Calomys callidus as a potential junin virus reservoir

Journal of Medical Virology 273238-243 ( 1989)

Calomys Callidus as a Potential Junin Virus Reservoir Cristina Videla, Adriana Kajon, Guadalupe Carballal, and Mercedes Weissenbacher Departarncvito de Microhiologia, Facultad de Medicina, Universidad cte Buenos Aires, Buenos Aires, Argentina The present study investigated whether C. callidus, a species belonging t o the Calomys genus, is capable of developing experimentally a persistent Junin virus (JV) infection. Newborn and adult cricetids were inoculated with the attenuated XJ-Clone 3 strain of JV by intracerebral or mucosal route. The present results indicate that the species is susceptible to J V infection, capable of shedding virus chronically through saliva and developing a persistent infection as shown by the detection of virus in brain tissue at 60 days post infection. These findings, and the fact that this cricetid shares its distribution areas with Calomys musculinus and Akodon azarae, support C. callidus as a potential JV reservoir.

KEY WORDS: Junin virus, natural reservoirs, Calomyscallidus, Argentine haemorrhagic fever

INTRODUCTION Argentine haemorrhagic fever ( AHF) is a n endemoepidemic disease whose etiological agent is Junin virus ( J V ) , which belongs to the Arenaviridae family. The endemic area is an agricultural and cattle-raising region in the provinces of Buenos Aires, Cordoba, Santa Fe, and La Pampa, having spread progressively to reach some 120,000 km2 a t present IMaiztegui e t al., 19861. The incidence of the disease is higher during the autumn months, the time when the density of rodent carriers of J V is greatest. The naturally infected species captured in the endemic area include Calomys musculinus, C. laucha, Akodon azarae, and Mus musculus [Sabattini et al., 1977, Sabatini and Contigiani, 19821. It appears that J V is not restricted to the area known to be endemic, as it has also been detected in rodents captured elsewhere. Thus, J V was isolated from a n A.azarae in Pila, Buenos Aires province, some 280 km from the epidemic focus ( J u n i n ) ,concurrently with the detection of antibodies in two local inhabitants [Weissenbacher e t al., 19851. Studies on experimental inoculation of the main known reservoirs, C. musculinus and C. laucha, with wild J V strains indicate that both hosts are able to c

1989 ALAN R. LISS, INC.

develop persistent infections with virus shedding through secretions ISabattini and Contigiani, 19821. The purpose of the present study was to determine if another Calomys species, C. callidus, was capable o f developing experimentally a persistent J V infection. Recently described a s a full species, the geographic distribution of this cricetid is not precisely known, but it has been captured both in the north and northeast of the country (Chaco and Del Palmar National Parks), a s have specimens of Calomys musculinus and Akodori azarae IVitullo et al., 19841 (Fig. 1). The results obtained suggest that C . callidus, at least theoretically, may participate in the natural cycle of J V transmission and point to the need to extend investigations to field studies in order to determine if this cricetid is infected naturally.

MATERIALS AND METHODS Virus XJ-Clone 3 J V strain was used, attenuated for humans, primates, and guinea pigs, with five passages in suckling mouse brain, with a titre of lo7 LD5{,/ml. Animals For viral titrations, 48- to 72-hour-old Rockland mice were used. C. callidus specimens were provided by the bioterium at the National Atomic Energy Commission, Argentina, where the colony had been started with pairs captured in the Del Palmar National Park (Colon, Entre Rios province) [Hodara et al., 19841. Viral Isolation Ten percent organ homogenates were prepared in Eagle medium plus 5% inactivated calf serum (ICS)

Accepted for publication September 22, 1988. Address reprint requests to Guadalupe Carballal, MD, Departamento de Microbiologia. Paraguay 2155 Piso 11. (1121I Buenos Aires, Argentina. Drs. Videla and Kajon a r e Fellows of t h e Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET,; Drs. Carballal and Weissenbacher a r e members of the Research ( h r c e r (CONICET).

J u n i n Virus i n Calomys Callidus

239

tralizing antibodies were determined by t h e constant serum-variable virus o r by variable serum-constant vir u s methods with XJ-Clone 3 as t h e challenge virus ILehmman-Grube et al., 19791.

