INFECTION AND IMMUNrrY, Dec. 1974, p. 1362-1369Copyright 0 1974 American Society for Microbiology

Vol. 10, No. 6Printed in U.SA.

Modoc Viral Infections in the Deer Mouse Peromyscusmaniculatus

JAMES W. DAVIS,I JAMES L. HARDY, AND WILLIAM C. REEVESSchool of Public Health, University of California, Berkeley, California 94720

Received for publication 24 June 1974

The pathogenesis of Modoc virus and its mechanism of transmission wereinvestigated in Peromyscus maniculatus gambeli (deer mouse) as a model tounderstand the natural history of this virus. Animals were readily infected by theintranasal or subcutaneous route of inoculation. Virus could be detected by directisolation techniques in many organs and body fluids during the first 7 to 9 daysafter intranasal inoculation. Increases in viral titers were detectable first in lungsand then the spleen, salivary-submaxillary glands, and lymph nodes. Viremiaswere low titered and ephemeral. Virus was recovered from urine and throat swabs4 to 6 days and 4 to 7 days after inoculation, respectively. Serum dilutionneutralization, hemagglutination inhibition, or complement fixation antibodieswere detected in sera of some animals 13 days after infection and in all animalsafter 20 days. Antibodies persisted for the 168-day period of observation.Persistent viral infection was demonstrable by in vitro culturing of lungs orpooled lungs, salivary glands, and kidneys from 14 of 69 mice that were sacrificedfrom 1 to 6 months after intranasal inoculation. Attempts to demonstratehorizontal or vertical transmission of Modoc virus among mice were generallyunsuccessful. Female deer mice infected with virus before mating passedmaternal antibodies to their young.

Modoc (MOD) virus is related antigenicallyto group B arboviruses but there is no evidencethat it has an arthropod vector (9, 16). Infec-tious virus is shed in the urine of hamsters formany weeks after infection and it was suggestedthat wild mammals might be in the basic hostsof this virus (10). Attempts to demonstrateeither horizontal or vertical transmission ofMOD virus in hamsters were unsuccessful (5);however, the hamster is not a natural host forthe virus. Thus, the deer mouse, Peromyscusmaniculatus gambeli, from which all strains ofMOD virus have been isolated (16), was se-lected as a natural host in which to extendpathogenesis studies ofMOD virus. (This paperis based on a portion of a dissertation submittedto the University of California in partial fulfill-ment of the requirement for the Ph.D. degree.)

MATERIALS AND METHODSVirus. The prototype strain (M544) of MOD virus

was isolated from the mammary glands of a lactatingdeer mouse trapped in Modoc County, Calif., duringJuly, 1958 (9). This virus was passaged intramuscu-larly three times in 6- to 8-week-old Syrian hamstersby using viremic blood as the inoculum for the secondand third passages (4). The stock virus used to infect

'Present address: Flow Labs, Inc., P. 0. Box 2226. 1710Chapman Avenue, Rockville, Md. 20852.

deer mice was prepared from viremic blood at thethird hamster passage level and titered 105 8 plaque-forming units (PFU) per ml of blood, etc. Stock virusused for the serum dilution neutralization (SDN) test,and for antigens in the hemagglutination inhibition(HI) and complement fixation (CF) tests, was pre-pared as 10% (wt/vol) homogenates of infected brainsfrom Swiss albino suckling mice inoculated intracere-brally (0.03 ml per mouse) with hamster passage 2virus.The procedures used to identify MOD virus and to

detect extraneous viruses in the viral stocks weredescribed previously (5).Deer mice. Colonized P. maniculatus gambeli were

obtained from D. Bruce Francy at the CommunicableDisease Center in Fort Collins, Colo. This colony wasinitiated in 1967 at the United States Public HealthService Plague Laboratory in San Francisco from wildmice trapped in Modoc County, Calif. The colony wasmaintained by random breeding. Approximately 50mice were shipped from Fort Collins to Berkeley inJanuary of 1970. Upon arrival, 15 females and 1 malewere examined for evidence of infection with MODvirus. No virus was isolated from cell cultures pre-pared from trypsinized lungs, spleens, salivary-sub-maxillary glands, kidneys, or pooled axial, inguinal,and popliteal lymph nodes. No antibodies to MODvirus were detected by HI, CF, or SDN tests in 1:10dilutions of sera from these 16 mice. After receipt atBerkeley, breeding pairs were maintained in individ-ual isocages in a building separate from that used for

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other viral research. The F1 and F. generations of theoriginal breeding stock were used for experimentalwork. All animals were tested for HI antibodies priorto experimental studies and none had MOD viralantibodies.

