Seneca Valley Virus Enters PK-15 Cells via Caveolae-Mediated Endocytosis and Macropinocytosis Dependent on Low-pH, Dynamin, Rab5, and Rab7.

Seneca Valley virus (SVV), a new pathogen resulting in porcine vesicular disease, is prevalent in pig herds worldwide. Although an understanding of SVV biology pathogenesis is crucial for preventing and controlling this disease, the molecular mechanisms for the entry and post-internalization of SVV, which represent crucial steps in viral infection, are not well characterized. In this study, specific inhibitors, Western blotting, and immunofluorescence detection revealed that SVV entry into PK-15 cells depends on low-pH conditions and dynamin. Furthermore, results showed that caveolae-mediated endocytosis (CavME) contributes crucially to the internalization of SVV, as evidenced by cholesterol depletion, downregulation of caveolin-1 expression by small interfering RNA knockdown, and overexpression of a caveolin-1 dominant negative (caveolin-1-DN) in SVV-infected PK-15 cells. However, SVV entry into PK-15 cells did not depend on clathrin-mediated endocytosis (CME). Furthermore, treatment with specific inhibitors demonstrated that SVV entry into PK-15 cells via macropinocytosis depended on the Na+/H+ exchanger (NHE), p21-activated kinase 1 (Pak1), and actin rearrangement, but not phosphatidylinositol 3-kinase (PI3K). Electron microscopy showed that SVV particles or proteins were localized in CavME and macropinocytosis. Finally, knockdown of GTPase Rab5 and Rab7 by siRNA significantly inhibited SVV replication, as determined by measuring viral genome copy numbers, viral protein expression, and viral titers. In this study, our results demonstrated that SVV utilizes caveolae-mediated endocytosis and macropinocytosis to enter PK-15 cells, dependent on low pH, dynamin, Rab5, and Rab7. IMPORTANCE Entry of virus into cells represents the initiation of a successful infection. As an emerging pathogen of porcine vesicular disease, clarification of the process of SVV entry into cells enables us to better understand the viral life cycle and pathogenesis. In this study, patterns of SVV internalization and key factors required were explored. We demonstrated for the first time that SVV entry into PK-15 cells via caveolae-mediated endocytosis and macropinocytosis requires Rab5 and Rab7 and is independent of clathrin-mediated endocytosis, and that low-pH conditions and dynamin are involved in the process of SVV internalization. This information increases our understanding of the patterns in which all members of the family Picornaviridae enter host cells, and provides new insights for preventing and controlling SVV infection.

Involvement of adaptor proteins in clathrin-mediated endocytosis of virus entry.

The first step in the initiation of effective viral infection is breaking through the cytomembrane to enter the cell. Clathrin-mediated endocytosis is a key vesicular trafficking process in which a variety of cargo molecules are transported from the outside to the inside of the cell. This process is hijacked by numerous families of enveloped or non-enveloped viruses, which use it to enter host cells, followed by trafficking to their replicating sites. Various adaptor proteins that assist in cargo selection, coat assembly, and clathrin-coated bud maturation are important in this process. Research data documented on the involvement of adaptor proteins, such as AP-2, Eps-15, Epsin1, and AP180/CALM, in the invasion of viruses via the clathrin-mediated endocytosis have provided novel insights into understanding the viral life cycle and have led to the development of novel therapeutics. Here, we summarize the latest discoveries on the role of these adaptor proteins in clathrin-mediated endocytosis of virus entry and also discuss the future trends in this field.

Equipment-free recombinase polymerase amplification assay using body heat for visual and rapid point-of-need detection of canine parvovirus 2.

A visible and equipment-free recombinase polymerase amplification assay combined with a lateral flow strip (LFS RPA) was developed to detect canine parvovirus type 2 (CPV-2), which is the etiological agent of canine parvovirus disease. The CPV-2 LFS RPA assay was developed based on the VP2 gene and is performed in a closed fist using body heat for 15 min; the products are visible to the naked eye on the LFS within 5 min. The assay could detect CPV-2a, CPV-2b and CPV-2c, and there was no cross-reaction with the other viruses tested. Using the standard CPV-2 DNA as a template, the analytical sensitivity was 1.0 × 102 copies per reaction, which was the same result as that of a real-time PCR. The assay performance was further evaluated by testing 60 canine fecal samples, and CPV-2 DNA was detected in 46 samples (76.7%, 46/60) by LFS RPA, which was the same result as that of the real-time PCR assay and higher than that of the SNAP method (48.3%, 29/60). The novel CPV-2 LFS RPA assay is an attractive and promising tool for rapid and convenient diagnosis of CPV disease, especially cage side and in underequipped laboratories.

