efficient induction of persistent and prenatal parvovirus infection in rats

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E L S E V I E R Virus Research 44 (1996) 67-78

Virus Research

Efficient induction of persistent and prenatal parvovirus infection in rats

Diane J. Gaertner l, Abigail L. Smith, Robert O. Jacoby*

Section o/ Comparative Medicine, Yale University School o1" Medicine, P.O. Box 208016, New Haven, CT 06520-8016, USA

Received 27 February 1996; accepted 4 June 1996

Abstract

Parvoviruses are prevalent and disruptive infectious agents of laboratory rats. Risks to rat-based research from infection are increased by the persistence of virus in immune rats and by prenatal transmission of infection. The mechanisms leading to viral persistence and prenatal infection are poorly understood and have been difficult to study for lack of reliable and humane induction methods. We report here protocols for inducing persistent and prenatal infection without causing clinical disease using the UMass strain of rat virus (RV), a common rat parvovirus. Infant rats inoculated by the oronasal route at 6 days of age had greater than 90% prevalence of persistent infection. RV-UMass also induced intrauterine infection in pregnant rats inoculated by the oronasal route. Inoculation of dams at gestation day 9 frequently caused severe disease in the fetuses whereas inoculation at gestation day 12 caused primarily asymptomatic fetal infection that persisted post partum. RV-UMass infection facilitates study of parvoviral- host interactions that are relevant to laboratory rats and which also may improve understanding of persistent and prenatal human parvovirus infection.

Keywordr: Model; Rat; Parvovirus; Infection; Persistence; Prenatal

1. Introduction

Rat virus (RV) is a common parvovirus of laboratory rats. Infection is a significant risk to research using rats because of its high prevalence

* Corresponding author. Tel,: + 1 203 7852525. Present address: Institute for Animal Studies, Albert Ein-

stein College of Medicine, 1300 Morris Park Avenue, Bronx. New York 10461-1602.

and disruptive effects which include lethal prenatal infection, illness and death in young rats and adventitious infection of cell lines and trans- plantable tumors (reviewed in Jacoby et al., 1979; Tattersall and Cotmore, 1986; Jacoby and Ball- Goodrich, 1995). Because parvoviruses preferen- tially target mitotically active cells, RV infection can potentially interfere with any biological response of rats that involves cell proliferation, such as assays commonly used in immunological re-

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68 D.J. GaerlHer cl a/. I iru,s Rcs~'arch 44 (199(>) 07 7,';

search (McKisic et al., 1995). Furthermore, parvoviruses are very stable (Yang et al., 1995) thereby increasing risks from infection for research and the health of laboratory rats.

Severe or lethal RV infection is attributable to lytic virus replication in mitotically active cells. Therefore, susceptibility is highest in fetal and infant rats (Kilham and Margolis, 1966, Jacoby et al., 1987). Clinically silent infection is, however, the most common result of exposure, irrespective of age. Silent infection in juvenile or adult rats is usually short-lived, whereas rats exposed to virus during infancy can develop persistent infection of variable duration, up to at least 6 months (Jacoby et al., 1991). The pathogenesis of persistent infection is obscure, but recent observations offer clues about relevant factors. Small amounts of virus appear adequate to sustain persistent infection. Thus, standard isolation techniques are inade- quate and explant cultures (which amplify infectious virus) are required to detect infectious virus in persistently infected rats (Paturzo et al., 1987). Additionally, in situ hybridization (ISH) has confirmed that viral DNA-positive cells are sparse in rats harboring RV for 4 or more weeks (Gaert- ner et al., 1993). They occur primarily in lymphoid tissues, endothelium and vascular muscle tunics, which are targets for virus during both acute and persistent infection. Persistent infection occurs in the presence of host immunity and it is not known how RV evades immune elimination. T lymphocytes (T cells) appear to be essential to prevent persistent infection, since inoculation of RV into athymic (T cell-deficient) adult rats in- variably results in persistence whereas euthymic adult rats develop self-limiting infection (Gaertner et al., 1989). This finding also indicates that ma- ture rats retain cells that can support persistent infection. Passive immunization of athymic rats with RV immune immunity to RV present before or virus (Gaertner et administered after

serum indicated that humoral prevents infection only if it is at the time of inoculation of

al., 1991, 1995). Immune serum inoculation of virus suppressed

infection, but did not eliminate it. Therefore, the integrity and timing of host immunity appear to be critical for determining the outcome of infection. Conversely, since RV can perturb immune

responses of adult rats (McKisic et al., 1995), it may impede anti-viral defenses, thereby influen~:- mg the severity oi duration of infection.

