Genetic characterization of four strains porcine circovirus-like viruses in pigs with diarrhea in Hunan Province of China.

In this study, we detected a circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA virus [named Po-Circo-like (PCL) virus] in intestinal tissue and fecal samples of pigs. PCL virus contains a single-stranded DNA genome, and ORF1 encodes the Rep and not the typical capsid protein encoded in PCV. The Rep protein may be responsible for viral genome replication. In addition, PCL virus may be one of the pathogens causing diarrhea symptoms in pigs. We identified four strains of PCL virus in two different pig farms with severe diarrhea outbreaks in Hunan Province, China. The strains in this study share 85.7-99.7% nucleic acid identity and 84.7-100% amino acid identity with Rep of the reference strains. A multiple sequence alignment of these PCL viruses and Bo-Circo-like CH showed a identity of 93.2% for the Rep protein, and the nucleotide identity was 86.7-89.3%. Moreover, Bo-Circo-like CH and HN75, HN39-01, HN39-02 had similar stem-loop sequences. In conclusion, the present study is the first detailed report of the PCL virus in Hunan provinces, which is a potential new virus in pigs that might be involved in cross-species transmission. Further investigation is needed to determine the pathogenesis of this virus and its epidemiologic impact.

A Method for the Analysis of African Swine Fever by Viral Metagenomic Sequencing.

In 2018, there was an outbreak of African swine fever (ASF) in China, which spread to other provinces in the following 3 years and severely damaged China's pig industry. ASF is caused by the African swine fever virus (ASFV). Given that the genome of the African swine fever virus is very complex and whole genome information is currently inadequate, it is important to efficiently obtain virus genome sequences for genomic and epidemiological studies. The prevalent ASFV strains have low genetic variability; therefore, whole genome sequencing analysis provides a basis for the study of ASFV. We provide a method for the efficient sequencing of whole genomes, which requires only a small number of tissues. The database construction method was selected according to the genomic types of ASFV, and the whole ASFV genome was obtained through data filtering, host sequence removal, virus classification, data assembly, virus sequence identification, statistical analysis, gene prediction, and functional analysis. Our proposed method will facilitate ASFV genome sequencing and novel virus discovery.

Development of a Dual Fluorescent Microsphere Immunological Assay for Detection of Pseudorabies Virus gE and gB IgG Antibodies.

Pseudorabies, also known as Aujezsky's disease, is an acute viral infection caused by pseudorabies virus (PRV). Swine are one of the natural hosts of pseudorabies and the disease causes huge economic losses in the pig industry. The establishment of a differential diagnosis technique that can distinguish between wild-type infection and vaccinated responses and monitor vaccine-induced immunoglobulin G(IgG) is crucial for the eventual eradication of pseudorabies. The aim of this study was to develop a rapid dual detection method for PRV gE and gB protein IgG antibodies with high specificity and sensitivity. PRV gE codons at amino acid residues (aa) 52-238 and gB codons at aa 539-741 were expressed to obtain recombinant PRV gE and gB proteins via a pMAL-c5x vector. After purification with Qiagen Ni-nitrilotriacetic acid (NTA) agarose affinity chromatography, the two proteins were analyzed via SDS-PAGE and immunoblotting assays. Two single fluorescent-microsphere immunoassays (FMIAs) were established by coupling two recombinant proteins (gE and gB) to magnetic microbeads, and an effective dual FMIA was developed by integrating the two single assays. Optimal serum dilution for each assay, correlation with other common swine virus-positive sera, and comparison with ELISA for two PRV antigens were tested for validation. Compared with ELISA, the specificity and sensitivity were 99.26% and 92.3% for gE IgG antibody detection, and 95.74% and 96.3% for the gB IgG antibody detection via dual FMIA. We provide a new method for monitoring PRV protective antibodies in vaccinated pigs and differentiating wild-type PRV infection from vaccinated responses simultaneously.

First report and genetic diversity of porcine bufavirus in China.

