Proteome Analysis Reveals Syndecan 1 Regulates Porcine Sapelovirus Replication.

Porcine sapelovirus A (PSV) is a single stranded, positive-sense, non-enveloped RNA virus that causes enteritis, pneumonia, polioencephalomyelitis, and reproductive disorders in pigs. Research on PSV infection and interaction with host cells is unclear. In this study, we applied tandem mass tag proteomics analysis to investigate the differentially expressed proteins (DEPs) in PSV-infected pig kidney (PK)-15 cells and explored the interactions between PSV and host cells. Here we mapped 181 DEPs, including 59 up-regulated and 122 down-regulated DEPs. Among them, osteopontin (SPP1), induced protein with tetratricopeptide repeats 5 (IFIT5), ISG15 ubiquitin-like modifier (ISG15), vinculin (VCL), and syndecan-1 (SDC1) were verified significantly changed using RT-qPCR. Additionally, overexpression of SDC1 promoted PSV viral protein (VP)1 synthesis and virus titer, and silencing of SDC1 revealed the opposite results. Our findings show that SDC1 is a novel host protein and plays crucial roles in regulating PSV replication.

Entry of sapelovirus into IPEC-J2 cells is dependent on caveolae-mediated endocytosis.

BACKGROUND: Porcine sapelovirus (PSV), a species of the genus Sapelovirus within the family Picornaviridae, are a significant cause of enteritis, pneumonia, polioencephalomyelitis and reproductive disorders in pigs. However, the life cycle of PSV on the molecular level is largely unknown. METHODS: Here, we used chemical inhibitors, RNA interference, and overexpression of dominant negative (DN) mutant plasmids to verify the roles of distinct endocytic pathways involved in PSV entry into porcine small intestinal epithelial cell line (IPEC-J2). RESULTS: Our experiments indicated that PSV infection was inhibited when cells were pre-treated with NH4Cl or chloroquine. Inhibitors nystatin, methyl-ß-cyclodextrin, dynasore and wortmannin dramatically reduced PSV entry efficiency, whereas the inhibitors chlorpromazine and EIPA had no effect. Furthermore, overexpression caveolin DN mutant and siRNA against caveolin also decreased virus titers and VP1 protein synthesis, whereas overexpression EPS15 DN mutant and siRNA against EPS15 did not reduce virus infection. CONCLUSIONS: Our findings suggest that PSV entry into IPEC-J2 cells depends on caveolae/lipid raft mediated-endocytosis, that is pH-dependent and requires dynamin and PI3K but is independent of clathrin and macropinocytosis.

Gorham's disease: treatment with an autologous iliac bone graft and a reverse total shoulder arthroplasty.

BACKGROUND: Gorham's disease (GSD) is a rare osteolytic disease with unclear etiology, and no known prevention or effective treatment. Here we report a new surgical treatment for a case of GSD in September 2017. CASE PRESENTATION: We report GSD in a 52-year-old woman. She had disappearance of her humeral head and a defect of the glenoid bone in her left shoulder joint, which were serious obstacles to joint function. We used an autologous iliac bone graft to repair the glenoid bone defect and a reverse total shoulder arthroplasty. After surgery, humeral osteolysis did not continue, and her shoulder function recovered well. CONCLUSIONS: This case suggests that autologous bone grafting can still be used to treat GSD despite it being an osteolytic disease. The successful treatment suggests that this method could be used for GSD in other bones.

Genomic organization and recombination analysis of a porcine sapovirus identified from a piglet with diarrhea in China.

