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
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ABSTRACT |
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INTRODUCTION |
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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.
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METHODS |
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All plasmid stocks were prepared using the Plasmid Maxi kit (Qiagen) and concentrations were determined by averaging the A260 (1=50 µg ml1) 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. 175750) 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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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 68 weeks, were purchased from Harlan SpragueDawley. 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 2453 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 (56 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 0·20·3 µg total DNA (30 00040 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.
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RESULTS |
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DISCUSSION |
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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 virushost 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 68 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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 5 May 2005;
accepted 13 July 2005.
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