The ability of RV to cross the placenta adds significantly to its impact on rat-based research and to the difficulties of eliminating it from brecd- ing colonies. The course of prenatal RV infection appears to depend on virus Stl"am and dose, and the stage of pregnancy at which dams are cxposcd (Jacoby et al., 1988; Kilham and Ferm. 1%4: Kilham and Margolis, 1966, 1969). Previous studies suggested that prenatal infection can be lethal or non-lethal, but the factors that determine these outcomes are unclear. Additionally. the course of non-lethal intrauterine infection during post-natal life has not been assessed.

In-depth studies of persistent and prenatal RV infection have been hampered by a lack o[" reliable models. Prior studies with a pathogenic lield isolate, designated RV-Y, yielded considerable vari- ability in the induction of both types of refection (Jacoby et al., 1988, 1991). Furthermore, circum- stances leading to persistent RV-Y infection (inoculation of 2 day-old rats) also caused a high incidence of lethal infections. Therefore, we sought conditions that would reproducibly cause prenatal and persistent infection, while avoiding clinical disease. We report here that an isolate designated RV-UMass (Guberski et al., 1991), which also is highly pathogenic lk~r infant rats, induced persistent infection reproducibly and asymptomatically when infant rats were inoculated by a natural (oronasal) route at 6 day's of age rather than at 2 days of age. RV-UMass also effectively induced prenatal infection after oronasal inoculation of pregnant dams. Further- more, inoculation conditions could be manipu- lated to favor non-lethal infection.

2. Materials and methods

2.1. Animals

Specific pathogen-free Sprague-Dawley (SD) (Taconic, Germantown, NY) and athymic Rowett nude (rnu/rnu) rats (Rattus norvegicus) (Animal Genetics and Production Branch, National Insti-

D.J. Gaermer et al. / Virus Research 44 (1996) 67 78 69

tutes of Health and Biological Testing Branch, National Cancer Institute, Bethesda, MD) were housed and husbanded in Micro-isolator R cages (Lab Products, Maywood, NJ) as previously described (Jacoby et al., 1987). Rats were inoculated as infants (2 or 6 days old), as juveniles (4 weeks old) or as pregnant adults. Randomly selected SD rats were tested before inoculation and at the end of each experiment for antibodies to common murine viruses and Mycoplasma pulmonis by im- munofluorescence assay (Smith, 1983). SD sentinel rats housed in open cages in the same room with athymic rats also were tested serologically to confirm that experimental rats remained specific pathogen-free. Only inoculated and contact-exposed rats seroconverted to RV, and all rats remained seronegative for other agents. All animal experimentation was approved by the Yale Animal Care and Use Committee.

2.3. Necropsy and tissue collection

Infant and juvenile rats were euthanatized with carbon dioxide gas and bled by cardiocentesis. Serum was separated and stored at - 2 0 ° C . Tis- sue samples from brain, lung, liver, spleen, kidney, lymph nodes, abdominal vessels, and the gastrointestinal and reproductive tracts were fixed overnight in periodate-lysine-paraformaldehyde (McLean and Nakane, 1974), rinsed in phos- phate-buffered saline (PBS) and embedded in paraffin. Fragments of spleen, kidney and lung were explanted at the time of necropsy. Pregnant rats were injected intraperitonealty (i.p.) with hep- arin (200 IU/kg) 30 min prior to euthanasia. They were then deeply anesthetized with pentobarbital sodium and the left horn of the uterus was re- moved aseptically. Individual placentas and fetal spleens and kidneys were explanted from 4 5 fetuses from each rat. The rats were perfused with saline and then with fixative.

2.2. Virus, animal inoculation and assay for infectious virus

2.4. Histopathology and in situ hybridization (ISH)

Plaque-purified RV-UMass was obtained from Dr. Arthur Like at the University of Massachu- setts Medical Center, Worcester, MA and two stocks were prepared: the first (stock 1) by two passages in 324K cells (Jacoby et al., 1987) and the second (stock 2) by a passage in 324K cells followed by a passage in N R K cells (Guberski et al., 1991). The stocks had titers in N R K cells of 106~ TCIDs0/ml and 108.0 TCIDso/ml, respec- tively. Infant rats were inoculated oronasally (ON) with virus stock 1 by placing 10 pl of an appropriate dilution of virus on the external nares and in the mouth. Juvenile rats were inoculated in the same way with 20 /zl per site. Time-mated pregnant rats were inoculated ON with virus stock 2, 50/~1 per site, or intravenously (IV) with 100 /tl of stock 2. Tissues from inoculated rats were tested for infectious RV by explantation of multiple 1 mm tissue fragments that were cultured for 3 weeks and frozen. Explant lysates were then inoculated onto monolayer cultures of N R K cells that were held for 6 days and observed for cyto- pathic effect (Paturzo et al., 1987).