Experimental Design

Fig. I . Endemic area IMaiztegui e t al.. 19861. F\U Geographical distribution [Cahrera. 1961 I. Capture zone of(’ cnllitftr.s IVitullo et al.. 19x41. Insert: Argentina in South America.

a n d 1% antibiotics a n d centrifuged 40 minutes at 10,000 g; t h e s u p e r n a t a n t s were inoculated in suckling mice by intracerebral ( I C ) route o r in Vero cell monolayers. Faucial a n d ocular samples, collected with swabs moistened in Eagle medium plus 5% ICS a n d 2% antibiotics, were placed in 0.5 ml of t h e s a m e medium a n d then inoculated IC in suckling mice. For persistence studies, brain coculture was performed on Vero cells [Boxaca et al., 19841, with three weekly subcultures, followed by IC inoculation of supernatants in suckling mice. Viral isolation in suckling mice was considered positive when t h e mice died between 10 a n d 2 1 days post infection (PI)with typical neurological signs I Weissenbacher et al., 19871. T h e development of characteristic J V C P E in Vero cells was observed at 11 days PI. TCID,,, o r LD,,, were calculated by t h e Reed-Muench method [Reed a n d Muench, 19381.

Experimental infection with J V w a s performed in newborn a n d a d u l t C. callidus by IC a n d intranasal routes, whereas females in contact with their infected litters also were studied. Experimental intracerebral infection. Inoculation was carried o u t in 28 newborn C . callidus (48-72 hours old) a n d in 14 adults (60-90 d a y s old) with 10’ LDSo XJ-Clone 3. Illness a n d mortality were recorded daily. Survivors were killed at 2 months PI. Brain a n d blood samples for viral isolation a n d s e r u m for antibody determination were obtained. T h r e e a d u l t c. callidus t h a t became ill were killed at 13, 14, a n d 17 d a y s PI a n d fauces a n d brain were searched for virus. Six noninfected newborn a n d one a d u l t C. callidus were used as controls. Experimental infection by mucosal route. Intranasal inoculation was performed in 2 8 C. callidits 48-72 hours old a n d in 7 a d u l t s with 10:’ LDso XJClone 3. Illness a n d mortality were recorded daily, a n d body weight in newborns was obtained once a week. Overtly ill animals ( t w o adults a n d five newborn) were killed a n d samples stored at -70°C for brain viral isolation. In newborn C. callidus, fauces were swabbed at 10, 20, 30, 45, a n d 60 d a y s PI; in t h r e e animals, ocular swabbing was performed at 20 days PI. Survivors were killed at 2 months PI to harvest brain a n d salivary gland samples for direct viral isolation in suckling mouse, a n d serum for determination of neutralizing and/or IFA, as well as brain impression s m e a r s for a n tigen detection. Brain samples from seven newborn a n d t h r e e a d u l t animals killed at 2 months PI were also tested by coculture. Five noninoculated newborns a n d two adults were employed as controls. Study o f females in contact with infected litters. Four mothers in contact with their litters inoculated with XJ-Clone 3 strain were studied to determine whether or not infection had been acquired. T h r e e females were killed at 7 months post contact a n d one at 2 months. Brain samples for viral isolation, s e r u m for antibody determination, a n d brain impression s m e a r s for antigen detection were obtained.

Immunofluorescent (IF) Studies Brain impression smears were obtained at t h e time of killing a n d fixed in cold acetone for 10 minutes. To detect viral antigens, s m e a r s were treated with a n a n RESULTS ti-JV rat hyperimmune serum, followed by fluoresceinInoculation by Intracerebral Route labelled goat antimouse immunoglobulins (GIBCO). Nonimmune rat serum was used as control. Readings In newborn animals, 35% (10/28) were overtly ill were performed with Carl Zeiss epimicroscope with a n from 11-14 days PI, exhibiting ataxia, ocular occlusion, gait lateralization, a n d stunted growth as comHBO,,, mercury lamp. pared with controls. Serology Rodents t h a t became ill failed to recover a n d e i t h e r S e r u m immunofluorescent antibodies ( I F A )were de- died or were killed at 2 months PI. Mortality w a s 17% termined by indirect I F employing BHK/21 cells per- from 11-21 days PI. Virus isolation studies were not sistently infected with J V [Carballal et al., 19801. Neu- performed during t h e acute stage, b u t brain virus w a s

Videla et at.

