Toward a vaccine against Argentine hemorrhagic fever

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Toward a Vaccine against Argentine

Hemorrhagic Fever1

JULIO

G. BARRERA

0~02 & KELLY T. MCKEE,

JR.~

A vaccine against Argentine hemorrhagic feven the “ma1 de 10s rastrojos” of the pampas, has been a dream of physicians and scientists involved with the disease since its recognition in the 1950s. Several killed and live immunogens have been produced and tested in pursuit of this goal, none of which has proved suitable for

widespread human use. Recently, a new live-attenuated Junin virus vaccine, Candid #I, was developed through a cooperative international effort. Testing conducted to date indicates that this vaccine holds considerable promise.

A

rgentine hemorrhagic fever (AHF) is

a clinically severe, rodent-borne zoonosis restricted in nature to the fertile

pampas of north-central Argentina. The

causative agent, Junin virus, is an arena-

virus that induces chronic infections

among reservoir rodents of the family

Cricetidae and is transmitted to man via fomites and aerosols of contaminated ex- creta. The region’s rural inhabitants and agricultural workers constitute the population at greatest risk (I). Cases of AHF, typically fatal among 5% to 30% of those

afflicted and generally more severe in

older persons, are recognized throughout the year; but the vast majority of the illnesses occur during a well-defined epi-

‘This article will also be published in Spanish in the Boletin de la Oficina Sanitaria Panamericana, vol. 110. Correspondence should be addressed to Dr. McKee, Division of Medicine, USAMRIID, Ft. De- trick, Frederick, Maryland, 21701-5011, USA. The views of the authors do not purport to reflect the views of the U.S. Department of the Army or De- partment of Defense.

2The Salk Institute Government Services Division, Swiftwater, Pennsvlvania, U.S.A.

aDivision of Medicine, United States Army Medical Research Institute of Infectious Diseases, Fort De- trick, Frederick, Maryland, U.S.A.

demic season that lasts from February through July and coincides with the annual grain harvests.

AHF epidemiology indicates an ex-

panding disease with changing inci-

dence. When the disease first was recognized in the 195Os, its endemic area was limited to a region 16,000 km2 within the province of Buenos Aires (I). Since then it has spread to involve three adjacent

provinces; it currently covers an area

nearly eight times that originally de-

scribed (2), and appears destined to con-

tinue extending outward in a north-

easterly direction (3).

During the 1960s and 197Os, epidemic outbreaks exceeding 1,000 cases per year were frequent. Since 1979, however, the annual incidence has rarely exceeded 400 cases (4; J. I. Maiztegui, unpublished data). One reason is that the continually advancing disease front has created “his- toric” areas where AHF has been seen for 10 years or more. While cases con- tinue to occur in such regions, the annual disease incidence is much lower than that seen in more recently affected regions

(2). Curiously, this phenomenon occurs

despite a continued population suscep-

tibility that exceeds 95% (5).

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HISTORY OF VACCINE

DEVELOPMENT

Inactivated Vaccines

The need for a widely available vaccine to protect against Al% has long been recognized. Initial efforts focused on for-

malin-inactivated mouse brain prepara-

tions (6, 7) that induced neutralizing

antibodies and protected mice and

guinea pigs against virulent Junin challenge (Table 1). One such product was inoculated into more than 15,000 persons between 1959 and 1962, although its efficacy was never assessed (8). However, the need to administer 3-4 weekly doses

with or without a “booster,” together

with concerns about the virus substrate (i.e., mouse brain), severely limited these vaccines’ potential for general use. Re- cently, some of these deficiencies were

addressed through production of a cell

culture-derived, formalin-inactivated im-

munogen. Neutralizing antibodies were

induced successfully with this product; however, vaccinated animals failed to re- sist challenge with a virulent Junin virus strain (9).

Additional inactivation strategies have

included exposure of Junin virus to

photoactive dyes (10, Zl), ultraviolet irra- diation (E’), heating, and acetone (23). Though at least one resulting product has

been administered to humans in limited studies (Table l), all such products have proved of limited utility. A subunit im-

munogen composed of purified G38

envelope glycoprotein emulsified in

Freund’s complete adjuvant has induced neutralizing antibodies in rabbits and has protected guinea pigs against virulent Ju- nin virus challenge (14). While this approach has shown some limited promise, additional studies are still needed to as- sess its potential for human vaccination.