Infection of deer mice. Deer mice (4 to 8 weeksold) were usually inoculated intranasally (IN) with300 to 1,500 PFU of virus contained in 0.05 ml ofstabilizing diluent. Mice were maintained individ-ually in isocages unless otherwise indicated. Thestabilizing medium consisted of 0.75% bovine al-bumin (fraction V) in 0.05 M H,BO2-0.12 M NaClat pH 9.0.

In one experiment, groups of seven to eight miceeach were inoculated IN or subcutaneosuly (SC; 0.1ml per mouse) with serial 10-fold dilutions of virus.Mean infective doses were calculated by the methodof Reed and Muench (12).

Collection of specimens. Mice were bled from theorbital sinuses to obtain samples for viwemia andantibody determinations. Blood was collected in aPasteur pipette, and five drops (approximately 0.25ml) were added to 1 ml of viral stabilizing medium togive a 1:5 dilution of blood.

At selected intervals after infection, mice weresacrificed and various organs were removed. Portionsof each organ were tested directly for virus as 10%homogenates in stabilizing medium and after cultur-ing in vitro. Organs from which in vitro cultures wereprepared included pooled lymph nodes (axial, ingui-nal, and popliteal), salivary-submaxillary glands,spleens, kidneys, lungs, and bone marrow. In additionto these organs, 10% homogenates were prepared fordirect viral isolation attempts from liver, brain, spinalcord, and heart. Urine was collected by opening thebladder and rinsing it with 1 ml of stabilizing diluent.Throat swabs taken immediately before sacrifice wereimmersed in 1 ml of stabilizing diluent.

In vitro cultures were prepared from enzymaticallydispersed cells or tissue fragments as described previ-ously (5). At 14 days after initiation, cultures werefrozen and thawed to release virus, and fluids fromthree cultures prepared from each organ were mixedwith an equal volume of stabilizing medium for viralassay.

All samples that were to be assayed for virus werestored at -70 C. Serum samples for antibody testswere stored at -20 C.

Viral assay. Virus was assayed by plaquing onVero cells that were grown in 60-mm plastic petridishes in a medium consisting of 5% fetal calf serum,in Eagle autoclavable minimal essential mediumprepared in Earle balanced salt solution (4). Afteradsorption of virus to cells in 0.2-ml volumes for 1.5 to2 h at 37 C, cultures were rinsed with Dulbeccophosphate-buffered saline at pH 7.4 pt.nd overlaid with5 to 6 ml of medium that consisted of 1.5% methylcellulose (4,000 centipoises) in autoclavable minimalessential medium in Earle balanced salt solution plus2% heat inactivated (56 C for 30 min) fetal calf serum.To visualize plaques, cultures were overlaid after 4days incubation at 37 C with 6 ml of the samemedium except that it contained neutral red(1:36,000) and the methylcellulose was replaced by

1% purified agar. Plaques were counted 3 days laterand viral titers were expressed in PFU per gram oftissue or milliliter of blood or cell culture fluid.

All cell culture media contained 1.6 g of NaHCO,per liter, 100 U of penicillin per ml, and 100 mg ofstreptomycin per ml. Cultures were incubated at 37 Cin a humidified atmosphere of 5% CO, in air.

Serological tests. Antibody to MOD virus wasmeasured by microtest adaptations of the SDN (6),HI (13), and CF (1) tests. Hemagglutinins and CFantigens were prepared by the sucrose-acetone tech-nique (3) from brains of infected Swiss albino sucklingmice. Sera used in the HI test were extracted withacetone and adsorbed with goose erythrocytes (3).SDN, CF, and HI titers of 1:10 were consideredpositive for antibodies.

Vertical transmission in deer mice. Five femalemice were inoculated IN with MOD virus 17 to 54days before they littered. One baby was removed onthe day of birth from each litter, sacrificed, and testedfor virus both by direct isolation from a 10% homoge-nate and after culturing in vitro. Blood and serumsamples were taken from the remaining offspring at 2to 27 days after they were weaned. Viral isolation wasattempted after in vitro culturing of selected organsfrom six progeny at 37 to 47 days after weaning.

