Article available online at http://www.idealibrary.com on Microbial Pathogenesis 2001; 30: 000–000

PATHOGENESISMICROBIAL

doi:10.1006/mpat.2001.0441

Susceptibility of mouse mammary glands tomurine gammaherpesvirus 72 (MHV-72)infection: evidence of MHV-72 transmissionvia breast milkHana Raslovaab, Monique Berebbib, Julius Rajcania, Alain Sarasinb,Jan Matisb & Marcela Kudelovaa

aInstitute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, 842 45 Bratislava,Slovak Republic and bC.N.R.S., Institut de Recherche sur le Cancer, UPR 2169-7, Rue Guy Moquet,94801 Villejuif Cedex, France

(Received October 2, 2000; accepted in revised form March 9, 2001)

Murine gammaherpesvirus 72 (MHV-72) is a virus of wild rodents and serves as a convenient smallanimal model to understand the pathogenesis of Epstein–Barr virus (EBV) and human herpesvirus8 (HHV8) infection. In laboratory mice MHV-72 causes an acute infection of lung epithelial cellsand establishes the latency in B lymphocytes. In this study, we investigated athymic nude andimmunocompetent mice for distribution of virus in organs after infection with MHV-72. Ten daysfollowing subcutaneous dorsal injection of nude mice, virus replicated in lungs, lymphoid organs,salivary glands and also in mammary glands. The virus titre decreased by day 21 post-infection informer tissues, but increased in mammary glands. Presence of virus DNA sequences was detectedin the lymphoid and non-lymphoid tissues until the death of the animals (about 1 month post-infection). Infection of immunocompetent mice with MHV-72 induced replication of virus up to 42days post-infection in mammary glands reaching the highest level of infectious virus at day 8 post-infection. These data show that there is latent infection in mice never detected before. Moreover,virus DNA was detected using nested PCR (by amplification of a portion of gp150 gene sequence)in the mammary glands and the milk of mouse mothers infected with MHV-72 2 days beforedelivery. We demonstrated the presence of virus DNA also in the milk removed from the stomachof non-infected newborn mice, which were nourished by infected mothers (wet-nurses) for 1 or 2days. The failure to detect the virus DNA in newborn mice lungs confirmed that they did notbecome infected from wet-nurses by the intranasal route. This suggests that MHV may be naturallytransmitted to newborn mice via breast milk. 2001 Academic Press

Key words: murine herpesvirus 4 (MHV) strain 72, murine gammaherpesvirus, nude mice, milk, virustransmission, mammary glands.

∗Author for correspondence. E-mail: virukude@savba.sk

0882–4010/01/000000+00 $35.00/0 2001 Academic Press

2 H. Raslova et al.

macrophages were found to represent the majorIntroductionreservoir of latent MHV-68 in peritoneal cells [9,16] as they harboured MHV-68 genome-positive

Murine herpesvirus 72 (MHV-72), similar to the cells with a 10 times higher frequency than theMHV-68 strain, is a natural pathogen isolated B-cell-enriched peritoneal exudate cells [16].from the wild murid rodent Chlethrionomys glare- We recently showed that MHV-72 infection ofolus [1]. Analysis and sequencing of the MHV- athymic BALB/c nude mice generated atypical68 genome has shown that several ORFs are in B lymphocytes in the blood and in the spleenpart homologous with or closely related to the [17]. Here we focus on tissue specificity and onDNA sequence of gammaherpesviruses such as the presence of MHV-72 in various organs of T-HHV 8, EBV and herpesvirus saimiri (HVS) cell deficient nude mice. The finding that the[2–5]. The polypeptide profile of MHV-72 has nude mice mammary glands were highly sus-been found to be closely related to that of ceptible to polyoma virus infection and trans-MHV68, suggesting that both isolates comprise formation [18, 19] led us to investigate thethe same virus species [6]. Both MHV isolates mammary glands of nude mice infected withare able to establish productive infection in MHV-72. These results demonstrate efficientfibroblast and epithelial cell lines derived from MHV-72 replication in mammary glands. Toseveral mammalian species, including humans determine if the susceptibility of the glandular[1, 7]. After intranasal or oral inoculations, MHV- epithelial cells to MHV infection is specific for68 spreads to lungs and then via the hemato- nude mice only, experiments with immuno-genous route to adrenal glands, spleen, liver, competent mice were also performed. Finally,kidney, heart muscle, lymph nodes, thymus and the MHV transmission via breast milk as a nat-bone marrow [8–11]. As demonstrated in im- ural route of infection was confirmed.munocompetent mice infected by the intranasalroute, the lungs are the main tissue productively

Resultsinfected with MHV, which replicates in the al-veolar epithelium and in mononuclear cells [8,

MHV-72 infection of nude mice11]. The acute infection in the lungs resolveswithin 7–10 days post-infection. Beyond this

