Isolation and Characterization of Swinepox Virus from Outbreak in Russia.

Swinepox virus (SWPV) is the only member of the Suipoxvirus genus of the Poxviridae family and is an etiologic agent of a worldwide disease specific for domestic and wild pigs. SWPV outbreaks are sporadically recorded in different regions of Russia. In 2013, an outbreak of the disease causing skin lesions was registered on a pig farm in Russia. The presence of SWPV in the scab samples was assessed by in-house real-time PCR, reference PCR amplification, and nucleotide sequencing of the viral late transcription factor-3 (VLTF-3) gene and was then confirmed by virus isolation. Thus, the in-house real-time PCR proposed in this study could serve as a useful tool for the rapid specific detection of the swinepox virus. In the study, it has been demonstrated for the first time that nasal and oral swabs can be used for PCR diagnosis of the disease and for swinepox virus isolation. Phylogenetic analysis revealed that the isolated virus was closely related to SWPV isolates registered in Germany, USA, and Brazil, and slightly differed from the Indian isolates. During experimental infection of pigs, a low pathogenicity of the Russian isolate was observed. Our data provides the first report on the isolation and characterization of swinepox virus in Russia.

Deletion of the CD2 Gene in the Virulent ASFV Congo Strain Affects Viremia in Domestic Swine, but Not the Virulence.

African swine fever (ASF) is an infectious disease that causes the most significant losses to the pig industry. One of the effective methods for combating this disease could be the development of vaccines. To date, experimental vaccines based on the use of live attenuated strains of the ASF virus (ASFV) obtained by the deletion of viral genes responsible for virulence are the most effective. Deletion of the EP402R gene encoding a CD2-like protein led to the attenuation of various strains of the ASFV, although the degree of attenuation varies among different isolates. Here we have shown that the deletion of the EP402R gene from the genome of a high-virulent Congo isolate did not change either the virulence of the virus or its ability to replicate in the swine macrophage cell cultures in vitro. However, in vivo, animals infected with ΔCongo-v_CD2v had a delay in the onset of the disease and viremia compared to animals infected with the parental strain. Thus, deletion of the CD2 gene in different isolates of the ASFV has a different effect on the virulence of the virus, depending on its genetic background.

Comparison of Attenuated and Virulent Strains of African Swine Fever Virus Genotype I and Serogroup 2.

African swine fever (ASF) is a contagious disease of pigs caused by the ASF virus (ASFV). The main problem in the field of ASF control is the lack of vaccines. Attempts to obtain vaccines by attenuating the ASFV on cultured cell lines led to the production of attenuated viruses, some of which provided protection against infection with a homologous virus. Here we report on the biological and genomic features of the attenuated Congo-a (KK262) virus compared to its virulent homologue Congo-v (K49). Our results showed differences in in vivo replication and virulence of Congo-a. However, the attenuation of the K49 virus did not affect its ability to replicate in vitro in the primary culture of pig macrophages. Complete genome sequencing of the attenuated KK262 strain revealed an 8,8 kb deletion in the left variable region of the genome compared to the virulent homologue K49. This deletion concerned five genes of MGF360 and three genes of MGF505. In addition, three inserts in the B602L gene, genetic changes in intergenic regions and missense mutations in eight genes were detected. The data obtained contribute to a better understanding of ASFV attenuation and identification of potential virulence genes for further development of effective vaccines.

Analysis of gene expression in monocytes of immunized pigs after infection with homologous or heterologous African swine fever virus.

African swine fever is a deadly disease of pigs caused by the large DNA virus (ASFV). Despite intensive research, little is known about the molecular mechanisms of ASFV pathogenesis. Transcriptome analysis of host and viral genes in infected macrophages revealed changes in expression of genes involved in various biological processes, including immune response, inflammatory response and apoptosis. To understand the mechanisms of virus pathogenesis, we used transcriptome analysis to identify the differences in gene expression between peripheral blood monocytes (PBMCs) isolated from pigs immunized with attenuated Congo ASFV strain (KK262), and then infected in vitro with virulent homologous Congo strain (K49) or heterologous Mozambique strain (M78). We found that overexpression of IFN-γ was detected only in cells infected with M78, although the expression of interferon-stimulated genes was increased in both types of cells. In addition, up-regulation of pro-inflammatory cytokines and chemokines was found in PBMCs infected with the heterologous strain M78, in contrast to the cells infected with K49. These data may indicate the beginning of an early immune response in cells infected with a heterologous, but not homologous strain. Transcriptome analysis revealed down-regulation of genes involved in endocytosis and phagocytosis in cells infected with the K49 strain, but not in PBMCs infected with M78. On the contrary, we detected activation of endoplasmic reticulum stress response genes in cells infected with a homologous strain, but not in cells infected with a heterologous strain. This study is the first attempt to determine the differences in the response to ASF infection between homologous and heterologous strains at the cellular level. Our results showed that not only genes of the immune response, but also genes involved in endocytosis and cellular stress response may be important for the formation of cross-protective immunity. This data may be useful for vaccine development or testing of candidate vaccines.

