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Pavio N.,Laboratory for Animal Health | Pavio N.,French National Institute for Agricultural Research | Pavio N.,National Veterinary School of Alfort | Meng X.-J.,Center for Molecular Medicine and Infectious Diseases | And 3 more authors.
Current Opinion in Virology | Year: 2015

The concept of zoonotic viral hepatitis E has emerged a few years ago following the discovery of animal strains of hepatitis E virus (HEV), closely related to human HEV, in countries where sporadic cases of hepatitis E were autochthonous. Recent advances in the identification of animal reservoirs of HEV have confirmed that strains circulating in domestic and wild pigs are genetically related to strains identified in indigenous human cases. The demonstration of HEV contamination in the food chain or pork products has indicated that HEV is frequently a foodborne zoonotic pathogen. Direct contacts with infected animals, consumption of contaminated animal meat or meat products are all potential means of zoonotic HEV transmission. The recent identification of numerous other genetically diverse HEV strains from various animal species poses additional potential concerns for HEV zoonotic infection. © 2014 Published by Elsevier B.V.


Chafin C.B.,Center for Molecular Medicine and Infectious Diseases | Regna N.L.,Center for Molecular Medicine and Infectious Diseases | Dai R.,Center for Molecular Medicine and Infectious Diseases | Caudell D.L.,Center for Molecular Medicine and Infectious Diseases | Reilly C.M.,Center for Molecular Medicine and Infectious Diseases
Autoimmunity | Year: 2013

Recent evidence supports a role for epigenetic alterations in the pathogenesis of systemic lupus erythematosus (SLE). MicroRNAs (miRNAs or miRs) are endogenous epigenetic regulators whose expression is altered in many diseases, including SLE. IL-6 is an inflammatory cytokine produced by mesangial cells during lupus nephritis (LN). IL-6 contains a potential binding site for miRNA-let-7a (let-7a) in its 3′ untranslated region (UTR). We found let-7a expression was significantly increased in the mesangial cells of pre-diseased and actively diseased New Zealand Black/White (NZB/W) mice compared to age-matched New Zealand White (NZW) mice. Overexpression of let-7a in vitro increased IL-6 production in stimulated mesangial cells compared to non-transfected controls. Inhibition of let-7a did not significantly affect immune-stimulated IL-6 production. When stimulated mesangial cells overexpressing let-7a were treated with the transcription inhibitor Actinomycin D (ActD), IL-6 was degraded faster, consistent with the direct targeting of the 3′ UTR of IL-6 by let-7a. Overexpression of let-7a increased the expression of tristetraprolin (TTP), an RNA-binding protein (RBP) that has 5 potential binding regions in the 3′ UTR of IL-6. ActD inhibited the transcription of proteins including TTP that may contribute to the let-7a-mediated increase in immune-stimulated IL-6 production. These data show that NZB/W mice have higher let-7a expression than NZW mice and that increased let-7a expression in vitro increases IL-6 production in stimulated mesangial cells. Further studies examining the role of let-7a expression in inflammation are warranted. © 2013 Informa UK Ltd.


Chafin C.B.,Virginia State University | Chafin C.B.,Center for Molecular Medicine and Infectious Diseases | Regna N.L.,Virginia State University | Caudell D.L.,Wake forest University | Reilly C.M.,Virginia State University
Cellular and Molecular Immunology | Year: 2014

