Quantification of vertical transmission of Murine polyoma virus by real-time quantitative PCR

Shaojie Zhang, Adrienne L. McNees and Janet S. Butel

Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, MS BCM385, Houston, TX 77030, USA

Correspondence
Janet S. Butel
jbutel{at}bcm.edu


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Pathogenesis studies of viral infections in vivo require sensitive assay methods. A sensitive and specific real-time quantitative PCR (RQ-PCR) assay was developed to detect Murine polyoma virus (MuPyV) DNA sequences. A quantitative assay to measure the single-copy murine wild-type p53 gene was developed to normalize viral gene copies to cell numbers. Both assays were sensitive over a seven-log dynamic range, with a reproducible detection limit of 10 copies per reaction. To determine viral loads and tissue distribution following vertical transmission of MuPyV, pregnant BALB/c mice were inoculated intraperitoneally with virus in late pregnancy. Progeny animals born to infected mothers were followed for 21 days. Viral loads in four tissues (salivary gland, kidney, liver and spleen) were highest at 7 days after birth and dropped to low levels by 14 and 21 days of age, with loads ranging from 5 to 2 million MuPyV copies per 103 cells. Significant animal-to-animal variation occurred. Fourteen of 21 (67 %) progeny were virus-positive in one or more tissue samples. Transplacental transmission was observed in 6/7 (86 %) litters. Infected fetuses per positive litter ranged from 1/7 (14 %) to 5/6 (83 %) with viral loads ranging from 5 to 25 417 MuPyV copies per 1000 fetal cells. Maternal tissues and blood were frequently highly positive 2 days after inoculation, but viral loads were low by day 14. This study demonstrated the vertical transmission, including transplacental transmission, of MuPyV following acute infection of pregnant mice. It should be considered that there is a possibility that other polyomaviruses, including those in humans, may be vertically transmitted.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Polyomaviruses are now recognized as human pathogens, especially in hosts with compromised immune function. However, the precise mode of polyomavirus transmission between hosts is unsettled, with vertical transmission being a possible mechanism under certain conditions. The quantitative dynamics of infection in different tissues following infection are unknown for most polyomaviruses.

Murine polyoma virus (MuPyV) is thought to be transmitted primarily via urine from adults to newborn mice that experience primary infections in the respiratory tract, followed by persistent inapparent infections in the kidneys (Gottlieb & Villarreal, 2001). However, it has been reported that transplacental transmission can occur following MuPyV infection of pregnant mice (McCance & Mims, 1977) or rats (Verhagen et al., 1993), with tumours resulting in progeny in the latter study. There has also been a description of transplacental transmission of SV40 in hamsters, resulting in tumours in the offspring (Rachlin et al., 1988). There are anecdotal observations suggesting that human polyomavirus BKV may be transmitted transplacentally (Taguchi et al., 1975; Pietropaolo et al., 1998), as well as a reported isolation of SV40 from a newborn child (Brandner et al., 1977). Finally, SV40 was isolated from a juvenile rhesus monkey that was separated from his mother soon after birth and hand-raised by humans (Lednicky et al., 1998). Together, these observations indicate that polyomaviruses can be vertically transmitted.

