A review on polysaccharides from Artemisia sphaerocephala Krasch seeds, their extraction, modification, structure, and applications.

Artemisia sphaerocephala Krasch (ASK) is an important member of Compositae (Asteraceae) family. Its seeds have been widely used as traditional medicine and to improve the quality of food. Water soluble and water insoluble polysaccharides are found in the seeds of this plant. Research has been conducted on the extraction of polysaccharides, their modification and determination of their structure. To date different techniques for extraction purposes have been applied which are reviewed here. Antioxidant, antidiabetic, anti-obesogenic, antitumor, and immunomodulatory activities have been explored using in vivo and in vitro methods. Moreover, these polysaccharides have been used as packaging material and as a sensing component for monitoring the freshness of packaged food. Some experimental results have shown that the quality of foods is also improved by using them as a food additive. We have also indicated some of the potential areas that are needed to be explored.

Past, present and future of hepatitis E virus infection: Zoonotic perspectives.

The origin of hepatitis E virus (HEV) is not fully understood, but it is considered an emerging zoonotic pathogen. To date, HEV has been isolated from many animal species. The family Hepeviridae consists of two genera. The genus Orthohepevirus includes four distinct species (A, B, C, and D), each with distinct genotypes. Within the Orthohepevirus A species, HEV-1 and HEV-2 host ranges are restricted to humans, whereas genotypes 3 and 4 primarily infect a wide range of diverse animal species, in addition to being zoonotic to humans. Swine and wild boar species were previously thought to be the primary natural HEV reservoir, but recently rabbits have also been identified as major carriers. Moreover, increasing the number of HEV infections within the food supply chain underscore the important role of farming and food processing practices in limiting virus transmission. Notably, a Chinese commercial vaccine has the potential to protect humans and possibly animal reservoirs from HEV infection. This review summarizes the status of HEV infection worldwide in different animal species and outlines various modes of zoonotic transmission, with reference to cross-species transmission and recent vaccine developments.

Evaluation of recombinant Chinese avian hepatitis E virus (CaHEV) ORF2 and ORF3 proteins for protection of chickens against CaHEV infection.

Avian hepatitis E virus (HEV) is the etiologic agent of big liver and spleen disease in chickens. In 2010, the Chinese avian HEV (CaHEV) strain was isolated from chickens and demonstrated to cause the decreased egg production in layer hens. No avian HEV commercial vaccine has yet been developed to prevent virus infection in China. In this study, recombinant CaHEV truncated ORF2 and complete ORF3 proteins were evaluated separately for immunoprotection of chickens against CaHEV infection. First, truncated ORF2 and complete ORF3 proteins were expressed in Escherichia coli. Next, 48 specific-pathogen-free chickens were randomly divided into three groups. One group was immunized with truncated ORF2 protein, the second group was immunized with recombinant ORF3 protein, while the third group (control) was mock-immunized with PBS. After booster immunization, chickens in all three groups were challenged intravenously with CaHEV infectious stock and assessed for viremia, fecal virus shedding, seroconversion, and gross hepatic lesions. In the ORF2 protein-immunized group, no chickens showed evidence of avian HEV infection. In the ORF3 protein-immunized group, nine chickens exhibited viremia and seven had fecal virus shedding. In the control group, all 16 chickens showed viremia and fecal virus shedding. However, the durations in chickens from the ORF3 protein group (2-4weeks) were shorter than the ones from the control group (4-8weeks). Moreover, no gross liver lesions emerged in the ORF2 protein group, while lesions observed in the ORF3 protein group were milder than in controls. Therefore, the ORF2 protein can confer complete immunoprotection against chicken CaHEV infection, while the ORF3 protein only confers partial immunoprotection.

Seroprevalence of avian hepatitis E virus and avian leucosis virus subgroup J in chicken flocks with hepatitis syndrome, China.

