Ferret hepatitis E virus (HEV), a novel hepatitis E-like virus, has been identified in ferrets in The Netherlands. Due to the lack of a cell-culture system for ferret HEV, the antigenicity, pathogenicity and epidemiology of this virus have remained unclear. In the present study, we used a recombinant baculovirus expression system to express the 112-N-terminus and 47-C-terminus-amino-acid-truncated ferret HEV ORF2 protein in insect Tn5 cells, and found that a large amount of a 53 kDa protein (F-p53) was expressed and efficiently released into the supernatant. Electron microscopic analysis revealed that F-p53 was self-assembled into virus-like particles (ferret HEV-LPs). These ferret HEV-LPs were estimated to be 24 nm in diameter, which is similar to the size of G1, G3, G4 and rat HEV-LPs derived from both the N-terminus- and C-terminus-truncated constructs. Antigenic analysis demonstrated that ferret HEV-LPs were cross-reactive with G1, G3, G4 and rat HEVs, and rat HEV and ferret HEV showed a stronger cross-reactivity to each other than either did to human HEV genotypes. However, the antibody against ferret HEV-LPs does not neutralize G3 HEV, suggesting that the serotypes of these two HEVs are different. An ELISA for detection of anti-ferret HEV IgG and IgM antibodies was established using ferret HEV-LPs as antigen, and this assay system will be useful for monitoring ferret HEV infection in ferrets as well as other animals. In addition, analysis of ferret HEV RNA detected in ferret sera collected from a breeding colony in the USA revealed the genetic diversity of ferret HEV.

Genotype 1 hepatitis E virus (HEV-1), unlike other genotypes of HEV, has a unique small open reading frame known as ORF4 whose function is not yet known. ORF4 is located in an out-framed manner in the middle of ORF1, which encodes putative 90 to 158 amino acids depending on the strains. To explore the role of ORF4 in HEV-1 replication and infection, we cloned the complete genome of wild-type HEV-1 downstream of a T7 RNA polymerase promoter, and the following ORF4 mutant constructs were prepared: the first construct had TTG instead of the initiation codon ATG (A2836T), introducing an M→L mutation in ORF4 and a D→V mutation in ORF1. The second construct had ACG instead of the ATG codon (T2837C), introducing an M→T mutation in ORF4. The third construct had ACG instead of the second in-frame ATG codon (T2885C), introducing an M→T mutation in ORF4. The fourth construct contained two mutations (T2837C and T2885C) accompanying two M→T mutations in ORF4. For the latter three constructs, the accompanied mutations introduced in ORF1 were all synonymous changes. The capped entire genomic RNAs were generated by in vitro transcription and used to transfect PLC/PRF/5 cells. Three mRNAs containing synonymous mutations in ORF1, i.e., T2837C^RNA, T2885C^RNA, and T2837C/T2885C^RNA, replicated normally in PLC/PRF/5 cells and generated infectious viruses that successfully infected Mongolian gerbils as the wild-type HEV-1 did. In contrast, the mutant RNA, i.e., A2836T^RNA, accompanying an amino acid change (D937V) in ORF1 generated infectious viruses upon transfection, but they replicated slower than the wild-type HEV-1 and failed to infect Mongolian gerbils. No putative viral protein(s) derived from ORF4 were detected in the wild-type HEV-1- as well as the mutant virus-infected PLC/PRF/5 cells by Western blot analysis using a high-titer anti-HEV-1 IgG antibody. These results demonstrated that the ORF4-defective HEV-1s had the ability to replicate in the cultured cells, and that these defective viruses had the ability to infect Mongolian gerbils unless the overlapping ORF1 was accompanied by non-synonymous mutation(s), confirming that ORF4 is not essential in the replication and infection of HEV-1.