To quantify transmission and characterize the quantitative tissue distribution of a polyomavirus following vertical transmission in a susceptible host, we utilized the MuPyV model in mice. A sensitive and specific real-time quantitative PCR (RQ-PCR) assay for MuPyV and an RQ-PCR assay for the single-copy murine wild-type p53 gene were developed so that viral gene copy numbers could be quantified and normalized to cell numbers in different murine tissue specimens.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Plasmid DNA standards.
The target sequences used for quantification of viral and cellular genes included the N termini of the T-antigens (T-ag) of MuPyV (large, middle and small) and sequences of intron 3 within the murine wild-type p53 gene. A plasmid encoding the complete MuPyV genome, strain A2 (from T. Benjamin, Department of Pathology, Harvard Medical School, Boston, USA), was generated by cloning the genome into pBluescript II KS+ (Stratagene) at the EcoRI site. To quantify cell numbers based on genome equivalents, amplification of the p53 gene intron 3 sequence (a sequence not encoded in the p53 pseudogene present in the murine genome) was used as a cellular normalization standard to allow calculations of viral genome copies per murine cell. Amplification of a cellular gene also serves as a control for the suitability of a test DNA sample for gene amplification. A plasmid encoding this sequence (pGene Switch/V5-HisA-53) (Invitrogen) was constructed using standard techniques. PCR amplification of the p53 gene from the LTR-XA construct (Hinds et al., 1989) (obtained from G. Lozano, Department of Molecular Genetics, University of Texas MD Anderson Cancer Center, Houston, USA) was performed using primers that span a BamHI restriction sequence 5'-TATGGATCCGTTATGGTTATGACTGCCATGGAGGAGTCA-3' and an EcoRI restriction sequence 5'-TATGAATTCGTCTGAGTCAGGCCCCACTTT-3'. Restriction digests of the amplicons and the pGene Switch/V5-HisA-53 plasmid were prepared using BamHI and EcoRI under conditions supplied by the manufacturer (New England BioLabs), and the resulting fragments were purified using the QIAquick Gel Extraction kit (Qiagen). Competent DH5{alpha} cells were transformed by the ligated constructs and resulting clones were confirmed by restriction digestion and sequence analyses.

All plasmid stocks were prepared using the Plasmid Maxi kit (Qiagen) and concentrations were determined by averaging the A260 (1=50 µg ml–1) of multiple dilutions. The copy number of each plasmid was calculated based on its molecular mass. Plasmid standards were made by preparing serial 10-fold dilutions in a final volume of 1 ml sterile DNase-free water (Invitrogen), such that 10 µl standard added to a 50 µl real-time PCR reaction contained input copy numbers ranging from 107 copies to 100 copy. These standards were analysed in duplicate in each RQ-PCR assay.

Primers and Taqman probes.
Using Primer Express software, primers and probes were designed to amplify sequences within the T-ag gene (no. 175–750) for MuPyV and sequences of the murine wild-type p53 gene. Sequences are shown in Table 1. The oligonucleotides were synthesized by Applied Biosystems. The probes specific to the T-ag gene and the p53 gene were labelled with FAM (6-carboxyfluorescein) at the 5' ends and with MGB (minor groove binder) at the 3' ends. All primers and probes were received in the PCR clean rooms core facility at the Department of Molecular Virology and Microbiology, Baylor College of Medicine. The primers were reconstituted with sterile water (Gibco) and were aliquoted and stored at –20 °C in small volumes to minimize multiple uses of these reagents.


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Table 1. Sequences of primers and probes used in PCR assays

 
PCR conditions.
RQ-PCR was performed using the PRISM 7000 Sequence Detection system (Applied Biosystems) according to the manufacturer's recommendations. Using either strip tubes or 96-well plates and caps of optical grade (Applied Biosystems), 50 µl PCR reactions were prepared containing 900 nM each primer, 100 nM TaqMan FAM-MGB probe and 25 µl 2x TaqMan Universal PCR Master mix (Applied Biosystems). PCR reactions were prepared in the PCR clean rooms core facility using positive-displacement pipetters and barrier tips. Ten microlitres of standard plasmid dilutions or positive control samples and test DNA samples were added outside the core facility after the tubes containing the master mix and negative controls were sealed. RQ-PCR reaction conditions for amplification of all target genes were as follows: 50 °C for 2 min, denaturing at 95 °C for 10 min and 40 cycles of denaturing at 95 °C for 15 s followed by annealing and extension at 60 °C for 1 min. Amplification data were measured as an increase in reporter fluorescence generated by cleavage of the reporter dye FAM from the 5' end of the probe by the TaqMan exonuclease activity and were collected in real-time and analysed by the Sequence Detection system software. The increase in reporter fluorescence is normalized to the passive reference dye ROX (carboxy-X-rhodamine) to correct for volume fluctuations that may occur during preparation or the reaction. The threshold cycle number, CT, is the cycle in which the normalized reporter fluorescence increases 10 standard deviations above the mean background fluorescence detected in the first 15 cycles. For each target gene, reporter fluorescence was detected at a CT of approximately 16 for reactions containing 107 input copies. This is consistent with the calculation that 40 cycles of efficient PCR will generate approximately 5x1011 copies from one starting molecule and that 1x107 copies is predicted to be detected at cycle number 16.