BACKGROUND: From 2014 to 2015 in China, many broiler breeder and layer hen flocks exhibited a decrease in egg production and some chickens developed hepatitis syndrome including hepatomegaly, hepatic necrosis and hemorrhage. Avian hepatitis E virus (HEV) and avian leucosis virus subgroup J (ALV-J) both cause decreasing in egg production, hepatomegaly and hepatic hemorrhage in broiler breeder and layer hens. In the study, the seroprevalence of avian HEV and ALV-J in these flocks emerging the disease from Shandong and Shaanxi provinces were investigated. RESULTS: A total of 1995 serum samples were collected from 14 flocks with hepatitis syndrome in Shandong and Shaanxi provinces, China. Antibodies against avian HEV and ALV-J in these serum samples were detected using iELISAs. The seroprevalence of anti-avian HEV antibodies (35.09%) was significantly higher than that of anti-ALV-J antibodies (2.16%) (p = 0.00). Moreover, the 43 serum samples positive for anti-ALV-J antibodies were all also positive for anti-avian HEV antibodies. In a comparison of both provinces, Shandong chickens exhibited a significantly higher seroprevalence of anti-avian HEV antibodies (42.16%) than Shaanxi chickens (26%) (p = 0.00). In addition, the detection of avian HEV RNA and ALV-J cDNA in the liver samples from the flocks of two provinces also showed the same results of the seroprevalence. CONCLUSIONS: In the present study, the results showed that avian HEV infection is widely prevalent and ALV-J infection is endemic in the flocks with hepatitis syndrome from Shandong and Shaanxi provinces of China. These results suggested that avian HEV infection may be the major cause of increased egg drop and hepatitis syndrome observed during the last 2 years in China. These results should be useful to guide development of prevention and control measures to control the diseases within chicken flocks in China.

Effects of Carbopol® 934 proportion on nanoemulsion gel for topical and transdermal drug delivery: a skin permeation study.

Nanoemulsions (NEs) are used as transdermal drug delivery systems for systematic therapeutic purposes. We hypothesized that the skin permeation profile of an NE could be modulated by incorporating it into a hydrogel containing differing proportions of thickening agent. The objectives of this study were as follows: 1) to determine the stability and skin irritability of NE gels (NGs) containing 1%, 2%, and 3% (w/w) Carbopol® 934 (CP934) (termed NG1, NG2, and NG3, respectively); 2) to compare the skin permeation profiles and drug deposition patterns of the NGs; and 3) to visualize the drug delivery routes of the NGs. Terbinafine and citral were incorporated into the NGs as model drugs. Ex vivo skin permeation tests indicated that the percutaneous flux rates of terbinafine decreased in the order NE (215 µg/cm2) > NG1 (213 µg/cm2) > NG2 (123 µg/cm2) > NG3 (74.3 µg/cm2). The flux rates of citral decreased in the order NE (1,026 µg/cm2) > NG1 (1,021 µg/cm2) > NG2 (541 µg/cm2) > NG3 (353 µg/cm2). The NGs accumulated greater amounts of the drugs in the stratum corneum and less in the epidermis/dermis than did the NE (P<0.05) over a period of 12 h. Laser scanning confocal microscopy indicated that the NGs altered the main drug delivery routes from skin appendages to intercellular paths. Histological images suggested that perturbations to the skin structure, specifically the size of the epidermal intercellular spaces and the separation distance of dermal collagen bundles, could be significantly minimized by increasing the proportion of CP934. These results suggest that adjustments of the CP934 proportions can be used to modulate the skin permeation profiles of NGs for specific therapeutic purposes.

MYH9 is an Essential Factor for Porcine Reproductive and Respiratory Syndrome Virus Infection.

Porcine reproductive and respiratory syndrome (PRRS) caused by the PRRS virus (PRRSV) is an important swine disease worldwide. PRRSV has a limited tropism for certain cells, which may at least in part be attributed to the expression of the necessary cellular molecules serving as the virus receptors or factors on host cells for virus binding or entry. However, these molecules conferring PRRSV infection have not been fully characterized. Here we show the identification of non-muscle myosin heavy chain 9 (MYH9) as an essential factor for PRRSV infection using the anti-idiotypic antibody specific to the PRRSV glycoprotein GP5. MYH9 physically interacts with the PRRSV GP5 protein via its C-terminal domain and confers susceptibility of cells to PRRSV infection. These findings indicate that MYH9 is an essential factor for PRRSV infection and provide new insights into PRRSV-host interactions and viral entry, potentially facilitating development of control strategies for this important swine disease.

A Novel Blocking ELISA for Detection of Antibodies against Hepatitis E Virus in Domestic Pigs.