Update in the methodology of the chronic stress paradigm: internal control matters.

To date, the reliability of induction of a depressive-like state using chronic stress models is confronted by many methodological limitations. We believe that the modifications to the stress paradigm in mice proposed herein allow some of these limitations to be overcome. Here, we discuss a variant of the standard stress paradigm, which results in anhedonia. This anhedonic state was defined by a decrease in sucrose preference that was not exhibited by all animals. As such, we propose the use of non-anhedonic, stressed mice as an internal control in experimental mouse models of depression. The application of an internal control for the effects of stress, along with optimized behavioural testing, can enable the analysis of biological correlates of stress-induced anhedonia versus the consequences of stress alone in a chronic-stress depression model. This is illustrated, for instance, by distinct physiological and molecular profiles in anhedonic and non-anhedonic groups subjected to stress. These results argue for the use of a subgroup of individuals who are negative for the induction of a depressive phenotype during experimental paradigms of depression as an internal control, for more refined modeling of this disorder in animals.

Growth Kinetics and Protective Efficacy of Attenuated ASFV Strain Congo with Deletion of the EP402 Gene.

African swine fever (ASF) is an emerging disease threat to the swine industry worldwide. There is no vaccine against ASF, and progress is hindered by a lack of knowledge concerning the extent of ASFV strain diversity and the viral antigens conferring type-specific protective immunity in pigs. We have previously demonstrated that homologous ASFV serotype-specific proteins CD2v (EP402R) and/or C-type lectin are required for protection against challenge with the virulent ASFV strain Congo (Genotype I, Serogroup 2), and we have identified T-cell epitopes on CD2v which may be associated with serotype-specific protection. Here, using a cell-culture adapted derivative of the ASFV strain Congo (Congo-a) with specific deletion of the EP402R gene (ΔCongoCD2v) in swine vaccination/challenge experiments, we demonstrated that deletion of the EP402R gene results in the failure of ΔCongoCD2v to induce protection against challenge with the virulent strain Congo (Congo-v). While ΔCongoCD2v growth kinetics in COS-1 cells and primary swine macrophage culture were almost identical to parental Congo-a, replication of ΔCongoCD2v in vivo was significantly reduced compared with parental Congo-a. Our data support the idea that the CD2v protein is important for the ability of homologous live-attenuated vaccines to induce protective immunity against the ASFV strain Congo challenge in vivo.

Transforming growth factor-beta1-mediated Slug and Snail transcription factor up-regulation reduces the density of Langerhans cells in epithelial metaplasia by affecting E-cadherin expression.

Epithelial metaplasia (EpM) is an acquired tissue abnormality resulting from the transformation of epithelium into another tissue with a different structure and function. This adaptative process is associated with an increased frequency of (pre)cancerous lesions. We propose that EpM is involved in cancer development by altering the expression of adhesion molecules important for cell-mediated antitumor immunity. Langerhans cells (LCs) are intraepithelial dendritic cells that initiate immune responses against viral or tumor antigens on both skin and mucosal surfaces. In the present study, we showed by immunohistology that the density of CD1a(+) LCs is reduced in EpM of the uterine cervix compared with native squamous epithelium and that the low number of LCs observed in EpM correlates with the down-regulation of cell-surface E-cadherin. We also demonstrated that transforming growth factor-beta1 is not only overexpressed in metaplastic tissues but also reduces E-cadherin expression in keratinocytes in vitro by inducing the promoter activity of Slug and Snail transcription factors. Finally, we showed that in vitro-generated LCs adhere poorly to keratinocytes transfected with either Slug or Snail DNA. These data suggest that transforming growth factor-beta1 indirectly reduces antigen-presenting cell density in EpM by affecting E-cadherin expression, which might explain the increased susceptibility of abnormal tissue differentiation to the development of cancer by the establishment of local immunodeficiency responsible for EpM tumorigenesis.

Phage T4 endonuclease SegD that is similar to group I intron endonucleases does not initiate homing of its own gene.