Three groups of 10 nude BALB/c female micetime persistent and latent infection in this organwere inoculated subcutaneously with differentcan be found [8, 10]. In addition, life-long latentdoses of MHV-72 (104, 105 and 106 pfu/mouse).infection can be established in lymphoid organs,In infected animals, examined once a week,peripheral blood lymphocytes and mononuclearsevere clinical symptoms developed (hunchedcells [9, 12], providing an animal model for theback, inactivity, severe weakness and ema-study of gammaherpesvirus pathogenesis.ciation) and all the animals died within 1 monthSimilarly to the EBV, the lymphoid B cellpost-infection, whatever dose had been in-compartment is so far considered as the majoroculated. Thereafter, an additional group of ninesite of MHV-68 and MHV72 latency [9, 12].nude mice were inoculated with 106 pfu/mouse.However, the later results on MHV infection ofThree virus infected and one mock-infected micemature B cells deficient mice raised doubts aboutwere killed on days 4, 10 and 21 post-infection,this finding [13, 14]. Usherwood et al. [13] showedrespectively. Samples of lungs, spleen, lymphthat the clearance of infectious virus from lungsnodes, liver, heart, bone marrow, kidney, mam-was delayed to 10–17 days post-infection, andmary and salivary glands were removed andthey could find no evidence of infection of theinvestigated for infectious virus by plaque assayspleen or of other organs, either by reactivation[procedure (a)] at 10 and 21 days post-infection,assay or by using a sensitive nested PCR method.and for the presence of virus DNA by SouthernHowever, they confirmed previously describedblot analysis (4, 10 and 21 days post-infection).long-term persistence of MHV-68 DNA in lungs

[10, 15]. Furthermore, the acute MHV-68 splenicinfection was shown to be dependent on B- Detection of infectious virus in nude micecell maturation, since in mice depleted of Blymphocytes no viral particles could be detected, By 10 days after infection, high titres of infectiousthough latent virus was still present [14]. These virus (up to 250 pfu/mg tissue) could be de-results demonstrated that MHV latency is tected in lymph nodes and lungs (Table 1) fol-

lowed by a five-fold decrease of virus titre atnot exclusively B-cell dependent. Moreover,

MHV-72 transmission via breast milk 3

Table 1. Detection of MHV-72 in various organs of infected female nude mice

Organs

Days Mammary Heart Salivary Lymph Liver Kidney Spleen Lungs Bonepost-infection glands glands nodes marrow

Plaque assay∗10 30 0 23 250 0 27 50 250 1021 83 2 18 50 6 30 10 20 1

Southern blot analysis†4 − − − nd − − − − −

10 + − + nd − + + + ++21 + − + ++ + ++ ++ ++ +

For the plaque assay the limit of detection was 1 pfu/mg of tissue and data shown are the average of three experiments.∗ pfu/1 mg of tissue.† For details see Fig. 2.nd=not done.

day 21 post-infection. Similar decline in virus Presence of infectious virus in nude micemammary glandstitre was observed in the spleen (from 50 to

10 pfu/mg) and in the bone marrows (from 10to 1 pfu/mg) between 10 and 21 days post- Mammary glands of all MHV-72 infected nude

mice showed hyperplasia. As demonstrated, theinfection. In the kidney and salivary glands thevirus titre remained unchanged at day 10 and primary epithelial cell line, originating from ex-

planted hyperplastic mammary glands which21 post-infection (27 and 30 pfu/mg of kidneyand 23 and 18 pfu/mg of salivary glands), while had been removed from a nude mouse at day

21 post-infection [Fig. 3(a)], released infectiousin the mammary glands it increased by morethan two-fold, reaching 83 pfu/mg at day 21 virus into media. The released virus induced

typical CPE on normal murine mammary glandspost-infection. Only low titres of infectious viruscould be detected in the heart and liver (2 and cell line [Fig. 3(b)]. Subsequently to the pro-

pagation of infectious virus, the primary cell6 pfu/mg of tissue) but only at day 21 post-infection (Table 1). line died.

MHV-72 spread to mammary glands ofDetection of virus DNA in nude miceimmunocompetent mice

The BamHI/E fragment [Fig. 1(a)] from MHV-72 DNA was cloned into the plasmic pUC19 as To ascertain if mammary glands of immuno-

competent mice are susceptible to MHV-72 in-previously described [20], then labelled and usedas a probe for Southern blot analysis. At day 4 fection, 12 mice were infected with the dose of

106 pfu/mouse. Mammary glands removed frompost-infection, the MHV-72 DNA sequences inquestion did not hybridize with the total DNA three mice at days 3, 8, 15 and 42 post-infection

were evaluated for the presence of virus by theextracted from mouse organs listed in Table 1.At day 10 post-infection, virus DNA sequences plaque assay [procedure (b)]. The highest level

of infectious virus was detected at day 8 post-were found in all organs examined except theheart and liver. While the heart remained neg- infection (625 pfu/mg). The virus was still in-

completely cleared from mammary glands atative 21 days post-infection, the liver showedslight positivity (Table 1 and Fig. 2). In all other day 15 post-infection, since a low amount of

infectious MHV was detected already 6 weeksorgans increased amounts of virus DNA weredetected compared with day 10 post-infection after infection suggesting persistent replication

(Table 2). These results confirmed that the MHV-(data not shown). One week later, at around 1month post-infection, all the infected nude mice 72 was also able to replicate in the mammary

glands of immunocompetent mice.were dead.

