Generation of High-Quality African Swine Fever Virus Complete Genome from Field Samples by Next-Generation Sequencing.

African swine fever (ASF) is a lethal contagious viral disease of domestic pigs and wild boars caused by the African swine fever virus (ASFV). The pandemic spread of ASF has caused severe effects on the global pig industry. Whole-genome sequencing provides crucial information for virus strain characterization, epidemiology analysis and vaccine development. Here, we evaluated the performance of next-generation sequencing (NGS) in generating ASFV genome sequences from clinical samples. Thirty-four ASFV-positive field samples including spleen, lymph node, lung, liver and blood with a range of Ct values from 14.73 to 25.95 were sequenced. For different tissue samples collected from the same sick pigs, the proportion of ASFV reads obtained from the spleen samples was 3.69-9.86 times higher than other tissues. For the high-viral-load spleen samples (Ct < 20), a minimum of a 99.8% breadth of ≥10× coverage was revealed for all the samples. For the spleen samples with Ct ≥ 20, 6/12 samples had a minimum of a 99.8% breadth of ≥10× coverage. A high average depth of sequencing coverage was also achieved from the blood samples. According to our results, high-quality ASFV whole-genome sequences could be obtained from the spleen or blood samples with Ct < 20. The high-quality ASFV genome sequence generated in this study was further used for the high-resolution phylogenetic analysis of the ASFV genomes in the early stage of the ASF epidemic in China. Our study demonstrates that NGS may act as a useful tool for efficient ASFV genome characterization, providing valuable information for disease control.

Tetrandrine (TET) inhibits African swine fever virus entry into cells by blocking the PI3K/Akt pathway.

African Swine Fever Virus (ASFV) infection causes an acute and highly contagious disease in swine, resulting in significant economic losses and societal harm worldwide. Currently, there are no effective vaccines or antiviral drugs available for ASFV. Tetrandrine (TET) is extracted from the traditional Chinese herb Stephania tetrandrae, possesses diverse biological functions such as anti-inflammatory, anti-tumor, and antiviral activities. The study comprehensively evaluated the anti-ASFV effect of TET and validated it through biological assays. The dose-dependent inhibition of TET against ASFV was confirmed and a novel mechanism of TET's anti-ASFV activity was elucidated. TET effectively inhibits ASFV during internalization by blocking macropinocytosis through the inhibition of the PI3K/Akt pathway. The specific inhibitor LY294002, targeting the PI3K/Akt pathway, exhibits similar antiviral activity against ASFV as TET. Furthermore, the inhibitory effect of TET against other viruses such as Lumpy Skin Disease Virus (LSDV) and Porcine Epidemic Diarrhea Virus (PEDV) was also identified. Our findings suggest that TET effectively inhibits ASFV and reveal the potential for broad-spectrum antiviral drugs targeting the PI3K/Akt pathway.

Revealing common differential mRNAs, signaling pathways, and immune cells in blood, glomeruli, and tubulointerstitium of lupus nephritis patients based on transcriptomic data.

Lupus nephritis (LN) is a potentially fatal autoimmune disease. The purpose of this study was to find potential key molecular markers of LN to aid in the early diagnosis and management of the disease. Datasets GSE99967_blood, GSE32591_glomeruli, and GSE32591_tubulointerstitium were included in this study. Differentially expressed mRNAs (DEmRNAs) were identified between the normal control and LN groups using the limma package in R. Common DEmRNAs in the three datasets were taken. Subsequently, functional enrichment analysis, immune correlation analysis, receiver operating characteristic (ROC) curve analysis and real-time polymerase chain reaction (RT-PCR) verification were performed. In this study, 11 common DEmRNAs were obtained and all of them were up-regulated. In protein-protein interaction (PPI) networks, we found that MX dynamin like GTPase 1 (MX1) and radical S-adenosyl methionine domain containing 2 (RSAD2) had the highest interaction score (0.997). Functional enrichment analysis revealed that MX1 and RSAD2 were enriched in influenza A and hepatitis C signaling pathways. The area under the curve (AUC) values of interferon-induced protein 44 (IFI44) and MX1 in GSE32591_glomeruli and GSE32591_tubulointerstitium datasets are 1, which is worthy of further study on their diagnostic value and molecular mechanism. The xCell analysis showed abnormal distribution of granulocyte-macrophage progenitor (GMP) cells in blood, glomeruli, and tubulointerstitium. Pearson's correlation analysis found that GMP cells were significantly correlated with lactotransferrin (LTF) and cell cycle. Identification of common DEmRNAs and key pathways in the blood, glomeruli, and tubulointerstitium of patients with LN provides potential research directions for exploring the molecular mechanisms of the disease.

