Valosin-containing protein (VCP/p97) is responsible for the endocytotic trafficking of classical swine fever virus.

Classical swine fever virus (CSFV), a member of the Flaviviridae enveloped RNA virus family, results in an epidemic disease that brings serious economic losses to the pig industry worldwide. Valosin-containing protein (VCP/p97), a multifunctional active protein in cells, is related to the life activities of many viruses. However, the role of VCP in CSFV infection remains unknown. In this study, it was first found that treatment of VCP inhibitors impaired CSFV propagation. Furthermore, overexpression or knockdown of VCP showed that it was essential for CSFV infection. Moreover, confocal microscopy and immunoprecipitation assay showed that VCP was recruited for intracellular transport from early endosomes to lysosomes. Importantly, knockdown of VCP prevented CSFV to release from early endosomes, suggesting that VCP is a key factor for CSFV trafficking. Taken together, our findings first demonstrate that the endocytosis of CSFV into PK-15 cells requires the participation of VCP, providing the alternative approach for the discovery of novel anti-flaviviridae drugs.

Rab22a cooperates with Rab5 and NS4B in classical swine fever virus entry process.

Classical swine fever virus (CSFV) is an ancient and economically important flavivirus that utilizes a Rab5-dependent endocytic pathway to enter host cells. Rab22a is a small GTPase that cooperates with Rab5 in the regulation of early endosome dynamics. Until now, the role of Rab22a in the flavivirus life cycle has been poorly defined. In this study, we systematically analyzed the role of Rab22a in CSFV proliferation and internalization using multiple viral replication analyses in combination with the overexpression, knockdown, and mutation of Rab22a, and found that Rab22a is involved in the entry process of CSFV. Confocal microscopy results showed that Rab22a colocalized with virus particles during the early phase of infection. Furthermore, by using glutathione S-transferase pull-down and co-immunoprecipitation assays, we verified the interaction between Rab22a and CSFV non-structural protein NS4B, and determined that NS4B can only bind to wild-type Rab22a, but not to the mutants Q64L and S19N. In addition, we explored the relationship between Rab22a, Rab5 and NS4B in CSFV internalization, and found out that these three proteins bind in early endosomes, and then through a Rab22a-Rab5-NS4B cascade allows the entry of CSFV. Taken together, our findings highlight the role of Rab proteins in CSFV internalization, and extend the understanding of the life cycle of flaviviruses.

Seroprevalence of severe fever with thrombocytopenia syndrome virus in animals in Kagoshima Prefecture, Japan, and development of Gaussia luciferase immunoprecipitation system to detect specific IgG antibodies.

We conducted a seroprevalence investigation of the healthy population of animals in Kagoshima Prefecture, an area in which severe fever with thrombocytopenia syndrome (SFTS) is endemic. Of 104 domestic cat and 114 dog samples, 2 (1.9%) and 11 (9.6%) were positive for anti-SFTS virus (SFTSV) IgG by indirect ELISA, respectively. Viral RNA was detected in one dog (0.9%) by RT-PCR. Of the 102 wild boar (Sus scrofa) and 107 deer (Cervus nippon) samples tested, 55 (53.9%) and 37 (34.7%) were positive for anti-SFTSV IgG, respectively. Only one wild boar (1.0%) was positive for viral RNA. Although symptomatic SFTSV infections in domestic cats have increased in this area, the seroprevalence of the healthy population of domestic cats tends to be lower than those of other animals. We developed a Gaussia luciferase immunoprecipitation system (GLIPS) using mammalian cells expressing a recombinant SFTSV nucleoprotein (SFTSV-rNP) for the detection of SFTSV-specific antibodies in samples from various animal species. The sensitivity of the assay was highly consistent with that of indirect ELISA, indicating that it could serve as a useful tool for a large-scale surveillance of SFTSV across multiple species of animals.

Development of molecular and histological methods to evaluate stress oxidative biomarkers in sea bass (Dicentrarchus labrax).

