Quantification of IFN-λ, IFN-α5, and IFN-β transcripts in virus-infected brains and livers.

<p>Histograms show the number of IFN cDNA copies per 10⁶ β-actin cDNA copies, determined by real-time PCR, after reverse transcription of RNA extracted from the brain and liver of mice infected with different RNA viruses, in different experimental settings (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000017#ppat-1000017-t002" target="_blank">Table 2</a>). A, B, F, G, H: mean and standard deviation of groups of mice. C–E: data from individual mice. Background amplification in mock-infected mice (not shown) was less than 1 copy of IFN-β or IFN-λ, and less than 10 copies of IFN-α5 cDNA, per 10⁶ copies of β-actin cDNA.</p

IFN-α and IFN-λ responding cells in the kidney adipose tissue and in the brain.

<p>Mx1 expression, detected by immunohistochemistry (as white nuclear spots), 7 days after electroinjection of a plasmid coding for MuIFN-α6T or MuIFN-λ3. A–C–E: Mx1/WT mouse electroinjected with a plasmid coding for IFN-α6T. B–D–F: Mx1/IFNAR1-KO mouse electroinjected with a plasmid coding for IFN-λ3. A–B: sections showing the kidney adipose tissue. C–D: brain sections showing the choroid plexus of the 4th ventricle. E–F: Higher magnification of the choroid plexus. G: Cartoon showing the structural organization of the choroid plexus.</p

Relative expression of the various IFN-α subtypes in MHV-A59 infected livers.

<p>IFN-α coding sequences were amplified by RT-PCR using a primer mixture designed to amplify equally the different murine IFN-α subtypes <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000017#ppat.1000017-Delhaye1" target="_blank">[33]</a>. PCR products were then cloned and individual clones were sequenced. The histogram shows the percentage of sequences from 2 mice (50 and 53 sequences) corresponding to each IFN-α subtype.</p

Primers sequences and PCR conditions used.

a<p>Genbank accession number (<a href="http://www.ncbi.nlm.nih.gov/Genbank/" target="_blank">http://www.ncbi.nlm.nih.gov/Genbank/</a>).</p>b<p>(s) sense primer; (as) antisense primer.</p

Mouse infections with RNA viruses.

a<p>1 day post-infection was reported to correspond to the peak of LDV replication and of IFN expression in vivo.</p>b<p>Mice infected with highly neurovirulent viruses were sampled when signs of encephalitis were prominent (generally less than 24 hours before death).</p>c<p>The DA1 strain of TMEV produces a transient encephalitis lasting about 1 or 2 weeks. In mice with the H-2 b haplotype, the virus is then rapidly cleared by the cytolytic T lymphocyte response <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000017#ppat.1000017-Brahic1" target="_blank">[40]</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000017#ppat.1000017-Roos1" target="_blank">[41]</a>. Mice were sampled at 5 days post-infection, a time-point representative of the acute phase of infection.</p>d<p>Preliminary RT-PCR experiments failed to reveal a clear difference in IFN expression and viral load between 129/Sv mice infected i.p. for 2 days and for 7 days. Only mice with amounts of MHV-A59 detectable by conventional RT-PCR were taken into account.</p>e<p>These samples from TMEV (GDVII strain) and LACVdelNSs infected brains were from a previous work <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000017#ppat.1000017-Delhaye1" target="_blank">[33]</a>.</p

Mx1 gene expression induced by systemic IFN-α and IFN-λ, in organs of IFNAR1-positive and IFNAR1-deficient mice.

<p>Mx1 gene transcription was analyzed by real-time RT-PCR, 7 days after electroinjection of plasmid coding for MuIFN-α6T, MuIFN-λ3 or the empty vector (mock) in 6 week-old Mx1-positive mice (2 BALB.A2G-Mx1 and 2 B6.A2G-Mx1 mice, grouped as Mx1/WT mice) or in 8 week-old Mx1/IFNAR1-KO mice. Results are expressed as Mx1 cDNA copies per β-actin cDNA copy. Graphs present results for individual mice and the mean for each group, for one representative experiment.</p

IFN-α and IFN-λ responding cells in the kidney.

<p>Mx1 expression, detected by immunohistochemistry (white nuclear spots), 7 days after electroinjection of a plasmid coding for MuIFN-α6T or MuIFN-λ3. Sections of the kidney from: A. control Mx1/WT mouse electroinjected with the empty vector. Note that few cells (mostly endothelial cells) were weakly Mx1-positive. B. control Mx1/IFNAR1-KO mouse electroinjected with a plasmid coding for IFN-α6T. C–E–G: Mx1/WT mouse electroinjected with a plasmid coding for IFN-α6T. D–F–H: Mx1/IFNAR1-KO mouse electroinjected with a plasmid coding for IFN-λ3.</p

Induction of Mx1 gene expression in response to circulating IFNs.

a<p>Mx1 induction (mean±SD) by IFNs:  = Mx1 expression determined by real-time RT-PCR in organs of mice electroinjected with the plasmid expressing the indicated IFN divided by Mx1 expression in the corresponding organ of mice electroinjected with the empty vector.</p>b<p>BALB.A2G-Mx1 mice.</p>c<p>BALB.A2G-Mx1 (n = 2) and B6.A2G-Mx1 (n = 2).</p

OASl2 expression in different tissues after electroinjection of plasmids coding for IFN-α or IFN-λ.

<p>OASl2 transcripts detected by real-time RT-PCR, 7 days after electroinjection of plasmid coding for MuIFN-α6T, MuIFN-α6T/D78N, MuIFN-λ3 or the empty vector (mock), in 7 week-old FVB/N mice. Results are expressed as OASl2 cDNA copies per β-actin cDNA copy. Graphs present results for individual mice and the mean for each group, for one representative experiment.</p

Relative type III and type I IFN gene transcription in the brain and in the liver of infected C57BL/6 mice.

a<p>Calculations were as followed: for each mouse, the ratio between type III and type I IFN transcripts (IFN-λ/IFN-α or IFN-λ/IFN-β was calculated for the liver and for the brain. Mean ratios were then calculated for liver samples and for brain samples.</p>b<p>Mann-Whitney p values testing whether the mean ratio between type III and type I expression measured in the liver differs significantly from that measured in the brain.</p