Visual and equipment-free reverse transcription recombinase polymerase amplification method for rapid detection of foot-and-mouth disease virus.

BACKGROUND: Foot-and-mouth disease (FMD), which is caused by foot-and-mouth disease virus (FMDV), is a highly contagious tansboundary disease of cloven-hoofed animals and causes devastating economic damages. Accurate, rapid and simple detection of FMDV is critical to containing an FMD outbreak. Recombinase polymerase amplification (RPA) has been explored for detection of diverse pathogens because of its accuracy, rapidness and simplicity. A visible and equipment-free reverse-transcription recombinase polymerase amplification assay combined with lateral flow strip (LFS RT-RPA) was developed to detect the FMDV using primers and LF probe specific for the 3D gene. RESULTS: The FMDV LFS RT-RPA assay was performed successfully in a closed fist using body heat for 15 min, and the products were visible on the LFS inspected by the naked eyes within 2 min. The assay could detect FMDV serotypes O, A and Asia1, and there were no cross-reactions with vesicular stomatitis virus (VSV), encephalomyocarditis virus (EMCV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 (PCV2) and pseudorabies virus (PRV). The analytical sensitivity was 1.0 × 102 copies in vitro transcribed FMDV RNA per reaction, which was the same as a real-time RT-PCR. For the 55 samples, FMDV RNA positive rate was 45.5% (25/55) by LFS RT-RPA and 52.7% (29/55) by real-time RT-PCR. For the LFS RT-RPA assay, the positive and negative predicative values were 100% and 80%, respectively. CONCLUSIONS: The performance of the LFS RT-RPA assay was comparable to real-time RT-PCR, while the LFS RT-RPA assay was much faster and easier to be performed. The developed FMDV LFS RT-RPA assay provides an attractive and promising tool for rapid and reliable detection of FMDV in under-equipped laboratory and at point-of-need facility, which is of great significance in FMD control in low resource settings.

Antimicrobial activity and safety evaluation of peptides isolated from the hemoglobin of chickens.

BACKGROUND: Hemoglobin is a rich source of biological peptides. As a byproduct and even wastewater of poultry-slaughtering facilities, chicken blood is one of the most abundant source of hemoglobin. RESULTS: In this study, the chicken hemoglobin antimicrobial peptides (CHAP) were isolated and the antimicrobial and bactericidal activities were tested by the agarose diffusion assay, minimum inhibitory concentration (MIC) analysis, minimal bactericidal concentration (MBC) analysis, and time-dependent inhibitory and bactericidal assays. The results demonstrated that CHAP had potent and rapid antimicrobial activity against 19 bacterial strains, including 9 multidrug-resistant bacterial strains. Bacterial biofilm and NaCl permeability assays, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were further performed to detect the mechanism of its antimicrobial effect. Additionally, CHAP showed low hemolytic activity, embryo toxicity, and high stability in different temperatures and animal plasma. CONCLUSION: CHAP may have great potential for expanding production and development value in animal medication, the breeding industry and environment protection.

Fatal disease associated with Swine Hepatitis E virus and Porcine circovirus 2 co-infection in four weaned pigs in China.