Five micron paraffin sections were mounted on RNAse-free slides coated with aminoalkylsilane (Henderson, 1989). Strand-specific 35S-labeled RNA probes were transcribed from a 1700 bp fragment spanning map units 0.19-0.52 from the 3' end of the RV-Y genome that had been cloned into PGEM3ZF (Promega, Madison, WI) (Gaert- ner et al., 1993). This region of the RV-UMass genome encodes nonstructural proteins which are highly conserved among the rodent parvoviruses (Ball-Goodrich, 1995). The plus sense probe detects virion and replicating form (RF) DNA while the minus sense probe detects RF DNA and mRNA. Such probes can, therefore, help to dif- ferentiate sites of virus replication from sites of virus sequestration (Bloom et al., 1989). Hy- bridizations were performed according to the method of Stoler and Broker as previously reported (Stoler and Broker, 1986: Gaertner et al., 1993) and tissues were counterstained with hema- toxylin and eosin and examined by light microscopy. RV-positive and RV-negative control slides were included in each hybridization. Back-

70 D.J. Gaermer ~'I a/. 17ru.~ Re.~carch 44 (1996) 67 7X

ground counts were consistently more than 4 grains per cell. Tissue sections were classified as containing no signal or mild (1 5 positive cells), moderate (5 50 positive cells), or high ( > 50) numbers of positive cells per low power field. Histopathological examinations also were performed on these sections.

2.5. Serology

Individual sera were diluted 1:10 in sterile 0.15 M saline, heat-treated (60°C for 20 min) and tested for RV-Y antibody by indirect immu- nofluorescence assay (Smith, 1983).

3. Results

3. I. R V- U M a s s injection oj' inJ'ant rats

The median infectious dose (IDs0) and median lethal dose (LDs0) for RV-UMass stock 1 were determined in 2-day old rats to establish a virus dose for subsequent experiments. Fourteen litters of SD rats were inoculated by the oronasal route with stock virus diluted 10 2.0_10 ,~.0 Mortality was recorded for 3 weeks and moribund rats were euthanatized and recorded as deaths. Sera from surviving rats were tested for RV antibody. The IDs0 was calculated from the total morbidity (seroconversions, clinical disease and deaths). The IDs0 dose was 10 7.0 per ml and the LDso dose was 10 5.3 per ml.

To determine if acute and persistent infection induced by RV-UMass resembled that previously reported for RV-Y, 2 day-old SD rats were inoculated with 20 TCIDso of RV-UMass. Groups of six rats were examined at weeks 1 and 2 after inoculation (acute phase) and at weeks 4 and 8 (persistent phase). Rats were weaned at 3 weeks of age, then individually housed to prevent cross- infection.

Clinical signs of acute RV infection occurred 9 -19 days after inoculation and consisted of stunted growth, anorexia, diarrhea, icterus, ataxia and high mortality. The lesions and distribution of RV-UMass D N A closely resembled that found previously during acute RV-Y infection of infant

rats (Gaertner et al., 1993), but RV-DNA-pos imc cells occurred at higher prevalence especiall> in lymphoid tissues and muscular tunics of blood vessels and intestines (Fig. 1). Vascular endolhe- lium was RV positive in all affected tissues and was often associated with focal hemorrhage.

Infectious RV-UMass was detected in ten of eleven rats tested at 4 or 8 weeks after inoculation (Table 1). Viral D N A was found in multiple tissues at 4 weeks, especially among endothelial cells and myocytes. Hybridization of tissues col- lected at 8 weeks with a minus-sense probe detected an occasional cell containing replicating virus. Contact transmission of RV-UMass by a separate group of rats continued through week 9, but infectious virus was recovered fi'om eight of ten inoculated rats through week t3, with kidney being infected more frequently than spleen or lung among tissues examined by explant culture (Table 2). Therefore, as previously found with RV-'f . infectious virus persisted in rats that had ceased to transmit infection.

Lung

Liver

Spleen

L Nodes

Thymus

Kidney

Bra}n

Heart

Vessels

S Intestine

L Intestine

Reproductive organs

PI Week

1 4 8 1 4 8

2 Days 4 Weeks

Age @ inoculation

Key

None

Low

Moderate

High

Fig. 1. Prevalence of cells with in situ hybridization (ISH) signal for parvovirus virion DNA (RV) in tissues of groups of six Sprague Dawley rats inoculated oronasally with RV- UMass at 2 days or 4 weeks of age. Tissue sections were classified as containing no signal or mild (1 5 positive cells), moderate (5 50 positive cells), or high (> 50) numbers of positive cells per low power field.

D.J. Gaertner et al. / Virus Research 44 (1996) 67.-78 71

Table 1 Detection of infectious virus in selected tissues of Sprague Dawley rats inoculated with the rat parvovirus RV-UMass by the oronasal route

Age at inoculatiow' Necropsy at week Virus-positive tissues

Spleen Kidney Lung

Total infected rats

2 days

4 weeks

I 5/5 5/5 5/5 5:5 2 5/5 5:5 5/5 5/5 4 3/'3 3/3 3/3 3/3 8 2/8 7/8 4/8 7/8

1 5/6 4/6 4/6 5/6 4 5/6 3/6 5/6 5/6 8 1/6 0,/6 0/6 0/6

~ Two day-old rats were inoculated with 20 TCIDs0 and 4 week-old rats were inoculated with 800 TCIDs~ ~.

