Characterization and Comparison of SLAM/CD150 in Free-Ranging Coyotes, Raccoons, and Skunks in Illinois for Elucidation of Canine Distemper Virus Disease

Canine distemper virus (CDV) is a cause of significant disease in canids and increasingly recognized as a multi-host pathogen, particularly of non-canid families within Carnivora. CDV outbreaks in sympatric mesocarnivores are routinely diagnosed in the Forest Preserve District of Cook County, Illinois. CDV is diagnosed more commonly and the disease more severe in raccoons and striped skunks than in coyotes. Research in other species suggests host cell receptors may play a role in variable disease outcome, particularly, the signaling lymphocyte activation molecule (SLAM) located on lymphoid cells. To evaluate receptor differences, partial SLAM genes were sequenced, and predicted amino acid (AA) sequences and structural models of the proposed viral interface assessed. Of 263 aligned nucleotide base pairs, 36 differed between species with 24/36 differences between canid and non-canids. Raccoon and skunk predicted AA sequences had higher homology than coyote and raccoon/skunk sequences and 8/11 residue differences were between coyote and raccoons/skunks. Though protein structure was similar, few residue differences were associated with charge and electrostatic potential surface alterations between canids and non-canids. RNAScope®(Advanced Cell Diagnostics, Silicon Valley, USA) ISH revealed low levels of expression that did not differ significantly between species or tissue type. Results suggest that differences in host receptors may impact species-specific disease manifestation

Hepatic deficiency of the pioneer transcription factor FoxA restricts hepatitis B virus biosynthesis by the developmental regulation of viral DNA methylation.

The FoxA family of pioneer transcription factors regulates hepatitis B virus (HBV) transcription, and hence viral replication. Hepatocyte-specific FoxA-deficiency in the HBV transgenic mouse model of chronic infection prevents the transcription of the viral DNA genome as a result of the failure of the developmentally controlled conversion of 5-methylcytosine residues to cytosine during postnatal hepatic maturation. These observations suggest that pioneer transcription factors such as FoxA, which mark genes for expression at subsequent developmental steps in the cellular differentiation program, mediate their effects by reversing the DNA methylation status of their target genes to permit their ensuing expression when the appropriate tissue-specific transcription factor combinations arise during development. Furthermore, as the FoxA-deficient HBV transgenic mice are viable, the specific developmental timing, abundance and isoform type of pioneer factor expression must permit all essential liver gene expression to occur at a level sufficient to support adequate liver function. This implies that pioneer transcription factors can recognize and mark their target genes in distinct developmental manners dependent upon, at least in part, the concentration and affinity of FoxA for its binding sites within enhancer and promoter regulatory sequence elements. This selective marking of cellular genes for expression by the FoxA pioneer factor compared to HBV may offer the opportunity for the specific silencing of HBV gene expression and hence the resolution of chronic HBV infections which are responsible for approximately one million deaths worldwide annually due to liver cirrhosis and hepatocellular carcinoma

Effects of FoxA deletion on liver FoxA1, FoxA2, FoxA3 and FoxO1 transcript levels, liver size and serum HBeAg in adult HBV transgenic mice.

<p>Quantitative analysis of the (A) FoxA1, (B) FoxA2, (C) FoxA3 and (D) FoxO1 transcripts by RT-qPCR in the HBV transgenic mice. The GAPDH transcript was used as an internal control for the quantitation of the FoxA1, FoxA2, FoxA3 and FoxO1 RNAs. The mean relative FoxA1, FoxA2, FoxA3 and FoxO1 transcript levels plus standard deviations derived from male and female FoxA-expressing (HBVFoxA2^fl/flAlbCre(-), HBVFoxA1^fl/flFoxA2^fl/flAlbCre(-) and HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(-)) and FoxA-deleted (HBVFoxA2^fl/flAlbCre(+), HBVFoxA1^fl/flFoxA2^fl/flAlbCre(+) and HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(+)) HBV transgenic mice is shown. The levels of the transcripts which are statistically significantly different between Cre(-) and Cre(+) HBV transgenic mice by a Student’s t-test (p<0.05) are indicated with an asterisk (*). Similar quantitative analysis of (E) liver size and (F) serum HBeAg levels is also shown. Average number of mice per group was 6.5±1.9 (Range: 3–9).</p

Histological analysis of liver samples from adult HBV transgenic mice.

<p>Control FoxA-expressing (HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(-), panels A-C) and FoxA-deleted (HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(+), panels D-F) HBV transgenic mice are indicated. Immunohistochemical staining indicates the presence of nuclear HBcAg throughout the liver whereas cytoplasmic staining is located primarily in the centrolobular hepatocytes in control mice (panel A) whereas HBcAg is minimally detectable in the FoxA-deleted mice (panel D). Hematoxylin and eosin (H&E) staining indicates biliary epithelial proliferation in the FoxA-deleted mice (panel E) which is absent in the control mice (panel B). Trichrome (TC) staining indicates bridging portal fibrosis in the FoxA-deleted mice (panel F) which is absent in the control mice (panel C). The black size bar is 100 μm.</p

Effect of FoxA-deletion on HBV DNA methylation distribution in adult mouse liver.

