Understanding the textural enhancement of low-salt myofibrillar protein gels filled with pea protein pre-emulsions through interfacial behavior: Effects of structural modification and oil phase polarity.

This study investigated the effects of pea protein pre-emulsions containing triglyceride- or diglyceride-oil on the emulsifying and gelling properties of low-salt myofibrillar protein (MP). Pea protein isolates treated with pH12-shifting (PPIpH) or ultrasonication (PPIU) demonstrated superior initial interfacial adsorption and higher final interfacial pressure than native pea protein. Within MP/PPI blends, an increased ratio of MP led to a decrease in interfacial pressure, while simultaneously enhancing film elasticity at both polar and non-polar interfaces. Polar diglyceride promoted protein adsorption and fostered interfacial interactions between modified pea proteins and MP, enhancing the cross-linking of transglutaminase (TG) in the composite emulsion gels. Combining diglyceride-type PPIU and PPIpH emulsions with TG increased gel strength to 0.58 N and 0.63 N, respectively, from an initial 0.33 N, yielding a denser protein network with uniformly dispersed oil droplets. Therefore, the utilization of diglyceride and modified PPI can serve as structural enhancers in comminuted meat products.

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.

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.

Molecular Detection and Genetic Characterization of Japanese Encephalitis Virus in Animals from 11 Provinces in China.

Japanese encephalitis virus (JEV), which uses a mosquito primary vector and swine as a reservoir host, poses a significant risk to human and animal health. JEV can be detected in cattle, goats and dogs. A molecular epidemiological survey of JEV was conducted in 3105 mammals from five species, swine, fox, racoon dog, yak and goat, and 17,300 mosquitoes from 11 Chinese provinces. JEV was detected in pigs from Heilongjiang (12/328, 3.66%), Jilin (17/642, 2.65%), Shandong (14/832, 1.68%), Guangxi (8/278, 2.88%) and Inner Mongolia (9/952, 0.94%); in goats (1/51, 1.96%) from Tibet; and mosquitoes (6/131, 4.58%) from Yunnan. A total of 13 JEV envelope (E) gene sequences were amplified in pigs from Heilongjiang (5/13), Jilin (2/13) and Guangxi (6/13). Swine had the highest JEV infection rate of any animal species, and the highest infection rates were found in Heilongjiang. Phylogenetic analysis indicated that the predominant strain in Northern China was genotype I. Mutations were found at residues 76, 95, 123, 138, 244, 474 and 475 of E protein but all sequences had predicted glycosylation sites at 'N154. Three strains lacked the threonine 76 phosphorylation site from non-specific (unsp) and protein kinase G (PKG) site predictions; one lacked the threonine 186 phosphorylation site from protein kinase II (CKII) prediction; and one lacked the tyrosine 90 phosphorylation site from epidermal growth factor receptor (EGFR) prediction. The aim of the current study was to contribute to JEV prevention and control through the characterization of its molecular epidemiology and prediction of functional changes due to E-protein mutations.

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.

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.

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.

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.

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.

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.

Bioactive isoquinoline alkaloids from Corydalis saxicola.

Twelve isoquinoline alkaloids including two new nitro-containing tetrahydroprotoberberines, (-)-2,9-dihydroxyl-3,11-dimethoxy-1,10-dinitrotetrahydroprotoberberine (1) and (+)-4-nitroisoapocavidine (2), were isolated from the whole plant of Corydalis saxicola Bunting. The structures of the new compounds were established by spectroscopic analysis and chemical evidence. The inhibitory activity of these isolates against cholinesterase and canine parvovirus were evaluated. Compounds 1 and 1A, (+)-1-nitroapocavidine (5), berberine (8), palmatine (9), dehydrocavidine (10), and sanguinarine (11) showed potent inhibitory activity against acetylcholinesterase with IC(50) values of less than 10 µM, while only compound 1 possessed weak activity against canine parvovirus. Structure-activity studies demonstrated that the nitro substituents at ring A in the tetrahydroprotoberberines led to an increase in the anti-acetylcholinesterase activity.

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.