In aquaculture, fish species may experience stressful episodes caused by poor farming conditions. The exponential increase of global aquaculture has raised the number of research studies aimed at demonstrating the sensitivity of aquatic animals in confined environments. The development of a real-time PCR and immunohistochemistry methods were investigated to evaluate the presence, localization, and quantity of biomarkers of oxidative stress in European sea bass (Dicentrarchus labrax). In particular, stress tests such as manipulation and temperature changes were conducted through molecular methods to identify the expression level of heat shock protein 70 (HSP70) in stressed animals compared with a control group. The immunohistochemical technique was also applied to locate and study the trends-levels of nitrotyrosine (NT), heat shock protein 70 (HSP70), malondialdehyde (MDA), and 4-hydroxy-2-nonenal (HNE) in different tissues from stressed animals and control group. The presence of the rodlet cell (RCs) was evaluated by histology in both a control and stressed group. Our results show that the real-time PCR method developed is specific for the evaluated target gene and that manipulation and temperature increase are strong stressors for animals. Relative quantification data revealed a gene expression increase of HSP70 in the stressed group of animals compared to the control group. The antibodies used for the immunohistochemical staining were efficient, and it was possible to appreciate the increase of immunoprecipitates in European sea bass either manipulated or stressed by temperature increase. The present study can be a starting point to allow the quantification of HSP70 and the identification of other stress biomarkers in D. labrax.

Establishment of rat anti-canine DEP domain containing 1B (DEPDC1B) monoclonal antibodies.

DEP domain-containing 1B (DEPDC1B) is involved in the regulation of cell de-adhesion and actin cytoskeleton activity during the G2/M transition of the cell cycle, and its overexpression has been proven to be associated with cancer progression in several human cancers. Canine DEPDC1B was identified as a gene that was overexpressed in canine lymphoma tissues in our previous study. However, in dogs, the protein expression of DEPDC1B remains to be determined due to the lack of a specific monoclonal antibody. Here, we developed rat monoclonal antibodies against canine DEPDC1B and characterized their applicability for immunodetection assays. Our findings demonstrated that these antibodies are functional and can be important tools to investigate the precise role of DEPDC1B in canine tumors.

Comprehensive analysis of the ubiquitome in rabies virus-infected brain tissue of Mus musculus.

Ubiquitination is an important post-translational modification (PTM) that plays a key role in almost every aspect of cellular processes and many signaling pathways in eukaryotes. In this study, we performed a quantitative ubiquitome study to identify the global change of ubiquitination induced by rabies virus (RABV) infection in the mouse brain tissue. 4,243 ubiquitinated sites, mapping to 1,626 proteins were identified; using a cutoff of fold change >2, 644 and 70 ubiquitinated proteins were up- and down-regulated, respectively. GO analysis indicated that the differentially ubiquitinated proteins (DUPs) were significantly enriched in the myelin sheath of cells and binding activity. KEGG pathway analysis indicated that the identified proteins were related to biosynthesis of amino acids. Of note, ubiquitination was observed on all five RABV proteins by both proteomics and biochemical approaches. Our study revealed the global ubiquitome of RABV-infected mice and provides a valuable resource for investigating the pathogenic mechanisms of RABV.

Identification of determinants that mediate binding between Tembusu virus and the cellular receptor heat shock protein A9.

Heat shock protein A9 (HSPA9), a member of the heat shock protein family, is a putative receptor for Tembusu virus (TMUV). By using Western blot and co-immunoprecipitation assays, E protein domains I and II were identified as the functional domains that facilitate HSPA9 binding. Twenty-five overlapping peptides covering domain I and domain II sequences were synthesized and analyzed by using an HSPA9 binding assay. Two peptides showed the capability of binding to HSPA9. Dot blot assay of truncated peptides indicated that amino acid residues 19 to 22 and 245 to 252 of E protein constitute the minimal motifs required for TMUV binding to HSPA9. Importantly, peptides harboring those two minimal motifs could effectively inhibit TMUV infection. Our results provide insight into TMUV-receptor interaction, thereby creating opportunities for elucidating the mechanism of TMUV entry.

Membrane-associated GRP78 helps subgroup J avian leucosis virus enter cells.

