1. Microbiology and Infectious Disease
Download icon

Sexual transmission of murine papillomavirus (MmuPV1) in Mus musculus

  1. Megan E Spurgeon
  2. Paul F Lambert  Is a corresponding author
  1. University of Wisconsin-Madison School of Medicine and Public Health, United States
Short Report
Cite this article as: eLife 2019;8:e50056 doi: 10.7554/eLife.50056
3 figures, 2 tables and 1 additional file

Figures

Rationale and experimental design for MmuPV1 sexual transmission studies.

(A) A full-slide scan of a representative H and E-stained female reproductive tract from a Donor infected for 4 months with MmuPV1+UV+E2 with anatomical regions labeled. On the right, higher magnification images of the cervicovaginal fornix (inset) stained with H and E (left) or immunofluorescence for keratin (KRT; red) and MmuPV1 L1 capsid protein (L1; green). (B) Schematic of MmuPV1 sexual transmission experimental design. Mice infected or potentially infected are indicated in red. (C) DNA was isolated from cervicovaginal lavage samples from a group of representative MmuPV1+UV+E2-infected females that were used as Donors in Experiments 3 and 4. Lavages were conducted at the onset of Experiment 3 (4 months post-infection), the onset of Experiment 4 (8 months post-infection) and Experiment four endpoint (10 months post-infection. DNA was analyzed by PCR for the MmuPV1 E2 gene (top) or for the p53 gene (bottom) to verify DNA presence/quality.

https://doi.org/10.7554/eLife.50056.003
Evidence for sexual transmission: Assessment of MmuPV1 infection status in female Recipient reproductive organs.

(A) DNA was isolated from cervicovaginal lavage (CVL) samples collected from a group of representative Recipient female mice at three different occasions. The numerical mouse identifiers correspond to mice listed in Table 1. The three different CVL time points are as follows (time is listed in weeks following introduction to male Breeder): Mock Recipient mice #1–3 and Recipients #1–3 from Experiment 1 (CVL1: 6 weeks, CVL2: 13 weeks, CVL3: 17 weeks), Recipient mice #5, #8, and #9 from Experiment 2 (CVL1: 8 weeks, CVL2: 11 weeks, CVL3: 13 weeks), Recipient mouse #16 from Experiment 3* (CVL1: 3 weeks, CVL2: 4.5 weeks, CVL3: 9 weeks), and Recipient mice #19, #21, and #22 from Experiment 4 (CVL1: 4.5 weeks, CVL2: 7 weeks, CVL3: 9 weeks). DNA was analyzed by PCR for the MmuPV1 E2 gene (top) or for the p53 gene (bottom) to verify DNA presence/quality. (B) Incidence of MmuPV1 infection via sexual transmission in Recipient females as determined by CVL for MmuPV1 E2 gene. (C) Schematic of co-habitation study in which each co-housed pair consisted of an experimentally MmuPV1-infected female mouse and an uninfected female mouse. After 3 weeks of co-habitation, DNA isolated from cervicovaginal lavages was analyzed by PCR for the MmuPV1 E2 gene (top) or for the p53 gene (bottom) to verify DNA presence/quality. (D) Full-slide scans of the female reproductive tract harvested from Recipient Mouse #3 with a prolonged MmuPV1 infection as a result of sexual contact. Tissue is stained with H and E (left) or for the MmuPV1 E4 viral transcript using RNAscope (right). Higher magnification images of the infected regions of epithelia are shown stained with H and E (top), RNAscope for the MmuPV1 E4 transcript (middle), and the MmuPV1 L1 protein (green) and keratin 14 (red) by immunofluorescence (bottom). White arrow indicates junction between uninfected and MmuPV1-infected epithelia. All scale bars = 100 µM.

https://doi.org/10.7554/eLife.50056.005
Male Breeders harbor infections in their reproductive organs.