3.2. R V- UMass infection o f juvenile rats

Juvenile rats were inoculated oronasally route with 800 TCIDs0 of RV-UMass and examined at 1, 4 or 8 weeks. All rats seroconverted, but remained clinically normal. Explant cultures indicated that the prevalence of infection was high at 1 and 4 weeks, but low at 8 weeks. (Table 1). The frequency of RV-DNA-positive cells during acute infection (1 week after inoculation) was low compared with that found in rats inoculated with RV-UMass as infants, but greater than previously found in juvenile rats inoculated with RV-Y (Gaertner et al., 1993) (Fig. 1). Positive cells were detected most often in the cervical and mesenteric lymph nodes during acute infection, but were less common at 4 and 8 weeks.

3.3. Induction o f persistent R V infection

The foregoing results suggested that RV- UMass could be used to induce persistent infection efficiently and humanely if the severe effects of acute infection during infancy were alleviated. Preliminary trials to circumvent pathogenic infection and enhance persistent infection had begun earlier using RV-Y as the inoculum. Varying factors had been tested, including rat strain, age at inoculation, virus dose and the role of passive immunization (data not shown). Although none of these variations yielded a completely satisfac- tory model they demonstrated that resistance to

lethal RV infection by 6 days of age with older rats remaining partially susceptible to persistent infection.

Responses of 6 day-old euthymic and athymic infants to RV-UMass were compared in a preliminary experiment to determine if persistent infection was preceded by clinical disease and if T cells were essential for resistance to lethal infection. No clinical signs were observed in either group and all rats remained infected 4 and 8 weeks after inoculation (Table 3). The distribution of RV-DNA- positive cells in euthymic rats 1, 4 and 8 weeks after inoculation was similar to that previously found in euthymic rats inoculated at 2 days of age (Fig. 2). The number of positive cells was, however, higher in athymic rats than in euthymic rats. During persistent infection (4 and 8 weeks) signal in both types of rats occurred predominantly in lymphoid tissues and blood vessels (Fig. 3). The intestinal muscle tunics were also involved, especially in the athymic rats. However, no lesions were found in either group during acute or persistent infection. These results showed that RV- UMass could induce persistent infection without causing clinical disease and that age-related resistance to disease was not T lymphocyte-dependent. To confirm the reproducibility of the model, 16 additional euthymic infants were inoculated with RV-UMass at 6 days of age. All rats remained clinically normal and 15 had an infectious virus in one or more explanted tissues after 8 weeks, kidney being the most consistently infected (Table 3).

72 D.J. (;at'rtncr el al. l'irt~.~ Rcscardl 44 (19961 67 7.~'

Table 2 Contact transmission of infection by Sprague Dawley rats inoculated ~ith 5 ) T ( ' I D , , , of the rat put'~o~iru> l~,\"-t:M>s b\ ,h,'

oronasal route at 2 days of age'

Transmission to sentinel rats Infectious virus in index rats

Exposure week Seroconversion Spleen Kidney Lullg Total ruts

5 10/10 4+4 4 4 4 4 4 4 9 4/10 I/5 55 O5 5 5

13 0,10 4'10 610 2 l0 8 l0

" All figures indicate proportion of affected rats or tissues relatNe to the tolal number of tissues or ruts examined.

3.4. Induction of prenatal in/ection

Our prior studies with RV-Y indicated that prenatal infection was most consistent when large doses of virus were inoculated IV during mid-gestation (Jacoby et al., 1988). Therefore, initial at- tempts to induce prenatal infection with RV-UMass used similar conditions. Pregnant dams were inoculated IV with 10 7.o TCIDso of RV-UMass stock 2. When dams were inoculated at gestation day 12, fetuses from eight of nine litters developed multi-systemic necrosis within 5 7 days, and infectious virus was detected in each of 25 fetuses from 9 litters (Table 4). By contrast, dams inoculated at gestation day 18 bore clinically normal litters. However, virus was recovered from eight of 12 pups necropsied at 2 days post-partum and from eight of 24 offspring at 3 weeks of age.

To determine if administration of RV-UMass by a natural route caused prenatal infection, pregnant dams were inoculated ON with 10 v° TCIDs0 of virus at gestation day 9 or 12 and necropsied 10 days later. Each of five litters from dams inoculated at 9 days had severe multi-systemic necrosis and virus was isolated from one or more fetuses from each dam. Three of six dams inoculated at gestation day 12 had fetuses that ap- peared clinically normal and three remaining dams had both dead and normal fetuses. How- ever, fetuses in all litters were infected (Table 4). Thus, RV-UMass caused prenatal infection reproducibly after inoculation of dams by a natural route, but the pathogenicity for fetuses depended on the timing of inoculation. The results also implied that surviving progeny might remain in-

fected. To assess this possibility, two pregnant dams were inoculated ON with 10 '> TCID> of RV-UMass at gestation day 12. They bore clinically normal litters, but nine of 14 pups tested by explant culture had infectious virus 3 weeks after birth, 4 5 weeks after the dams were inocuhtted.