Homing endonucleases are a group of site-specific endonucleases that initiate homing, a nonreciprocal transfer of its own gene into a new allele lacking this gene. This work describes a novel phage T4 endonuclease, SegD, which is homologous to the GIY-YIG family of homing endonucleases. Like other T4 homing endonucleases SegD recognizes an extended, 16bp long, site, cleaves it asymmetrically to form 3'-protruding ends and digests both unmodified DNA and modified T-even phage DNA with similar efficiencies. Surprisingly, we revealed that SegD cleavage site was identical in the genomes of segD- and segD+ phages. We found that segD gene was expressed during the T4 developmental cycle. Nevertheless, endonuclease SegD was not able to initiate homing of its own gene as well as genetic recombination between phages in its site inserted into the rII locus.

A new dimethyl sulfoxide-based method for gene promoter methylation detection.

The identification of gene promoter methylation is a useful tool for the molecular diagnosis of human diseases. We have developed a new PCR-based technique for detecting the methylation status of CpG islands of gene promoters. This new method, named methyl-sensitive dimethyl sulfoxide-PCR (Ms-DMSO-PCR), is based on the finding that methylated and unmethylated DNAs show a different sensitivity to the amount of DMSO used in the PCR reaction. For the amplification of methylated DNA, more DMSO is required in comparison to unmethylated DNA. This finding resulted in the development of a simple PCR screening of CpG islands with addition of DMSO in the range from 0 to 8% (v/v), and the same pair of primers is sufficient for distinguishing hyper- or hypomethylated gene promoters from normally methylated sequences. This new technique is a one-step procedure and does not require any modifications of DNA or expensive equipment. Therefore, Ms-DMSO-PCR has the potential to be widely used for clinical applications as well in basic research.

Role of hormone cofactors in the human papillomavirus-induced carcinogenesis of the uterine cervix.

If human papillomavirus (HPV) is necessary for the development of (pre)neoplastic lesions of the uterine cervix, it is not sufficient. Among the cofactors involved in the malignant transformation of cells infected by HPV, sex hormones may facilitate the cervical carcinogenesis by different mechanisms, including the induction of squamous metaplasia in the transformation zone of the cervix, interactions between steroid hormones and HPV gene expression and alterations of the local immune microenvironment.

Single substitution in bacteriophage T4 RNase H alters the ratio between its exo- and endonuclease activities.

The article describes substitutions in bacteriophage T4 RNase H which provide so called das-effect. Phage T4 DNA arrest suppression (das) mutations have been described to be capable of partially suppressing the phage DNA arrest phenotype caused by a dysfunction in genes 46 and/or 47 (also known as Mre11/Rad50 complex). Genetic mapping of das13 (one of the das mutations) has shown it to be in the region of the rnh gene encoding RNase H. Here we report that Das13 mutant of RNase H has substitutions of valine 43 and leucine 242 with isoleucines. To investigate the influence of these mutations on RNase H nuclease properties we have designed a novel in vitro assay that allows us to separate and quantify exo- or endonuclease activities of flap endonuclease. The nuclease assay in vitro showed that V43I substitution increased the ratio between exonuclease/endonuclease activities of RNase H whereas L242I substitution did not affect the nuclease activity of RNase H in vitro. However, both mutations were necessary for the full das effect in vivo. Molecular modelling of the nuclease structure suggests that V43I substitution may lead to disposition of H4 helix, responsible for the interaction with the first base pairs of 5'end of branched DNA. These structural changes may affect unwinding of the first base pairs of gapped or nicked DNA generating a short flap and therefore may stabilize the DNA-enzyme complex. L242I substitution did not affect the structure of RNase H and its role in providing das-effect remains unclear.

Mutant ubiquitin (UBB+1) associated with neurodegenerative disorders is hydrolyzed by ubiquitin C-terminal hydrolase L3 (UCH-L3).

Mutant ubiquitin (UBB(+1)) accumulates in the hallmarks of tauopathies and polyglutamine diseases. We show that the deubiquitinating enzyme YUH1 of Saccharomyces cerevisiae and its mouse and human ortholog UCH-L3 are able to hydrolyze the C-terminal extension of UBB(+1). This yields another dysfunctional ubiquitin molecule (UB(G76Y)) with biochemical properties similar to full length UBB(+1). UBB(+1) may be detected in post-mortem tissue due to impaired C-terminal truncation of UBB(+1). Although the level of UCH-L3 protein in several neurodegenerative diseases is unchanged, we show that in vitro oxidation of recombinant UCH-L3 impairs its deubiquitinating activity. We postulate that impaired UCH-L3 function may contribute to the accumulation of full length UBB(+1) in various pathologies.