Conventional PCR reactions for the detection of MuPyV regulatory region sequences were performed as described previously (Shearer et al., 2005), using a Perkin-Elmer GeneAmp PCR system 2400 thermocycler. Each PCR reaction contained 2 µg tissue DNA (about 3x105 cell equivalents). Primers used for amplification of the viral regulatory region (designed by J. A. Lednicky) are shown in Table 1. The nucleotide numbering is according to MuPyV, strain A2 (GenBank accession no. NC_001515). Primers were obtained from Integrated DNA Technologies. The cycling conditions began with a 5 min denaturation step at 94 °C, followed by 45 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s and ending with 7 min at 72 °C. Ten microlitres of the reaction was analysed on a 2 % agarose gel and visualized by ethidium bromide staining.

Virus.
A seed stock of MuPyV, strain A2, was obtained from T. Benjamin and propagated in NIH3T3 cells grown in Dulbecco's modified Eagle's medium (DMEM) and 10 % fetal calf serum (FCS) (Hyclone). Monolayers were infected followed by the addition of DMEM and 2 % FCS. After cytopathic effects were observed, sodium bicarbonate was added and the cells were harvested by scraping and pelleted by centrifugation. The resulting cell pellets were suspended in 1/20 volume of supernatant and subjected to three cycles of freezing and thawing. Cell debris was removed by centrifugation, and the supernatants were aliquoted and stored at –20 °C as the MuPyV stock. The titre of a MuPyV stock was determined by measuring haemagglutination units (HAU) using a 0·3 % (v/v) suspension of guinea pig erythrocytes (Colorado Serum Company) in PBS (Ludlow & Consigli, 1987). The HAU titre was determined as the reciprocal of the highest dilution that produced detectable agglutination.

Mice.
Pregnant BALB/c mice at mid-gestation, aged 6–8 weeks, were purchased from Harlan Sprague–Dawley. Animal protocols were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine, and animals were maintained in compliance with relevant federal regulations, guidelines and policies. Pregnant mice were housed individually in the biohazard facility at the Center for Comparative Medicine at Baylor College of Medicine, which is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. One to two days after arrival the mice were injected intraperitoneally with 125 HAU of MuPyV. This corresponded to 3·8x108 MuPyV genomes (RQ-PCR analysis of the virus stock indicated 3x106 viral genomes per HAU). Pregnant mice were sacrificed 24–53 h post-inoculation (p.i.) and their fetuses harvested. In parallel experiments, litters were delivered by MuPyV-inoculated females (2 days p.i.) and progeny mice were sacrificed at 3, 7, 14 and 21 days post-parturition (5–6 mice at each collection point). Tissues harvested from the progeny mice included salivary gland (parotid and submandibular glands were pooled), kidney, liver and spleen. These same tissues, plus mammary glands and blood, were harvested from the maternal mice. Blood was collected into EDTA (lavender-top) tubes (Sarstedt) and blood cells and plasma were separated by centrifugation.

