Genome Characterization of a Pathogenic Porcine Rotavirus B Strain Identified in Buryat Republic, Russia in 2015

Citation: Alekseev, K.P.; Penin, A.A.; Mukhin, A.N.; Khametova, K.M.; Grebennikova, T.V.; Yuzhakov, A.G.; Moskvina, A.S.; Musienko, M.I.; Raev, S.A.; Mishin, A.M.; Kotelnikov, A.P.; Verkhovsky, O.A.; Aliper, T.I.; Nepoklonov, E.A.; Herrera-Ibata, D.M.; Shepherd, F.K.; Marthaler, D.G. Genome Characterization of a Pathogenic Porcine Rotavirus B Strain Identified in Buryat Republic, Russia in 2015. Pathogens 2018, 7, 46.An outbreak of enteric disease of unknown etiology with 60% morbidity and 8% mortality in weaning piglets occurred in November 2015 on a farm in Buryat Republic, Russia. Metagenomic sequencing revealed the presence of rotavirus B in feces from diseased piglets while no other pathogens were identified. Clinical disease was reproduced in experimentally infected piglets, yielding the 11 RVB gene segments for strain Buryat15, with an RVB genotype constellation of G12-P[4]-I13-R4-C4-M4-A8-N10-T4-E4-H7. This genotype constellation has also been identified in the United States. While the Buryat15 VP7 protein lacked unique amino acid differences in the predicted neutralizing epitopes compared to the previously published swine RVB G12 strains, this report of RVB in Russian swine increases our epidemiological knowledge on the global prevalence and genetic diversity of RVB

Metagenomic sequencing generates the whole genomes of porcine rotavirus A, C, and H from the United States.

The genus Rotavirus comprises eight species, designated A to H, and two recently identified tentative species I in dogs and J in bats. Species Rotavirus A, B, C and H (RVA, RVB, RVC and RVH) have been detected in humans and animals. While human and animal RVA are well characterized and defined, complete porcine genome sequences in the GenBank are limited compared to human strains. Here, we used a metagenomic approach to sequence the 11 segments of RVA, RVC and RVH strains from piglets in the United States (US) and explore the evolutionary relations of these RV species. Metagenomics identified Astroviridae, Picornaviridae, Caliciviridae, Coronoviridae in samples MN9.65 and OK5.68 while Picobirnaviridae and Arteriviridae were only identified in sample OK5.68. Whole genome sequencing and phylogenetic analyses identified multiple genotypes with the RVA of strain MN9.65 and OK5.68, with the genome constellation of G5/G9-P[7]/P[13]-I5/I5- R1/R1-C1-M1-A8-N1-T7-E1/E1-H1 and G5/G9-P[6]/P[7]-I5-R1/R1-C1-M1-A8-N1-T1/T7-E1/E1-H1, respectively. The RVA strains had a complex evolutionary relationship with other mammalian strains. The RVC strain OK5.68 had a genome constellation of G9-P[6]-I1-R1-C5-M6-A5-N1-T1-E1-H1, and shared an evolutionary relationship with porcine strains from the US. The RVH strains MN9.65 and OK5.68 had the genome constellation of G5-P1-I1-R1-C1-M1-A5-N1-T1-E4-H1 and G5-P1-I1-R1-C1-M1-A5-N1-T1-E1-H1, indicating multiple RVH genome constellations are circulating in the US. These findings allow us to understand the complexity of the enteric virome, develop improved screening methods for RVC and RVH strains, facilitate expanded rotavirus surveillance in pigs, and increase our understanding of the origin and evolution of rotavirus species

Novel Mammalian orthorubulavirus 5 Discovered as Accidental Cell Culture Contaminant

A distinct Russian Mammalian orthorubulavirus 5 (PIV5) was detected in cell culture exhibiting cytopathic effect and hypothesized to be contaminated by a scientist with respiratory symptoms. The identification of the divergent strain indicated a lack of knowledge on the diversity of PIV5 strains and calls for surveillance of global PIV5 strains

Identification of C/EBP alpha as a novel target of the HPV8 E6 protein regulating miR-203 in human keratinocytes

Patients suffering from Epidermodysplasia verruciformis (EV), a rare inherited skin disease, display a particular susceptibility to persistent infection with cutaneous genus beta-human papillomavirus (beta-HPV), such as HPV type 8. They have a high risk to develop nonmelanoma skin cancer at sun-exposed sites. In various models evidence is emerging that cutaneous HPV E6 proteins disturb epidermal homeostasis and support carcinogenesis, however, the underlying mechanisms are not fully understood as yet. In this study we demonstrate that microRNA-203 (miR-203), a key regulator of epidermal proliferation and differentiation, is strongly down-regulated in HPV8-positive EV-lesions. We provide evidence that CCAAT/enhancer-binding protein a (C/EBP alpha), a differentiation-regulating transcription factor and suppressor of UV-induced skin carcinogenesis, directly binds the miR-203 gene within its hairpin region and thereby induces miR-203 transcription. Our data further demonstrate that the HPV8 E6 protein significantly suppresses this novel C/EBP alpha/mir-203-pathway. As a consequence, the miR-203 target.Np63a, a proliferation-inducing transcription factor, is up-regulated, while the differentiation factor involucrin is suppressed. HPV8 E6 specifically down-regulates C/EBP alpha but not C/EBP beta expression at the transcriptional level. As shown in knock-down experiments, C/EBP alpha is regulated by the acetyltransferase p300, a well-described target of cutaneous E6 proteins. Notably, p300 bound significantly less to the C/EBP alpha regulatory region in HPV8 E6 expressing keratinocytes than in control cells as demonstrated by chromatin immunoprecipitation. In situ analysis confirmed congruent suprabasal expression patterns of C/EBP alpha and miR-203 in non-lesional skin of EV-patients. In HPV8-positive EV-lesions both factors are potently down-regulated in vivo further supporting our in vitro data. In conclusion our study has unraveled a novel p300/C/EBP alpha/mir203- dependent mechanism, by which the cutaneous HPV8 E6 protein may expand p63-positive cells in the epidermis of EV-patients and disturbs fundamental keratinocyte functions. This may drive HPV-mediated pathogenesis and may potentially also pave the way for skin carcinogenesis in EV-patients

p63 and miR-203 expression are altered in HPV8 E6 expressing HaCaT cells.

