ARGENTINE HEMORRHAGIC FEVER:

CURRENT KNOWLEDGE

by

Norma E. Mettier, M.D.

PAN AMERICAN HEALTH ORGANIZATION

Pan American Sanitary Bureau, Regional Office of the

WORLD HEALTH ORGANIZATION

1969

ARGENTINE HEMORRHAGIC FEVER:

CURRENT KNOWLEDGE

by

Norma E. Mettier, M.D.

Scientific Publication No. 183

PAN AMERICAN HEALTH ORGANIZATIONPan American Sanitary Bureau, Regional Office of the

WORLD HEALTH ORGANIZATION525 Twenty-third Street, N.W.Washington, D.C. 20037, U.S.A.

1969

NOTE

One of the commitments of the Pan American Health Organization,carried out through its Department of Research Development and Co-ordination, is to improve communication among biomedical scientists.In view of the scientific and public health importance of Argentinehemorrhagic fever---a disease that has been under study for over tenyears by diferent teams of investigators-it seemed that a summary ofcurrent knowledge in this field would be of considerable interest tophysicians and epidemiologists throughout the Americas. The presentmonograph, which is completed by an exhaustive bibliography, was pre-pared by Dr. Norma E. Mettler, formerly of the Department of Micro-biology and Parasitology of the School of Medicine, University of BuenosAires, Argentina, and at present at the Department of Epidemiology andPublic Health of the Yale University School of Medicine, New Haven,Connecticut, U.S.A.

CONTENTS

Page

1. Historical Background ..................................................... 1

2. General Ecology of Argentina ............................ ......... 5

3. Epidemiological Observations .......................................... 11

4. The Disease ...................................................................... 15

5. Junín Virus ...................................................................... 22

6. Immunological Investigation ............................................ 30

7. Isolation and Identification of Junín Virus ............... ...... 33

8. Special Study of the 1963 Outbreak ................................ 36

9. Recent Extension of AHF to New Areas .......................... 42

10. Final Considerations ........................................................ 43

REFERENCES .......................................................................... 45

iii

Figures and Tables

Page

Figure 1 Province of Buenos Aires, Argentina: Northwestern Parishes in Which AHFWas First Recorded ................................................................................................. 2

Figure 2 Argentina: Major Political Subdivisions; Principal Rivers, Roads, and Rail-roads .......................................................................................................................... 6

Figure 3 Argentine Hemorrhagic Fever: Morbidity and Mortality Curves, 1959, 1960,and 1961 .................................................................................................................. 12

Figure 4 Curves Comparing Registered Cases of AHF with Serological Results, 1963 36

Table 1 Clinically Diagnosed Cases of AHF: Geographical and Chronological Dis-tribution, 1963 ......................................................................................................... 37

Table 2 Cases of AHF with Serological Conversion for Junín Virus as Against All AHFCases Serologically Studied, 1963 .......................................................................... 38

Table 3 AHF Cases Serologically Negative with Respect to Junín Virus: Cases withAntibodies for Group B Arboviruses as Against Total Junín-Negative CasesTested for Group B, 1963 ...................................................................................... 40

Figure 5 Histogram of Registered Cases of AHF and Serological Results, 1963 .............. 41

Table 4 Presence of Junín Virus CF Antibodies in Persons Who Had AHF fromOne to Ten Years Before Serological Test ........................................................... 43

V

1. HlISTORICAL BACKGROUND

The endemo-epidemic disease that is thesubject of this essay has been observed fora great many years in Argentina, in thenorthwestern part of the Province of BuenosAires. Currently named Argentine hemor-rhagic fever (AHF), after the country inwhich it was first described, it has beenknown in the past by a variety of othernames-enfermedad del sello, mal de losrastrojos ("stubble disease"), mal de O'Hig-gins ("O'Higgins' disease"), enfermedad deJunín ("Junín disease"), gripón ("severeflu"), and so on.

It is difficult if not impossible to deter-mine the exact year in which this maladywas first observed. According to the datacollected by Martínez Pintos (54), epi-demics of variable intensity, reaching theirpeak in the fall of the year (April and May)and sometimes showing a high rate of mor-tality, have been recorded since 1943. Pre-liminary observations were made at the Juliode Vedia Hospital in Nueve de Julio Parish,Province of Buenos Aires (Fig. 1), wherethe clinical symptoms were thought to bedue to "malignant grippe."

The first mention of the disease in theliterature comes from Arribalzaga (5), whoreported "a new epidemic disease of un-known etiology: nephrotoxic, leukopenic,and [characterized by] enanthematous hyper-thermia." The first cases were observed inthe city of Bragado, Province of BuenosAires, in 1953 and 1954. The incidence washighest among rural laborers, and the firstpatients were potato harvesters who had beenworking in the outskirts of Bragado andAlberti. Later the epidemics spread to the

urban areas. The 1953 epidemic startedwith sporadic cases early in the year, rapidlycame to a peak in April and May, andtapered off slowly, to disappear after Au-gust. The following year morbidity waslower but the characteristics were basicallythe same. Cases did not break out simul-taneously in the same household or in insti-tutions. Males were more frequently affectedthan females, and only a few cases in chil-dren, 10 to 12 years old, were observed. Atthe time of the epidemics, Arribalzagalooked, without success, for epizootic dis-eases in the afflicted area and for changes inweather or sanitary conditions that might ac-count for the outbreak. Attempts to isolateviruses, Leptospira, or bacteria from patientswere also unsuccessful. He concluded, onthe basis of differential diagnosis, that avirus was probably responsible for the dis-ease. It was postulated that the source ofcontamination was something in the field,since all the cases but two failed to provideevidence of direct human-to-human trans-mission (5).

In 1956, Domingo Duva published a re-port (34) on 25 patients whom he attendedpersonally from February to September1953 and from April to August 1954 inMechita, a small town in Bragado Parish.Mechita, with a population of 2,150, is atypical rural area with unpaved streets andidle land even in the center of town. Of the25 patients, 5 were youngsters between 12and 16, and the remaining 20 were adults-16 men and 4 women. None of them hadbeen working in the fields. The cases werescattered about the town. Pigs, rabbits, dogs,

1

FIG. 1. PROVINCE OF BUENOS AIRES, ARGENTINA: NORTHWESTERN PARISHES IN

WHICH AHF WAS FIRST RECORDED

61° 60°

DE MAYO

¡ 340

35°

61°

o60

(REPRODUCED FROM PIROSKY, et al, 19591

2

ALEM

350

and an unusual number of rats were foundnearby the patients' homes. Remarking onthe lack of interhuman contagion, Duva con-sidered the possibility of a leptospiral in-fection.

Though there are no publications or offi-cial records on cases during 1955, 1956, or1957, physicians in the endemic areaclaimed that indeed there had been casesevery year and that their attempts to reportthem to public authorities had met with un-successful results. The situation came to ahead, however, in 1958. It so happened thatthe Minister of Public Health, a physicianwith knowledge of the problem, had land-holdings in the endemic area. The epidemicthat year was more severe than ever, andit received wide publicity through newspa-pers, radio, and television. All these cir-cumstances combined to press the publichealth authorities into action.

At last local physicians had support fromthe public and the government. Work couldstart on looking for the etiology of the dis-ease and developing preventive measures.Commissions were organized. One, headedby Dr. Pirosky, was appointed by the Na-tional Ministry of Public Health; another,under Dr. Martínez Pintos, was set up bythe Provincial Ministry of Public Health;a third was organized by the Medical Schoolof Buenos Aires University at the invitationof local medical groups in the endemic area.This last team was made up of personnelfrom the Department of Infectious Diseases,then headed by Dr. Humberto Ruggiero, andthe Department of Microbiology and Parasi-tology, under Dr. Daniel Greenway. Thepresent author was engaged in a full-timeprogram of teaching and research at the lat-ter department during this period.

Field trips from the city of Buenos Aires,where the laboratories were located, to theendemic area, especially the Junín RegionalHospital, were conducted to collect mate-rials for the isolation of infectious agents.Blood, spinal fluid, urine, feces, and pieces

of organs from fatal cases were inoculatedin experimental animals and in media forgrowing bacteria and fungi. The search wascentered on pathogenic microorganisms,viruses and Leptospira in particular, thatwould be most likely to be involved becauseof the epidemiological pattern.

Guinea pigs proved to be the most sus-ceptible of the animals used. They developeda pathological picture similar to the humandisease, with exaggerated hemorrhagic man-ifestations.

From three patients the Buenos AiresUniversity team isolated three strains of anagent-in one instance from the blood, inanother from the urine (a case with hema-turia), and in the third from organs of afatal case (81). The agent was named"Junín virus," because the work had beenperformed with materials collected from theRegional Hospital in the city of Junín. Inde-pendently, Dr. Pirosky's team isolated avirus with which it was possible to reproducethe disease in man (93). Even though theagent isolated by the Buenos Aires Univer-sity group was also capable of producing thedisease in humans (108), it was not until1961 that the viruses isolated by both teamswere compared to determine whether theywere one single agent or different virusesproducing similar clinical manifestations(69).

Studies directed toward finding Junín virusin nature were started, and successful isola-tions were achieved with wild mice (79) andmites (85). The agent's presence in wildmice and in an arthropod, plus its sensitivityto sodium desoxycholate and the lack of in-terhuman infection, gave reason to place itin the arthropod-borne group of viruses.

Junín was tested against all the otherviruses in the collection of The RockefellerFoundation Virus Laboratories and found tobear an antigenic relationship to a strain ofTacaribe virus isolated from bats in Port ofSpain, Trinidad, in 1956 (32). Since

3

Tacaribe had primacy, a new complex withthis name was born. Further isolations ofJunín from patients and wild animals in na-ture by different laboratories resulted in thecollection of some 100 or more strains.

Shortly after the isolation of Junín virus,an epidemic with clinical symptomatologysimilar to that of AHF appeared in Bolivia.Serological study showed the presence ofantibodies against Junín virus in convales-cents (129). The following year, in a sim-ilar outbreak in San Joaquín Valley, Bolivia,another virus was isolated and namedMachupo. This agent showed a relationshipto Junín in the complement-fixation test butnot in the serum neutralization test. (46).

Still another agent, designated Portillovirus, was isolated at the Children's Hospitalin the city of Buenos Aires in 1963 in thecourse of an etiological study of a children'sdisease known as uremic-hemolytic syn-drome. This virus, shown to be related toTacaribe and Junín by complement-fixation(70), is currently under study at YaleUniversity to fully determine its relationshipto the other members of the Tacaribe group.

Shortly after the designation of Portillovirus, the Belém Laboratories in Brazil an-nounced that their team had isolated a virusrelated to the Tacaribe group from wildrodents. This agent was recognized as a newmember and given the name Amaparí (87).In 1966, Pichinde virus, also related to thegroup, was isolated from wild rodents inColombia. This agent is currently under

study at the World Reference Center forArboviruses at Yale University (122). Andfinally, not long ago the Communicable Dis-ease Center in Atlanta, Georgia, isolated twostrains from wild rodents trapped in Tampa,Florida, and found the virus to be relatedto Amaparí and Pichinde. It has been namedTamiami (Chamberlain, personal communi-cation).

Thus, the Tacaribe group now has the fol-lowing seven members: Tacaribe, Junín,Portillo, Machupo, Amaparí, Pichinde, andTamiami. Further investigations will definethe relationships among the various membersof this group. In the course of isolatingthese agents, two important facts have beenbrought to light. One is that viruses fromthe Tacaribe group have been isolated fromwild rodents in the United States. The otheris that the uremic-hemolytic syndrome, aclinical condition of unknown etiology re-ported in the United States (60), has thesame clinical manifestations as the diseasein Argentina which has been proven in atleast 60 per cent of the cases studied to becaused by an agent of the Tacaribe group(66). Clearly, there is a need for betterdissemination of knowledge about humandisease produced by viruses of the Tacaribegroup. The present paper is intended tomake available in English a summary of theknowledge acquired to date about Argentinehemorrhagic fever-which has been tracedin at least 70 per cent of the cases to Junínvirus.

4

2. GENERAL ECOLOGY OF ARGENTINA

Geography

Argentina owes its wide range of climateto sharp variations in altitude and a latitudi-nal extension of over 33 degrees, from21°55'S to 55°3'S. The wedge-shaped con-tinental territory, 3,700 km long and 1,700km across at its widest point, covers ap-proximately 2,800,000 km2 (Fig. 2).

The Andean mountain chain, which ex-tends the entire length of South America,forms a natural division between Argentinaand Chile. In the north, the Andean rangesextend east through approximately one thirdof the Argentine territory, but farther souththe width of the mountainous border dimin-ishes sharply. All the territory east of thischain, comprising by far the greater part ofthe country, has the character of a plain,rising from sea level at the Atlantic coast tothe Andean foothills. The highest elevationin the Argentine Andes is the Aconcagua, a7,021-meter peak north of the Río Negro.In the Province of Córdoba there are threeshort parallel ranges belonging to anotherformation older than the Andes.

In all parts of the republic, January is thewarmnest month of the year, and June andJuly are the coolest.

The main watersheds belong to the LaPlata system, which is made up of theParaná, Uruguay, Pilcomayo, Bermejo, Para-guay, and La Plata rivers. Other importantrivers are the Colorado, the Negro, theChubut, and the Deseado, which form thePatagonian system and flow into the Atlantic.In the interior there is the Andean snow-fedDesaguadero system. And finally, there is

the Dulce river, which starts in the highlands,undergoes various name changes, and finallyflows into the Mar Chiquita lagoon in theProvince of Córdoba.

The railroad system, which has a total of44,000 km of track, stems outward fromthe port of Buenos Aires and connects withlines in neighboring Chile, Bolivia, andParaguay. The main roads also fan outwardfrom the capital. Neither the railroads northe highways seem to bear any direct rela-tion to the spread of AHF, though there arenumerous fences along these routes, andalso between fields, that constitute an im-portant shelter for rodents.

There is aviation service to all the mniajorcities of the interior. Exchanges with inter-national service are made at the heavilytrafficked Ezeiza Airport, which is situateda few miles from the city of Buenos Aires.

Administratively, Argentina is divided intoa federal district, 22 provinces, and a terri-tory. The Federal District (city of BuenosAires) is the political, economic, and cul-tural capital of the country. The provincesmost affected by AHF are Buenos Aires,Santa Fe, La Pampa, and Córdoba.

The Argentine people, some 23,000,000strong, are generally first, second, or thirdgeneration Europeans, the heaviest migra-tions having come from Spain and Italy,with smaller groups from Germany, England,France, Belgium, Holland, and Switzerland.Spanish is the national language-and there-fore the language in which much of the exist-ing literature on AHF has been published.In general, the country is sparsely populated,with an average density of about seven per-

5

FIG. 2. ARGENTINA: MAJOR POLITICAL SUBDIVISIONS; PRINCIPAL RIVERS,

ROADS, AND RAILROADS

CORRIENT

ARGENTINA

· CITY

RIVER

ROAD

t: ;:;aii : RAILROAD-

................... PROVINCE BOUNDARY

-..-.- INTERNATIONAL BOUNDARY

6

sons per square kilometer, but there is a highconcentration in Greater Buenos Aires,where some 7,000,000 people have theirhomes.

Economy

Argentina is essentially an agriculturalcountry. Around 5,220,000 tons of corn areharvested each year from 3,420,000 hectares,mainly in the AHF endemic area. Wheat iscultivated on about 5,250,000 hectares andyields over 5,000,000 tons a year. Nearlyhalf of this produce-2,570,000 tons ofcorn and 2,500,000 tons of wheat-is ex-ported annually. Flax is abundant, and Ar-gentina is the first producer of this oleaginousplant in the world. Oats, barley, and rye arealso cultivated on a broad scale.

The bulk of the nation's agricultural prod-ucts, with certain exceptions, come from theAHF endemic area. The exceptions aresugar cane, from Tucumán, Salta, and Jujuy;cotton, from Chaco, Formosa, Corrientes,and Santiago del Estero; grapes, from Men-doza, San Juan, and the Río Negro Valley;and citrus fruits, apples, and pears from RíoNegro, Entre Ríos, Corrientes, Misiones,Salta, Tucumán, and Jujuy.

Forests cover more than 100,000 hec-tares. Quebracho extract and timber are themost important forest products, and theycome mainly from the Province of Misiones.

Argentina's fishing industry is greatly un-derexploited. Though the country has a"marine pampa" extending along the At-lantic coast from the La Plata river to theBeagle canal and covering an area of1,000,000 km2, there are only a few fishingcenters-among the most important, Mardel Plata, Necochea, and Rawson (Chubut).

The country also has vast mineral wealthpotential, but so far the only important ef-forts at exploitation have been limited topetroleum and iron, mainly in Patagonia andin the northwestern provinces-Salta, Jujuy,and Mendoza.

Meat, grapes, wine, sugar, oil, yerba mate(Paraguayan tea), and textile manufacturesare old and characteristic Argentine in-dustries. Heavy and light industries, such asthe manufacture of cars, refrigerators, tele-vision sets, and petrochemical products-plastics, synthetic detergents, india rubber,insecticides, synthetic fibers, and the like-have been developed since World War II.

Cattle raising, which already thrived dur-ing the Spanish colonial period, is the oldestand most important rural activity. Argentinais the world's number one exporter of meat.Of approximately 43,500,000 head of cattle,most are concentrated in the provinces ofBuenos Aires, Santa Fe, Corrientes, andEntre Ríos. Sheep raising is extensive mainlyin the southern provinces. In all, the countryhas about 50,000,000 sheep, and its woolexports place it internationally in third place,after Australia and New Zealand. There areabout 7,300,000 horses, which are raisedmainly in the provinces of Buenos Aires,Santa Fe, and La Pampa.

Endemic Area

General description

The AHF endemic area basically includesthe provinces of Buenos Aires and LaPampa, the southern part of Santa Fe, andthe eastern part of Córdoba. It is situated inthe richest part of the country, commonlyreferred to as the Pampa region. This dis-trict includes the most populous provincesand is scored by many principal and sec-ondary roads and railways. It has a tem-perate, healthful climate. Though it enjoysa large amount of sunshine, generally thereis adequate rainfall as well. The area extendsover nearly 10 degrees of latitude, but theclimate shows little variation. It never snows.Light frost may sometimes occur during thecold months, but the vegetation is neverfrozen.

7

The advent of European civilization inArgentina produced a marked change in theflora of this region. Useful trees and plantsfrom every part of the world-cereals,alfalfa, grasses, and all varieties of fruit-were introduced. The Australian eucalyptus,in particular, thrives in the Pampa. Othervarieties that have been planted extensivelyinclude the acacia, the sycamore, the para-dise tree, and several types of evergreens.The trees are especially seen close to homesin the rural areas.