BACKGROUND: In recent decades, Porcine circovirus 2 (PCV2) infection has been recognized as the causative agent of postweaning multisystemic wasting syndrome, and has become a threat to the swine industry. Hepatitis E virus (HEV) is another high prevalent pathogen in swine in many regions of the world. PCV2 and HEV are both highly prevalent in pig farms in China. CASE PRESENTATION: In this study, we characterized the HEV and PCV2 co-infection in 2-3 month-old piglets, based on pathogen identification and the pathological changes observed, in Hebei Province, China. The pathological changes were severe, and general hyperemia, hemorrhage, inflammatory cell infiltration, and necrosis were evident in the tissues of dead swine. PCR was used to identify the pathogen and we tested for eight viruses (HEV, Porcine reproductive and respiratory syndrome virus, PCV2, Classical swine fever virus, Porcine epidemic diarrhea virus, Transmissible gastroenteritis coronavirus, Porcine parvovirus and Pseudorabies virus) that are prevalent in Chinese pig farms. The livers, kidneys, spleens, and other organs of the necropsied swine were positive for HEV and/or PCV2. Immunohistochemical staining showed HEV- and PCV2-antigen-positive signals in the livers, kidneys, lungs, lymph nodes, and intestine. CONCLUSION: HEV and PCV2 co-infection in piglets was detected in four out of seven dead pigs from two pig farms in Hebei, China, producing severe pathological changes. The natural co-infection of HEV and PCV2 in pigs in China has rarely been reported. We speculate that co-infection with PCV2 and HEV may bring some negative effect on pig production and recommend that more attention should be paid to this phenomenon.

Nucleolar Phosphoprotein NPM1 Interacts With Porcine Circovirus Type 3 Cap Protein and Facilitates Viral Replication.

Porcine circovirus type 3 (PCV3) is a recently discovered virus with potentially significant implications on the global swine industry. PCV3 replication involves the entry of the viral capsid (Cap) protein with nucleolar localization signals into the nucleus. Using liquid chromatography-mass spectrometry analysis, nucleolar phosphoprotein NPM1 was identified as one of the cellular proteins bound to PCV3 Cap. Co-immunoprecipitation demonstrated that PCV3 Cap interacts directly with NPM1, where the region binding with NPM1 is mapped to amino acid residues 1-38 of Cap. Upon co-transfection, the expression of Cap protein promoted the redistribution of NPM1, which translocated from the nucleus to the cytoplasm and colocalized with Cap in cultured PK15 cells. NPM1 expression was upregulated and translocated from the nucleus to the cytoplasm in PCV3-infected cells, upon siRNA-mediated depletion, or upon treatment with NPM1 inhibitor in PK15 cells with impaired PCV3 replication, as evidenced by decreased levels of viral DNA synthesis and protein expression. By contrast, the replication of PCV3 was enhanced in stably NPM1-expressing cells via a lentivirus-delivered system. Taken together, these findings indicate that NPM1 interacts with PCV3 Cap and plays a crucial role in PCV3 replication.

Porcine Circovirus Type 3 Enters Into PK15 Cells Through Clathrin- and Dynamin-2-Mediated Endocytosis in a Rab5/Rab7 and pH-Dependent Fashion.

Porcine circovirus type 3 (PCV3) invades multiple tissues and organs of pigs of different ages and are widely spread throughout pig farms, emerging as an important viral pathogen that can potentially damage the pig industry worldwide. Since PCV3 is a newly discovered virus, many aspects of its life cycle remain unknown. Porcine kidney epithelial cells are important host targets for PCV3. Here, we used systematic approaches to dissect the molecular mechanisms underlying the cell entry and intracellular trafficking of PCV3 in PK15 cells, a cell line of porcine kidney epithelial origin. A large number of PCV3 viral particles were found to colocalize with clathrin but not caveolin-1 after entry, and PCV3 infection was significantly decreased when treated with chlorpromazine, dynasore, knockdown of clathrin heavy chain expression via RNA interference, or overexpression of a dominant-negative mutant of EPS15 in PCV3-infected cells. After internalization, the viral particles were further observed to colocalize with Rab5 and Rab7, and knockdown of both expression by RNA interference significantly inhibited PCV3 replication. We also found that PCV3 infection was impeded by ammonium chloride treatment, which indicated the requirement of an acidic environment for viral entry. Taken together, our findings demonstrate that PCV3 enters PK15 cells through a clathrin- and dynamin-2-mediated endocytic pathway, which requires early and late endosomal trafficking, as well as an acidic environment, providing an insightful theoretical basis for further understanding the PCV3 life cycle and its pathogenesis.

Hepatitis E Virus Induces Brain Injury Probably Associated With Mitochondrial Apoptosis.