Hepatitis E virus (HEV) infects both humans and animals, with an overall human mortality rate generally less than 1%, but as high as 20% among pregnant women. HEV strains fall into 4 major genotypes. Zoonotic genotypes 3 and 4 associate with sporadic human and animal HEV cases in many industrialized countries. To date, collective evidence implicates pigs as the main HEV reservoir, justifying the importance of monitoring HEV infection rates in pig herds to prevent human illness. Due to the lack of a robust in vitro cell culture system for viral propagation, no "gold standard" assay has yet been developed to detect HEV infection in domestic pigs. 1E4, a monoclonal antibody (mAb) specific for the C-terminal 268 amino acids of HEV genotype 4 ORF2 capsid protein (sORF2-C), was generated and conjugated to horseradish peroxidase (HRP) for use in a blocking ELISA (bELISA). Optimal sORF2-C coating antigen concentration (8 µg/ml), HRP-1E4 dilution (1:1000), and test pig serum dilution (1:20) were determined using a checkerboard titration test. A cut-off value of 16.9% was chosen to differentiate between positive vs. negative sera after mean percent inhibition (PI) testing of 230 negative pig sera. Compared with the indirect ELISA (iELISA), western blot, and a commercial ELISA kit for detecting anti-HEV antibodies in human sera, the bELISA showed no statistical differences and statistically high coincidence of 93.23%, 92%, and 95% with the other tests, respectively. A blocking ELISA (bELISA) for detecting anti-HEV antibodies in pig serum samples was developed with high sensitivity and high specificity comparable to that of the indirect ELISA. The bELISA results exhibited high agreement with iELISA, western blot, and a commercial ELISA kit designed to detect human anti-HEV antibodies. Therefore, bELISA should serve as an ideal method for large-scale serological investigation of anti-HEV antibodies in domestic pigs.

Rescue and evaluation of a recombinant PRRSV expressing porcine Interleukin-4.

BACKGROUND: The current vaccines for porcine reproductive and respiratory syndrome virus (PRRSV) have failed to provide broad protection against infection by various strains of PRRSV. Porcine Interleukin-4 (pIL-4) plays an important role in the regulation of the immune response and has been used previously as an immunological adjuvant. The objective of this study was to construct a recombinant PRRSV expressing pIL-4 and to evaluate the immune response of the recombinant virus in piglets. METHODS: The pIL-4 gene was inserted in the PRRSV (CH-1R strain) infectious clone by overlap PCR. Indirect immunofluorescence assay (IFA) and Western blotting were used to confirm the recombinant virus. The stability of the recombinant virus was assessed by DNA sequencing and IFA after 15 passages in vitro. Recombinant virus was injected into pigs and efficacy of immune protection was evaluated in comparison with the parental virus. RESULTS: The recombinant virus (CH-1R/pIL-4) was successfully rescued and shown to have similar growth kinetics as the parental virus. The recombinant virus was stable for 15 passages in cell culture. Pigs vaccinated with CH-1R/pIL-4 produced a similar humoral response to the response elicited by parental virus, but IL-4 level in the supernatant of PBMCs from pigs vaccinated with CH-1R/pIL-4 was significantly higher than the parent virus at 28 days post-immunization (DPI). Flow cytometric (FCM) analysis showed that the percentage of CD4(+)CD8(+) double positive T (DPT) cells in the CH-1R/pIL-4 vaccinated group was significantly higher than the parental virus at 3 and 7 Days Post-Challenge (DPC), and the IL-4 level in the blood significantly increased at 7 DPC. However, the viral load and histopathology did not show significant difference between the two groups. CONCLUSIONS: A recombinant PRRSV expressing porcine IL-4 was rescued and it remained genetically stable in vitro. The recombinant virus induced higher DPT ratios and IL-4 levels in the blood after HP-PRRSV challenge compared to the parental virus in piglets. However, it did not significantly improve protection efficacy of PRRSV vaccine.

Antigenic properties of avian hepatitis E virus capsid protein.

Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease and hepatitis-splenomegaly syndrome in chickens, and is genetically and antigenically related to mammalian HEVs. HEV capsid protein contains immunodominant epitopes and induces a protective humoral immune response. A better understanding of the antigenic composition of this protein is critically important for the development of effective vaccine and sensitive and specific serological assays. To date, six linear antigenic domains (I-VI) have been characterized in avian HEV capsid protein and analyzed for their applications in the serological diagnosis and vaccine design. Domains I and V induce strong immune response in chickens and are common to avian, human, and swine HEVs, indicating that the shared epitopes hampering differential diagnosis of avian HEV infection. Domains III and IV are not immunodominant and elicit a weak immune response. Domain VI, located in the N-terminal region of the capsid protein, can also trigger an intense immune response, but the anti-domain VI antibodies are transient. The protection analysis showed that the truncated capsid protein containing the C-terminal 268 amino acid residues expressed by the bacterial system can provide protective immunity against avian HEV infection in chickens. However, the synthetic peptides incorporating the different linear antigenic domains (I-VI) and epitopes are non-protective. The antigenic composition of avian HEV capsid protein is altogether complex. To develop an effective vaccine and accurate serological diagnostic methods, more conformational antigenic domains or epitopes are to be characterized in detail.

Development and evaluation of a SYBR Green real-time RT-PCR assay for detection of avian hepatitis E virus.

BACKGROUND: Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease, as well as hepatitis-splenomegaly syndrome in chickens. To date, conventional reverse transcriptase polymerase chain reaction (RT-PCR) and nested RT-PCR methods have been used for the diagnosis of avian HEV infection in chickens. However, these assays are time consuming, inconvenient, and cannot detect the virus quantitatively. In this study, a rapid and sensitive SYBR Green real-time RT-PCR assay was developed to detect avian HEV RNA quantitatively in serum, liver, spleen, and fecal samples from chickens. RESULTS: Based on the sequence of the most conserved HEV gene, ORF3, the primers for the assay were designed, and the standard plasmid was constructed. The detection limit of the assay was shown to be 10 copies/µl of standard plasmid/reaction, with a corresponding cycle-threshold value of 29.3. The standard curve exhibited a dynamic linear range across at least 7 log units of DNA copy number. The specificity and reproducibility of this assay was high, showing that the assay detected avian HEV RNA specifically and with little variability. Compared to conventional RT-PCR, the current assay is more sensitive for detecting avian HEV in serum, liver, spleen, and fecal samples from chickens. CONCLUSIONS: A rapid, specific, and reproducible SYBR Green real-time RT-PCR assay was developed for the diagnosis of avian HEV infection in chickens. This assay can accurately detect avian HEV RNA in serum, liver, spleen, and fecal samples with more sensitivity than conventional RT-PCR.

Characterization of Two Novel Linear B-Cell Epitopes in the Capsid Protein of Avian Hepatitis E Virus (HEV) That Are Common to Avian, Swine, and Human HEVs.

UNLABELLED: Antisera raised against the avian hepatitis E virus (HEV) capsid protein are cross-reactive with human and swine HEV capsid proteins. In this study, two monoclonal antibodies (MAbs) against the avian HEV capsid protein, namely, 3E8 and 1B5, were shown to cross-react with the swine HEV capsid protein. The motifs involved in binding both MAbs were identified and characterized using phage display biopanning, peptide synthesis, and truncated or mutated protein expression, along with indirect enzyme-linked immunosorbent assay (ELISA) and Western blotting. The results showed that the I/VPHD motif is a necessary core sequence and that P and H are two key amino acids for recognition by MAb 3E8. The VKLYM/TS motif is the minimal amino acid sequence necessary for recognition by MAb 1B5. Cross-reactivity between the two epitopes and antibodies against avian, swine, and human HEVs in sera showed that both epitopes are common to avian, swine, and human HEVs. In addition, amino acid sequence alignment of the capsid proteins revealed that the key motifs of both novel epitopes are the same in HEVs from different animal species, predicting that they may be common to HEV isolates from boars, rabbits, rats, ferrets, mongooses, deer, and camels as well. Protein modeling analysis showed that both epitopes are at least partially exposed on the surface of the HEV capsid protein. Protective capacity analysis demonstrated that the two epitopes are nonprotective against avian HEV infection in chickens. Collectively, these studies characterize two novel linear B-cell epitopes common to avian, swine, and human HEVs, which furthers the understanding of HEV capsid protein antigenic structure. IMPORTANCE: More and more evidence indicates that the host range diversity of hepatitis E virus (HEV) is a global public health concern. A better understanding of the antigenic structure of the HEV capsid protein may improve disease diagnosis and prevention. In this study, binding site mapping and localization as well as the antigenic biology of two novel linear B-cell epitopes common to several different species of HEV were characterized. These findings partially reveal the antigenic structure of the HEV capsid protein and provide potential applications for the development of diagnostics and interventions for HEV infection.