4 H. Raslova et al.

120kb

0

(a) (b)

10 20 30 40 50 60 70 80 90 100 110

P1A1 IHGMFRA2UKQP2

NJBESLOT

D CBum repeat

18 21 22

17 19 20ORFs

gpF4 gpR2

gpF7 gpRBamHI

M7 (1448 bp)

poly A

Figure 1. BamHI restriction map of MHV-68 according to Virgin et al. [6]. (a) Fragment E was cloned andused as a probe. ORF 17 is coding for the capsid protein, ORF 19 for the tegument protein, ORF 21 for thethymidine kinase, ORF 22 for the glycoprotein H. ORFs 18 and 20 correspond to unknown products. (b)Localization of nested PCR primer sets within the glycoprotein 150 gene (M7) sequence situated in BamHI/Q-K fragment of virus genome.

MHV-72 transmission via the breast milk mice (I1–I3) were infected 2 days before deliverywith 106 pfu/mouse. Two non-infected pregnantmice (NI4, NI5) were used as a control. Together,To determine whether the MHV-72 could be

transmitted via breast milk, three gestational four mouse babies born of infected mothers (I1a,I1b, I2a and I3a), six ones born of non-infectedmothers but nourished by infected mothers (wet-nurses) (NIa, NIb, NIc, NId, NIe, NIf) and onecontrol mouse baby (NIg) were investigated forthe presence virus by nested PCR (summarizedin Tables 3 and 4).

The virus DNA sequences were found at 10

Table 2. Time course of MHV-72 titres detected inmammary glands of immunocompetent mice aftersubcutaneous dorsal inoculation

Days post-infection Plaque assay∗

3 10±3

7.35.6

3.6

2.9

2.3

Kb NMuM

G

Splee

n

LungM

amm

ary g

lands

Liver

Lymph

. nod

es

Saliva

ry gl

ands

Kidney

Bone

8 625±18Figure 2. Detection of MHV-72 DNA sequences in 15 23±5different nude mice tissues at 21 days post-infection. 42 22±4Southern blot analysis of total DNA (8–10 �g/lane)digested with BamHI and probed with the 32P-labelled The limit of detection was 1 pfu/mg of tissue and datapUC/BamHI/E vector. Normal murine mammary shown are the average of experiments performed on threegland cell line (NMuMG) DNA was used as a neg- mice.

∗ pfu/mg of tissue.ative control. Standard ladder bands are on the left.

MHV-72 transmission via breast milk 5

(a) (b)

(c)

Figure 3. Cytopathic effect on murine mammary gland cells. (a) Primary epithelial culture originating fromhyperplastic mammary glands of nude mouse infected with the MHV-72 for 21 days; (b) CPE on normalmurine mammary gland (NMuMG) cell line induced by the supernatant from (a) observed after 36 h ofcultivation; (c) mock infected NMuMG cell line.

days post-infection in breast milk and mammaryglands of two infected mothers (wet-nurses) (I1, Table 4. The MHV-72 transmission via breast milkI2) and also in the lungs of mother I1. The failure detected by nested PCRto detect the virus DNA sequence in the lungsof infected mother I2 could be explained by Wet-nursesmore effective clearance of MHV-72 from its

Mouse babies I1 I2 I3respiratory tract [Table 3(a) and Fig. 4(a)–(c)].

NIaa lungs −st.c. +liver ndTable 3. Detection of MHV-72 by nested PCR in

organs and body fluids of immunocompetent mice NIba lungs −st.c. +

(a) Infected mothers liver ndNIca lungs −I1 I2

st.c. −liver ndLungs + −

Mammary glands + + NIda lungs −Milk + + st.c. +

liver nd(b) Mouse babies

NIea lungs +st.c. +I1aa I1ba I2aa I3aa NIgb

liver ndLungs − − − − − NIfb lungs −Stomach − − + + − st.c. +content liver −Liver nd nd nd − −

Mouse babies born of non-infected mothers and nourishedby infected wet-nurses: a for 2 days, b for 1 days (also seea Born of and suckling infected mothers.

b Born of and suckling non-infected mothers. Fig. 4).nd=not done.nd=not done.