(A) Incidence of MmuPV1 infection via sexual transmission in male Breeders as determined by staining tissue for MmuPV1 E4 transcript using RNAscope. (B) Various regions of the penis in MmuPV1-positive male Breeders stained with H and E or RNAscope for MmuPV1 E4 viral transcripts. Lower magnification images on the left for Male #1 include inset boxes (red) indicating the region staining positive for E4, which is shown on the far right. Higher magnification H and E-stained image of the region is shown in the center. Top: Male #1 (did not transmit to Recipient) with MmuPV1-positive region in the glans penile epithelium (red inset). Bottom: Male #5 (transmitted to Recipient) with MmuPV1-positive region in the glans penile epithelium stained for L1 (green) and K14 (KRT; red) (white inset; left), H and E (red inset; middle), and E4 by RNAscope (red inset; right). (C) Tissue from Male #3 (transmitted to Recipient #3 shown in Figure 2C) is stained with H and E (left two columns) or for the MmuPV1 E4 viral transcript using RNAscope (right) three panels). Lower magnification images on the left include inset boxes (red) indicating the region staining positive for E4. Higher magnification images of the infected regions of epithelia are shown stained with H and E and RNAscope for the MmuPV1 E4 transcript (bottom). In the RNAscope analysis, slides were left untreated, treated with DNase to remove any signal from viral DNA, and with DNase+RNase to verify signal is specific for viral RNA transcripts. Top: Male #3 (transmitted to Recipient) with MmuPV1-positive region in the mump ridge groove of the glans penis (red inset). Bottom: Male #3 (transmitted to Recipient) with MmuPV1-positive region in the prepuce (foreskin)/inner preputial space (red inset). All scale bars = 100 µM.

https://doi.org/10.7554/eLife.50056.006

Tables

Table 1
Overview of MmuPV1 sexual transmission experiments and results
https://doi.org/10.7554/eLife.50056.004
Expt.Experimental
Conditions
Treatment of
DONOR Female
DONOR pregnancyInfection status of MALE
BREEDER
Infection status of RECIPIENT
female
RECIPIENT
pregnancy

