Lymphocytic Choriomeningitis Virus Infection Fetal ... · viremia. Hamsters with persistently high levels ofviremia and viruria devel-oped chronic glomerulonephritis and widespread

INFECTION AND IMMUNITY, Mar. 1976, p. 967-981Copyright © 1976 American Society for Microbiology

Vol. 13, No. 3Printed in U.S.A.

Lymphocytic Choriomeningitis Virus Infection in Fetal,Newborn, and Young Adult Syrian Hamsters

(Mesocricetus auratus)JOHN C. PARKER,* HOWARD J. IGEL,' ROBERTA K. REYNOLDS,2 ANDREW M. LEWIS, JR.,3 AND

WALLACE P. ROWE3Microbiological Associates, Bethesda, Maryland 20014

Received for publication 29 July 1975

The pathogenesis of lymphocytic choriomeningitis virus infection in fetal,newborn, and young adult hamsters was studied. Infected newborn hamstersinitially developed a persistent viremia and viruria with titers often in excess of104.0 mean infectious doses/0.03 ml of blood or urine. After week 12 two differentpatterns of infection became evident. Approximately one-half of the hamsterseventually cleared the infection, whereas the others developed a chronic pro-gressive and ultimately fatal disease characterized by continuous high-titeredviremia and viruria and high titers of virus in their tissues. Complement-fixingantibody and, to a lesser degree, virus-neutralizing antibody coexisted with theviremia. Hamsters with persistently high levels of viremia and viruria devel-oped chronic glomerulonephritis and widespread vasculitis, whereas hamstersthat cleared their infections did not develop these lesions. Litters of hamstersborn to viremic mothers were invariably infected. Litter sizes were small andbreeding effectiveness was reduced; however, vertical, congenital infection wassuccessfully passed through three generations. The course of infection in thecongenitally infected hamsters was similar to that in newborn infected ham-sters, with all animals producing complement-fixing antibody, some animalsbeing capable of clearing the viremia and remaining healthy, and other animalshaving persistent viremia and fatal disease. Inoculated young adult hamstersdid not become diseased, developed viremia and viruria which persisted up to 3and 6 months, respectively, and developed complement-fixing antibody by 10days after infection. The prolonged urinary excretion of large amounts of lym-phocytic choriomeningitis virus by asymptomatic, chronically infected hamstersis an important public health consideration when dealing with potential humaninfection.

The mouse lymphocytic choriomeningitis(LCM) virus model has been utilized exten-sively in the study of virus-induced immuno-pathological diseases (18, 30, 31, 36, 43). Al-though other animal species are known to besusceptible to LCM virus, including humans,hamsters, monkeys, dogs, guinea pigs, rats,rabbits, and chickens (30, 48, 50), until recentlyit was generally believed that the only signifi-cant natural reservoirs ofLCM virus were wildhouse mice (Mus musculus) or certain coloniesof commercially reared laboratory mice. In1965, an epizootic of LCM virus infection oflaboratory hamsters in the United States was

' Present address: Children's Hospital of Akron, Akron,Ohio 44308.

2 Present address: Viral Carcinogenesis Branch, Na-tional Cancer Institute, Bethesda, Md. 20014.

3Present address: Laboratory of Viral Diseases, Na-tional Institute of Allergy and Infectious Diseases, Be-thesda, Md. 20014.

reported (3, 33), which was responsible for 23human cases. Since that time an additional 167human cases have been reported which wereacquired via contact with infected hamsters (4,8, 11, 19, 25, 39). LCM virus infections arefrequently found in commercial hamster colo-nies in Germany (1, 14), and it is presentlyestimated that sale of these animals as pets isresponsible for approximately 1,000 humancases in that country each year (1). The firstinfected commercial hamster colony in theUnited States was reported in 1974 (4-8, 11).During our initial study of LCM virus infec-

tion in hamsters (33) we discovered that a num-ber of hamsters infected during the first day oflife had persistent viremia and viruria and anincreasing mortality beginning around 5months of age. These findings together with theincreased number of hamster-associated hu-man LCM virus infections and the general lack

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968 PARKER ET AL.

of information regarding the pathogenesis ofinfection in the hamster prompted this work.The pathogenesis of LCM infection in the fetal,newborn, and adult Syrian hamster was stud-ied, with emphasis on the multiplication anddistribution of the virus, the immune response,and the histopathology. A common feature ofinfection was a chronic viremia and viruria,which persisted more than a year in some in-fected newborns and for 3 to 6 months in someinfected adults. In infected newborns approxi-mately half of the hamsters cleared their infec-tions, whereas the remainder continued to beviremic and viruric, developed vasculitis andchronic progressive glomerulonephritis, anddied of renal failure at about 1 year of age.

MATERIALS AND METHODS

Hamsters. Random-bred Syrian golden hamsterswere obtained from the National Institutes ofHealth, Bethesda, Md. This colony was not infectedwith LCM virus. LCM virus-infected hamsters werehoused in flexible Trexler-type or rigid plastic germ-free isolators (Germfree Laboratories, Inc., Miami,Fla.) and were maintained using standard germfreetechniques (41) modified to provide operator protec-tion from LCM infection. All contaminated materi-als were removed from the isolators via the exit portthrough a plastic sleeve and into a large plastic (12mil) bag attached to the sleeve. The bag wassprayed with peracetic acid mist, garotted with tapeas close as possible to the trap, cut free from theisolator, and steam sterilized before disposal. Ham-sters inside the isolators were housed in disposableplastic cages (Maryland Plastics, New York, N.Y.)with perforated stainless-steel tops. Apples wereused for the water source and diet was steam steri-lized (Charles River pre-fortified rat-mouse formula7RF, Agway, Inc., Syracuse, N.Y.).