In winter the land is plowed in prepara-tion for spring planting. In the spring thereare fields of flax and wheat, and in the sum-mer, corn. Alfalfa and pasture are seen atall times of the year. The fields are fre-quently from 50 to 80 hectares in size. Dur-ing summers with humidity and abundantrain-for example, the summer of 1964grassland vegetation is luxuriant. This con-dition is accompanied by an increase in thewild rodent population, because the vegeta-tion provides abundant food as well as pro-tection against predators such as owls andhawks. If the rodent population gets toohigh, epizootics and intra- and interspecificfighting-similar to the 1958 Microtus crashin Oregon-develop among these animals bythe end of the winter (30).

Most of the towns in the endemic area aresmall. Generally the streets are unpaved andthere is no running water or sewage disposalsystem. Distances between large cities withproper sanitary facilities are usually quitegreat.

The rural dwellings, scattered over thelarge areas between the towns, are typicallymade of adobe, rarely of concrete. Thefloors and patios are made of brick or plainearth; roofs are of wood and zinc; and theinsulation usually consists of adobe, straw,or sometimes wattle and daub. A homegenerally has one or two bedrooms and anadjoining room that serves as kitchen anddining room. Tin sheds, chicken coops, andpigpens are found near the house. The

privy installed over a cesspool only a fewfeet from the house completes this unit.Drinking water is obtained from a pump,well, spring, or cistern. The boundaries be-tween these dwellings and the open fieldsare wire fences, reed grass, or shrubs anddo not form a barrier for wild rodents.

The corn and sunflower harvest coincideswith the autumnal peak of the AHF epi-demic cycles (April and June). The manualharvesting of the crops employs a greatnumber of laborers from the town or fromother provinces, and usually an entire fam-ily, including women and children, par-ticipates. During this time the laborers, mostoften coming from other places, build hutsof maize stems and straw on the groundover the field where the crop is beingharvested. Thus, both their working andleisure time is spent in intimate proximitywith the wild rodents.

Fauna: wild and domestic

The Pampa has a great variety of birdlife, including hawks, falcons, owls, herons,storks, swans, partridge, plovers, ducks,chajas, and many other species known bynative names. Frogs, toads, and ants areubiquitous.

The search for the etiological agent ofAHF in the animal reservoir was concen-trated mainly on wild rodents because oftheir high population density in the cornfields.

Field rats and mice (suborder Myomorpha,family Cricetidae) are present not only inthe cornfields but also in the untilled landssurrounding the towns in the endemic area.There are many South American species inthe Myomorpha suborder, among themAkodon arenicola hunteri, Akodon areni-cola beatus, Hesperomys murillus murillus,Hesperomys laucha, and Oryzomys flavescens(54). Akodon arenicola hunteri stays awayfrom the human habitat; even when allkinds of food are available it prefers to eatdead congener, regardless of whether it is

8

in captivity or in a wild environment.,Hesperomys laucha seems to be presentonly in the eastern Pampa in the Province.of Buenos Aires (A. Cabrera and J. Yepes).Hesperomys bimaculatus bonaerensis is con-sidered a geographic form of H. bimaculatusbimaculatus (A. Cabrera et al.). It is foundalmost exclusively in the northeastern partof the Province of Buenos Aires. Hesperomysmurillus murillus is a species of the Argen-tine Pampa that inhabits the Province ofBuenos Aires and eastern Córdoba. In thecentral provinces and toward the southwest,another subspecies, H. murillus cordobensis,is recognized (A. Cabrera and J. Yepes).Oryzomys flavences is present in a large areaof Argentina and the southern part ofUruguay, except at high altitudes.

In addition to Cricetidae and nativeSouth American mice, there are also ratsand mice imported from Europe. Theselatter rodents reproduce in such numbers inthe endemic area that they are consideredreal pests.

The Rattus genus is represented by twospecies, the black rat (Rattus rattus rattus)and the Norwegian rat (Rattus norvegicus).These species are enemies and never livetogether. They migrate en masse during thesummertime and are not even stopped byrivers.

With respect to naturalized exotic speciesof Muridae, Mus musculus musculus hasbeen reported to be a plague in the Provinceof Buenos Aires (R. Ringuelet and R. H.Aramburu, 1957). It lives close to man,invading human habitations and nesting inthe furniture or in quiet places of the house.It eats food found in people's homes-fruit,cheese, candy, and the like, and it alsochews on materials such as wood, paper,and clothing to make a powder for its nest.It is also found away from houses in bignests in the stubble. Two other species ofthe genus Mus were described by José Yepesin 1935: M. musculus Linn. and M. mus-culus brevirostis Waterh. The first is found

throughout the entire country, and the sec-ond in the eastern part, close to La Platariver.

Other rodents abundant in the area in-clude those of the Caviidae family, Cabiinaesubfamily. Cavia pamparum is a plague inthe Pampa. It is similar to the commonguinea pig, but lives in the wild state. Othermembers of this family are Microcaviaaustralis and Galea musteloides littoralis;both shun man.

As a consequence of the discovery ofsylvatic plague in Argentina by Dr. de laBarrera, many studies dealing with the dis-tribution of wild animals in the country wereconducted. These studies are very useful,especially in showing the distribution of therodents from which Junín virus has beenisolated (119).

Other wild animals very common in theendemic area are Ctenomydae porteonsiporteonsi and the skunk, Conepatus sulfo-cans gibsoni, usually seen along the road-sides. From the order Marsupialia there areopossums, of which the Didelphis para-guayensis paraguayensis and the red opos-sum, or Lutreolina crassicaudata bonari, arereal pests. Among the bats, Myotis albes-cens, Histiotus montanus, Lasiurus borealisblosseoillii, Lasiurus cinereus villosissius,and Tadarida brasiliensis have been recog-nized in Argentina. The hare, orderLagomorfos, family Leporidae, Lepus euro-paeus europaeus is extremely abundant, andso is the partridge. Both species are avidlyhunted as food delicacies. In the process,there is ample opportunity for the hunters tocome in contact with reservoirs of AHF.

Among the less common species are theviscacha (Lagostomus maximus maximus),the quirquincho (family Dasypodidae,species Chaetaphractus villosus, and thepampean mulita (Masypus septemcinetus),which are seen less frequently every day.The same is true of the coypu (Myocastorcoypus bonariensis), the common fox

9

(Pseudalopex gymnocercus), and the minorferret (Galictis cuja huronax). The "cat ofthe straw" (Lynchailurus pajeros pajeros)and the Oncijelis geoftroyi have almost dis-appeared (54).

Dogs and cats, the good friends of man,are always present in the home.

As stated before, cattle, horses, sheep,and pigs exist in large numbers and consti-tute one of the area's major sources ofwealth. The poultry usually found close to

homes are chickens, hens, ducks, turkeys,and geese.

As a rule, both wild and domestic animalshave a great number of ectoparasites-mites,fleas, and the like. In the Province ofBuenos Aires at least 6 families, 8 subfam-ilies, 12 genera, and 19 species of fleas havebeen identified (20).

Although the area has an abundance ofmosquitoes, no attempts at virus isolationhave been made.

10

3. EPIDEMIOLOGICAL OBSERVATIONS

The high incidence of AHF among cornharvesters is so well recognized that theaffliction is called "stubble disease." Thoughthe present trend in the area is towardmechanized picking, there are times when itis impossible to harvest corn with machines.This is the case, for example, when theplants have fallen over because of strongwinds.

Although AHF bears an apparent re-lationship to the rural environment, fre-quently there are patients who have not leftthe towns or cities in the epidemic area.These urban areas, however, are not freeof rodents.

It is noteworthy that the annual period ofthe epidemics coincides with the rainy sea-son and that, in a given outbreak, weeklyand monthly fluctuations in the number ofpatients interned at the public medicalcenters appear to be related to climatic fac-tors, especially temperature and rainfall (54).

The morbidity data since 1958 show con-siderable fluctuation in the annual incidenceof the disease:

Year Number Mortalityof cases (% of cases)

1958 283 191959 1,027 6.311960 335 6.271961 816 7.841962 362 ?1963 653 4.421964 3,026 ?1965 148 ?1966 643 ?1967 until Sept. 6 1,059 8.69

Mortality, however, has shown a tendencyto decline, thanks to improved diagnosticand therapeutic measures that permit earlyrecognition of the disease and timely treat-ment of its severe and typical forms. De-layed diagnosis of severe cases accounts forthe high mortality reported during the firstlarge epidemic in 1958. Also, in theseearlier years, cases of medium and mildseverity may have escaped detection. Thus,in more recent years, as the total number ofreported cases increased, the proportion ofdeaths became lower.

In general, the first cases occur late inMarch, the epidemic reaches its peak inMay and June, and after that the cases de-crease steadily during July and remain verylow in August and September, when themorbidity curve drops to nearly zero (Fig.3). It should be kept in mind that this dis-tribution corresponds to the fall and wintermonths in the Southern Hemisphere. Onlysporadic cases have been observed duringthe rest of the year.

Simultaneous outbreaks within a groupof people working in the same field is a fre-quent observation and suggests the likeli-hood of infection from a common source.Human-to-human transmission has beenalmost entirely ruled out. Epidemiologicalobservations have shown that human sub-jects are not sources of infection for thehealthy population around them. Even withpatients in the same room or ward in hos-pitals, only rarely has the disease developedamong the medical staff or other persons inclose contact with the patients (111). Norcould any data be found to support the pos-

11

FIG. 3. ARGENTINE HEMORRHAGIC FEVER: MORBIDITY

1959, 1960, AND 1961AND MORTALITY CURVES,

1961MORBIDITY 816

.............. MORTALITY 64

1960 MORBIDITY 335

.............. MORTALITY 21

1959 MOR

.............. MORRBIDITY 1027

RTALITY 65

.DAYS 1 10 |O 20 1 |0lO 20 31 20 2 2 0 | 20 | 31 10 | 20 1 30 lo1 20 1 1 1 20 | lO 31 20 2

M.IONTHS JANUARY FEBRUARY 1 MARCH APRIL1 MAY JUNE JULY AUGUST SEPTEMUER OCTOSER NOVEIBER

Z ,I E 2 | 6 8I 1 2 4 | 4 6 82 10 125 10 68 40 52 64 42 39 23 1 13 II 1TOTAL D.ATHS 10_ - I ' I " I lo I 22 1" 5 2 , _

(Reproduced from Martínez Pintos, 1962)

12

U,4

U

ugu

AM

z

M,I

UM.

oegrU

z

=EZj 40

20

120

#AVWvi

u

U-

LUJ

Zz

100

80

60

40

20

ur _ _ _

sible role of food or drinking water in thetransmission of AHF. When the diseaseappears in an area, it becomes an endemicfocus for several years thereafter, suggestingthat the source of infection is something thatremains in the environment once it is estab-lished.

The endemic nature of AHF in certainlocalities in the Province of Buenos Aires iswell explained by the presence of rodents in-fected with the virus (79, 84, 112). Thesame is true for the Province of Córdoba(26, 124).

There is a definite parallel between thedensity of rodents and the incidence of AHF.Seasonally, the rodent population is highestin autumn and winter, which is when theAHF epidemics are at their peak, and lowestin summer, when only sporadic cases areobserved (30). Also, the lands directlyassociated with AHF infection are those de-voted to field crops, where rodents are mostpopulous, rather than cattle-raising lands.The great majority of cases occur amongpeople living in primitive conditions out inthe open, where contact with rodents isfacilitated. Disease-afflicted homes are fre-quently located on the border of uninhabitedgrasslands or open fields. The few casesobserved among people living in cities maybe accounted for by brief trips to thecountryside.

Repeated isolations of Junín virus fromwild rodents (26, 79, 84, 91, 112), as wellas several isolations from their ectoparasites(4, 85, 89), point convincingly to a connec-tion between the appearance of AHF inhumans and the presence of rodents in-fected with a virus from the zoonosesgroup, the disease probably being trans-mitted through the animals' excreta or theirblood-sucking ectoparasites.

In 1959, 449 wild rodents were trappedin the endemic area. Of these, 52.8 percent were Mus musculus; 24.4 per cent,Hesperomys laucha (Dumarest); 11.1 percent, Akodon arenicola; 6.2 per cent,

Oryzomys fiavences (Waterhouse); and 5.3per cent, Rattus sp. Ten strains of Junínvirus were isolated from the Mus musculus,one from the Hesperomys laucha, and onefrom the Akodon arenicola (79). In theProvince of Córdoba, 46 strains were iso-lated from Calomys laucha alone, and addi-tional strains were taken from Akodonazarae, Akodon obscurus, Calomys muscu-linus, and Oryzomys flavences (26). Otherworkers have isolated Junín virus from wildguinea pigs, or Cavia pamparum (cuis), andfrom hares (Lepus europaeus) as well(4, 22). There may be an even widerdistribution in nature, but not many attemptsto isolate virus from other sources havebeen made to date.

With mouse ectoparasites, it was demon-strated by Pirosky and co-workers in 1959that mites (Mesostigmata without furtheridentification) were able to transmit theJunín virus infection experimentally (89).Further investigations in mites yieldedJunín virus from Echinolaelaps echidninus(Berlese) taken from nests of naturally in-fected Mus musculus (85). Another suc-cessful isolation from mites was made in1965, this time from Eubrachilaelapsrotundus taken from Akodon azarae (4).

Research has not yet shown exactly howthe virus is transmitted from infectedrodents to healthy people. It is not clearwhether they become infected by contactwith the rodents' excreted urine, or by theirectoparasites (Gamesoid mites), fromwhich the virus has been isolated. Amongthe rodents themselves, it is most probablethat circulation of the virus is maintained bythe ectoparasites of the animals servingas vectors. Man can be infected withoutthe intervention of arthropods; this has beenproven by numerous Junín virus infectionsamong laboratory workers. But whether ornot this is true in the natural environmnentno one knows, even though a number ofhypotheses may be formulated. If thehuman infections acquired in the field come

13

from an environment contaminated with theurine of infected rodents, without any inter-vention of arthropods, the lack of infectionin summertime could be explained by thenatural sterilization of the field by ultravioletradiation from the sun.

Although there are few well-documentedinstances of human-to-human transmission,there is a theoretical possibility that with in-creased adaptation to man and the develop-ment of persistent chronic infection thevirus may propagate by human contact inthe absence of an arthropod vector.

From the public health standpoint, AHFis not the most serious problem in Argen-tina. In addition to the summer diarrheaand other diseases of children that constitutea major problem throughout all of SouthAmerica, Chagas' disease and tuberculosisinvolve a great number of adults, and thesequelae usually cause permanent handi-caps. However, the fact that AHF afflictsworkers in the farthest reaches of the countryat harvest time gives it great economic, aswell as social and political, importance.

14

4. THE DISEASE

Clinical Description

AHF is an acute infectious disease of 7to 15 days' duration followed by a longconvalescent period of approximately onemonth; with few exceptions, there are nosequelae.

For the most part, the persons who con-tract the disease are newcomers in theendemic area who have contact with thefield. The patients are usually males-cornharvesters or people involved in other ruralactivities. The disease is unkown or un-recognized in infants, and only few cases inchildren have been recorded. Morbidity ishigher in males than in females, the ratiobeing approximately 5 to 1.

The clinical symptoms are not alwaysuniform, but the typical case is diagnosedreadily; the local physicians were so im-pressed by the signs and symptoms that itwas named for its hallmark (enfermedad delsello). In a fully developed, "classical" setof symptoms and laboratory findings, thecomplex includes involvement of the renal,cardiovascular, hematic, and nervous sys-tems.

This essay synthesizes the observations onthe clinical characteristics of the diseasemade by different teams of investigatorsfrom 1955 up to the present (3, 5, 35, 40,57, 62, 74, 90, 91, 105, 106, 109, 110,113, 114), plus the personal observations ofthe author as part of a team and/or as anindividual physician.

In general, the disease is considered tohave mild, moderate, and severe forms,though sometimes the characteristics over-

lap. After an asymptomatic incubationperiod of 7 to 16 days, three stages can bedifferentiated: the initial period, the acutephase, and convalescence. In 3 to 30 percent of the patients, depending on theseverity of the epidemic, death occurs be-fore the convalescent period. Althoughthese three states are not found in all casesof AHF, the phasic course of the disease isbeyond doubt, and the suggested division indistinct periods should lead to a betterunderstanding of the essence of the path-ological changes and their sequential appear-ance.

In the first stage-the initial or invasionperiod-the usual onset is vague withgrippe-like symptoms, such as malaise,fatigue, chills, and lumbar backache. Inaround 5 per cent of the cases, however, thefirst symptoms appear suddenly with severeprostration and lumbar and limb pains.Fever and accentuated asthenia are alwayspresent regardless of the type of onset(113). The bulbar and palpebral conjunc-tivas are injected without any secretion ortearing. The face becomes edematous andflushed, as in measles. Headache, postorbitalpain on moving the eyeballs, and sub-palpebral edema are almost constant. Stiff-ness is present in the neck and the musclesof the costovertebral canals. The patientsometimes complains of feeling as if he werestrapped down to a pale.

The patient is dull, drowsy, confused, andoften apathetic. He does not respond im-mediately to questions. Tremor of thetongue during its protrusion is accentuated,as in general paresis of neurosyphilis. The

15

patient's gait is unsteady, and active move-ment is slow. Frequently there is tremor ofthe upper extremities when the arms areextended forward at shoulder level, as inhyperthyroidism. Another conmmon symptomis noninflammatory painless generalizediymphadenopathy.

Enanthemas are present on the buccalmucosa. At the juncture of the soft and hardpalate one can usually see a number of smallhemorrhages, petechiae, and/or some twentyto thirty microvesicles of clear content,without color change in the surroundingarea. They are not painful; they are presentfrom four to six days, increasing in numberduring the acute phase (114).

Halitosis, anorexia, thirst, nausea, vomit-ing, and epigastralgia are sometimes present.Constipation is frequent, and only in a fewpatients does one observe a brief period ofdiarrhea. The feces in these cases are fluid-homogeneous, without mucus, but fre-quently with an admixture of fresh bloodand sometimes melena. The diarrhea laststwo or three days and usually ceases with-out any therapeutic measures. Meteorismand cecal gurgling have been noted. Inwomen, there may be uterine hemorrhages.Bradycardia with arterial hypotension ispresent in 90 per cent of the cases (113),and dizziness is observed in a large propor-tion as well. As a rule, the spleen and liverare not enlarged; palpation in the kidneyarea, however, is very painful.

The initial or invasion period lasts twoto four days, and passage to the fully devel-oped, or acute, stage is accompanied by anaccentuation of the same symptomatology.

