacciniain Quadrumana. J. Med. Res. 9:223-359.

15. Casey, H. W., J. M. Woodruff, and W. I.Butcher. 1967. Electron microscopy of a be-nign epidermal pox disease of rhesus monkeys.Amer. J. Pathol. 51:431-446.

16. Cho, C. T. Biological activity of monkeypoxvirus: studies on growth characteristics in cellculture, pathogenesis in monkeys and modifi-cation of infection by methisazone. Disserta-tion Abstracts International, vol. 31, no. 11, p.6773-B. University Microfilms, Ann Arbor,Mich.

17. Cho, C. T., C. R. Bolano, P. S. Kamitsuka, andH. A. Wenner. 1970. Methisazone and mon-keypox virus: studies in cell cultures, chickembryos, mice and monkeys. Amer. J. Epidem-iol. 92:137-144.

18. Cho, C. T., C. Liu, and H. A. Wenner. 1972.Monkeypox virus: responses of infection inchick embryos. Proc. Soc. Exp. Biol. Med.139:1206-1212.

19. Cho, C. T., T. Locke, and H. A. Wenner. 1970.Viremia and virus measurements of rabbitpoxin CV-1 cells. Appl. Microbiol. 19:791-794.

20. Cho, C. T., and H. A. Wenner. 1972. In vitrogrowth characteristics of monkeypox virus.Proc. Soc. Exp. Biol. Med. 139:916-920.

21. Chu, C. M. 1948. Studies on vaccinia hemag-glutinin. I. Some physicochemical properties.J. Hyg. 46:42-48.

22. Crandell, R. A., H. W. Casey, and W. B. Brum-low. 1969. Studies of a newly recognized pox-virus of monkeys. J. Infect. Dis. 119:80-88.

23. Dales, S. 1963. The uptake and development ofvaccinia virus in strain L cells followed withlabeled viral deoxyribonucleic acid. J. CellBiol. 18:51-72.

24. Daniel, M. D., A. Karpas, L. V. Melendez, N. W.King, and R. D. Hunt. 1967. Isolation ofherpes-T virus from a spontaneous disease insquirrel monkeys (Saimiri sciureus). Arch. Ge-

samte Virusforsch. 22:324-331.25. Downie, A. W. 1965. Poxvirus group, p. 932-964

In F. L. Horsfall and J. Tamm (ed.), Viral andrickettsial infections of man. 4th ed., J. B.Lippincott Co. Philadelphia.

26. Downie, A. W., C. H. Taylor-Robinson, A. E.Caunt, G. S. Nelson, P. E. C. Manson-Bahr,and T. C. H. Matthews. 1971. Tanapox: a newdisease caused by a pox virus. Brit. Med. J.1:363-368.

27. Douglas, J. D., K. N. Tanner, J. R. Prine, D. C.Van Riper, and S. K. Derwelis. 1967. Mollus-cum contagiosum in chimpanzees. J. Amer.Vet. Med. Ass. 151:901-904.

28. Fenner, F. 1949. Mouse-pox (infectious ectro-melia of mice). A review. J. Immunol.63:341-373.

29. Fenner, F. 1958. The biological characteristics ofseveral strains of vaccinia, cowpox and rabbit-pox viruses. Virology 5:502-529.

30. Furness, G., and J. S. Younger. 1959. One-stepgrowth curves of vaccinia virus in cultures ofmonkey kidney cells. Virology 9:386-395.

31. Gispen, R., J. D. Verlinde, and P. Zwart. 1967.Histopathological and virological studies onmonkeypox. Arch. Virusforsch. 21:205-216.

32. Grace, J. T., and E. A. Mirand. 1963. Humansusceptibility to a simian tumor virus. Ann.N.Y. Acad. Sci. 108:1123-1128.

33. Grace, J. T., E. A. Mirand, S. J. Millan, and R.S. Metzger. 1962. Experimental studies onhuman tumors. Fed Proc. 21:32-36.

34. Hahon, N. 1958. Cytopathogenicity and propaga-tion of variola virus in tissue culture. J. Immu-nol. 81:426-432.

35. Hahon, N., and B. J. Wilson. 1960. Pathogenesisof variola in Macaca irus monkeys. Amer. J.Hyg. 71:69-80.

36. Hahon, N. 1961. Smallpox and related poxvirusinfections in the simian host. Bacteriol. Rev.25:459-481.

37. Hahon, N., and M. H. McGavran. 1961. Air-borne infectivity of the variola-vaccinia groupof poxviruses for the cynomolgus monkey. Ma-caca irus. J. Infect. Dis. 109:294-298.