Mice. Random-bred Swiss Webster mice were ob-tained from the National Laboratory Animal Co.,Creve Coeur, Mo. This mouse colony was not in-fected with LCM virus. Mice 21 to 28 days of agewere used in all virus infectivity titrations and forfootpad neutralization assays.LCM virus. (i) Fortner strain. The Fortner strain

of LCM virus was isolated from an LCM-contami-nated hamster tumor designated Fortner fibrosar-coma no. 2 (15, 16, 33). The tumor was passaged ineither newborn or weanling age hamsters by subcu-taneous transplant of tumor fragments. These ham-sters were housed in germfree isolators. Tumorsmeasuring between 1 and 2 cm were excised andpooled, and a 10% suspension in Eagle basalmedium with Earles blanced salt solution (EBME)was prepared. After two freeze-thaw cycles, the sus-pension was clarified by centrifugation at 1,000 x gfor 30 min, and the supernatant fluid was filteredthrough a 1.2-,um membrane filter (Millipore Corp.)to remove any remaining viable tumor cells. Thesuspension was stored at -90 C.

(ii) Strain CA 1371. The mouse brain-passaged

INFECT. IMMUN.

strain CA 1371 of LCM virus, a mouse meningo-tropic strain, was used as challenge virus in infec-tivity assays and in neutralization tests.

(iii) Certification. The Fortner and CA 1371 LCMvirus strains were not contaminated with the fol-lowing viruses: pneumonia virus of mice, reovirus3, Theiler's GD VII, Sendai, simian virus 5, minutevirus of mice (MVM), Kilham rat virus, H-1, mouseadenovirus, mouse hepatitis virus (MHV), polyoma,K, lactic dehydrogenase.

Virus titrations. (i) Blood. Hamster blood wasobtained from the retro-orbital venous plexus withheparinized capillary tubes (Arthur H. Thomas,Philadelphia, Pa.), frozen and thawed once, andscreened in mice for LCM virus at a 1:2 dilution ortitrated. Virus dilutions were made in EBME con-taining 20% veal infusion broth. Three mice wereinoculated by the intracranial route with 0.03 ml ofeach 10-fold dilution and were observed for deathsthrough day 14. Mice surviving at day 14 were chal-lenged intracranially with 1,000 mean lethal doses/0.03 ml of LCM virus CA 1371. Mice that had origi-nally received a sublethal immunizing dose sur-vived the challenge, whereas nonimmunized micedied within 8 days. The number of mice that haddied within the original 14 days after inoculationplus the number that survived challenge were com-bined, and the infectivity dose (ID-,,,/0.03 ml) wascalculated (32). Urine or blood specimens containingvirus at a 1:2 dilution and not at a 1:10 dilution wererecorded as trace positive.

(ii) Urine. Hamster urine was diluted and ti-trated following the same virus titration techniqueused for the blood.

(iii) Organs. Organs were homogenized and pre-pared as 10% clarified suspensions in EBME. Ten-fold dilutions were inoculated into weanling mice(0.03 ml intracranially, and mean lethal dose andID.;,, titers were calculated as outlined above.

Histopathology. At intervals of approximately 3to 10 weeks, four to eight of the neonatally infectedhamsters were sacrificed for histopathological ex-amination. At the time of sacrifice, blood, serum,and urine were collected. Tissues including kidney,liver, brain, lung, pancreas, uterus, and heart wereremoved and fixed in 10% neutral formalin. Tissueswere embedded in paraffin, sectioned at 5 ,tm, andstained with hematoxylin and eosin.

Serology. (i) CF. Sera or plasmas were tested forcomplement-fixing (CF) antibody by the microtitertechnique (45) using the optimal dilution of antigenand two units of complement (32). Antigen consistedof soluble CF antigen prepared from the Fortnerfibrosarcoma tumors. Twenty-percent tumor sus-pensions were prepared in EBME, and infectiousvirus was removed by centrifugation at 100,000 x gfor 90 min. These soluble CF antigen preparationsusually titered 1:16 to 1:32 against a standard anti-LCM guinea pig serum. Zones of antigen and/orantibody excess and anticomplementary sera werefrequently encountered with the hamster sera andnecessitated careful interpretation of the CF tests.At the completion of the experiment, all sera wereretested together and many were titrated by theblock titration technique (32). The end point titer

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LCM VIRUS INFECTION IN HAMSTERS

was the highest serum dilution fixing sufficientcomplement to prevent the lysis of 75% or greater ofthe sensitized sheep erythrocytes.