6 H. Raslova et al.

(c) 1

(a) 1

1

(b) 1

(d)

2 23 34 45 56 67 78 89 910 1011 1112 1213 1314 14 15

2 3 4 5 6 7 8 9 10 11 12 13 14 152 3 4 5 6 7 8 9 10 11 12

Figure 4. Agarose gels showing the detection of MHV-72 gp150 gene by nested PCR yielding 640 bp productin organs and body fluids of infected and non-infected immunocompetent mice (for more details see Fig.5). (a) Infected mouse I1, her own babies I1a, I1b and non-infected mouse babies after 2 days nutritivecontact with mouse I1. Lanes: 1, 100 bp ladder; 2, NMuMG cells infected with MHV-72 (positive control); 3,I1 milk; 4, I1 mammary glands (mam.gl.); 5, I1 lungs; 6, I1a stomach content (st.c.); 7, I1a lungs; 8, I1b st.c.;9, I1b lungs; 10, I1+NIa st.c.; 11, I1+NIa lungs; 12, I1+NIb st.c.; 13, I1+NIb lungs; 14, H2O. (b) Infectedmouse I2, her own baby I2a and non-infected mouse babies after 2 days nutritive contact with mouse I2.Lanes: 1, 100 bp ladder; 2, NMuMG cells infected with MHV-72 (positive control); 3, I2 milk; 4, I2 mam.gl.;5, I2 lungs; 6, I2a st.c.; 7, I2a lungs; 8, I2+NIc st.c.; 9, I2+NIc lungs; 10, I2+NId st.c.; 11, I2+NId lungs; 12,H2O; 13, 100 bp ladder; 14, I2+NIe st.c.; 15, I2+NIe lungs. (c) Mouse baby (NIg) born of non-infected mouseNI5, mouse babies born of infected mouse I3, non-infected mouse babies after 1 day nutritive contact withmouse I3. Lanes: 1, 100 bp ladder; 2, NMuMG cells infected with MHV-72 (positive control); 3, NIg st.c.; 4,NIg lungs; 5, NIg liver; 6, I3a st.c.; 7, I3a lungs; 8, I3a liver; 9, I3+NIf st.c.; 10, I3+NIf lungs; 11, I3+NIfliver; 12, H2O. (d) BamHI digestion (yielding 442 and 198 bp fragments) of nested PCR products from positivesamples in (a)–(c). Lanes: 1, 100 bp ladder; 2, NMuMG cells infected with MHV-72 (positive control); 3, I1milk; 4, I1 mam.gl.; 5, I1 lungs; 6, I1+NIa st.c.; 7, I1+NIb st.c.; 8, I3a st.c.; 9, I3+NIf st.c.; 10, I2 milk; 11, I2mam.gl.; 12, I2a st.c.; 13, I2+NId st.c.; 14, I2+NIe st.c.; 15, I3+NIe lungs.

The stomach content, the lungs and the liver contact of newborn mice of non-infected motherswith infected mothers (as wet-nurses) feasiblefrom 5 day old mouse babies (I1a, I1b, I2a, I3a)

born of infected mothers were also examined for 1 day (I3+NIf) or 2 days (I1+NIa, I1+NIb,I2+NIc, I2+NId, I2+NIe). One newborn[Table 3(b)]. In the mouse baby born of infected

mother I2 (I2a), which was analysed at day 8 mouse (NIg) was used as a control (Fig. 5). Afternutritive contact for 1 day with a wet-nurse atpost-infection, the virus DNA sequences were

detected in the stomach content but not in the day 7 post-infection or 2 days with wet-nursesat day 8 post-infection, respectively, the stomachlungs [Fig. 4(b)]. No virus DNA sequence was

found either in the lungs or in the stomach content, the lungs and the liver of mouse babieswere investigated by nested PCR (summarizedcontents of two mouse babies born of infected

mother I1 (I1a, I1b) at day 8 post-infection [Fig. in Table 4). The MHV-72 DNA sequences foundin the stomach content of five mouse babies4(a)]. In the mouse baby born of infected mother

I3 (I3a), analysed 7 days post-infection, the virus (I1+NIa, I1+NIb, I2+NId, I2+NIe, I3+NIf)confirmed the MHV-72 transmission via breastDNA sequence was detected in the stomach

content, but not in lungs and liver [Fig. 4(c)]. milk of infected wet-nurses in the course of day7 up to 10 post-infection, i.e. during the acuteAt 5 or 6 days after the delivery, we had taken

away the newborn mice of three infected mice phase of infection [Table 4, Fig. 4(a)–(c)]. Thelungs from the four mouse babies testedfrom their mothers. Then we made a nutritive

MHV-72 transmission via breast milk 7

: non-infected mother (NI);

NI4(a)

NI5(b)

NIc NIgNId NIeNIbNIa NIf

+

I3 + NIf++

delivery

I3

I2I1

I2 + NIeI1 + NIa I1 + NIb I2 + NIc I2 + NId

: mouse baby born of non-infected mother;

: infected mother (wet-nurse) (I);

: nutritive contact

analysis of lungs andstomach content

after 2 days of nutritive contact

analysis of lungs,stomach content

and liver after 1 dayof nutritive contact

analysis of lungs,stomach content

and liver(negative control)

Figure 5. Schedule of experiments to prove the MHV-72 transmission via milk of infected wet-nurses (in thecourse of day 7 up to 10 post-infection) to non-infected mouse babies. A nutritive contact was made feasiblewith (a) wet-nurses I1 and I2 (from day 8–10 post-infection) and (b) wet-nurse I3 (from day 7 to 8 post-infection).