(# if > 1)
# Positive CVL
(total # of CVLs) in
RECIPIENT
Female
13 weeks breeding; Recipients untreated prior to breeding, placed on E2 8 weeks after introduction of male.No Virus #1YesNegativeNegativeYes0 (4)
No Virus #2NoNegativeNegativeYes0 (4)
No Virus #3YesNegativeNegativeYes0 (4)
33 weeks breeding; Recipients untreated prior to breeding.No Virus #4YesNegativeNegativeYes0 (3)
No Virus #5YesNegativeNegativeNo0 (3)
No Virus #6YesNegativeNegativeNo0 (3)
4Prolonged Donor and Recipient breeding with male (8 weeks). Recipients untreated prior to breeding.No Virus #7YesNegativeNegativeYes (2)0 (4)
No Virus #8YesNegativeNegativeYes (2)0 (4)
No Virus #9YesNegativeNegativeYes (2)0 (4)
13 weeks breeding, Recipients untreated prior to breeding, placed on E2 8 weeks after introduction of male.MmuPV1+UV+E2 #1NoPositiveNegativeYes0 (4)
MmuPV1+UV+E2 #2YesNegativeNegativeYes0 (4)
MmuPV1+UV+E2 #3YesPositivePositive (Prolonged)Yes3 (4)
23 weeks breeding, Recipients untreated prior to breeding, placed on E2 8 weeks after introduction of male.MmuPV1+UV+E2 #4NoNegativeNegativeYes0 (5)
MmuPV1+UV+E2 #5NoPositivePositive (Transient)Yes1 (5)
MmuPV1+UV+E2 #6NoNegativeNegativeYes0 (5)
MmuPV1+UV+E2 #7NoNegativeNegativeYes0 (5)
MmuPV1+UV+E2 #8NoNegativeNegativeYes0 (5)
MmuPV1+UV+E2 #9YesNegativePositive (Transient)Yes1 (5)
33 weeks breeding; Recipients untreated prior to breeding, not treated with E2.MmuPV1+UV+E2 #10NoPositiveNegativeYes0 (3)
MmuPV1+UV+E2 #11NoNegativeNegativeYes (2)0 (3)
MmuPV1+UV+E2 #12YesNegativeNegativeYes0 (3)
3*3 weeks breeding; female Recipients treated with Depo-Provera 5d prior to breeding, not treated with E2.MmuPV1+UV+E2 #13NoNegativeNegativeNo0 (3)
MmuPV1+UV+E2 #14NoNegativeNegativeNo0 (3)
MmuPV1+UV+E2 #15YesNegativeNegativeNo0 (3)
MmuPV1+UV+E2 #16YesNegativePositive (Prolonged)No2 (3)
4Prolonged Donor and Recipient breeding with male (8 weeks). Recipients untreated prior to breeding, not treated with E2.MmuPV1+UV+E2 #17NoNegativeNegativeYes (2)0 (4)
MmuPV1+UV+E2 #18NoNegativeNegativeYes (2)0 (4)
MmuPV1+UV+E2 #19YesNegativePositive (Prolonged)Yes (3)3 (4)
MmuPV1+UV+E2 #20NoNegativeNegativeYes0 (4)
MmuPV1+UV+E2 #21NoNegativePositive (Transient)Yes (3)1 (4)
MmuPV1+UV+E2 #22NoNegativePositive (Prolonged)Yes (2)2 (4)
Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain backgroundFVB/NTaconic BiosciencesRRID:IMSR_TAC:fvbMales (n = 31)
Females (n = 31)
Strain, strain backgroundMmuPV1Joh et al., 2012
Uberoi et al., 2016
GenBank: GU808564.1In-lab stock ‘AU 11/13’, pAU.4
AntibodyAnti-MusPV1 L1
(rabbit polyclonal immune serum)
Chris Buck, NCI/NIHIF (1:5000)
AntibodyAnti-K14
(rabbit polyclonal)
BioLegendCat#905301;
RRID:AB_2565048
IF (1:1000)
Sequence-based reagentMmuPV1_E2_1Hu et al., 2015
Cladel et al., 2017a
Spurgeon et al., 2019
PCR primersGCCCGAAGACAACACCGCCACG
Sequenced-based reagentMmuPV1_E2_2Hu et al., 2015
Cladel et al., 2017a
Spurgeon et al., 2019
PCR primersCCTCCGCCTCGTCCCCAAATGG
Sequenced-based reagentp53-1Spurgeon et al., 2019PCR primersTATACTCAGAGCCGGCCT
Sequenced-based reagentp53-2Spurgeon et al., 2019PCR primersACAGCGTGGTGGTACCTTAT
Sequenced-based reagentp53-3Spurgeon et al., 2019PCR primersTCCTCGTGCTTTACGGTATC
Sequenced-based reagentMusPV-E4Xue et al., 2017RNAscope probe
Cat#473281
Commercial assay or kitRNAscope 2.5 HD Detection Kit BrownACDBioCat# 322300
Chemical compound, drugTyramide signal amplification (TSA)-related reagentsOnline protocol: https://doi.org/10.17504/protocols.io.i8cchsw
Chemical compound, drugmedroxyprogesterone acetateAmphastar PharmaceuticalsDepo-Provera3 mg/animal, subcutaneous injection
Chemical compound, drugNonoxynol-9 (4%)Options ConceptrolCat#24714950 µl/mouse, intravaginal
Chemical compound, drugCarboxyl methylcellulose (4%)Sigma AldrichCat# C488825 µl/mouse, intravaginal
Chemical compound, drug17β-estradiol pellet, 0.05 mg/60 daysInnovative Research of AmericaCat#SE-1210.05 mg/60 days, subcutaneous pellet
OtherHematoxylin QSVectorCat#H-3404Counterstain
OtherShandon Instant HematoxylinThermo FisherCat#6765015H and E stain
OtherEosinSigma AldrichCat#E4382H and E stain
OtherDNase IThermo Fisher ScientificCat#EN052120 units/sample
OtherRNase AQiagenCat#1006657500 µg/sample
OtherRNase T1FermentasCat#EN05422000 units/sample

Data availability

All data generated and analyzed during this study are included in the manuscript and supporting files.

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)