Characterization of self-assembled virus-like particles of rat hepatitis E virus generated by recombinant baculoviruses

Abstract

Hepatitis E virus (HEV) is a causative agent of hepatitis E. Recently, a novel hepatitis E-like virus was isolated from Norway rats in Germany. However, the antigenicity, pathogenicity and epidemiology of this virus are unclear because of the lack of a cell-culture system in which to grow it. In this study, an N-terminally truncated ORF2 protein was expressed in insect Tn5 cells using a recombinant baculovirus expression system and a large amount of 53 kDa protein was expressed and efficiently released into the supernatant. Electron microscopic analyses of the purified 53 kDa protein revealed that the protein self-assembled into two types of empty HEV-like particles (rat HEVLPs). The smaller rat HEVLPs were estimated to be 24 nm in diameter, which is similar to the size of genotype G1, G3 and G4 HEVLPs. The larger rat HEVLPs were estimated to measure 35 nm in diameter, which is similar to the size of native rat HEV particles. An ELISA to detect antibodies was established using rat HEVLPs as the antigens, which demonstrated that rat HEVLPs were cross-reactive with G1, G3 and G4 HEVs. Detection of IgG and IgM antibodies was performed by examination of 139 serum samples from wild rats trapped in Vietnam, and it was found that 20.9 % (29/139) and 3.6 % (5/139) of the samples were positive for IgG and IgM, respectively. In addition, rat HEV RNA was detected in one rat serum sample that was positive for IgM. These results indicated that rat HEV is widespread and is transmitted among wild rats.

Construction and characterization of an infectious cDNA clone of rat hepatitis E virus

“…An antigen capture ELISA was used to detect rat HEV antigen. Briefly, duplicate wells of flat-bottom 96-well polystyrene microplates (Dynex) were coated with 100 ml coating buffer (0.1 M carbonate/ bicarbonate buffer, pH 9.6) containing a 1 : 1000 dilution of a hyperimmune serum elicited in a rabbit with rat HEV-like particles (Li et al, 2011). The coating was performed at 4 uC overnight.…”

“…The whole-genome sequence of rat HEV present in selected samples was analysed by next-generation sequencing. Virus particles were enriched from the samples by caesium chloride density-gradient centrifugation (Li et al, 2011). A 200 bp fragment library was constructed for each sample preparation using a NEBNext Ultra RNA Library Prep kit for Illumina version 2.0 (New England Biolabs) according to the manufacturer's instructions.…”

“…Antirat HEV IgG was detected by ELISA as described previously (Li et al, 2011). An antigen capture ELISA was used to detect rat HEV antigen.…”

The development of a recombinant hepatitis E vaccine HEV 239

“…With truncations in the 111 N-terminal aa and the 53 C-terminal aa, the protein can self-assemble into a T D 1 icosahedral VLP via a baculovirus / insect cell expression system. 10 The VLP form of genotypes 3 and 4 can be crystallized to allow the structural determination at a resolution of 3.5 A . 11,12 The analysis of these structures has revealed 3 functional domains of pORF2, including the S domain (aa 118-313), which contributes to the 5-fold interaction and the formation of the basal shell; the P1 domain (aa 314-453), which is important for the 3-fold interaction; and the P2 domain (aa 454-606), which is involved in the 2-fold interaction and is responsible for host interactions.…”

Construction of an Infectious cDNA Clone of a Swine Genotype 3 HEV Strain Isolated in Shanghai, China

“…Epidemics of genotype 3 have been reported in many regions including North America, Europe and some Asian countries [23,24,25,26,27,28], and of genotype 4 in several Asian countries. Recently, several other novel hepeviruses have been isolated from HEV infections of rabbits [29], rats [30,31,32,33,34], bats [35] and cut-throat trout [36]. Among the four mammalian genotypes, genotypes 1 and 2 have so far been restricted to humans and associated with large waterborne epidemics [37], while accumulating evidence indicates that genotypes 3 and 4 are common in both humans and animals, and are responsible for sporadic cases of hepatitis E [30,38,39].…”