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.

Long non-coding RNA LOC103222771 promotes infection of porcine reproductive and respiratory syndrome virus in Marc-145 cells by downregulating Claudin-4.

Porcine reproductive and respiratory syndrome (PRRS) is an important swine disease caused by infection of porcine reproductive and respiratory syndrome virus (PRRSV), which leads to huge loss in swine industry. How to effectively control PRRS is challenging. Long non-coding RNA (lncRNA) are key regulator of viral infections and anti-virus immunological responses, therefore, further understanding of lncRNAs will aid to identification of novel regulators of viral infections and better design of prevention and control strategies to viral infection related diseases and immune disorders. We demonstrated that PRRSV infection upregulated the expression of lncRNA LOC103222771 in Marc-145 cells and porcine alveolar macrophage cells (PAMs) and that LOC103222771 is mainly located in cytoplasm. Knockdown of LOC103222771 could inhibit the PRRSV infection in Marc-145 cells. RNA-seq analysis and subsequent validation revealed increased expression of Claudin-4 (CLDN4) in Marc-145 when LOC103222771 was specifically downregulated，suggesting that LOC103222771 might be an upstream regulator of CLDN4, an important component of tight junctions for establishment of the paracellular barrier that controls the flow of molecules in the intercellular space between epithelial cells. We and others showed that Downregulation of CLDN4 could boost the infection of PRRSV. Collectively, LOC103222771/CLDN4 signal axis might be a novel mechanism of PRRSV pathogenesis, implying a potential therapeutic target against PRRSV infection.

High Genetic Diversity of Porcine Sapovirus From Diarrheic Piglets in Yunnan Province, China.

As one of the most important enteric viruses, sapovirus (SaV) can infect humans and a variety of animals. Until now, 19 SaV genogroups have been identified, among which 4 from human (GI, GII, GIV, and GV) and 8 from swine (GIII, GV-GXI). Porcine sapovirus (PoSaV) GIII has been prevalent in China; however, the status of PoSaV infection in Yunnan province remains unknown. In this study, 202 fecal samples were collected from piglets associated with outbreaks of acute diarrhea in Yunnan between January and May 2020. PoSaV detection revealed that the total PoSaV infection rate in Yunnan was 35.2%, with 21 PoSaV strains determined and phylogenetically analyzed. The phylogenetic tree analyses demonstrated that twenty PoSaV strains belonged to GIII and fell into five genotypes, whereas one PoSaV strain (YNQB) belonged to GV. Sequence alignments revealed deletions in VP2 region in 10 of the 20 GIII strains, as well as deletions and insertions in VP1 region of the GV strain (YNQB). Furthermore, genomic recombination analyses showed that two GIII strains (YNAN and YNJD) were recombinants, closely related to reference sequences MK965898 and LC215880, MK965898 and FJ387164, respectively. In summary, PoSaV-GIII strains were identified in Yunnan in 2020, and for the first time, a PoSaV-GV strain was identified from China, whereas the comprehensive analyses illustrated high genetic diversity of Yunnan PoSaV strains. This study may shed new light on the current PoSaV infections in Yunnan and pave the way toward further control of the PoSaV infections in China.

Comparative Analyses of Antibiotic Resistance Genes in Jejunum Microbiota of Pigs in Different Areas.

Antibiotic resistance genes (ARGs) are emerging environmental contaminants that threaten human and animal health. Intestinal microbiota may be an important ARGs repository, and intensive animal farming is a likely contributor to the environmental burden of ARGs. Using metagenomic sequencing, we investigated the structure, function, and drug resistance of the jejunal microbial community in Landrace (LA, Kunming), Saba (SB, Kunming), Dahe (DH, Qujing), and Diannan small-ear piglets (DS, Xishuangbanna) from different areas in Yunnan Province, China. Remarkable differences in jejunal microbial diversity among the different pig breeds, while the microbial composition of pig breeds in close areas tends to be similar. Functional analysis showed that there were abundant metabolic pathways and carbohydrate enzymes in all samples. In total, 32,487 ARGs were detected in all samples, which showed resistance to 38 categories of drugs. The abundance of ARGs in jejunum was not significantly different between LA and SB from the same area, but significantly different between DS, DH and LA or SB from different areas. Therefore, the abundance of ARGs was little affected by pig breeds and microorganism community structure, but it was closely related to geographical location. In addition, as a probiotic, Lactobacillus amylovorus is also an important ARGs producing bacterium. Our results revealed the antibiotic exposure and intestinal microbial resistance of farms in the study areas, which could provide basic knowledge and potential strategies for rational use of antibiotics and reducing the risk of ARGs transmission in animal husbandry.