The fully developed disease usuallypresents hemorrhagic injury characterizedby bleeding of the gums, epistaxis, and insome few severe cases, hematemesis, melena,and enterorrhagia. A rash first appears onthe trunk, primarily in the axillae and onthe chest, and spreads to the extremities. Itis usually petechial, and in some cases con-

fluent, so that large areas of the skin assumea purpuric appearance.

The fever is of a remittent type; the pulseis slow compared with the temperature rise;and arterial hypotension, usually moderate,is observed in all patients. If the blood pres-sure drops precipitousiy, this is a sign ofgrave prognostic significance, particularly ifassociated with oliguria. Marked decreasein diuresis, sometimes resulting in completeanuria in the second period, as often ob-served in 1958, may be found (109). Inthese cases, the oliguria or anuria is fol-lowed by prolonged polyuria; in others, thedevelopment of acute renal failure leads touremic coma, followed by death. Signs in-dicating progressive central nervous systeminvolvement, ranging from disorientationand delirium through convulsions to stuporand finally coma, as terminal manifestations,are frequently seen in severe cases. A char-acteristic odor, different from that of anyother ward in the same hospital, is presentwherever AHF patients are confined.

Although the patient is extremely thirsty,he refuses to drink, in order to avoid spasmsof the laryngeal and pharyngeal muscles, asobserved in rabies.

When the disease is at its height, thepatient's temperature may reach 39° to40°C. In this stage, cutaneous hyper-esthesia and contraction of the musclesunder the skin are observed upon stimula-tion. Photophobia, strabismus, and nystag-mus may also be seen.

In some patients, gastrointestinal dis-orders are conspicuous. The tongue is dry,and coated with a thick brown layer.Halitosis, with an odor of decay, especiallyin cases with bleeding of the gums, oralmucosa, and nasopharynx, is usuallypresent.

Changes in the respiratory organs areusually slight and infrequent; the most com-mon finding is a dry cough.

The final, or convalescent, period beginswith a drop in temperature and usually

16

lasts two to four weeks, or even longer insevere cases. During the recovery period,the fever diminishes by lysis. Hyperemia ofthe face and mucous membranes disappearsand the hemorrhages on the skin are reab-sorbed. The blood pressure and pulse ratebecome normal, and only changes detectablein the clinical laboratory may remain.Physical examination may only reveal a gen-eral weakness and a tendency to fatigue,which can persist for some weeks.

During this period some patients have aspecial gait similar to that presented bydengue. Usually in convalescence there isa partial and gradual loss of hair, followedeventually by the growth of new hair to sup-plant what was lost. This is especially notedamong women. Even if all the hair ischanged, there is never a time of completealopecia.

The recovery is usually without sequelae,though there have been a few cases in whichparkinsonism was present for one or twoyears and others in which albuminuria per-sisted for a long time. Uncomplicated casesare seldom fatal; the prognosis is negativeonly in cases with serious nervous disturb-ances or severe hemorrhagic manifestationsor both-especially when there are hema-temesis, melena, and enterorrhagias-andalso in persons with alcoholic habits, and inthe aged. During the recovery period latentchronic infections such as amebiasis mayexacerbate.

In 1.2 to 1.8 per cent of the cases, re-lapses were observed 15 days or a monthafter the original onset, with repetition ofthe previous symniptomatology (106).

Diagnosis

Differential diagnosis

AHF must be differentiated from otheracute infectious diseases that share some ofthe same symptomatology, such as influenza,

rickettsiosis, leptospirosis, toxoplasmosis,and typhus.

Laboratory investigation is necessary inorder to make a differential diagnosis.Sometimes it is impossible to attempt todifferentiate this disease from others onclinical grounds alone. Etiological investi-gation through serology and isolation of theinfectious agent will confirm or reject anyof the diagnoses mentioned above.

Clinical laboratory diagnosis

The routine laboratory procedures, suchas complete blood count and urinalysis, areof great value in diagnosis. Generally, aclinical picture such as that described in theprevious section, associated with thrombocy-topenia, leukopenia, and special cells in theurine sediment, will be labeled as AHF untilsuch time as a contrary diagnosis can bedemonstrated.

Blood

Regardless of the severity of the disease,the laboratory findings will generally reveala normal or lowered erythrocyte sedimenta-tion rate during the first week (1 to 7 mmin the first hour, using Westergren andWintrobe). This value rises during the sec-ond and third week, particularly if bacterialcomplications develop. In mild cases it re-mains normal. The hematocrit shows nor-mal value rises, except in cases of severedehydration, where the increase in cells isproportionate to the degree of hemoconcen-tration.

The number of red cells is usually normalat all stages of the disease, except when thehemorrhagic manifestations are exaggeratedand there is consequent anemia in propor-tion to the blood loss.

Leukopenia due to granulopenia is invari-ably found. The total white blood cellcount begins to fall by the second day offever and drops to a low of 1,900 to 3,000by the fourth or fifth day after onset. Caseswith values as low as 400 leukocytes per

17

mm3 have been observed. The leukopeniais accompanied by aneosinophilia andneutropenia (5, 91, 109, 126).

The neutrophils suffer severe alterations,with marked anisocytosis and toxic granu-lations. There are a great number ofneutrophils with crooked-shaped nuciei. Thenuclei of the neutrophilic granulocytes aremarginated.

There is nuclear chromodiffusion, some-times accompanied by pycnotic condensa-tions of the chromatin, with partial break-age of the nuclear membrane, which give theappearance of flaked nuclei.

At the peak of infection, atypical mono-nuclears, some of them showing vacuola-tion of nuclei and cytoplasm similar tohystiocytes, are characteristic findings (126).

Recovery starts at the same time that theoliguria disappears, the number of whiteblood cells begins to increase either slowlyor suddenly, and leukocytosis appears. Thecorresponding normal white cell differen-tial count signifies the reappearance ofeosinophils and monocytes with normalnuclei and the disappearance of any toxicgranulations in neutrophils and plasmaticcells that may have been present. Anyearlier increase in white cells than whatwould be expected from the normal courseof disease suggests a focus of bacterial infec-tion somewhere in the organism.

The number of platelets decreases somrne-what during the initial stage, and even moresharply at the height of the disease. Valuesas low as 6,000 per mm3 have been en-countered. The average in most cases is be-tween 30,000 and 40,000 per mm3 . Giantand morphologically abnormal platelets maybe seen on smears. During convalescencethe number returns to normal.

The coagulation time is longer than nor-mal. In severe cases, periods of as much asan hour, with the blood kept in a tube at37°C, have been observed. Clot retractionis incomplete (110, 127). Using moresophisticated techniques such as the throm-

boelastographic method, it was demon-strated that 65 per cent of the patientspresent hypocoagulability during the acutestage, while in convalescence the tracingsare normal in 77 per cent of the cases, andsometimes even a reactional hypercoagula-bility is present. Oniy in a very few casesdoes the hypocoagulability persist in con-valescence. The hypocoagulability is trans-lated by the lengthening of "r" and "k" andthe narrowing of "am" (100). Fibrinogen isfound to be lowered in cases of markedhypocoagulability, and occasionally fibrin-olysis is observed.

Bleeding time is increased. The plasmaprothrombin time and concentration arenormal.

The ionic calcium is normal or loweredduring the initial and acute periods, andalways normal during convalescence.

The total proteins in blood plasma are inthe lower normal range. The zonal elec-trophoresis of the proteins generally showshypoalbuminemia with a moderate increasein alpha 1 and 2 globulins, a normobetaglobulin level, and a normogamma globulinlevel. During convalescence the hypo-albuminemia still remains, but the gammaglobulin increases, and thus the inversion ofthe proteic rate does so too (37, 107).

The total lipemia and cholesterolemiamay reach very low values; cholesterolranges of from 58 to 138 mg/100 ml havebeen observed. The direct bilirubin isnegative and the indirect is in the normalrange, only having shown to be increased ina few fatal cases. Liver function tests,thymol turbidity, and Hanger's test also givenormal results.

The electrolytes are without severe alter-ations except in grave cases where hy-ponatremia and hypochloremia, accom-panied by a slight decrease in the CO2combining power, have been observed.Potassium is normal or increased. Bloodsugar is increased to values of from 120 to150 mg/100 ml, but it gradually returns to

18

normal in days or weeks. In some patients,values as high as 200 mg/100 ml late in theacute period, followed by a sudden normal-ization of the sugar level in about two days,have been reported.

The urea is normal or increased; cases of40 mg/100 ml, with oliguria or anuriapresent, have been recorded. When theanuria or oliguria last, the values dropsharply to normal in one or two days.

The white cell count, the differentialwhite cell count, the number of platelets,and bleeding and coagulation times normal-ize their values independently of the evolu-tion of the disease, whether toward cure ordeath.

In bone marrow there are macrophageswith leukocytes phagocyted-a fact thatmay account for the central destruction ofwhite cells reported by Vucetich (126).During convalescence the red cells of thebone marrow reveal hypofunction.

CSF spinal fluid findings have beennormal even in the presence of spinalmuscular contraction.

Urine

Albuminuria begins to show in the initialperiod and increases to 0.10 to 0.50g/1000 ml during the acute stage. Thosepatients whose initial albuminuria disappearshave a second rise to 3 or 4 g/1000 ml last-ing one or two days, with normalization im-mediately thereafter.

Cylindruria is present early in the disease,sometimes preceding albuminuria. Theamount of cylinders is unrelated to the de-gree of albuminuria. In general, the cylindersare hyaline, hyaline-granulose, and epithe-lial; some are erythrocytic.

Vacuolated epithelial cells are a character-istic finding in the urine and are used as acriterion for diagnosis. First described byMilani, these cells were studied in detail byPalatnik (75, 76). They are 18 to 45 u insize-about twice the size of a lymphocyte.They have hyaline, intensely eosinophilic

inclusions, which stand out sharply in thecytoplasm. The inclusions-round, half-moon, or ring shaped-are 10 to 26 l insize. In the later stages of the disease largeinclusion bodies, along with granular inclu-sion forms, are observed in the cytoplasm.The inclusion maintains a stable configura-tion during the initial and acute stages. Atthe beginning of convalescence the formsdecrease in size and number. Soon there-after, they disappear, and they are not pres-ent in the late stage of convalescence, afterthe disease is over, or in the case of relapse.The inclusions are Fuelgen negative, butthey have Fuelgen positive corpuscles; withPAS they present a typical and stablemorphology. Palatnik suggests that thegranules represent viral material and thatthe inclusions are related to the intracellularcycle of the virus.

Pyocytes and erythrocytes are often seenthroughout the entire course of the disease,and for this reason a daily check of theurine is advisable. Macrohematuria wasobserved in only a few cases. The 17-ketosteroids are lowered, especially insevere cases.

ECG

Modifications of the ECG were found in71 per cent of the patients. The most char-acteristic alterations consisted of an elevatedST interval, with upper concavity, followedby an exaggerated T wave. In 20 per centof the patients a flat or inverted T wave wasfound. Sometimes the P wave had lowvoltage and the PR interval was extended.The QT interval was found to extend mod-erately in 14 per cent of the patients. The Uwave was found in 9 per cent of the cases(101).

Low voltage was an infrequent mani-festation, as were nodal rhythm and ven-tricular extrasystoles. No bundle branchheart blocks were observed. An electrobal-listocardiographic study of eleven con-valescents showed normal variants of the

19

cardiogram in nine cases and probablemnyocardial injury in two of the cases (86).

Pathology

Pauioogical cuhanges at various stages ofthe disease have been described by Riveroet al. (97) and Polak and Jufe (94). Thelesions or changes were not specific for AHFand had features similar to those of otherdiseases and infections accompanied byhemorrhagic diatheses, particularly otherhemorrhagic fevers (48, 118).

Postmortem examination reveals alter-ations corresponding to the lesions producedby the virus itself or to intercurrent infec-tions associated with the cause of death.The alterations produced by the virus itselfcorrespond to perturbations of the bloodcapillary system, reactivation of the reticu-loendothelial system, and areas of tissuedegeneration, as described below.

Gross lesions

The gross anatomy shows general conges-tion and various degrees of hemorrhageson the skin and in some of the organs, espe-cially the lung and spleen. Generalizedlymphadenopathy appears in almost everycase. Infiltration resembling that of bron-chopneumonia, necrotic areas in theparenchyma of the lung, and even pleuraleffusion have been seen. Renal damage, asin nephrosis with acute glomerulitis, isusually observed.

Microscopic lesions

The dominant changes are vascular;vasodilation, with congestion, edema, andhemorrhages, is the main presenting symp-tom. The chief abnormality occurs in andabout the small blood vessels and consistsof endothelial swelling, perivascular edema,and diapedetic hemorrhages. There is acellular reaction characterized by hyper-plasia in the reticulohistiocytary system of

the lymph nodes, tonsils, spleen, and bonemarrow; in the bone marrow there are nomature neutrophil or eosinophil leukocytes.

Dystrophic changes are present in theparenchyma of the organs and in the bloodvessels. In the liver, hepatic cells showgranular degeneration of the cytopiasm. Tnecentral vein is dilated, and the sinusoids con-tain a deposit of bile pigment. Edema isobserved in the connective tissue surround-ing the dilated central vein, and lymphocyticinfiltration is seen in some of the Kiernan'sspaces.

The kidney alterations are mainly in theproximal tubules, where the epithelial cellsshow alteration of the nucleus, granularcytoplasm, and an indefinite border. Thelumina are obstructed by hyaline and cel-lular casts. Acute glomerulitis is present,with general congestion, microscopic hem-orrhages, and edema, as in the other organs.The microdissection of the tubules done byAoki et al. (2) showed renal alterationssimilar to those observed with Koreanhemorrhagic fever.

In the spleen, hyaline degeneration of thecorpuscular arteries, with focal modificationsin the sinuses, is observed.

In the lungs, inspecific bronchopneumonicfoci and interalveolar hemorrhages arepresent.

There is congestion with marked menin-geal edema in the brain. Rovere et al.claimed the presence of Lafora's amyloidbodies in the cerebral tissue (98, 99).

When intercurrent infections are thecause of death, the pathology corresponds toalterations caused by the virus itself at thestage of the disease when death occurred,plus the changes corresponding to the inter-current infections.

Treatment

Symptomatic treatment and good nursingcare are of the utmost importance. There is

20

no specific treatment except for serumtransfusions (vide infra). It is imperativeto put the patient in bed as soon as possible.The most severe cases appear in personswho continue to work in spite of generalmalaise and muscular pain. Early rest helpsto prevent the disease from becomingsevere.

Even though the dry tongue and skin mayjustify the ample use of fluids in treatment,it is necessary to bear in mind the renal in-volvement and observe caution in admin-istering liquids. Only in severe cases wherevomiting and absolute anorexia are presentare parenteral fluids advisable, and thenacid-base determinations should be used forcontrol. Otherwise, parenteral injectionsmust be avoided. They can provide meansof entry for secondary bacterial infections,and, in addition, because of the hemorrhagicdiathesis present in these patients, they cancause hematic collections at the place wherethe needle is introduced.

The administration of convalescent serumfrom other patients, though not necessarywith mild attacks, is advisable in severecases. Treatment with convalescent serumis routine in the endemic area, and some-times its administration can have spectacularresults. Within a matter of hours, the con-dition of a severely ill, semicomatose patientcan be reversed. The experience of localphysicians has shown that this treatmentworks better when instituted very early inthe disease-no more than five days after

onset. From 250 to 500 cc of serum is useddepending on the individual patient and theseverity of the case. Interestingly enough,even though convalescent serum is admin-istered routinely in Argentina for treatmentof AHF, to date no cases of hepatitis havebeen observed among the patients. As arule, a patient's friends who have alreadyhad AHF donate their blood on such occa-sions.

The administration of oral antibiotics isconsidered advisable, particularly after ob-serving the lesions produced by Junín virusin the intestinal walls of experimentalguinea pigs. These lesions facilitate the pas-sage of bacteria from the intestine to theblood stream. Medication with antibioticsshould help to prevent such complications.

Fresh blood transfusion is advisable insevere hemorrhagic cases. If the bloodcomes from a person with antibodies againstJunín virus there will be the added advantageof neutralizing the virus infectivity, but ifantibodies are not present, at least the resti-tution of volemia and the administration ofplatelets, white and red cells, and all theother factors needed for coagulation willhelp return the organism to its equilibrium.In general, no more than 500 cc of wholeblood is given at one time; transfusions arerepeated if necessary.

More detailed information about treat-ment and the effects of specific drugs can befound in the literature (18, 56). Basically,the criteria outlined above are applied.

21

5. JUNIN VIRUS

An infectious agent of AHF was first iso-lated from victims of the 1958 outbreak.Three different strains were obtained: onefrom the blood of a patient in the acute stage;a second from another patient's urine, whichcontained a large amount of blood; and athird from a suspension of viscera (brain,liver, spleen, and kidney) taken at thenecropsy of a fatal case. The strains wereisolated by inoculation of guinea pigs. Eventhough many hosts were used in the labora-tory in an effort to isolate the causative agent,the guinea pigs were the first to show a re-sponse characterized by death with hem-orrhagic manifestations resembling those pre-sented in human beings (62). Indeed, thehemorrhagic characteristics were exagger-ated. All three strains behaved similarly inthis host.

Intracerebral inoculation of a pool of in-fected guinea pig organs in suckling miceproduced ataxia, spasms, and occasionallyparalysis of the hind legs. The infectedmouse brain fixed complement with humanconvalescent serum but not with serum takenfrom a patient in the acute stage of thedisease.

Since the agent passed through a Cham-berlain L5 candle, failed to multiply in cell-free media, and could not be seen under thevisible light microscope, it was considered tobe a virus. It was given the name Junín,after the city where the materials had beencollected. The etiological role of the viruswas subsequently demonstrated by inocula-tion of infectious material in a volunteer andby multiple accidental infections in labora-tory workers, some of them fatal. In the

same epidemic, Pirosky and his group (91),working independently, also isolated severalstrains of a virus. One of the members ofthis team reproduced the disease in himselfby autoinoculation. Further studies showedthat the viruses isolated by the two teamswere indistinguishable by complement-fixa-tion (69).

Physical and Chemical Properties

The virus is very sensitive to acidification.When kept on dry ice, unless it is perfectlysealed, the diffusion of CO2 into the ampoulewill cause it to become inactivated.

Infected organs, or suspensions of in-fected organs in a pH 7.2 buffer with 0.75per cent bovine albumin, can be stored foryears at -70°C in an electrically operatedfreezer without loss of infectivity. If thematerial is frozen and thawed, the infectivetiter of the virus drops with each step.Lyophilization of infected suspensions hasproved to be a good method of stock con-servation, since the decrease of virus titer isminimal.