Hepatitis E virus (HEV) infection has been associated with extrahepatic manifestations, particularly neurological disorders. Although it has been reported that HEV infection induced hepatocyte apoptosis associated with mitochondria injury, activation of mitochondrial apoptotic pathway in the central nervous system during HEV infection was not clearly understood. In this study, the induction of mitochondrial apoptosis-associated proteins and pro-inflammatory cytokines were detected in HEV infected Mongolian gerbil model and primary human brain microvascular endothelial cells (HBMVECs). Mitochondrial exhibited fragments with loss of cristae and matrix in HEV infected brain tissue by transmission electron microscope (TEM). In vitro studies showed that expression of NADPH oxidase 4 (NOX4) was significantly increased in HEV infected HBMVECs (p < 0.05), while ATP5A1 was significantly decreased (p < 0.01). Expressions of pro-apoptotic proteins were further evaluated. Bax was significantly increased in both HEV infected brain tissues and HBMVECs (p < 0.01). In vivo studies showed that caspase-9 and caspase-3 were activated after HEV inoculation (p < 0.01), associated with PCNA overexpression as response to apoptosis. Cytokines were measured to evaluate tissue inflammatory levels. Results showed that the release of TNFα and IL-1ß were significantly increased after HEV infection (p < 0.01), which might be attributed to microglia activation characterized by high level of IBA1 expression (p < 0.01). Taken together, these data support that HEV infection induces high levels of pro-inflammatory cytokines, associated with mitochondria-mediated apoptosis. The results provide new insight into mechanisms of extra-hepatic injury of HEV infection, especially in the central nervous system.

Brain Infection by Hepatitis E Virus Probably via Damage of the Blood-Brain Barrier Due to Alterations of Tight Junction Proteins.

Extrahepatic injury, particularly neurologic dysfunctions such as Guillain-Barré syndrome, neurologic amyotrophy, and encephalitis/meningoencephalitis/myositis were associated with HEV infection, which was supported by both clinical and laboratory studies. Thus, it is crucial to figure out how the virus invades into the central nervous system (CNS). In this study, CNS lesions were determined in rabbits and Mongolian gerbils inoculated with genotype 4 HEV. Junctional proteins were detected in HEV infected primary human brain microvascular cells (HBMVCs). Viral encephalitis associated perivascular cuffs of lymphocytes and microglial nodules were observed in HEV infected rabbits. Both positive- and negative-strand of HEV RNA was detected in brain and spinal cord in rabbits intraperitoneally infected with HEV at 28 dpi (days postinoculation), but not in rabbits gavaged with HEV. HEV ORF2 protein was further examined in both brain and spinal cord sections of infected rabbits, with positive signals located mainly in neural cells and perivascular areas. Ultrastructural study showed thickened and reduplicated basement membranes of capillary endothelium in HEV RNA positive brain tissues. In vitro study showed loss of tight junction proteins including Claudin5, Occludin, and ZO-1 (zonula occludens-1) in HBMVCs inoculated with HEV for 48 h. These findings indicated that disruption of the blood-brain barrier (BBB) might be potential mechanisms of HEV invasion into the CNS. It provides new insights to further study HEV associated neurologic disorders and will be helpful for seeking potential therapeutics for HEV infection in the future.

Hepatitis E Virus Induces Hepatocyte Apoptosis via Mitochondrial Pathway in Mongolian Gerbils.

Previous studies demonstrated that Mongolian gerbils can be infected by hepatitis E virus (HEV), which induces the hepatic injury. Here, the mitochondria in hepatocytes from HEV-infected gerbils were considerably swollen, thin cristae. After HEV infection, the activity of superoxide dismutase significantly decreased (p < 0.01), while malondialdehyde concentrations significantly increased, compared with those in the control group (p < 0.01). Adenosine triphosphatase levels decreased significantly in the hepatocyte of the inoculated groups, compared with those in control group (p < 0.05) at days 21, 28, 42 post-inoculation (dpi) as well. Furthermore, the levels of ATP synthetase ATP5A1 significantly decreased during HEV infection, compared with those in the control group (p < 0.05). According to the TdT mediated dUTP nick end labeling (TUNEL) detection, TUNEL positive hepatocytes increased in the inoculated group, compared with that in the control group (p < 0.05). Up-regulation of the mitochondrion-mediated apoptosis regulating proteins, Bax and Bcl-2, in the HEV-infected gerbils (p < 0.05) was observed. However, cytochrome c levels in mitochondria decreased, while this molecule was detected in the cytoplasm of the infected animals, in contrast to that in the control group. Apaf-1, and active caspase-9 and -3 levels were shown to be significantly higher in the inoculated group compared with those in the control group (p < 0.05). Taken together, our results demonstrated that HEV infection induces hepatocyte injuries and activity of the mitochondrial apoptotic pathway, which trigger the hepatocyte apoptosis in Mongolian gerbils.