38. Hall, A. S., and W. P. McNulty. 1967. A conta-gious pox disease in monkeys. J. Amer. Vet.Med. Ass. 151:833-838.

39. Heberling, R. L., J. Eichberg, and S. S. Kalter.1972. The course of monkeypox virus infectionin conventional and gnotobiotic baboons(Papio cynocephalus). Abstr. Annu. MeetingAmer. Soc. Microbiol., p. 233.

40. Heberling, R. L., and S. S. Kalter. 1971. Induc-tion, course and transmissibility of monkeypoxin the baboon (Papio cynocephalus). J. Infect.Dis. 124:33-38.

41. Henderson, D. A. 1971. Official Records of theWorld Health Organization, no. 194, p. 408.

42. Horgan, E. S., and M. A. Haseeb. 1939. Crossimmunity experiments in monkeys betweenvariola, alastrim and vaccinia. J. Hyg.39:615-637.

43. Hull, R. N. 1969. The significance of simianviruses to the monkey colony and the labora-

16 BACTERIOL. REV.

on March 9, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

MONKEYPOX VIRUS

tory investigator. Ann. N.Y. Acad. Sci.162:472-482.

44. Joklik, W. K. 1966. The poxviruses. Bacteriol.Rev. 30:33-66.

45. Kalter, S. S., and R. L. Heberling. 1971. Com-parative virology of primates. Bacteriol. Rev.35:310-364.

46. Loh, P. C., and J. L. Riggs. 1961. Demonstrationof the sequential development of vaccinialantigens and virus in infected cells: observa-tions with cytochemical and differentialfluorescent procedures. J. Exp. Med.114:149-160.

47. Magrath, G. B., and W. R. Brinckerhoff. 1904.On experimental variola in the monkey. J.Med. Res. 6:230-246.

48. Marennikova, S. S., E. B. Gurvich, and E. M.Shelukhina. 1971. Comparison of the proper-ties of five pox virus stains isolated frommonkeys. Arch. Gesamte. Virusforsch.33:201-210.

49. McConnell, S. J., Y. F. Herman, D. E. Mattson,and L. Erickson. 1962. Monkeypox disease inirradiated cynomolgus monkeys. Nature(London) 195:1128-1129.

50. McConnell, S., Y. F. Herman, D. E. Mattson, D.L. Huxsoll, C. M. Lang, and R. H. Yager. 1964.Protection of rhesus monkeys against mon-keypox by vaccinia virus immunization. Amer.J. Vet. Res. 25:192-195.

51. McConnell, S. J., R. 0. Spertzel, D. L. Huxsoll,L. H. Elliott, and R. H. Yager. 1964. Plaquemorphology of monkeypox virus as an aid tostrain identification. J. Bacteriol. 87:238-239.

52. McConnell, S., R. L. Hickman, W. L. Wooding,and D. L. Huxsoll. 1968. Monkeypox: experi-mental infection in chimpanzee (Pan satyrus)and immunization with vaccinia virus. Amer.J. Vet. Res. 29:1675-1680.

53. McNulty, W. P., W. C. Lobitz, F. Hu, C. A.Maruffo, and A. S. Hall. 1968. A pox disease inmonkeys transmitted to man. Arch. Dermatol.97:286-293.

54. Melnick, J. L. 1971. Classification and nomen-clature of animal viruses. Progr. Med. Virol.13:462-484.

55. Middelkamp, J. N., and H. A. Wenner. 1969.Electron microscopy of monkeypox virus inepidermal lesions. Bacteriol. Proc., p. 190.

56. Mika, L. A., and J. B. Pirsch. 1960. Differentia-tion of variola from other members of thepoxvirus group by the plaque technique. J.Bacteriol. 80:861-863.

57. Nicholas, A. H., and W. P. McNulty. 1968. Invitro characteristics of a poxvirus isolated fromrhesus monkeys. Nature (London) 217:745-746.

58. Noble, J., Jr. 1970. A study of new and old worldmonkeys to determine the likelihood of a si-mian reservoir of smallpox. Bull. WHO42:509-514.

59. Noble, J., and J. A. Rich. 1969. Transmission ofsmallpox by contact and by aerosol routes inMacaca irus. Bull. WHO 40:279-286.

