Genomic Characteristics of Feline Anelloviruses Isolated from Domestic Cats in Shanghai, China.

Viral metagenomics techniques allow the high-throughput discovery of possible pathogens carried by companion animals from their feces and other excreta. In this study, the viral metagenomics of 22 groups of fecal samples from domestic cats revealed a high prevalence of feline anelloviruses (FcTTV) infection in domestic cats in Shanghai, China. Serum samples from 30 cat individuals were further detected by polymerase chain reaction, and an average positive rate of 36.67% (11/30) of FcTTV infection was found. Next, the full-length sequences of five Shanghai FcTTV variants were obtained and submitted to GenBank with access numbers OP186140 to OP186144. Phylogenetic analysis indicates that the Shanghai FcTTV variants have relatively consistent genomic characteristics, with two variants from Zhejiang 2019 and one variant from the Czech Republic 2010. The recombination event analysis of the variants showed that one variant (OP186141_SH-02) had a primary parental sequence derived from a variant (KM229764) from the Czech Republic in 2010, while the secondary parental sequence was derived from OP186140_SH-01. The results revealed that FcTTV infection is prevalent in domestic cats and that the use of viral metagenomics to rapidly identify some infecting viruses whose hosts lack clinical features would be an effective approach.

Identification and Genome Characterization of Novel Feline Parvovirus Strains Isolated in Shanghai, China.

Feline panleukopenia virus (FPV) is the causative agent of hemorrhagic gastroenteritis in feline animals. FPV has been evolving over time, and there have been several different strains of the virus identified. Some of these strains may be more virulent or more resistant to current vaccines than others, which highlights the importance of ongoing research and monitoring of FPV evolution. For FPV genetic evolution analysis, many studies focus on the main capsid protein (VP2), but limited information is available on the nonstructural gene NS1 and structural gene VP1. In the present study, we firstly isolated two novel FPV strains circulating in Shanghai, China, and performed full-length genome sequencing for the desired strains. Subsequently, we focused on analyzing the NS1, VP1 gene, and the encoding protein, and conducted a comparative analysis among the worldwide circulating FPV and Canine parvovirus Type 2 (CPV-2) strains, which included the strains isolated in this study. We found that the 2 structural viral proteins, VP1 and VP2, are splice variants, and VP1 has a 143 amino-acid-long N-terminal compared to VP2. Furthermore, phylogenetic analysis showed that divergent evolution between FPV and CPV-2 virus strains were clustered mostly by country and year of detection. In addition, much more continuous antigenic type changes happened in the process of CPV-2 circulating and evolution compared to FPV. These results stress the importance of the continuous study of viral evolution and provide a comprehensive perspective of the association between viral epidemiology and genetic evolution.

Phylogenetic Characteristics of Canine Parvovirus Type 2c Variant Endemic in Shanghai, China.

Canine parvovirus type 2 (CPV-2) has spread and mutated globally over the past 40 years. In the present study, 206 samples from dogs suspected of CPV-2 infection were collected from five veterinary clinics in Shanghai city, China. The average positive rate for CPV-2 was detected to be 40.78% using the PCR method. Using an F81 cell (feline kidney cell) culture, the isolates of three CPV-2c strains were obtained. The near full-length genome sequences of the isolates were determined and submitted to GenBank: CPV-SH2001 (MW650830), CPV-SH2002 (MW811188), and CPV-SH2003 (MW811189). By comparing the amino acid sequences of 12 CPV strains with those of 48 related strains retrieved from GenBank, all of the CPV strains from Shanghai were typed as belonging to a relatively new CPV-2c variant spreading in Asia, with typical amino acid residues (5Gly, 267Tyr, 324Ile, and 370Arg) in the VP2 protein. The divergence time of this new CPV-2c clade was estimated by the phylogenetic tree using the maximum likelihood and RelTime with Dated Tips (RTDT) approaches. Our results indicate that the 426 and 324 VP2 amino acid residues are under strong selection pressure with a posterior probability of 0.966 and 0.943, respectively. Therefore, this study provides insight into the phylogenetic characteristics of the current CPV-2c variant in Shanghai city, China.