Since Mongolian gerbils are broadly susceptible to hepatitis E virus (HEV), including genotypes 1, 4, 5, and 8 (HEV-1, HEV-5, HEV-5, and HEV-8) and rat HEV, they are a useful small animal model for HEV. However, we have observed that the subtypes HEV-3k and HEV-3ra in genotype 3 HEV (HEV-3) were not infected efficiently in the gerbils. A small-animal model for HEV-3 is also needed since HEV-3 is responsible for major zoonotic HEV infections. To investigate whether gerbils can be used as animal models for other subtypes of HEV-3, we injected gerbils with five HEV-3 subtypes (HEV-3b, -3e, -3f, -3k, and -3ra) and compared the infectivity of the subtypes. We detected viral RNA in the gerbils’ feces. High titers of anti-HEV IgG antibodies in serum were induced in all HEV-3b/ch-, HEV-3f-, and HEV-3e-injected gerbils. Especially, the HEV-3e-injected animals released high levels of viruses into their feces for an extended period. The virus replication was limited in the HEV-3b/wb-injected and HEV-3k-injected groups. Although viral RNA was detected in HEV-3ra-injected gerbils, the copy numbers in fecal specimens were low; no antibodies were detected in the sera. These results indicate that although HEV-3′s infectivity in gerbils depends on the subtype and strain, Mongolian gerbils have potential as a small-animal model for HEV-3. A further comparison of HEV-3e with different genotype strains (HEV-4i and HEV-5) and different genera (rat HEV) revealed different ALT elevations among the strains, and liver damage occurred in HEV-4i- and HEV-5-infected but not HEV-3e- or rat HEV-infected gerbils, demonstrating variable pathogenicity across HEVs from different genera and genotypes in Mongolian gerbils. HEV-4i- and HEV-5-infected Mongolian gerbils might be candidate animal models to examine HEV’s pathogenicity.

To be effective, a vaccine against human immunodeficiency virus type 1 (HIV-1) must induce virus-specific T-cell responses and it must be safe for use in humans. To address these issues, we developed a recombinant vaccinia virus DIs vaccine (rDIsSIVGag), which is nonreplicative in mammalian cells and expresses the full-lengthgaggene of simian immunodeficiency virus (SIV). Intravenous inoculation of 10⁶PFU of rDIsSIVGag in cynomologus macaques induced significant levels of gamma interferon (IFN-γ) spot-forming cells (SFC) specific for SIV Gag. Antigen-specific lymphocyte proliferative responses were also induced and were temporally associated with the peak of IFN-γ SFC activity in each macaque. In contrast, macaques immunized with a vector control (rDIsLacZ) showed no significant induction of antigen-specific immune responses. After challenge with a highly pathogenic simian-human immunodeficiency virus (SHIV), CD4⁺T lymphocytes were maintained in the peripheral blood and lymphoid tissues of the immunized macaques. The viral set point in plasma was also reduced in these animals, which may be related to the enhancement of virus-specific intracellular IFN-γ⁺CD8⁺cell numbers and increased antibody titers after SHIV challenge. These results demonstrate that recombinant DIs has potential for use as an HIV/AIDS vaccine.

The live-attenuated oral polio vaccine has long been used as the standard for polio prevention, but in order to minimize the emergence of pathogenic revertants, the inactivated polio vaccine (IPV), which is administered intramuscularly or subcutaneously, is being increasingly demanded worldwide. However, there is a global shortage of IPV, and its cost is an obstacle in developing countries. Therefore, dose-sparing with intradermal administration of IPV has been investigated. In this study, rats were immunized by intradermal (ID) and intra-muscular (IM) administration of Sabin-derived inactivated polio vaccine (sIPV) produced in Japan, and the immune responses were evaluated. The results showed that one-fifth (1/5)-dose of ID administration yielded neutralizing antibody titers comparable to the full-dose IM administration, whereas 1/5-dose of IM adminis-tration was less effective than the full dose. Furthermore, a vertical puncture-type ID injection device (Immucise) that was originally developed for humans was modified for rats, resulting in successful and stable ID adminis-tration into the thin skin of rats. Based on these results, the ID administration of sIPV using Immucise in clinical use is expected to offer benefits such as reduced amounts of vaccine per dose, cost-effectiveness, and thereby the feasibility of vaccination for more people.