Live Vaccines

The alternative to virus inactivation

and classical vaccine development in-

volves production of a live-attenuated immunogen. As Table 2 indicates, a num-

ber of such immunogens, derived from

both heterologous and homologous vi-

ruses, have been produced and tested.

Heterologous live vaccines. Viruses

comprising the family Arenaviridae fall

serologically into two groups: the so-

called “Old World” (lymphocytic cho-

riomeningitis, Lassa, Mopeia, Mobala,

and Ippy) and “New World” (Machupo,

Junin, Tacaribe, Amapari, Parana, Lat-

ino, Tamiami, Pichinde, and Flexal)

arenaviruses.

The type-member of the “New World” complex, Tacaribe virus (1.5), is closely

Table 1. Candidate Argentine hemorrhagic fever vaccines (inactivated).

Laboratory animal experience Human experience Type Strain used immunogenic Protective Immunogenic Protective

Formalin-

l

Multiple Not tested Yes (6) Not tested Not determined (8) killed MC2 Yes (7) Yes (7) Not tested

Not tested XJ Clone 3 Yes (9) No (9) Not tested Not tested

Photo-

i

Xl Not tested Partial (81 Not tested Not tested inactivated RC Not tested Yes (IS) No (18, 6W No (61)

Subunit MC, Yes (14) Yes (14) Not tested Not tested dBoosted seropositives.

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Table 2. Candidate Argentine hemorrhagic fever vaccines (live).

Strain Passage Laboratory animal experience Human experience Type used historya Immunogenic Protective Immunogenic Protective Heterologous Tacaribe Strain 11573 Yes ( 18-26) Yes (18-26) Not tested Not tested

x10 GP,. MB,, (43) Yes (43, 44) Yes (43, 44) Not tested Not tested XJ Cl 3 _{G”,, MB,,,} Yes (32) Yes (32) Yes (3 1, 34) Possibly (63) Homologous RMK,, HB,,

MA-l 11 3, MB,b

Candid #l GP,, MB,,, FRL,$ Yes (50, 52, 53) Yes (50, 52, 53) Yes (56, 57) Under study GP = guinea pig; MB = mouse brain; RMK = rhesus kidney cell culture; HB = hamster brain; MA-1 11 = rabbit kidney cell line; FRL = fetal rhesus lung cell culture. Subscripts indicate passage numbers.

bPortions of passage history uncertain; lineage reconstructed from written documents provided by Dr. N. Wiebegna and (31)

(see text).

cJ.C. Barrera Oro, manuscript in preparation.

related both serologically and bio-

chemically to Junin virus (26, 17). Cross- protection against Junin virus has been

demonstrated repeatedly in guinea pig

(28-22) and nonhuman primate (23-26)

model systems. Adverse reactions have not been reported among animals receiv- ing Tacaribe virus in doses suitable for

vaccination. A single human infection

with Tacaribe virus has been recognized, this being characterized by fever, head-

ache, and constitutional symptoms that

resulted in hospitalization (noted in 27). However, this infection resulted from exposure to laboratory virus preparations of possibly high titer, making generaliza-

tions regarding human pathogenicity of

Tacaribe difficult. There is no evidence that the virus induces chronic or persis- tent infections in mammalian models (25, reviewed in 17). Heterologous immunity induced by Tacaribe appears to be long lasting (25, 28). On the basis of these ob- servations, Tacaribe virus has been pro-

posed as a candidate heterologous AHF

vaccine (27, 25). No human studies have yet been conducted.