We previously identified chicken Annexin A2 (chANXA2) as a novel receptor for retrovirus avian leucosis virus subgroup J (ALV-J), using a DF1 cell line expressing the viral envelope (env) protein. To further probe whether other proteins participate in virus infection, we investigated several host proteins from co-immunoprecipitation with the DF1 cell line expressing viral env. Mass spectrometry analysis indicates that the chicken glucose-regulation protein 78 (chGRP78) of the DF1 membrane interacted with the ALV-J env protein. The results revealed that antibodies or siRNA to chGRP78 significantly inhibited ALV-J infection and replication, and over-expression of chGRP78 enabled the entry of ALV-J into non-susceptible cells. Taken together, these results are the first to report that chGRP78 functions to help ALV-J enter cells.

A Novel Detection Platform for Shrimp White Spot Syndrome Virus Using an ICP11-Dependent Immunomagnetic Reduction (IMR) Assay.

Shrimp white spot disease (WSD), which is caused by white spot syndrome virus (WSSV), is one of the world's most serious shrimp diseases. Our objective in this study was to use an immunomagnetic reduction (IMR) assay to develop a highly sensitive, automatic WSSV detection platform targeted against ICP11 (the most highly expressed WSSV protein). After characterizing the magnetic reagents (Fe3O4 magnetic nanoparticles coated with anti ICP11), the detection limit for ICP11 protein using IMR was approximately 2 x 10(-3) ng/ml, and the linear dynamic range of the assay was 0.1~1 x 10(6) ng/ml. In assays of ICP11 protein in pleopod protein lysates from healthy and WSSV-infected shrimp, IMR signals were successfully detected from shrimp with low WSSV genome copy numbers. We concluded that this IMR assay targeting ICP11 has potential for detecting the WSSV.

Genetic and serological surveillance for non-primate hepacivirus in horses in Japan.

Non-primate hepacivirus (NPHV) is a recently discovered homolog of the hepatitis C virus in horses. The frequency and distribution of NPHV infections among horses in Japan is unknown. In this study, serum samples from 453 horses across Japan were screened for NPHV RNA using real-time RT-PCR and anti-nonstructural 3 protein (NS3) antibodies using the Gaussia luciferase immunoprecipitation system assay. In order to monitor the course of NPHV infection in horses, we examined 31 stored samples (9 adult horses and 22 young horses) obtained one year ago and compared the results to the recent data. Stored sera from 7 mare-foal pairs were also examined. The NS3 region sequences of 14 NPHV strains from NPHV RNA positive serum samples were determined and analyzed phylogenically. Of the 453 serum samples tested, 33.55% were positive for anti-NS3 antibody and 13.68% were positive for NPHV RNA. We found a higher rate of NPHV RNA detection in serum obtained from young horses (1-2 years of age) than that of adults, in two geographically distinct areas. We observed higher variation in the course of infection over one year in young horses than in adult horses. The foals were infected with NPHV after the weaning period. Phylogenic analysis revealed that while NPHV NS3 genes isolated in Japan clustered with sequences previously classified as NPHV, but the genetic diversity of the Japanese NPHV strains we detected was not correlated with their geographic origin. In conclusion, Japanese horses exhibit a high prevalence of NPHV. Young age appears to be a risk factor for such viral infection in Japan, although the infectious route was not determined.

Genomic architecture of histone 3 lysine 27 trimethylation during late ovine skeletal muscle development.

The ruminant developmental transition from late foetus to lamb is associated with marked changes in skeletal muscle structure and function that reflect programming for new physiological demands following birth. To determine whether epigenetic changes are involved in this transition, we investigated the genomic architecture of the chromatin modification, histone 3 lysine 27 trimethylation (H3K27me3), which typically regulates early life developmental processes; however, its role in later life processes is unclear. Chromatin immunoprecipitation coupled with next-generation sequencing was used to map H3K27me3 nucleosomes in ovine longissimus lumborum skeletal muscle at 100 days of gestation and 12 weeks post-partum. In both states, H3K27me3 modification was associated with genes, transcription start sites and CpG islands and with transcriptional silencing. The H3K27me3 peaks consisted of two major categories, promoter specific and regional, with the latter the dominant feature. Genes encoding homeobox transcription factors regulating early life development and genes involved in neural functions, particularly gated ion channels, were strongly modified by H3K27me3. Gene promoters differentially modified by H3K27me3 in the foetus and lamb were enriched for gated ion channels, which may reflect changes in neuromuscular function. However, most modified genes showed no changes, indicating that H3K27me3 does not have a large role in late muscle maturation. Notably, promyogenic transcription factors were strongly modified with H3K27me3 but showed no differences between the late gestation foetus and lamb, likely reflecting their lack of involvement in the myofibre fusion process occurring in this transition. H3K27me3 is a major architectural feature of the epigenetic landscape of ruminant skeletal muscle, and it comments on gene transcription and gene function in the context of late skeletal muscle development.