Epidemiological investigation and genetic characterization of porcine astrovirus genotypes 2 and 5 in Yunnan province, China.

Astroviruses (AstVs) are among the most important viruses causing diarrhea in human infants and many animals, posing a threat to public health safety and a burden on the economy. Five porcine AstV (PAstV) genotypes have been identified in various countries, including China. However, the epidemiology of PAstV in Yunnan province, China, remains unknown. In this study, 489 fecal samples from pigs in all 16 prefectures/cities of Yunnan were collected between April and August of 2020 for epidemiological investigation. The total infection rate of PAstV-2 or PAstV-5 was 39.9%, with suckling piglets having the highest infection rate (62.3%). The ORF2 genes of seven PAstV-2 and 10 PAstV-5 isolates were sequenced and phylogenetically analyzed. In addition to coinfections with PAstV-2 and PAstV-5, coinfections of PAstV with other diarrhea-inducing viruses (e.g., porcine bocavirus) were also discovered. A comparison of ORF2-encoded capsid protein sequences revealed that there were multiple insertions and deletions in the seven Yunnan PAstV-2 sequences, while point mutations, but no deletions or insertions, were found in the 10 Yunnan PAstV-5 sequences, which were very similar to the reference sequences. This is the first epidemiological investigation and genetic characterization of PAstV-2 and PAstV-5 in Yunnan province, China, demonstrating the current PAstV infection situation in Yunnan.

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.

Small molecule screening identified cepharanthine as an inhibitor of porcine reproductive and respiratory syndrome virus infection in vitro by suppressing integrins/ILK/RACK1/PKCα/NF-κB signalling axis.

Porcine Reproductive and Respiratory Syndrome (PRRS) is a devastating disease among the most notorious threats to the swine industry worldwide and is characterized by respiratory distress and reproductive failure. Highly evolving porcine reproductive and respiratory syndrome virus (PRRSV) strains with complicated genetic diversity make the current vaccination strategy far from cost-effective and thus urge identification of potent lead candidates to provide prevention and treatment approaches. From an in vitro small molecule screening with the TargetMol Natural Compound Library comprising 623 small molecules, cytopathic effect (CPE) observations and RT-qPCR analysis of viral ORF7 gene expression identified cepharanthine (CEP) to be one of the most protent inhibitors of PRRSV infection in Marc-145 cells. When compared with tilmicosin, which is one of the most commonly used antibiotics in swine industry to inhibit infections, CEP more prominently inhibited PRRSV infection represented by both RNA and protein levels, further reduced the TCID50 by 5.6 times, and thus more remarkably protected Marc-145 cells against PRRSV infection. Mechanistically, western blot analyses of the Marc-145 cells and the porcine alveolar macrophages (PAMs) with or without CEP treatment and PRRSV infection at various time points revealed that CEP can inhibit the expression of integrins ß1 and ß3, integrin-linked kinase (ILK), RACK1 and PKCα, leading to NF-κB suppression and consequent alleviation of PRRSV infection. Collectively, our small molecule screening identified cepharanthine as an inhibitor of PRRSV infection in vitro by suppressing Integrins/ILK/RACK1/PKCα/NF-κB signalling axis, which may enlighten the deeper understanding of the molecular pathogenesis of PRRSV infection and more importantly, suggested CEP as a potential promising drug for PRRS control in veterinary clinics.

Inhibition of porcine reproductive and respiratory syndrome virus by PKC inhibitor dequalinium chloride in vitro.

As a severe disease characterized by reproductive failure and respiratory distress, porcine reproductive and respiratory syndrome (PRRS) is one of the most leading threats to the swine industry worldwide. Highly evolving porcine reproductive and respiratory syndrome virus (PRRSV) strains with distinct genetic diversity make the current vaccination strategy much less cost-effective and thus urge alternative protective host directed therapeutic approaches. RACK1-PKC-NF-κB signalling axis was suggested as a potential therapeutic target for PRRS control, therefore we tested the inhibitory effect of PKC inhibitor dequalinium chloride (DECA) on the PRRSV infection in vitro. RT-qPCR, western blot, Co-IP and cytopathic effect (CPE) observations revealed that DECA suppressed PRRSV infection and protected Marc-145 cells and porcine alveolar macrophages (PAMs) from severe cytopathic effects, by repressing the PKCα expression, the interaction between RACK1 and PKCα, and subsequently the NF-κB activation. In conclusion, the data presented in this study shed more light on deeper understanding of the molecular pathogenesis upon PRRSV infection and more importantly suggested DECA as a potential promising drug candidate for PRRS control.