(I1+NIa, I1+NIb, I2+NId, I3+NIf) remained To confirm that the nested PCR products ori-ginate from the MHV-72 gp150 gene, all positivenegative. Virus detected in the lungs of one

mouse baby (I2+NIe) suggests that the virus samples were digested by BamHI endonucleaseyielding the expected 442 and 198 bp fragmentscan disseminate from the gastrointestinal to the

respiratory tract. [Fig. 4(d)].

8 H. Raslova et al.

this epithelial tissue to the MHV infection isDiscussionspecific for nude mice, we also investigated themammary glands of immunocompetent mice.

It has been established that in immuno- In contrast to the results obtained in nude mice,competent mice [8–12] MHV spreads mainly via where acute virus replication increased untilthe blood stream and that the preferential sites their death, in immunocompetent mice a per-of infection are the lungs and the lymphoid sistent infection was established in mammaryorgans. Our results in nude mice showed that glands around 2 weeks post-infection. Reachingthe virus replicates in lymphoid organs, lungs, the highest level at day 8 post-infection in mam-salivary glands, kidney, heart, liver and mam- mary glands of immunocompetent mice, a lowmary glands. It should be stressed that infectious amount of infectious virus could already bevirus was present in the mammary glands of found 6 weeks post-infection.both nude and immunocompetent mice, but per- Some herpesviruses, e.g. human cyto-sistent infection could only be found in the latter. megalovirus, herpes simplex virus, varicella-However, it cannot be excluded that the only zoster virus and Epstein–Barr virus have beenreason nude mice do not have persistent in- associated with congenital or perinatal diseasefection is that they died within 1 month. Pre- [24–28]. The study of mother–to–child verticalviously, Usherwood et al. [13] and Weck et al. EBV transmission proved by the means of nested[14] did not find detectable infectious MHV-68 PCR strongly supported the possibility of EBVin the spleen of B cell deficient mice at any transmission in utero or during delivery [29].time after infection, indicating that mature B HCMV has been shown to be shed into thelymphocytes are indispensable for acute splenic breast milk of seropositive women 1 month afterinfection. Yet, the spleen cells of these mice delivery [30]. Here we demonstrated that MHVharboured latent virus 6 weeks post-infection, can be transmitted via breast milk during acutethus demonstrating that MHV-68 establishes lat- infection of mammary glands. In the stomachency in other cell types than mature B lym- content of five mouse babies (I1+NIa, I1+NIb,phocytes. Weck et al. [16] tried to determine I2+NId, I3+NIf) nourished by three differentwhether hematopoietic cell types, other than B infected wet-nurses (I1, I2 and I3) MHV DNAcells, carried latent MHV-68. Their experiments sequences were found in the absence of prenataldemonstrated MHV-68 latency in two different transmission and of intranasal infection. Whilehematopoietic cell types, macrophages and B virus DNA was detected in the milk and mam-cells, and suggested that bone marrow was a mary glands of both infected mouse mothers (I1reservoir for latent MHV-68. In our T cell de- and I2) at day 10 post-infection, the lungs wereficient nude mice model, the acute infection positive in one (I1) and negative in another ofappeared 4 days post-infection and decreased them (I2). This finding could be explained byafter 10 days post-infection in all lymphoid or- faster clearance of virus from the respiratorygans, while a high level of virus DNA sequences tract of mouse I2 compared with mouse I1.could be detected in the lungs and in lymph Two mouse babies of mouse I1 harboured virusnodes up until the animals died. However, the neither in the lungs or in stomach content, whilevirus which gets cleared from nude mice lungs in the mouse baby born of infected mother I2,to a great extent, but not completely, is also fully the virus was detected in the stomach content.cleared from all non-lymphoid organs. These This supports the notion that unequal amountsresults are in agreement with the finding that of virus might be present in the milk of botheven after subcutaneous dorsal injection, the mouse mothers, the mouse I2 being more ex-main site of productive virus replication remains tensively infected and shedding more virus intothe lungs [10, 21]. milk during the nursing period (2–8 days post-

Our study is the first to report the presence of infection). It should be mentioned that the mouseMHV-72 in mammary glands. It was previously babies born of infected mice were killed andreported that gammaherpesvirus EBV could be investigated after 6 days suckling (at day 8 post-associated with human breast cancer [22, 23]. infection), 2 days before the milk of their mothersThe results, presented here, could corroborate was tested. At day 8 post-infection, when thesuch hypothesis in that the infectious virus and highest level of infectious virus in mammaryMHV-72 DNA sequences were detected in the glands was detected by us, a nutritive contactnude mice mammary glands exhibiting signs of of non-infected mouse babies with infected

mothers I1 and I2 (now as wet-nurses) had beenhyperplasia. To determine if susceptibility of