(ii) Neutralization. Neutralizing antibody wasassayed by the footpad technique (24). Sera to betested were diluted in twofold steps, mixed withequal volumes of virus (final concentration was 100times more than the amount of LCM virus CA 1371in 0.03 ml required to give maximal pad swelling in10 days), and incubated at room temperature for 1 h,and 0.03 ml of each dilution was inoculated into thefootpad of the hind leg of four 21-day-old mice. Theopposite footpad was inoculated with normal ham-ster serum plus diluent. The degree of swelling ofthe footpad was estimated 8 to 10 days after inocula-tion and graded on a 0 to 4+ scale, with 4+ signify-ing maximum swelling. A 0 to 1 + reading was con-sidered positive for the presence of neutralizing an-tibody.

(iii) Immunofluorescent staining. Hamster tis-sue sections were cut (6 ,um) on an Internationalcryostat model CTI. Sections were fixed and stainedusing the procedure and reagents kindly furnishedby Roger E. Wilsnack (52).

RESULTSNeonatal infection. (i) Course of infection.

Within 24 h after birth, litters from 13 hamsterswere inoculated with 0.1 ml of Fortner strainLCM virus containing 105.8 ID50/0.03 ml. Sevenlitters were inoculated by the subcutaneousroute and six by the intraperitoneal route; be-cause there was no difference in the outcome ofinfection, the data for the two groups have beencombined for presentation here. Two uninfectedlitters were held as controls. One hundred andseventy-two hamsters were inoculated withLCM virus, and 112 survived to weaning at 3weeks of age. All of the early deaths occurredduring the first 10 days after birth, and themajority was the result of cannibalism duringthe first 48 h. It is not known if any of thesedeaths were the result of the LCM virus infec-tion.From week 7 through 42 of infection some of

the hamsters began to show signs of illness. Inthe earliest stages the animals became less ac-tive, their hair became disarranged, and theyhad an ungroomed appearance. As the diseaseprogressed the hamsters lost weight, assumedhunched postures, developed blepharitis, andin general gave an appearance similar to thecondition described in mice as late or wastingdisease (21). The condition of these hamstersslowly deteriorated until they became mori-bund several weeks or months after the firstsigns of clinical disease.Of the 57 hamsters alive at 42 weeks, 26 (46%)

were ill, whereas the remaining 31 (54%) werenormal, without any apparent clinical effects ofthe infection. Neither litter origin nor sex had

any influence on the clinical outcome of infec-tion.At 3- to 10-week intervals, beginning at 4

weeks of age, blood for virus and CF antibodytesting was obtained from each hamster. Urinesamples also were obtained from some ham-sters and titrated for virus. At each time inter-val, except for week 94 when only one hamsterwas examined, four to eight hamsters were sac-rificed for histopathological examination andfor titration of virus in organs; generally, equalnumbers of clinically well and ill hamsterswere chosen for sacrifice at each time point. Forthe purpose of data analysis, the animals canbe grouped into five catagories: (i) seriallytested animals alive at 42 weeks that showed noclinical illness = healthy group (31 hamsters);(ii) serially tested animals alive at 42 weeks butthat had developed the typical clinical disease= diseased group (26 hamsters); (iii) clinicallyill hamsters selected for sacrifice before 42weeks (19 hamsters); (iv) clinically well ani-mals sacrificed before 42 weeks (12 hamsters);and (v) 24 animals lost from the longitudinalstudy because of accidental deaths or use inbreeding experiments. Since category (iv) ani-mals might have developed illness if they hadbeen allowed to live, they do not represent ahomogeneous group, and their virological dataare not presented here. In comparing the longi-tudinal virological and serological findings inthe healthy and diseased hamsters, only cate-gories (i) and (ii) are used.The comparative virology of these two groups

of hamsters is shown in Table 1 and Fig. 1 and2. Through week 7 of infection, the virology andappearance of all the hamsters was similar.Viremia was present in all animals, with titersranging from 102-2 to 1050° ID50/0.03 ml of blood.In the group of diseased hamsters, CF antibodytiters increased steadily through week 16 ofinfection and viremia titers decreased slightly;however, after week 16 this pattern began toreverse, and viremia titers increased and CFantibody titers decreased. Titers of virus in theurines showed a small but continuous increasethrough week 59. One diseased hamster thatlived for 94 weeks still had a urine virus titer of104-0 ID.,,,/0.03 ml of urine. All of the diseasedhamsters remained viremic through at leastweek 49, at which time they had an averageviremia titer of 104-3 ID50/0.03 ml of blood. Serafrom these hamsters were frequently anticom-plementary, probably as the result of circulat-ing antigen-antibody complexes, which boundcomplement. After week 59, the data compiledon the diseased hamsters are probably biasedby the small sample size, since only a few of the

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TABLE 1. Course ofLCM infection in hamsters inoculated less than 24 h after birth

Clinical Weeks of infectionAssay | conditior| Palrameter

4 7 12 16 21 25 33 42 495968 78 91-94

Viremia Diseased No. tested 26 26 26 24 2 26 26 26 21 13 5 1% Positive 100 100 100 100 100 100 100 100 100 92 80 100Virus titee Nl"' 4.0 3.7 3.4 3.8 4.1 4.2 4.2 4.3 4.0 4.1 4.3

Healthy No. tested 31 31 31 31 14 18 31 31 21 24 16 12% Positive 100 100 84 55 21 28 6 6 5 8 0 0Virus titer NT 4.5 2.9 2.1 1.3 1.8 1.0 NT TraceNT 0 0