DNA extraction of tissues.
Total DNA was isolated from weighed organ tissues from newborn and adult mice, from plasma and from individual fetuses using a proteinase K and phenol/chloroform protocol as described previously (Lednicky & Butel, 1998; Shearer et al., 2005). DNA was recovered from white blood cells (WBC) using the Amplicor Whole Blood Specimen Preparation kit (Roche Diagnostics). DNA concentrations were determined as described above and DNA samples were diluted such that 10 µl added to an RQ-PCR reaction contained 0·2–0·3 µg total DNA (30 000–40 000 cell equivalents per reaction). Earlier experiments had determined the optimal amount of sample DNA to be added to the RQ-PCR reaction in order to reduce interassay variability and maximize the accuracy of quantification. All samples were analysed by RQ-PCR for MuPyV T-ag and cellular p53 DNAs.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Absolute quantification of MuPyV and p53 in RQ-PCR assays
To quantify MuPyV loads in infected mice, the T-ag region was chosen as the target gene. Primer pairs and fluorescent probes (Table 1) were designed to detect amplification of a conserved region in the T-ag gene and a unique sequence within intron 3 of the murine wild-type p53 gene. Absolute quantification of the target genes was determined using dilutions of standard plasmids encoding the MuPyV T-ag gene and murine p53 gene sequences as described in Methods. Representative amplification of the standard dilutions for each target gene is shown as the change in normalized fluorescent reporter signal, {Delta}Rn, plotted against the cycle number of the reaction (Fig. 1). In both assays there was no increase in the fluorescence above the threshold in reactions containing the non-template controls. These representative plots indicate that for each target gene 100 copy is detected. However, this was not observed in all assays, although 101 copies were regularly detected, so the detection limit of the assays used as described was 10 copies per reaction for both the viral and cellular genes.



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Fig. 1. Amplification plots showing the normalized reporter fluorescence ({Delta}Rn) plotted against the cycle number for the standard dilution series for each target gene. Plasmids encoding the T-ag gene sequences from MuPyV (a) or the murine p53 gene sequences (b) were prepared and diluted as described in Methods and analysed by real-time PCR. The log10 of the input copy number of each standard is indicated on the amplification plot. These data are representative of multiple experiments. NTC, Non-template controls.

 
Absolute quantification of viral gene copies was determined from standard curves generated by plotting the log10 of the known input gene copy number of the standard dilution series against the CT value observed in the RQ-PCR analysis. Representative standard curves generated from three independent experiments for each target gene are shown in Fig. 2. These data show that results for the standard dilutions for all target genes were precise and consistent and there was little intra- and interassay variability.



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Fig. 2. Standard curves generated by plotting the observed CT value against the log10 of the input copy number of standard plasmid DNA encoding T-ag gene sequences of MuPyV (a) and murine p53 gene sequences (b). These data show representative standard curves from independent experiments used to determine quantities in unknown samples.

 
Sensitivity of RQ-PCR for MuPyV sequences
Conventional PCR techniques have been applied by us and others in studies of MuPyV infections (Berke & Dalianis, 2000; Shearer et al., 2005). To compare the detection sensitivity of conventional PCR and agarose gel protocols to that of RQ-PCR, reactions were prepared using the plasmid standard. Primers specific to the MuPyV regulatory region were used and reactions were carried out as described in Methods. Following resolution of reaction products on a 2 % agarose gel, the lowest input copy number detected was 104 viral copies per reaction (Fig. 3, lane 7). An input copy number of 10 was reproducibly detected in RQ-PCR reactions (Fig. 1a). Thus, the sensitivity of detection of the RQ-PCR assay was increased by up to three logs as compared with the conventional PCR protocol.



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Fig. 3. Detection of MuPyV using conventional PCR and agarose gel analysis. As described in Methods, 10 µl of the standard plasmid dilutions were amplified using primers specific to the MuPyV regulatory region. Ten µl of the 50 µl reaction was analysed on a 2 % agarose gel with ethidium bromide staining.

 
Quantification of target genes was affected by the amount of sample DNA added to the RQ-PCR reaction. Amplification of p53 was inhibited in some samples at input concentrations greater than 0·2 µg of sample DNA per reaction, whereas detection of the cellular gene in other samples was less sensitive to this apparent inhibition (Fig. 4). As observed for detection of the cellular gene, amplification of the viral T-ag gene was inhibited frequently in the presence of more than 0·4 µg of total DNA (data not shown). These results suggest that quantification of target genes may be more consistent and accurate when 0·2–0·3 µg of input DNA is added per reaction.