MHV-72 transmission via breast milk 9

made feasible. In the presented study the MHV- can be helpful for better understanding of ver-tical herpesvirus infection via breast milk.72 was transmitted via milk from infected wet-

nurses to five mouse babies after 1 or 2 dayssuckling. It was previously shown that followingintranasal infection titres of infectious virus are

Materials and Methodspeaking in the lungs at days 6–10 post-infection,but it could be detected there at the first day

Virus and cellspost-infection [8, 21]. On the other hand, a doseneeded for such early appearance of virus in the

Murine gammaherpesvirus strain 72 was kindlylungs is minimally 5×105 pfu/mouse. However,provided by J. Mistrikova from the Faculty ofto avoid the risk of intranasal infection of mouseNatural Sciences, Comenius University, Slovakbabies from infected wet-nurses, we chose 1 orRepublic. Stable normal murine mammary gland2 day nursing intervals only. Four mouse babiescell line (NMuMG, ATCC CRL-1636) was prop-born of a non-infected mother and nourishedagated in Dulbecco’s modified Eagle’s mediumby infected wet-nurses had been shown to be(DMEM, Gibco) supplemented with 10% (v/v)virus negative in the lungs (I1+NIa, I1+NIb, of fetal calf serum (FCS). The MHV-72 clone 8I2+NId, I3+NIf), but virus positive in stomach [1] was propagated in NMuMG cells at a lowcontent. Not only in the stomach content but multiplicity (0.1 pfu/cell). The virus was har-also in the lungs from one mouse baby (I2+NIe) vested from cells and supernatants at 4–6 daysthe virus DNA sequences were found. Even post-infection. The suspension was clarified bythough Mistrikova et al. [9] for MHV-72 and low speed centrifugation and then purified on

Usherwood et al. [13] for MHV-68 showed that a continuous (10/60%) sucrose gradient. Stockafter intranasal MHV infection of mouse the virus (titre 109–1010 pfu/ml) was stored at−70°Cantibody titre rose starting on day 7 post-in- until use.fection, the antibody titre in milk of infectedwet-nurses is probably too low for virus neut-ralization at day 10 post-infection. Thus, the Animalsvirus could be capable of disseminating fromgastric to respiratory tract. Moreover, an intra- Female athymic nude mice (BALB/c nu/nu),gastrical route of MHV-68 infection was recently non-gestational and gestational immuno-shown, by Peacock and Bost [31], to result in competent mice (BALB/c) were purchased fromsystemic lymphocytosis. The MHV-68 RNA and IFFA CREDO (Domaine des Oncins, BP 0109,DNA but not latent virus could be detected in L’Arbresle cedex, France) and were usedintestinal epithelial cells as long as 30 days post- throughout. The animals were kept in an isolatorinfection, i.e. at the time when the antibody titre under germfree conditions. Food and sawdustreaches its plateau suggesting that some of these bedding were sterilized by the irradiation andcells might be persistently infected. To prove the the drinking water was autoclaved.establishment of persistent infection of intestinal All non-gestational mice were inoculated atepithelial cells of neonates suckling MHV in- the age of 5 weeks by a single subcutaneousfected mother needs further investigation. dorsal injection of MHV-72 (104–106 pfu/mouse).

Results presented here are generally in agree- The gestational mice were infected subcutan-ment with studies of EBV transmission via breast eously 2 days before delivery with a dose ofmilk by Junker et al. [32], showing the possibility 106 pfu/mouse.of natural MHV transmission from infectedmothers to newborn mice during breast feeding.They approve the Junker’s hypothesis [32] that Plaque assaybreast milk may be a significant source for earlyEBV infection of infants. However, the data of Plaque assays were performed in 24 well tissueseroepidemiologic study in Japan [33] indicated culture plates on NMuMG cells (1.5×105 cells/that breast milk is not a crucial source of early well). (a) Serial 10-fold dilutions of differentEBV or HHV-6 infection in infancy. Further study nude mouse tissue (lungs, spleen, lymph nodes,is under way to investigate the possibility of liver, heart, bone marrow, kidney, mammary andMHV-72 transmission from latently infected salivary glands) homogenates were prepared in

isotonic medium (DMEM) and plated on themice to breast-fed newborn mice. This approach

10 H. Raslova et al.

NMuMG monolayers. The virus titres were was up to 5.109 cpm/�g DNA. Total DNAs isol-ated from various organs [as described aboveevaluated as previously described [11]. (b) To

increase the sensibility of the plaque assay (a), in procedure (a)] were digested with BamHIendonuclease (New England Biolabs, Bethesdathe mammary glands of immunocompetent mice

were mechanically disrupted in hypotonic so- Research Laboratory), separated by electro-phoresis in a 0.8% agarose gel at 50 V for 10 hlution (three times diluted DMEM) and ho-

mogenized using a Potter homogenizator. Serial in 1×TBE buffer [35] and then blotted on anitrocellulose membrane (Schleicher & Schuell).10-fold dilutions of the homogenates were plated

on the NMuMG monolayers and the virus titres After hybridization to the 32P-labelled MHV-72BamHI/E probe under high stringency con-were evaluated as described above.ditions [36], the membrane was exposed on X-ray film (KODAK) for 1 day at −70°C.