Virus in urine Diseased No. tested 6 2 4 7 13 4 4 4 2 1% Positive 100 100 100 100 100 100 100 100 100 100Virus titer 4.0 4.6 4.4 5.0 4.6 5.4 5.4 5.4 4.0 4.0

Healthy No. tested 5 2 7 6 5 4 4 3 2 14% Positive 100 100 100 100 100 75 100 67 0 0Virus titer 4.2 4.6 3.9 3.2 2.9 2.9 1.5 1.4 0 0

CF antibody Diseased No. tested 26 26 24 26 26 26 26 26 22 12 6 4% Positive 54 85 96 96 88 77 77 77 64 75 67 50Antibody titer' 20 68 121 134 87 88 67 47 40 40 79 158

Healthy No. tested 31 31 19 30 30 31 31 31 29 24 18 16 12% Positive 68 87 100 100 97 100 97 97 97 100 89 94 92Antibody titer 23 105 340 355 10 237 166 98 92 55 54 55 75

Diseased, 26 hamsters that developed clinical disease (by week 42). Healthy, 31 hamsters that never developed disease.bUsually three to five positive hamsters were titrated. Titers are expressed as the mean log, IDdO/0.03 ml of blood or

urine.c NT, Not titrated.dReciprocal of geometric mean serum dilution.

diseased hamsters remained in the experimentat this time.

In the group of hamsters designated ashealthy, CF antibody titers were consistentlyhigher than CF titers in diseased hamsters,peaked at week 16 at a titer of 1:355, and thengradually declined to plateau at a mean titer ofapproximately 1:55. Viremia titers of thehealthy hamsters decreased rapidly after week12, and only two hamsters (6%) were viremic at33 weeks; at this time all of the 26 diseasedhamsters were still viremic. Viruria persistedlonger than viremia in the healthy animals,and two of three hamsters still were excretingvirus in their urine after 59 weeks of infection(102.2 and 100.8 ID-,,/0.03 ml of urine).Three representative hamsters from the in-

fected newborn group on which serial bleedinghad been throughout the experiment weretested for neutralizing antibody (Table 2). Onehamster, L5H2, which cleared its viremia byweek 12 and its viruria by week 42, developeddetectable CF and neutralizing antibody byweek 7 that persisted through 78 weeks. On theother hand, neither of the two diseased ham-sters tested, one of which did have detectableCF antibody, ever had high titers of circulatingneutralizing antibody. Again this may havebeen the result of the large quantity of circulat-ing virus rather than lack of adequate antibodyproduction.

(ii) LCM virus titers in the organs of ham-sters infected as newborns. Organs from ham-sters selected for pathology were titrated fortheir infectious virus content. Results fromthose diseased at the time of sacrifice (cate-gories [ii] and [iii]) are shown in Table 3, andresults on hamsters that remained healthy for42 weeks or more (category [iv]) are shown inTable 4. From hamsters with signs of diseasethat were sacrificed for histology, the followingwere titrated for infectious virus: blood, urine,and a portion of the kidney, brain, lung, andliver (Table 3). In the diseased hamsters, hightiters of virus were found in the organsthroughout life. Virus titers were highest inthe kidney, slightly lower in the liver, lungs,and urine, and lowest in the blood and brain.

In the healthy animals, tissue extracts weregenerally noninfectious or contained only lowtiters of virus (Table 4); kidney and urinetended to be positive more often than did blood,lung, liver, or brain.

(iii) Histopathology of neonatally infectedhamsters. The pathology of hamsters infectedneonatally with LCM virus was followed from 4through 94 weeks of infection. A retrospectivecorrelation of the pathological findings with theclinical, viral, and antibody status of each ham-ster revealed two distinct types of infection.Those animals that cleared their initial viremiaand viruria and remained healthy developed

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LCM VIRUS INFECTION IN HAMSTERS 971

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WEEKS OF INFECTION

FIG. 1. Course of LCM virus infection in hamsters inoculated within 24 h after birth. Symbols: 0, CFantibody titer (reciprocal of geometric mean); 0, viremia titer (mean log,); A, viruria titer (mean log,,).Approximately halfof the group of infected hamsters was able to suppress and clear their viremia and viruriaand remained healthy throughout their lives, whereas the other hamsters (46%) continued to be viremic andviruric and developed a fatal disease.

only moderate and transient lymphocytic infil-tration of the viscera, whereas those hamsterswith persistent viremia, viruria, and clinicalillness showed persistent lymphocytic inflam-mation, widespread vasculitis, and progressiveglomerulonephritis. Uninoculated control ham-sters examined throughout the experimentshowed no significant inflammatory reactionsand no evidence of vasculitis or glomerulone-phritis.

During the first few weeks of infection allanimals showed similar histological findings.At 4 weeks there was minimal evidence of aninflammatory response. By 7 weeks there were

scattered lymphocytic infiltrates, primarily inthe liver and kidney. At weeks 12 and 16 postin-fection, the healthy hamsters showed moderatenodular lymphocytic infiltrates of the liver andkidney and scattered lymphocytic inflamma-tion of pancreas, lung, and meninges. These

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972 PARKER ET AL.