Secreted phosphoprotein-1 accelerates the progression of human colorectal cancer through activating ß-catenin signaling.

Colorectal cancer (CRC) is a common malignant tumor of the digestive tract and one of the leading causes of cancer-associated mortality. Secreted phosphoprotein-1 (SPP-1) is overexpressed in CRC and promotes cancer progression, but the underlying mechanisms underlying SPP-1 function remain unclear. The present study aimed to explore the effects of Wnt/ß-catenin signaling in SPP-1-induced CRC progression. The expression patterns of SPP-1 in CRC tissues were examined using reverse transcription-quantitative (RT-q)PCR, western blotting and immunohistochemistry. SPP-1 expression in cells was assessed using RT-qPCR and western blotting. Cell-Counting Kit-8, flow cytometry and tumor-burdened mice experiments were used to determine cell proliferation, apoptosis and in vivo tumor formation abilities. The results showed that SPP-1 expression was markedly elevated in CRC tissues and cells compared with that in normal colorectal tissues and cells. High expression of SPP-1 was associated with advanced clinical process and low overall survival rate in patients with CRC. Besides, SPP-1 could interact with ß-catenin and positively regulated ß-catenin protein expression, and enhanced its nuclear accumulation. Moreover, SPP-1-upregulation significantly enhanced cell proliferation and in vivo tumor formation ability, and reduced apoptosis, whereas these effects were all abolished when ß-catenin was silenced. Overall, the present study revealed that SPP-1 promoted the progression of CRC in a ß-catenin-dependent manner.

Genomic Characteristics and Selection Signatures in Indigenous Chongming White Goat (Capra hircus).

The Chongming white goat (CM) is an indigenous goat breed exhibits unique traits that are adapted to the local environment and artificial selection. By performing whole-genome re-sequencing, we generated 14-20× coverage sequences from 10 domestic goat breeds to explore the genomic characteristics and selection signatures of the CM breed. We identified a total of 23,508,551 single-nucleotide polymorphisms (SNPs) and 2,830,800 insertion-deletion mutations (indels) after read mapping and variant calling. We further specifically identified 1.2% SNPs (271,713) and 0.9% indels (24,843) unique to the CM breed in comparison with the other nine goat breeds. Missense (SIFT < 0.05), frameshift, splice-site, start-loss, stop-loss, and stop-gain variants were identified in 183 protein-coding genes of the CM breed. Of the 183, 36 genes, including AP4E1, FSHR, COL11A2, and DYSF, are involved in phenotype ontology terms related to the nervous system, short stature, and skeletal muscle morphology. Moreover, based on genome-wide F ST and pooled heterozygosity (Hp) calculation, we further identified selection signature genes between the CM and the other nine goat breeds. These genes are significantly associated with the nervous system (C2CD3, DNAJB13, UCP2, ZMYND11, CEP126, SCAPER, and TSHR), growth (UCP2, UCP3, TSHR, FGFR1, ERLIN2, and ZNF703), and coat color (KITLG, ASIP, AHCY, RALY, and MC1R). Our results suggest that the CM breed may be differentiated from other goat breeds in terms of nervous system owing to natural or artificial selection. The whole-genome analysis provides an improved understanding of genetic diversity and trait exploration for this indigenous goat breed.

[Enhanced recovery after preserving the left colonic artery during laparoscopic anterior resection for rectal cancer].

