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. 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 exhibited 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 usually widespread and is transmitted among wild rats. Introduction Hepatitis E virus (HEV) is the causative agent of hepatitis E, a viral disease that manifests as acute hepatitis (Emerson & Purcell, 2003). The disease represents an important public health problem in developing countries and is transmitted primarily by the faecalCoral route (Balayan in the family (Emerson and 123 from and (nuclear polyhedrosis virus DNA (BaculoGold 21100D; Pharmingen) and either pVL1393-ORF2 or pVL1393-ORF2 by a Lipofectin-mediated method as specified by the manufacturer (Gibco-BRL). The cells were incubated at 26.5 C in TC-100 medium (Gibco-BRL) supplemented with 8?% FBS and 0.26?% bactotryptose phosphate broth (Difco Laboratories). The recombinant virus was plaque purified three times in Sf9 cells and designated Ac[ORF2] and Ac[ORF2], respectively. To achieve large-scale expression, Blonanserin an insect cell line from for 60 min. The supernatant was then spun at 32?000 r.p.m. for 3 h in a Beckman SW32Ti rotor, and the resulting pellet was resuspended in EX-CELL 405 medium at 4 C overnight. For sucrose-gradient centrifugation, 1 ml of each sample was laid on top of a 10C40?% (w/w) gradient and centrifuged at 32?000 r.p.m. for 2 h in a Beckman SW55Ti rotor. For CsCl-gradient centrifugation, 4.5 ml of each sample was mixed with 2.1 g CsCl and centrifuged at 35?000 r.p.m. for 24 h at 10 C in the same rotor. The gradient was fractionated into 250 l aliquots, and each fraction was weighed to estimate the buoyant density and isopycnic point. Each fraction was diluted with EX-CELL 405 medium and centrifuged for Blonanserin 2 h at 50?000 r.p.m. in a Beckman TLA55 rotor to sediment the HEVLPs. Electron microscopy. Purified HEVLPs were placed Pax1 on a carbon-coated grid for 45 s, rinsed with distilled water, stained with a 2?% uranyl acetate solution and examined under a JEOL TEM-1400 electron microscope operating at 80 kV. N-terminal amino acid sequence analysis. The proteins separated by SDS-PAGE were visualized by staining with GelCode Blue Staining Reagent (Pierce) and purified by sucrose-gradient centrifugation. N-terminal amino acid microsequencing was carried out using 100 pmol protein by Edman automated degradation on an Applied Biosystems Model 477 Protein Sequencer. Hyperimmune sera. Rabbits were immunized with rat, G1, G3 and G4 HEVLPs. Immunization was performed by one percutaneous injection of purified HEVLPs with a dose of 500 g per rabbit. Rats were immunized with the recombinant rat HEVLPs by intramuscular injection at a dose of 200 g per rat, and booster injections were carried out at 4 and 6 weeks after the first injection with half doses of rat HEVLPs. All of the injections, including booster injections, were carried out without adjuvant. Immunized animals were bled 3 weeks after the last injection. Rat serum samples. A total of 130 serum samples from laboratory rats (Wistar; Japan SLC) were collected at the Division for Experimental Animal Research of the National Institute of Infectious Diseases of Blonanserin Japan. A total of 139 serum samples from wild rats were collected in Vietnam (39 samples were collected in 2009 2009 in Haiphong, and 64 and 36 sera were collected in Hanoi and Haiphong in 2011, respectively). With regard to the rat species sampled, 123 were identified as and 16 were identified as and were performed with an unpaired Students em t /em -test. Acknowledgements The authors would like to thank Tomoko Mizoguchi for secretarial work. This study was supported in part by grants for Research on Emerging and Re-emerging Infectious Diseases, Research on Hepatitis and Research on Food Safety from the Ministry of Health, Labour, and Welfare of Japan. This work also was supported in part by the Japan Initiative for Global Research Network.