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hamsters that cleared their infection, recovered, and remained healthy; 0, hamsters with chronic viremia andviruria that developed disease. All of the diseased hamsters remained viruric and most were viremic all oftheir lives, whereas many of the hamsters without disease had cleared their viremia by 33 weeks and theirviruria by 80 weeks after infection. Both healthy and diseased hamsters developed CF antibody.

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TABLE 2. Development ofCF and neutralization antibody in three hamsters infected with LCM virus within24 h after birth

Hamster Weeks of infectionAssay

No. Condition 4 7 12 16 21 25 33 42 49 59 68 78 94

L5H2 Healthy CF antibody - 320" 640 640 320 640 320 160 160 160 80 160bNeut. antibody - 10 .40 240 .160 160 . 320 -320 -320 160 40 NTJViremia + + - - NT NT - - - - NT

L12H8 Diseased CF antibody - 20 320 40 80 160 80 40 40bNeut. antibody - +. - ++ + + + + +Viremia + + + + NT + + + +

LlH1O Diseased CF antibody - 20 - _ - _ - -Neut. antibody - ±+- _ -_ - _ _+ + 4-Viremia + + + + NT + + + + + NT + +

Reciprocal serum antibody titer.Sacrificed for histopathological study.

C NT, Not tested.d Partial inhibition of footpad swelling response at a 1:5 serum dilution.

TABLE 3. Virus titers in blood, urine, and organsfrom diseased hamsters infected with LCM virus

within 24 h after birth

Weeks of Mean virus titer" (log,o)infection Blood Urine Kidney Lung Liver Brain

7 4.1 4.0 5.2 5.4 4.4 4.412 2.1 5.1 4.5 3.8 2.916 3.9 4.6 5.3 4.2 4.2 3.921 3.4 4.1 5.1 4.5 3.8 3.426 3.8 3.9 5.1 4.4 4.1 3.233 4.1 5.0 4.6 4.9 5.5 3.839 5.5 5.0 5.5 4.242 4.3 5.2 5.4 4.2 5.0 3.949 4.4 5.3 5.5 4.9 5.2 3.559 3.8 5.5 5.5 5.5 5.5 3.374 3.9 4.0 5.5 4.5 3.9 4.294 4.2 4.0 5.5 5.5 5.0 4.8

Virus titers of one to five hamsters are ex-pressed as ID50/0.03 ml of blood, urine, or 10% organsuspensions. Titrations were carried through a10-; " dilution. For calculating mean titers whenend points exceeded the highest test dilution of10-i °, a titer of 10-;' was considered the end point.

changes subsided, and throughout the rest ofthe study the healthy group showed only slightscattered chronic inflammation of the visceraand no histological evidence of vascular or glo-merular disease.

In contrast, those animals that did not cleartheir infection began to show more markedpathological findings at 12 weeks after infec-tion. At this time, the lymphocytic infiltrateswere quite intense and widely distributedthroughout the organs, including liver, lung,pancreas, spleen, kidney, meninges, and brain.The inflammatory lesions were most marked in

the liver and consisted of large nodular peripor-tal aggregates of lymphocytes with a fewplasma cells. Subacute and chronic interstitialpancreatitis was quite striking and in somecases was accompanied by acinar atrophy andinterstitial fibrosis. In the brain there werefocal collections of lymphocytic cells in the me-ninges and a scattered but rather striking peri-vascular mononuclear infiltrate within the cor-tex. In addition to chronic interstitial nephritis,there were slight proliferative changes in theglomerular tufts and slight basement mem-brane thickening.At 16 weeks the inflammatory infiltrates

were generally much the same as noted at 12weeks. However, the character of the renal pa-thology had changed somewhat in that the in-flammatory infiltrate was less pronounced, butglomerular and renal vascular involvementhad become quite apparent. The glomerularlesion was primarily that of basement mem-brane thickening with occasional small foci offibrinoid degeneration in the glomerular tufts."Wire-loop" lesions similar to those seen in lu-pus erythematosis were apparent in some of theglomeruli. In addition, there were occasionalrenal arterioles, with thickening and hyaliniza-tion of the wall and eosinophilic smudgingresembling fibrinoid degeneration. Vascularlesions also were noted in the uterus, in whichrather striking changes of arterioles and smallarteries were apparent. These changes con-sisted of perivascular infiltrates of lympho-cytes, monocytes, and plasma cells, adventitialproliferation, and thickening, and fibrinoiddegeneration of the media of the vessels withconcomitant narrowing of the lumens.

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At 21 weeks the inflammatory infiltrates nant and most striking pathological findingwere seen in the same organs as previously was the above-described vascular disease. Thementioned and, in addition, were quite promi- widespread panarteritis involved the kidney,nent in the heart, skeletal muscle, and urinary uterus, heart (Fig. 3), pancreas, and sometimesbladder. Vascular changes in the kidney and the gonads, urinary bladder, intestine, anduterus were as noted at 16 weeks. skeletal muscle.From week 26 to 42 of infection the predomi- After week 42 only slight visceral vascular

TABLE 4. LCM virus titers in healthy hamsters 42 to 74 weeks after infection as newborns

Weeks of in- Parameter Blood Urine Kidney Lung Liver Brainfection

42-49 No. positive/no. tested 1/4 3/4 2/4 0/4 0/4 1/4Virus titer" Trace 2.0 2.4 _b - 1.0