Low-Dose X-Ray Increases Paracellular Permeability of Human Renal Glomerular Endothelial Cells.

Objective: Glomerular endothelium functions as a filtration barrier of metabolites in the kidney. Although X-ray irradiation modulated the permeability of the vascular endothelium, the response of human renal glomerular endothelial cells (HRGECs) to low-dose X-ray irradiation has not been investigated. We evaluated the impacts of low-dose X-ray irradiation on HRGECs and revealed the underlying mechanism. Methods: HRGECs were exposed to X-ray with doses of 0, 0.1, 0.5, 1.0, and 2.0 Gy. The proliferation, viability, and apoptosis of HRGECs were examined by MTT assay, trypan blue staining assay, and TUNEL staining, respectively. The paracellular permeability was assessed by paracellular permeability assay. The expression of VE-cadherin was investigated via immunofluorescence assay. Western blot and qRT-PCR detected the expression levels of VE-cadherin and CLDN5. Besides, the expression levels of pVE-cadherin (pY658), TGF-ß, TGF-ßRI, Src, p-Src, Smad2, p-Smad2, Smad3, p-Smad3, SNAIL, SLUG, and apoptosis-related proteins were tested by Western blot. Results: The proliferation, viability, and apoptosis of HRGECs were not affected by low-dose (<2.0 Gy) X-ray irradiation. X-ray irradiation dose-dependently reduced the level of VE-cadherin, and VE-cadherin and CLDN5 levels were reduced with X-ray irradiation. The levels of pY658, p-Src, p-Smad2, and p-Smad3 were upregulated with the increase in X-ray dose. Besides, the paracellular permeability of HRGECs was increased by even low-dose (<2.0 Gy) X-ray irradiation. Therefore, low-dose X-ray irradiation reduced the cumulative content of VE-cadherin and increased the level of pY658 via activation of the TGF-ß signaling pathway. Conclusion: Even though low-dose X-ray exposure had no impact on proliferation, viability, and apoptosis of HRGECs, it increased the paracellular permeability by deterioration and downregulation of VE-cadherin through stimulating the TGF-ß signaling pathway. This study built the framework for kidney response to low-dose irradiation exposure.

Time Series Data Prediction and Feature Analysis of Sports Dance Movements Based on Machine Learning.

Sports dance is a competition project and a kind of sports, with the characteristics of being smooth, generous, leisurely, and comfortable, dance steps, smooth movements, and flowing clouds, and it can give full play to the indoor space. In the light of the new era, sports dance is also playing an increasingly important role. Through the time series data and feature analysis of dance sports movements through machine learning, the internal information is mined to find the trends and laws. Machine learning in the era of big data is widely used in research as the main tool for data analysis and mining. The key difficulty of data mining has always been time series data. Machine learning refers to a method of using the resulting data in a computer to derive a certain model and then using this model to make predictions. The core is "using algorithms to parse data, learn from it, and then make decisions or predictions about new data."

Continuous surveillance revealing a wide distribution of class I Newcastle disease viruses in China from 2011 to 2020.