BACKGROUND: Sapovirus (SaV), a member of the family Caliciviridae, is an etiologic agent of gastroenteritis in humans and pigs. To date, both intra- and inter-genogroup recombinant strains have been reported in many countries except for China. Here, we report an intra-genogroup recombination of porcine SaV identified from a piglet with diarrhea of China. METHODS: A fecal sample from a 15-day-old piglet with diarrhea was collected from Shanghai, China. Common agents of gastroenteritis including porcine circovirus type 2, porcine rotavirus, porcine transmissible gastroenteritis virus, porcine SaV, porcine norovirus, and porcine epidemic diarrhea virus were detected by RT-PCR or PCR method. The complete genome of porcine SaV was then determined by RT-PCR method. Phylogenetic analyses based on the structural region and nonstructural (NS) region were carried out to group this SaV strain, and it was divided into different genotypes based on these two regions. Recombination analysis based on the genomic sequence was further performed to confirm this recombinant event and locate the breakpoint. RESULTS: All of the agents showed negative results except for SaV. Analysis of the complete genome sequence showed that this strain was 7387 nt long with two ORFs and belonged to SaV GIII. Phylogenetic analyses of the structural region (complete VP1 nucleotide sequences) grouped this strain into GIII-3, whereas of the nonstructural region (RdRp nucleotide sequences) grouped this strain into GIII-2. Recombination analysis based on the genomic sequence confirmed this recombinant event and identified two parental strains that were JJ259 (KT922089, GIII-2) and CH430 (KF204570, GIII-3). The breakpoint located at position 5139 nt of the genome (RdRp-capsid junction region). Etiologic analysis showed the fecal sample was negative with the common agents of gastroenteritis, except for porcine SaV, which suggested that this recombinant strain might lead to this piglet diarrhea. CONCLUSIONS: P2 strain was an intra-genogroup recombinant porcine SaV. To the best of our knowledge, this study would be the first report that intra-genogroup recombination of porcine SaV infection was identified in pig herd in China.

Comparison of Viromes in Ticks from Different Domestic Animals in China.

Ticks are involved in the transmission of various arboviruses and some tick-borne viruses pose significant threats to the health of humans or livestock. This study aimed to investigate the geographical distribution of tick species and tick-associated viruses in central and eastern China. Total 573 ticks from domestic animals including dogs, sheep and cattle were collected in 2017. Two genera of ticks were identified including Rhipicephalus and Haemaphysalis. Sequencing was performed on Miseq Illumina platform to characterize the tick viromes from the four different sampling locations. Following trimming, 13,640 reads were obtained and annotated to 19 virus families. From these sequences, above 37.74% of the viral reads were related to the RNA viruses. Virome comparison study revealed that the tick viral diversity was considerably different in the two identified tick genera. The viral diversity of R. microplus was significantly different from that of other Rhipicephalus species. On the other hand, substantial overlap in viral species was observed between the same genera. In addition, we found no evidence that the natural host played a major role in shaping virus diversity based on the comparison of their viromes. Rather, the geographic location seems to significantly influence the viral families. Phylogenetic study indicated that the novel negative-sense RNA viruses identified in this study was closely related to Bole tick virus 1 and 3 viruses. In conclusion, the present study provides a baseline for comparing viruses detected in ticks, according to species, natural hosts, and geographic locations.

Molecular cloning and differential expression patterns of the regulatory subunit B' gene of PP2A in goldfish, Carassius auratus.