Porcine RACK1 negatively regulates the infection of classical swine fever virus and the NF-κB activation in PK-15 cells.

Classical swine fever (CSF) is one of the main viral diseases of swine worldwide. The causative pathogen is CSF virus (CSFV), a small enveloped RNA virus of the genus Pestivirus. Activation of NF-κB is a hallmark of most viral infections and the viral pathogens frequently kidnap NF-κB pathway for their own advantages, however, it is unclear or even controversial about whether CSFV infection can activate NF-κB signal pathway. RACK1 was shown as an interacting host protein with CSFV NS5A protein, but no studies so far have clearly defined the role of RACK1 during CSFV infection and NF-κB activation. In this study, to properly address these open questions, using RT-qPCR, western blot, indirect fluorescence staining, siRNA knockdown and protein overexpression techniques, we demonstrated that CSFV infection reduced the RACK1 expression at both mRNA and protein levels in PK-15 cells. Downregulation of cellular RACK1 enhanced CSFV infection and subsequent NF-κB activation, while RACK1 overexpression inhibited CSFV infection and the NF-κB activation. In conclusion, RACK1 is a negative cellular regulator for CSFV infection and NF-κB activation in PK-15 cells. Our work addressed a novel aspect concerning the regulation of innate antiviral immune response during CSFV infection. This study may provide some insights into the molecular mechanisms of CSFV infection in swine. However, the elaborate mechanism by which CSFV regulates NF-κB activation and how RACK1 plays its roles in CSFV infection and NF-κB induction require further in-depth studies.

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.

The detection of canine circovirus in Guangxi, China.

Since the first description of canine circovirus (CanineCV)-associated infection, there have been several reports on the distribution of the disease in worldwide. To investigate the prevalence and genetic diversity of CanineCV in China, we conducted PCR screening of 1226 dog serum samples collected from different regions in mainland China between 2014 and 2016. CanineCV DNA was found in 81/926 serum samples from Guangxi Province. Furthermore, 25 full-length genomes of CanineCV from positive samples were sequenced and compared with CanineCV sequences in the GenBank database. Pairwise analysis showed that the determined genome sequences shared 84.9%-100% identity among themselves and 81.4%-90.5% with the other 28 sequences. Phylogenetic analysis revealed that the 52 viral genome sequences could be divided into two genotypes (CanineCV-1 and CanineCV-2). Analysis of the amino acid sequences of the capsid protein revealed the existence of 9 major regions of variation. The present work contributes to the understanding of CanineCV molecular epidemiology.

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.

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.

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.

Molecular epidemiology of pseudorabies virus in Yunnan and the sequence analysis of its gD gene.

Outbreaks of pseudorabies (PRs) have occurred in Yunnan, China, which caused significant economic loss. To determine the prevalence and origin of PR in Yunnan, especially among vaccinated pigs, overall 791 samples of blood, tissue, semen, and sera were analyzed by serological methods, PCR, and sequence analysis of gD gene. Detection with viral gI antibody or PCR showed that the yearly positive rates of PR virus (PRV) in Yunnan from 2010 to 2014 were 48.15, 21.26, 2.17, 5.22, and 0.35%, respectively, with an average of 15.43%. In general, the incidence declined through the period of 2010-2014 probably due to the application of PRV eradication strategies. A phylogenetic tree was constructed based on the complete sequence of gD gene, with all strains clustered into two independent clades, i.e., Asian and European-American clades. The virus isolates from Henan, Tianjin, Heilongjiang, Sichuan, Shandong, Fujian, Xinjiang, Hubei, Guangdong, and Yunnan fell into Asian group, which harbored South Korea isolate. Four Yunnan virus isolates together with South Korean Namyangju fell into in the European-American clade. It showed that PR was pandemic as there was not a clear clue about the geographical origin of the PRV isolates in China since 2010.

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 heterogeneity of swine hepatitis E virus isolates from Yunnan province, China in 2011-2012.