OBJECTIVE: To evaluate the postoperative outcomes of preserving the left colonic artery during laparoscopic anterior resection for rectal cancer. METHODS: The clinicopathologic data of 91 rectal cancer patients (pathologic Stage II) undergoing laparoscopic anterior resection was retrospectively analyzed. During the surgeries, the left colonic artery was preserved in 40 patients (preserved group) and ligated in 51 patients (unpreserved group). The operating time, intraoperative blood loss, time to first flatus and defecation, duration of postoperative abdominal distension and pain, number of retrieved lymph nodes, ileum fistulation and anatomical leakage rate were compared between the two groups. RESULTS: The surgeries were completed in all the 91 patients laparoscopically without conversion. There was no intraoperative complications including rectal perforation, injury to vessel or ureter in either group. The operating time, blood loss and number of retrieved lymph nodes were similar between the groups (P>0.05). Three patients in preserved group and 5 in ligation group received preventive ileum fistulation due to low rectal cancer. Anatomical leakage occurred in three patients of unpreserved group. The average duration of postoperative abdominal distension and pain was 2.14∓0.35 days in preserved group and 3.15∓0.42 days in ligation group. The time to first flatus and defecation was 37.15∓12.62 h and 3.16∓0.52 days in preserved group and 62.25∓11.75 h and 4.25∓0.75 days in ligation group. Postoperative hospital stay was 4.54∓0.42 days in preserved group and 6.23∓0.51 days in ligation group. Total hospitalization cost in the two groups was 34 525.32∓1206.36 Yuan and 41 215∓1051.32 Yuan, respectively. Significant differences were found the in duration of postoperative abdominal distension and pain, postoperative hospital stay, and total cost between the two groups (P<0.05). CONCLUSION: During laparoscopic anterior resection for rectal cancer, preserving the left colonic artery effectively ensures the blood supply to the anastomosis and the remaining descending colon to promote the recovery of the patients after surgery.

Function of CD163 fragments in porcine reproductive and respiratory syndrome virus infection.

Monocyte/macrophage scavenger receptor CD163 plays an important role in porcine reproductive and respiratory syndrome virus (PRRSV) infection. To identify the domains of CD163 involved in PRRSV infection, CD163 fragments P1 (1-798 bp), P2 (790-2046 bp), P3 (2023-3345 bp), Y-P1 (160-798 bp), Y-P2 (790-2046 bp) and Y-P3 (2143-3084 bp) were expressed by eukaryotic and prokaryotic expression systems, respectively. Infection experiments revealed that non-permissive BHK-21 cells transfected with pCD163 could be infected by PRRSV. However, cells with truncated CD163 (P1, P2, or P3) were not susceptible to PRRSV. Meanwhile, Y-P1, Y-P2, and Y-P3 were expressed in E. coli and antisera to these peptides were prepared in mice. A virus blocking test showed that Y-P2 protein and anti-Y-P2 mouse serum could block PRRSV infection in a dose-dependent manner, while Y-P3 protein could improve virus infection.

Molecular characterization of a virulent genotype VIId strain of Newcastle disease virus from farmed chickens in Shanghai.

A virulent Newcastle disease virus strain was isolated from diseased chickens in Shanghai, China. The isolated strain was initially characterized as highly virulent because of a short mean death time in embryonated chicken eggs and specific-pathogen-free chickens and was typed as neurotropic by intracloacal inoculation of chickens. The isolated strain had a dibasic amino acid motif in the fusion protein cleavage site sequence required for systemic replication in the host cell. The strain fell into subgenotype VIId by phylogenetic analysis of the fusion protein gene. Although these results demonstrated some sequence similarity between the isolated strain and strains responsible for outbreaks of Newcastle disease in China and Taiwan, the unusually high mortality (86.4%) set this strain aside from other VII strains. Finally, a cross-protection assay demonstrated that La Sota and clone 30 live vaccines could not protect chickens from infection with the isolated strain, with a zero survival rate being observed when chickens were challenged with a high dose of virulent VIId virus.

Detection of a porcine rotavirus strain with VP4, VP7 and NSP4 genes of different animal origins.