3.5. Pathology of prenatal inlection a/?er oronasa/ inoculation qlpregnaitt ~k#ns

Fetuses from dams inoculated at 9 days had multi-systemic necrosis and RV DNA in the lung, heart, liver, brain, intestine and lymphoid tissues. Endothelial cells and interstitial mesenchymal cells of the fetal placenta as well as umbilical vessels also were consistently virus-positive (Fig. 4). Severely affected placentas were also undergo- ing necrosis. By contrast, signal o1" lesions were rarely seen in the maternal placenta. Pathogenic infection in fetuses from dams inoculated on gestation day 12 resembled that just described. whereas normal-appearing fetuses did not have microscopic lesions and had few RV-DNA-positive cells even though they were commonly virus- positive by explant culture. Tissues fi'om three rats that remained infected three weeks post-par- turn, were examined by ISH, but no RV-DNA- positive cells were detected.

4. Discussion

RV presents significant risks to experimentation using rats (Campbell et al., 1977: Guberski et al.. 1991: Brown et al., 1993; McKisic et al., 1995) and persistence adds to risks from RV infection

D,J. Gaermer et al. ,' Virus Research 44 (/996) 67 78 73

Table 3 Detection of infectious virus in athymic and euthymic rats inoculated with oronasal route at 6 days of age

20 TCID¢ of the rat parvovirus RV-UMass by the

Experiment no. Type of rat Post-inoculation week Virus-positive

Spleen Kidney Lung Rats

I:' rnu (athymic) 4 33 3/3 3'3 3 3 8 5/5 5/5 5/5 5 5

SD (euthymic) 4 3/4 4/4 3/4 4 4 8 2,,'4 3/4 1/'4 3 4 ~

2 SD (euthymic) 8 6/16 13/16 5/16 1516

" The corresponding in situ hybridization results are summarized in Fig. 3. b The rat in which infectious virus was not detected following explantation of spleen, kidney and lung had virus-posilive cells (detected by in situ hybridization) in abdominal vessels and peripheral lymph nodes.

by prolonging opportunities for interference, especially if virus-susceptible cell populations are am- plified during experimentation. Earlier evidence suggested that RV infection can persist (Robey et al., 1968; Lipton et al., 1973). However, the most definitive evidence stems from a study in which 2 day-old rats were inoculated ON with RV-Y and remained infected for at least 6 months (Jacoby et al., 1991).

The current results describe the first model for

Lung

Liver

Spleen

L. Nodes

Kidney

Brain

Heart

Vessels

S. Intestine

L. Intestine

Reproductive organs

~st-in~ulationWeek

1 4 8 4 8

$D RNU

Key

None

Low

Moderate

High

Fig, 2. Prevalence of cells with ISH signal for RV virion in tissues of groups of six euthymic (SD) and athymic (RNU) rats inoculated oronasally with RV-UMass at 6 days of age. Tissue sections were classified as containing no signal or mild (1 5 positive cells), moderate (5 50 positive cells), or high ( > 50) numbers of positive cells per low power field.

reliable induction of persistent rat parvovirus infection in rats and extend our prior findings with RV-Y in at least three ways. First, a second field isolate of RV, RV-UMass, caused persistent infection, supporting the concept that persistence is a general feature of RV infection. Second, a higher proportion of rats infected with RV-UMass were persistently infected than previously found with RV-Y, implying that the frequency of persistent infection during natural outbreaks is likely to be RV strain-dependent. Third, the RV-UMass model confirms unpublished results showing that resistance to lethal infection develops during the first week of life although older rats remain susceptible to persistent infection. By 6 days of age both euthymic and athymic infant rats withstood a dose of virus that was lethal for 2 day-old rats and 30 of 32 developed persistent infection. Therefore, innate factors such as decreased mi- totic activity of cells in primary target tissues such as liver and brain during postnatal development may be more important than immunological maturation for the onset of resistance to clinical disease. Although the current study did not determine when resistance to persistent infection is fully developed in euthymic rats, the results suggest that the onset is gradual rather than abrupt, since even rats inoculated as young adults were often infected for at least 4 weeks.