Epigenetic factors, including altered microRNA (miRNA) expression, may contribute to aberrant immune cell function in systemic lupus erythematosus (SLE). MiRNA-let-7a (let-7a) has been shown to directly alter cell cycle progression and proinflammatory cytokine production. Due to the crucial role of let-7a in cell division and inflammation, we investigated let-7a-mediated proliferation and NFκB translocation in J774A.1 macrophages and MES 13 mesangial cells in vitro. In immune-stimulated cells transfected with let-7a, cell proliferation was significantly increased over time. There was a significant increase in the number of immune-stimulated cells in S and G 2 phases. Immune-stimulated cells overexpressing let-7a had increased nuclear translocation of NFκB. Bioinformatical analysis revealed that the E2F family, critical regulators of the G1-S transition, has potential binding sites for let-7a in their mRNA transcripts. Let-7a overexpression significantly increased the expression of the cell cycle activator E2F2 and increased retinoblastoma protein (Rb) phosphorylation in immune-stimulated cells. The cell cycle inhibitor E2F5 was significantly decreased in let-7a-transfected cells that were immune-stimulated. Bioinformatical analysis revealed E2F2 and NFκB are transcription factors predicted to regulate the let-7a promoter. We analyzed transcriptional regulation of let-7a by real-time RT-PCR using chromatin immunoprecipitation with E2F2 and NFκB antibodies. There was an increase in E2F2 and NFκB binding in DNA enriched for the let-7a promoter in immune-stimulated cells. Silencing E2F2 or NFκB significantly decreased let-7a expression and IL-6 production in immune-stimulated cells. Taken together, our results suggest that overexpression of let-7a may contribute to hyperplasia and the proinflammatory response in SLE. © 2014 CSI and USTC.


Maciel B.M.,University Estadual Of Santa Cruz | Dias J.C.T.,University Estadual Of Santa Cruz | Romano C.C.,University Estadual Of Santa Cruz | Sriranganathan N.,Center for Molecular Medicine and Infectious Diseases | And 2 more authors.
Genetics and Molecular Research | Year: 2011

Quantification of Salmonella in asymptomatic carrier animals can be used to assess microbial risk and monitor the level of contamination in domestic animals used for food production. We examined the sensitivity, specificity and accuracy of real-time qPCR, without pre-enrichment or selective enrichment stages, for the quantification of S. enterica serovar Enteritidis in resistant mice, as a model of asymptomatic carrier animal. The results were compared with those obtained by traditional bacteriological culture methods, the gold standard test. Two hundred and forty-three samples, including spleen, liver, mesenteric lymph nodes, portions of intestine, intestinal content of the ileocecal portion, and feces, were collected from a group of 27 C57BL/6 mice, that had been intragastrically inoculated with high doses of S. enterica serovar Enteritidis. The real-time qPCR assay presented a consistent linearity of the standard curve (r2 = 0.999), with very low differences between melting temperatures, and low coefficients of variation in intra- (<1%) and interassay (<2%) comparisons. The primers were highly specific; there was no amplification with other Salmonella serovars or with DNA from uninfected tissues and feces from mice. The detection limit of the technique was defined as 32 copies of S. enterica serovar Enteritidis. A sensitivity of 90%, a specificity of 77% and an accuracy of 79% were obtained. The higher sensitivity of PCR was reflected in a kappa coefficient of 0.41, showing moderate agreement between tests. We conclude that real-time qPCR is a good alternative for diagnostic scanning in asymptomatic carrier animals, due to its high sensitivity and rapidity. © FUNPEC-RP.


Caudell D.,Center for Molecular Medicine and Infectious Diseases | Caudell D.,U.S. National Institutes of Health | Harper D.P.,U.S. National Institutes of Health | Harper D.P.,Uniformed Services University of the Health Sciences | And 5 more authors.
Blood | Year: 2010

The t(10;11) translocation results in a CALM-AF10 fusion gene in a subset of leukemia patients. Expression of a CALM-AF10 transgene results in leukemia, with prolonged latency and incomplete penetrance, suggesting that additional events are necessary for leukemic transformation. CALM-AF10 mice infected with the MOL4070LTR retrovirus developed acute leukemia, and ligation-mediated polymerase chain reaction was used to identify retroviral insertions at 19 common insertion sites, including Zeb2, Nf1, Mn1, Evi1, Ift57, Mpl, Plag1, Kras, Erg, Vav1, and Gata1. A total of 26% (11 of 42) of the mice had retroviral integrations near Zeb2, a transcriptional corepressor leading to overexpression of the Zeb2-transcript. A total of 91% (10 of 11) of mice with Zeb2 insertions developed B-lineage acute lymphoblastic leukemia, suggesting that Zeb2 activation promotes the transformation of CALM-AF10 hematopoietic precursors toward B-lineage leukemias. More than half of the mice with Zeb2 integrations also had Nf1 integrations, suggesting cooperativity among CALM-AF10, Zeb2, and Ras pathway mutations. We searched for Nras, Kras, and Ptpn11 point mutations in the CALM-AF10 leukemic mice. Three mutations were identified, all of which occurred in mice with Zeb2 integrations, consistent with the hypothesis that Zeb2 and Ras pathway activation promotes B-lineage leukemic transformation in concert with CALM-AF10.