Nested PCR assay. Nested PCR assay was de-Explantation procedureveloped for detection of MHV-72 DNA in totalDNA isolated from various tissues and bodyMammary glands of nude mouse were removedfluids [as described above in procedure (b)]at 21 days post-infection and minced using scis-using two sets of primers. Two forward primers:sors under sterile conditions. Small piecesgpF7, 5′-GAAACAACCACCCCTTCCCAA-3′(1 mm3) of tissue were placed on Petri dishesfrom nt 70046 to 70066; gpF4, 5′-TCCCAA-and after 2 h adsorption, DMEM medium sup-CAAGAGGATG-3′ from nt 70138 to 70154; andplemented with 10% FCS was added. A freshtwo reversed primers: gp R, 5′-CTGTG-medium was replaced every 5 days during theGGTGCCCAGCGGAGG-3′ from nt 71056 to3 week period of cultivation. The removed71037; gp R2, 5′-TGCTGGTTGAGTGGTGGT-3′medium was tested for presence of infectiousfrom nt 70777 to 70760, were designated ac-virus on NMuMG cells. The inoculated cellscording to the sequence data of MHV-72 gly-were observed microscopically for virus CPE atcoprotein 150 gene (GenBank Acc No. AF294428)daily intervals for up to 6 days.which is homologous to those of strain 68 locatedin the BamHI/Q-K fragment region [Fig. 1(b)][6]. In comparison to MHV-68 no difference in

Isolation of total DNA from different organs the nucleotide sequences of primers used inof infected mice nested PCR assay were found. The length of

the first PCR product flanked by outer primers(a) DNA from lungs, spleen, lymph nodes, liver, (gpF7, gpR) was 1011 bp, while that amplified byheart, bone marrow, kidney, mammary and sa- nested inner primers (gpF4, gpR2) was 640 bp,livary glands of nude mice were isolated from respectively. The latter yielded a 442 bp and athe frozen organs reduced to powder in the 198 bp fragment after BamHI digestion. PCRpresence of dry ice in a homogenizer, as pre- reactions were performed under the followingviously described [34]. (b) Total DNA from conditions. The first PCR: the standard PCRlungs, liver, milk and stomach content of im- reaction mixture contained 100 ng denaturedmunocompetent mice were isolated using total DNA (prepared at 95°C for 10 min). ThisQIAmp Blood kit (Qiagen). template was cycled 35 times (95°C for 1 min,

52°C for 1 min and 72°C for 2 min) followed byone final cycle for 12 min at 72°C. Nested PCR:a 1/25 volume of the first PCR mixture wasMHV-72 DNA detection in different organsamplified in the standard PCR reaction mixtureand body fluids of infected micefor 40 cycles (95°C for 1 min, 57°C for 1 min and

Southern blot analysis. The MHV-68 restriction 72°C for 1 min) followed by one cycle of 12 minmap [2] and genome sequence data [6] were used at 72°C. The PCR products were separated byto identify the 7 kbp long BamHI/E fragment of electrophoresis in 1.5% agarose gel and visu-MHV-72 [Fig. 1(a)]. The fragment was cloned alized under UV light.into pUC19 as previously described [19], labelledin the presence of [�-32P]dCTP with the help of AcknowledgementsRandom Labelling kit (Boehringer, Mannheim),purified on Sephadex G-50 column and then This work was supported by a grant from the As-

sociation pour la recherche sur le cancer (Villejuif,used as a probe. The specific activity of probe

MHV-72 transmission via breast milk 11

16 Weck KE, Kim SS, Virgin IV HW, Speck SH. Macro-France) and by a grant No. 2/5054/98 of the Grantphages are the major reservoir of latent murine gamma-Agency for Science (Slovak Republic). We thank Drherpesvirus 68 in peritoneal cells. J Virol 1999; 73:Jela Mistrikova for the virus strain MHV-72.3268–73.

17 Raslova H, Mistrikova J, Kudelova M, et al. Immuno-phenotypic study of atypical lymphocytes generated inperipheral blood and spleen of nude mice after MHV-72 infection. Viral Immunol 2000; 13: 313–27.