In a separate experiment, female mice were inocu-lated IN during the first week after they littered.These females were not returned to their offspringuntil 8 h after inoculation. Shortly before they werereturned to their litters, the nasal and oral passages ofthese animals were washed with saline to removeresidual free virus. Blood samples were collected forviral and antibody tests from the offspring at variousintervals after they were weaned, and selected organswere taken from some of the progeny, cultured invitro, and tested for virus.One experiment was done to evaluate the possibil-

ity that suckling mice might become infected fromvirus in lactating fluids. Lactating mice were inocu-lated IN with virus 3 to 8 days after littering. Thesemothers were sacrificed 3 to 10 days after inoculation.Viral isolation was attempted from blood, and from10% homogenates and fluids of fragment cultures oflungs and mammary glands.

Horizontal transmission in deer mice. Horizontaltransmission was tested by caging normal mice withinfected mice at various times after IN inoculationwith MOD virus. Groups of either one normal and oneinfected or three normal and three infected mice werecaged together for periods of 5 to 28 days in isocageswith wood shavings as bedding. Inoculated mice weretested for HI antibody 28 days after inoculation. Thecontact mice were tested for HI antibody 28 days afterbeing separated from the infected mice.The possibility that infectious virus could be de-

tected in the urine or throats of infected mice wasevaluated. Mice were inoculated with virus, and urineand throat swab samples were collected each day from3 to 7 days after inoculation for viral assay. The urinesamples were collected while mice were held in a 7 Ccold room. Mice were placed on one-fourth-inch wiremesh screens atop funnels that conducted excretedfluids into tubes that contained 1 ml of the stabilizing

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medium. Portions of urine and throat swab samplesthat contained detectable virus when plaqued in Verocells were subsequently inoculated SC (0.1 ml) intonormal mice. One mouse was injected with eachthroat swab sample and three mice were inoculatedwith each urine sample. These mice were bled at 28days after inoculation and their sera were tested forHI antibody.

Horizontal transmission by cannibalism was evalu-ated using a third group of infected mice. Lungs wereremoved at selected intervals after infection, mincedwith scissors, and washed free of visible blood withphosphate-buffered saline. In vitro fragment cultureswere prepared from a portion of the lung fragmentsand the rest of each lung was force fed to a normalmouse using sterile forceps. These mice were testedfor development of HI antibody 28 days after beingfed lung tissue.

Identification of viral isolates. Virus that waspresent in one or more positive samples from eachinfected mouse was identified as MOD virus by thedirect fluorescent antibody technique using MODviral hyperimmune sera prepared in hamsters. Thispool of hyperimmune hamster sera was negative (titerof <1:4) by indirect fluorescent antibody tests forantibodies to lymphocytic choriomeningitis virus andtwo new viruses (a herpesvirus and a myxovirus)recently isolated from primary kidney cell cultures ofwild Peromyscus (personal communication, RichardW. Emmons, Infectious Disease Section, Calif. StateDepartment of Public Health).The pool of hyperimmune hamster sera was frac-

tionated and serum globulins were conjugated withfluorescein isothiocyanate as described by Cherry etal. (2) and modified by Lennette and associates (11).Cover-slip cultures of Vero cells were inoculated withviral specimens, incubated at 37 C for 3 to 4 days,fixed with acetone at - 20 C, and stored at - 70 Cuntil stained with the fluorescent antibody conjugate.

The technique described by Emmons and Lennette(7) was used to stain cells with a 1:20 dilution of thefluorescent antibody conjugate. Cover slips werecounterstained with a 0.1% solution of Evans blue.Specificity of the fluorescent antibody conjugate wasdemonstrated by its failure to stain uninfected Verocells and Vero cells infected with vesicular stomatitisvirus. Identification of three isolates from in vitrocultures of lungs of deer mice infected 3 monthspreviously with MOD virus was confirmed by SDNtests employing MOD hyperimmune sera prepared inrabbits.

RESULTS

Infection of deer mice. Mice (4 to 8 weeksold) were easily infected with MOD virus byboth the IN and SC routes of inoculation. Themean infective doses were 2.5 and 0.13 PFU forgroups of seven to eight deer mice inoculated INand SC, respectively, with serial 10-fold dilu-tions of MOD virus.Low titered viremias of 1 to 3 days duration

were detected 1 to 4 days after inoculation in 10of 13 and 2 of 8 mice that were inoculated INand SC, respectively, and subsequently devel-oped antibodies (Table 1). No viremia wasobserved after day 4 of inoculation.

