RACK1 promotes porcine reproductive and respiratory syndrome virus infection in Marc-145 cells through ERK1/2 activation.

Porcine reproductive and respiratory syndrome (PRRS) is an acute infectious disease that spreads rapidly among pigs and seriously threatens the pig industry. Activation of ERK1/2 is a hallmark of most viral infections. RACK1 interacts with a variety of kinases and membrane receptors that closely associated with viral infections and the development and progression of cancer. However, no studies have clearly defined whether RACK1 can regulate PRRSV infection through ERK1/2 activation. In our study, using RT-qPCR, immunoblotting, indirect fluorescent staining, siRNA knockdown and protein overexpression techniques, we found that downregulation of cellular RACK1 inhibited ERK1/2 activation and subsequently suppressed PRRSV infection, while overexpression of RACK1 enhanced ERK1/2 activation and PRRSV infection. Bioinformatic and Co-immunoprecipitation experimental analysis revealed that cellular RACK1 could interact with viral N protein to exert its function. We elaborated that RACK1 promoted PRRSV replication in Marc-145 cells through ERK1/2 activation. Our study provides new insights into regulating the innate antiviral immune responses during PRRSV infection and contributes to further understanding of the molecular mechanisms underlying PRRSV replication.

Factors influencing platelet isolation: a prospective multicenter study from Western China.

Tumor-educated platelets (TEPs) have been widely reported to have promising application potential; nonetheless, platelet isolation from peripheral blood is an important but neglected step in TEPs research for platelet-based liquid biopsy. In this article, we discussed some common influence factors for platelet isolation. To investigate the factors involved in platelet isolation, a prospective multicenter study was conducted on healthy Han Chinese adults (18 to 79 years of age). A total of 208 individuals were included in the final statistical analysis out of the 226 healthy volunteers who were prospectively enrolled from four hospitals. The primary study metric was the platelet recovery rate (PRR). The similar pattern was observed in the four hospitals, The PRR at room temperature (23°C±2°C) was slightly higher than the PRR at cold temperature (4°C±2°C). Moreover, the PRR gradually decreased as the storage time increased. The PRR for samples within 2 hours of storage is significantly higher than for samples beyond 2 hours (p < .05). Additionally, PRR was also affected by the equipment used in different centers. This study confirmed several factors that influence platelet isolation. In our study, we indicated that platelet isolation should be performed within two hours of peripheral blood draw and held at room temperature until isolation, and that centrifuge models should be fixed during the extraction process, which will further improve the research progress of platelet-based liquid biopsy in cancer.

What is the context? Globally, cancer is one of the leading cause of premature death. Early screening is important for cancer diagnosis and treatment and can even significantly lower cancer mortalityGlobally, cancer is one of the leading cause of premature death. Early screening is important for cancer diagnosis and treatment and can even significantly lower cancer mortalityFor the liquid biopsy, isolation is an important step. Early studies have explored the influencing factors of exosome, circulating tumor cells (CTCs), and other components extraction in liquid biopsy.Despite platelet also being an excellent source of liquid biopsy, few studies have explored the factors that influence platelet isolation.Considering the importance of platelet isolation in tumor-based platelet liquid biopsy, our aim is to optimize platelet isolation conditions as much as possible to obtain a high platelet recovery rate.What is new? In this study, we conducted a prospective multicenter study ofhealthy adults from four centers, combining whole blood with platelet-richplasma to investigate factors influencing platelet recovery rate (PRR) during platelet isolation.In our study, we indicated that platelet isolation should be performed within two hours at room temperature, and that centrifuge models should be fixed during the extraction process, which will further improve the research progress of platelet-based liquid biopsy in cancer.What is the impact? In future platelet-related studies, we should fix the sample storage temperature, storage time and centrifuge model in the process of platelet extraction, so as to reduce the variables affecting platelet extraction as much as possible and ensure the stable recovery rate of platelet extraction.

Clinlabomics: leveraging clinical laboratory data by data mining strategies.