The RV-UMass model provides a reproducible, asymptomatic persistent infection that can be evaluated sequentially using molecular hybridiza-

74 D..I. GaurlHcr ul a/. I iru.~ Rusuarch 44 (1996J ~7 7<\

Fig. 3. RV-DNA-positive cells in rats inoculated oronasally with RV-UMass and necropsied 8 weeks laler. 111 silu h}hridizatioll using a plus-sense "S-labeled RV probe. (a) Mesenteric artcr} from a euthymic (SD) rat with virus-posilivc cells in the endothelium (single arrow) and in the tunica media (double arrows), x 180. (b) Mesenteric arter 5 fl'om an athymic (rnu, rnu) rat x~ilh hear} labeling of endothelium and a I'ev,' virus-posilive cells in the lunica media (arrow}. x 90. (c) Mesenteric l x.mph node Iornl ~i eulhymic (SD) rat with virus-positive cells (exemplified at arrows) in a germinal center, x 360. (d) Mesenicric lymph node froln an ath}mic (rnu~rnu) rat with virus-posilixe cells ill a primary follicle, x I gll.

t i on , e x p l a n t c u l t u r e a n d i m m u n o l o g i c a l m a n i p u -

l a t i o n to r evea l h o w R V p r e v a i l s a g a i n s t a n al-

l i ance o f h o s t i m m u n i t y a n d t i s sue m a t u r a t i o n .

T h e n u m b e r o f R V - D N A - p o s i t i v e cells d e c r e a s e d

m o r e s h a r p l y in e u t h y m i c r a t s t h a n in a t h y m i c

ra t s b e t w e e n 1 a n d 4 weeks a f t e r i n o c u l a t i o n

s u p p o r t i n g the n o t i o n t h a t p e r s i s t e n t i n f e c t i o n in

e u t h y m i c r a t s is m o d u l a t e d , a t leas t in pa r t , by

D.J. Gaertner et al. / Virus Research 44 (1996) 67-78 75

Table 4 Effect of route and stage of pregnancy on intrauterine infection after inoculation of dams with 10 7.o TCIDs0 of the rat parvovirus RV-UMass

Virus inoculation

Route Gestation day

Days to necropsy Litters with clinical disease ~' Virus-positive fetuses

Intravenous 12 5 -7 8/9 25/25 18 7 0/7 8/12

24 b 0/7 8/22

Oronasal 9 10 5/5 9/9 12 10 3/6 t9/20

30 b 0/2 9/14

~' Proportion of animals affected/proportion of animals examined. b Infants were necropsied at 21 days of age.

host immunity. Additional comparisons in future studies should help to separate effects of immunological factors from those of innate factors during the development of persistent infection.

The threat to rat breeding colonies from prenatal rat parvovirus infection has been appreciated since Kilham and Margolis demonstrated the pathogenicity of RV for fetal rats 30 years ago (Kilham and Margolis, 1966). Reports of natural RV infection in breeding colonies have, however, often been anecdotal and based primarily on clinical signs such as production losses concurrent with seroconversions in rat colonies. This may indicate that intrauterine infections are rare, since RV strains are known to vary in their ability to cross the placenta (Kilham and Ferm, 1964; Kil- ham and Margolis, 1966, 1969; Novotny and Het- rick, 1970; Jacoby et al., 1988). Our results suggest that prenatal infection, like postnatal infection, can be silent. This finding was unexpected given the pathogenicity of RV-UMass and the susceptibility of mitotically active fetal tissues. The timing and route of inoculation of pregnant dams were at least two factors which strongly influenced the outcome of prenatal infection. Oronasal inoculation on gestation day 9 or IV inoculation on gestation day 12 typically caused lethal fetal infection whereas inoculation at later timepoints (ON inoculation at gestation day 12 or IV inoculation at gestation day 18) commonly led to silent infection. Since the efficacy of prenatal RV infection is also virus-dose dependent (Jacoby

et al., 1988), the findings support speculation that threshold concentrations of virus (i.e. viremia) are required to breach the placenta and to cause lethal infection of the fetus. They also imply that the threshold increases later in pregnancy. Prior studies indicate, not surprisingly, that such concentrations are more rapidly and easily achieved by parenteral rather than ON inoculation of pregnant dams (Kilham and Margolis, 1969). In addi- tion, the timing of maternal immunity could also influence the course and severity of infection by protecting the fetus from lethal disease, but not from silent (or persistent) infection, in much the same way that passively-acquired immunity pro- tects infant rats from lethal infection.