<p>HaCaT cells stably expressing HPV8 E6 or corresponding pLXSN control cells were investigated in different experiments. <b>(A)</b> p63 protein expression was analyzed by Western blot in relation to actin (shown is one experiment out of three), <b>(B)</b> ΔNp63α mRNA expression by qRT-PCR in relation to RPL13A. <b>(C)</b> Organotypic cultures were stained for p63 expression (brown). <b>(D)</b> Three independent cultures were quantified for p63-positive and -negative nuclei (per microscopic field in randomly selected areas, 200x). <b>(E)</b> MiR-203 expression was determined after treatment with Ca²⁺ for 72 h by Northern blot. 28S RNA served as loading control. One experiment out of three is shown. <b>(F)</b> The same RNA as in (E) was used to analyze miR-203 levels by qRT-PCR. Expression levels were normalized on RNU6B by 2^ΔΔCt. Mean values ± SD from <i>n</i> ≥ 3 experiments performed in duplicates are shown. (**p<0.01, ***p<0.001, ****p<0.0001)</p

HPV8 E6 involves the miR203/p63-pathway to reprogram keratinocyte functions.

<p><b>(A)</b> Cell cycle analysis of HPV8 E6 expressing and pLXSN control HaCaT cells by flow cytometry using propidium iodide staining. Shown are cell counts in the respective cell cycle phases in relation to the gated single cell population. <b>(B)</b> BrdU incorporation was measured in triplicates in HPV8 E6 expressing HaCaT cells and normalized to pLXSN-control. <b>(C)</b> HPV8 E6 expressing and pLXSN control HaCaT monolayer cultures were scratched. Pictures were taken at time points 0, 24 and 48 h. The area of the gap, indicated by dotted lines (left panel), was determined in relation to time point 0 h (right panel). HPV8 E6 expressing cells were scratched 24 h after 10 nM p63-siRNA (or si-control) <b>(D),</b> or 20 nM miR-203-mimic transfection (or control-mimic) <b>(E)</b> and analyzed as in (B). Scale bar: 200 μm. Mean values ± SD from <i>n</i> = 3 experiments are shown. (ns: not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001)</p

p300 regulates C/EBPα and miR-203 expression.

<p><b>(A)</b> NHK stably expressing HPV8 E6, mutant Δ8E6 (Δaa132-136) deficient in p300-binding, or pLXSN control cells were analyzed for C/EBPα expression by qRT-PCR. <b>(B)</b> C/EBPα protein expression was determined by IHC in HPV8 E6, Δ8E6 expressing HaCaT or control organotypic cultures (red). <b>(C)</b> In retrovirally infected NHK cells miR-203 expression was measured by qRT-PCR, <b>(D)</b> p63 and involucrin protein expression was determined by Western blot in relation to actin (shown is one representative blot out of three). <b>(E)</b> ΔNp63α and <b>(F)</b> involucrin mRNA expression levels were measured by qRT-PCR normalized to RPL13A. <b>(G)</b> NHK were transfected with 10 nM p300-specific siRNA (pool or two single siRNAs versus si-control), harvested 48 h later and expression of p300 mRNA, miR-203, C/EBPα and C/EBPβ mRNA were quantified by qRT-PCR as described. Mean values ± SD from <i>n</i> ≥ 3 experiments performed in duplicate are shown. <b>(H)</b> HPV8 E6 expressing HaCaT or pLXSN control cells were subjected to chromatin immunoprecipitation. Protein-genomic DNA complexes were precipitated with anti-p300 antibody. DNA was isolated, amplified by real-time PCR with primers specific for the nt -1533 to -1398 regulatory region of the <i>CEBPΑ</i> gene. The amount of target DNA precipitated with the isotype control antibody was set at 1. The mean values ± SD from 3 independent experiments are presented. (ns: not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).</p

p63 and miR-203 regulation in EV-lesions and by HPV8 oncoproteins in NHK in vitro.

<p>Sections of non-lesional skin <b>(A, C)</b> and HPV8-positive lesional skin <b>(B, D)</b> from EV-patients were stained using antibodies against p63 (A, B) or probes against miR-203 (C, D). NHK stably expressing HPV8 E6, E7, E6/E7 or the corresponding pLXSN control cells were analyzed for <b>(E)</b> p63 protein expression by Western blot in relation to actin expression (shown is one experiment out of three), <b>(F)</b> ΔNp63α mRNA or <b>(G)</b> miR-203 expression by qRT-PCR. ΔNp63α mRNA was determined in relation to RPL13A, miR-203 level to RNU6B by the 2^ΔΔCt method. In (F, G) mean values ± SD from <i>n</i> ≥ 3 experiments performed in duplicates are shown. (**p<0.01, ***p<0.001, ****p<0.0001)</p