The recent global focus on big data in medicine has been associated with the rise of artificial intelligence (AI) in diagnosis and decision-making following recent advances in computer technology. Up to now, AI has been applied to various aspects of medicine, including disease diagnosis, surveillance, treatment, predicting future risk, targeted interventions and understanding of the disease. There have been plenty of successful examples in medicine of using big data, such as radiology and pathology, ophthalmology cardiology and surgery. Combining medicine and AI has become a powerful tool to change health care, and even to change the nature of disease screening in clinical diagnosis. As all we know, clinical laboratories produce large amounts of testing data every day and the clinical laboratory data combined with AI may establish a new diagnosis and treatment has attracted wide attention. At present, a new concept of radiomics has been created for imaging data combined with AI, but a new definition of clinical laboratory data combined with AI has lacked so that many studies in this field cannot be accurately classified. Therefore, we propose a new concept of clinical laboratory omics (Clinlabomics) by combining clinical laboratory medicine and AI. Clinlabomics can use high-throughput methods to extract large amounts of feature data from blood, body fluids, secretions, excreta, and cast clinical laboratory test data. Then using the data statistics, machine learning, and other methods to read more undiscovered information. In this review, we have summarized the application of clinical laboratory data combined with AI in medical fields. Undeniable, the application of Clinlabomics is a method that can assist many fields of medicine but still requires further validation in a multi-center environment and laboratory.

The Clinical Application Value of the Prognostic Nutritional Index for the Overall Survival Prognosis of Patients with Esophageal Cancer: A Robust Real-World Observational Study in China.

Esophageal cancer is a kind of cancer with high morbidity and mortality, which is accompanied by a profound poor prognosis. A prognostic nutritional index, based on serum albumin levels and peripheral lymphocyte count, has been confirmed to be significantly associated with various cancers. This study was aimed at exploring the prognostic significance of PNI in the overall survival prognosis of patients with esophageal cancer. As a real-world study based on the big database, clinical data of 2661 patients with esophageal cancer were evaluated retrospectively, and the individuals were randomly divided into training and testing cohorts. In these two cohorts, patients are classified into a high-risk group (PNI < 49) and a low-risk group (PNI ≥ 49). Univariate and multivariate analyses were performed to analyze the independent risk factors for the prognosis of esophageal cancer patients by using the Cox proportional hazards regression model. In this study, whether in the training cohort or the testing cohort, according to the univariate analysis, gender, tumor size, tumor grade, T stage, N stage, M stage, TNM stage, and PNI were significantly correlated with overall survival. Furthermore, the multivariate analysis showed that gender, T stage, N stage, M stage, TNM stage, and PNI were independent prognostic risk factors for esophageal cancer. PNI can be regarded as an independent prognostic factor combined with gender, T stage, N stage, M stage, and TNM stage, and it might be a novel reliable biomarker for esophageal cancer.

Selection and Validation of Reference Genes for Pan-Cancer in Platelets Based on RNA-Sequence Data.

Many studies in recent years have demonstrated that some messenger RNA (mRNA) in platelets can be used as biomarkers for the diagnosis of pan-cancer. The quantitative real-time polymerase chain reaction (RT-qPCR) molecular technique is most commonly used to determine mRNA expression changes in platelets. Accurate and reliable relative RT-qPCR is highly dependent on reliable reference genes. However, there is no study to validate the reference gene in platelets for pan-cancer. Given that the expression of some commonly used reference genes is altered in certain conditions, selecting and verifying the most suitable reference gene for pan-cancer in platelets is necessary to diagnose early stage cancer. This study performed bioinformatics and functional analysis from the RNA-seq of platelets data set (GSE68086). We generated 95 candidate reference genes after the primary bioinformatics step. Seven reference genes (YWHAZ, GNAS, GAPDH, OAZ1, PTMA, B2M, and ACTB) were screened out among the 95 candidate reference genes from the data set of the platelets' transcriptome of pan-cancer and 73 commonly known reference genes. These candidate reference genes were verified by another platelets expression data set (GSE89843). Then, we used RT-qPCR to confirm the expression levels of these seven genes in pan-cancer patients and healthy individuals. These RT-qPCR results were analyzed using the internal stability analysis software programs (the comparative Delta CT method, geNorm, NormFinder, and BestKeeper) to rank the candidate genes in the order of decreasing stability. By contrast, the GAPDH gene was stably and constitutively expressed at high levels in all the tested samples. Therefore, GAPDH was recommended as the most suitable reference gene for platelet transcript analysis. In conclusion, our result may play an essential part in establishing a molecular diagnostic platform based on the platelets to diagnose pan-cancer.