The resistance to this virus at differenttemperatures has been studied, using M-199free of any serum as diluent and checking thevirus titer after exposure to 56 ° and 37°C.Ten minutes' exposure at 56°C decreased thetiter in the logarithmic expression by one;thirty minutes killed the virus totally. Afterone hour at 37°C the titer decreased by 0.09logs, after six hours by 0.37 logs, and aftertwenty-three hours by 0.56 logs.

The virus was shown to be very sensitiveto sodium desoxycholate, as is generally the

22

case with arboviruses. It is inactivated bytrypsin, chloroform, and ether. The op-timum pH for virus viability is on the alka-line side, close to neutrality (pH 7.1 to 7.4).It has more resistance to alkaline than toacid pH; in a diluent free of proteins, verylight acidity kills it. When protective sub-stances like bovine albumin or rabbit serumare added to the diluent, the lability of theagent is decreased, as is the case with manyother viruses. A phosphate buffer of pH 7.1to 7.4 with 0.75 per cent bovine albumin isroutinely used as diluent. A tenfold concen-tration of albumin in the same pH 7.2 bufferdid not prove to be better; on the contrary,the infectivity tended to decrease.

The effect of pH, temperature, and ultra-violet light on infected guinea pig plasma hasbeen studied. No significant decrease in in-fectivity was observed between pH 5.5 and9.5 after six hours at 4°C or between pH6.5 and 9 after eighteen hours at this sametemperature. However, a significant decreasewas found after eight days at 4°C, threedays at 25°C, twenty-six hours at 37°C, andten minutes at 56°C. With the ultravioletlight at a distance of 67 cm, a minimumexposure of thirty seconds was necessary toachieve significant inactivation; after fortyseconds, 99.9 per cent of the virus was in-activated (12).

Lajmanovich et al. (49) determined thesize of Junín virus, strain XJ, in the plasmaof infected guinea pigs by means of ultra-centrifugation with a Spinco preparation.Assuming spherical shape and a density of1.2 g per cc, they calculated that the diameterwas between 18 and 25 mi. The same sourceof virus was used to obtain a pellet, which,resuspended in 1/50 of the primitive volumein a phosphate buffer of pH 7.4, was chemi-cally analyzed for nucleic acid determination.The authors concluded that Junín is an RNAvirus (78).

Electron microscopic study of Junín viruswas attempted by Dr. Zaharzevski in infectedlymph nodes of guinea pigs, and images

compatible with the characteristics describedfor Junín virus were found (132).

Behavior in Animals andTissue Cultures

Experimental animals

Guinea pigs

As stated before, the guinea pig was thefirst laboratory animal to show characteristicresponses to the virus. After inoculation ofvirus materials by any of several routes (videinfra), it may become sick and die, usuallywithin 11 to 20 days and sometimes later,depending on the amount of virus present inthe inoculum. As a rule, animals that do notappear to have been given sufficient virus toproduce disease are held for 40 days beforethey are considered free from infection. Eventhen, when no apparent disease ensues, ad-ditional information can be obtained bytesting the sera of the survivors for comple-ment-fixing (CF) antibodies. A single injec-tion of mouse-adapted XJ strain, even inamounts of 100 mouse LD50 or less, pro-duces a high titer of CF antibodies in theguinea pigs. According to Casals (21), it ispossible, but unlikely, that this amount ofinert virus could stimulate the productionof CF antibodies; it seems reasonable tothink that the guinea pigs can suffer a non-fatal, inapparent infection. The XJ strain,originally highly pathogenic for guinea pigswhen inoculated intraperitoneally, appearsto lose this pathogenicity after about 40passages in newborn mice (69). The reasonfor this loss of pathogenicity is as yet unde-termined. It may be due to selection in themouse of a less virulent virus population, toenvironmental conditions such as diet andhandling of the guinea pigs, or to higher re-sistance of North American guinea pigs ascompared with the Argentine animals.

Intraperitoneal, intramuscular, and sub-cutaneous routes of inoculation are routinely

23

used, although other routes can be chosen.Intracerebral inoculation of guinea pigsusually gives a picture of encephalitis insteadof the hemorrhagic manifestations. Testsusing nebulization; corneal scarification; andintravenous, skin, and oral administrationhave also produced infection, even thoughmortality is lower. The virus usually tendsto be localized in the lymph nodes and thespleen. Fever begins on the seventh or eighthday after inoculation and remains high untilthe last day of life, when a marked hypo-thermia suddenly occurs. Loss of weight ispresent from the beginning and increasesafter the fever appears. Viremia is constant,and the titer continues to rise until death(13). The study of blood and bone marrowshows absolute leukopenia, relative and ab-solute lymphocytosis, decreased eosinophils,slight anemia (normochromic and normocy-tic), increased reticulocytes, increased al-kaline phosphatases in the leukocytes,inhibition of mature myeloblasts in the bonemarrow, and an increased red blood cellcount. From the first day of inoculation untilthe animal's death there is a decreasednumber of platelets (39).

Studies of coagulation factors with the XJstrain show increased deficiencies up untiljust before death. Coagulation time risesfrom the normal 3' 20" to 60' with lack ofclot retraction (19).

Histaminemia was determined one daybefore inoculation and again on the third,seventh, and tenth days thereafter. Un-inoculated controls were tested simul-taneously. It was found that a significantdecrease in histaminemia is apparent inanimals after the seventh day of infection;there is increased histaminemia, reachingnormal values, in sick animals just beforedeath; and there is no difference in his-tamine release between normal and infectedlung tissue (131).

A study of inter-guinea pig contaminationrate, using 16 strains of Junín virus, wasperformed by placing normal guinea pigs in

the same cage with the inoculated animalsand in neighboring cages. Although 100 percent of the inoculated guinea pigs died withthe characteristic picture, only 11.3 per centof the other guinea pigs in the same cagedied, and none of the normal animals inneighboring cages died (42). Since theelimination of Junín virus through the urineof infected guinea pigs starts to appear onthe seventh day after inoculation, contamina-tion of the uninoculated animals placed inthe same cage probably occurred throughcontact with the infected urine. Despitenumerous attempts, Junín virus has neverbeen isolated from the stools of infectedanimals.

Infected guinea pigs show alterations intheir immune response to other antigens,such as red cell preparations from differentspecies (83). They also manifest an in-crease in susceptibility to escherichia coliendotoxin administered by the parenteralroute (41).

A detailed study of the seric proteins ininfected guinea pigs was performed usingelectrophoresis on paper and a gel of poly-acrylamide (17).

The fairly precise relation between theamount of virus inoculated and the incuba-tion time between inoculation and appear-ance of a given symptom has long beenrecognized. In 1939, for example, with rab-bit papilloma, the time interval betweeninoculation in the rabbit skin and appearanceof papillomas is related to the concentrationof the inoculum by a mathematical formula(15). In the present case, a linear relation-ship between intramuscularly inoculateddoses of Junín virus and the time of theguinea pigs' death can be determined. How-ever, the formula cannot be applied with ahigh concentration of virus; it is useful onlywith dilutions starting at 10- 4 (27).

The gross anatomy of guinea pigs infectedwith Junín virus shows only conspicuoushemorrhagic manifestations, consisting ofpetechiae, ecchymosis, or hematic suffusions

24

on the skin, suprarenal glands, kidneys, andsmall and large intestines, and usually areasof lung hepatization. The liver and spleenare generally normal in size, except when along incubation period leads to fatty degen-eration of the liver and consequent enlarge-ment. Hypertrophy of Peyer's patches is aconstant finding. Bacteriological studies inthis animal, as in human beings, show nega-tive results (38).

The pathological picture of Junín virus inguinea pigs closely resembles that presentedby epizootic hemorrhagic disease in deer.This fact led to the development of a com-plement-fixing system with the New Jerseydeer strain for purposes of comparison withJunin virus. The two agents were found tobe completely different antigenically, how-ever (71).

Histological examination of different or-gans consistently shows general congestion,with microhemorrhages scattered through-out, as in human beings (9, 10). In sectionsof the CNS and ganglion stained with Seller'stechnique it is possible to observe intra-cytoplasmic inclusion bodies similar to thosedescribed in the urine of humans infectedwith this same virus (76) or with Portillovirus (70) and in mouse brain infected withTacaribe virus (32).

Mice

Mice, the animals most widely used forresearch in virology, have been very helpfulin the study of Junín virus. Indeed, impor-tance should be given not only to white micefor research but also to wild mice present inthe endemic area. When studies on thisvirus were first started, even though thedisease had been reproduced in guinea pigs,there was no way of ascertaining the rise ofantibodies in human convalescents untilnewborn mice were available as a host forthe neutralization test and as a source ofcomplement-fixing antigen from their in-fected brains (82). Intracerebral inocula-tion of a pool from infected guinea pig

organs in newborn mice produced a diseasenine days later, with signs of encephalitis,ataxia, tremors, and convulsions. In thosemice that survived one or two days longer,paralysis of the hind legs appeared as well.Newborn mice are also susceptible to thevirus inoculated intraperitoneally, althoughnot to the same extent as with the intra-cerebral route.

Adult mice are susceptible, too, but to alesser degree, and only by intracerebralinoculation, not intraperitoneal. The titer islower, infection is irregular and spotty, andsome mice survive without showing anysymptomatology.

The incubation period of the virus in new-born mice inoculated intracerebrally dependson the particular virus strain and the amountof virus present in the inoculum. With theprototype strain, the disease usually startsaround the eighth or ninth day and the miceare held for 21 days to determine the end-point in terms of LD5 o. With other strains,the incubation period is shorter (five days),but it is always necessary to keep the ani-mals 21 days to reach the endpoint, and insome cases 28 days, as observed by Sabat-tini (26).

Since newborn mice were found to be sus-ceptible to inoculation by more than oneroute, this host was used for measuring theneutralization of infectivity of human con-valescent sera. Infected mouse brain wastaken as the source of Junín virus. Later, thegrowth curve of the virus in the infectedmouse brain was studied by measuring theinfectivity in HeLa cells, and the optimumtime to harvest the brain for use as com-plement-fixing antigen was determined. Theprototype XJ strain was utilized in theassay (69).

A comparative study of Junín virusmultiplication in the brain of 24- and 72-hour-old suckling mice was carried out byinoculation of 10-2 10-0- 4, and 7 x 10-2particles from another strain of this virus.Both groups of mice were found equally sus-

25

ceptible, and there was a plateau at themaximum titer between the third and theeighth day after inoculation. At the highestlevel of inoculum, interference was also ob-served (47). The production of interferencein HeLa cells, using infected mouse brain asinoculum, especially in recently harvestedmaterial, was a very common finding whenthis type of work was done routinely.

Newborn mice are susceptible to naturalinfection as well. When exposed for 16 daysto 2,000 acarids collected from the nest ofa field mouse, newborn mice showed typicalsigns of infection. The virus responsible forthis infection was propagated in newbornmice and was shown to cross-react withhuman strains in complement-fixation tests(89).

Mice infected with strains of Junín virusshow signs of encephalitis regardless of theroute of inoculation; histologically, the brainis infiltrated with lymphocytes and perivas-cular cuffing (82). The alterations in thekidney, bone marrow, and blood vessels arevery slight compared with those found inman and guinea pigs (10).

Newborn mice have proved to be a goodhost for the primary isolation of Junín virus.Of 34 strains directly isolated in newbornmice, 22 were recovered in the first passage,1 after a blind passage, and 11 after twoblind passages (26). Investigators at theCórdoba Institute of Virology found new-born mice to be more effective than guineapigs for virus isolation.

The brain of newborn mice has been usedin an attempt to localize the viral antigen.The Weller and Coons method, usingfluorescent antibodies, was applied to brainsections taken from mice 11 days after inocu-lation. The results showed a large amountof fluorescent material, stained greenish-yellow, which corresponded to specific viralantigen particles (88).

A more exhaustive study of this problemis under way. In particular, efforts arefocused on determining the relationship be-

tween maximum fluorescence and the lengthof time after virus inoculation is completed,and on the use of this information as a toolfor earlier diagnosis of AHF (58).

Embryonated hens' eggs

The prototype XJ strain of Junín viruswas adapted to the chick embryo (61), anda study of the biological properties of thevirus adapted to this host was carried out(62). The adapted strain produced pockson the chorioallantoic membrane similar tothose presented by herpes simplex or small-pox. The specificity of the lesions was dem-onstrated by a neutralization test; sera takenfrom patients during the acute period of thedisease failed to neutralize the pocks, butconvalescent sera did. Furthermore, humangamma globulin taken from convalescentsera and labeled with lissamine-rhodamineB200 reacted specifically with infected mem-branes but failed to react with normal orwith Junín-virus-infected membranes pre-viously blocked with the same unlabeledgamma globulin.

Following inoculation of the chorioallan-toic membrane, the virus reached its maxi-mum titer in the embryo in five days. Thehighest infective titer was found in theamniotic fluid, followed in decreasing orderby the allantoic fluid, the brain, the chorioal-lantoic membranes, the yolk sac, the liver,and the heart. In the last-mentioned organthere were only traces of virus. The adaptedstrain was inoculated endovenously into theallantoic sac, the amniotic sac, and the yolksac to determine the best route for obtainingthe highest concentration of virus in theallantoic fluid harvested seven days later.The chorioallantoic membrane route gave thehighest concentration of virus in the fluid.

The virus adapted to eggs was unable toagglutinate red blood cells from one-day-oldor adult chickens, humans, mice, rats,guinea pigs, hamsters, or toads (Bufoarenarum), whether in saline or exposed topHs ranging from 5.8 to 7.6. Human sera

26

from cases of Korean hemorrhagic fever andColorado tick fever were unable to neutralizethe virus.

A description of the pathology of the virusin the different organs of the chick embryohas been reported (62). After 14 passagesin chick embryos, the virus showed nochange in pathogenicity for guinea pigs ornewborn mice. For that reason, no furtherstudies were performed.

Other laboratory animals

In rats, as in mice, Junín virus is patho-genic for the newborn. The localization ofthe virus in different regions of the brain hasbeen studied (29).

The newborn chick is also susceptible tointracerebral inoculation of the virus, show-ing paralysis followed by death (Villa Lucio,personal communication).

Junín virus is pathogenic for infant lab-oratory hamsters, but the infected hamster'sbrain is not as suitable as mouse brain forproduction of complement-fixing antigen,since the antigen appears in the brain onlyafter the onset of symptomatology (128).The distribution of the virus in hamsters wasstudied by Bruno Lobo et al. (14), usingfluorescent antibody techniques.

One primate, the marmoset, or titi, ofPanama (Saguinus geoffroyi), was found tohave great susceptibility to inoculation bythe peripheral route, not only to Junín butalso to the related Machupo, Tacaribe, andAmaparí viruses (77).

Wild animals

The isolation of virus from field mice(Hesperomys laucha laucha) was reportedby Pirosky et al. (92). Two out of 20 micecaptured in a maize plantation situatedwithin the epidemic area showed signs ofinfection after a period of seven days inisolation from other mice. A virus was iso-lated from brain tissue suspensions of thediseased mice by intracerebral inoculationinto newborn white mice. Antigens prepared

from infected mouse brain gave positivecomplement-fixation reactions with humanconvalescent sera from the epidemic understudy. No virus was found in the brains ofhealthy field mice, though their sera did showneutralizing antibodies that suggested recov-ery from natural infection.

On the basis of observations of wild micetrapped in the endemic area in 1959,Pirosky et al. claimed that these animals areinfected in nature in the same manner ashuman beings, showing illness followed byeither death or recovery. In the latter casethe sera were found to have neutralizingantibodies for the virus. As a consequence,the team concluded that wild mice cannot bethe reservoir of the virus in nature and thatit is more likely that mites, from which thevirus has been isolated (89, 92), are thereservoir.

In 1965 a new epidemic broke out in theProvince of Buenos Aires. It followeddirectly after an epizootic in a particularlyhigh population of wild rodents (125). Eventhough the virus apparently killed a goodnumber of the rodents, the survivors seemedto carry this virus, or at least a related one,chronically and asymptomatically (50).Hence, they cannot be definitely rejected asreservoirs.

Attempts on the part of the presentauthor's team to isolate viruses from wildrodents resulted in the recovery of tenstrains from Mus musculus, one fromHesperomys, and one from Akodon (79,84, 112). The trapped animals seemed tobe healthy. The strains were recovered frompools of organs (liver, kidney, brain, andspleen) and inoculated in guinea pigs. Fromguinea pigs, in turn, they were inoculatedintracerebrally into newborn mice. The in-fected mouse brain was used as a source ofcomplement-fixing antigen for virus identi-fication.

In the Province of Córdoba, which wasfirst recognized as an endemic area in 1963(123), an attempt to isolate viruses from

27

wild rodents yielded 46 strains of Junín fromCalomys laucha. In addition, four strains ofJunín and five strains of other viruses wereobtained from Akodon (26). The sameinvestigators tested the brain and viscera ofthe wild mice as complement-fixing antigenin the presence of a known hyperimmuneserum; 2 out of 35 Calomys were found tofix complement. The starting antigen was a20 per cent suspension of organs takenseparately. In one of the Calomys, the CFtiters were as follows: brain, 1:128; spleen,1:4; kidney, 1:8; and liver, 1:32. Strainsof Junín virus were also isolated from thesesame two Calomys. On the other hand, nocomplement-fixing antigen could be ob-tained in two other Calomys from whichvirus had been isolated. Thus, while the useof organs from wild rodents for the prepara-tion of CF antigens is easier and more directfor epidemiological purposes, it is less sensi-tive than virus isolation for studying the in-fective rate in wild animals (26).

Isolation of Junín virus from other wildanimals has been reported in Caviapamparum (cuis) and their flea ectoparasites(24), Mesostigmata without further identi-fication (89), Echinolaelaps echidninus(85), and Eubrachilaelaps rotundus (4).The hare (Lepus europeaus) has been foundinfected in nature; two attempts at virusisolation from this animal yielded two strainsof Junín virus (4).