240

TABLE I. Experimental Infection of Calomys Callidus With IO'LDr,,~of J u n i n Virus tXJ-Clone 3 ) by Intracerebral and Intranasal Route Animals tn)

Intracerebral Newborn (28) Adults

Morbidity tdays PI)

%'

Mortality (days PI)

% '

Brain virus isolation Acute stage 2 months PI

35 t 1 1-60)

17 ( 11-21)

ND"

119'

+

.3"

50 (12-55)

50 (14-55)

313

Oil 1

+

z3.7"

71 (15-30)

69 (15-21)

415

718

+ ',1180+'

212'

1I5

+

(14)

Intranasal Newborn

(28) Adults

Serology"

28 (19-22)

(7) "Neutralizing antibodies at 2 months P I . "ND. not done. 'Number positivehumber studied animals.

detected in one of nine a n i m a l s studied, at 2 months PI, by tissue culture inoculation. Neutralizing antibodies were detected in pooled sera, with indices greater t h a n 2. Seven a d u l t rodents (Table I ) presented signs of disease resembling those observed in newborns, from 2 weeks PI, including ocular occlusion a n d gait lateralization. Three animals were killed when ill at 13, 14, a n d 17 days PI, a n d four died from 14 to 55 days PI. Virus w a s isolated from fauces in two animals a n d from t h e brain in t h e t h r e e rodents killed d u r i n g t h e acute stage of infection. T h e seven survivors were killed at 2 months PI. No virus w a s isolated from brain o r from blood by tissue culture, a n d neutralizing antibodies were detected with indices ranging from 2 to 4.

.y -

"Neutralization index. "Neutralizing antibodies titre. 'Virus was also isolated from fauces.

Fig. 2. C . callrdus inoculated a t birth by mucosal route with XJClone 3 a t 16 days PI. Ruffled ha ir a nd ocular occlusion are evident.

Inoculation by Mucosal Route Twenty newborns showed signs of disease from 2 weeks PI, including ataxia, total o r partial ocular occlusion, ruffled h a i r (Fig. 21, a n d gait lateralization (Fig. 3 ) . Growth s t u n t i n g was shown by a lower vs. normal mean body weight, which became significant at 20 days PI. (Fig. 4). T h e eight survivors h a d recovered their weight to normal values at 2 months PI, when they were killed. Mortality for newborns was 69%,a n d t h e mean day of death was 22.44 2 11.41 ( m e a n 5 SD). Virus was isolated from pharyngeal swabs at 10, 20, 30, a n d 45 d a y s PI, a n d t h e percentage of isolation decreased with time. Besides, virus was also isolated from ocular swabs from two of t h e three anim.als studied t h a t presented ocular occlusion; in four of five rodents, virus was detected in t h e brain at 10-20 d a y s PI Fig. 3. C. callrdus inoculated a t birth by mucosal route with XJ(Table 11). Clone 3 a t 20 days PI. Lateralization d u e to hind-limb paresia is All eight survivors presenting no overt signs of dis- observed, ease were killed at 2 months PI. All six brain cocult u r e s proved positive. Virus was detected in five of seven brains a n d in five of seven salivary glands by t h e salivary gland in all eight survivors. Immunofluodirect isolation in suckling mice. Viral antigen was rescence andlor neutralizing antibodies were detected detected in four brain impression s m e a r s by I F (Table in all sera studied. 111).Therefore, J V was rescued from either t h e brain o r The virus isolated from t h e brains during t h e acute

Junin Virus in Calomys Callidus

24 1

sented ocular occlusion at 2 months PI and gait lateralization at 7 months, whereas female #4 exhibited lateralization at 2 months, when i t was killed. Virus was rescued from the brains of both females by suckling mouse inoculation. Furthermore, in one animal, viral antigen was detected in brain impression smears by immunofluorescence. In the two remaining females, no overt signs of disease were observed, and brain viral isolation proved negative. All four females developed humoral immune response as shown by the presence of IFA (Fig. 5).