A new classifier constructed with platelet features for malignant and benign pulmonary nodules based on prospective real-world data.

Objectives: As the pulmonary nodules were hard to be discriminated as benignancy or malignancy only based on imageology, a prospective and observational real-world research was devoted to develop and validate a predictive model for managing the diagnostic challenge. Methods: This study started in 2018, and a predictive model was constructed using eXtreme Gradient Boosting (XGBoost) based on computed tomographic, clinical, and platelet data of all the eligible patients. And the model was evaluated and compared with other common models using ROC curves, continuous net reclassification improvement (NRI), integrated discrimination improvement (IDI), and net benefit (NB). Subsequently, the model was validated in an external cohort. Results: The development group included 419 participants, while there were 62 participants in the external validation cohort. The most accurate XGBoost model called SCHC model including age, platelet counts in platelet rich plasma samples (pPLT), plateletcrit in platelet rich plasma samples (pPCT), nodule size, and plateletcrit in whole blood samples (bPCT). In the development group, the SCHC model performed well in whole group and subgroups. Compared with VA, MC, BU model, the SCHC model had a significant improvement in reclassification as assessed by the NRI and IDI, and could bring the patients more benefits. For the external validation, the model performed not as well. The algorithm of SCHC, VA, MC, and BU model were first integrated using a web tool (http://i.uestc.edu.cn/SCHC). Conclusions: In this study, a platelet feature-based model could facilitate the discrimination of early-stage malignancy from benignancy patients, to ensure accurate diagnosis and optimal management. This research also indicated that common laboratory results also had the potential in diagnosing cancers.

CircSEC24A promotes tumor progression through sequestering miR-455-3p in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) ranks third in the cause of death due to cancer. Circular RNA circSEC24 Homolog A (circSEC24A) has been uncovered to be upregulated in liver cancer. However, the function of circSEC24A in HCC is indistinct. We analyzed the microarray datasets GSE78520 and GSE94508 to search for differentially expressed circRNAs associated with HCC. Expression of circSEC24A, microRNA (miR)-455-3p, and protein phosphatase, Mg2+/Mn2+ dependent 1F (PPM1F) mRNA was detected by quantitative real-time polymerase chain reaction (RT-qPCR). Loss-of-function experiments were conducted to validate the biological function of circSEC24A in HCC cells in vitro and in vivo. Protein levels were evaluated by western blotting and immunohistochemistry (IHC). The relationship between circSEC24A or PPM1F and miR-455-3p was verified by a dual-luciferase reporter and/or RNA immunoprecipitation (RIP) assays. circSEC24A was overexpressed in HCC. circSEC24A silencing decreased xenograft tumor growth in vivo and repressed proliferation, metastasis, invasion, epithelial-to-mesenchymal transition (EMT), induced cell cycle arrest, and apoptosis of HCC cells in vitro. circSEC24A acted as a molecular sponge to sequester miR-455-3p, resulting in elevating the expression of PPM1F. miR-455-3p inhibitor reversed the suppressive impact of circSEC24A silencing on malignant behaviors of HCC cells. PPM1F overexpression offsets the inhibitory effect of miR-455-3p mimic on malignant behaviors of HCC cells. circSEC24A sponged miR-455-3p to elevate the PPM1F expression, resulting in accelerating malignant behaviors of HCC cells. The study provided a potential therapeutic target for patients with HCC.