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Fig. 4. Copy numbers of p53 detected in a range of concentrations of DNA template. Representative test samples (n=9) were tested by RQ-PCR by adding increasing amounts of DNA template to determine an optimal concentration for analysing all samples in this study. The samples analysed included total DNA extracted from fetal mice or organs of newborn mice. The p53 copy numbers shown are the mean and the standard error of the mean for three independent experiments. Sample 1, liver (n=3); sample 2, salivary gland (n=3); sample 3, embryo (n=3).

 
Vertical transmission of MuPyV to progeny of infected mothers
These RQ-PCR techniques were applied to study the dynamics of MuPyV infection following vertical transmission. Pregnant BALB/c mice were inoculated with 125 HAU of MuPyV, strain A2, in late pregnancy. Progeny animals born to these mothers were sacrificed on days 3, 7, 14 and 21 post-parturition (5–6 mice per time point). Total DNA was extracted from several tissues of progeny mice and viral gene copy numbers per cell were quantified using RQ-PCR (Table 2). Overall, 14/21 progeny (67 %) were found to be virus-infected. Of the samples collected from newborn mice sacrificed 3 days after birth, 35 % were positive with viral loads averaging 981 MuPyV copies per 1000 cells (range 5 to 4394 copies). Kidney and liver specimens contained the most viral genome copies. By day 7, 50 % of the tissues assayed contained detectable viral genomes, with increased viral loads that averaged 4·6x105 copies per 1000 cells (range 28 to 2x106 copies). Virus was widespread and relatively evenly distributed among the tissues analysed, with the liver and salivary gland specimens containing the highest MuPyV copy numbers. One of the mice sacrificed in this group contained very high viral genome copy numbers in all tissues, the only animal of the 21 progeny mice analysed that appeared to be exceptionally susceptible to the infection. If that animal was excluded from the analysis, then 6/16 (38 %) tissues were virus-positive at day 7 and the viral loads averaged 1·3x104 copies per 1000 cells (range 28 to 9x104 copies). Analysis of tissues collected from progeny animals at 14 and 21 days of age revealed that the viral burden had dropped substantially, with low copy numbers ranging from 5 to 138 per 1000 cells among the tissues examined. At these 2- and 3-week time points, salivary gland samples were more frequently positive than were samples from other organs. Samples from control animals were negative (data not shown). Collectively, these data demonstrate the vertical transmission of MuPyV to progeny mice following inoculation of a pregnant female. A transient increase in viral load occurs in multiple organs of these newborn mice, peaking about 7 days after birth, followed by persistence of low numbers of viral genomes through to, at least, day 21.


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Table 2. Viral loads over time in tissues of progeny mice following MuPyV infection of pregnant females: evidence of vertical transmission

 
Transplacental transmission of MuPyV
The vertical transmission of MuPyV to progeny animals, following inoculation of pregnant mice, could have occurred either before or after birth. The possibility of transplacental transmission in utero was next examined. Pregnant BALB/c mice were inoculated with MuPyV as described above. The mice were sacrificed 24–53 h later and the fetuses were harvested. Total DNA was isolated from individual fetuses and analysed by RQ-PCR (Table 3). A total of 39 fetuses from seven different litters were analysed. Virus transmission was detected in 6/7 (86 %) litters, as evidenced by one or more virus-positive fetuses in the litter. The number of infected fetuses per positive litter ranged from 1/7 (14 %) to 5/6 (83 %). Overall, 12/39 (31 %) fetuses tested positive. Viral loads ranged from 5 to 25 417 genome copies per 1000 fetal cells. This experiment showed that transplacental transmission of MuPyV can occur and is a relatively common event following acute infection of the mother during late pregnancy.


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Table 3. Viral loads in fetal mice following MuPyV infection of pregnant females: evidence of transplacental transmission

 
Distribution of MuPyV in maternal tissues
The maternal mice inoculated with MuPyV during pregnancy were sacrificed 1–25 days later, depending on the experimental analysis of their progeny (Tables 2 and 3). At the time of sacrifice maternal tissues were collected, total DNAs extracted and the viral genome content quantified by RQ-PCR (Table 4). Very little virus was detected in the animal sacrificed 1 day p.i. (mouse 1), whereas viral loads were high in most tissues by 2 days p.i. (mice 2–6). Of the tissues tested, the mammary gland contained some of the highest viral loads (MuPyV copies per 1000 cells) at this time. After 2 weeks p.i., the viral contents in maternal tissues were very low or undetectable (mice 7–9). Tissues from control, uninoculated mice were negative for viral DNA. It is noteworthy that mouse 6, the mother of fetal litter no. 6 that showed no evidence of virus transmission in utero (Table 3), was as highly infected as other pregnant mice that did transmit the virus transplacentally.


