Wenjie Gong

Genetic diversity of subgenotype 2.1 isolates of classical swine fever virus.

As the causative agent of classical swine fever, the economically devastating swine disease worldwide, classical swine fever virus (CSFV) is currently classified into the 11 subgenotypes, of which subgenotype 2.1 is distributed worldwide and showing more genetic diversity than other subgenotypes. Prior to this report, subgenotype 2.1 was divided into three sub-subgenotypes (2.1a-2.1c). To further analyze the genetic diversity of CSFV isolates in China, 39 CSFV isolates collected between 2004 and 2012 in two Chinese provinces Guangxi and Guangdong were sequenced and subjected to phylogenetic analysis together with reference sequences retrieved from GenBank. Phylogenetic analyses based on the 190-nt and/or 1119-nt full length E2 gene fragments showed that current CSFV subgenotype 2.1 virus isolates in the world could be divided into 10 sub-subgenotypes (2.1a-2.1j) and the 39 isolates collected in this study were grouped into 7 of them (2.1a-2.1c and 2.1g-2.1j). Among the 10 sub-subgenotypes, 2.1d-2.1j were newly identified. Sub-subgenotype 2.1d isolates were circulated only in India, however the rest 9 sub-subgenotypes were from China with some of them closely related to isolates from European and neighboring Asian countries. According to the temporal and spatial distribution of CSFV subgenotype 2.1 isolates, the newly classified 10 sub-subgenotypes were further categorized into three groups: dominant sub-subgenotype, minor sub-subgenotype and silent sub-subgenotype, and each sub-subgenotype can be found only in certain geographical areas. Taken together, this study reveals the complex genetic diversity of CSFV subgenotype 2.1 and improves our understanding about the epidemiological trends of CSFV subgenotype 2.1 in the world, particularly in China.

Serum Metabolomic Profiling of Piglets Infected with Virulent Classical Swine Fever Virus

Classical swine fever (CSF) is a highly contagious swine infectious disease and causes significant economic losses for the pig industry worldwide. The objective of this study was to determine whether small molecule metabolites contribute to the pathogenesis of CSF. Birefly, serum metabolomics of CSFV Shimen strain-infected piglets were analyzed by ultraperformance liquid chromatography/electrospray ionization time-of-flight mass spectrometry (UPLC/ESI-Q-TOF/MS) in combination with multivariate statistical analysis. In CSFV-infected piglets at days 3 and 7 post-infection changes were found in metabolites associated with several key metabolic pathways, including tryptophan catabolism and the kynurenine pathway, phenylalanine metabolism, fatty acid and lipid metabolism, the tricarboxylic acid and urea cycles, branched-chain amino acid metabolism, and nucleotide metabolism. Several pathways involved in energy metabolism including fatty acid biosynthesis and β-oxidation, branched-chain amino acid metabolism, and the tricarboxylic acid cycle were significantly inhibited. Changes were also observed in several metabolites exclusively associated with gut microbiota. The metabolomic profiles indicate that CSFV-host gut microbiome interactions play a role in the development of CSF.

Retrospective study of porcine circovirus type 2 infection reveals a novel genotype PCV2f

Porcine postweaning multisystemic wasting syndrome (PMWS) caused by porcine circovirus type 2 (PCV2) is a disease causing severe economic losses annually worldwide to the pig industry. PCV2 infection was first reported in China in 2000, and currently has three major genotypes, PCV2a, b and d, circulating in this country. To further elucidate the origin and prevalence of PCV2 in China, 123 clinical pig tissue samples collected in 25 provinces between 1990 and 1999 were analysed by PCV2-specific PCR, resulting in identification of 23 PCV2 strains collected between 1996 and 1999. Phylogenetic analysis based on the nucleotide sequences of open reading frame 2 (ORF2) showed that 20 of the 23 grouped within PCV2a, while the remaining three strains formed an independent clade, so far unreported and therefore named PCV2f. This genotype shared lower sequence identity with other known genotypes. This study provides further understanding of the genetic diversity and evolution of PCV2 and has tracked PCV2 infection in China back to 1996 rather than 2000.

Inhibition of Rabies Virus by 1,2,3,4,6-Penta-O-galloyl-β-d-Glucose Involves mTOR-Dependent Autophagy

The compound 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG), a gallotannin present in various plants such as Rhus chinensis Mill and Paeonia suffruticosa, has a broad spectrum of antiviral effects. The present study investigated its potency against infection of mice with rabies virus (RABV). Results demonstrated that PGG strongly inhibited virus titers (50-fold), viral mRNA expression (up to 90%), and protein synthesis in vitro. Importantly, we found that PGG not only suppressed viral adsorption and entry, but also directly inactivated RABV through suppression of autophagy by mediating activation of the mTOR-dependent autophagy signaling pathway. In vivo, PGG (10 mg/kg) alleviated the clinical symptoms and reduced the mortality of infected mice by 27.3%. Collectively, our results indicate that PGG has potent anti-RABV effect, and merits further investigation as an anti-RABV drug.