Tissue cultures

The virus proved capable of being propa-gated serially in HeLa cells. Appropriatecell viability during the long experimentalperiod was obtained by the use of a suitablecombination of growth-promoting and main-tenance media, as well as by frequent fluidchanges during the period of viral synthesis.A characteristic cytopathogenic effect wasobserved during the first passage and did notchange during ten consecutive transfers ofthe agent. Evidence that the cytopathogenic

agent was Junín virus was derived fromfluorescent antibody studies and from neu-tralization tests with sera from animals im-munized with virus that had been propagatedin newborn mice (67). Infected HeLa cellswere examined at intervals, stained withGiemsa, and treated with fluorescent anti-body. Foci of 1 to 30 cells (usually 6 to 10)appeared with basophilic cytoplasmic inclu-sions; these inclusions varied in size andshape, were often near the nucleus, and werefrequently surrounded by a clear halo. Afterthree days, cytopathic changes become vis-ible without staining. These consisted offoci of shrunken granular cells that fell offthe glass. At this stage the inclusions werelarger. During the next 24 hours the focienlarged and became confluent. By theseventh day destruction of the cell sheet wasmarked, although islands of unaffected cellspersisted and giant cells could be seen.Fluorescent antibody studies two days afterinoculation showed antigen in small roundcytoplasmic inclusions in individual cells.Groups of cells containing antigen were alsoseen, and the infection appeared to bespreading from one infected cell to surround-ing cells. Antigen was not observed in thenucleus. The subsequent stages resembledthose shown by Giemsa staining; the inclu-sion visible by both methods appeared tocorrespond (95).

Partial regeneration of the cells occurred21 days after inoculation. The cultures re-mained chronically infected, and up to 3 logTC 50 per ml could be found after 60 days.Serial passages of three strains of Junín viruswere accomplished without difficulty. Usuallythe passages were carried out at seven-dayintervals, and from 69 to 79 passages wereundergone (16). Attempts with other tissuecultures resulted in reproduction of Junínvirus in the kidney cells of guinea pigs, rab-bits, and hamsters, and in the fibroblasticgrowth of chicken and mouse embryo. Un-der direct microscopic observation, however,no cytopathogenic effect was present (33).

28

Plaque production of this virus in Rhesusmnonkey kidney cell monolayers was reportedby Henderson and Downs (44). Plaque as-says were performed using the Hsiung-Melnick overlay supplemented with lacto-albumin hydrolysate and yeast extract, asrecommended by Coleman. Plaques startedto develop by the sixth to eighth day, but atthis early stage they appeared as small, ir-regularly shaped clusters rather than as dis-crete, clear areas.

Clear plaque areas that could be counteddirectly or as clusters developed progres-sively by the ninth or eleventh day. Tacaribevirus behaved similarly in this tissue culture,and a study of the antigenic relationshipbetween these two agents using the plaqueneutralization test was carried out. The end-points were clear-cut, and there appearedto be a one-way cross.

With the MA-111 cell line in cell mono-layers under a fluid medium of M-199 with2 per cent fetal calf serum added, maximumvirus titers of 6 to 7 loglo plaque-formingunits (PFU) were obtained between two andfive days after inoculation, depending on theamount of virus in the dose. With a singleagar overlay, as described by Henderson andDowns, Junín, like Tacaribe and Machupo,produced clear plaques between six to tendays after inoculation (128).

The reproduction of Junín virus in a con-tinuous Vero cell line of kidney tissuecultures from the African green monkey(Cercopithecus aethiops) was reported byRhim et al. (96) and is now used routinelyby Dr. Buckley at the YARU unit at Yale

for all the viruses of the Tacaribe group.With the same maintenance medium as re-ported for the HeLa cell line (67), thecytopathogenic effect is evident at three tofive days after inoculation.

Personal experience has shown that acomplement-fixing antigen can be obtainedfrom infected Vero cells. The infected tissueis frozen and thawed twice and then centri-fuged for one hour at 40,000 rpm. Afterthis, the sediment is resuspended in a salineor Veronal buffer of 1:50 or 1:100 thevolume of the initial infected material.Homogenization of the sediment is done byslight sonication. At the same time, uninocu-lated Vero cells are treated in the same wayas a normal control. Titers of 1:64 or more,depending on the amount of virus presentin the infected tissue and on the concentra-tion of the resuspension fluid, are obtained,and no anticomplementarity has been found.

With the Dulbeco method, Vero cells be-haved similarly to the Rhesus monkey kid-ney cell monolayers in the studies by Hender-son and Downs (44). Observations werealso made of the PR5 strain from infectedmouse brain; when diluted 10- °' r it yieldedfrom 50 to 100 plaques. The cell monolayerwas stained with neutral red five days afterinoculation, and two days later the finalreading was taken.

With Junín virus, the plaque method,using Vero cells and agar overlay, givesmore clear-cut results than fluid cultures intubes; it also avoids the cell regenerationseen with the latter.

29

6. IMMUNOLOGICAL INVESTIGATION

Characterization: Tacaribe Group

The method introduced by Clarke andCasals for the study of arboviruses, espe-cially the sucrose-acetone extraction of in-fected mouse brain as complement-fixingantigen, proved to be the most effective tech-nique for the identification and immunologi-cal characterization of Junín virus (25).While the antigen prepared by this methodnever showed hemagglutinating propertieswith any of the strains of Junín virus tested,it gave high titers in the complement-fixationtest, especially when the infected mousebrain was harvested at the peak of virusreproduction (69). This material permitteda comparative study with all the other virusesavailable at The Rockefeller FoundationVirus Laboratory, currently the YARU unitat Yale University.

Hyperimmune serum for the homologoussystem is obtained by immunization of four-to eight-week-old mice, as is done routinelyin arbovirus research. It is now possibleto obtain large volumes of ascitic fluid,which is as good as serum for serologicalwork, by virus immunization followed by theuse of the relatively nonvirulent ascites cellneoplasm Sarcoma 180/TG. The titers ofthe ascitic fluid antibodies compare favor-ably with those of the sera, and more than30 times as much antibody can be obtainedper mouse (116).

In exhaustive complement-fixation tests,Junín antigens were tried against hyperim-mune sera for practically all known arbo-viruses, as well as a few other agents thatwere included for purposes of a differential

diagnosis-herpes, Q fever, rickettsialpox,murine typhus, Newcastle disease, mouseencephalomyelitis, and encephalomyocar-ditis. The only serum that cross-reacted withthe Junín CF antigen was that of Tacaribe, avirus repeatedly isolated from bats and oncefrom mosquitoes by Downs et al. (32) inTrindidad. This serum, with a homologoustiter of 1:256, reacted against Junín antigenwith a titer of 1:32. Not only did the mouseimmune serum cross-react with the antigen,but human AHF convalescent sera reactedwith the antigen from Trinidad as well.

To determine the relationship more pre-cisely, two neutralization tests were per-formed, one with Junín virus and the otherwith Tacaribe virus, using the same speci-mens of immune and control sera. A Junínguinea pig serum with a titer of 1:32 against10,000 TCD 50 of homologous virus failed toprotect against Tacaribe virus, and aTacaribe serum, which at constant dilution1:2 protected against 100 TC50 of homol-ogous virus, failed to show any protectionagainst Junín virus. Thus it was demon-strated that Junín and Tacaribe are two dis-tinct agents (69). Other investigators usingthe same CF technique to compare Tacaribevirus with strains of Junín virus from differ-ent epidemics arrived at the same results-that is to say, they found the same type ofdifferences in serum titer between thehomologous and heterologous Junín andTacaribe systems (1).

Although some clinical resemblance mayexist between the Korean and Argentinehemorrhagic fevers, sera from six patientsconvalescing from the former failed to react

30

to a Junín complement-fixing antigen (69).In the absence of a hemagglutinating

antigen for Junín, sera known to have CF orneutralizing antibodies for this virus weretested against hemagglutinating antigensprepared from the following arboviruses:group A: Mayaro, Eastern equine encepha-litis (EEE), Western equine encephalitis(WEE), Venezuelan equine encephalitis(VEE), Aura, and Una; group B: St. Louisencephalitis (SLE), Ilheus, Powassan, Bus-suquara, yellow fever, dengue Type II,Japanese B encephalitis (JBE), CentralEuropean tick-borne encephalitis, louping-ill,Omsk hemorrhagic fever, and Russianspring-summer encephalitis (RSSE); groupC: Marituba, Oriboca, and Caraparú; andBunyamwera, Germiston, Cache Valley,Guaroa, California encephalitis (CE),Tahyna; Guama, Koongol, Witwatersrand,Ketapang, Bakau, Neapolitan sandfly fever,Icoaraci, Akabane, Ingwavuma (SA AN4165), Sathuperi, Manzanilla, Tacaiuma,and Bwamba. No cross-reactions were ob-served.

Further studies to discover possibleantigenic relationships with viruses otherthan those from the arthropod-borne groupwere performed with measles virus andmouse adenovirus (43). With the formerthe complement-fixation and measles hemag-glutination-inhibition tests were used. Nocross-reaction between Junín and measlescould be found by complement-fixation,using either of the heterologous systems forcomparison, or by hemagglutination-inhibi-tion using measles virus as antigen. Withmouse adenovirus, no cross-reaction witheither of the antigens against either of theheterologous systems could be detected. Thereason for studying these two antigenicrelationships was that both measles virus(45) and mouse adenovirus (43) had beenreported to be capable of propagating innewborn mice when inoculated intracere-brally, with behavior similar to that observedfor Junín. Moreover, the inclusion bodies

found in the cells of the urine sediment inboth Junín and measles infections are simi-lar. And finally, Bergold has observed thatthe image of Tacaribe group viruses re-sembles that of the adenoviruses under theelectron microscope; they have the samenumber of capsomers (7).

Extending the study of antigenic relation-ships between Junín virus and other path-ogenic organisms, sera from AHF con-valescents with known titers against Junínand experimental sera obtained in miceusing the XJ and RP strains of Junín werechecked by microagglutination againstLeptospira from the following groups:icterohaemnorrhagiae, javanica, canicola,ballum, pyrogenes, cynopteri, autumnalis,pomona, australis, grippotyphosa, hebdo-madis, bataviae, hyos, biflexa, and sema-ranga. No ability to agglutinate Leptospirawas shown by any of the sera tested exceptthe XJ mouse immune serum, which re-acted to Leptospira ballum, strain Mus 127.The same reaction, in a lesser degree how-ever, was shown by the sera of normal micefrom the same colony used as controls(68).

In short, up to now the only agents thathave reacted to Junín virus are the othermembers of the Tacaribe group, with whichit shares a common complement-fixingantigen. Junín can be differentiated by theneutralization test and, according to Cua-drado and Casals (31), by immunolectro-phoresis on agar. This latter method seemsto provide a means for detecting specificdifferences among Amaparí, Junín, andTacaribe viruses. With this technique,Cuadrado found Portillo virus to be differentfrom Junín (personal communication).

Experimental Vaccination

Owing to the seriousness of the diseasecaused by Junín virus and the consequentpublic pressure for action, attempts havebeen made to develop a vaccine to help pro-

31

tect the exposed population in the endemicarea.

A formalin-inactivated vaccine was pre-pared from mouse brain tissue infected witha pool of several strains of Junín virus. Thevaccine, produced and distributed by theNational Institute of Microbioiogy, BuenosAires, was given to more than 15,000 per-sons beginning in May 1959. The regularcourse of vaccination consisted of three in-jections of 1 cc each, followed by a boostereach subsequent year. Some vaccinees re-ceived only one injection, others as many asseven. The vaccination program waseventually discontinued for administrativereasons, and no evaluation of the efficacy ofthe vaccine was ever carried out.

Since Junín and Tacaribe viruses werefound to be antigenically related, while thelatter lacked pathogenicity for guinea pigs,several investigators started looking into thepossibility of using Tacaribe virus as a vac-cine against infection from Junín (80, 120).

Adult guinea pigs immunized with oneinoculation of 300,000 PFU of Tacaribevirus were protected against challenge withJunín virus 14 days later (120). Theguinea pigs immunized with Tacaribe virushad complement-fixing antibodies againstTacaribe and Junín virus at 14 days, thelevel against Junín virus being lower.Neutralizing antibodies against Tacaribevirus were detectable at 14 days, butneutralizing antibodies against Junín viruswere negligible even after 49 days.

In another experiment (28), guinea pigswere inoculated with live Tacaribe virus bythe intramuscular route. It was possible toisolate infectious virus from the lymph nodesfor a period of 48 days after inoculation,although not consistently. Virus was presentin the serum on the seventh day and in theliver on the thirteenth day. There was noimnimunity against challenge with Junín virusin the first three days, partial resistance ap-peared after seven days, and complete im-munity was established thereafter. Comple-ment-fixing antibodies against Tacaribe andJunín viruses were detected on the thirteenthday, the titer against the homologous anti-gen being higher.

The development of immunity againstJunín virus infection after inoculation withTacaribe virus has been explained by inter-ference rather than other mechanisms.

Thus, in another experiment (128), pas-sive immunization against Tacaribe virus insuckling hamsters was obtained through im-munization of the mothers, but the passiveantibodies against Tacaribe virus did notafford protection when the hamsters werechallenged with Junín virus. In still aanother study, guinea pigs infected withTacaribe virus developed CF antibodies,but not neutralizing antibodies, againstJunín (120).

Interference between Junín and otherantigenically unrelated viruses, such as VEE,has been demonstrated using the whitemouse as host (59).

7. ISOLATION AND IDENTIFICATION OF

JUNIN VIRUS

Special Precautions

As a rule, no special precautions beyondthose routinely followed in the handling ofarboviruses are available. Infections inlaboratory workers and animal keepers arevery comrnmon in places where Junín virus ispresent, and unfortunately they can be quitesevere. It is thought that these laboratoryinfections are contracted either from aerosolin the environment, as is the case with manyother arthropod-borne viruses, or throughcuts or minor abrasions in the skin (102,111). It is advisable, therefore, to sendmaterials for etiologic diagnosis to lab-oratories specialized in this kind of research,at least until vaccination for laboratoryworkers against this virus can be performed.In Argentina, where the disease is endemrnic,there are at least three centers-two ofthem in the city of Buenos Aires and one inthe city of Córdoba-where this kind of in-vestigation can be done.

In the United States, where the diseasecould well appear in a person coming fromthe endemic area-whether an Americancitizen returning from a business or pleasuretrip, or an Argentine entering as an immi-grant or a visitor-it is necessary to knowwhat institution should take care of this kindof research. When there is a suspicion ofAHF, even if the patient has not been outof the country, an explanation of the cir-cumstances should be sent to the NationalCommunicable Disease Center in Atlanta,Georgia, or to the WHO Reference Center

of the Yale Arbovirus Research Unit(YARU) at Yale University, New Haven,Connecticut, for clarification of the etiology.

Clinical Sources of Materialsfor Testing

AHF is a systemic disease in which theinfectious agent is present in the bloodalmost from the beginning, and usually theviremia persists throughout the febrileperiod (11). It is diagnosed etiologicallyby recovery and identification of the virusfrom the patient's blood, urine, and organs,and/or by demonstration of a rise in anti-body titer in serum during convalescence.

Blood and pieces of organs from fatalcases are most suitable for virus isolation.Successful results have been achieved withserum stored at -70°C for periods of a yearor longer. The virus has also been isolatedfrom urine, which seems to be a good sourceas well. It has never been isolated fromfeces.

On primary isolation, certain strains showa selective pathogenicity for particular hosts.This is an important diagnostic consider-ation, since a strain can be lethal for aguinea pig, for instance, and show no lethalinfection in suckling mice.

As a matter of routine, guinea pigs andnewborn mice are simultaneously inoculatedfor virus isolation. Two or four guinea pigsand at least two litters of mice are used foreach attempt. The guinea pigs, preferably

33

between 250 and 350 g, are inoculated bythe intraperitoneal route (0.15 to 1.0 cc) orintramuscularly or subcutaneously (0.3 to0.5 cc) with original material from a patient.The usual material is whole blood, serum, orclots macerated in saline or some otherdiluent. lf the disease had reached a suffi-ciently advanced stage in the patient,neutralizing antibodies or other inhibitorsmay be present in the serum. For thisreason, it is advisable to use not only un-diluted serum or whole blood, but tenfolddilutions of the same materials as well, so asto dissociate the virus-antibody complex. Inmany instances, guinea pigs show no signsof illness after inoculation with undilutedblood, whereas disease becomes definitelyapparent after a 10-2 or greater dilution.The same precautions should be taken withmice, and intracerebral and subcutaneousinjection should be done simultaneously inany one animal.

When urine is used, the guinea pigsshould be inoculated by the intramuscularor subcutaneous routes.

It is usually at least a month, sometimeslonger, before a viral diagnosis can bepinned down, especially when it is necessaryto wait for the convalescent serum sampleto demonstrate a rise in antibodies againstthe virus.

Procedures such as the fluorescent anti-body technique, while available as an inves-tigator's tool, have not yet been applied inthe routine diagnosis of Junín infections.The presence in the patient's urine of giantcells, presumably containing viral antigen,could provide a likely means for detectingJunín virus during the acute stage of thedisease.

With routine serology techniques, thespecific diagnosis can be speeded up bysacrificing some of the inoculated sucklingmice on the seventh or eighth day and usingtheir infected brains as crude antigen in theform of a 10 or 20 per cent suspension insaline or Veronal buffer. After light cen-

trifugation, the supernatant acts as a goodcomplement-fixing antigen, with a titer onlyone or two dilutions lower than that obtainedwith the sucrose-acetone treatment. If theantigen from the suspect material (and notthe controls) reacts against a known serumwith a good titer for Juníin, t.is means thatthe agent belongs to the Tacaribe group; ifit does not react, a blind passage in othermice can then be performed. Serologytechniques are essential for the diagnosis ofJunín virus, because many agents can killmice or guinea pigs after similar incubationperiods. Since guinea pigs are not a goodsource for antigen, infection with Junínvirus in this animal can be shown in twoways: (1) if it becomes sick or dies, bloodor pools of organs may be transferred tonewborn mice by the intracerebral route toobtain an antigen for the CF test; or (2)serum from a surviving animal may betested for CF antibodies against Junín. Ifthe complement-fixation test is positive, thenthe virus is a member of the Tacaribe group(Junín, Machupo, Portillo, Amaparí,Pichinde, Tamiami, or Tacaribe), and aneutralization test will be necessary to deter-mine its precise identity.

Neutralization and Complement-Fixation Tests

For final identification, neutralization ofthe suspected isolate by specific Junín im-mune serum is required.

The isolated strain is diluted serially intenfold steps to an appropriate endpoint(usually l10-10). A prototype strain ofJunín virus is treated in the same way. Eachdilution of each virus strain is distributedinto three rows of test tubes in 0.2 ccamounts. To the first row an equal amountof Junín immune serum, undiluted and un-heated, is added; to the second, normalserum from the same host as the Junín im-mune serum (usually mice or guinea pig

34

serum), also undiluted and unheated, is

added. To the third row, diluent is added.Incubation is carried out at 37°C for one

hour, after which the chosen hosts areinoculated with the contents of each rowseparately.