Presence and distribution of urocortin and corticotrophin-releasing hormone receptors in the bovine thyroid gland.

Urocortin (UCN), a 40 amino acid peptide, is a corticotrophin-releasing hormone (CRH)-related peptide. The biological actions of CRH family peptides are mediated via two types of G-protein-coupled receptors, CRH type 1 (CRHR1) and CRH type 2 (CRHR2). The aim of this study was to investigate the expression of UCN, CRHR1 and CRHR2 by immunoprecipitation, Western blot, immunohistochemistry and RT-PCR in the bovine thyroid gland. Immunoprecipitation and Western blot analysis showed that tissue extracts reacted with the anti-UCN, anti-CRHR1 and anti-CRHR2 antibodies. RT-PCR experiments demonstrated that mRNAs of UCN, CRHR1 and CRHR2 were expressed. UCN immunoreactivity (IR) and CRHR2-IR were found in the thyroid follicular and parafollicular cells and CRHR1-IR in the smooth muscle of the blood vessels. These results suggest that a regulatory system exists in the bovine thyroid gland based on UCN, CRHR1 and CRHR2 and that UCN plays a role in the regulation of thyroid physiological functions through an autocrine/paracrine mechanism.

Breed-dependent transcriptional regulation of phosphoenolpyruvate carboxylase, cytosolic form, expression in the liver of broiler chickens.

Hepatic gluconeogenesis is the main source of glucose during chicken embryonic development, and it plays a major role in glucose homeostasis for developing embryos. Phosphoenolpyruvate carboxylase (PEPCK) catalyzes the rate-limiting step of gluconeogenesis, yet how hepatic PEPCK expression is differentially regulated between chicken breeds remains elusive. In this study, fertile eggs from a slow-growing Chinese Yellow Feathered Chicken and a fast-growing White Recessive Rock Chicken were incubated under the same standard conditions, and serum and liver samples were collected on embryonic d 18 (18E). The fast-growing breed had a significantly higher fetal weight (P < 0.01) and serum glucose concentration (P < 0.05) compared with the slow-growing breed. The fast-growing breed also had significantly higher hepatic mRNA expression levels of the cystolic form of PEPCK (PEPCK-c; P < 0.05) and significantly higher hepatic mRNA and protein expression levels of cAMP response element binding protein 1 (CREB-1; P < 0.05). Moreover, the binding of phosphorylated CREB-1 to the PEPCK-c promoter tended to be higher in the fast-growing breed (P = 0.08). Breed-specific epigenetic modifications of the PEPCK-c promoter were also observed; the fast-growing breed demonstrated lower CpG methylation (P < 0.05) and histone H3 (P < 0.05) levels but more histone H3 acetylation (H3ac) and histone H3 lysine 27 trimethylation (H3K27me3; P < 0.05) compared with the slow-growing breed. Our results suggest that hepatic PEPCK-c expression is transcriptionally regulated in a breed-specific manner and that fast- and slow-growing broiler chicken fetuses exhibit different epigenetic modifications on their PEPCK-c promoter regions.

Factors influencing measurement of serum iron concentration in dogs: diurnal variation and hyperferritinemia.

We evaluated diurnal variation and hyperferritinemia as factors that influence the values of serum iron concentration in dogs, using the International Committee for Standardization in Hematology (ICSH) colorimetric method. Serum iron levels were significantly higher in the morning than in the evening in 6 clinically healthy beagle dogs, and the maximum decrease in serum iron concentration was 47.3%. Moreover, the change in serum iron concentrations in 22 clinical canine cases with various serum ferritin levels was evaluated by immunoprecipitation of ferritin. The rate of decline in the serum iron concentrations positively correlated with serum ferritin levels (r=0.48, P=0.024). These results show that it is necessary to consider the sampling time and serum ferritin level for accurate interpretation of serum iron concentrations in dogs.