It is well established that the protein serine/threonine phosphatase 2A (PP2A) plays very important roles in many different cellular processes, including cell proliferation and differentiation, gene expression, neurotransmission, apoptosis, and aging. PP2A consists of three heterogenic subunits: the scaffold subunit A, the catalytic subunit C, and the regulatory subunit B. While both the scaffold and the catalytic subunits contain only two forms, at least four families of the regulatory subunits, B, B', B'', and B''' have been identified. These regulatory subunits from different families are encoded by different genes and bear other functions besides directing the specificity of PP2A. To study the functions of the regulatory subunits of PP2A in lower vertebrates, we have cloned the full-length cDNA sequence of the gene encoding the regulatory subunit B'delta of PP2A from gold fish, Carassius auratus using 3'-RACE and 5'-RACE cloning strategies. Our results revealed that the full-length B'delta cDNA contains 2415 bp and encodes a protein of 555 amino acids. The B'delta protein displays a very high level of sequence identity with the B'delta regulatory subunit from other species of vertebrates. Regarding its expression pattern, RT-PCR revealed that the highest level of mRNA was detected in brain, a less level detected in liver, spermary, ovary, kidney and gill, and the lowest level detected in the fin. During different developmental stages of gold fish, the highest level of mRNA expression was detected at the stages of two-cell, multiple-cell, blastula and gastrula, and a decreased level of B'gamma mRNA was detected in other developmental stages. At the protein level, the highest expression level of B'delta protein was found in spermary, ovary, brain and heart, a less amount found in liver and the lowest level detected in kidney, gill and fin. Developmentally, B'delta protein was strongly expressed at the stages of two-cell, multiple-cell, blastula, gastrula, neurula, and optic vesicle, and then decreased at the stages of brain differentiation and eye pigmentation. These results suggest that B'delta appears to play a very important role during gold fish development and also in adult tissue homeostasis.

Porcine sapelovirus enters PK-15 cells via caveolae-dependent endocytosis and requires Rab7 and Rab11.

To comprehensively understand the endocytosis of Sapelovirus A (PSV) entry into PK-15 cells, we studied PSV infection in the context of cell perturbations through drug inhibition, siRNA silencing and overexpression of dominant negative (DN) mutants. We showed here that PSV infection of PK-15 cells was unaffected by pretreated with chlorpromazine, EIPA, knockdown of the clathrin heavy chain or overexpression of Eps15 DN mutant. Conversely, PSV infection was sensitive to NH4Cl, chloroquine, dynasore, nystatin, MßCD and wortmannin with reduced PSV VP1 expression levels and virus titer. Additionally, PSV invasion leaded to rapid actin rearrangement and disruption of the cellular actin network enhanced PSV infection. After internalization the virus was transported to late endosomes and/or cycling endosomes that requires the participation of Rab7 and Rab11. Our findings demonstrate that PSV uses caveolae-dependent endocytosis as the predominant entry portal into PK-15 cells which requires low pH, dynamin, Rab7 and Rab11.

Molecular Cloning of the Genes Encoding the PR55/Bß/δ Regulatory Subunits for PP-2A and Analysis of Their Functions in Regulating Development of Goldfish, Carassius auratus.

The protein phosphatase-2A (PP-2A), one of the major phosphatases in eukaryotes, is a heterotrimer, consisting of a scaffold A subunit, a catalytic C subunit and a regulatory B subunit. Previous studies have shown that besides regulating specific PP-2A activity, various B subunits encoded by more than 16 different genes, may have other functions. To explore the possible roles of the regulatory subunits of PP-2A in vertebrate development, we have cloned the PR55/B family regulatory subunits: ß and δ, analyzed their tissue specific and developmental expression patterns in Goldfish ( Carassius auratus). Our results revealed that the full-length cDNA for PR55/Bß consists of 1940 bp with an open reading frame of 1332 nucleotides coding for a deduced protein of 443 amino acids. The full length PR55/Bδ cDNA is 2163 bp containing an open reading frame of 1347 nucleotides encoding a deduced protein of 448 amino acids. The two isoforms of PR55/B display high levels of sequence identity with their counterparts in other species. The PR55/Bß mRNA and protein are detected in brain and heart. In contrast, the PR55/Bδ is expressed in all 9 tissues examined at both mRNA and protein levels. During development of goldfish, the mRNAs for PR55/Bß and PR55/Bδ show distinct patterns. At the protein level, PR55/Bδ is expressed at all developmental stages examined, suggesting its important role in regulating goldfish development. Expression of the PR55/Bδ anti-sense RNA leads to significant downregulation of PR55/Bδ proteins and caused severe abnormality in goldfish trunk and eye development. Together, our results suggested that PR55/Bδ plays an important role in governing normal trunk and eye formation during goldfish development.