60. Peters, J. C. 1966. Eine "Monkey-Pox" enzootie

im affenhaus des Tiergartens "Blijdorp."Kleint.-Prax. 11:65-71.

61. Peters, J. C. 1966. A monkeypox-enzooty in theBlijdorp Zoo. Tijdschr, Diergeneesk91 :387-391.

62. Pirsch, J. B., L. A. Mika, and E. H. Purlson.1963. Growth characteristics of variola virus intissue culture. J. Infect. Dis. 113:170-187.

63. Prier, J. E., and R. M. Sauer. 1960. A pox diseaseof monkeys. Ann. N.Y. Acad. Sci. 85:951-959.

64. Prier, J. E., R. M. Sauer, R. G. Malsberger, andJ. M. Sillaman. 1960. Studies on a pox diseaseof monkeys. II. Isolation of the etiologic agent.Amer. J. Vet. Res. 21:381-384.

65. Rhodes, A. J., and C. E. van Rooyen. 1968. Thepoxvirus group: general properties, p. 299-311.In Textbook of virology. The Williams & Wilk-ins Co., Baltimore.

66. Rouhandeh, H., R. Engler, M. T. A. Fouad, andL. L. Sells. 1967. Properties of monkeypoxvirus. Arch. Virusforsch. 20:363-373.

67. Sauer, R. M., J. E. Prier, R. S. Buchanan, A. A.Creamer, and H. C. Fegley. 1960. Studies on apox disease of monkeys. I. Pathology. Amer. J.Vet. Res. 21:377-380.

68. Sheek, M. R., and W. E. Magee. 1961. Anautoradiographic study on the intracellulardevelopment of vaccinia virus. Virology15:146-163.

69. Smith, K. O., and D. G. Sharp. 1960. Interactionof virus with cells in tissue cultures. I. Adsorp-tion on and growth of vaccinia virus in L cells.Virology 11:519-532.

70. von Magnus, P., E. K. Anderson, K. B. Peterson,and A. Birch-Anderson. 1959. A pox-like dis-ease in cynomolgus monkeys. Acta Pathol.Microbiol. Scand. 46:156-176.

71. Wenner, H. A., C. R. Bolano, C. T. Cho, and P.S. Kamitsuka. 1969. Studies on the pathogen-esis of monkeypox. HI. Histopathological le-sions and sites of immunofluorescence. Arch.Gesamte Virusforsch. 27:179-197.

72. Wenner, H. A., C. Bolano, C. T. Cho, and P. S.Kamitsuka. 1969. Monkeypox. IV. Modifica-tion of disease pattern by antilymphocyticsera. J. Infect. Dis. 120:318-331.

73. Wenner, H. A., C. T. Cho, C. R. Bolano, and P.S. Kamitsuka. 1969. Studies on the pathogen-esis of monkeypox. II. Dose-response and virusdispersion. Arch. Gesamte Virusforsch.27:166-178.

74. Wenner, H. A., P. S. Kamitsuka, F. D. Macas-aet, and P. Kidd. 1967. Pathogenesis of mon-keypox, p. 40-44. Antimicrobiol. Ag. Chemo-ther. 1966. American Society for Microbiology,Ann Arbor, Mich.

75. Wenner, H. A., F. D. Macasaet, P. S. Kamit-suka, and P. Kidd. 1968. Monkeypox. I. Clini-cal, virologic and immunologic studies. Amer.J. Epidemiol. 87:551-566.

76. Westwood, J. C. N., E. A. Boulter, E. T. W.Bowen, and H. B. Maber. 1966. Experimentalrespiratory infection with poxviruses. I. Clini-cal, virological and epidemiological studies.Brit. J. Exp. Pathol. 47:453-465.

VOL. 37, 1973 17

on March 9, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

CHO AND WENNER

77. Woodroofe, G. J., and F. Fenner. 1962. Serologi-cal relationships within the pox virus group:

an antigen common to all members of thegroup. Virology 16:334-341.

78. Woodson, B. 1968. Recent progress in poxvirus

BACTERIOL. REV.

research. Bacteriol. Rev. 32:127-137.79. Yau, T., and H. Rouhandeh. 1970. Uncoating of

monkeypox virus (MPV) in continuous Africangreen monkey kidney cell line, CV-1. Bacteriol.Proc., p. 204.

18

on March 9, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from