BACKGROUND: Bufavirus is a newly discovered zoonotic virus reported in numerous mammals and humans. However, the epidemiological and genetic characteristics of porcine bufaviruses (PBuVs) in China remain unclear. METHODS: To detect PBuVs in China, 384 samples (92 fecal and 292 serum specimens) were collected from 2017 to 2018, covering six provinces in China, and were evaluated by nested PCR. Further, the positive samples from different provinces were selected to obtain the complete genome of Chinese PBuVs. RESULTS: The prevalence rate of PBuV was 16.7% in Chinese domestic pigs in the Guangdong, Guangxi, Fujian, Jiangxi, Anhui, and Henan provinces. Additionally, the positive rate of fecal specimens was higher than that of the serum samples. Next, we sequenced nine near-complete genomes of Chinese field PBuV strains from different provinces. Homology and phylogenetic analyses indicated that Chinese PBuVs have high genetic variation (93.3-99.2%), showed higher nucleotide identity with an Austrian PBuV strain (KU867071.1), and developed into different branches within the same cluster. CONCLUSION: To our knowledge, this is the first report on PBuV in China, expanding the geographic boundaries of PBuV circulation. Our data demonstrate that PBuVs are widely distributed in the six Chinese provinces. Moreover, these Chinese PBuVs exhibit genetic variation and continuous evolution characteristics. Taken together, our findings provide a foundation for future studies on bufaviruses.

Ginsenoside Rg1 Suppresses Type 2 PRRSV Infection via NF-κB Signaling Pathway In Vitro, and Provides Partial Protection against HP-PRRSV in Piglet.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a huge threat to the modern pig industry, and current vaccine prevention strategies could not provide full protection against it. Therefore, exploring new anti-PRRSV strategies is urgently needed. Ginsenoside Rg1, derived from ginseng and notoginseng, is shown to exert anti-inflammatory, neuronal apoptosis-suppressing and anti-oxidant effects. Here we demonstrate Rg1-inhibited PRRSV infection both in Marc-145 cells and porcine alveolar macrophages (PAMs) in a dose-dependent manner. Rg1 treatment affected multiple steps of the PRRSV lifecycle, including virus attachment, replication and release at concentrations of 10 or 50 µM. Meanwhile, Rg1 exhibited broad inhibitory activities against Type 2 PRRSV, including highly pathogenic PRRSV (HP-PRRSV) XH-GD and JXA1, NADC-30-like strain HNLY and classical strain VR2332. Mechanistically, Rg1 reduced mRNA levels of the pro-inflammatory cytokines, including IL-1ß, IL-8, IL-6 and TNF-α, and decreased NF-κB signaling activation triggered by PRRSV infection. Furthermore, 4-week old piglets intramuscularly treated with Rg1 after being challenged with the HP-PRRSV JXA1 strain display moderate lung injury, decreased viral load in serum and tissues, and an improved survival rate. Collectively, our study provides research basis and supportive clinical data for using Ginsenoside Rg1 in PRRSV therapies in swine.

Phylogeography, phylodynamics and the recent outbreak of lineage 3 porcine reproductive and respiratory syndrome viruses in China.

Novel highly pathogenic porcine reproductive and respiratory syndrome viruses (PRRSVs) have attracted increasing attention owing to their continual high emergence and recent re-emergence. Recently, lineage 3 PRRSVs, belonging to the type 2 viruses, with novel characteristics and increased virulence have been continuously re-emerging in China, thereby posing a great threat to pig farming. However, available information about lineage 3 is limited. Here, we carried out molecular epidemiological investigations for PRRSV surveillance in most regions of China from 2007 to 2017. More than 3,000 samples were obtained, amounting to 73 sequences of lineage 3 viruses. The origin, demographic history and spread pattern of lineage 3 PRRSVs were investigated combining with the database globally. Phylogeography and phylodynamic analyses within a Bayesian statistical framework revealed that lineage 3 viruses originated in Taiwan. Followed by subsequent propagation to different areas and geography, it dichotomized into two endemic clusters. South China has become an epicentre for these viruses, which diffused into China's interiors in recent years. Furthermore, viral dispersal route analysis revealed the risk of viral diffusion. Overall, the origin, epidemic history and geographical evolution of lineage 3 PRRSVs were comprehensively analysed in this study. In particular, the epicentre of southern China and the diffusion routines of the viruses are highlighted in this study, and the possible continuous transmission of the novel lineages poses the biggest threat to pig farmers.

