Characteristics and Functions of HEV Proteins.

Hepatitis E virus (HEV) is a non-enveloped virus containing a single-stranded, positive-sense RNA genome of 7.2 kb, which consists of a 5' non-coding region, three open reading frames (ORFs), and a 3' non-coding region. ORF1 is diverse between genotypes and encodes the nonstructural proteins, which include the enzymes needed for virus replication. In addition to its role in virus replication, the function of ORF1 is relevant to viral adaption in culture and may also relate to virus infection and HEV pathogenicity. ORF2 protein is the capsid protein, which is about 660 amino acids in length. It not only protects the integrity of the viral genome, but is also involved in many important physiological activities, such as virus assembly, infection, host interaction, and innate immune response. The main immune epitopes, especially neutralizing epitopes, are located on ORF2 protein, which is a candidate antigen for vaccine development. ORF3 protein is a phosphoprotein of 113 or 114 amino acids with a molecular weight of 13 kDa with multiple functions that can also induce strong immune reactivity. A novel ORF4 has been identified only in genotype 1 HEV and its translation promotes viral replication.

Asialoglycoprotein receptor facilitates infection of PLC/PRF/5 cells by HEV through interaction with ORF2.

Although the biological and epidemiological features of hepatitis E virus (HEV) have been studied extensively in recent years, the mechanism by which HEV infects cells is still poorly understood. In this study, coimmunoprecipitation, pull-down, and ELISA were used to show that the HEV ORF2 protein interacts directly with the ectodomain of both ASGR1 and ASGR2. Susceptibility to HEV correlated positively with the expression level of surface asialoglycoprotein receptor (ASGPR) in cell lines. ASGPR-directed small interfering RNA (siRNA) in HEV-infected PLC/PRF/5 cells had no significant effect on HEV release, suggesting that ASGPR mainly regulates the viral binding and entry steps. Both the purified ASGPR ectodomain and anti-ASGPR antibodies disturbed the binding of HEV to PLC/PRF/5 cells. The classic ASGPR ligands asialofetuin, asialoganglioside, and fibronectin competitively inhibited the binding of HEV to hepatocytes in the presence of calcium. HeLa cell lines stably expressing ASGPR displayed increased HEV-binding capacity, whereas ASGPR-knockout PLC/PRF/5 cell lines had lower HEV-binding capacity. Thus, our study demonstrates that ASGPR is involved in and facilitates HEV infection by binding to ORF2. J. Med. Virol. 88:2186-2195, 2016. © 2016 Wiley Periodicals, Inc.

Characteristics and Functions of HEV Proteins.

Hepatitis E virus (HEV) is a non-enveloped virus containing a single-stranded, positive-sense RNA genome of 7.2 kb, which consists of a 5' noncoding region, three open reading frames (ORFs), and a 3' noncoding region. ORF1 is diverse between genotypes and encodes the nonstructural proteins, which include the enzymes needed for virus replication. In addition to its role in virus replication, the function of ORF1 is relevant to viral adaption in cultured cells and may also relate to virus infection and HEV pathogenicity. ORF2 protein is the capsid protein, which is about 660 amino acids in length. It not only protects the integrity of the viral genome but is also involved in many important physiological activities, such as virus assembly, infection, and host interaction. The main immune epitopes, especially neutralizing epitopes, are located on ORF2 protein, which is a candidate antigen for vaccine development. ORF3 protein is a phosphoprotein of 113 or 114 amino acids with a molecular weight of 13 kDa with multiple functions that can also induce strong immune reactivity.

Characteristics of bacterial community in eyelashes of patients with Demodex blepharitis.