59-74 No. positive/no. tested 0/3 1/3 0/2 0/3 0/2 0/3Virus titee" - 0.75 - - - -

" Mean virus titer (log,,,) of positive hamsters.b < 10 - (.3

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disease was present, and the most significantfindings become progressive chronic renal dis-ease. Grossly, the kidneys were pale and uni-formly granular. Microscopically, the picturewas that of chronic glomerulonephropathy withlobular simplification of the tufts, crescenticadhesions, basement membrane thickening,and glomerular sclerosis and obliteration (Fig.4). Renal arteriolar disease was frequently as-sociated with the glomerular changes. Irregu-lar granular deposits ofgamma globulin, and toa lesser extent LCM virus-specific antigen,were seen by immunofluorescence along theglomerular basement membranes and the wallsof involved arterioles at the height of the dis-ease.

Significant differences in the histology of the

lymphoid tissues of the infected hamsters,either healthy or sick, from that of the uninocu-lated control hamsters were not apparent inthis study.

Congenital infection. In a preliminary studya pregnant, viremic hamster that had been in-fected with LCM virus at birth was sacrificed,and her litter of three pups was removed byhysterectomy. A 10% suspension of the pooledviscera from these embryos had a virus titer of10J3 ID.;,/0.03 ml. To further study the congeni-tal infection, breeding pairs of neonatally in-fected hamsters were established. From thesepairings 15 pregnant hamsters resulted.Whereas all of these females were at one timeviremic, at the time of pregnancy 10 alreadyhad cleared their infections. These 10 females

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FIG. 4. Chronic glomerulonephropathy of a clinically ill hamster 10 months after LCM virus infection.There is marked glomerular hyalinization, basement membrane thickening, and arteriolar thickening,hyalinization, and chronic perivascular inflammation. Hematoxylin and eosin stain, -250 x.

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976 PARKER ET AL.

gave birth to 54 normal appearing pups, none ofwhich had viremia or virus in tissue extracts.The remaining five pregnant hamsters were

viremic during pregnancy and gave birth to 19pups. All three hamsters from one litter andone to three sucklings from each of the remain-ing four litters were sacrificed on the day ofbirth, and pools of the viscera from each litterwere titrated for LCM virus. All of the fivesuspensions contained from 104-- to 10) ID.-,, ofLCM virus/0.03 ml. The hamsters in two of thefour litters were never healthy and died withina few days after birth. Ten hamsters from theremaining litters were weaned and all wereviremic. At 8, 12, 16, and 20 weeks of age themean viremia titers of these hamsters were

1036, 103-6, 1032, and 103-' ID5,,/0.03 ml of blood,and their mean viruria titers were 103-1, 104-4,103, and 104.0 ID.-,,/0.03 ml of urine, respec-tively. Of eight hamsters tested at week 20, sixwere viremic and all eight were viruric. Threeof four hamsters tested at 70 weeks were stillviremic (104.0 ID-.,,/0.03 ml of blood).The breeding history of one of the female

hamsters (L1OH2), mated to an infected male,is illustrated in Table 5. This female was vi-remic during the first of her two pregnancies,and all hamsters in her first litter were born

infected. Two of the infected female F, litter-mates were bred by a littermate, and they like-wise gave birth to LCM-infected litters (F2 gen-eration). One of these infected littermates(F2H1O) was bred and also gave birth to an

infected litter (F:1).The course of infection in congenitally in-

fected hamsters was similar to that in hamstersinoculated with LCM neonatally, in that somewere able to clear their infections and did notdevelop disease whereas others remained vi-remic and developed a chronic, progressive dis-ease. The pathological lesions found in congeni-tally infected hamsters with progressive dis-ease were identical to those described in neo-natally infected hamsters with progressive dis-ease. The congenital infection seemed to havean adverse effect on breeding success in thatsmaller numbers of hamsters were born andfewer survived to weaning age, thus reducingthe number available to breed and perpetuatethe congenital infection. However, it was possi-ble to serially transmit the congenital infectionfor at least three generations.Adult infection. Twenty-five 3-week-old

hamsters were inoculated intraperitoneallywith 0.1 ml of a 10% cell-free suspension ofFortner LCM virus containing 106.3 ID,(-/O. 1 ml.

TABLE 5. Congenital LCM virus infection through three generations

Mother's virology during Litter's virology after birthpregnancy

Litter Female Virusno. parent Age Vire- CF an- No.

(weeks) mia ti- tibody born No. TitePb (mean) History of littertee, titer positive blood tissue

1 L1OH2" 12 1.8 40 7 7 4.3 F, Six weaned: (1 sacrificed)2 cleared virus, normal4 developed disease and died at

75-92 weeks of age.2 L1OH2 17 -d 320 10 0 - F, Nine weaned: (1 sacrificed)

9 normal

3 F1H3 10 4.5 80 7 7 .5.5e F2 Sacrificed4 F1H3 13 3.5 160 7 7 5.1 F2 Five weaned: (2 sacrificed)

2 cleared virus by week 173 remained viremic at week 17

5 FlHl 10 3.8 1,280 3 3 .4.5 F2 Three weaned:All cleared virus by week 30

6 F2H10 15 3.8 1,280 3 3 4.2e F:i Sacrificed

Log,(, ID5,/0.03 ml of blood.Log,,, ID50/0.03 ml of blood or tissue of hamsters tested at 4 weeks of age except litters 3 and 6.L1OH2 was inoculated with LCM virus when less than 24 h of age. This female never developed any

clinical signs of illness and cleared her viremia when 16 weeks of age. Her first litter was born when she wasviremic and her second litter was born when she was not viremic.

d( <10-03.Litters 3 and 6 were sacrificed on the day of birth, and the viscera were pooled and titrated for virus.