Juhan N.M.,Center for Molecular Medicine and Infectious Diseases | LeRoith T.,Center for Molecular Medicine and Infectious Diseases | Opriessnig T.,Iowa State University | Meng X.J.,Center for Molecular Medicine and Infectious Diseases
Virus Research | Year: 2010

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases (PCVAD) in pigs. The open reading frame (ORF) 3 of PCV2 reportedly induces apoptosis and is associated with PCV2 pathogenicity. In this study, we first created an ORF3-null PCV2 mutant (muPCV2) by site-directed mutagenesis and demonstrated that the dimerized plasmid DNA of muPCV2 clone is infectious when injected intramuscularly (I.M.) into pigs. Subsequently, by using a well-characterized pig model we compared the pathogenicity of the muPCV2 and the wildtype PCV2. Thirty-one pigs were divided into 3 groups of 11, 10, and 10 each: group 1 pigs were each inoculated I.M. with PBS buffer as negative controls, group 2 pigs each with 200 μg of muPCV2 infectious DNA clone, and group 3 pigs each with 200 μg of wildtype PCV2 infectious DNA clone. Blood was collected prior to inoculation and weekly thereafter, and tested for PCV2 antibodies by ELISA and serum viral DNA loads by quantitative PCR. All pigs were necropsied at 35 days post-inoculation. The results showed that pigs inoculated with muPCV2 had a delayed seroconversion and lower serum viral load. However, there was no significant difference in the average scores of the histological or gross lesions or the amount of PCV2-specific antigen in tissues between wildtype PCV2- and muPCV2-inoculated groups. Thus, the data from this study do not fully support the conclusion of a previous report regarding PCV2 attenuation by abrogation of ORF3 although the results did show that ORF3 is dispensable for PCV2 replication in pigs. © 2009 Elsevier B.V. All rights reserved.


Roop II R.M.,East Carolina University | Caswell C.C.,Center for Molecular Medicine and Infectious Diseases
Cell Host and Microbe | Year: 2013

Studies described by Eisele et al. (2013) and Xavier et al. (2013) in this issue of Cell Host & Microbe show that the bacterial pathogens Salmonella and Brucella exploit the increased levels of glucose present in alternatively activated macrophages to sustain chronic infections in experimentally infected mice. © 2013 Elsevier Inc.


Rajasekaran P.,Center for Molecular Medicine and Infectious Diseases | Surendran N.,University of Maryland Baltimore County | Seleem M.N.,Virginia Polytechnic Institute and State University | Sriranganathan N.,Center for Molecular Medicine and Infectious Diseases | And 2 more authors.
Vaccine | Year: 2011

Infection by members of the Gram-negative bacterial genus Brucella causes brucellosis in a variety of mammals. Brucellosis in swine remains a challenge, as there is no vaccine in the USA approved for use in swine against brucellosis. Here, we developed an improved recombinant Brucella abortus vaccine strain RB51 that could afford protection against Brucella suis infection by over-expressing genes encoding homologous proteins: L7/L12 ribosomal protein, Cu/Zn superoxide dismutase [SOD] and glycosyl-transferase [WboA]. Using strain RB51. leuB as a platform and an antibiotic-resistance marker free plasmid, strains RB51. leuB/SOD, RB51. leuB/SOD/L7/L12 and RB51. leuB/SOD/WboA were constructed to over-express the antigens: SOD alone, SOD and ribosomal protein L7/L12 or SOD and glycosyl-transferase, respectively. The ability of these vaccine candidates to protect against a virulent B. suis challenge were evaluated in a mouse model. All vaccine groups protected mice significantly (P<0.05) when compared to the control group. Within the vaccine groups, the mice vaccinated with strain RB51. leuB/SOD/WboA were significantly better protected than those that were vaccinated with either strain RB51. leuB/SOD or RB51. leuB/SOD/L7/L12. These results suggest that Brucella antigens can be over-expressed in strain RB51. leuB and elicit protective immune responses against brucellosis. Since the plasmid over-expressing homologous antigens does not carry an antibiotic resistance gene, it complies with federal regulations and therefore could be used to develop safer multi-species vaccines for prevention of brucellosis caused by other species of Brucella. © 2011 Elsevier Ltd.