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Table 4. Viral loads in maternal tissues and blood following MuPyV infection of pregnant females

 
Blood of the maternal animals was also collected at sacrifice. WBC and plasma were separated, DNAs extracted and viral sequences detected by RQ-PCR. MuPyV genome content was determined per 1000 WBC and per microlitre of plasma (Table 4). High levels of virus were present in the plasma at 1 day p.i., in contrast to minimal amounts of virus in the various tissues. By 2 days p.i., virus was detectable in both WBC and plasma of all animals; levels of virus then dropped significantly by 2 weeks p.i. The amount of virus detected in the blood did not correlate with viral loads in different tissues of a given animal, suggesting that the tissue-associated virus did not reflect circulating blood-associated virus.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
This report describes the development of sensitive and quantitative real-time PCR assays for MuPyV and the murine wild-type p53 gene. Other studies have used real-time PCR for MuPyV (Heidari et al., 2000; Klucky et al., 2004), but to the best of our knowledge this combination of assays has not been described previously for the murine system. The assays are sensitive over a seven-log dynamic range and have a detection limit of 10 copies per reaction for both the viral and cellular genes. These characteristics are similar to those of the RQ-PCR assays recently developed for the human polyomaviruses (BKV, JCV and SV40) and the human RNase P cellular gene (McNees et al., 2005). The RQ-PCR assay described here is up to three logs more sensitive for detection of MuPyV than the conventional PCR protocol. This increased sensitivity permits more informative data to be obtained from pathogenesis studies where the amount of virus present in tissues in infected hosts may be very low. The ability to quantify both virus copy numbers and cell numbers in the same sample allows the calculation of viral genomes per cell, providing a means of normalization for comparison among different animals and different tissue specimens. However, this assay is not able to determine the specific cell types infected within a tissue, how many cells in a positive tissue are infected, or whether the viral copies detected represent virus replication in that tissue or simple accumulation of genomes.

The patterns of MuPyV infection observed in the progeny animals born to mothers infected during pregnancy (Table 2) were similar to those that had been described following inoculation of newborn BALB/c mice. In general, the infection spread rapidly to multiple organs, peaking in intensity about 1 week p.i., and then was cleared from most organs in another week's time, presumably due to the maturation of the immune response (Dubensky & Villarreal, 1984; Wirth et al., 1992; Gottlieb & Villarreal, 2000; Berke & Dalianis, 2000). The kidney is considered to be the major site of MuPyV persistence (Rowe et al., 1960; Dubensky & Villarreal, 1984). The sensitive RQ-PCR assay employed in this study provides additional insights. It appears that very low copy numbers of MuPyV may remain associated with more tissues than previously suspected, with the salivary gland being the most commonly infected organ by day 21. The low viral loads detected in some animals would be below the limits of detection of most assays used in previous pathogenesis studies.

As observed by others who have analysed MuPyV infections in vivo (Heidari et al., 2000), there is significant animal-to-animal variation, even when inbred litter mates are characterized. The observed individual variation emphasizes the complexity of the polyoma virus–host interaction. This was most evident in our study by the litter sacrificed at day 7 post-birth in which one litter mate had much higher viral loads than other animals in all tissues examined.