The difference between the virus titers inthe immune serum and the normal serumwill reveal the extent of specific neutraliza-tion; the difference between the titers in theserum and the diluent will show the extent

of nonspecific inhibition. The set with theprototype strain of Junín virus will indicatehomologous protection of the serum usedfor virus identification.

The endpoint in each group is consideredto be the dose sufficient to infect or kill 50per cent of the host (mice, guinea pigs, tis-

sue culture). It may also be taken as the

average plaque-forming unit per ml in eachgroup. A difference of 2.5 1oglo between the

two groups is usually considered significant,but identification can be suggested by a

smaller difference.A number of facts must be taken into con-

sideration in performing the neutralizationtest. In the first place, newborn mice are

more suitable than guinea pigs for reasons of

economy and space. If newborn mice are

going to be used, it is best to keep only eight

mice in each litter to avoid overcrowding,since they must be observed for 21 days to

reach the endpoint in the case of determin-ing LD50 for titration. Newborn mice can

also be used to determine the ID 50. For this

purpose, all the animals are sacrificed on the

seventh or eighth day after inoculation, and

a crude antigen is prepared by diluting

macerated mouse brain in 10 or 20 per cent

saline or Veronal buffer. A complement-

fixation test is then performed against a

Junín immune serum with a high titer and

another negative control (Shope, 1967). The

mouse brain having a positive reaction to

the immune serum will be considered to be

infected with Junín virus, and the negativemice to be uninfected. The titration is done

in the same way as before, and in this case

the units will be ID50. The same procedurecannot be used with guinea pigs because this

animal is not a good producer of Junín CF

antigen. The measurement of antibodiesaround the thirtieth day after inoculation of

the virus serum mixtures is not reliable be-

cause there is great variation in individualresponse to the same inoculum, and even

though the virus in the mixture may havebeen neutralized or killed, it may still be a

good antigen capable of inducing an immuneresponse in the guinea pig.

To diagnose a Junín virus infection in

Argentina, it is only necessary for the iso-

lated virus to react by CF with Junín anti-sera, or else for paired sera to show serologi-

cal conversion with Junín antigen. For thisreason, it is not known for sure whether the

100 or more strains of Junín virus mentionedin Chapter 1 are really one single agent, or

whether they are different viruses with a

common complement-fixing antigen.Human convalescent sera from Junín in-

fection have antibodies against Tacaribevirus as well, but to a lesser degree (69);

human convalescent sera from cases of

uremic-hemolytic syndrome have antibodiesagainst Junín virus; and human convalescentsera from Bolivian hemorrhagic fever have

antibodies against Machupo and Junínviruses and to a lesser extent against Tacaribevirus (51). It would not be surprising,therefore, if a careful study of the many virus

strains currently labeled Junín were to show

that some of them are Machupo, Amaparí,or new entities in the group.

The isolation of Junín virus from human

beings directly in tissue cultures has not beenreported, even though theoretically there is

no reason why this could not be done.

To develop the system-antigen, serum,

or ascitic fluid-in mice for comparison be-tween viruses in the Tacaribe group or

between strains of the same virus, the currentmethodology for arbovirus research is fol-

lowed (130).

35

8. SPECIAL STUDY OF THE 1963 OUTBREAK

From the time that Junín virus was orig-inally isolated up to 1963, no comprehensiveserological study of an entire epidemic hadbeen carried out to discover the exact role ofJunín virus. Only scattered data on the riseof antibodies against this agent were avail-able.

At the beginning of 1963, a well-plannedserological study was started to determine therelationship of Junín virus to all the clinicallydiagnosed cases of AHF. For this purpose,two or three serum samples were taken fromeach patient for antibody investigation-oneat the time of the initial medical examination,a second thirty days later, and in many in-stances a third two months after the onset(63). In addition to the serum samples, epi-demiological data were collected on eachpatient at the first medical examination. Thisinformation included the patient's full name,sex, age, places of residence and work, andprevious history of vaccination. With respectto the current illness, the date of onset, thedegree of severity, and the presence or ab-sence of similar cases within the same house-hold were recorded. Finally, the number ofthe patient's record in the hospital was noted.These data, together with detailed findingsfrom all the tests performed, were assembledin a professorship thesis for the MedicalSchool of the University of Buenos Aires(64).

Four public medical centers located inJunín, Rojas, Chacabuco, and Salto tookpart in the program. From the 653 clinicallydiagnosed cases reported during that year(Table 1), it was possible to obtain 430paired sera. Curves showing morbidity,

number of cases studied through serology,and number of cases with serological con-version for Junín during the year are pre-sented in Figure 4. In the complement-fixation test, 69.55 per cent of the pairedsera studied showed serological conversionfor Junín virus (72). The geographicaldistribution of these positive cases is shownin Table 2.

The paired sera without serological con-version for Junín, plus 40 other paired serawith conversion for Junín virus used as acontrol, were tested by hemagglutination-in-

FIo. 4. CURVES COMPARING REGISTERED CASES OFAHF wITH SEROLOGICAL RESULTS, 1963

240

220

2ee I

2l0

ion

160

<u 140

0 120

It'0

MONTH

- CASES OF ANF REGISTERED

-- CASES SEROLOGICALLY STUDIED

---- C ASES WITH SEROLOGICAL CONVERSION FOR JUNIN VIRUS

36

.-0 " o I ee k^ m " m w 0 ,l e m u- -, 0 t-.

IS-oS

6Pr8t

£s» ti I I ¡ 1 - 1st-OII I I

LE9 '"1* ¡ I

6£-8£zc

TLz-9 _ o ' o I

Sc*t ti ^ i

Et~~ -= ti <> ¡ -q*

61-81

LI-91

¡ ¡

¡ ¡

--

-l

-

- I1 1

I sJ

I 4

I M

-m r o CI

00 t - - NI

\0 00-CN" 1 1 - 1 1rn - - ¡

10 I,1 t " ¡ - ¡ ¡ ¡ ¡

'ti

1 =

1 1

-I

" ~ M"- e1* 1~

I 1~ e

¡ - ~ cmq

III

I I 1

§ .,o,

o~~~° - 37 2 E 2

37

a

Umoului

¡ ¡

1 1

1 1

-I

e,;

.a

o

.0

o

t~M,u

u]o

'0

o

o)

2

o¢

El

K e

a Ern

M 1

P m

I1 1

1

1

1

1

1

1

1 1

41-r- o k^0

" - W \ CO co XD _I X s kn ~= 00 00 <40 - - - -- -,- - - 'r -

r- C-1 O m 0% 0 - cq m <lI - = -0 a > w000\ -o 0 c4 m -4

00

<~1

m

<40

1-4

mr

11 1 11

11 1'11

I1 I 1

-1i~ : ii- > --

4. l---- 4. -4O cl0

C> e I qI

-- 4 - - - N _ , m

0s rq -4 -4

0ci .>0 0 _

0 0 <D c o t

v -000

o 0 0\ eene

ci ~ ~ ~ c

c oci < 4 0 I 1 C I I

ci <4 0% 'tcooci

_O r _i _ 4

¡ Ie 1

I Ikn

1l

1 11 1

l1 1

¡ 1 1¡ ¡

¡ ¡1~I

1 ¡1

o N o

¡II I I I 1

1 1 "-I 1--

-I I ¡ -I D

II I I I I

¡ ' ' ¡I I IW O

01 014o 0 o ¢ 2.

38o -l 2 -- "-' ' - ,

38

.4

.Ja0apun

6P'8P

L"91'

£'{-o:,'

L£-9E

6Z-8¿

LZ-9Z

1Z-01

61-81

LI-91

*S51t1

m

o

0\

o«

u)

<

z

o

¢

o

z

o

;>

u

i

0

zo

rA

Z

©1..>

u

©

4

04

o

rA

0

L)i

01

01

1-

*0loa,

.1

2

o

40

o

et8"0

*0o.

Z1

x0

4>z z

0

al

4>40

'0.

* -e

hibition, using the techniques of Clarke andCasals (25), against the following arbo-viruses: from group A: EEE, WEE, Mu-cambo, and Mayaro; and from group B:SLE, yellow fever, dengue Type II, Omskhemorrhagic fever, Ilheus, and KyasanurForest disease (65). In the 40 paired serawith conversion for Junín virus, no conver-sions for arboviruses from groups A or Bwere found. In those without conversion forJunín, no antibodies against group A werefound, but group B told a different story:some true conversions for SLE virus antigenand some activity against other members ofgroup B were found; however, the responsi-ble virus was not determined. In some in-stances it was noticed that a high antibodylevel against SLE was present in the firstsample but that the titer against the samevirus in the second sample, taken usually amonth later, showed a decrease of twofolddilutions or greater. This decline in thehemagglutination-inhibition (HI) antibodylevel was good confirmatory evidence of arecent infection with one of the group Barthropod-borne viruses. Indeed, parallelfindings were made during an outbreak ofSLE in Tampa, Florida (36).

In summary, of all the cases studied, 69.55per cent showed conversion for Junín virusby CF; 20.32 per cent showed activity forgroup B arboviruses, and 10.13 per cent hadno antibodies against any of the virusestested.

Lack of serological conversion by the CFtest in 30 per cent of the patients could atfirst be attributed to poor sensitivity of thereaction; however, in view of the HI test re-sults with group B antigens, this assumptionappears unlikely. It would seem that someother viruses causing the same clinical pictureas AHF are involved.

The presence of SLE virus, or at least avery closely related agent, has already beendemonstrated in serological surveys (73,

115). However, the clinical effects of thisvirus in man have never been described inArgentina; even though cases of encephalitisoccur, virological diagnosis is rarely avail-able.

It was unexpected to find SLE or a closelyrelated agent producing a hemorrhagic dis-ease. Since no neutralization tests were per-formed, and only a few members of the groupB arboviruses were included in the serologi-cal study, some doubt arose as to the identityof the virus, but it is known from the samestudy that the agent is neither Omsk hem-orrhagic fever nor Kyasanur Forest disease.

Attempts to isolate viruses from acute-period sera (kept at -70°C) from AHFpatients with conversion for group B arbo-viruses but not for Junín yielded three in-fectious agents. The investigators at YARUdemonstrated that two of the strains wereSLE. The third agent is still under study.It is different from SLE, but still closelyrelated; it may be Ilheus.

Cases clinically diagnosed as AHF withserological conversion for SLE or someother group B arbovirus occurred at randomover the entire epidemic area and throughoutthe full course of the outbreak (Fig. 5 andTable 3). These cases were treated at sev-eral separate medical centers, and apparentlynone of the attending physicians saw anydifference from patients with a clinical diag-nosis of AHF who showed conversion forJunín. The physician in charge of the RojasCenter stated that in his judgment there wasno distinction in the severity of the illnessbetween the two groups of patients. Heobserved severe, mild, and doubtful cases inboth of them (personal communication).

Information was exchanged with theauthor on the various clinical manifestationsof AHF-the hemorrhagic, nervous, and

39

r- - =

m1~ " -

5''~

¡- ¡

II1

III

II1

1 II

Yllllk^ o = -

I IIII

IIIII¡ ¡ ¡ 1 1

1 1 1 1 1

I I

_lI.Cl II5 1 ¡- 1 1 ¡ ¡

e 1 > 1 1 1 I ¡

Cq

cq Im I II

V. t t- ,

IIIIIIb oI I I I

j:: I;I I II

ala

E-'

g~ k^ 5I I

1 ¡

II1

III

II

II

¡ 1 ¡

II

II

III

¡III

III¡

1 1 1

1 1 1

1 1 1

1 1 1

1 1 1

1 1 1

I 1-1

¡II

¡ II1

¡ II1

III1II 1 -

l 11 1 1 1

¡ I I ¡ I I I ¡

0> 0

o o .-

0 C *~. Cd 00 E L °

40

3on

o

e4= 0

-4 u'0 c

o 00,,

~ _

E0>

bt0>'-0)0

E-o'-

00>A

* 43-

alapufl

<0

E n

>oo

Zm

¢n <

<) 0

,3

x E

£L-9£

£E-_Z

LZ-9Z

£-Ztr

6I-81

L*-91

*SI-tV

I

I

I

1_

FIG. 5. HISTOGRAM OF REGISTERED CASES OF AHF

AND SEROLOGICAL RESULTS, 1963

24O

2t20

200

14I1240

ou

£:E

z

120

10o

lo

Io

40

20

o

MONTH

- REGISTE RED CASES

~ CASES WITH CONVERSION FOR JUNIN VIRUS

~ C ASES WITH ANTIBODIES AGAINST GROUP B ARBOVIRUSES

E CASES WITHOUT ANTIBODIES AGAINST ANTIGENS TESTED

mixed forms. There are no data as yet toshow whether the cases produced by SLEvirus remain in the nervous form (103, 104)or in the hemorrhagic, or whether they canoccur with the signs of both.

Further studies on this matter will becarried out.

Cases from the same cornfield, breakingout during the same week and even on thesame day, and treated at the same publicmedical center, are produced sometimes byJunín and other times by a group B arbo-virus.

With this new knowledge about theetiology of AHF, it will be necessary to lookfor viruses in mosquitoes from the endemicarea and to explore other ways in which SLEvirus can be transmitted to human beings.

Later, in 1964, at least one case of AHFwithout conversion for Junín or a group Barbovirus showed conversion for Maguari(BEAR 7272) from the Bunyamwera group(66). Since this antigen was not used in the1963 tests, it could not be correlated with the10.13 per cent of cases that had no anti-bodies for Junín or group B.

41

9. RECENT EXTENSION OF AHF TO NEW AREAS

Between 1955 and 1963, AHF was con-sidered to be endemic only in the Provinceof Buenos Aires in an area approximately20,000 km 2 (53, 55) with a population ofabout 250,000. In 1963, a new endemicarea was recognized in the Province ofCórdoba when 14 cases were registered.The complement-fixation test showed that 9out of 13 were infected with Junín virus.

Vanella studied 76 cases in the Provinceof Córdoba over the period 1963-1964, andhis clinical and epidemiological findings havebeen presented in a report (123). The lat-est data available from the Córdoba endemicarea, where the cases are increasing eachyear, showed a morbidity index of 60:100,000 and a mortality rate of 6 per cent.

In 1963, the main focus of the epidemicwas in Salto, a parish in the Province ofBuenos Aires where the disease had not beenrecognized before. In 1967, however, theepidemic was centered in the parish of Per-gamino (also in the Province of BuenosAires), where scattered cases had been ob-

served in previous years. Only five casesfrom this area were clinically recognized in1963; two of these were studied serologi-cally, and one of them proved to be a Juníninfection. The most recent news on morbid-ity and distribution of the disease in theProvince of Buenos Aires appeared in LaNación on September 6, 1967. This reportstates officially that in 1967 Pergamino had331 cases and 32 deaths; Rojas, 195 and30 deaths; Salto, 167 and 12; Junín, 123and 3; Nueve de Julio, 8 and 1; Chacabuco,100 and 3; Carlos Casares, 3 and 1; Bragado,9 and 3; Bartolomé Mitre, 87 and 7; Alberti,7 and no deaths; Capitán Sarmiento, 3 andno deaths; and Colón, 26 and no deaths.

Since 1964, the Provinces of La Pampaand Santa Fe have had scattered cases inzones nearby the Province of Buenos Aires.

From all this data, it is clear that the en-demic area is expanding every year and atthe same time coming increasingly closer tothe populous cities of Buenos Aires andCórdoba.

42

10. FINAL CONSIDERATIONS

AHF and its associated Tacaribe-groupviruses have become a new public healthproblem in the Americas during the lastdecade (117). Numerous efforts have beenmade to clarify the epidemic-epizootic as-pects of the disease and to explore possibleapproaches to immunoprophylaxis.

Among the many Argentine institutionsthat are devoting concentrated efforts to thestudy of the disease are the National Minis-try of Public Health, through the MalbranInstitute (4, 8, 91, 121); the National Insti-tute of Agricultural Technology (INTA),through the Zoonoses Institute (22, 23); theBuenos Aires Provincial Ministry of PublicHealth (54, 57); the Córdoba ProvincialMinistry of Public Health, through the Cór-doba Institute of Virology (26, 124); andthe University of Buenos Aires, through theSchool of Medicine (40, 72). In addition,individual physicians in the endemic area are

doing everything they can to help combat thedisease.

In response to a request for sera from per-sons with previous history of AHF for CFtesting purposes, a local physician fromJunín drew blood from all the patients hehad attended from 1953 to 1962. In thisway, 147 sera were obtained, and the resultsof this study are shown in Table 4. Factorstaken into account were the number of yearssince the disease occurred and the patient'splace of residence.

Although the study of the 1963 epidemicdid show that 70 per cent of the AHF caseshad CF antibodies against Junín virus duringthe convalescent period, this retrospectivesurvey could not provide very much furtherconcrete data on the subject because of in-sufficient knowledge about CF reactionagainst Junín when patients were in con-valescence.

TABLE 4. PRESENCE OF JUNíN VIRUS CF ANTIBODIES IN PERSONS WHO HAD AHFFROM ONE TO TEN YEARS BEFORE SEROLOGICAL TEST *

No. of Years Number of Cases Positive by CF/Number of Cases Studied in Each Parishafter

AHF Junin Rojas Chacabuco Bragado Viamonte Chivilcoy Lincoln Total

Totals 25/54 1/4 15/33 9/28 13/24 3/3 0/1 66/1471 year 2/8 0/1 4/8 1/4 4/7 - - 11/282 years 6/12 1/2 2/5 3/4 3/4 2/2 - 17/293 years 8/17 - 3/5 1/8 1/3 - - 13/334 years 8/11 0/1 3/8 3/6 4/5 1/1 0/1 19/335 years 1/5 - 3/6 0/2 1/3 - - 5/16From 6 to10 years 0/1 - 0/1 1/4 0/2 - - 1/8

* Based on blood samples provided by C. Magnonf.

43

The information in Table 4 does not in-clude the former patients' sex, because only9 of the 147 subjects were females-5 posi-tive and 4 negative. Of the remaining 138males, 61 showed antibodies in the comple-ment-fixation test and 77 did not. Since noserological tests were performed with thesepatients' sera at the time of the illness, thepresent results cannot be interpreted as nec-essarily proving the persistence of antibodiesto Junín virus. This is especially true sinceit is now known that some of the casesclinically labeled as AHF show conversionto viruses other than Junín. It has also beenshown that with laboratory-acquired infec-tions the CF antibodies disappear within ayear (6). Furthermore, it is possible thatcontinuing residence of former patients inthe endemic area may expose them to newcontacts with the virus, thus creating abooster effect.