All infected mice had developed HI, CF, andSDN antibodies to MOD virus by 20 days afterinoculation (Table 2). Peak antibody titers wereobtained by 27 days after inoculation by CF andSDN tests and 112 days by the HI test. All theanimals tested had antibody titers of 1:10 orgreater by all three tests from day 20 throughday 168 after inoculation.

Viral spread and persistence. A preliminary

TABLE 1. Viremias in Peromyscus maniculatus inoculated intranasally or subcutaneously with MOD virusa

No. of mice devel- Log,. PFU of virus/ml of blood in mice with detectable viremiasRoute of inoculation loping detectable by days after inoculationcviremias/no.

infected' 1 2 3 4 5 6

Intranasal 10/13 1.1 1.1 <1.1<1.1 3.4 <1.1<1.1 2.9 <1.1

<1.1 ~~~~~1.71.

<1.1 2.4 <.

3.1<11<.2.4<11<.

<1.1 2.4 <.

Subcutaneous 2/8 2.5 <1.1 <.______________ ____ ______ ____ _ ______ 2.8 <1.1 <.

a Each mouse was inoculated with 300 to 500 PFU of virus.'All mice developed HI antibody by 28 days after inoculation, including individuals that did not have

detectable viremias.c Mice were bled on altemate days starting 1 or 2 days after inoculation.

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experiment was done to determine whethervirus persisted in immune animals. For thisstudy, in vitro cultures were prepared frompooled kidneys, lungs, and salivary-submaxil-lary glands of each of 25 mice at 28 days afterinoculation. Virus was isolated from cultures of3 of 12 mice inoculated SC and 3 of 13 miceinoculated IN. On the basis of these preliminaryresults, an experiment was set up to measure

TABLE 2. Development of HI, CF, and SDNantibodies in Peromyscus maniculatus inoculated

intranasally with MOD virus

Day HI titer" CF titerb SDN titer"after_infec-tiona GM Range GM Range GM Range

0 0 0 06 0 0 1.6 <10-1013 6.3 < 10-10 2.5 < 10-10 13.2 < 10-2020 15.2 10-20 11.5 10-20 20.0 10-4027 15.2 10-20 15.2 10-20 30.3 10-4056 69.6 40-80 15.2 10-20 15.2 10-2084 69.6 40-80 13.2 10-20 15.2 10-20112 91.9 80-160 13.2 10-20 15.2 10-20140 60.6 20-160 11.2 10-20 11.5 10-20168 60.6 40-160 13.2 10-20 13.2 10-40

a Sera were obtained from five deer mice at each sampleperiod.

° Serum samples collected from 0 to 27 days after inocula-tion were tested separately from samples collected from 56 to168 days after inoculation. GM, Geometric mean titer.

the initial spread of virus in organs of miceinoculated IN and then to determine the per-sistence of virus in various organs. The sensitiv-ity of the direct viral isolation and in vitroculture techniques for detection of persistentviral infections was also evaluated.

Virus was recovered by direct isolation frommany organs and fluids of mice during the first9 days after IN inoculation (Table 3). Lungswere found infected from immediately afterinoculation to 9 days after inoculation. Virusappeared in the spleens, salivary-submaxillaryglands, kidneys, and lymph nodes by 2 or 3 daysafter inoculation and remained detectable inthese organs through 7, 8, or 9 days. Viremiaswere detectable from 2 to 4 days after infectionand virurias from 4 to 6 days. Virus wasobtained from throat swabs immediately afterinoculation and again from 4 to 6 days afterinoculation. Sporadic viral isolations were madefrom homogenates of brain, spinal cord, heart,and liver, and from fluids of bone marrowcultures.

Viral titers in organs and fluids during thefirst 10 days after infection are depicted in Fig.1. The highest titers were detected in the lungs,spleens, salivary-submaxillary glands, andlymph nodes. Peak titers occurred after 2 or 3days in lungs and after 6 days in spleens,salivary-submaxillary glands, and lymph nodes.