18 Berebbi M, Dandolo L, Hassoun J, Bernard AM,ReferencesBlangy D. Specific tissue targeting of polyoma virusoncogenecity in athymic nude mice. Oncogene 1988;

1 Blaskovic D, Stancekova M, Svobodova J, Mistrikova J. 2: 149–56.Isolation of five strains of herpesvirus from two species 19 Demengeot J, Jacquemier J, Torrente M, Blangy D, Be-of free living small rodents. Acta Virol 1980; 24: 468. rebbi M. Pattern of polyomavirus replication from in-

2 Efstathiou S, Ho YM, Minso AC. Cloning and molecular fection until tumor formation in the organs of athymiccharacterization of the murine herpesvirus 68 genome. nu/nu mice. J Virol 1990; 64: 5633–9.J Gen Virol 1990; 71: 1355–64. 20 Raslova H, Matis J, Rezuchova I, Macakova K, Berebbi

3 Efstathiou S, Ho YM, Hall S, Styles CJ, Scott SD, Com- M, Kudelova M. The bystander effect mediated by thepels U. Murine herpesvirus 68 is genetically related new murine gammaherpesvirus 72-thymidine kinase/to the gammaherpesviruses Epstein–Barr virus and 5′-fluoro-2′deoxyuridine (MHV72-TK/5-FUdR) systemherpesvirus saimiri. J Gen Virol 1990; 71: 1365–72. in vitro. Antiviral Chemistry and Chemother 2000; 11:

4 Mackett M, Stewart JP, Pepper S de V, et al. Genetic 273–82.content and preliminary transcription analysis of a 21 Kulkarni AB, Holmes KL, Fredericson TN, Hartley JW,representative region of murine gamma-herpesvirus 68. Morse III HC. Characteristics of a murine gamma-J Gen Virol 1997; 78: 1425–33. herpesvirus infection immunocompromised mice. In

5 Virgin HW, Latreille P, Wamsley P, et al. Complete Vivo 1997; 11: 281–92.sequence and genomic analysis of murine gamma- 22 Labrecque LG, Barnes DM, Fentiman IS, Griffin BE.herpesvirus 68. J Virol 1997; 71: 5894–904. Epstein–Barr virus in epithelial cell tumors: a breast

6 Reichel M, Matis J, Mistrikova J, Lesso J. The analysis cancer study. Cancer Res 1995; 55: 39–459.of polypeptides in the nuclei and cytoplasm of cells 23 Bonnet M, Guinebretiere JM, Kremmer E, et al. Detectioninfected with murine herpesvirus 72. J Gen Virol 1994; of Epstein–Barr virus in invasive breast cancer. J Natl75: 1259–65. Cancer Inst 1999; 91: 1376–81.7 Svobodova J, Blaskovic D, Mistrikova J. Growth char- 24 Grant S, Edmond E, Syme J. A prospective study ofacteristics of herpesviruses isolated from free living cytomegalovirus infection in pregnancy. I. Laboratorysmall rodents. Acta Virol 1982; 26: 256–63. evidence of congenital infection following maternal8 Sunil-Chandra NP, Efstathiou S, Arno J, Nash AA. primary and reactivated infection. J Infect 1981; 3:Virological and pathological features of mice infected 24–31.with murine gammaherpesvirus 68. J Gen Virol 1992;

25 Nahmias AJ, Josey WE, Naib ZM, Freeman MG, Fer-73: 2347–56.nandez RJ, Wheeler JH. Perinatal risk associated with9 Mistrikova J, Remenova A, Lesso J, Stancekova M.maternal genital herpes simplex virus infection. Am JReplication and persistence of murine herpesvirus 72 inObstet Gynecol 1971; 110: 825–37.lymphatic system and in peripheral blood mononuclear

26 Stagno S, Whitley RJ. Herpesvirus infection of preg-cells of Balb/c mice. Acta Virol 1994; 38: 151–6.10.nancy. Part II: herpes simplex virus and varicella-zoster-10 Rajcani J, Blaskovic D, Svobodova J, Ciampor F,virus infections. N Engl J Med 1985; 313: 1327–30.Huckova D, Stanekova D. Pathogenesis of acute and

27 Joncas JH, Alfieri C, Leyritz-Wills M, et al. Simultaneouspersistent murine herpesvirus infection in mice. Actacongenital infection in Epstein–Barr virus and cyto-Virol 1985; 29: 51–60.megalovirus. N Engl J Med 1981; 304: 1399–403.11 Rajcani J, Bustamante de Contreras LR, Svobodova J.

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MHV-72 transmission via breast milk 13

AUTHOR QUERIES

Title page - ? which author should be marked with an ∗ as the author for correspondence.Please provide full locations of all suppliers.Please check Table 1 carefully: re-arranged it.Reversed Figs 4 and 5 as need to be numbered in sequence in the text.Please supply the full page range for Reference 1.Please supply originals for Figs 2, 3 and 4.Is the use of bold and italics correct throughout. eg. I3+NIf, I1+N1a etc?See copy for Text, Legends and Tables.