Earlier studies using conventional histopathology and virus isolation have intimated that the placenta is the primary barrier against fetal RV infection as well as the initial target for intrauterine infection (Kilham and Margolis, 1969). Cur- rent ISH results also suggest that placental infection preceded or was concurrent with fetal infection, irrespective of the route by which dams were inoculated. They also revealed that endothelial cells were a common target in both sites, indicating that endothelial infection may be a primary pathway for fetal infection. Thus, the outcome of prenatal infection is likely to be influenced by an interplay of factors including placental maturation, the course and intensity of maternal viremia, and the development of maternal immunity. Perhaps the most important finding

76 D.J. Ga~,rlzwr ('l a/. l iru.~ Rc.~carc/1 44 11990) (~7 v,s'

Fig. 4. RV-DNA-posit ive cells in placental and fetal tissues fl-om SD fats inoculated oronasall 5 xvith RV-Y at 0 daxs of gestalion and necropsied 10 days later. In situ hybridization using a plus-sense "S-labeled RV probe. (a) Fetal placenta alld ulnbilical ~c,,,scl. Note labeling of endothelium exemplified at arrow. × 90. (b) Higher magnitication of fetal placen'ta x~ith endothelial labeling exemplified at arrows), x 360. (c) Virus-positive cells in felal mesenteric lymph node. * 90. (d) Virus-positive cells in l)tal Iher. Hepatocytes (single arrow) and endothelium (double arrows) arc labeled, x IN().

in this series of experiments is that prenatal exposure can lead to persistent infection in rats that remain clinically normal.

While the rat models described here may have primary value for clarifying the impact of RV

infection on research animals, they also have no- table similarities to parvovirus infections of other species (Johnson, 1971; Smith et al., 1993: Jacoby et al., 1995). The most provocative comparison is to human parvovirus B19, which also causes pre-

D.J. Gaermer et al. / Virus Research 44 (1996) 67 78 77

natal and persistent infection (Jordan and Sever, 1994: Koch et al., 1993: Morey et al., 1992; Faden et al., 1992). Further analysis of the rat models should emphasize their potential for understanding this increasingly common human health prob- lem.

Acknowledgements

The authors express their appreciation for the outstanding technical assistance of Frank X. Paturzo and Elizabeth A. Johnson. This research was supported by Grant RR04047 from the United States Public Health Service.

References

Bali-Goodrich, L.J. (1995) Personal communication. Bloom, M.E., Atexandersen, S., Mori, S. and Wolfinbarger,

J.B. (1989) Analysis of parvovirus infections using strand- specific hybridization probes. Virus Res. 14, 1 26.

Brown, D.W., Welsh, R.M. and Like, A.A. (1993) Infection of peripancreatic lymph nodes but not islets precedes Kilham rat virus-induced diabetes in BB/Wor rats. J. Virol. 67. 5873 5878.

Campbell, Jr.. D.A., Staal, S.P., Manders, E. K., Bonnard. G.D., Oldham, R.K. Salzman, L.A. and Herberman, R.B. (1977) Inhibition of in vitro proliferative responses by in vivo passaged rat 13762 mammary carcinoma cells. II. Evidence that Kilham rat virus is responsible for the inhibitory effect. Cell. lmmunol. 3, 378-391.

Faden, H., Gary, G.W. and Anderson, L.J. (1992) Chronic parvovirus infection in a presumably immunologically healthy woman. Clin. Infect. Dis. 15, 595-7.

Gaertner, D.J., Jacoby, R.O., Johnson, E.A., Paturzo, F.X.. Smith, A.L. and Brandsma, J.L. (1993) Characterization of acute rat parvovirus infection by in situ hybridization. Virus Res. 28, 1 18.

Gaertner, DJ., Jacoby, R.O., Johnson, E.A., Paturzo, F.X and Smith, A.L (1995) Persistent rat virus infection in juvenile athymic rats and its modulation by immune serum. Lab. Anim. Sci. 45, 249 253.

Gaertner. D.J., Jacoby, R.O., Paturzo F.X., Johnson, E.A., Brandsma. J.L. and Smith, A.L. (1991) Modulation of lethal and persistent rat parvovirus infection by antibody. Arch. Virol. 118, 1 9.

Gaertner, D.J., Jacoby, R.O., Smith, A.L., Ardito, R.B. and Paturzo, F.X. (1989) Persistence of rat parvovirus in athymic rats. Arch. Virol. 10, 259-. 268.

Guberski, D.L., Thomas, V.A. and Shek, W.R. (1991) Induc- tion of type I diabetes by Kilham's rat virus in diabetes-re- sistant BBWor rats. Science 254, 1010 1013.

Henderson, C. (1989) Aminoalkylsilane:an inexpensive simple preparation for slide adhesion. J. Histotech. 12, 123 124.

Jacoby, R.O. and Ball-Goodrich, L.J. (1995) Parvovirus infections of mice and rats. Sem. Virol. 6, 329 337.

Jacoby, R.O., Bhan, P.N., Gaertner, D.J., Smith, A.L. and Johnson, E.A. (1987) The pathogenesis of rat virus infection in infant and juvenile rats after oronasal inoculation. Arch. Virol. 95, 251 270.

Jacoby, R.O., Bhatt, P.N., and Jonas, A.M.. (1979) Viral disease. In: HJ Baker, JR Lindsey and SH Weisbroth (Eds.), The Laboratory Rat: Biology and Diseases, Vol. I. Academic Press, New York, pp. 272 306.