TABLE 3. Occurrence of MOD virus in organs and fluids of experimentally infected Peromyscus maniculatusduring the first 10 days after infectiona

No. of isolations/no. of organ or fluid samples tested

Lung

2/32/32/36/62/33/33/32/61/32/30/3

Spleen

0/30/31/36/63/33/33/34/61/30/30/3

Sali-vary-sub-maxil-lary

glands

0/30/31/36/63/33/33/35/61/30/30/3

0/30/30/32/61/32/33/32/60/30/30/3

0/30/31/32/61/30/31/31/60/31/30/3

0/30/32/32/33/30/30/30/30/30/30/3

0/30/30/30/31/30/31/30/30/30/30/3

0/30/30/30/30/31/30/30/30/30/30/3

0/30/30/31/30/30/31/30/30/30/30/3

0/30/30/30/30/31/30/30/30/30/30/3

0/30/30/30/31/32/32/30/30/30/30/3

3/30/30/30/32/33/32/31/30/30/30/3

0/30/31/30/30/30/33/31/30/30/30/3

aMice were inoculated intranasally with 300 to 500 PFU of virus. Isolation of virus was made by direct

inoculation of Vero cells with body fluids or tissue homogenates.'Axial, inguinal, and popliteal lymph nodes were pooled.c Urine was collected from each bladder by cutting it open and then rinsing it with 1 ml of stabilizing

medium.d Viruses isolated from the bone marrow were recovered from the fluids of bone marrow tissue cultures.

eAnimals were sacrificed immediately after inoculation.

Day afterinoculation

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In vitro cultures were prepared individuallyfrom portions of lungs, kidneys, spleens, sali-vary-submaxillary glands, and lymph nodes of 6and 30 mice that were sacrificed 1 to 7 and 14 to177 days after inoculation, respectively (Table4). During the first week of infection, virus was

LUNG

6.0 -a0 4.0- 2m 2.0

0 2 4 6 810

SALIVARY - SUBMAXILLARYGLANDS

2 4 6 8 10

8.0 _ SPLEEN

LYMPH NODES (AXIAL,6.0 INGUINAL, AND POPLITEAL)

i22.0 8 1

2 4 6 8 10 2 4 6 8 10

4.0 BLOOD

2.0 -4 _ _

2 4 6 8 10

BRAIN

2.0 L__

2 4 6 8 10

LIVER

2.0

2 4 6 8 10

DAYS AFTER

4.0 KIDNEY

2.0L- SiA

2 4 6 8 10

HEART

2.0 =_ _ _ ___

o 001;xI2 4 6 8 10

SPINAL CORD

2.0 C-- -T1---o A:/\/\ _a

2 4 6 8 10

INOCULATION

FIG. 1. Titers of MOD virus in the organs andblood of Peromyscus maniculatus during the first 10days after intranasal inoculation. Symbols: ,mean titer for three to six mice; I,range of titers;- - -, minimal titer detectable.

recovered in 18 of 29 attempts from fragmentcultures and 16 of 29 attempts from organhomogenates. In contrast, cell cultures grown invitro from the trypsinized portions of each organwere less sensitive for detecting virus as viruswas isolated in only 9 of 30 attempts.From 14 to 177 days after infection, virus was

recovered from 8 of 30 lungs by in vitro culturetechniques but no virus was detectable in ho-mogenates of the same lungs (Table 4). Of theeight isolations made from in vitro culturedlungs, four came from both fragment and tryp-sinized cell cultures, three from only fragmentcell cultures, and one from only trypsinized cellcultures. No virus was recovered from in vitrocultures of salivary-submaxillary glands,spleen, kidneys, and lymph nodes from these 30mice. Homogenates of these four organs werenot tested for virus. In addition, direct viralisolation attempts were made on urines, bloods,and throat swab samples from these 30 miceand no virus was isolated.A total of 17 MOD viral isolates were made

from lungs or pooled salivary glands-lungs-kid-neys cultured in vitro from 97 deer mice thatwere sacrificed from 14 to 366 days after inocula-tion and were circulating antibodies to MODvirus at the time of sacrifice. Persistent infec-tions were demonstrated in three of three miceat 2 weeks after infection, 7 of 32 mice at 1month, 6 of 29 mice at 3 months, 1 of 8 mice at6 months, and none of 27 mice at 1 year.