Homologous live vaccines. A fortuitous

observation at the Rockefeller Founda-

tion Virus Laboratories provided the key to developing a live-attenuated homolo-

gous vaccine. Investigators there noted

that the prototype XJ strain of Junin virus

120 Bulletin of PAHO 25(2), 1992

seemed to lose its pathogenicity for

guinea pigs after about 40 passages

through newborn mouse brain (29)-al-

though this was thought initially to be due to host resistance rather than viral attenuation (30). Subsequently, a virus pool was created by Wiebenga at the U.S. National Institutes of Health by passing a plaque-purified isolate (purified in pri- mary rhesus monkey kidney cells) from mouse brain passage 14 of XJ into suckling hamster brain. This strain, desig- nated XJ Clone 3 (N. Wiebenga, written

communication), was passed further in

continuous MA-104 and MA-111 cells,

one passage (from MA-111 cells) being further passed in suckling mouse brain

and evaluated as a prospective vaccine

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XJ Clone 3 proved attenuated and immunogenic after intraperitoneal inoculation of guinea pigs, and was found to

protect animals against virulent virus

challenge (32). Its safety for man then

was evaluated in seven individuals in-

volved in Junin virus research. Mild,

transitory clinical reactions occurred

among the volunteer group (33), and

neutralizing antibodies were elicited in all (34).

On the basis of these encouraging re- sults, XJ Clone 3 was administered to an

additional 629 Argentine volunteers in

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were recorded in 75.6% of 213 intensively

studied individuals: 44% developed fe-

ver, often accompanied by asthenia,

headache, retro-ocular pain, and myalgia which appeared 3-10 days after immu-

nization and resolved spontaneously

within 3 days; 29.1% developed asthenia, headache with retro-ocular pain, and myalgia unaccompanied by fever that lasted 1 to 4 days; and 2.4% developed a local reaction at the site of inoculation that dis- appeared within 48 hours. The remaining

volunteers (24.4%) remained asympto-

matic (35). Out of 57 individuals given

hematologic analyses, 29 (50.9%) dis-

played mild leukopenia, 5 (8.8%) mildly decreased platelet counts, and 11(19.2%) both; no alterations were found in the remaining 12 (21.1%) (31, 35). Neutralizing antibodies were found in 97% of 165 individuals examined 1-3 months after vac-

cination (31). Follow-up studies con-

ducted 7-9 years later on 267 of these volunteers revealed no long-term side ef- fects; 153 of 165 (90%) retained measur- able neutralizing antibodies against Junin virus (31).

Because of uncertainties in the passage history of XJ Clone 3 (its previous pas-

sage through heteroploid cells and

mouse brain substrates), administration of the vaccine was discontinued in 1971

(17). Further characterization of this

product continued, however, and addi-

tional passages of the prototype XJ strain were evaluated for attenuation.

It was clear that XJ Clone 3 was signifi-

cantly attenuated for guinea pigs when

compared with naturally occurring Junin virus strains (36). Nevertheless, residual

peripheral virulence, neurotropism, and

neurovirulence could be demonstrated in

these animals and in primates (37-39).

Moreover, by using sensitive tech-

niques-such as co-cultivation and im-

munoperoxidase staining for antigens-

persistence of the virus in guinea pig tis-

sues could be demonstrated for up to

three months (39-41), and antigen could be found in primate organs over one year after infection (42).

Another attenuated Junin virus strain,

derived from prototype XJ and desig-

nated XJO, was maintained in newborn

mouse brain and not passaged in cell culture (43). Although XJO reportedly con- ferred complete protection against viru-

lent Junin virus challenge for guinea

pigs, it also exhibited persistence, neuro-

tropism, and neurovirulence similar to

those exhibited by XJ Clone 3 (41,43-47).

CANDID #1

Encouraging discussions held in 1976

during the First International Seminar on

Hemorrhagic Fevers Produced by Arena-

viruses (48) led to a joint effort directed at developing an acceptable live-attenuated Junin virus vaccine against AHF. This was begun as a joint effort in 1979 by the U.S. Army Medical Research Institute of

Infectious Diseases and the Argentine

Ministry of Health and Social Action, under auspices of the United Nations De-

velopment Program and PAHO. The

goals were straightforward: identify a virus at least as attenuated as XJ Clone 3 for humans with an acceptable passage history; adapt the virus to replication in cer- tifiable cell substrates; select attenuated variants with minimal residual virulence that are immunogenic and confer protec-

tion upon laboratory animals; and de-

velop and test this candidate vaccine in human volunteers under the supervision

of U.S., Argentine, and PAHO regula-

tory agencies.