Classical swine fever virus infection suppresses claudin-1 expression to facilitate its replication in PK-15 cells.

Classical swine fever (CSF) is one of the most epidemic viral diseases in swine industry. The causative pathogen is CSF virus (CSFV), a small enveloped RNA virus of Flaviviridae family. Claudin-1 was reported to be involved in the infections of a number of viruses, including many from Flaviviridae family, but no studies have investigated the role of porcine claudin-1 during CSFV infection in PK-15 cells. In this study, on the one hand, we demonstrated that CSFV infection reduced the claudin-1 expression at both mRNA and protein levels; on the other hand, CSFV infection was enhanced after claudin-1 knockdown, but inhibited by claudin-1 overexpression in a dose-dependent manner. Furthermore, negative correlation was demonstrated between the claudin-1 expression and CSFV titer. In conclusion, claudin-1 might be a barrier for CSFV infection in PK-15 cells, while CSFV bypasses the barrier through lysosome mediated degradation of claudin-1, which could be repressed by bafilomycin A1. Although the elaborate mechanisms how claudin-1 plays its roles in CSFV infection require further investigations, this study may advance our understanding of the molecular host-pathogen interaction mechanisms underlying CSFV infection and suggests enhancement of porcine claudin-1 as a potential preventive or therapeutic strategy for CSF control.

Normal reference intervals of prognostic nutritional index in healthy adults: A large multi-center observational study from Western China.

BACKGROUND: It has been widely reported that the prognostic nutritional index (PNI) played a pivotal role in nutritional assessment of surgical patients and tumor prognosis. In order to improve the accuracy of evaluation in Western China, we established reference intervals (RIs) of PNI in healthy controls. METHODS: A retrospective cohort study on healthy ethnic Han adults (18-79 years) was conducted to explore the influences of age, gender, study centers, and instruments on PNI and to establish RIs. The data came from a healthy routine examination center database and laboratory information system (LIS) of four centers in Western China, and there were 200 persons selected randomly for verification of RIs. RESULTS: Five thousand eight hundred and thirty-nine healthy candidates were enrolled. PNI showed a marked gender dependence, and males had significantly higher PNI than females across all ages (p < 0.01). We found that PNI is significantly different between age groups (p < 0.01), the value of PNI tended to decrease with age increasing. There is also an obvious influence of centers and instruments on PNI (p < 0.01). CONCLUSIONS: We established reference intervals of PNI in healthy Han Chinese population in Western China and validated successfully. Further established RIs will lead to better standardizations of PNI for clinical applications.

Integration of platelet features in blood and platelet rich plasma for detection of lung cancer.

OBJECTIVES: To determine whether the integration platelet features in blood and platelet rich plasma can establish a model to diagnose lung cancer and colon cancer, even differentiate lung malignancy from lung benign diseases. METHODS: 245 individuals including 159 lung cancer and 86 normal participants were divided into the training cohort and testing cohort randomly. Then, 32 colon cancers, 37 lung cancers, and 21 benign patients were enrolled into validate cohort. The whole blood and corresponding platelet rich plasma (PRP) samples from all participants were prospectively collected, and the platelet features were determined. The features which are statistically significant at the univariate analysis in the training cohort and reported significant features were entered the diagnostic model. A receiver operator characteristic (ROC) curve was drawn to evaluate the accuracy of the model in each cohort. RESULTS: In the training cohort, multiple platelet features were significantly different in lung cancer patients, including MPV in whole blood, MPV, and platelet count in PRP and platelet recovery rate (PRR). For the training cohort, the diagnostic model for lung cancer performed well (AUC = 0.92). The probability distribution of lung cancers and controls in testing cohort were also separated well by the diagnostic model (AUC = 0.79). The diagnostic model for colon cancer also performed well (AUC = 0.79). The model also has a potential value in differentiating the lung malignancy from the benign (AUC = 0.69). CONCLUSION: The PRR was first raised and used in the detection of lung cancer. This study identified a diagnostic model based on PRR and other platelet features in whole blood and PRP samples with the potential to distinguish patients with lung cancer or colon cancer from healthy controls. The model could also be used to distinguish between lung cancer from the benign disease.