Evaluation of an incubation instrument-free reverse transcription recombinase polymerase amplification assay for rapid and point-of-need detection of canine distemper virus.

Canine distemper, caused by Canine distemper virus (CDV), is a highly contagious and fatal systemic disease in free-living and captive carnivores worldwide. Accurate, rapid and simple detection of CDV is critical to improve disease management and prevent outbreaks. In this study, a visible and incubation instrument-free reverse-transcription recombinase polymerase amplification assay combined with lateral flow strip (LFS RT-RPA) was developed to detect CDV using primers and lateral flow (LF) probe specific for the nucleocapsid (N) protein gene. The CDV LFS RT-RPA assay was performed in a closed fist using body heat for 15 min, and the products were visible to the naked eyes on the LFS within 5 min. The assay could detect CDV, and there was no cross-reaction with the other viruses tested. Using the in vitro transcribed CDV RNA as template, the analytical sensitivity was 9.4 × 101 copies per reaction, which was the same result as that of a real-time RT-PCR. The assay performance was further evaluated by testing 32 nasal/oropharyngeal swab samples, and CDV RNA positive rate was 62.0% (20/32) by LFS RT-RPA, which was the same result as that of the real-time RT-PCR assay. The performance of the LFS RT-RPA was comparable to real-time RT-PCR, while the LFS RT-RPA assay was much faster and easier to perform. The novel CDV LFS RT-RPA assay provides an attractive and promising tool for rapid and reliable detection of CDV in the underequipped laboratory and point-of-need facility, which is of great significance in CD control in low resource settings.

A real-time loop-mediated isothermal amplification method for rapid detection of Lawsonia intracellularis in porcine fecal samples.

Porcine proliferative enteritis is a common diarrheal disease characterized by thickening of the intestinal mucosa in swine due to enterocyte proliferation, which is caused by Lawsonia intracellularis. In this study, a real-time loop-mediated isothermal amplification (LAMP) assay was developed to detect L. intracellularis based on the conserved region of the 16S ribosomal RNA gene. The optimal reaction conditions of the real-time LAMP was 65 °C for 60 min. The LAMP products could be detected by both real-time turbidity and direct visual inspection. The assay was specific for L. intracellularis, as no cross-reaction was observed with other pathogens. The detection limit of the real-time LAMP assay was 1.4 × 10-1pg of L. intracellularis DNA, which was the same as that of real-time PCR and approximately 100 times more sensitive than that of conventional PCR. Of the 136 clinical samples, L. intracellularis DNA was identified in 60 samples by real-time LAMP, which was the same as real-time PCR and higher than conventional PCR (36.8%, 50/136). The specific, sensitive and rapid real-time LAMP assay developed in this study could be a useful alternative tool in point-of-care (POC) diagnosis of L. intracellularis infection.

Increased Mast Cell Activation in Mongolian Gerbils Infected by Hepatitis E Virus.

Recently, mechanism study of hepatitis E virus (HEV) infection has attracted an increasing attention because of the growing rate of the acute hepatitis caused by the virus over the world. As an important initiate in the inflammation, mast cells (MCs) play a critical role in maintaining a healthy physiology. However, the function of the MCs in the acute hepatitis caused by HEV is still unclear. In the present study, mongolian gerbils infected by HEV were used as an animal model to evaluate the role of MCs in the HEV infection. The positive ELISA and RT-PCR results showed the gerbils was successfully infected with HEV. The number of mast cell in the liver and the small intestine in the infected animals were growing higher significantly than the control group. In addition, higher expression of the tryptase and 5-HT in the liver and the intestine detected by immunohistochemical method and western blot also indicate the activation of MCs in the infection. These results suggest that MCs play an important role in the hepatitis E.