The risk-based active surveillance for Newcastle disease virus (NDV) was carried out in China from 2011 to 2020. A total of 110,018 swabs were collected from 28 provinces. 2,389 class I NDVs were isolated and identified by RT-PCR and sequencing. The average annual positivity rate of class I NDVs from 2011 to 2020 was 2.17%. In the last 10 years, the positivity rate was highest in 2011 (4.76%), and has since decreased. Most viruses were isolated from chickens, while others were collected from ducks, geese and pigeons, as well as from the environment. The positivity rates for class I NDVs in poultry ranged from 0.55% to 2.40%. The viruses were isolated from 373 sampling sites in 24 provinces, mainly in East, Central, South and Southwest China. The positivity rates of NDVs in wholesale markets (51.58%) and retail markets (42.83%) were much higher than those in poultry farms (7.14%) and slaughterhouses (3.85%). Phylogenetic analyses showed that most isolates belonged to sub-genotype 1.1.2, while only 22 viruses belonged to sub-genotype 1.2, indicating the viruses in sub-genotype 1.1.2 were the predominant strains in China. The F and HN genes of six strains in the two sub-genotypes were sequenced and analyzed. The cleavage sites of F protein in the six viruses were 112ERQER/L117, 112ERQGR/L117 or 112GRQERL117, which were typical of low virulence NDV. Several mutations were identified in the functional domains of F and HN proteins, including fusion peptide, heptad repeat region, transmembrane domains and neutralizing epitopes. This study revealed the distribution, genetic and phylogenetic characteristics of class I NDVs in China, and could help us to better understand the epidemiological context of class I NDVs in China.

Comparison between class I NDV and class II NDV in aerosol transmission under experimental condition.

Recent epidemiological surveys have shown that class I Newcastle disease virus (NDV) is widely distributed in China. However, little is currently known about its transmission. Therefore, in this study, we compared the transmission of class I and class II NDV. Specific-pathogen-free chickens were divided into a class I NDV inoculation group and an aerosol-exposed infection group and kept in 2 separate isolators (A and B, respectively) that were connected with an airtight plastic pipe. After inoculation, air samples were collected regularly with an All-Glass Impinger-30 (Liaoyang, China), and the airborne virus contents were analyzed using the plaque count method. In addition, oral and cloacal swabs were collected regularly to detect virus shedding using quantitative reverse transcription PCR. Similar trials were conducted simultaneously with class II NDV in isolators C and D. We consistently detected class I NDV aerosols in both isolators A and B up to 40 D post-inoculation (dpi). The aerosol concentration reached a maximum of 13.81 × 103 plague-forming units per cubic meter of air at 18 dpi and was significantly higher than that of class II NDV at 21 and 24 dpi. We also detected class I virus shedding from 2 to 40 dpi in the inoculated chickens and from 7 to 40 D post-aerosol-exposed infection in the aerosol-exposed chickens. This phenomenon may explain why class I NDV has been the primary epidemic strain of NDV in recent years.

Molecular characterization of new emerging sub-genotype VIIh Newcastle disease viruses in China.

Newcastle disease (ND) has been enzootic in China for several decades since the first recognition of the disease in 1946 in China. Continuous surveillance revealed that the sub-genotype VIId Newcastle disease virus (NDV) has been predominantly responsible for most of ND outbreak in China in recent years. But in the present study, three virulent NDVs isolated from poultry in southern China were classified as sub-genotype VIIh, which is highly related to the viruses circulating in some Southeast Asia countries. Continuous isolation of genotype VIIh NDV strains in the region suggests its panzootic potential. This is the first report of the sub-genotype VIIh NDVs in domestic poultry in China. The complete genome length of the three isolates was 15,192 nucleotides, and the motif at the cleavage site of F protein was 112RRRRR/F117 or 112RRRKR/F117, which was typical of virulent NDV. Phylogenetic analysis based on the F gene revealed that the three viruses had close relationship with the sub-genotype VIIh virus isolated from wild bird in 2011 in China. These viruses might have formed a stable lineage in poultry during 2012-2016 and have the potential to cause enzootic in China. Our study revealed the genetic and phylogenetic characteristics of the three sub-genotype VIIh isolates, which could help us to better understand the epidemiological context of these viruses.

Elevated levels of serum sclerostin are linked to adverse cardiovascular outcomes in peritoneal dialysis patients.