The phosphorylation of the N protein could affect PRRSV virulence in vivo.

The porcine respiratory and reproductive syndrome virus (PRRSV) nucleocapsid (N) protein is a multiphosphorylated protein.It has been proved that the phosphorylation of N protein could regulate the growth ability of PRRSV in Marc-145 cells. However, further investigation is needed to determine whether phosphorylation of the N protein could affect PRRSV virulence in piglets. In this study, we confirmed that the mutations could impair PRRSV replication ability in porcine primary macrophages (PAMs) as they did in Marc-145 cells. The animal experiments suggested that the pathogenicity of the mutated virus (A105-120) was significantly reduced compared with parent strain (XH-GD). Our results suggested that the phosphorylation of the N protein contributes to virus replication and virulence. This study is the first to identify a specific modification involved in PRRSV pathogenicity. Mutation of PTMs sites is also a novel way to attenuate PRRSV virulence. The mutations could be a marker in a vaccine. In conclusion, our study will improve our understanding of the molecular mechanisms of PRRSV pathogenicity.

Emergence of novel recombination lineage 3 of porcine reproductive and respiratory syndrome viruses in Southern China.

Lineage 3 of porcine reproductive and respiratory syndrome viruses, which belong to North America type 2, has a long epidemic history in China. The novel lineage 3 viruses constantly emerging in recent years are characterized by a high detection rate and significant pathogenicity. In this study, we investigated the prevalence of lineage 3 in southern China and selected two isolated strains for genome and virulence analyses. A cross-sectional epidemiology investigation indicated that the prevalence of lineage 3 antigens was 35.68% (95% CI: 27.6-44.3%) among 227 samples collected from over 100 infected farms from January 2016 to July 2017 in southern China. Two novel isolates of lineage 3 were selected. After 20 passages, Marc-145 cells were not susceptible to those viruses. Full-length genome analysis indicated that the two strains share 95.2% homology with each other and 95.7%-96.2% with highly pathogenic porcine reproductive and respiratory syndrome viruses (HP-PRRSVs; JXA1-like strain, lineage 8.7). Phylogenetic and molecular evolutionary results showed that for the two isolates, HP-PRRSV provides most of the ORF1 gene. Animal experiment revealed discrepancies in virulence between the strains. Although challenge resulted in 100% morbidity, the isolate carrying most of the HP-PRRSV ORF1 caused severe clinical symptoms and 40% mortality, whereas the other isolate containing part of the ORF1 gene caused no mortality. Overall, these findings suggest that lineage 3 viruses might be commonly circulating in most of southern China. Frequent recombination events within HP-PRRSVs of this lineage with changing virulence could represent potential threats to the pig industry.

[Effects of Wogonin on Apoptosis,Invasion,Migration and Wnt/ß-Catenin Signaling Pathway of Gastric Cancer Cells SGC7901].