BACKGROUND: Demodex blepharitis (DB) is a common disease of the ocular surface. The characteristics of the bacterial community in eyelash roots after Demodex infestation are still unknown. Knowledge of the characteristics of the bacterial community of eyelash follicles in patients with DB can provide valuable insights for guiding the diagnosis and treatment of DB. METHODS: Twenty-five patients with DB (DB group) and 21 non-DB volunteers (control group) were enrolled in the study. Eyelashes from the upper eyelid of the right eye were sampled, and 16S ribosomal DNA (rDNA) sequencing was performed to determine the V3-V4 regions of the microbial 16S rDNA gene within 1 month of infestation. The sequencing data of the two groups were analyzed and compared. The effect of the bacterium Burkholderia on the survival of Demodex mites was evaluated using Demodex obtained from 12 patients with DB other that the patients in the DB group. RESULTS: A total of 31 phyla and 862 genera were identified in the DB and control groups. The five most abundant phyla in the two groups were Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and Cyanobacteria. The abundance of Actinomycetes was significantly higher in the DB group than in the control group. At the genus level, the five most abundant genera in the two groups were Pseudomonas, Burkholderia-Caballeronia-Paraburkholderia, Rolstonia and Acinetobacter; Clostridium sensu stricto 1 was abundant in the control group and Corynebacterium_1 was abundant in the DB group. Compared with the control group, the abundance of Burkholderia-Caballeronia-Paraburkholderia was 2.36-fold lower in the DB group. Linear discriminant analysis Effect Size (LEfSe) analysis revealed Burkholderia-Caballeronia-Paraburkholderia, SC_I_84_unclassified, Nonmyxobacteria and Succinvibrio to be the major biomarkers in the control group and Catenibacterium and Lachnospiraceae NK4A136 group to be the major biomarkers in the DB group. To explore the performance of these optimal marker models, receiver operational characteristic curve analysis was performed, and the average area under the curve value of Burkholderia-Caballeronia-Paraburkholderia was 0.7448. Burkholderia cepacia isolated from normal human eyelashes was fermented, and the Demodex mites isolated from patient eyelashes were cultured together with its fermented supernatant. The results showed that the fermentation supernatant could significantly reduce the survival time of the Demodex mites, suggesting the potential therapeutic value of this bacterium against Demodex. CONCLUSIONS: The composition of the bacterial community in the eyelashes of DB patients differed from that in eyelashes of healthy volunteers, revealing a decrease in bacterial diversity in infested eyelashes. This decrease may be related to the occurrence and development of DB. The supernatant of Burkholderia cepacia culture medium was found to inhibit the growth of Demodex in eyelash hair follicles, providing a new insight with potential applications for the clinical treatment of Demodex infestation.

Development of in vitro and in vivo neutralization assays based on the pseudotyped H7N9 virus.

H7N9 viral infections pose a great threat to both animal and human health. This avian virus cannot be handled in level 2 biocontainment laboratories, substantially hindering evaluation of prophylactic vaccines and therapeutic agents. Here, we report a high-titer pseudoviral system with a bioluminescent reporter gene, enabling us to visually and quantitatively conduct analyses of virus replications in both tissue cultures and animals. For evaluation of immunogenicity of H7N9 vaccines, we developed an in vitro assay for neutralizing antibody measurement based on the pseudoviral system; results generated by the in vitro assay were found to be strongly correlated with those by either hemagglutination inhibition (HI) or micro-neutralization (MN) assay. Furthermore, we injected the viruses into Balb/c mice and observed dynamic distributions of the viruses in the animals, which provides an ideal imaging model for quantitative analyses of prophylactic and therapeutic monoclonal antibodies. Taken together, the pseudoviral systems reported here could be of great value for both in vitro and in vivo evaluations of vaccines and antiviral agents without the need of wild type H7N9 virus.

Multiplex Cell-Free DNA Reference Materials for Quality Control of Next-Generation Sequencing-Based In Vitro Diagnostic Tests of Colorectal Cancer Tolerance.