Swine liver methylomes of Berkshire, Duroc and Landrace breeds by MeDIPS.

Using a methyl-DNA immunoprecipitation technique in combination with next-generation deep sequencing, we conducted comprehensive DNA methylation profiling of liver genomes from three pig breeds: Berkshire, Duroc and Landrace. The profiles revealed that the distribution patterns of methylation signals along the genome are conserved among the three pig breeds. Specifically, many signals in coding genes were found in introns, and most signals in the repetitive elements were identified in non-long terminal repeat (LTR) retrotransposons such as long and short interspersed repetitive elements, implying a significant association with alternative splicing and expression of retrotransposable elements respectively. Differentially methylated regions among the three pig breeds were identified in the non-LTR retrotransposons, suggesting that they may lead to differential retrotransposable element activity. Altogether, this study provides advanced swine methylome data and valuable resources for understanding the function of DNA methylation in the evolutionary divergence of different pig breeds.

Identification and in vitro characterization of a Marek's disease virus-encoded Ribonucleotide reductase.

Marek's disease virus (MDV) encodes a ribonucleotide reductase (RR), a key regulatory enzyme in the DNA synthesis pathway. The gene coding for the RR of MDV is located in the unique long (UL) region of the genome. The large subunit is encoded by UL39 (RR1) and is predicted to comprise 860 amino acids whereas the small subunit encoded by UL40 (RR2) is predicted to be 343 amino acids long. Immunoprecipitation analysis of MDV-1 (GA strain)-infected cells with T81, a monoclonal antibody specific for RR of MDV, identified two major proteins of 90,000 and 40,000 daltons, corresponding to RR1 and RR2, respectively. In addition, RR was abundantly expressed in the cytoplasm of cells infected with 51 strains of MDV belonging to MDV serotypes 1, 2, and 3 as demonstrated by immunofluorescence staining. Northern blot analysis of RNA extracted from MDV-infected cells showed a major band of around 4.4 kb in size corresponding to the RR1 and RR2 transcripts. In vivo, RR was abundantly expressed in lymphoid organs and feather follicle epithelium of MDV-infected chickens during early cytolytic infection, as determined by immunohistochemistry. There was, however, no expression of RR in MDV-induced tumors in lymphoid organs. The abundant expression of RR in MDV-infected chicken may suggest an important role of RR in the conversion of ribonucleotides to deoxyribonucleotides for MDV DNA synthesis.

Evidence that calf bronchopneumonia may be accompanied by increased sialylation of circulating immune complexes' IgG.

Immune complexes (IC) could have an important role in the pathogenesis of pre-ruminant calves' bronchopneumonia. IC are potent activators of complement and neutrophils and they might be responsible for immune protection, as well as for pulmonary damage. Immunoglobulin G (IgG), as constituents of IC, initiates the effector phase of immune response through binding of Fcγ and complement receptors. The oligosaccharide moieties expressed on IgG can modulate their antigen affinity and effector function. Structural characteristics of IgG molecules from IC in the pre-ruminant calves have not been studied in detail. The aim of our study was to determine if the glycosylation profile of IgG from circulating IC (CIC) in calves with bronchopneumonia differed from those of healthy control calves. A total number of 13 Holstein-Friesian calves, at the age of three months were included in the study. All calves were clinically examined by a veterinarian. Calves were classified by signs of respiratory disease in two groups: healthy (n=6) and diseased (n=7) calves. The CIC from calves' sera were isolated by the polyethylene glycol precipitation (PEG) method. IgG molecules were isolated from PEG precipitates by Protein G affinity method. The level of expression and localization N-acetylglucosamine, galactose, sialic acid, and fucose within the isolated IgG was determined by lectin blot assay. Calves with bronchopneumonia had a statistically significantly increased level of CIC. IgG molecules were isolated from CIC of both healthy and diseased calves. Several other proteins in complex with IgG were detected in both groups of animals. The isolated IgG heavy chains of healthy calves expressed N-acetylglucosamine, galactose, sialic acid, and fucose. The light chains of IgG expressed N-acetylglucosamine, sialic acid, and fucose whereas galactose was not detected in healthy calves. In diseased animals, galactose was detected on light chains, and both heavy and light IgG chains were more sialylated. Proteins in complex with IgG were also lectin reactive, and their glycosylation in diseased animals was different compared to healthy controls. Increased sialylation is a characteristic of anti-inflammatory IgG. The increased sialylation of IgG from CIC in bronchopneumonia might be an attempt of immune system of calves to protect lung tissues against damages provoked by activated cells and secreted pro-inflammatory cytokines. At the same time, increased IgG sialylation could explain the inability of calves' immune system to initiate the process of antigen elimination by activation of Fcγ receptors.