Vertical transmission. Six litters, whichincluded 20 babies, were born to five mothersthat had been infected with MOD virus 17 to 54days prior to littering. One baby was sacrificedfrom each litter on the day of birth and tested

TABLE 4. Comparative sensitivities of three isolation procedures for detection of MOD virus in organs ofPeromyscus maniculatus in the early (1 to 7 days) and late (14 to 177 days) periods after IN inoculation

No. of virus isolates/no. of organs tested

3 to 7 days after inoculation 14 to 177 days after inoculation

Organ Direct isola- Cells from Direct isola- Cells fromtion from trypsinized Organ frag- tion from trypsinized Orafagorgan organs growing ments growing organ organs growing ments growing

suspensions in vitro in vitro suspensions in vitro in vitro

Lung ............... 3/6 4/6 5/5 0/30 5/30a 7/30aSalivary-submaxil-

lary glands ....... 5/5 2/6 6/6 NTb 0/30 0/30Spleen ............. 5/6 2/6 5/6 NT 0/30 0/30Kidney ............ 2/6 1/6 1/6 NT 0/30 0/30Axial, inguinal, and

popliteal lymphnodes ........... 1/6 0/6 1/6 NT 0/30 0/30

aVirus was recovered from lungs of eight animals: four from both trypsinized and fragment cell cultures,three from only fragment cell cultures, and one from only trypsinized cell cultures.

b NT, Not tested.

0wn

IL0

w

CL

1.)0

0

0

a

0

IL0.

0

0-i

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for virus both by direct isolation and afterculturing in vitro. No virus was isolated. Bloodsamples were collected from the 14 remainingoffspring at 2 to 10 days after they were weanedat 21 days of age. Virus was not detected in anyof the blood samples. HI antibody titers of 1:20*or greater were detected in the sera from 11 ofthese 14 offspring at 2 to 7 days after weaning,but no antibody was detected in 1:10 dilutionsof serum taken subsequently from these miceat 15 to 27 days after they were weaned. Theseresults suggested that the antibodies detectedduring the first week after weaning may havebeen maternal antibodies. The blood and indi-vidually cultured spleens, salivary-submaxillaryglands, lungs, kidneys, and pooled axial, ingui-nal, and popliteal lymph nodes of six offspringwere tested for virus at 37 to 47 days after wean-ing, but no virus was recovered.In a second experiment, seven female mice

were infected with virus during the first weekafter littering. At 17 days postweaning, bloodsamples were taken from all 19 of the offspringin the litters, but virus was not isolated fromany of these samples. Five of the offspring hadHI antibody titers of 1:20 or greater at 14 dayspostweaning, and four of these offspring re-

mained positive for antibodies when tested 38days or later after weaning. Three of the micewith persisting antibody titers were born to thesame female mouse. The fourth offspring whichremained positive for HI antibodies had one

sibling which did not have detectable antibodyat 14 days or later after weaning. Virus was notisolated from the blood or from cultured organs

(spleens, salivary-submaxillary glands, lungs,kidneys, and pooled axial, inguinal, and poplit-eal lymph nodes) of three mice with persistingantibodies or of six other offspring that werenegative for HI antibodies when tested 39 to 49days postweaning. The fourth mouse whichremained HI positive was kept alive and had an

HI titer of 1:40 when tested at 98 days afterweaning.The possibility that babies might become

infected from virus in lactating fluids frominfected mothers during the acute phase ofinfection was evaluated by inoculating fourlactating females with virus and then sacrificingthem at various times up to 10 days afterinoculation. Virus was recovered by both directisolation and after in vitro cultivation of bothmammary glands and lungs from each of thethree female mice that were sacrificed 3, 5, or 7days after infection. Viral titers in the mam-mary tissues ranged from 104 to 106 PFU/g. Allthree mice were viremic when sacrificed. The

fourth mouse was sacrificed 10 days after infec-tion and virus was obtained only from thecultured lungs.Horizontal transmission. None of the 10

males paired with inoculated females in thevertical transmission experiments became in-fected. The results of a more extensive study toevaluate horizontal transmission of MOD virusfrom infected mice to normal mice are shown inTable 5. Only one of 38 normal mice becameinfected after being caged individually withmice that had been inoculated 2 to 7 dayspreviously. No normal mice became infectedwhen caged singly, or in groups of three withequal numbers of the inoculated animals atintervals between 28 and 112 days after inocula-tion.An experiment was done to determine

whether virus recovered from throat swab andurine specimens was infectious for normal mice.Throat swabs and urine samples were collecteddaily from each of five mice from 3 to 7 daysafter inoculation. Virus was recovered by directisolation in Vero cells from 14 of 25 throat swabspecimens and 3 of 25 urine samples. Virus wasobtained in throat swab samples of all five miceand in urine samples of two mice. Twelve of the14 positive throat swab specimens and two ofthe three urine samples were infectious fornormal deer mice inoculated SC with a portionof each sample as determined by the develop-ment of HI antibody.