PURPOSE: To investigate the correlation between elevated serum sclerostin levels and chronic kidney disease outcomes for patients receiving peritoneal dialysis (PD). METHODS: We performed a prospective observational study in stable PD patients. Serum sclerostin levels were determined via enzyme immunoassay, and median levels of sclerostin were used to divide patients into high and low sclerostin groups. New-onset cardiovascular events (CVEs) and cardiovascular mortality were evaluated during a 6-year follow-up period. RESULTS: Ninety-eight patients [mean age 52.5 ± 10.9 years, 49% males, 21.4% diabetic, median dialysis vintage 40.7 (range 17.9-72.2) months] were recruited. Compared with those in the low sclerostin group, patients in the high sclerostin group demonstrated higher levels of total-cholesterol, NT-proBNP, and osteoprotegerin (all P < 0.05). During the 6-year study period, 25 CVEs and 17 cardiovascular deaths occurred in the high sclerostin group, whereas 11 CVEs and four cardiovascular deaths occurred in the low sclerostin group. A Cox regression analysis determined that high sclerostin levels significantly increased the risk for CVEs (HR 2.475, 95% CI 1.116-5.489, P = 0.026) and cardiovascular death (HR 3.484, 95% CI1.134-10.706, P = 0.029), after multiple adjustments were made. CONCLUSIONS: Our data suggest that high sclerostin levels may predict the onset of CVEs and cardiovascular mortality among PD patients.

Six-minute walk test predicts all-cause mortality and technique failure in ambulatory peritoneal dialysis patients.

AIM: This study investigated the associated factors of 6-min walk test (6MWT) and its predictive value of outcome in patients undergoing peritoneal dialysis (PD). METHODS: This is a single centre prospective observational cohort study. Stable ambulatory PD patients in our centre between 1 May 2010 and 30 April 2011 were enrolled in this study. All included subjects performed 6MWT, and 6-min walk distances (6MWDs) were recorded. Patients were divided into two groups according to 6MWD and prospectively followed up until death, cessation of PD or to the end of the study (30 September 2012). RESULTS: A total of 145 patients were enrolled, including 63 (43%) males. Multiple stepwise regression showed that age (ß = -0.295, P = 0.001), diastolic blood pressure (DBP) (ß = 0.292, P = 0.001), left ventricular ejection fraction (LVEF) (ß = 0.198, P = 0.019) were independently associated with lower 6MWD. By the end of the study, six (8%) patients died in long 6MWD group while 15 (20%) died in the short 6MWD group, a significantly lower patient survival was observed in short 6MWD group (Log-rank = 4.983, P = 0.026). Patients with short 6MWD also showed inferior technique survival (Log-rank = 4.838, P = 0.028). There was no significant difference in peritonitis-free survival between the two groups (Log-rank = 0.801, P = 0.371). However, more patients in short 6MWD group had been transferred to hemodialysis due to peritonitis (25% vs 4.2%, P = 0.013). CONCLUSION: Age, diastolic blood pressure, LVEF are independent associated factors of 6MWD in patients undergoing PD. Having the advantages of easy applicability and safety, 6MWT may be proposed as an important predictor of outcome in ambulatory PD patients.

Genomic Characterizations of a Newcastle Disease Virus Isolated from Ducks in Live Bird Markets in China.

One class I Newcastle disease virus (NDV), designated as duck/Guangxi/1261/2015 (GX1261), was isolated from asymptomatic ducks in live bird markets (LBM) from southern China during the national active surveillance for NDVs in 2015. The complete genome length of GX1261 isolate was 15,198 nucleotides with the gene order of 3'-NP-P-M-F-HN-L-5'. The motif at the cleavage site of F protein was 112ERQER/L117, which was typical of low virulence NDV. Several mutations were identified in the functional domains of F and HN proteins, including fusion peptide, heptad repeat region, transmembrane domains and neutralizing epitopes. Phylogenetic analysis based on the complete F gene revealed that the isolate was clustered into sub-genotype 1c in class I, and showed a high level of similarity with the strains isolated from waterfowl in the United States of America. This is the first report of this kind of virus in the mainland of China. These results demonstrated that GX1261-like viruses might exist in asymptomatic waterfowl, and remain undetected or unidentified. Thus, more investigation needs to be done in order to identify the source of the virus. This study revealed the genetic and phylogenetic characteristics of GX1261 isolate and could help us to better understand the epidemiological context of class I NDV in China.