Integrin ß3, a RACK1 interacting protein, is critical for porcine reproductive and respiratory syndrome virus infection and NF-κB activation in Marc-145 cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the pathogen of porcine reproductive and respiratory syndrome (PRRS), which is one of the most economically harmful diseases in modern pig production worldwide. Receptor of activated protein C kinase 1 (RACK1) was previously shown to be indispensable for the PRRSV replication and NF-κB activation in Marc-145 cells. Here we identified a membrane protein, integrin ß3 (ITGB3), as a RACK1-interacting protein. PRRSV infection in Marc-145 cells upregulated the ITGB3 expression. Abrogation of ITGB3 by siRNA knockdown or antibody blocking inhibited PRRSV infection and NF-κB activation, while on the other hand, overexpression of ITGB3 enhanced PRRSV infection and NF-κB activation. Furthermore, inhibition of ITGB3 alleviated the cytopathic effects and reduced the TCID50 titer in Marc-145 cells. We also showed that RACK1 and ITGB3 were NF-κB target genes during PRRSV infection, and that they regulated each other. Our data indicated that ITGB3, presumably as a co-receptor, played an imperative role during PRRSV infection and NF-κB activation in Marc-145 cells. PRRSV infection activates a positive feedback loop involving the activation of NF-κB and upregulation of ITGB3 and RACK1 in Marc-145 cells. The findings would advance our elaborated understanding of the molecular host-pathogen interaction mechanisms underlying PRRSV infection in swine and suggest ITGB3 and NF-κB signaling pathway as potential therapeutic targets for PRRS control.

Overexpression of RACK1 enhanced the replication of porcine reproductive and respiratory syndrome virus in Marc-145 cells and promoted the NF-κB activation via upregulating the expression and phosphorylation of TRAF2.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative pathogen for porcine reproductive and respiratory syndrome (PRRS), which lead to huge loss to porcine industry. RACK1 (receptor of activated protein C kinase 1) was first identified as a receptor for protein kinase C. Mounting evidence demonstrated that RACK1 played diverse roles in NF-κB activation and virus infections. We previously reported that siRNA knockdown of RACK1 inhibited PRRSV replication in Marc-145 cells, abrogated NF-κB activation induced by PRRSV infection and reduced the viral titer. Here we established a Marc-145 cell line which could stably overexpress RACK1 to consolidate our findings. Based on the data from RT-qPCR, western blot, immunofluorescence staining, cytopathic effects and viral titer analysis, we concluded that overexpression of RACK1 could enhance the replication of PRRSV in Marc-145 cells and promote the NF-κB activation via upregulating TRAF2 expression and its phosphorylation. Marc-145 cells overexpressing RACK1exhibited severe cytopathic effects post infection with PRRSV and elevated the viral titer. Taken together, RACK1 plays an essential role for PRRSV replication in Marc-145 cells and NF-κB activation. The results presented here shed more light on the understanding of the molecular mechanisms underlying PRRSV infection and its subsequent NF-κB activation. Therefore, we anticipate RACK1 as a promising target for PRRS control.

Epidemiology and phylogeny of spike gene of porcine epidemic diarrhea virus from Yunnan, China.

Porcine epidemic diarrhea (PED) causes acute enteric disease and yellowish watery diarrhea, making piglets fast dehydration to death. PED threatens pig industry and leads to substantial economic losses. After the first reports, PED in Yunnan province, China was again identified in 2013 during an epidemiological survey, with follow-up data showing an overall positive rate of 17.47% during 2013-2017, lower than that in other provinces in China. The complete S gene of porcine epidemic diarrhea virus (PEDV) is 4149-4158 bp long. Phylogenetic analysis of S gene was performed using 9 new isolates from Yunnan province, China, together with 225 full-length S genes available in GenBank. The nine Yunnan isolates were clustered into classical G1b and pandemic G2a groups, indicating new variants have been emerging in Yunnan province. When taking the previously submitted 3 isolates from China into consideration, all the 12 isolates were clustered into 4 groups, i.e., G1a, G1b, G2a and G2b, suggesting that a highly diverse and complex clustering might result from co-infections in more than 13 provinces in China, as well in South Korea, Japan, Vietnam, Thai and USA. Identification of new types of PEDV strains would stimulate the development of effective vaccines for the prevention and control of PED in a more precise manner.