Rapid detection of transmissible gastroenteritis virus in swine small intestine samples using real-time reverse transcription recombinase polymerase amplification.

A rapid and specific real-time reverse-transcription recombinase polymerase amplification assay (RT-RPA) was developed to detect the transmissible gastroenteritis virus (TGEV) in this study. The primers and exo probe were designed to be specific for a portion of spike (S) gene conserved in TGEV, but absent in the closely related porcine respiratory coronavirus (PRCV). The amplification was performed at 40 °C for 20 min. The assay could only detect the TGEV, and there was no cross-reaction with other pathogens tested. Using the in vitro transcribed TGEV RNA as template, the limit of detection of the developed RT-RPA was 100 copies per reaction. The assay performance was evaluated by testing 76 clinical samples by RT-RPA and a real-time RT-PCR. Fourteen samples were TGEV RNA positive in RT-RPA (18.4%, 14/76), which were also positive in the real-time RT-PCR. The diagnostic agreement between the two assays was 100% (76/76). The R2 value of RT-RPA and real-time RT-PCR was 0.959 by linear regression analysis. The developed RT-RPA assay provides a useful alternative tool for rapid, simple and reliable detection of TGEV in resource-limited diagnostic laboratories and on-site facilities.

Replication of hepatitis E virus in the ovary and promotion of oocyte apoptosis in rabbits infected with HEV-4.

Hepatitis E virus (HEV) infection can induce infertility and miscarriage in pregnant women and infect neonates through vertical transmission. However, the mechanism of infertility and vertical transmission remains unclear. In the present study, we evaluated the replication of HEV in the ovary and structural and molecular changes induced by HEV after intraperitoneal injection of HEV in rabbits. Positive- and negative-strand HEV RNA was detected in the ovaries at 28 and 49 days post-infection. Positive HEV open reading frames 2 and 3 signals were observed in the ovaries by immunohistochemistry staining. Histopathological changes of ovarian tissues were observed, including scattered cell necrosis and lymphocyte infiltration. The ratio of normal follicles decreased, whereas the ratio of atresia follicles increased in the HEV RNA-positive ovaries compared to the control group by counting the number of follicles at all levels. In addition, TUNEL results showed that apoptosis in follicle cells and oocytes was promoted by HEV infection. These results suggest that the ovary is one of the replication sites of HEV and that the expression of HEV RNA and antigen in ovarian tissue caused structural and molecular changes that promoted germ cell apoptosis. HEV can infect and replicate in the ovum at different stages, which is a novel mechanism for HEV vertical transmission.

Detection of Genotype 4 Swine Hepatitis E Virus in Systemic Tissues in Cross-Species Infected Rabbits.

Increasing evidence demonstrates that hepatitis E virus (HEV) can be transmitted across species. According to previous reports, swine HEV has two genotypes, genotype 3 and 4, and both can infect humans by the fecal-oral route. Thus, it is crucial for the control of HEV zoonotic transmission to evaluate the dynamics of viral shedding and distribution in different tissues during cross-species infection by HEV. In this study, rabbits were infected with genotype 4 swine HEV by the intraperitoneal route. The results showed that HEV RNA not only shed in the feces but also in the saliva of some rabbits during infection with swine HEV. Viremia appeared late after infection, and anti-HEV IgG was not obvious until the appearance of high viremia levels. After the rabbits were euthanized, a histopathological examination showed that the livers developed overt hepatitis accompanied by an elevation of alanine aminotransferase (ALT) and aspartate transaminase (AST). Furthermore, HEV RNA was detected in various tissues, especially in the salivary glands and tonsils. Subsequently, negative-stranded HEV RNA was practiced in tissues with positive HEV RNA, which demonstrated that HEV replicated in the tissues. Next, we harvested additional tissues from the liver, salivary gland, tonsil, spleen, thymus gland, lymph node and intestine, which are known as replication sites of swine HEV. Additionally, we also observed the HEV antigen distributed in the organs above through immunohistochemical staining. These results demonstrate that rabbits could be used as an animal model for researching cross-species infection of genotype 4 HEV. It is also noteworthy that HEV can shed in the saliva and presents the risk of droplet transmission. These new data provide valuable information for understanding cross-species infection by HEV.