Genomic Characterizations of Six Pigeon Paramyxovirus Type 1 Viruses Isolated from Live Bird Markets in China during 2011 to 2013.

The genomes of six pigeon paramyxovirus type 1 (PPMV-1) isolated from symptomless pigeons in live poultry markets during the national active surveillance from 2011 to 2013 were sequenced and analyzed in this study. The complete genome lengths of all isolates were 15,192 nucleotides with the gene order of 3'-NP-P-M-F-HN-L-5'. All isolates had the same motif of 112RRQKRF117 at the cleavage site of the fusion protein, which was typical of velogenic Newcastle disease virus (NDV). Several mutations were identified in the functional domains of F and HN proteins, including fusion peptide, heptad repeat region, transmembrane domains and neutralizing epitopes. Phylogenetic analysis based on sequences of complete genomes and six genes revealed that all isolates belonged to genotype VI in class II, but at least 2 sub-genotypes were identified. Most isolates were placed into sub-genotype VIb with the exception of pi/GX/1015/13, which was classified in sub-genotype VIa. The obvious antigenic difference between PPMV-1 isolates and La Sota strain was found based on the R-value calculated by cross hemagglutination inhibition (HI) assay. These results provided the evidence that PPMV-1 could be detected from healthy pigeons, and our study may be useful in designing vaccines used in pigeon, and developing molecular diagnostic tools to monitor and prevent future PPMV-1 outbreaks.

[Detection of an NA gene molecular marker in H7N9 subtype avian influenza viruses by pyrosequencing].

This study aimed to establish a method for the detection and identification of H7N9 avian influenza viruses based on the NA gene by pyrosequencing. According to the published NA gene sequences of the avian influenza A (H7N9) virus, a 15-nt deletion was found in the NA gene of H7N9 avian influenza viruses. The 15-nt deletion of the NA gene was targeted as the molecular marker for the rapid detection and identification of H7N9 avian influenza viruses by pyrosequencing. Three H7N9 avian influenza virus isolates underwent pyrosequencing using the same assay, and were proven to have the same 15-nt deletion. Pyrosequencing technology based on the NA gene molecular marker can be used to identify H7N9 avian influenza viruses.

Genome Sequences of an H5N1 Highly Pathogenic Avian Influenza Virus Isolated from Vaccinated Layers in China in 2012.

An H5N1 virus was isolated from vaccinated layers during an outbreak of highly pathogenic avian influenza (HPAI) in Ningxia, China, in 2012. Phylogenetic analysis revealed that the virus is a novel variant in clade 7.2, and the outbreak likely resulted from mutations in the viral hemagglutinin (HA) gene.

Complete genome sequences of new emerging newcastle disease virus strains isolated from china.

Five Newcastle disease virus strains isolated from geese were classified into a new genotype, designated genotype XII. The complete genome sequences of two strains indicated that these viruses were distinct from viruses of genotype VII. More investigations need to be conducted for us to understand the origin of these new strains.

[Establishment of reverse genetics system for class I NDV08-004 strain].

Based on the genomic sequence of NDV08-004 strain (GenBank accession number FJ794269), seven pairs of primers were designed to amplify the genomic fragments by RT-PCR and cloned into pGEM-Teasy vector. The fragments (named A to G) were sub-cloned into transcription vector pOLTV5 according to the universal RE site and the plasmid named NDV08-004-pO which contained the full length cDNA of NDV08-004 strain was constructed. Three helper plasmids (pCI-NP, pCI-P and pCI-L) together with NDV08-004-pO were co-transfected into BSR T7/5 cells, and the transfection supernatant was inoculated into SPF embryonated eggs to rescue the virus. The virus was rescued successfully and identified by HA and RT-PCR and sequencing. The rescue system constructed in this study provided a good foundation for the further related research.

Evaluating viral interference between Influenza virus and Newcastle disease virus using real-time reverse transcription-polymerase chain reaction in chicken eggs.

