Hepatitis E Virus Produced from Cell Culture Has a Lipid Envelope

<div><p>The absence of a productive cell culture system hampered detailed analysis of the structure and protein composition of the hepatitis E virion. In this study, hepatitis E virus from a robust HEV cell culture system and from the feces of infected monkeys at the peak of virus excretion was purified by ultra-centrifugation. The common feature of the two samples after ultracentrifugation was that the ORF2 protein mainly remained in the top fractions. The ORF2 protein from cell culture system was glycosylated, with an apparent molecular weight of 88 kDa, and was not infectious in PLC/PRF/5 cells. The ORF2 protein in this fraction can bind to and protect HEV RNA from digestion by RNase A. The RNA-ORF2 product has a similar sedimentation coefficient to the virus from feces. The viral RNA in the cell culture supernatant was mainly in the fraction of 1.15g/cm³ but that from the feces was mainly in the fraction of 1.21 g/cm³. Both were infectious in PLC/PRF/5 cells. And the fraction in the middle of the gradient (1.06g/cm³) from the cell culture supernatant,but not that from the feces, also has ORF2 protein and HEV RNA but was not infectious in PLC/PRF/5.The infectious RNA-rich fraction from the cell culture contained ORF3 protein and lipid but the corresponding fraction from feces had no lipid and little ORF3 protein. The lipid on the surface of the virus has no effect on its binding to cells but the ORF3 protein interferes with binding. The result suggests that most of the secreted ORF2 protein is not associated with HEV RNA and that hepatitis E virus produced in cell culture differs in structure from the virus found in feces in that it has a lipid envelope.</p></div

The analysis of the fraction rich inHEV RNA.

<p>(A) The density-gradient fraction of virus from different sources and with different treatments. The X-axis is the fraction number and the right Y-axis is the density of the fraction. The left Y-axis is the HEV RNA content. (B) The binding ability of virus to anti-ORF3 MAb, the Y-axis is the percentage of binding HEV RNA to total HEV RNA of sample. The * represents the P value of HEV virus in the cell culture supernatant with NP40 treatment compared to the same sample without NP40 treatment. P value is less than 0.05. (C) The binding effect of virus to cells, the X-axis is the time post incubation with samples and the Y-axis was the binding RNA content to cells. (D) The fraction 15 of cell culture supernatant was detected by western blotting with an anti-ORF3 MAb.</p

A and B are density-gradient fraction of HEV in cell culture supernatant (A) and feces (B).

<p>The X-axis shows the fraction number, the left Y-axis is the ORF2 protein content (μg/ fraction) and the RNA content (10⁵copies/fraction of cell culture supernatant HEV and 10⁶ copies/fraction of feces HEV) in the fraction and the right Y-axis is the density of the fraction. C and D are quantification of HEV RNA in culture supernatant of PLC/PRF/5 cells incubated with virus from cell culture (C) and from feces (D). The X-axis is the time post incubation and the Y-axis is the HEV RNA concentration.</p

Specific Competitive Inhibition EMSA of HEV RNA and captured ORF2 protein.

<p>The biotin-RNA without ORF2 protein was as negative control. The assay was carried out as described under “Materials and Methods”. The non-biotin labeled RNA in lane 2 and 3 were 4 fold and 8 fold to biotin labeled RNA. The lane 4 was shown the nonspecific competitive inhibition of 0.2μg total PLC/PRF/5 cell RNA.</p

The standard curve of ORF2protein (A) and HEV RNA (B).

<p>(A): The X-axis is the concentration of ORF2 protein standard and Y-axis is the value of OD₄₅₀. (B): X-axis is the concentration of HEV RNA standard and Y-axis is the Ct value of real time RT-PCR.</p

Antigenic variations of recent street rabies virus

ABSTRACTThe genetic and/or antigenic differences between street rabies virus (RABV) and vaccine strains could potentially affect effectiveness of rabies vaccines. As such, it is important to continue monitoring the glycoprotein (G) of the street isolates. All RABVG sequences in public database were retrieved and analysed. Using a pseudovirus system, we investigated 99 naturally occurring mutants for their reactivities to well-characterized neutralizing monoclonal antibodies (mAbs) and vaccine-induced antisera. A divergence in G sequences was found between vaccine strains and recent street isolates, with mutants demonstrating resistance to neutralizing mAbs and vaccine-induced antibodies. Moreover, antigenic variants were observed in a wide range of animal hosts and geographic locations, with most of them emerging since 2010. As the number of antigenic variants has increased in recent years, close monitoring on street isolates should be strengthened