Hepatitis E Virus Genotype 4 Sequences Detected in Sewage from Treatment Plants of China.

The aim of this study was to investigate the occurrence of hepatitis E virus (HEV) in sewage samples in Shen Zhen, China. Sewage samples were collected from 152 sewage plants including livestock sewage, domestic sewage and treated sewage from May to July of 2015. Two of 152 samples were HEV positive (1.32%) from the livestock sewage plants. Partial ORF2 fragments of HEV were sequenced and a phylogenetic tree was constructed using MEGA5.1. Blast and phylogenetic analyses showed that both of these two sequences belonged to HEV Genotype 4. To the best of our knowledge, this is the first study on the molecular characterization of HEV in wastewater in China and the first time to detect Genotype 4 in the sewage. Results from this study indicate that the possibilities of sporadic infections of HEV should be emphasized because virus still has the possibility to be circulating in the sewage in China.

Antigen detection and apoptosis in Mongolian gerbil's kidney experimentally intraperitoneally infected by swine hepatitis E virus.

We examined the effect of hepatitis E virus (HEV) on the renal tissue pathogenesis, morphological damages and related molecular mechanisms following swine HEV suspension intraperitoneally inoculation in Mongolian gerbils. The microscopic and ultramicroscopic analyses of kidney tissue structure were carried out at different points after inoculation of HEV. The immunohistochemistry, real-time PCR and Western blot were performed to explore the molecular mechanisms associated with HEV presence in the renal tissues. Real-time PCR revealed that the copies of HEV RNA in the kidney were detected at 7 dpi, and peaked at 14 dpi at a concentration was 7.18 logs g(-1), with detection of HEV ORF2 antigen by immunohistochemistry. Hematoxylin and eosin (HE) staining showed pathological lesions including glomerular atrophy, degeneration, edema and necrosis of renal tubular epithelial cells and Mallory and Sirius red staining indicated the presence of collagen fibers and fibrosis in kidney tissues of inoculated gerbils. Ultrastructural studies of basal membrane of renal tubules demonstrated the rough and uneven with mitochondria swelling and vacuolation in the tissues of HEV inoculated animals. Similarly, significantly higher number of (TUNEL)-positive cells were seen in renal tubule tissues compared to control group. Moreover, immuno histochemical results indicated that significant increase expression of the B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), FAS and Caspase-3 in HEV inoculated Mongolian gerbils at each time points. Relative mRNA expression by real-time PCR revealed a significantly higher (P<0.05) mRNA level of BAX, Bcl-2 and caspase-3 transcription in HEV inoculated Mongolian gerbils. Our results demonstrates that activation of mitochondria and Caspase-3 protease might be induced the apoptosis which subsequently cause the necrosis and cell death of renal epithelial cells during acute phase of HEV infection in HEV inoculated Mongolian gerbils.

Development of a vivo rabbit ligated intestinal Loop Model for HCMV infection.

BACKGROUND: Human Cytomegalovirus (HCMV) infections can be found throughout the body, especially in epithelial tissue. Animal model was established by inoculation of HCMV (strain AD-169) or coinoculation with Hepatitis E virus (HEV) into the ligated sacculus rotundus and vermiform appendix in living rabbits. The specimens were collected from animals sacrificed 1 and a half hours after infection. RESULTS: The virus was found to be capable of reproducing in these specimens through RT-PCR and Western-blot. Severe inflammation damage was found in HCMV-infected tissue. The viral protein could be detected in high amounts in the mucosal epithelium and lamina propria by immunohistochemistry and immunofluorescense. Moreover, there are strong positive signals in lymphocytes, macrophages, and lymphoid follicles. Quantitative statistics indicate that lymphocytes among epithlium cells increased significantly in viral infection groups. CONCLUSIONS: The results showed that HCMV or HEV + HCMV can efficiently infect in rabbits by vivo ligated intestine loop inoculation. The present study successfully developed an infective model in vivo rabbit ligated intestinal Loop for HCMV pathogenesis study. This rabbit model can be helpful for understanding modulation of the gut immune system with HCMV infection.