Jacoby, R.O., Gaertner, D.J., Bhatt, P.N., Paturzo, F.X. and Smith, A.L. (1988)Transmission of experimentally-induced rat virus infection. Lab. Anim. Sci. 38. I I 14.

Jacoby, R.O.. Johnson, E.A., Bali-Goodrich, LJ., Smith, A.L. and McKisic, M.D. (1995)Characterization of mouse parvovirus infection by in situ hybridization. J Virol. 69, 3915 3919.

Jacoby, R.O., Johnson, E.A., Paturzo, F.X., Gaertner, D.G., Brandsma. J.L. and Smith, A.L. (19911 Persistent rat parvovirus infection in individually housed rats, Arch. Virol. 117, 193 205.

Johnson, R.H. ( 1971 ) Transplacental infection ot piglets with a porcine parvovirus. Res. Vet. Sci. 12, 570 572.

Jordan, E.K. and Sever, J.L. (1994) Fetal damage caused by parvo~iral infections. Reproduct. Toxicol. 8, 161 189.

Kilham, L. and Ferm, V.H. (1964) Rat virus (RV) infection of pregnant, fetal and newborn rats. Proc. Soc. Exp. Bio. Med. 117, 874 879.

Kilham, L. and Margolis, G. (1966) Spontaneous hepatitis and cerebellar hypoplasia in suckling rats due to congenital infection with rat virus. Am. J. Pathol. 49, 457 475.

Kilham, L. and Margolis, G. (1969) Transplacental infection o1 rats and hamsters induced by oral and parenteral inocu- hitions of H-I and rat viruses (RV), Teralology 2. I11 124.

Koch, W.C., Adler, S.P. and tlarger, J. (1993) Intrauterine parvovirus B19 infection may cause an asymptomatic or recurrent postnatal infection. Pediair. Infect. Dis. J. 12, 747 50.

Lipton, H., Nathanson. N. andHoudus, J. (1973) Enteric transmission of parvoviruses: pathogenesis of rat virus infection in adult rats. Am. J. Epidemiol. 6, 443 446.

McKisic, M.D., Paturzo, F.X., Gaertner. [).J., Jacoby. R.O. and Smith, A.L. 11995) A nonlethal rat parvovirus infection suppresses rat T cell effector functions. ,1. hnmunol. 155, 3979 3986.

McLean, I.W. and Nakane, P.K. (1974) Periodate-lysine- paraformaldehyde fixative. A new fixative for immunoelec- tron microscopy. J. Histochem. Cytochem. 22, 1077 1083.

Morey, A.L., Keeling, J.W., Porter, H.J. and Fleming, K.A. (1992) Clinical and histopathological features of parvovirus BI9 infection in the human fetus. Br. J. Obstet. Gynaecol. 99, 566 574.

Novotny, J.F. and Hetrick, F.M. {1970) Pathogenesis and transmission of Kilham rat virus infection m rats. Infect. lmmunol. 2, 298 303.

78 D.J . GuerlHct" ~'¢ u/. l iru.~ R~'seurcll 44 (1996) 67 7S

Paturzo, F.X., Jacoby, R.O., Bhatt, P.N., Smith, A.L., Gaert- net, D.G. and Ardito, R.B. (1987) Persistence of rat virus in seropositive rats as detected by explant culture. Arch. Virol. 95, 137 142.

Robey, R.E., Woodman, D.R. and Hctrick, F.M. (1968) Stud- ies on the natural infection of rats with the Kilham rat virus. Am. J. Epidemioh 88. 139 14.

Smith, A.L. (1983) Response of weanling randombred mice to inoculation with minute virus of mice. Lab. Anim. Sci, 33, 37 39.

Smith, A.L., Jacoby, R.O., Johnson, E.A.. Paturzo, F.X. and Bhatt. P.N. (1993) In vivo studies with an 'orphan ' parvovirus of mice. Lab. Anita. Sci. 43, 175 182.

Stoler, M. and Broker, T. 11986) In situ hybridization detection of human papillomavirus I)NAs and messenger R N,-\s m genital condylonaas and a cervical carcmonaa flu1/i. Pathol. 17. 1250 1258.

Tattersall, P. and Cotmore. S.[:. ( 1 9 8 6 ) T h e rodent pal-. voviruses. In: Bhatt. P.N., R.O. ,lucoby, H.C. Morse, , \ ~ . New (Eds), Viral and Mycoplasmal InfectioNs ol t.zlbol'a- tory Rats: Effects on Research. Academic Pres',. Orlando. pp. 3{)5 348.

Yang, F.-('., Pulurzo, F.X. and Jacoby, R.O. (1995) Environ- mental stability and transmission of rat virus. I.ab. \ h i m Sci. 45, 140 144.

efficient induction of persistent and prenatal parvovirus infection in rats

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