BACKGROUND: Hepatitis E is a disease of major public-health concern mainly in developing countries. Although molecular and sero-epidemiological investigations of HEV have been performed in many provinces in China, the epidemiological data from Yunnan Province are limited and genotypes are not be fully characterized. In this study the prevalence and characteristics of hepatitis E virus (HEV) detected in pigs from Yunnan province, China was evaluated. RESULTS: A total of 13 out of 187 pig fecal samples collected in 2011 revealed HEV positive results; likewise, 7 out of 69 samples collected in 2012 exhibited positive results. These findings indicated a total prevalence of 7.8% (20/256). Phylogenetic and molecular evolutionary analysis results revealed that nine strains were found in the samples obtained in 2011, in which 87.1% to 99.4% nucleotide sequence identity was shared among these strains; and 77.0% to 81.9%, 52.2% to 53.6%, 77.0% to 88.2% and 77.9% to 96.8% nucleotide sequence identities were shared with strains representing genotypes 1, 2, 3, and 4. Five strains were detected in the samples obtained in 2012, in which 94.2% to 99.3% nucleotide sequence identity was shared among the strains, and 81.0% to 82.5%, 81.8% to 83.2%, 81.0% to 92.7% and 81.0% to 97.8% nucleotide sequence identities were shared with strains representing the genotypes 1, 2, 3, and 4. CONCLUSIONS: Analysis of fourteen detected HEV strains revealed that three of them were subtype 4d, two were subtype 4b; the nine remaining isolated strains were subtype 4 h. These results indicated that the prevalence of HEV in the swine herds of Yunnan was quite high, additional public-health concerns should focus on pork safety.

Chemical constituents of Arisaema franchetianum tubers.

A novel pyrrolidine alkaloid, (2R*,3S*,5S*)-N,2-dimethyl-3-hydroxy-5-(10-phenyldecyl)pyrrolidine (1), and 17 known compounds were isolated from Arisaema franchetianum Engl. (Araceae) tubers. The 17 compounds were bergenin (2), emodin (3), caffeic acid (4), nobiletin (5), 3-O-ß-d-galactopyranosyl-hederagenin 28-O-ß-d-xylopyranosyl(1 â†’ 6)-ß-d-galactopyranosyl ester (6), coniferin (7), qingyangshengenin (8), methylconiferin (9), syringaresinol 4'-O-ß-d-glucopyranoside (10), gagaminine (11), perlolyrine (12), (S)-1-(1'-hydroxyethyl)-ß-carboline (13), 1-(ß-carboline-1-yl)-3,4,5-trihydroxy-1-pentanone (14), 1-methoxycarbonyl-ß-carboline (15), indolo[2,3-α]carbazole (16), 4-hydroxycinnamic acid methyl ester (17), and methyl 4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-(hydroxymethyl)ethyl] ferulate (18). The inhibitory activities of compound 1 and its N-methyl derivative (1a) against porcine respiratory and reproductive syndrome virus (PRRSV), human leukemic K562 cells, and human breast cancer MCF-7 cells were evaluated. Compounds 1 [50% inhibited concentration (IC(50)) = 12.5 ± 0.6 µM] and 1a (IC(50) = 15.7 ± 0.9 µM) were cytotoxic against K562 cells. Compound 1a also had a weak effect on PRRSV with an IC(50) value of 31.9 ± 6.0 µM [selectivity index (SI) = 18.7].

Identification of porcine reproductive and respiratory syndrome virus inhibitors through an oriented screening on natural products.

Porcine reproductive and respiratory syndrome(PRRS) caused by porcine reproductive and respiratory syndrome virus(PRRSV) is one of the most infectious diseases in the swine industry worldwide, causing big economic losses. Vaccines are major weapons against PRRSV, however, current available vaccines have several limitations. Developing chemical drugs as alternatives is required. On the basis of traditional medical knowledge, we purposely selected 15 natural products originated from Chinese herbs with anti-infectious effects. Their antiviral activities were evaluated by PRRSV-induced cytopathic effect(CPE) on MARC-145 cells and reverse transcription polymerase chain reaction(RT-PCR) assay. Compounds ethoxysanguinarine(EOSG) and atractylodinol were found to be the hits which could significantly reduce PRRSV-associated CPE with 50% inhibited concentration(IC50) values of 7.9 and 39.4 µmol/L, respectively. Meanwhile, compounds ethoxysanguinarine and atractylodinol significantly decreased mRNA expression of ORF7 gene in a dose-dependent manner. Study results suggest that compounds ethoxysanguinarine and atractylodinol may be useful anti-PRRSV drugs for swine industry or the hits for further lead optimization.