Titer is expressed as mean mouse ID5r,/0.03 ml of a 10% tissue suspension.

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Unfortunately, within the first 4 weeks of theexperiment, the hamsters contracted "wet-tail"disease (46), and 9 of the 25 hamsters died atthis time. Whether the LCM infection contrib-uted to any of these deaths is not known. Dur-ing the course of this study some additionalhamsters were removed for other experimentsnot presented here.At intervals through 30 weeks of infection,

the hamsters were orbitally bled, and theplasma was tested for CF antibody and bloodwas titrated for virus (Table 6). Urine also wascollected and titrated for virus.After week 4 of infection all of the hamsters

were healthy and none ever developed clinicaldisease. CF antibody appeared by day 10 in allhamsters, reached its peak titer at 60 days, and

then declined slowly; however, all of the ham-sters remained positive through 210 days (Table6; Fig. 5). Viremia was of relatively short dura-tion compared to neonatally or congenitally in-fected hamsters, and most hamsters hadcleared their viremia by 60 days. This was alsotrue of the infection in other major organs,except for the kidney in which a prolongedinfection occurred (Table 7). Urines from two ofsix hamsters tested contained virus 180 daysafter infection; the titers were 103-` and 100-'ID-,,/0.03 ml of urine.

DISCUSSIONNaturally occurring LCM virus infection in

hamsters is now recognized as an importantfactor in the overall epizootiology of LCM virus

TABLE 6. Course ofLCM virus infection in hamsters inoculated at 3 weeks of age

CF antibody Virus

Days of in- Blood Urinefection No. % Posi- Titer

tested tive (mean)" No. % Posi- Titerb No. % Posi- Titerbtested tive (ID,() tested tive (ID50)

10 19 100 66 19 100 4.830 14 100 234 14 86 1.6 14 100 3.860 14 100 286 14 29 Trace 11 91 3.190 10 100 91 10 10 Trace 10 60 2.4

120 10 100 85 10 0 -C 10 40 1.7150 7 100 44 6 33 2.0180 7 100 54 6 33 2.2210 5 100 69

Geometric mean titer, reciprocal of dilution.b Mean titer of positive hamsters only; log1), ID50/0.03 ml of blood or urine.c <10-° 3-

I-

z

0cc

0

00

m

0 30 60 90 120 150DAYS OF INFECTION

FIG. 5. LCM virus infection in young adult hamsters. Symbols: Cgeometric mean); 0, viremia titer (mean log,,,); A, viruria (mean log,).

320

-160

I-

00co

z

ox

), CF antibody titer (reciprocal of

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TABLE 7. LCM virus titers in blood, urine, and organs of hamsters inoculated at 3 weeks of age

Day after in- Hat CF anti- Virus titersbfection body tite!dl Blood Urine Kidney Liver Lung Brain

10 H10 80 .5.5 NT' .4.5 .5.5 5.0 4.8H13 80 -5.5 NT .5.5 4.2 .5.5 5.0

60 H21 320 -d 2.5 4.5 - - -H2 40 Trace 4.2 3.8H4 320 - 2.5 3.8 - - -

Reciprocal of serum dilution.bLog1o ID50/0.03 ml of blood or urine and 10% tissue suspension of kidney, liver, lung, and brain." NT, Not tested.d - <10-0.1

infections in laboratory animals and as an im-portant source of infection to humans (1, 2, 4, 8,11, 19, 33, 39, 40). Earlier reports dealing withexperimental LCM virus infection in hamsterswere often vague or conflicting. Volkert andHannover Larsen (48) reported high mortalityin newborn hamsters within 3 weeks after inoc-ulation, and, whereas viremia and CF antibodycoexisted in survivors, viremia usually per-sisted less than 3 months. In adult hamsters,Rhodes and Chapman (42) observed disease anddeath within 17 days after intracranial inocula-tion; however, Smadel and Wall (47), with theexception of three questionable animals, didnot observe disease in hamsters after intracra-nial inoculation. The hamsters cleared theirinfections after week 4 and developed CF andneutralizing antibody, which persisted for sev-eral months. Other investigators also did notobserve disease in infected adult hamsters (2,33, 50, 51). In the present study we did notobserve any definite LCM virus-induced clini-cal disease after intraperitoneal inoculation ofweanling hamsters; most animals had clearedtheir viremias after 2 months, but in some ani-mals kidney infections with viruria persistedfor 6 months. All hamsters developed CF anti-body, which persisted throughout the 6-monthtest period.

In newborn hamsters infected with the Fort-ner strain of LCM virus, about half of the ham-sters cleared their viremias within 8 monthsafter inoculation, whereas the remainder con-tinued to be viremic and viruric and developedvasculitis and chronic progressive glomerulone-phritis, and died, presumably of renal failure,at about 1 year of age. All of the hamstersappeared to be capable of a humoral antibodyresponse, although levels of both CF and neu-tralizing antibody were low and more difficultto detect in viremic hamsters.

