Two strains of epidemic hemorrhagic fever (EHF) virus were isolated from the lung tissues of Apodemus agrarius mice that were captured in an area where EHF is endemic. The strains were isolated by passages in A. agrarius mice from a nonendemic area. Identification of the isolates by usual procedures was confirmed by repeated blind tests with coded sera. Contamination with certain known viruses such as reovirus, adenovirus (types 3 and 7), and other pathogens, such as murine typhus rickettsiae and Leptospira, which may be naturally present in wild rodents, appeared to have been ruled out. The antigen slides made from these isolates are in use in the specific diagnosis and seroepidemiologic studies of EHF. The first successful application is the serodiagnosis of a mild type of hemorrhagic fever that occurs with characteristic epidemiologic features in certain provinces of China.

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In February and December of 2019 separately, two large-scale outbreaks of diarrhea were observed in the same swine farm with 3000 sows in Shanghai, China. We successfully isolated two PEDV isolates (strain shxx1902 and shxx1912) from clinical samples in this farm using suspension Vero cells in February and December, respectively. The third PEDV strain (SH1302) tested positive in the other farm of Shanghai in 2013 was also isolated using suspension Vero cells. The three isolates were better adapted to growth in adherent Vero cells through serial passages in the suspension Vero cells. The three isolated strains were detected positive by an immune- fluorescence assay and observed through electron microscopy. Phylogenetic analysis of the complete genomic sequence demonstrated that shxx1902(the 5th passage) and shxx1912(the 5th passage) clustered with a new GII subgroup (GII-c), which consisted of SINDEL strains from America (e.g., OH851), and their S gene belonged to GII-a. Both the strains(the 35th passage) have occurred dramatic changes in their genomes compared with the 5th passage. The 5th and 35th SH1302 belonged to GI-b genotype. The anti-N protein antibody titer of the strain shxx1902 was elevated to the same level as the vaccine strain(CV777) in mice. The use of the suspension Vero cells to isolate and propagate PEDV provide an effective approach for studies of the epidemiological characteristics and vaccine development of this virus.

In this study, one G2c-subtype strain of porcine epidemic diarrhea virus (PEDV) (SHXX1902 strain) was isolated from clinical samples in suspended Vero cells, which was different from the genotype of the commercial AJ1102 vaccine. As a result, we determined the pathogenicity of different passages’ isolates (SHXX1902 strain) and compared the immunogenicity of G2c-subtype strain (SHXX1902 strain) with the commercial AJ1102 vaccine. The viral titer reached 107 50% tissue culture infectious dose (TCID50)/ml, which met the requirement for seed virus replication during vaccine development. Five-day-old piglets were orally infected with viruses from passages P5 and P35 to determine the pathogenicity and immunogenicity of different passages. Pregnant sows were immunized with inactivated SHXX1902-P5 or the commercial AJ1102 vaccine (first immunized with an attenuated vaccine and then boosted with an inactivated vaccine) to study the influence of the culture method on the immunogenicity of the strain. The median pig diarrhea dose (PDD50) and the median lethal dose (LD50) of the P5 virus were 102.00 and 102.84 TCID50/ml, respectively. All five piglets infected with the SHXX1902-P5 virus shed the virus 24 h after vaccination, whereas only two of the five piglets treated with the SHXX1902-P35 virus shed the virus 48 h after vaccination. The SHXX1902-P35 virus was partially attenuated in the 5-day-old piglets. Inactivated SHXX1902-P5 induced PEDV-specific immunoglobulin G (IgG) antibody responses equivalent to those induced by AJ1102 after infection in sow serum. However, the IgA titer induced by AJ1102 was much higher than that induced by inactivated SHXX1902-P5 since the boost immunization. On days 5 and 7 after farrowing, the IgA titers were similar among the immunized groups. Our study highlights that serial passage can lead to the attenuation of G2c-subtype strain. The immunogenicity of the inactivated strain was similar to the commercial vaccine. Our observation helped conceptualize appropriate study designs for the PEDV vaccine.