Characterization of an anti-rainbow trout (Oncorhynchus mykiss) CD3ε monoclonal antibody.

This study characterizes a monoclonal antibody (mAb) produced against the cytoplasmic tail region of the epsilon chain of the CD3 (CD3ε) transmembrane protein found on T lymphocytes of rainbow trout (Oncorhynchus mykiss). Flow cytometry and fluorescent microscopy conducted on trout leukocytes with the anti-trout CD3ε mAb showed a distinctive population of IgM(-) CD3e(+) lymphocytes fitting the expected profile of T-cells. Immunoprecipitation of lysates derived from trout lymphocytes revealed a 19 kDa protein and peptide analysis confirmed its specificity for CD3ɛ. In vitro proliferation assays with T-cell mitogens, ConA and PHA, resulted in a 3 fold increase in the percentage of CD3ɛ+ lymphocytes compared to LPS and control cultures. The mAb characterized in this study will be useful in further elucidation for both the role and distribution of T lymphocytes in the teleost immune system.

Immunoprecipitation of equine CD molecules using anti-human MABs previously analyzed by flow cytometry and immunohistochemistry.

Earlier studies investigating the cross-reactivity of antibodies submitted to the HLDA8 had used flow cytometry as a method of choice to screen mAbs for reactivity with equine leukocytes, including two-color flow-cytometry to characterize the lymphocyte population they detect. In addition, immuno-histochemistry (IHC) was used to detect distribution of positive cells in lymphoid tissue sections. In this study we performed immunoprecipitation (IP) to complement the previous results and add valuable information regarding the molecules detected by the cross-reacting antibodies. Surface molecules from primary equine PBMC or the equine cell line T8888 were biotinylated prior to precipitation to determine the molecular weight of the corresponding molecules in a western blot using streptavidin-AP. 21 out of 24 mAbs precipitated the molecules with a MW corresponding to its human orthologue. Positive mAbs were directed against CD2, CD5, CD11a, CD11b, CD14, CD18, CD21, CD44, CD83, CD91, CD172a, MHCI and MHCII. Three mAbs directed against CD49d, CD163, and CD206 which were unambiguously identified earlier by flow cytometry failed to immunoprecipitate the corresponding CD molecule. MAbs detecting CD molecules which are expressed internally like CD68 and mAbs of IgM class could not be included into this approach.

Vaccine-induced antibodies linked to bovine neonatal pancytopenia (BNP) recognize cattle major histocompatibility complex class I (MHC I).

A mysterious disease affecting calves, named bovine neonatal pancytopenia (BNP), emerged in 2007 in several European countries. Epidemiological studies revealed a connection between BNP and vaccination with an inactivated vaccine against bovine virus diarrhea (BVD). Alloantibodies reacting with blood leukocytes of calves were detected in serum and colostrum of dams, which have given birth to calves affected by BNP. To understand the linkage between vaccination and the development of alloantibodies, we determined the antigens reacting with these alloantibodies. Immunoprecipitation of surface proteins from bovine leukocytes and kidney cells using sera from dams with a confirmed case of BNP in their gestation history reacted with two dominant protein species of 44 and 12 kDa. These proteins were not detected by sera from dams, free of BVDV and not vaccinated against BVD, and from sera of animals vaccinated with a different inactivated BVD vaccine. The 44 kDa protein was identified by mass spectrometry analysis as MHC I, the other as ß-2-microglobulin. The presence of major histocompatibility complex class I (MHC I) in the vaccine was confirmed by Western blot using a MHC I specific monoclonal antibody. A model of BNP pathogenesis is proposed.