This study confirmed that MuPyV can be transmitted in utero following acute infection of the pregnant female. In the original report of transplacental transmission of MuPyV (McCance & Mims, 1977), infectivity assays were used to detect virus. When pregnant CD-1 mice were inoculated intraperitoneally with 5x108 TCID50 of MuPyV on day 10 of pregnancy, all pools of fetuses tested 5 days later were virus-positive, as were all 30 fetuses assayed individually. In another experiment in that study, mice were inoculated on day 15 of gestation and the kidneys of the newborns were taken within 10 h of birth. Pooled kidneys from one of three litters contained detectable infectious virus. In this current study involving BALB/c mice inoculated in late pregnancy, RQ-PCR assays showed that transplacental transmission occurred in 6/7 (86 %) litters (Table 3). The number of infected fetuses per litter ranged from 14 to 83 % with wide variation in the amount of virus per infected fetus (5 to 25 417 copies per 103 fetal cells). Overall, 12/39 (31 %) fetuses were virus-positive. Important unanswered questions are what determines the frequency of transmission among litters and to specific fetuses within a given litter.

Previous studies have established that adult mice are much less susceptible to MuPyV infection than newborn animals (Rowe et al., 1960; Wirth et al., 1992, 1997; Atencio et al., 1993; Gottlieb & Villarreal, 2000; Berke & Dalianis, 2000). However, it has been noted that virus replication can occur in animals beyond 3 weeks of age (Wirth et al., 1997; Heidari et al., 2000). The maternal animals in this study, inoculated at 6–8 weeks of age, appeared to support MuPyV infection in numerous tissues 2 days later (Table 4). It has been reported that adult murine kidneys that had been damaged became able to support acute MuPyV infection (Atencio et al., 1993). Although the kidneys of the adult females in this study had not been damaged, they did appear to be infected. It is not known if the kidneys of these animals would have remained persistently infected long-term, as the experiment was terminated. As predicted by earlier studies, the acutely infected adult mice cleared the infection from all tissues within 3 weeks so that only very small numbers of viral genomes remained detectable. It was evident that the tissues in the mothers that might serve as sources of virus for transmission to newborns were infected at the time they gave birth, including salivary gland (saliva), kidney (urine) and mammary gland (milk). Inhalation is presumed to be the main route of natural infection (Gottlieb & Villarreal, 2001). It has been demonstrated that lungs become resistant to infection by intranasal inoculation between 2 and 4 days after birth (Gottlieb & Villarreal, 2000), so natural infections must occur to newborns.

Importantly for consideration of the process of transplacental transmission, virus was detected in the blood of maternal mice (Table 4). We anticipate that blood would be the likely route of infection in utero. Although it has been reported that MuPyV is rarely detected in the blood of infected normal adult animals (Wirth et al., 1992; Berke & Dalianis, 2000), the sensitive RQ-PCR assay revealed viral presence in blood soon after infection. It is likely that the chance of transplacental transmission is related to the occurrence of an acute viral infection.

Previous observations (Streuli et al., 1990) noted that some uninoculated mice may harbour latent infections with MuPyV. In this study, all tissues tested from control, uninoculated animals were virus-negative, ruling out naturally occurring latent infections or theoretical germ-line transmission of viral sequences.

It has been suggested that the ability to induce a high tumour profile is correlated with the ability of a virus strain to establish a disseminated productive infection (Dubensky et al., 1991). Mice infected before birth in this study had disseminated infections but no animals were held long-term to observe for tumour development. Whether animals infected in utero are more or less tumour-prone than those infected naturally as neonates is unknown.

The demonstration of vertical transmission, including transplacental transmission, of MuPyV in the murine model raises the possibility that human polyomaviruses might similarly be vertically transmitted, under the appropriate conditions. It will be important to determine if humans infected as very young infants are at elevated risk of polyomavirus-associated disease. Sensitive and specific molecular assays, such as RQ-PCR, can reveal low-level infections in tissues and specimens that would be undetectable by other methods. Future studies should address what levels of viral loads are biologically meaningful in different tissues infected with polyomaviruses.


   ACKNOWLEDGEMENTS
 
We thank Dr Tom Benjamin for the gift of murine polyoma virus. This study was supported in part by National Aeronautics and Space Administration Cooperative Agreement NCC 9-58 (Project No. IIH00403) and by grant CA09197 from the National Cancer Institute.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 5 May 2005; accepted 13 July 2005.



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