Kenney S.P.,Center for Molecular Medicine and Infectious Diseases | Meng X.-J.,Center for Molecular Medicine and Infectious Diseases
PLoS ONE | Year: 2015

Human ribosomal protein S17 (RPS17) is mutated in Diamond-Blackfan Anemia (DBA), a bone marrow disorder that fails to produce sufficient red blood cells leading to anemia. Recently, an RPS17 protein sequence was also found to be naturally inserted in the genome of hepatitis E virus (HEV) from patients chronically-infected by HEV. The role of RPS17 in HEV replication and pathogenesis remains unknown due to the lack of knowledge about how RPS17 functions at a molecular level. Understanding the biological function of RPS17 is critical for elucidating its role in virus infection and DBA disease processes. In this study we probed the subcellular distribution of normal and mutant RPS17 proteins in a human liver cell line (Huh7). RPS17 was primarily detected within the nucleus, and more specifically within the nucleoli. Using a transient expression system in which RPS17 or truncations were expressed as fusions with enhanced yellow fluorescent protein (eYFP), we were able to identify and map, for the first time, two separate nuclear localization signals (NLSs), one to the first 13 amino acids of the amino-terminus of RPS17 and the other within amino acids 30-60. Additionally, we mapped amino acid sequences required for nucleolar accumulation of RPS17 to amino acids 60-70. Amino acids 60-70 possess a di-RG motif that may be necessary for nucleolar retention of RPS17. The results from this study enhance our knowledge of RSP17 and will facilitate future mechanistic studies about the roles of RSP17 in hepatitis E and DBA disease processes. © 2015 Kenney, Meng.


Meng X.J.,Center for Molecular Medicine and Infectious Diseases
Veterinary Microbiology | Year: 2010

Hepatitis E virus (HEV) is a small, non-enveloped, single-strand, positive-sense RNA virus of approximately 7.2 kb in size. HEV is classified in the family Hepeviridae consisting of four recognized major genotypes that infect humans and other animals. Genotypes 1 and 2 HEV are restricted to humans and often associated with large outbreaks and epidemics in developing countries with poor sanitation conditions, whereas genotypes 3 and 4 HEV infect humans, pigs and other animal species and are responsible for sporadic cases of hepatitis E in both developing and industrialized countries. The avian HEV associated with Hepatitis-Splenomegaly syndrome in chickens is genetically and antigenically related to mammalian HEV, and likely represents a new genus in the family. There exist three open reading frames in HEV genome: ORF1 encodes non-structural proteins, ORF2 encodes the capsid protein, and the ORF3 encodes a small phosphoprotein. ORF2 and ORF3 are translated from a single bicistronic mRNA, and overlap each other but neither overlaps ORF1. Due to the lack of an efficient cell culture system and a practical animal model for HEV, the mechanisms of HEV replication and pathogenesis are poorly understood. The recent identification and characterization of animal strains of HEV from pigs and chickens and the demonstrated ability of cross-species infection by these animal strains raise potential public health concerns for zoonotic HEV transmission. It has been shown that the genotypes 3 and 4 HEV strains from pigs can infect humans, and vice versa. Accumulating evidence indicated that hepatitis E is a zoonotic disease, and swine and perhaps other animal species are reservoirs for HEV. A vaccine against HEV is not yet available. © 2009 Elsevier B.V. All rights reserved.

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