Toward the development of a one-dose classical swine fever subunit vaccine: antigen titration, immunity onset, and duration of immunity

Highly contagious classical swine fever (CSF) remains a major trade and health problem in the pig industry, resulting in large economic losses worldwide. In CSF-endemic countries, attenuated CSF virus (CSFV) vaccines have been routinely used to control the disease. However, eradication of CSFV in a geographical area would require permanent reduction to zero presence of the virus. It is therefore of paramount importance to develop a safe, potent, and non-infectious CSF vaccine. We have previously reported on a cost-effective CSF E2 subunit vaccine, KNB-E2, which can protect against CSF symptoms in a single dose containing 75 µg of recombinant CSFV glycoprotein E2. In this study, we report on a series of animal studies undertaken to elucidate further the efficacy of KNB-E2. We found that pigs vaccinated with a single KNB-E2 dose containing 25 µg of recombinant CSFV glycoprotein E2 were protected from clinical symptoms of CSF. In addition, KNB-E2-mediated reduction of CSF symptoms was observed at two weeks post-vaccination and the vaccinated pigs continued to exhibit reduced CSF clinical signs when virus challenged at two months and four months post-vaccination. These results suggest that KNB-E2 effectively reduces CSF clinical signs, indicating the potential of this vaccine for safely minimizing CSF-related losses.

Identification of cleavage of NS5A of C-strain classical swine fever virus

NS5A is a multifunctional non-structural protein of classical swine fever virus (CSFV) that plays an important role in viral replication, but how it exerts its functions is unknown. Here, we report the cleavage of NS5A of the vaccine C-strain, resulting in two truncated forms (b and c). Further experiments using calpain- and caspase-family-specific inhibitors, followed by a caspase-6-specific shRNAs and inhibitor, showed that the cleavage of C-strain NS5A to produce truncated form c is mediated by caspase-6, mapping to 272DTTD275, while the cleavage producing truncated form b is probably mediated by another unknown protease. shRNA-mediated downregulation of caspase-6 and blocking of enzyme activity in ST cells significantly impaired genome replication and virus production, indicating that NS5A cleavage is required for CSFV replication.

Construction of Recombinant Porcine Reproductive and Respiratory Syndrome Virus Expressing CSFV E2 Glycoprotein

Classical Swine Fever (CSF) and Porcine Reproductive and Respiratory Syndrome (PRRS) are two highly contagious infectious diseases caused by CSF virus (CSFV) and PRRS virus (PRRSV), respectively. Recombinant PRRSV expressing CSFV E2 glycoprotein could be used for the development of bivalent vaccine, antiviral drug or antibody screening assays against PRRSV and CSFV. In this study, a recombinant PRRSV expressing CSFV E2 glycoprotein (p129-CSFV-E2) was constructed. The E2 gene from CSFV C-strain vaccine was cloned and inserted between ORF1b and ORF2 gene of the PRRSV P129 strain. An additional transcriptional regulatory sequence 6 (TRS6) was inserted following the CSFV E2 for driving the transcription of ORF2. The construct efficiently produced progeny viruses and the expressed CSFV E2 protein was detected by immune staining of infected MARC145 cells.The growth ability of the p129-CSFV-E2 virus is comparable to the parental p129 virus. The genetic stability and stable expression of CSFV E2 of P129-CSFV-E2 virus could reach 11 passages in cell culture. The results showed that CSFV E2 glycoprotein could be expressed as a separated subgenomic unit in the PRRSV genome. The recombinant P129-CSFV-E2 virus can be useful for the development of novel vaccines, cell-based high throughput antiviral drug and antibody screening system against PRRSV and CSFV.

Complete genome sequence of a sub-subgenotype 2.1i isolate of classical swine fever virus from China

ABSTRACT The complete genome sequence of a sub-subgenotype 2.1i isolate of classical swine fever virus (CSFV), GD317/2011, was determined. Notably, GD317/2011 is distant from the sub-subgenotype 2.1b isolate HEBZ at genes of Erns, E1, E2, P7, NS2, NS5A and the 3′-nontranslated region (3′-NTR) but is closely related to that at genes of Npro, Core, NS3, NS4A, NS4B, and NS5B.