Objective: To explore the effects of wogonin on the apoptosis,invasion,migration and Wnt / ß-catenin signaling pathway of gastric cancer cells SGC7901. Methods: Three common gastric cancer cell lines( SGC7901,BGC-823 and MKN-45) were conventionally cultured to logarithmic growth. The cell proliferation was detected by MTT assay after treatment with different concentrations of wogonin( 0,20,50,100 and 200 µmol / L) for 24,48,72 and 96 h. The apoptosis rate, migration and invasion ability of SGC7901 cells were detected by Annexin V-FITC / PI annexin double staining flow cytometry, scratch test and transwell cell invasion assay, respectively. After treament with different concentrations of wogonin for 48 h,the protein levels of ß-catenin, C-myc and Cyclin D1 were detected by Western blotting. Results: Wogonin within the range of 20 ~ 200 mol / L could inhibit the proliferation of SGC7901,BGC-823 and MKN-45 cells in a dose-and time-dependent manner. Compared with 0 µmol / L wogonin group,After treament with different concentrations of wogonin for 24 h and 48 h,SGC7901 cells had elevated apoptosis rate and decreased migration distance and number of penetrating membrane cell in the rest concentrations( P < 0. 05). The protein levels of ß-catenin,C-myc and Cyclin D1 were lower in 20 ~ 200µmol /L wogonin groups than 0 µmol /L wogonin group except for the concentration of 20 µmol /L wogonin groups( P < 0. 05),and the effects in a dose-dependent manner. Conclusion: Wogonin has the ability to inhibit the proliferation of gastric cancer cells,and can induce apoptosis and inhibit cell migration and invasion,which may be related to the inhibition of Wnt / ß-catenin signaling pathway activation.

[Study on composition of essential oil in above-ground and root of Bupleurum malconense and root of B. chinense by AMDIS and retention index].

Chemical constituents of the essential oil in above-ground and root of Bupleurum malconense and root of B. chinense were investigated by GC-MS compiled with automated mass spectral deconvolution and identification system (AMDIS) and retention index. The results showd that the components of essential oil in B. malconense have some similarities with the one in B. chinense, and both of them have the higher content of caryophyllene oxide which is an active component of anti-inflammatory and analgesic. These results suggested that as a local substitute, B. malconense has a certain scientific basis of the treatment for cold fever.

Antirheumatoid Arthritis Activities and Chemical Compositions of Phenolic Compounds-Rich Fraction from Urtica atrichocaulis, an Endemic Plant to China.

Urtica atrichocaulis, an endemic plant to China, is commonly used to treat rheumatoid arthritis even though its pharmaceutical activities and chemical constituents were not studied. Herein, we reported our investigations on the chemical compositions of the phenolic compounds-rich fraction from U. atrichocaulis (TFUA) and their antirheumatoid arthritis activities. We found that the TFUA significantly inhibited the adjuvant-induced rats arthritis, carrageenin-induced rats paw edema, cotton pellet-induced mice granuloma, and the acetic acid-induced mice writhing response. Our phytochemical investigations on the TFUA resulted in the first-time isolation and identification of 17 phenolic constituents and a bis (5-formylfurfuryl) ether. The extensive HPLC analysis also revealed the chemical compositions of TFUA. Our further biological evaluation of the main phenolic components, individually and collectively, indicated that the antirheumatoid arthritis activities of TFUA were the combined effect of multiple phenolic constituents.

[Ttextual research of Cannabis sativa varieties and medicinal part].

OBJECTIVE: To determine the medicinal part and varieties of Cannabis Sativa through herbal textual research to Provide bibliographic reference for clinical application. METHOD: Herbal textual research of C. Sativa from ancient herbal works and modern data analysis. RESULT: Through the herbal textual research, the plant of the C. sativa, for Fructus Cannabis used now is identical with that described in ancient herbal literatures. People did not make a sharp distinction on medicinal part of C. sativa in the early stage literatures, female inflorescence and unripe fruit, fruit and kernel of seed were all used. Since Taohongjing realized the toxicity ofpericarp, all the herbal and prescription works indicate that the pericarp shall be removed before usage and only the kernel can be used. However, in modem literatures, both fruit and kernel can be used as medicinal part. CONCLUSION: The plants for Fructus Cannabis described in modern and ancient literatures are identical. The base of the original plant is the same either in ancient or modern. And the toxicity of the fruit is more than that of the kernel. The kernel is the exact medicinal part of C. Sativa.

[Fingerprint analysis of Dipsacus asperoides by HPLC].