Although no neutralization tests were per-formed with these 147 sera, neutralizingantibodies have been detected as long as14 years after an illness (6). However, sinceno neutralization-test surveys are availableto show the rate of antibody prevalenceamong persons living in the endemic areawho have never had the clinical syndrome,it is hard to interpret such long-lastingantibodies.

There are instances of persons who havehad AHF more than once. A patient fromJunín was hospitalized three times, in threedifferent years, with a diagnosis of AHF. Ifneutralizing antibodies persist for life, thenthere must be more than one virus involvedin the etiology, as the serological findingsfrom the epidemic seen to indicate. Eachsubsequent epidemic brings additional in-formation about serological conversion forviruses other than Junín.

Prevention of human infection can be ac-

complished in several ways. One method isto reduce the population of infected animalsin the endemic area. The spreading ofrodenticides and acaricides from helicoptershas not been well received. And with rea-son: the first big AHF epidemic, in 1958,appeared shortly after fumigation of the areawith dieldrin and, since the etiological agentof the disease was unknown, many peoplefelt that this toxic substance was to blame forthe disease, or at least for creating a pre-disposition thereto (52). This may be acoincidence, but it is true that fumigationwith dieldrin could trigger a biological im-balance, as has been claimed by many Ar-gentine investigators, and it is hard to provethat this is not so.

Another approach to reducing human in-fection would be to introduce mechanizedequipment for picking corn. Corn harvesters,however, are not the only people who con-tract the disease, and even if mechanicalcorn pickers were available, there are timeswhen harvesting has to be done by hand.

Still another avenue, and one worthy ofserious consideration, is to immunize thepopulation at risk. However, a vaccine hasyet to be developed. A problem is to deter-mine what will go into it. If the serologicalfindings and the results of the virus isolationattempts from the 1963 epidemic are car-ried through, then it would appear as thoughthe vaccine would have to contain agents ofSLE and some other group B arboviruses, inaddition to Junín. Moreover, many Junínvirus strains would have to be studied com-paratively by neutralization tests to see ifthey are alike.

Once all this information is in hand, at-tention will still have to be given to the de-velopment of a faster and more economicalmethod than the neutralization test for evalu-ating the vaccine's effectiveness in man.

44

REFERENCES

1. ADLER, E., and A. PARODI1965 Relación antigénica de 7 cepas de virus Junín aisladas en

epidemias de diferentes años. Rev Soc Argent Biol 41: 102-109.

2. AoI, A., H. H. D'ALoIsIo, and R. B. PoDio1960 Microdisección renal en la enfermedad de O'Higgins. Rev

Med de Córdoba 48: 121-135.

3. ALVAREZ AMBROSETTI, E., F. A. CINTORA, R. LOCICERO, C. MAGNONI,H. MILANI, and R. VACCAREZZA1959 Fiebre hemorrágica epidémica; observaciones clínicas. Día

Med (B Aires) 31: 232.

4. ARGENTINA. INSTITUTO NACIONAL DE MICROBIOLOGIA1966 Investigaciones sobre fiebre hemorrágica argentina. Publi-

cación de la Comisión Nacional Coordinadora para Estudioy Lucha Contra Fiebre Hemorrágica Argentina. BuenosAires, Secretaría de Estado de Salud Pública.

5. ARRIBALZAGA, R. A.1955 Una nueva enfermedad epidémica a germen desconocido;

hipertermia nefrotóxica, leucopénica y enantemática. DíaMed 27: 1204-1210.

6. BARRERA ORO, J. G.1963 Discussion on "Hemorrhagic Fevers in South America." Pro-

ceedings, Seventh International Congresses on Tropical Medi-cine and Malaria 3: 301-302.

7. BERGOLD, G. H.1966 Electron Microscopic Investigation of Some Arboviruses.

Abstract of Papers, Ninth International Congress for Micro-biology (Moscow), p. 455.

8. BERRIA, M. I., L. F. GUTMAN FRUGONE, R. GIROLA, and J. G. BARRERAOROIn press Estudios inmunológicos con virus Junín. Medicina (B Aires).

9. BESUCHIO, S. C.1966a Anatomía comparada de órganos de cobayos inoculados con

virus Junín. Comisón Nacional Coordinadora para Estudioy Lucha contra la Fiebre Hemorrágica Argentina. BuenosAires, Secretaría de Estado da Salud Pública.

10. BESUCHIO, S. C.1966b La patología comparada de la fiebre hemorrágica argentina.

Comisión Nacional Coordinadora para Estudio y Luchacontra la Fiebre Hemorrágica Argentina. Buenos Aires,Secretaría de Estado de Salud Pública.

11. BoXACA, M. C., L. B. DE GUERRERO, A. S. PARODI, H. R. RUGIERO, andS. GONZALEZ CAPA1965 Viremia en enfermos de fiebre hemorrágica. Rev Asoc Med

Argent 79: 230-238.

45

12. BOXACA, M. C., A. S. PARODI, C. COTO, and S. GONZALEZ1963 Effects of pH, Temperature and UV Radiation on Junín

Virus. Proceedings, Seventh International Congresses onTropical Medicine and Malaria 3: 319-320.

13. BOXACA, M. C., A. S. PARODI, H. RUGIERO, and R. BLAY1961 Fiebre hemorrágica experimental en el cobayo (virus Junín).

Rev Soc Argent Biol 37: 170-179.14. BRUNO LOBO, M., G. GOMEZ, P. A. WEBB, and K. M. JOHNSON

1967 Pathogenesis of Junín Virus in Hamsters. Program, SixteenthAnnual Meeting of the American Society of Tropical Medi-cine and Hygiene 155-156.

15. BRYAN, W. R., and J. W. BEARD1939 Estimation of purified papilloma virus protein by infectivity

measurements. J Infect Dis 65: 306-321.16. BUCKLEY, S.M.

1964 Junín and Tacaribe Work in HeLa Cells. Amer J Trop Med14: 792-794.

17. BUDZCO, B.1965 Electroforesis de las proteínas séricas de cobayos inoculados

con virus Junín (en papel y gel de poliacrilamida) Rev SocArgent Biol 41: 92.

18. CALDANI, L. A.1964 Vitamina B; en mal de los rastrojos. Día Med 36: 281.

19. CANAVERI, A. M., A. S. PARODI, and A. PAVLOVSKY1963 Factors of Coagulation in Experimental Hemorrhagic Dis-

ease in the Guinea Pig (Virus Junín, Strain XJ). Proceed-ings, Seventh International Congresses on Tropical Medicineand Malaria 3: 325-326.

20. CAPRI, J. J.1959 Lista de pulgas de la Provincia de Buenos Aires. Actas Prim

Jorn Entomoepidem Argent (B Aires).21. CASALS, J.

1965 Serological Studies on Junín and Tacaribe Viruses. Amer JTrop Med 14: 794-796.

22. CEDRO, V. C. F., C. DE PEREZ ARRIETA, R. A. CACCHIONE, J. CRESPO,E. S. CASCELLI, R. J. ROVERE, J. A. Ruis Y ORMAECHEA, E. ROSASCOSTA, E. S. MARTINEZ, and C. R. RAMIS1966 Aportes al estudio de la fiebre hemorrágica argentina. Publi-

cación de la Comisión Nacional Coordinadora para Estudioy Lucha contra Fiebre Hemorrágica Argentina. Buenos Aires,Secretaría de Estado de Salud Pública.

23. CEDRO, V. C. F., L. J. VILLA, R. A. CACCHIONE, J. A. CRESPO, N. RossI,R. J. ROVERE, F. J. PROHASKA, M. J. D. BULGINI, E. S. CASCELLI, C. B.DE PEREZ ARRIETA, J. E. CALCAGNO, and E. S. MARTINEZ1961 Fiebre endemo-epidémica del noroeste de la Provincia de

Buenos Aires (Primer informe). Rev Invest Ganaderas 12:155-181.

24. CEDRO, V. C. F., L. J. VILLA, R. A. CACCHIONE, J. A. CRESPO, R. J.ROVERE, E. S. CASCELLI, C. B. P. ARRIETA, M. J. BULGINI, J. A. R.COSTAS, and E. S. MARTINEZ1963 Contribución al estudio de la fiebre endemoepidémica del

noroeste de la Provincia de Buenos Aires. Proceedings, Sev-enth International Congresses on Tropical Medicine andMalaria 3: 331.

46

25. CLARKE, D. H., and J. CASALS

1958 Techniques for Hemagglutination-Inhibition with Arthropod-Borne Viruses. Amer J Trop Med 7: 571-573

26. CORDOBA. INSTITUTO DE VIROLOGIA

1966 Virus Junín en la Provincia de Córdoba. Publicación de laComisión Nacional Coordinadora para Estudio y Luchacontra Fiebre Hemorrágica Argentina. Buenos Aires, Secre-taría de Estado de Salud Pública.

27. COTo, C. E., A. S. PARODI, and S. LAJMANOVICH1963 Relationship Between Inoculated Doses of Junín Virus and

Time of Death in Guinea Pigs. Proceedings, Seventh Inter-national Congresses on Tropical Medicine and Malaria 3:324-325.

28. COTO, C. E., E. REY, and A. S. PARODI1967 Tacaribe Virus Infection of Guinea Pigs. Arch Ges Virus-

/orschung 20: 81-86.

29. COTO, C. E., L. ZAHARZEUSKIJ, and A. S. PARODI1965 Localización sub-celular del virus Junín en el cerebro del

ratón lactante; estudio de infectividad. Rev Soc Argent Biol41: 128-134.

30. CRESPO, J. A.1966 Ecología de una comunidad de roedores silvestres en el Par-

tido de Rojas, Provincia de Buenos Aires. Comisión NacionalCoordinadora para Estudio y Lucha contra Fiebre Hemorrá-gica Argentina. Buenos Aires, Secretaría de Estado de SaludPública.

31. CUADRADO, R. R., and J. CASALS

1967 Differentiation of Arboviruses by Immunoelectrophoresis.J Immun 98: 314-320.

32. DOWNS, W. G., C. R. ANDERSON, L. SPENCE, T. H. G. AITKIN, and A. H.GREENHALL

1963 Tacaribe Virus; a New Agent Isolated from Artibeus Batsand Mosquitoes in Trinidad, West Indies. Amer J Trop Med12: 640-646.

33. DUNAYEVICH, H., J. R. PERIES, I. PIROSKY, J. B. DE DUNAYEVICH, andG. CORONADO1962 Multiplicación del virus causal de la virosis hemorrágica del

noroeste bonaerense (virus Junín) en cultivos primarios detejidos. Rev Soc Argent Biol 38: 140-143.

34. DuvA, D. J. A.1956 Leptospirosis a forma gripotifosa. Día Med 28: 2463.

35. DuvA, D. J. A.1963 Experiencia clínica y terapéutica en el tratamiento del mal

del noroeste de la Provincia de Buenos Aires. Día Med 35:1612-1619.

36. FLORIDA. STATE BOARD OF HEALTH

1961 A Symposium: Florida State Board of Health; Tampa BayArea Arbovirus Investigations (1959).

37. FRATTINI, J. F.1959 Fiebre hemorrágica epidémica; investigaciones del medio

interno. Día Med 31: 242-244.

47

38. FRIOERIO, M., N. R. NOTA, D. J. GREENWAY,A. S. PARODI, H. R. RUGIERO,N. E. METTLER, E. RIVERO, F. GARZON, L. B. DE GUERRERO, andM. C. BOXACA1959 Fiebre hemorrágica epidémica; investigaeiones bacterioló-

gicas. Día Med 31: 10.

39. GONZALES, S. M., and J. D. MEJSZENKIER1964 Variaciones hematológicas en los cobayos con fiebre hemorrá-

gica experimental (virus Junín). Rev Soc Argent Biol 40:392-399.

40. GREENWAY, D. J., H. R. RUGIERO, A. S. PARODI, M. FRIGERIO, E. RIVERO,J. M. DE LA BARRERA, F. GARZON, M. BOXACA, N. E. METTLER, L. B.DE GUERRERO, and N. NOTA1959 Epidemic Hemorrhagic Fever in Argentina. Public Health

Rep 74: 1011-1014.

41. GRISTEIN, S., and A. S. PARODI1964 Acción de la endotoxina E. coli sobre los cobayos infectados

con virus Junín. Actas del Congreso de Fisiología (Santiago).

42. GUERRERO, L. B. DE, M. C. BOXACA, and A. S. PARODI1965 Fiebre hemorrágica experimental en cobayos (virus Junín);

contagio y eliminación de virus. Rev Asoc Med Argent 79:271-274.

43. HARTLEY, J. W., and W. P. ROWE1960 A New Mouse Virus Apparently Related to the Adenovirus

Group. Virology 11: 645-647.

44. HENDERSON, J. R., and W. G. DOWNS1965 Junín and Tacaribe Plaque Production in Rhesus Monkey

Kidney Cell Monolayers. Amer J Trop Med 14: 796-799.

45. IMAGAWA, D. T., and J. M. ADAMS1958 Propagation of Measles Virus in Suckling Mice. Proc Soc

Exp Biol Med 98: 567-569.

46. JOHNSON, K. M., N. H. WIEBENGA, R. B. MACKENZIE, M. L. KUNS, N. M.TAURASO, A. SHELOKOV, P. A. WEBB, G. JUSTINES, and H. K. BEYE.1965 Virus Isolations from Human Cases of Hemorrhagic Fever

in Bolivia. Proc Soc Exp Biol Med 118: 113-118.

47. KLIN, S., M. DUNAYEVICH, J. R. PERIES, and I. PIROSKY1962 Multiplicación del virus causal de la virosis hemorrágica del

noroeste bonaerense (virus Junín) en el cerebro del ratónlactante. Rev Soc Argent Biol 38: 135-139.

48. KREMERS, M. Y.1965 Arthropod-Borne Viral Hemorrhagic Fevers, Part II. Inter-

national Pathology 6: 99-103.

49. LAJMANOVICH, S., C. E. COTO, A. S. PARODI, and S. GONZALEZ1963 Ultracentrifugation and Identification of the Nucleic Acid

of Junín Virus. Proceedings, Seventh International Con-gresses on Tropical Medicine and Malaria 3: 323-324.

50. MACKENZIE, R. B.1965 Prolonged Urine Excretion of Machupo Virus by Rodents;

Slow Virus Infections. In D. C. Gajdusek, et al. (eds.),National Institute of Neurological Diseases and BlindnessMonograph No. 2, Washington, D. C., U.S. Department ofHealth, Education, and Welfare, pp. 155-156. (Public HealthService Publication No. 1378)

48

51. MACKENZIE, R. B., P. A. WEBB, and K. M. JOHNSON1965 Detection of Complement-Fixing Antibody After Bolivian

Hemorrhagic Fever, Employing Machupo, Junín andTacaribe Virus Antigens. Amer J. Trop Med 14: 1079-1084.

52. MARGNI, R. A., J. C. FERRARIO, J. M. PRADO, and F. LOMBAN1958 El mal de O'Higgins; un estado tóxico predisponente. Día

Med 30: 2522.

53. MARTINEZ PINTOS, I. F.1960 Mal de los rastrojos. An Com Invest Cient 1: 1-13.

54. MARTINEZ PINTOS, I. F.1962 Epidemiología del "mal de los rastrojos." An Com Invest

Cient 3: 9-102.

55. MARTINEZ PINTOS, I. F.1963 Fiebre hemorrágica argentina. Proceedings, Seventh Inter-

national Congresses on Tropical Medicine and Malaria 3:297-298.

56. MARTINEZ PINTOS, I. F., H. C. GUARINOS, L. A. CZEPLOWODSKI, H. D.MOLTENI, C. O. PETRILLO, S. MELI, and F. JASCHEK1961 Nuestra experiencia en el tratamiento del "mral de los rastro-

jos." Semana Médica 118: 856-863.

57. MARTINEZ PINTOS, I. F., H. C. GUARINOS, F. R. J. JASCHEK, and H. D.MOLTENI

1963 Formas clínicas de la fiebre hemorrágica del noroeste de laProvincia de Buenos Aires. Proceedings, Seventh Interna-tional Congresses of Tropical Medicine and Malaria 3: 318-319.

58. MARTINEZ SEGOVIA, Z. M. DE, F. GRAZIOLI, M. E. G. DE GARRE, and J. P.BOZZINI

1966 Aplicación de la inmunofluorescencia al diagnóstico de lafiebre hemorrágica argentina. Ciencia e Investigación 22:316-319.

59. MARTINEZ SEGOVIA, Z. M. DE, and S. KLIN1961 Interferencia entre los agentes causales de la encefalitis equina

venezolana y virosis hemorrágicia del noroeste bonaerense.I. Ensayos en ratones blancos. Acta Kravsi Cuad Inst NacMicrobiol (B Aires) 3: 62, 67-68.

60. McQuIGGAN, M. C., W. J. OLIVER, E. R. LITTLER, and J. C. CERNEY1965 Hemolytic-Uremic Syndrome. JAMA 191 (10): 787-792.

61. METTLER, N. E.1959 Adaptación al huevo del agente etiológico de la fiebre hemo-

rrágica epidémica del noroeste de la Provincia de BuenosAires. Rev Soc Argent Biol 35: 295-299.

62. METTLER, N. E.1960 Adaptación del virus Junín al embrión de pollo (Fiebre

Hemorrágica Argentina) y estudio de sus propiedades bio-ó16gicas. Thesis, Facultad de Medicina, Universidad de Buenos

Aires, Argentina.

63. METTLER, N. E.1963 Discussion on Hemorrhagic Fevers. Proceedings, Seventh

International Congresses on Tropical Medicine and Malaria3: 309.

49

64. METTLER, N. E.1964 Estudio serológico de la epidemia de fiebre hemorrágica

argentina del año 1963. Thesis, Facultad de Medicina, Uni-versidad de Buenos Aires, Argentina.

65. METTLER, N. E.1966a Estudio realizado con los sueros de la epidemia de fiebre

hemorrágica argentina (1963) que no presentaron conversiónserológica para virus Junín. Medicina 26: 161i-i69.

66. METTLER, N. E.1966b Studies about Arbovirus in Man in Argentina. Distributed

at the Eleventh International Congress of Microbiology(Moscow).

67. METTLER, N. E., S. M. BUCKLEY, and J. CASALS1961 Propagation of Junín Virus, the Etiological Agent of Argen-

tinian Hemorrhagic Fever, in HeLa Cell Cultures. Proc SocExp Biol Med 107: 684-688.

68. METTLER, N. E., R. A. CACHIONE, E. CASCELLI, and E. S. MARTINEZ

1965 Estudio de relaciones antigénicas entre arbovirus y lepto-spiras. Rev Med Vet (B Aires) 47: 123-124.

69. METTLER, N. E., J. CASALS, and R. E. SHOPE

1963 Study of the Antigenic Relationships between Junín Virus,the Etiological Agent of Argentinian Hemorrhagic Fever, andOther Arthropod-borne Viruses Amer J Trop Med 12: 647-652.