Two approaches to vaccine develop-

ment were undertaken concurrently. The

first, identification of an already attenuated Junin virus strain that could be used as a parent virus for further modification, provided a direct route to a highly attenuated virus that could be evaluated for

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acceptability as a vaccine candidate by

appropriate methods. The second en-

tailed isolation and passage in certified cell substrates of a novel, naturally occurring Junin virus strain in order to enrich for attenuated variants. The latter effort yielded viruses with reduced but still sig- nificant virulence, and this approach was abandoned.

The development initiative culminated in the production of Candid #1 vaccine.

The virus strain involved was derived

from a documented (mouse brain and

guinea pig passaged) descendant of prototype XJ Junin virus that had been passaged and cloned in certified diploid mammalian cells (Figure 1) (49; J.G. Bar- rera Or0 et al, manuscript in preparation) .

Candid #l is more attenuated for newborn mice and guinea pigs than XJ Clone 3 (50; J.G. Barrera Oro et al, manuscript

in preparation). Neuroinvasiveness and

neurovirulence in nonhuman primates

are minimal, being less than what is exhibited by XJ Clone 3 (51-53). Candid #l

has demonstrated immunogenicity and

protective efficacy in both guinea pigs and nonhuman primates (26, 50, 52, 53).

Parodl

Strain XJ

-1

Passaqes

Casals

XJll -L

GPp2MBpll

USAMRIID

XJ43 -1

GPp2MBp43

Barrera Or0

XJ44 -1

GPp2MBp44

Candld #l GPp2MBp44FRLplS

Figure 1. Passage history of Candid #l Junin virus vaccine. GPp = guinea pig passage, MBp

= newborn mouse brain passage, FRLp = fetal rhesus lung cell culture passage.

It has also been found to protect ma-

caques against heterologous challenge

with virulent Machupo virus, the closely related etiologic agent of Bolivian hemorrhagic fever (54, 55).

Candid #1 has been evaluated for

safety and immunogenicity in more than

200 human volunteers from both the U.S. and Argentina (56, 57). To date, no signif- icant adverse reactions related to vaccination have been observed, and more than 95% of the recipients have developed an-

tibodies after immunization. The vac-

cine’s efficacy is currently being studied by means of a large, double-blind, pla- cebo-controlled field trial in Argentina’s Santa Fe Province (J.I. Maiztegui, K.T. McKee, Jr., personal communication).

CONCLUSIONS

The history of vaccination against AHF spans a period of more than 30 years. During that time, a variety of classical ap-

proaches to vaccine development have

been employed in an attempt to produce a safe and effective human immunogen. Development of the current most promis- ing candidate, Candid #l, has depended upon experience gleaned from years of

experimentation in laboratories and

clinics on two continents. Specifically, ex- tensive testing of XJ Clone 3 in animal models and humans provided a wealth of scientific information permitting continued modification of the prototype XJ Ju- nin virus strain to ultimately produce Candid #l.

If successful, Candid #l will become the first vaccine widely employed against an arenavirus. It thus appears that experience gained in the e forts to develop it P

should have broader applicability to

other arenaviruses pathogenic for man,

most notably Lassa virus. Recently, vaccine research on Lassa fever prophylaxis has focused upon molecular cloning and vector insertion (58-61). While this excit-

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ing new approach holds great promise for vaccine development, it has not yet

produced a product suitable for human

use. Should the potential of this new 10.

technology fail to be realized, the experience gained through Junin virus vaccine

development will be of great utility in 11.

planning and implementing a classical

Lassa vaccine program.

12.

REFERENCES

1.

2.

3.

4.

5.

6.

7.

8.

9.

Maiztegui JI. Clinical and epidemiological patterns of Argentine haemorrhagic fever. Bull WHO. 1975;52:567-75.

Maiztegui J, Feuillade M, Briggiler A. Progressive extension of the endemic area and changing incidence of Argentine hemorrhagic fever. Med Microbial Immu- nol. 1986;175:149-52.