In vitro inhibition of porcine reproductive and respiratory syndrome virus replication by short antisense oligonucleotides with locked nucleic acid modification.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS), which is currently insufficiently controlled. From a previous small-scale screen we identified an effective DNA-based short antisense oligonucleotide (AS-ON) targeting viral NSP9, which could inhibit PRRSV replication in both Marc-145 cells and pulmonary alveolar macrophages (PAMs). The objective of this study was to explore the strategy of incorporating locked nucleic acids (LNAs) to achieve better inhibition of PRRSV replication in vitro. METHODS: The effective DNA-based AS-ON (YN8) was modified with LNAs at both ends as gap-mer (LNA-YN8-A) or as mix-mer (LNA-YN8-B). Marc-145 cells or PAMs were infected with PRRSV and subsequently transfected. RESULTS: Compared with the DNA-based YN8 control, the two AS-ONs modified with LNAs were found to be significantly more effective in decreasing the cytopathic effect (CPE) induced by PRRSV and thus in maintaining cell viability. LNA modifications conferred longer lifetimes to the AS-ON in the cell culture model. Viral ORF7 levels were more significantly reduced at both RNA and protein levels as shown by quantitative PCR, western blot and indirect immunofluorescence staining. Moreover, transfection with LNA modified AS-ON reduced the PRRSV titer by 10-fold compared with the YN8 control. CONCLUSION: Taken together, incorporation of LNA into AS-ON technology holds higher therapeutic promise for PRRS control.

RACK1 is indispensable for porcine reproductive and respiratory syndrome virus replication and NF-κB activation in Marc-145 cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS), which is currently insufficiently controlled. RACK1 (receptor of activated protein C kinase 1) was first identified as a receptor for protein kinase C, with increasing evidence showing that the functionally conserved RACK1 plays important roles in cancer development, NF-κB activation and various virus infections. However, the roles of RACK1 during PRRSV infection in Marc-145 cells have not been described yet. Here we demonstrated that infection of Marc-145 cells with the highly pathogenic PRRSV strain YN-1 from our lab led to activation of NF-κB and upregulation of RACK1 expression. The siRNA knockdown of RACK1 inhibited PRRSV replication in Marc-145 cells, abrogated NF-κB activation induced by PRRSV infection and reduced the viral titer. Furthermore, knockdown of RACK1 could inhibit an ongoing PRRSV infection. We found that RACK1 is highly conserved across different species based on the phylogenetic analysis of mRNA and deduced amino acid sequences. Taken together, RACK1 plays an indispensable role for PRRSV replication in Marc-145 cells and NF-κB activation. The results would advance our further understanding of the molecular mechanisms underlying PRRSV infection in swine and indicate RACK1 as a promising potential therapeutic target.