Congenitally infected hamsters respondedsimilarly; some were capable of clearing theirinfections, whereas some were not and devel-

oped a chronic wasting disease. Impairment ofthe reproductive performance, also reported formice (35), was observed in that it was difficultto obtain full-term pregnant hamsters, litterswere small, runts were common, and littergrowth was slow. It is clear that infected femalehamsters can serially transmit the infection totheir offspring via the congenital route.The type and degree of LCM virus-induced

disease in mice is influenced by a variety offactors, which include the strain of mouse (37),the strain of virus (13, 30), the dose of virus (23,29, 48), the age of the mouse (26), the route ofinoculation (22, 23, 29, 48), thymectomy (44), X-ray irradiation (43), and immunosuppression(17, 20). It is likely that some or all of theseparameters also would influence the course ofinfection in hamsters. In fact, preliminary datafrom our laboratory suggest that LCM virus CA1371, a mouse neurotropic strain, produces anacutely lethal infection in newborn Syriangolden hamsters, whereas the Fortner straindoes not. Also, other experiments suggest thatthe strain of hamster may influence the courseof infection and that small doses of FortnerLCM virus in newborn hamsters produce onlythe self-limiting form of infection. It is notewor-thy that chronic subclinical infections with pro-longed viruria like that observed in this studyoften occur in natural infections (1, 3, 4, 19, 33)and, further, that in commercial breeding colo-nies in Germany, where enzootic infection hasbecome established, some chronically infectedhamsters develop a runt disease which may besimilar to the disease we observed in this study(14).A few statements with regard to the public

health consideration of LCM virus infection inhamsters are appropriate. The prolonged infec-tion in the kidneys and excretion of largeamounts of LCM virus in the urine of chroni-cally infected hamsters is clearly a primarysource of virus for human infection. This urine-virus biohazard is enhanced because most


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chronically infected hamsters show no outwardsigns of infection, and the ones that do showsigns of disease do so only after many weeks or

months of infection.Like LCM virus, other arenaviruses are ro-

dent associated, and chronic subclinical infec-tions with horizontal and vertical transmis-sions are the basic mechanisms of infection. Inmany respects, Machupo virus infection in itsnatural host (28), Calomys callosus, resemblesLCM virus infection in hamsters. Newborn-in-fected Calomys do not develop acute disease butbecome chronically infected with viremia andviruria, which persist at least 6 months in theabsence of neutralizing antibody. Also, theseanimals have a reduced reproductive potential.Calomys infected at 9 days of age or older do notdevelop acute disease; however, two differentpatterns develop at approximately 13 weeksafter infection (28). Half of the animals losetheir viremia and develop neutralizing anti-body but have chronic kidney infection and vir-uria; the other half continue to have persistentviremia in the absence of neutralizing anti-body. Calomys, however, apparently do not de-velop a chronic progressive glomerulonephritis.In hamsters, Machupo virus produces lethalinfection in newborns and chronic asympto-matic infections in adults (27). In adult ham-sters, viruria occurs through 72 weeks of infec-tion and neutralizing and CF antibodies de-velop by 12 weeks and persist through 46 weeks(27).

Substantial information is available to sug-gest that it is the T lymphoid cell-mediatedimmune response that is responsible for theelimination of LCM virus from infected mice(34, 49), and that T lymphoid cells are the effec-tor cells responsible for the acute and fatalneurological disease in adult mice (12, 30),whereas humoral antibodies are implicated inthe slowly developing chronic renal disease (30,38). Interference with the cell-mediated im-mune response has a decisive enhancing effecton the establishment of persistent infections (9,34, 43, 44). In chronically infected mice, theability to synthesize anti-LCM gamma globulinis reduced (38), and the glomerulonephritis ofchronic LCM disease is mediated through an

immune complex pathogenesis (10, 30). Thisalso appears to occur in the kidneys of chroni-cally infected diseased hamsters, as suggestedby the deposits of viral antigen and gammaglobulin in the damaged glomeruli and renalarteriolar walls. It is not known whether theabsence of significant serum-neutralizing anti-body titers or reduction in CF antibody titersobserved in diseased hamsters was due to ex-

cess circulating antigen or to an impairment orhyporesponsiveness in the hamsters' ability tosynthesize anti-LCM gamma globulin; how-ever, CF antibodies were demonstrated in bothrecovered and diseased hamsters. In diseasedhamsters, complete or partial suppression ofthe cell-mediated immune response could ac-count for the lack of viral clearance and chronicinfection. Further studies on the function andmaturation of the immune system in the imma-ture hamster will be required to define thefactors determining whether the animal wasable to suppress the infection and remainhealthy or would undergo continued infectionwith subsequent disease and death.

ACKNOWLEDGMENTS

We wish to acknowledge the excellent technical assist-ance of Wade Gibson, William Warfield, and William Fox,and the secretarial assistance of Barbara Twombly andVirginia Alary. We are also grateful to Fritz Lehmann-Grube for his helpful discussion of the manuscript.

This research was supported by Public Health Servicecontracts SA-43-ph-4356, SA-43-67-700, and NO1-CP-33288from the National Cancer Institute.

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