Mills J, Ellis B, Maiztegui J, Ksiazek T, McKee K, Childs J. Prevalence of Junin virus antigen and antibody among rodents in endemic and non-endemic areas of Argentine hemorrhagic fever. Hon- olulu, Hawaii: 38th Annual Meeting of the American Society of Tropical Medi- cine and Hygiene; 1989. (Abstract). Carballal G, Videla CM, Merani MS. Epi- demiology of Argentine hemorrhagic fever. Eur J Epidemiol. 1988;4:259-74.

Weissenbacher MC, Sabattini MS, Avila MM, et al. Junin virus activity in two rural populations of the Argentine hemorrhagic fever (AHF) endemic area. ] Med Viral. 1983;12:273-280.

Pirosky I, Martini P, Zuccarini J, et al. Vir- osis hemorrrigica de1 noroeste bonaerense (endemo-epidemica, febril, exantemitica y leucop&ica): V, la vacuna especffica y ~43apaci6n. Orientac Med. 1959; 8:

Barrera Oro JG, Girola RA, Frugone G. Estudios immunoldgicos con virus Junfn: II, inmunidad adquirida por cobayos inoc- ulados con virus inactivado por formol. Medicina (Buenos Aires). 1967;27:279-82. Mettler N. Argentine hemorrhagic f#er: cur-

rent knowledge. Washington, DC: Pan American Health Organization; 1969. (PAHO scientific publication no. 183). Videla C, Carballal G, Remorini P, La

13.

14.

15.

16.

17.

18.

19.

20.

21.

Torre J. Formalin inactivated Junin virus: immunogenicity and protection assays. I Med Viral. 1989;29:215-20.

Parodi AS, de Guerrero LB, Weissen- bather M. Fiebre hemorr&ica Argentina: vacunaci6n con virus Junfn inactivado. Cienc Invest. 1965;21:132-33.

De Martinez Segovia ZM, Arguelles G, Tokman A. Capacidad antigknica de1 virus Junfn inactivado mediante oxidaci6n fotodin&mica. Medicina (Buenos Aires). 1980;40:156-60.

D’Aiutolo AC, Lampuri JS, Coto CE. Re- activaci6n de1 virus Junfn inactivado por la luz ultravioleta. Medicina (Buenos Aires). 1979;39:801.

Carballal G, Videla C, Oubiiia JR, Frigerio MJ. Antigenos inactivados de virus Junin. Medicina (Buenos Aires). 1985;45:153-58. Cresta 8, Padula P, de Martinez Segovia ZM. Biological properties of Junin virus proteins: I, identification of the immunogenic glycoprotein. Intervirology. 1980; 13:284-88.

Downs WC, Anderson CR, Aitken THG, Spence L, Greenhall AH. Tacaribe virus, a new agent isolated from Artibeus bats and mosquitos in Trinidad. Am ] Trop Med Hyg. 1963;12:639-46.

Damonte EB, Mersich SE, Candurra NA, Coto CE. Cross reactivity between Junin and Tacaribe viruses as determined by neutralization test and immunoprecipita- tion. Med Microbial Immunol. 1986; 175:85-88.

Weissenbacher MC, Laguens RF’, Coto CE. Argentine hemorrhagic fever. Curr Top Microbial Immunol. 1987;134:79-116. Parodi AS, Coto CE. Inmunizacidn de cobayos contra el virus Junin por inocula- cidn de1 virus Tacaribe. Medicina (Buenos Aires). 1964;24:151-53.

Tauraso N, Shelokov A. Protection against Junin virus by immunization with live Tacaribe virus. Proc Sot Exp Biol Med. 1965;119:608-11.

Weissenbacher MC, Coto CE, Calello MA. Cross-protection between Tacaribe complex viruses: presence of neutralizing antibodies against Junin virus (Argentine hemorrhagic fever) in guinea pigs infected with Tacaribe virus. Interoirology. 1975-76;6:42-49.

Coto CE, Damonte EB, Calello MA, Weissenbacher MC. Protection of guinea

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?igs inoculated with Tacaribe virus

igainst’lethal doses of Junin virus. J Infect

3is. 1980;141:389-93.

