Hiroshi Aoki

Congenital tremor (CT) in piglets was first reported in 1922, and although the causative pathogen was unknown for many years, atypical porcine pestivirus (APPV) was recently shown to be the cause. APPV is difficult to isolate, and there have been few reports of APPV isolated from field materials. Here, we successfully isolated infectious particles from a tonsillar emulsion from a CT-affected piglet using the established swine-kidney-derived cell line SK-L. In addition, we produced APPV artificially using these cells. Thus, SK-L cells are useful for both isolation and artificial production of APPV.

Classical swine fever (CSF) live vaccine used in Japan, GPE- strain, is produced using guinea pig kidney (GPK)-derived primary culture cells. This means that a large number of guinea pigs are used to generate the primary GPK cells needed to produce the CSF live vaccine, and alternative solution is desired. Hence, we established two GPK cell lines capable of culturing the GPE- strain: spontaneously immortalized GPK (GPK-SI) cells were generated by repeated passaging of primary GPK cells, and the other cell line, artificially immortalized GPK (GPK-AI) cells, were obtained by introducing the SV40 large T antigen gene into primary GPK cells. Both cell lines were susceptible to the GPE- virus, and the virus grew more efficiently in GPK-SI cells at 37°C. When the culture temperature was set to 30°C, the virus titer reached 104.8 50% Tissue Culture Infectious Dose (TCID50)/mL in GPK-SI cells 7 days after virus inoculation at a multiplicity of infection (MOI) of 1, which was equivalent to that in cells cultured at 37°C. When the virus was inoculated at MOI <1, the virus titer 7 days after inoculation was higher when cultured at 30°C than when cultured at 37°C in both cell lines, reaching 105.63 TCID50/mL in GPK-SI cells. These results indicate that GPK-SI and GPK-AI cells can potentially replace primary GPK cells for the production of CSF live vaccines. This could also contribute to stable CSF vaccine production and animal welfare.

A novel picornavirus was isolated from the faeces of a diarrhoeic cow using MA-104 cells at the third blind passage. This virus, named Den1/2021/JPN, was completely sequenced using total RNA from the cell culture supernatant by deep sequencing. The genome of Den1/2021/JPN had a standard picornavirus genome organisation with conserved picornaviral motifs. The 5' untranslated region harboured a type-II internal ribosomal entry site. Den1/2021/JPN was most closely related to a bovine parechovirus (Bo_ParV) named cow/2018/4, which has been recently identified in publicly available databases. Phylogenetic analyses and pairwise sequence comparison revealed that Den1/2021/JPN and Bo_ParV cow/2018/4 clustered with parechoviruses and were most closely related to Parechovirus E identified in birds of prey, exhibiting nucleotide sequence similarity of 64.2-64.5 %, 58.6-59.7 % and 66.3-66.4 % in the polyprotein, P1 and 2C+3 CD coding regions, respectively. This study presents the first report on the isolation of Bo_ParV. Den1/2021/JPN and Bo_ParV cow/2018/4, which are candidates for a novel species in the genus Parechovirus.

Bovine-derived cultured cells, including Madin-Darby bovine kidney cells, are used worldwide; however, lipofection tend to result in low transfection efficiency, which has impeded the progress of veterinary research. We performed experiments to confirm the lipofection efficiency of bovine-derived cultured cells, to identify cells that suitable for lipofection. Several bovine tissues (endometrium, testis, ear tissue and foetal muscle) were collected, and primary cultured cells were prepared. Lipofection assay showed that only bovine endometrium (BE)-derived cells could be transfected efficiently (50‒70%). BE cells can be divided into at least two types of cell populations (BE-1 and BE-2). The BE-1 cells, which were suitable for lipofection, were obtained by passages at short intervals and were negative for cytokeratin- and positive for vimentin-expression; the BE-2 cells did not have these characteristics and were not suitable for lipofection. Furthermore, the BE-1 cells and artificially immortalised cells of BE-1, iBE-1 cells, were utilised in a reporter assay requiring the introduction of multiple DNAs. Endometrial tissues can be collected from living cows, and BE-1 cells can be obtained easily by controlling passaging timing. The production of BE-1 cells and sharing the methods required to prepare them will contribute to the development of veterinary research.