DISCUSSION Experimental inoculation of newborn and adult Calomys callidus with attenuated XJ-Clone 3 strain of J V demonstrated that this species is susceptible to infection and furthermore is capable of developing longlasting infection with virus shedding through saliva. Fig. 4. Newborn C callrdus inoculated by mucosal route with XJExcept for Tacaribe virus, all of the arenaviruses Clone 3. Infected animal with stunting growth and normal with the known to date persist in nature in rodents belonging to same age a r e observed. the Muridae and Cricetidae families Ireviewed by Howard, 19861. In the case of JV, the virus has been isolated in naTABLE 11. Experimental Infection of Newborn Calomys (,'allidus With Junin Virus (XJ-Clone:,)by Intranasal Route ture mainly from two cricetid species, Calom-ysspp. and Akodon spp. [Sabattini e t al., 1977; Sabattini and ConViral isolation" from: tigiani, 1982 [. When experimentally inoculated with Davs PI Pharvngeal swabs Ocular swabs Brain wild J V strain, Calomys musculinus and C. laucha de10 8110" ND 212 velop persistent asymptomatic infections with virus 20 911 1 213 213 shedding through secretions [Sabattini et al., 1977; Sa:%0-45 319 ND ND battini and Contigiani, 19821. On the other hand, the 60 013 ND 718" XJ-Clone 3 strain, attenuated for the guinea pig, 'In suckling mouse. proved pathogenic for natural J V reservoirs such as C . "Number posit i v e h u m ber tested. ND. not done. musculinus ILampuri e t al., 19821 and Akodon azarae "l'erformed by coculture and,or in suckling mouse (see details in Ta[Videla e t al., 19861 as well as for other cricetids, inble 1111. cluding A . molinae [Carballal e t al., 19861 and A . dolores (unpublished data). Infection of C. musculinus a t up to 10 days of age stage and at 2 months PI was shown to be JV, as it was with XJ-Clone 3 led to severe neurological disease, neutralized by anti-JV rat serum in tissue culture. with mortality exceeding 50%. Persistent infection in Among inoculated adult animals, two of seven pre- the brain was detected, accompanied by meningoensented ocular occlusion and gait lateralization a t 12 cephalitis in the presence of neutralizing antibodies. days PI and were killed at 19-22 days PI. Virus was Adults were susceptible to infection by mucosal route, isolated from fauces in one and from brains in both with mortality reaching 50% [Laguens e t al., 1982; animals by suckling mouse inoculation. Lampuri e t al., personal communication 1. In the five survivors, viral isolation from brains was Newborn Akodon molinae infected with XJ-Clone 3 attempted by both coculture and suckling mouse inoc- presented 60% mortality, and survivors developed a ulation. J V was recovered from one of the three cocul- long-lasting infection, whereas adults proved scarcely tured brains at 60 days PI. Neutralizing antibodies susceptible to infection with this strain [Carballal e t were detected in pooled sera with a n index of 3 (Table al., 19861. I ). Results achieved in this study with C. callidus reVirus was not detected in fauces or brains in nonin- semble the findings for the main reservoir C. musculifected animals at 15, 20, and 60 days PI. nus and in A . molinae inoculated with XJ-Clone 3, as newborn C . callidus proved susceptible to infection by Study on Females in Contact With Their both the IC and mucosal route, presenting 20 and 70% Infected Litters mortality, respectively. Likewise, signs of disease obFour females t h a t remained in contact with their served were similar to those described for C . musculiXJ-Clone-3-infected litters were studied: three had lit- nus as regards neurological findings and lower body ters inoculated by the mucosal route ( # l , #2, & #3) weight ILampuri e t al., 1982; Vitullo e t al., 19881. and one by IC route (#4)(Table IV). Female #1 pre- Moreover, C:3H/ST mice persistently infected with G

Videla et al.

242

TABLE 111. Newborn Caloniys Callidus Inoculated With 10.’ LD,,, of J u n i n Virus t XJ-Clone.,) by Intranasal Route: Study of Eight Survivors a t 2 Months PI. Viral isolation“ ( logll, LD51,/rnl) Brain Salivary gland 2.7 2.7 2.7 ND 2.7 2.3 ND 2.3 3.5 2.3 2.7 2.3

Animal no. 1

2 3 4 5 6 7 8 Controls 1 noninfected

Antigen in brain (IF)

+ N D‘ + + ND +

Serology 1120’’

NL)

’

1/so”

ND 1/80’’

iv‘

+

1/5“ .lWl

ND

1

.‘In suckling mouse. ”Neutra I i xi ng a n t i hod ies ‘ND. not done. “IF antibodies.