Since mice can be cannibalistic and thereforecould become infected in nature by ingestion ofinfected tissues of other mice or rodents, aportion of lungs removed from each of 3 and 18mice at 4 and 54 days after inoculation, respec-tively, were fed to normal deer mice. None of thenormal mice developed HI antibodies by 28days after ingestion even though virus was

TABLE 5. Summary of attempts to demonstratehorizontal transmission ofMOD virus from infected

to normal Peromyscus maniculatusa

Ratio of Days after Fraction ofinfected to inoculation No. of normal micenormal mice mice were groups becomingin each cage caged together infected

1:1 2-7 38 1/381:1 28-35 51 0/511:1 56-63 42 0/423:3 84-112 13 0/34o

a Development of HI titer of 1:20 or greater by 28days after inoculation was considered evidence ofinfection.

b Six infected and eight contact mice were dead ormissing 28 days after the animals were caged together.

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recovered from fragment cultures prepared fromthree of the three lungs taken 4 days afterinfection and 3 of the 18 lungs removed 54 daysafter infection.

DISCUSSIONDeer mice were found to be highly susceptible

to infection with MOD virus by both IN and SCroutes of inoculation. Inoculation by the INroute was chosen for experimental studies sinceMOD virus was found to be excreted in urine ofhamsters, and this suggested that natural infec-tion might occur by contact of nasal or oralpassages of susceptible animals with virus shedby infected animals (9).Deer mice developed viremias of low titer and

short duration after inoculation with MODvirus. Neutralizing antibodies were detected thefirst or second week after inoculation and re-mained at measurable titers at least for 168days. During the first nine days after IN inocu-lation, virus was recovered regularly by directisolation from the lungs, spleens, salivary-sub-maxillary glands, and lymph nodes. Occasionalisolations were made from the kidneys duringthis period, but the titers were close to thedetectable level. The apparent infection of thekidneys may have resulted from glomerulartrapping of virus or virus-antibody complexesinstead of actual viral replication in this organ.Recovery of virus in throat swabs of mice from 4to 7 days after inoculation coincided with maxi-mal viral titers in salivary-submaxillary glands.MOD virus was recovered from in vitro cul-

tures prepared from lungs of some mice 10 to177 days after inoculation but not from homoge-nates of the same organs. Twenty-one percent ofthe cultured lungs (6 of 29) yielded MOD virusat 3 months after the mice were infected. Theproportion of mice that harbored latent virusmight have been greater than detected, sincethe in vitro techniques failed to recover virusfrom some organs that were taken from micesacrificed during the first week after inoculationand these organs yielded virus by direct viralisolation procedures.

Since MOD virus persisted in infected micefor several months after inoculation, an experi-ment was done to determine whether infectedfemale mice could infect their progeny. How-ever, no evidence of vertical transmission wasobtained with female mice that were infectedwith virus from 17 to 54 days before giving birth.Neutralizing antibodies in the blood of infectedfemale mice may have prevented virus fromreaching the embryos. Antibodies to MOD viruswere detected in weanling mice from theseimmune mothers, but the antibodies disap-

peared during the first month of life and areassumed to have been of maternal origin. Asimilar pattern of antibody occurrence has beenobserved for maternal antibodies in the progenyof female mice infected with lymphocytic cho-riomeningitis virus (8). The fall in antibodytiters in the young deer mice was consistentwith the reported half-life of humoral anti-bodies in other mice (14).Female mice that were inoculated with MOD

virus during the first postnatal week apparentlytransmitted virus to some progeny. The detec-tion of viral antibody in sera taken from off-spring 38 or more days after weaning wasconsidered evidence of transmission. None ofthe males in contact with infected femalesbecame infected, which suggested that the viralsuspension used to inoculate the female micewas not directly responsible for the observedtransmission to progeny. However, sucklingmice may be more susceptible than adult males.Vertical transmission might have occurred fromlactating fluids that contained virus and whichwas ingested by the nursing offspring. MODvirus was isolated from the mammary glands ofa second group of lactating mice during the firstweek after inoculation. Similar viral isolationshave been made from mammary glands ofexperimentally infected laboratory mice (9).