A new rotavirus strain, sh0902, was detected in diarrheic piglets on a farm in Shanghai, China, and its genotype was characterized as G1P[7]. Analysis of the VP4, VP7 and NSP4 genes demonstrated VP4 homology to bovine and swine rotavirus strains; the nucleotide (nt) and amino acid (aa) identities were 99.7% and 99.5%, respectively. The VP7 gene was highly homologous to that of a giant panda rotavirus strain, with 98.5% similarity at the nt level and 99% similarity at the aa level. The nucleotide sequence of the NSP4 gene displayed high homology to human rotavirus strain R479, with 99.7% identity at the nt level and 99.3% identity at the aa level. This is the first report of an unusual porcine rotavirus strain with VP4, VP7 and NSP4 genes that are highly homologous to bovine, swine, giant panda and human strains isolated at geographically distant sites (South Korea, China and India). Our data indicate that rotaviruses have circulated among humans and animals and undergone genome reassortment.

Detecting Newcastle disease virus in combination of RT-PCR with red blood cell absorption.

Reverse transcription-polymerase chain reaction (RT-PCR) has limited sensitivity when treating complicated samples, such as feces, waste-water in farms, and nucleic acids, protein rich tissue samples, all the factors may interfere with the sensitivity of PCR test or generate false results. In this study, we developed a sensitive RT-PCR, combination of red blood cell adsorption, for detecting Newcastle disease virus (NDV). One pair of primers which was highly homologous to three NDV pathotypes was designed according to the consensus nucleocapsid protein (NP) gene sequence. To eliminate the interfere of microbes and toxic substances, we concentrated and purified NDV from varied samples utilizing the ability of NDV binding red blood cells (RBCs). The RT-PCR coupled with red blood cell adsorption was much more sensitive in comparison with regular RT-PCR. The approach could also be used to detect other viruses with the property of hemagglutination, such as influenza viruses.

Efficient silencing of gene expression by an ASON-bulge-DNAzyme complex.

BACKGROUND: DNAzymes are DNA molecules that can directly cleave cognate mRNA, and have been developed to silence gene expression for research and clinical purposes. The advantage of DNAzymes over ribozymes is that they are inexpensive to produce and exhibit good stability. The "10-23 DNA enzyme" is composed of a catalytic domain of 15 deoxynucleotides, flanked by two substrate-recognition domains of approximately eight nucleotides in each direction, which provides the complementary sequence required for specific binding to RNA substrates. However, these eight nucleotides might not afford sufficient binding energy to hold the RNA substrate along with the DNAzyme, which would interfere with the efficiency of the DNAzyme or cause side effects, such as the cleavage of non-cognate mRNAs. METHODOLOGY: In this study, we inserted a nonpairing bulge at the 5' end of the "10-23 DNA enzyme" to enhance its efficiency and specificity. Different sizes of bulges were inserted at different positions in the 5' end of the DNAzyme. The non-matching bulge will avoid strong binding between the DNAzyme and target mRNA, which may interfere with the efficiency of the DNAzyme. CONCLUSIONS: Our novel DNAzyme constructs could efficiently silence the expression of target genes, proving a powerful tool for gene silencing. The results showed that the six oligo bulge was the most effective when the six oligo bulge was 12-15 bp away from the core catalytic domain.

Molecular characterization of porcine circovirus 2 isolated from diseased pigs co-infected with porcine reproductive and respiratory syndrome virus.

In this study, we isolated a porcine circovirus 2 (PCV2) strain from piglets co-infected with porcine reproductive and respiratory syndrome virus (PRRSV). The complete genome of this strain was sequenced, phylogenetic and polymorphic analyses were carried out. BLAST searches revealed the highest sequence identity (99.5% nt and 99.3% aa) to Guangxi strain EF675230. The phylogenetic tree showed that clustering of the isolates didn't strongly correlate to geographical distribution. Polymorphic analyses demonstrated that the amino acids at most of the polymorphic sites in Open Reading Frame 1(ORF1) and 2 (ORF2)belong to the same amino acid group according to chemical or structural properties, and revealed that highly polymorphic regions overlapped with the known immunoreactive epitopes of ORF2.

[Expression in Escherichia coli, purification and enzymatic properties of porcine urate oxidase].