<p>The CpG DNA methylation frequency distribution across the 11, 38 and 14 sites within HBV nucleotide coordinates 341–711, 1215–1629 and 2131–2441, respectively, from male (A, C and E) and female (B, D and F) FoxA-expressing (HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(-); M A1A2A3Cre(-) and F A1A2A3Cre(-)) and FoxA-deleted (HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(+); M A1A2A3Cre(+) and F A1A2A3Cre(+)) HBV transgenic mice is shown.</p

Effects of FoxA deletion on liver TNFα, 2OAS, IL6, TGFβ1, TGFβ2, TGFβ3, αSMA, Col1A1, CK19 and CK20 transcript levels in adult HBV transgenic mice.

<p>Quantitative analysis of the (A) TNFα, (B) 2OAS, (C) IL6, (D) TGFβ1, (E) TGFβ2, (F) TGFβ3, (G) αSMA, (H) Col1A1, (I) CK19 and (J) CK20 transcripts by RT-qPCR in the HBV transgenic mice. The GAPDH transcript was used as an internal control for the quantitation of the TNFα, 2OAS, IL6, TGFβ1, TGFβ2, TGFβ3, αSMA, Col1A1, CK19 and CK20 RNAs. The mean relative TNFα, 2OAS, IL6, TGFβ1, TGFβ2, TGFβ3, αSMA, Col1A1, CK19 and CK20 transcript levels plus standard deviations derived from male and female FoxA-expressing (HBVFoxA2^fl/flAlbCre(-), HBVFoxA1^fl/flFoxA2^fl/flAlbCre(-) and HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(-)) and FoxA-deleted (HBVFoxA2^fl/flAlbCre(+), HBVFoxA1^fl/flFoxA2^fl/flAlbCre(+) and HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(+)) HBV transgenic mice is shown. The levels of the transcripts which are statistically significantly different between Cre(-) and Cre(+) HBV transgenic mice by a Student’s t-test (p<0.05) are indicated with an asterisk (*). Average number of mice per group was 6.5±1.8 (Range: 4–9).</p

DNA (Southern) filter hybridization analysis of HBV DNA replication intermediates in the livers of adult HBV transgenic mice.

<p>(A) DNA (Southern) filter hybridization analysis of representative mice of each sex and genotype are shown. Noncontiguous lanes from multiple analysis are presented. The probe used was HBVayw genomic DNA. FoxA-expressing (HBVFoxA2^fl/flAlbCre(-), HBVFoxA1^fl/flFoxA2^fl/flAlbCre(-) and HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(-)) and FoxA-deleted (HBVFoxA2^fl/flAlbCre(+), HBVFoxA1^fl/flFoxA2^fl/flAlbCre(+) and HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(+)) HBV transgenic mice are indicated (Genotype A2, A1A2 and A1A2A3, respectively). The HBV transgene (Tg) was used as an internal control for the quantitation of the HBV replication intermediates. Tg = HBV transgene; RC = HBV relaxed circular replication intermediates; SS = HBV single stranded replication intermediates. (B) Quantitative analysis of the HBV DNA replication intermediate (RI) levels in HBV transgenic mice. The mean DNA replication intermediate levels plus standard deviations are indicated. Average number of mice per group was 6.7±1.8 (Range: 4–9). The levels of replication intermediates which are statistically significantly different between Cre(-) and Cre(+) HBV transgenic mice by a Student’s t-test (p<0.05) are indicated with an asterisk (*).</p

Tissue-specific and developmental regulation of HBV DNA methylation distribution.

<p>The CpG DNA methylation frequency distribution across the 11, 38 and 13 sites within HBV nucleotide coordinates 341–711, 1215–1629 and 2264–2474, respectively, from male (A-C) and female (D-F) wild-type HBV transgenic mouse liver, kidney, muscle, spleen, lung and brain DNA is shown. The CpG DNA methylation frequency distribution across the 11, 38 and 13 sites within HBV nucleotide coordinates 341–711, 1215–1629 and 2264–2474, respectively, from three independent 0.5 day old neonatal (G-I) wild-type HBV transgenic mouse liver DNA is shown.</p

Effect of FoxA-deletion on HBV DNA methylation in adult mouse liver.

<p>The percentage of CpG DNA methylation at each position within the HBV genome from male (A) and female (B) FoxA-expressing (HBVFoxA2^fl/flAlbCre(-), HBVFoxA1^fl/flFoxA2^fl/flAlbCre(-) and HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(-)) and FoxA-deleted (HBVFoxA2^fl/flAlbCre(+), HBVFoxA1^fl/flFoxA2^fl/flAlbCre(+) and HBVFoxA1^fl/flFoxA2^fl/flFoxA3^+/-AlbCre(+)) HBV transgenic mice is shown. The positions of the viral transcription initiation sites for the X-gene (X RNA), the precore/pregenomic transcripts (C RNA), the large surface antigen transcript (PS RNA) and the middle/major surface antigen transcript (S RNA) are shown. The locations of the FoxA binding sites and CpG island within the HBV genome are also indicated. (C) The average percent methylation of the CpG sites spanning nucleotide coordinate 1–706 is correlated with the level of serum HBeAg.</p