OBJECTIVE: To establish the HPLC fingerprints of Dipsacus asperoides for reflecting the internal information, evaluating its internal quality. METHODS: 20 batches of D. asperoides were collected from different place with the HPLC fingerprints method. Chromatographic column: Welchrom-C18 (250 mm x 4. 6 mm, 5 microm), mobile phase: acetonitrile and water (gradient elution), flow rate: 1.0 mL/min, detection wavelength: 212 nm, column temperature: 35 degrees C. RESULTS: The common mode of HPLC fingerprint was established and similar degrees to D. asperoides from different areas were compared. CONCLUSION: The method is stable, reliable, and with full information which can be used for quality evaluation, quality control item and crude drug identification of D. asperoides.

[HPLC fingerprint of Codonopsis tangshen from different habitats].

OBJECTIVE: To establish a sensitive and specific HPLC method for quality control of Codonopsis tangshen. METHOD: The samples were determined with the dereloped method. By the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2004A), mean chromatogram was generated as the representative standard fingerprint and the similarity of each chromatogram against the mean chromatogram was also calculated. Samples was clustered using principal component analysis (PCA) based on the ratio of characteristic peaks and standard peak. The chromatographic fingerprinting of C. tangshen, showing 7 characteristic peaks, was established from 29 habitats of C. tangshen. RESULT: The similarity of the chromatographic fingerprints from the 29 samples was over 0.8 and 23 samples were classified into one group based on hierarchical cluster, which showed the quality of C. tangshen from different habitats was in good consistency. CONCLUSION: The method can be applied for quality assessment of C. tangshen.

[Characteristics of element contents and their influence factors in Codonopsis tangshen].

OBJECTIVE: To study the characteristics of element contents and the influencing factors in Codonopsis tangshen. METHOD: The contents of elements were determined and the data was statistically analyzed. RESULT: The variation coefficients of elements in C. tangshen were lower than those in soil, and variation coefficients of C. tangshen from different habitats were lower than 50%. CONCLUSION: The absorption of nutritional elements in C. tangshen was related to content and condition of soil nutrients, and climate etc.

[Morphological and histological studies of Herpetospermum pedunculosum seeds and other substitutes].

OBJECTIVE: To provide new evidences for the identification of Herpetospermum pedunculosum seeds and other substitutes. METHOD: Microscopic examination of the cross sections and powers of the seeds of H. pedunculosum, Momordica charantia, Thladiantha setispina and M. cohinchinensis were made to characterize the drug materials. RESULT AND CONCLUSION: The external and internal structure of T. setispina and M. charantia were studied for the first time. Two types of different branched starry sclereids was found in M. cohinchinensis. Comparative studies on external and internal structure were carried out on seeds of the 4 species. As a result, they could be distinguished by external characteristics such as shape, size and color of their seeds, and by internal structure such as size and type of sclereid, epidermis and sub-epidermis cell of seed coat.

[Literature data investigation in semem of Herpetospermum pedunculosum].

Based on literature data, related specimens, commercial samples and field investigation, botanical origin of "bolingguazi" was clarified. Boling guazi was found to be used as a Tibetan medicine in China from 8th century, it was derived from Herpetospermum pedunculosum of cucurbitaceae, and main species of bolingguazi used in most areas of China were H. pednculosura, while seeds of Momordica charantia, Thladiantha setispina and M. cohinchinensis were also available in some areas of China.

[Conservation of endangered species resources of Tibetan medicine in China].

OBJECTIVE: To further investigate and discuss the cause of species endangerment, the status and present problem of conservation of traditional Tibetan medicine in China. METHOD: Previous relevant investigations and literatures were summed up in the field. The present situation of conservation of traditional Tibetan medicine was analyzed. RESULT: The status of endangered resources, cause of species endangerment, the conserving status and conserving measures etc were elaborated. The classification was made and suggestion of species conservation of traditional Tibetan medicine were put forward. CONCLUSION: The endangered species conservation of traditional Tibetan medicine was carried out by building protective area of endangered species resources and plant garden, setting up germplasm bank, developing the domestication and cultivation of Tibetan medicinal herbs most in use, strengthening the investigation and study of endangered species, launching exchange and cooperation of conservation techniques on endangered species, enhancing the protective awareness of endangered species traditional Tibetan medicine etc. By so doing we can facilitate the sustainable development of traditional Tibetan medicine in China.