22. 3amonte EB, Calello MA, Coto CE, Weissenbacher MC. Inmunizacion de co- >ayos contra la fiebre hemorragica Argen- bina con virus Tacaribe replicado en cd-

ulas diploides humanas. Medicina (Buenos Aires). 1981;41:467-70.

23. Weissenbacher MC, Coto CE, Calello MA, Rondinone SN, Damonte EB, Frigerio MJ. Cross-protection in nonhuman primates against Argentine hemorrhagic fever. In- fect Immun. 1982;35:425-30.

24. Samoilovich SR, Pecci Saavedra J, Frig- erio MJ, Weissenbacher MC. Nasal and intrathalamic inoculations of primates with Tacaribe virus: protection against Ar- gentine hemorrhagic fever and absence of neurovirulence. Acfa Virol. 1984;28: 277-81.

25. Samoilovich SR, Calello MA, Laguens RR Weissenbacher MC. Long-term protection against Argentine hemorrhagic fever in Tacaribe virus infected marmosets: vir- ologic and histopathologic findings. J Med Virol. 1988;24:229-36.

26. Kenyon RH, McKee K, Barrera Oro J, Ragland D, Crabbs C, Higgins Y. Protec- tive efficacies in rhesus monkeys of Can- did 1 strain Junin virus (JV) and Tacaribe virus against aerosol challenge with virulent JV Honolulu, Hawaii: 38th Annual Meeting of the American Society of Tropi- cal Medicine and Hygiene; 1989. (Abstract #136).

27. Peters CJ, Jahrling PB, Liu CT, Kenyon RH, McKee KT Jr, Barrera Oro JG. Experi- mental studies of arenaviral hemorrhagic fevers. Curr Top Microbial lmmunol. 1987;134:5-68.

28. Weissenbacher MC, Damonte EB. Fiebre hemorragica Argentina. Adel Microbial Enferm Infect. 1983;2:119-71.

29. Mettler NE, Casals J, Shope RE. Study of the antigenic relationships between Junin virus, the etiological agent of Argentinean hemorrhagic fever, and other arthropod- borne viruses. Am j Trop Med Hyg. 1963;12:647-52.

30. Casals J. Serological studies on Junin and Tacaribe viruses. Am J Trop Med Hyg. 1965; 14:794-95.

31. Ruggiero HA, Magnoni C, Guerrero LB 32.

33.

34.

35.

36.

37.

38.

39.

40.

de, et al. Persistence of antibodies and clinical evaluation in volunteers 7 to 9 years following the vaccination against Argentine hemorrhagic fever. J Med Virol. 1981;7:227-32.

Guerrero LB de, Weissenbacher MC, Par- odi AS. Inmunizacion contra la fiebre hemorrdgica Argentina con una cepa atenuada de1 virus Junin: I, estudio de una cepa modificada de1 virus Junfn, inmunizacion de cobayos. Medicina (Buenos Aires). 1969;29:1-5.

Ruggiero HR, Astarloa L, Gonzalez Cam- baceres C, Maglio F, Squassi G. In- munizacion contra la fiebre hemorrdgica Argentina con una cepa atenuada de virus Junfn: II, inmunizacion de voluntarios, anglisis clinic0 y de1 laboratorio. Medicina (Buenos Aires). 1969;29:81-87.

Weissenbacher M, Guerrero LB de, Help G, Parodi AS. Inmunizacion contra la fiebre hemorragica Argentina con una cepa atenuada de virus Junin: III, reac- ciones serologicas en voluntarios. Medic- ina (Buenos Aires). 1969;29:88-92.

Ruggiero HA, Magnoni C, Cintora FA, et al. Inmunizacion contra la fiebre hemorrdgica Argentina con una cepa atenuada de virus Junfn: anAlisis de 636 vacunados. Prensa Med Argenf. 1974;61:231-40. Contigiani MS, Sabattini MS. Virulencia diferencial de cepas de virus Junfn por marcadores biologicos en ratones y cobayos. Medicina (Buenos Aires). 1977; 37:244-51.