The aims of this research were to construct prokaryotic expression vector containing the gene of porcine urate oxidase (pUOX), optimize the conditions of the expression of pUOX in recombinant Escherichia coli BL21(DE3), and analyze the in vitro activity and the enzymological properties of pUOX. The pUOX gene was amplified by RT-PCR from the extracted total RNA of porcine liver, and was inserted into the prokaryotic expression vector pET30a(+) to construct a recombinant expression vector pET30a(+)/pUOX. We identified the recombinant vector by endonuclease digestion and sequence analysis. The pUOX gene was amplified and cloned into the vector pET30a(+) successfully. And then the recombinant vector was transformed into E. coli BL21(DE3). The expression of pUOX with a molecular of approximately 41 kD was induced by IPTG. We also optimized the expression conditions of the recombinant protein. The recombinant protein was mostly located in the cytoplasm and it was insoluble. After the inclusion body was solved in 8 mol/L urea and refolding in 2 mol/L urea, the recombinant protein was collected and purified by Ni2+-NTA column. This recombinant protein had a specific activity of 50.61 IU/mg and showed similar properties of optimum temperature and thermal stability, base on the enzymatic assay and analysis of enzymological properties. These results would help to analyze the in vivo activity by testing animal.

GB virus B disrupts RIG-I signaling by NS3/4A-mediated cleavage of the adaptor protein MAVS.

Understanding the mechanisms of hepatitis C virus (HCV) pathogenesis and persistence has been hampered by the lack of small, convenient animal models. GB virus B (GBV-B) is phylogenetically the closest related virus to HCV. It causes generally acute and occasionally chronic hepatitis in small primates and is used as a surrogate model for HCV. It is not known, however, whether GBV-B has evolved strategies to circumvent host innate defenses similar to those of HCV, a property that may contribute to HCV persistence in vivo. We show here in cultured tamarin hepatocytes that GBV-B NS3/4A protease, but not a related catalytically inactive mutant, effectively blocks innate intracellular antiviral responses signaled through the RNA helicase, retinoic acid-inducible gene I (RIG-I), an essential sensor molecule that initiates host defenses against many RNA viruses, including HCV. GBV-B NS3/4A protease specifically cleaves mitochondrial antiviral signaling protein (MAVS; also known as IPS-1/Cardif/VISA) and dislodges it from mitochondria, thereby disrupting its function as a RIG-I adaptor and blocking downstream activation of both interferon regulatory factor 3 and nuclear factor kappa B. MAVS cleavage and abrogation of virus-induced interferon responses were also observed in Huh7 cells supporting autonomous replication of subgenomic GBV-B RNAs. Our data indicate that, as in the case of HCV, GBV-B has evolved to utilize its major protease to disrupt RIG-I signaling and impede innate antiviral defenses. These data provide further support for the use of GBV-B infection in small primates as an accurate surrogate model for deciphering virus-host interactions in hepacivirus pathogenesis.

Recombinant bivalent vaccine against foot-and-mouth disease virus serotype O/A infection in guinea pig.

In this study, two DNA fragments encoding amino acid (141-160)-(21-140)-(141-160) of the VP1 of FMDV (foot-and-mouth disease virus) serotype O and (138-160)-(21-40)-(138-160) of the serotype A FMDV were chemically synthesized. These two tandem-repeat fragments were ligated and transfected into prokaryotic expression vector pTrcHis A to construct pTH-O-A. The other vector called pTH-O-scIgG-A was constructed similarly only that the two tandem-repeat DNA fragments were linked by the bovine-IgG heavy chain coding sequence. Guinea pigs immunized with the two bivalent vaccines pTH-O-A and pTH-O-scIgG-A showed both specific antibody activity and T cell proliferation responses. FMDV challenge tests showed that 85% and 70% of guinea pigs vaccinated twice with 200 mg of the fusion protein of pTH-O-A were protected from FMDV serotype O and serotype A infection respectively. 70% and 57% of the guinea pigs immunized with the fusion protein of pTH-O-scIgG-A were protected from FMDV serotype O and serotype A infection respectively.