The dynamics of a Chinese porcine G9P[23] rotavirus production in MA-104 cells and intestines of 3-day-old piglets

Abstract

Rotavirus A (RVA) G9 genotype is recognized as an emerging genotype which is spreading worldwide, however, our knowledge on pathogenicity of this virus is limited. In this study, porcine RVA strain HN03 was successfully isolated on MA-104 cells, and the isolate was propagated continuously for 7 passages after a virus cloning at passage 3. The virus titers from 4 to 10 passages ranged from 10^7.1 to 10^8.1 TCID₅₀/ml. The growth curve of HN03 strain in cell culture was determined, and the virus production dynamics was confirmed by immunoperoxidase monolayer assay (IPMA). Sequence and phylogenetic analyses based on full-length VP7 and partial VP4 genes indicated that HN03 strain belongs to genotype G9P[23]. In addition, the sixth passage of strain HN03 in cell culture was subjected to 3-day-old piglets. All infected piglets developed severe watery diarrhea within 24 hr post-inoculation (hpi), but recovered from disease after 72 hpi. RVA antigen could be detected by IHC in the cytoplasm of villous enterocytes as early as 2 hr after appearance of clinical symptoms and virus antigen load kept increasing in the next 30 hr. The dynamics of RVA HN03 strain proliferation on cells and in pigs extended our understanding of rotavirus pathogenicity.

Keywords: G9 genotype; G9P[23]; pathogenicity; porcine rotavirus.

Fig. 1.

Growth curve of cell-adapted porcine RVA strain HN03 in MA-104 cell culture. Data was presented as mean ± SD by triplicates.

Fig. 2.

Detection of porcine RVA antigen from MA-104 cell cultures by IPMA at different time points. Panels A−I represent 6, 9, 12, 15, 18, 21, 24, 27 and 30 hpi, respectively, and panel J represents MA-104 cells without infection at 30 dpi. Magnification 200 ×. A–J: Bar=50 μm.

Fig. 3.

Phylogenetic relationship among porcine RVA HN03 in this study and reference sequences in GenBank, as inferred by neighbor-joining analyses of the full-length VP7 (A) and partial VP4 (B) nucleotide sequences based on distance calculated using the Kimura 2-parameter model. Bootstrap values (in percentage) above 50 from 1,000 pseudoreplicates are shown. The isolate in this study is marked by closed circle.

Fig. 4.

Immunohistochemistry staining of small-intestine sections from infected and control pigs. Porcine RVA antigen (brown reaction product) was detected in the villous enterocytes. Magnification 200 ×. Bar=50 μm.