BACKGROUND: Simultaneous and sequential allantoic cavity inoculations of Specific-pathogen-free (SPF) chicken eggs with Influenza virus (AIV) and Newcastle disease virus (NDV) demonstrated that the interaction of AIV and NDV during co-infection was variable. Our research revisited the replication interference potential of AIV and NDV using real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) for AIV and NDV to specifically detect the viral genomes in mixed infections. RESULTS: Data from this survey showed that when different doses of NDV (Lasota or F48E8) and AIV (F98 or H5N1) were simultaneously inoculated into embryonating chicken eggs (ECE), interference with the growth of NDV occurred, while interference with the growth of AIV did not occur. When equal amount of the two viruses were sequentially employed, the degree of interference was dependent upon the time of superinfection and the virulence of virus. CONCLUSION: AIV have a negative impact on NDV growth if they are inoculated simultaneously or sequentially and that the degree of interference depended upon the quantity and relative virulence of the virus strains used; however, interference with AIV was not observed. Only if NDV were inoculated at an earlier time will NDV able to interfere with the growth of AIV.

Multiplex RT-PCR for rapid detection and differentiation of class I and class II Newcastle disease viruses.

A multiplex RT-PCR was developed for detection and differentiation of class I and class II strains of Newcastle disease virus (NDV). The method was shown to have high specificity and sensitivity. The results obtained from the multiplex RT-PCR for a total of 67 NDV field isolates obtained in 2009 were consistent with those obtained by nucleotide sequencing and phylogenetic analysis. A phylogenetic tree based on the partial sequences of the F gene revealed that the 67 field isolates of NDV could be divided into two classes. Twenty-seven NDV isolates were grouped into class I, and two genotypes were identified. Most of the class I isolates were determined to be of genotype 3, with the exception of isolate NDV09-034, which belonged to genotype 2. Forty class II NDV isolates were divided into three genotypes, namely genotype VII (27 isolates), genotype I (2 isolates) and genotype II (11 isolates). Isolates of genotypes I and II in class II were shown to be related to commercial vaccine strains used commonly in China. All isolates of genotype VII were predicted to be virulent, on the basis of the sequence motif at the cleavage site of the F gene. This genotype has become predominantly responsible for most outbreaks of ND in China in recent years. In conclusion, this multiplex RT-PCR provides a new assay for rapid detection and differentiation of both classes of NDV isolates.

[Detection of molecular markers of amantadine resistance in avian influenza viruses by pyrosequencing].

Mutation in any of five key amino acid residues (at positions 26, 27, 30, 31 and 34) within the M2 protein of influenza A viruses leads to resistance against the amantodine class of anti-influenza drugs. In this study, a pyrosequencing method was described to rapidly detect established five molecular markers of resistance to M2 blockers, amantadine. The residues L26, V27, A30, S31 and G34 in the M2 protein were targeted for pyrosequencing, and 94 avian influenza viruses were used to perform the amantadine resistance analysis. Our results showed that most of avian influenza viruses were amantadine resistant, Mutations V27I and S31N were founded in these isolates.

[Detection of molecular markers of amantadine resistance in swine influenza viruses by pyrosequencing].

OBJECTIVE: To evaluate the resistance against the adamantine of multi-genotype (H1N1, H3N2 and H9N2) swine influenza viruses isolated from China in recent years by pyrosequencing. METHODS: Mutation in one of five key amino acid residues (positions 26, 27, 30, 31 and 34) within the M2 protein of influenza A viruses, leading to resistance against the adamantine class of anti-influenza drugs. The residues L26, V27, A30, S31, and G34 in the M2 protein were targeted for pyrosequencing,and 10 swine influenza viruses obtained from China during 2004 to 2008 were used to perform the amantadine resistance analysis. RESULTS: All 5 H1N1 swine influenza viruses were adamantine resistance, three mutations were founded in these isolates, namely V27T, V27I and S31N. Other five isolates, including four H3N2 and one H9N2 swine influenza virus, were proved to be sensitive to amantadine. CONCLUSION: Pyrosequencing technology based on the M2 gene can be used to determine the amantadine resistance for multi-genotype swine influenza viruses.