Avila MM, Samoilovich SR, Weissen- bather MC. Infection de1 cobayo con la cepa atenuada de1 virus Junfn XJCl,. Me- dicina (Buenos Aires). 1979;39:597-603. Carballal G, Cossio PM, de la Vega MI, et al. Neurovirulencia de la cepa XJCI, de virus Junfn en un primate sudamericano. Cordoba, Argentina: III Congreso Argen- tino de Microbiologfa; 1982. (Abstract #186).

Laguens RM, Avila MM, Samoilovich SR, Weissenbacher MC, Laguens RI?. Patho- genicity of an attenuated strain (XJCl,) of Junin virus: morphological and virological

studies in experimentally infected guinea pigs. Infervirology. 1983;20:195-201. Avila MM, Laguens RM, Laguens Rl’, Weissenbacher MC. Selectividad tisular e indicadores de virulencia de tres cepas de1

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virus Junk Medicina (Buenos Aires). 1981;41:157-66.

41. Malumbres E, Boxaca MC, Guerrero LB de, Berria M, Lascano EF. Persistence of attenuated Junin virus strains in guinea pigs infected by IM or IC routes. 1 Infect Dis. 1984;149:1022.

42. Avila MM, Frigerio MJ, Weber EL, et al. Attenuated Junin virus infection in Cal- lifhrixjacchus. 1 Med Viral. 1985;15:93-100. 43. Guerrero LB de, Boxaca MC. Estudio pre-

liminar de una variante atenuada de1 virus Junfn derivada de la cepa prototipo

XJ.

Medicina (Buenos Aires). 1980; 40:267-74.

44. Boxaca MC, Guerrero LB de, Weber EL, Malumbres E. Protection inducida en cobayo por la variante XJa de1 virus Junfn. Medicina (Buenos Aires). 1981;41:25-34. 45. Guerrero LB de, Boxaca MC, Rabinovich

RD, Malumbres E. Evolution de la infec- cion en cobayos infectados con la variante atenuada XJ, de1 virus Junfn. Rev Argenf Microbial. 1983;15:205-12.

46. Boxaca MC, Guerrero LB de, Malumbres E. Modification of Junin virus neurotropism in the guinea pig model. Acfa Viral. 1984;28:198-203.

47. Guerrero LB de, Boxaca MC, Malumbres E, Mercedes Gomez M de las. Patho- genesis of attenuated Junin virus in the guinea pig model. 1 Med Virol. 1985; 15:197-202.

48. Eddy GA, Barrera Oro JG. Discusion general. Medicina (Buenos Aires). 1977; 37(Suppl3):257-59.

49. Barrera Oro JG, Eddy GA. Characteristics of candidate live attenuated Junin virus vaccine. Banff, Alberta, Canada: Interna- tional Conference on Comparative Virol- ogy; 1982. (Abstract #S4-10).

50. Lupton Hw, Cole FE, Moe J, et al. Safety and efficacy of a live, attenuated vaccine against Argentine hemorrhagic fever. Sendai, Tanan: Sixth International Con- gress of I Virology; 1984. (Abstract #P27-22).

51. McKee KT Jr, Green DE, Mahlandt BG, et al. Infection of Cebus monkeys with Junin virus. Medicina (Buenos Aires). 1985; 45~144-52.

52. McKee KT, Barrera Oro JG, Kuehne AI, Spisso J, Mahlandt BG. Immunogenicity and protective capacity of a live, attenu-

ated Argentine hemorrhagic fever (AHF) vaccine in primates. Calgary, Canada: XI International Congress for Tropical Medi- cine and Malaria; 16-22 September 1984. (Abstract).

53. Barrera Oro JG, McKee KT Jr, Kuehne AI, et al. Preclinical trials of a live, attenuated Junin virus vaccine in rhesus macaques. Paris, France: Institut Pasteur Sympo- sium on Vaccines and Vaccinations; 1985. (Abstract #l?28).

54. Lupton Hw, Jahrling PB, Barrera Oro JG, Peters CJ. Cross-protection against Ma- chupo virus with Candid #l live-attenuated Junin virus vaccine: II, postchallenge virological and immunological findings. Mar de1 Plata, Argentina: Second Interna- tional Conference on the Impact of Viral Diseases on the Development of Latin American Countries and the Caribbean Region; 1988. (Abstract #PE2).