Inhibition of porcine reproductive and respiratory syndrome virus replication in vitro using DNA-based short antisense oligonucleotides.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is caused by porcine reproductive and respiratory syndrome virus (PRRSV) and is an economically important disease in swine-producing areas. The objective of this study was to screen for effective antisense oligonucleotides (AS-ONs) which could inhibit PRRSV replication in MARC-145 cells and in pulmonary alveolar macrophages (PAM). RESULTS: Nine short AS-ON sequences against the well-conserved regions of PRRSV (5'-UTR, NSP9, ORF5 and ORF7) were selected. When MARC-145 cells or PAM were infected with PRRSV followed by transfection with AS-ONs, four AS-ON sequences targeting 5'-UTR, ORF5 or NSP9 were found to be the most effective oligonucleotides in decreasing the cytopathic effect (CPE) induced by PRRSV infection. Quantitative PCR and indirect immunofluorescence staining confirmed that ORF7 levels were significantly reduced both at RNA and protein levels. The PRRSV titration data furthermore indicated that transfection with AS-ON YN8 could reduce the PRRSV titer by 1000-fold compared with controls. CONCLUSION: The results presented here indicate that DNA-based antisense oligonucleotides can effectively inhibit PRRSV replication in MARC-145 cells and in PAM. Furthermore, comparing with the reported hit rates (approximately 10-30 %), we achieved a higher success rate (44 %). The strategy we took to design the antisense sequences might be applied to select AS-ONs that more efficiently reduce the expression of target genes.

Genetic diversity of the ORF5 gene of porcine reproductive and respiratory syndrome virus isolates in southwest China from 2007 to 2009.

To gain insight into the molecular epidemiology and possible mechanisms of genetic variation of porcine reproductive and respiratory syndrome (PRRS) in Yunnan Province of China, the ORF5 gene of 32 PRRSV isolates from clinical samples collected from 2007 to 2009 were sequenced and analyzed. Nucleotide and amino acid analyses were carried out on 32 isolates and representative strains of the North American genotype, European genotype and two representative Chinese isolates. Results revealed that these isolates share 86.9-99.0% nucleotide and 87.5-98.0% amino acid identity with VR-2332 the prototypical North American PRRSV, 61.7-62.9% and 54.3-57.8% with Lelystad virus (LV) the representative strain of European genotype, 91.2-95.4% and 90.0-94.5% with CH-1a that was isolated in mainland China in 1996, 88.1-99.3% and 85.5-99.0% with JX-A1 the representative strain of High pathogenic PRRSV in China, and 86.2-99.8% and 85.5-100.0% between isolated strains of different years, respectively. Phylogenetic analysis revealed that all 32 PRRSV isolates belonged to the North American genotype and were further divided into two different subgenotypes. Subgenotype 1 comprised twenty two Yunnan isolates which divided into two branches. Subgenotype 2 comprised ten isolates which closely related to the RespPRRS vaccine and its parent strain VR-2332. The functional domains of GP5 such as the signal peptide, ectodomain, transmembrane regions and endodomain were identified and some motifs in GP5 with known functions, such as primary neutralizing epitope (PNE) and decoy epitope were also further analyzed. Our study shown the great genetic diversity of PRRSV in southwest China, rendering the guide for control and prevention of this disease.

Seroprevalence and molecular detection of hepatitis E virus in Yunnan Province, China.

In this study, the prevalence and characteristics of hepatitis E virus (HEV) in pigs and the general population in the Yunnan province, China, were evaluated. Nine hundred sixty sera, 95 liver and 60 feces samples were randomly collected from pig farms and abattoirs, in addition 173 human sera were sampled in the provincial capital city for a serological survey and an RT-nPCR assay. The screening results showed that among 621 samples collected from five pig farms, the HEV-specific IgG positive rate ranged from 73.2% to 83.5%, and the overall seroprevalence was 78.9% (490/621). A further analysis revealed that the seroprevalence increased with age. The positive rate of human serum samples was 39.9% (69/173). HEV RNA was detected in five swine feces, six swine liver and one anti-HEV-IgM-positive human serum sample by RT-nPCR. Sequence and alignment of the 348-nt PCR-amplified products of 12 HEV strains identified nine distinct nucleotide sequences. Phylogenetic and molecular evolutionary analysis revealed that these nine sequences shared 84.2% to 100.0% nucleotide sequence identity with each other, with all isolates belonging to genotype 4 HEV and clustering with other Chinese swine and human HEV sequences determined earlier. This study results suggest that the prevalence of genotype 4 HEV is serious, both in pig herds and in the human population, and authorities should pay more attention to the prevalence of HEV in southwest China.