70. METTLER, N. E., C. A. GIANANTONIO, and A. S. PARODI1963 Aislamiento del agente causal del síndrome urémico-hemo-

lítico Proceedings, Seventh International Congresses on Tropi-cal Medicine and Malaria 3: 326-327.

71. METTLER, N. E., L. G. MACNAMARA, and R. E. SHOPE1962 The Propagation of the Virus of Epizootic Hemorrhagic Dis-

ease of Deer in Newborn Mice and HeLa Cells J Exp Med116: 665-678.

72. METTLER, N. E., and A. S. PARODI1965 Estudio serológico de la epidemia de fiebre hemorrágica

argentina del año 1963 Prensa Med Argent 52: 1479-1482.73. METTLER, N. E., A. S. PARODI, and J. CASALS

1963 Survey for Antibodies Against Arthropod-Borne Viruses inMan in Argentina Amer J Trop Med 12: 653-656.

74. MOLTENI, H. D., H. C. GUARINOS, C. O. PETRILLO, and F. JASCHEK

1961 Estudio clínico estadístico sobre 338 pacientes afectados porla fiebre hemorrágica epidémica del noroeste de la Provinciade Buenos Aires Semana Médica 118: 838-855.

75. PALATNIK, M.1960 Inclusión citoplasmática en las células urinarias de los pa-

cientes afectados por la fiebre hemorrágica epidémica delnoroeste bonaerense Semana Médica 116: 1081-1084.

76. PALATNIK, M.1963 Inclusiones citoplasmáticas en las células urinarias de la fiebre

hemorrágica argentina. Proceedings, Seventh InternationalCongresses on Tropical Medicine and Malaria 3: 317.

77. PAN AMERICAN HEALTH ORGANIZATION

1965 Annual Report of the Director. Washington, D. C., p. 54.(Official Document No. 70)

50

78. PARODI, A. S.1963 Discussion on "Hemorrhagic Fevers in South America." Pro-

ceedings, Seventh International Congresses on Tropical Medi-cine and Malaria 3: 300-301.

79. PARODI, A. S., J. M. DE LA BARRERA, H. R. RUGIERO, D. J. GREENWAY,M. YERGA, N. METTLER, M. BOXACA, and M. J. FRIGERIO

1959 Los reservorios del virus de la fiebre hemorrágica epidémicade la Provincia de Buenos Aires Prensa Med Argent 46:554-556.

80. PARODI, A. S., and C. E. COTO1964 Inmunización de cobayos contra el virus Junín por inocula-

ción del virus Tacaribe Medicina (B Aires) 24: 151-153.81. PARODI, A. S., D. J. GREENWAY, H. R. RUGIERO, E. RIVERO, M. FRIGERIO,

J. M. DE LA BARRERA, N. METTLER, F. GARZON, M. BOXACA, L. DEGUERRERO, and N. NOTA1958 Sobre la etiología del brote epidémico de Junín. Día Med

30: 2300-2302.82. PARODI, A. S., D. J. GREENWAY, H. R. RUGIERO, E. RIVERO, M. J.

FRIGERIO, N. E. METTLER, F. GARZON, M. BOXACA, L. DE GUERRERO,and N. NOTA1959 La etiología de la fiebre hemorrágica epidémica de la Provin-

cia de Buenos Aires Día Med 31: 249-250.

83. PARODI, A. S., N. R. NOTA, L. B. DE GUERRERO, M. J. FRIGERIO, M.WIESSENBACHER, and E. REY1967 Inhibition of Immune Response in Experimental Hemorrhagic

Fever (Junín Virus) Acta Virol (Praha) 11: 120-125.84. PARODI, A. S., H. R. RUGIERO, D. J. GREENWAY, N. E. METTLER, and

M. BOXACA1961 El aislamineto del virus Junín en roedores de zonas no epi-

démicas. Prensa Med Argent 48: 2321-2322.85. PARODI, A. S., H. R. RUGIERO, D. J. GREENWAY, N. METTLER, A. MARTI-

NEZ, M. BOXACA, and J. M. DE LA BARRERA1959 Aislamineto del virus Junín (F.H.E.) de los ácaros de la

zona epidémica [Echinolaelaps echidninus (Berlese)]. PrensaMed Argent 46: 2242-2244.

86. PEREIRA TORRES, R., V. A. J. ALBERTI, A. ROMORINI, and R. O. PEREZ1965 Modificaciones electrobalistocardiográficas en la virosis

hemorrágica argentina Rev Asoc Med Argent 79: 492-495.87. PINHEIRO, F. P., R. E. SHOPE, A. H. P. DE ANDRADE, G. DE BENSABATH,

G. V. CACIOS, and J. CASALS1966 Amaparí; a New Virus of the Tacaribe Group from Rodents,

and Mites of Amapá Territory, Brazil. Proc Soc Exp BiolMed 122: 531.

88. PIROSKY, I., A. TEYSSIE, B. AYERRA DE HOLSTEIN, A. RODRIGUEZ, andM. D'EMPAIRE1964 Virosis hemorrágica del noroeste bonaerense. VI. Visuali-

zación del antigeno viral específico mediante la técnica delos anticuerpos fluorescentes. Acta Kravsi Cuad Inst NacMicrobiol (B Aires) 3: 64-65.

89. PIROSKY, I., J. ZUCCARINI, E. A. MOLINELLI, and A. DI PIETRO

1959 Virosis hemorrágica del noroeste bonaerense; endemo-epi-démica, febril, enantemática y leucopénica. II. Recuperacióndel virus causal a partir de ácaros (Mesostigmata) capturados

51

(1958) en la zona epidémica. Orientación Médica 8 (340):156.

90. PIRosKY, I., J. ZUCCARINI, E. A. MOLINELLI, A. DI PIETRO, J. G. BARRERAORO, and P. MARTINI1959 Virosis hemorrágica del noroeste bonaerense; endemo-epi-

démica, febril, enantemática y leucopénica. III. Reseña delas principales observaciones e investigaciones realizadas porla Comisión Nacional ad hoc designada para estudiar in situel brote epidémico de 1958. Orientación Médica 8(341):171-180 and 8(346): 303-311.

91. PIROSKY, I., J. ZUCCARINI, E. A. MOLINELLI, A. DI PIETRO, J. G. BARRERAORO, P. MARTINI, and A. R. COPELLO1959 Virosis hemorrágica del noroeste bonaerense; endemo-epi-

démica, febril, enantemática y leucopénica. Buenos Aires,Instituto Nacional de Microbiología, Ministerio de AsistenciaSocial y Salud Pública. 197 p.

92. PIROSKY, I., J. ZUCCARINI, E. A. MOLINELLI, A. DI PIETRO, J. G. BARRERAORO, P. MARTINI, L. MARTOS, and M. D'EMPAIRE1959 Virosis hemorrágica del noroeste bonaerense; endemo-epi-

démica, febril, enantemática y leucopénica. IV. Investi-gaciones virológicas de orden epidemiológica (primera nota).Orientación Médica 8(361): 708-710.

93. PIROSKY, I., J. ZUCCARINI, E. A. MOLINELLI, A. DI PIETRO, P. MARTINI,B. FERREYRA, L. F. GUTMAN FRUGONE, and T. VAZOQUEZ1959 Virosis hemorrágica del noroeste bonaerense; endemo-epi-

démica, febril, enantemática y leucopénica. I. La primerainoculación experimental al hombre. Orientación Médica8(339): 144-148.

94. POLAK, M., and R. JUPE1961 Anatomía patológica del "mal de los rastrojos." Semana

Médica 118: 864-869.

95. RAPP, F., and S. M. BUCKLEY1962 Studies with the Etiologic Agent of Argentinian Epidemic

Hemorrhagic Fever (Junín Virus). Amer J Path 40: 63-75.

96. RHIM JOHNG, S., S. BUNSITU, and N. H. WIEBENGA1967 Growth of Junín Virus; the Etiological Agent of Argentinian

Hemorrhagic Fever in Cell Cultures Arch Ges Virusforsch21: 243-252.

97. RIVERO, E., D. J. GREENWAY, H. R. RUGIERO, A. S. PARODI, M. FRIGERIO,N. METTLER, M. BOXACA, N. NOTA, L. DE GUERRERO, and F. GARZON1959 Fiebre hemorrágica epidémica; anatomía patológica. Dia

Med 31: 246.

98. ROVERE, R. J., and J. I. ORMAECHEA1963 Estudio anatomohistopatológico de la encefalopatía de la

fiebre epidémica del noroeste de la Provincia de Buenos Aires(R.A.) A. Estudio anatomohistopatológico general. B. Loscuerpos amiláceos de Lafora. C. Los corpúsculos intranu-cleares de la neurona. Semana Médica 123: 1117-1122.

99. ROVERE, R. J., Y. RUTZ, and J. I. ORMAECHEA1963 Estudio anatomohistopatológico de la encefalopatía de la

fiebre epidémica del noroeste de la Provincia de Buenos Aires.Semana Médica 123: 1003-1010.

52

100. RUGOGIERO, H. A., H. R. RUGIERO, F. A. CINTORA, C. MAONoNI, L.ASTARLOA, C. E. GONZALEZ CAMBACERES, F. MAGLIO, G. SQUASSI, L.LEVERONE, H. M. GHIGLIAZZA, and E. BELLEGARDE

1967 Estudio tromboelastográfico. Rev Asoc Med Argent 81: 71-77.101. RUGGIERO, H., H. R. RUGIERO, F. A. CINTORA, C. MAGNONI, F. MAGLIO,

C. GONZALEZ CAMBACERES, L. ASTARLOA, and G. SQUASSI1964 Fiebre hemorrágica argentina. III. Aparato cardiovascular.

Rev Asoc Med Argent 78: 360-371.102. RUGIERO, H. R., E. ASTARLOA, H. RUGGIERO, L. N. ASTARLOA, H. Gmo-

LIAZZA, C. GONZALEZ CAMBACERES, F. MAGLIO, and G. SQUASSI

1965 Fiebre hemorrágica argentina; inoculación accidental y con-sideraciones médico-legales. Rev Asoc Med Argent 79: 536-538.

103. RUGIERO, H. R., E. B. CASTIGLIONE, F. MAGLIO, and F. M. C. CAMBA-CERES

1962 Fiebre hemorrágica epidémica; un caso de interés clínico-epidemiológico. Rev Asoc Med Argent 76: 145-152.

104. RUGIERO, H. R., F. A. CINTORA, E. LIBONATTI, C. MAGNONI, E. CASTIO-LIONE, and R. LOCICERO1960 Formas nerviosas de la fiebre hemorrágica epidémica. Prensa

Med Argent 47: 1845-1849.

105. RUGIERO, H. R., F. A. CINTORA, C. MAGNONI, R. LOCICERO, and H.MILANI

1950 Fiebre hemorrágica epidémica; diagnóstico precoz. PrensaMed Argent 46: 980-984.

106. RUGIERO, H. R., F. A. CINTORA, C. MAGNONI, H. RUGGIERO, C. GONZALEZCAMBACERES, F. MAGLIO, L. ASTARLOA, G. SQUASSI, A. GIACOSA, andD. FERNANDEZ1964 Fiebre hemorrágica argentina. IV. Formas clínicas. Rev

Asoc Med Argent 78: 500-510.

107. RUGIERO, H. R., A. GIACOSA, D. FERNANDEZ, H. A. RUGGIERO, F. MAOLiO,C. GONZALEZ, L. ASTARLOA, G. SQUASSI, F. CINTORA, and C. MAGNONI1965 Fiebre hemorrágica argentina. VI. Alteraciones de las pro-

teínas séricas. Rev Asoc Med Argent 79: 153-157.

108. RUGIERO, H. R., D. J. GREENWAY, A. S. PARODI, F. R. LOMBAN, M.FRIGERIO, A. C. PERCERUTTI, and M. BOXACA1959 Inoculación voluntaria del virus de la fiebre hemorrágica

epidémica; estudio clínico y etiológico. Día Med 31: 218.

109. RUGIERa, H. R., D. J. GREENWAY, A. S. PARODI, E. RIVERO, M. FIGERIO,N. E. METTLER, M. BOXACA, N. NOTA, L. DE GUERRERO, and F.GARZON1959 Fiebre hemorrágica epidémica; síntesis clínico-evolutiva. Rev

Asoc Med Argent 31: 236.

110. RUGIERO, H. R., H. MILANI, A. GIACOSA, D. FERNANDEZ, F. A. CINTORA,C. MAGNONI, H. RUGGIERO, L. ASTARLOA, F. MAGLIO, C. GONZALEZCAMBACERES, and G. SQUASSI1964 Fiebre hemorrágica argentina. V. Laboratorio clínico, im-

portancia diagnóstica y correlación clínica. Rev Asoc MedArgent 78: 611-618.

111. RUGIERO, H. R., A. S. PARODI, H. GOTTA, M. BOXACA, A. J. OLIVARI, andE. GONZALEZ1962 Fiebre hemorrágica epidémica. Infección de laboratorio y

pasaje interhumano. Rev Asoc Med Argent 76: 413417.

53

112. RUGIERO, H. R., A. S. PARODI, D. J. GREENWAY, J. M. DE LA BARRERA,M. YERGA, N. METTLER, and M. BOXACA1959 Consideraciones sobre el hallazgo del virus de fiebre hemorrá-

gica epidémica en roedores de zonas epidémicas y no epi-démicas de la Provincia de Buenos Aires. Pensa Med Argent46: 2009-2014.

113. RUGIERO, H. R., A. S. PARODI, H. G. RUGGIERO, N. METTLER, M. BOXACA,A. L. DE GUERRERO, A. CINTORA, C. MAGNONI, H. MILANI, F. MAGLIO,C. GONZALEZ CAMBACERES, L. ASTARLOA, G. SQUASSI, D. FERNANDEZ,and A. GIACOSA1964 Fiebre hemorrágica argentina. I. Período de incubación e

invasión. Rev Asoc Med Argent 78: 221-226.114. RUGIERO, H. R., H. RUGGIERO, C. GONZALEZ CAMBACERES, F. A. CIN-

TORA, F. MAGLIO, C. MAGNONI, I. ASTARLOA, G. SQuAssi, A. GIACOSA,and D. FERNANDEZ1964 Fiebre hemorrágica argentina. II. Estudio clínico descriptivo.

Rev Asoc Med Argent 78: 281-294.115. SABATTINI, M. S., R. E. SHOPE, and J. M. VANELLA

1965 Serological survey in Córdoba Province, Argentina. Amer JTrop Med 14: 1073-1078.

116. SARTORELLI, A. C., D. S. FISHER, and W. G. DOWNS1965 Use of Sarcoma 180/TG to Prepare Hyperimmune Ascitic

Fluid in the Mouse. J Immun 96: 676.117. SHELOKOV, A.

1964 Hemorrhagic Fever in the Americas; a Perspective. Amer JTrop Med 14: 790-792.

118. SMORODINTSEV, A. A., L. I. KAZBINTSER, and V. G. CHUDAKOV1964 Virus Hemorrhagic Fevers. Published for the National

Library of Medicine, U.S. Public Health Service, through theNational Science Foundation, Washington, D. C., by theIsrael Program for Scientific Translations.

119. SORIA, M. R., andN. A. PossI DE CAPRI1959 Trayectoria de la peste en la Argentina. Actas Prim Jorn

Entomoepidem Argent (B Aires).120. TARAUSO, N., and A. SHELOKOV

1965 Protection Against Junín Virus by Immunization with LiveTacaribe Virus. Proc Soc Exp Biol Med 119: 608-611.

121. TEYSSIE, A., B. A. HOLSTEIN, and M. D'EMPAIRE1962 Studies on the Action of "Virosis Hemorrágica del N.O.

Bonaerense" on Ehrlich Ascitic Tumor. International CancerCongress (Moscow), p. 545.

122. TRAPIDO, H., and C. SAN MARTIN1967 Pichinde Virus; a Preliminary Report on a New Virus from

Colombia Related to the Tacaribe Group. Presented at theSixteenth Annual Meeting of the American Society of Trop-ical Medicine and Hygiene.

123. VANELLA, J. M.1964 Epidemiología de la fiebre hemorrágica. Semana Médica

125: 1502.124. VANELLA, J. M., L. E. GONZALEZ, S. PAGLINI, and A. MARQUEZ

1964 Evidencia de laboratorio de actividad del virus de Junín enel sudeste de Córdoba; hipótesis sobre su epidemiología. DíaMed 36: 290-291.

54

125. VILCHES, A., J. BARRERA ORO, and L. GUTMAN FRUGONE1965 Una nueva área epidémica de fiebre hemorrágica argentina

iniciada a continuación de una epizootia en población aumen-tada de roedores silvestres. Seg Jorn EntomoepidemiolArgent 2: 9.

126. VUCETICH, M., I. F. MARTINEZ PINTOS, M. PALATNIK, G. D. ANDERSON,A. T. KIENZELMAN, H. C. GUARINOS, C. PETRILLO, F. R. J.JASCHEK, H. D. MOLTENI, and C. DuPUY

1963 Alteraciones hematológicas en la fiebre hemorrágica argen-tina. Proceedings, Seventh International Congresses on Tropi-cal Medicine and Malaria 3: 317-318.

127. VUCETICH, M., M. PALATNIK, G. ANDERSON, H. GUARINOS, and C.PETRILLO

1960 Contribución al estudio de la hemostasia en el mal de losrastrojos. An Comn Invest Cient 27p.

128. WIEBENGA, N. H.1965 Immunologic Studies of Tacaribe, Junín, and Machupo

Viruses. Amer J Trop Med 14: 802-808.129. WIEBENGA, N. H., A. SHELOKOV, C. J. GIBBS, JR., and R. B. MACKENZIE

1964 Epidemic Hemmorrhagic Fever in Bolivia. II. Demonstra-tion of Complement-Fixing Antibody in Patients' Sera withJunín Virus Antigen. Amer J Trop Med 13: 626-628.

130. WORLD HEALTH ORGANIZATION1967 Arboviruses and Human Disease. Geneva. (Technical Re-

port Series, No. 369).131. YANOVSKY, J. F.

1965 Fiebre hemorrágica experimental; histaminemia en cobayosinoculados con el virus Junín. Rev Soc Argent Biol 41: 162-165.

132. ZAHARZEVSKI, J. L., and S. PARODI1966 Microscopia electrónica de la acción citopatológica del virus

Junín en el ganglio linfático del cobayo. Medicina (B Aires)24: 210.

55