Background: Liquid biopsies based on next-generation sequencing (NGS) assays are confronted with more opportunities and challenges. Widespread clinical implementation of NGS-based cancer in vitro diagnostic tests (IVDs) highlighted the urgency to establish reference materials (RMs) which could provide full control of the process from nucleic acid extraction to test report generation. Quality control based on cell-free DNA (cfDNA) RMs is especially important for liquid biopsies. Methods: Here, we used genomic DNA from thirteen cell lines to establish four negative cfDNA RMs (N1-N4) and four multiplex cfDNA RMs (L1-L4) at serial allelic frequencies ranging from approximately 2% to 0.1%. All the cfDNA RMs were quantified and validated via both droplet digital polymerase chain reaction (ddPCR) and NGS. These RMs were distributed to eight domestic manufacturers to collaboratively evaluate the performance of several domestic NGS-based cancer IVDs covering four major NGS platforms (NextSeq, HiSeq, Ion Proton, and BGISEQ). Results: Each multiplex RM has eleven colorectal cancer-related mutations, including six KRAS mutations (G12S, G12C, G12D, G12A, G12V, and G13D), three NRAS mutations (G12D, Q61R, and Q61K), one PIK3CA mutation (H1047R), and one BRAF mutation (V600E). Each mutation in the cfDNA RMs was quantified and validated via both ddPCR and NGS, showing the good relevance of mutant allelic frequency. These RMs were distributed to eight domestic manufacturers for collaborative evaluation. All eight manufacturers provided similar results by domestic NGS-based cancer IVDs, except for manufacturer #5. The coefficient of variation (CV) was increased with decreasing mutant allelic frequency, and poor repetition occurred when the allelic frequency was lower than 0.5%. Conclusions: These results indicated that these cfDNA RMs would be pivotal for NGS-based cancer IVDs, especially for liquid biopsies of colorectal cancer-related mutations and would guide the further development of RMs covering more onco-related mutations.

Quality Control of Next-generation Sequencing-based In vitro Diagnostic Test for Onco-relevant Mutations Using Multiplex Reference Materials in Plasma.

Background: Widespread clinical implementation of next-generation sequencing (NGS)-based cancer in vitro diagnostic tests (IVDs) highlighted the urgency to establish reference materials which could provide full control of the process from nucleic acid extraction to test report generation. The formalin-fixed, paraffin-embedded (FFPE) tissue and blood plasma containing circulating tumor deoxyribonucleic acid (ctDNA) were mostly used for clinically detecting onco-relevant mutations. Methods: We respectively developed multiplex FFPE and plasma reference materials covering three clinically onco-relevant mutations within the epidermal growth factor receptor (EGFR) gene at serial allelic frequencies. All reference materials were quantified and validated via droplet digital polymerase chain reaction (ddPCR), and then were distributed to eight domestic manufacturers for the collaborative evaluation of the performance of several domestic NGS-based cancer IVDs covering four major NGS platforms (NextSeq, HiSeq, Ion Proton and BGISEQ). Results: All expected mutations except one at extremely low allelic frequencies were detected, despite some differences in coefficient of variation (CV) which increased with the decrease of allelic frequency (CVs ranging from 18% to 106%). It was worth noting that the CV value seemed to correlate with a particular mutation as well. The repeatability of determination of different mutations was L858R>T790M>19del. Conclusions: The results indicated our reference materials would be pivotal for quality control of NGS-based cancer IVDs and would guide the further development of reference materials covering more onco-relevant mutations.

Systematic identification of hepatitis E virus ORF2 interactome reveals that TMEM134 engages in ORF2-mediated NF-κB pathway.

Hepatitis E virus (HEV) is the causative agent of acute hepatitis E. Open reading frame 2 (ORF2) encodes the capsid protein of HEV, which is the main structural protein and may participate, together with the host factors, in viral entry and egress. However, it is still not clear which host proteins are involved in the interaction with ORF2 and what the functions of these ORF2-interacting proteins are. In this study, we have applied a split-ubiquitin yeast two-hybrid screening approach in combination with the pull-down and coimmunoprecipitation assays, identified and validated multiple interacting partners of ORF2 of genotype 1 and 4, which have diverse biological functions. Among these novel candidates that have not been previously reported, we have found that 20 of them are located in endoplasmic reticulum. TMEM134, which interacts and co-localizes with ORF2 in the endoplasmic reticulum, negatively regulates ORF2-mediated inhibition of the NF-κB signaling pathway. Our study for the first time has systematically mapped the ORF2 interactome in two genotypes of HEV, providing a new insight of understanding the virus-host interaction during the pathogenesis of HEV, and may offer potential therapeutic targets to intervene the HEV life cycle.