TABLE I\’. Female Calorn.vs Callidus Naturally Infected in Contact With Their J u n i n Virus Inoculated Litters” Animal no. 1 2 3

4

Mortality Yes No No Yes

Killed (months PI) 7

Brain virus“ (Log,,lLD,,,/rnl~ + . 2.7

Brain virus antigen t

7

7 2

IFA” t t t

+

’

2.7

ND

t

’Animals no. 1 , 2 , and :I had 2 month?;’ contact with intracerebrallv J V inoculated litters. No 4 had 2 months’ contact with intracerebr;illy JV inoculated litter. ,'isolation in suckling mouse. ”In serum diluted 1 5

LCM presented s t u n t i n g of growth associated with a growth hormone defect. Immunochemical a n d electron microscope studies demonstrated t h a t virus replication was restricted primariiy to t h e cells secreting growthhormone [Oldstone e t al., 19821. Ocular occlusion, together with viral isolation from ocular swabs, h a s also been described for t h e s a m e J V s t r a i n in guinea pigs after intranasal inoculation ISamoilovich et al., 19831. Similar findings were observed for congenital reovirus infections in mice /Hashimi et al., 19661. Newborn C . callidus inoculated by t h e mucosal route developed a long-lasting infection shown by viral isolation from t h e brain a n d salivary gland at u p to 2 months PI in t h e presence of circulating antibodies. Although virus could not be detected in swabs at 2 months, t h e fact t h a t viral infectivity was found in salivary gland points to likely virus shedding through s a liva. T h e salivary glands have been shown to be heavily infected in C. musculinus naturally infected with wild J V s t r a i n IMartinez Peralta et al., 19781. Adult C. callidus is also susceptible to infection, presenting signs of disease resembling those observed in newborn animals. Likewise, virus was isolated from t h e brains a n d fauces d u r i n g t h e acute stage, a n d survivors developed persistent infection, as virus could be isolated in one animal inoculated by t h e mucosal route at 2 months PI a n d in two females naturally infected by

Fig. 5. Indirect immunofluorescence in :I BHKQl cell llne persistently infected with J u n i n virus. Se rum from (’. ccillrtfus infected with jun& virus at 2 month postinfection. The resrtlon was revealed with a fluoresccin labelled antimouse total IFA antiserum

Junin Virus in Calornys Callidus

243

contact with their inoculated offspring at 2 a n d 7 months 'I' This latter finding demonstrates transmission, as litters inoculated by both t h e I c a n d mucosal routes infected their progenitors, most likely through saliva. Similar results have been reported for t h e main reservoir C. musculinus, infected either with t h e XJ-Clone 3 s t r a i n ILampuri et al., 19851 o r with wild J V s t r a i n ISabattini et al., 19771. Because o u r study ended at 2 months PI, it is unknown if virus persists longer in t h e Organism o r if t h e immune response observed leads to viral clearance. Although o u r findings point to C.callidus as a potential J V reservoir, factors such as geographical distribution, behaviour, and ecology of this species should also be borne in mind. C. callidus was originally described by Thomas as a subspecies ofC, ue,lustus [ Cabrera, 1961I, Chromosomic differences sufficient to reproductive isolation between t h e two species h a s been recently shown, so t h a t C, callidus h a s been proposed as a full species IVitullo et al., 19841. As to its geographical distribution, in 1961, Cabrera located t h e habitat of C, cal1idu.s in t h e Argentinian Mesopotamia E~~~~Rios and corrientes provinces) a n d in S a n t a Fe province. However, recently, Gal[idus also h a s been captured 600 k m northwest from the Mesopotamia, i n Chaco province IVitullo et al,, 19841, showing t h a t this species might be more widespread t h a n previously reported. U p to now, t h i s speties has not been found within the AHF endemic area; however, owing to t h e scanty ecological studies, its presence cannot be ruled out in this region. As a m a t t e r Of the area Of Santa Fe province makes up p a r t of t h e known endemic a r e a for A H F IMaiztegui e t al., 19861. summing up the results, callidus is capable of developing persistent infection a n d spreading virus through saliva, t h u s allowing horizontal transmission. i n theory* it may participate i n the chain of J V transmission. T h e present findings m u s t be confirmed by field work to determine if t h i s cricetid is naturally infected in t h e wild state.

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ACKNOWLEDGMENTS The authors thank D ~ .Susana ~ ~ Carlos~ Quintas, a n d Vida Hodara for providing t h e experimental animals. This work was supported by grants from CONICET, Fundacion Emilio Ocampo, a n d t h e University of Buenos Aires. REFERENCES 13oxaca MC'. Gomez MM. Berria MI, Iacono KF I 19841: Transplacental infection in guinea pigs inoculated with a n a t t e n u a t e d s t r a i n of J u n i n virus. Intervirology 21:178-l80. Cabrera A I 1961 I: Catdlngo d e 10s mamiferns d e America del Sud.

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