55. Jahrling PB, Trotter RW, Barrera Oro JG, et al. Cross-protection against Machupo virus with Candid #1 Junin virus vaccine: III, postchallenge clinical findings. Mar de1 Mata, Argentina: Second Interna- tional Conference on the Impact of Viral Diseases on the Development of Latin American Countries and the Caribbean Region; 1988. (Abstract #PE3).

56. MacDonald C, McKee K, Peters C, Fein- sod F, Cosgriff T, Barrera Oro J. Initial clinical assessment of humans inoculated with a live-attenuated Junin virus vaccine. Edmonton, Alberta, Canada: VII Interna- tional Congress of Virology; 1987. (Ab- stract #R3.27).

57. Maiztegui JI, McKee KT Jr. Inoculation of human volunteers with a vaccine against Argentine hemorrhagic fever. Banff, Al- berta, Canada: Sixth International Con- ference on Comparative and Applied Vir- ology; 1989. (Abstract #S4).

58. Clegg JC, Lloyd G. Vaccinia recombinant expressing Lassa-virus internal nucleo- capsid protein protects guinea pigs against Lassa fever. Lancef. 1987; 2(8552):186-88.

59. Auperin DD, Esposito JJ, Lange JV, et al. Construction of a recombinant vaccinia virus expressing the Lassa virus glycoprotein gene and protection of guinea pigs from a lethal Lassa virus infection. Virus Res. 1988;9:233-48.

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60. Morrison HG, Bauer SF’, Lange JV, Es- posito JJ, McCormick JB, Auperin DD. Protection of guinea pigs from Lassa fever by vaccinia virus recombinants expressing the nucleoprotein or the envelope gly- coproteins of Lassa virus. Virology.

1989;171:179-88.

a recombinant vaccinia virus containing the Lassa virus glycoprotein gene. Proc

Nat1 Acad Sci USA. 1989;86:317-21. 62. Guerrero LB de. Vacunas experimentales

contra la fiebre hemorrsigica Argentina.

Medicina (Buenos Aires) 1977;37(Suppl 3):252-259.

61. Fisher-Hoch SF’, McCormick JB, Auperin 63. Johnson KM. Status of arenavirus vac- D, et al. Protection of rhesus monkevs cines and their application. Bull WHO.

from fatal Lassa fever by vaccination w&h 1975;52:729-35. - -

Cholera Takes Hold in the Americas

The epidemic of cholera that is currently raging in South America has spread from its initial focus on the Pacific coast of Peru to areas throughout that country and to parts of Chile, Colombia, and Ecuador. From the start of the epidemic in January to 2 May 1991, almost 175,000 cases, of which 1,390 were fatal, had been notified by health authorities in those four countries, as follows: 169,255 cases and 1,244 deaths in Peru, 5,005 cases (1,107 confirmed) and 140 deaths in Ecuador, 189 cases and 5 deaths in Colombia, and 26 cases and 1 death in Chile. In addition, five cases have been reported from Amazonas State in Brazil, and five imported cases have occurred in the United States.

Cholera is an acute bacterial enteric disease, usually of sudden onset, that causes profuse watery stools, vomiting, rapid dehydration, acidosis, and circulatory collapse. Mild cases may be indistinguishable from other types of diarrhea except by fecal cultures. Without treatment, the case- fatality rate of serious cholera can reach 50%. With adequate treatment, mortality is around 1%. Management of the disease focuses on replacing fluids and electrolytes.

The prognosis for the course of the epidemic in the affected countries and in other countries in the Region is guarded, since it is not possible to prevent the spread of cholera from one country to another. The living con- ditions of the population are a crucial factor in determining the intensity with which cholera epidemics spread.

A comprehensive report on cholera in the Americas will appear in the next issue of the Bulletin ufPAH0.

Source: WHO, wkly Epidemiol Ret 66(18):132,1991; and PAHO, Epidemiol Bull 12(l): 1,2,14-15,199l.