Infectious virus was isolated from throatswabs and urine of mice during the first weekafter infection. Despite this availability of virus,horizontal transmission was demonstrated inonly 1 of 38 normal mice caged with infectedmice during the first 7 days after inoculationand did not occur at all later. The low titers ofviruria may have been quickly inactivated byadsorption onto the bedding, and the conditionsof caging may not have favored transmission ofthe virus found in the throat swabs. Deer miceare reported to live in large families during thewinter (10), and in such crowded conditionsthat virus might be transmitted more readilythan under the conditions of this experiment.In nature these mice often fight to protect terri-torial rights (15) and this behavior might aid inthe spread of virus.The very limited demonstration of transmis-

sion of MOD virus from infected to susceptiblemice did not support the hypothesis that thedeer mouse is the basic host or reservoir for thisagent. The strain of mice used in this study hadbeen maintained in laboratory colonies for sev-eral years and might have responded differentlyto viral infection than would wild mice. It is alsopossible that experimental conditions may nothave successfully reproduced the natural envi-ronment in which transmission occurs.

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MODOC VIRAL INFECTION IN DEER MICE

ACKNOWLEDGMENTS

This investigation was supported by Public Health Serviceresearch grant AI 03028 from the National Institute of Allergyand Infectious Diseases and general research support grant5-SO1-RR-05441 from the Division of Research Resources.We wish to express our sincere appreciation to Maxwell S.

Redfearn for maintenance of the deer mouse colony and toSarah Presser for technical assistance.

LITERATURE CITED

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2. Cherry, W. B., M. Goldmon, T. R. Carski, and M. D.Moody. 1960. Fluorescent antibody techniques. PublicHealth Monograph No. 729. U.S. Government PrintingOffice, Washington, D.C.

3. Clarke, D. H., and J. Casals. 1958. Techniques forhemagglutination and hemagglutination-inhibitionwith arthropod-borne viruses. Amer. J. Trop. Med.Hyg. 7:561-573.

4. Davis, J. W., and J. L. Hardy. 1973. In vitro studies withModoc virus in Vero cells: plaque assay and kinetics ofgrowth, neutralization, and thermal inactivation.Appl. Microbiol. 26:344-348.

5. Davis, J. W., and J. L. Hardy. 1974. Characterization ofpersistent Modoc viral infections in the Syrian ham-ster. Infect. Immunity 10:328-334.

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plaque neutralization method for arboviruses. Proc.Soc. Exp. Biol. Med. 125:741-747.

7. Emmons, R. W., and E. H. Lennette. 1966. Immunofluo-rescent staining in the laboratory diagnosis of Coloradotick fever. J. Lab. Clin. Med. 68:923-929.

8. Hotchin, J. 1971. Persistent and slow viruses. Monogr.Virol. 3:1-211.

9. Johnson, H. N. 1967. Ecological implications of antigeni-cally related mammalian viruses for which arthropodvectors are unknown and avian associated soft tickviruses. Jap. J. Med. Sci. Biol. 20:160-166.

10. Johnson, H. N. 1970. Long-term persistence of Modocvirus in hamster-kidney cells: in vivo and in vitrodemonstration. Amer. J. Trop. Med. Hyg. 19:537-539.

11. Lennette, E. H., J. D. Woodie, K. Nakamura, and R. L.Magoffin. 1965. The diagnosis of rabies by fluorescentantibody method (FRA) employing immune hamsterserum. Health Lab. Sci. 2:24-34.

12. Reed, L. J., and H. A. Muench. 1938. A simple method ofestimating fifty per cent endpoints. Amer. J. Hyg.27:493-497.

13. Sever, J. L. 1962. Application of a microtechnique to viralserological investigations. J. Immunol. 88:320-329.

14. Soloman, J. B. 1971. Foetal and neonatal immunology.Front. Biol. 20:1-381.

15. Stickel, L. F. 1968. Home range and travels, p. 373-411.In J. A. King (ed.), Biology of Peromyscus (Rodentia).The American Society of Mammalists, Stillwater, Okla.

16. Taylor, R. M. (compiler). 1967. Catalogue of arthropod-borne viruses of the world, ed. 1. U.S. GovernmentPrinting Office, No. 1760. Washington, D.C.

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