Ex Vivo Propagation of Pinctada Birnavirus Using Mantle Tissue Fragment Culture: Application for Measuring Replication at Different Temperatures, TCID50 Assay, and UV Sensitivity

Figures

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  Figure 1. The image on the left shows the shell of a pearl oyster with the visceral mass removed. The image on the right shows the mantle region excised for this study. The regions are color-coded as follows: White: marginal zone, gray: pallial zone, dark gray: central zone.

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  Figure 2. Top panel: The PiBV copy number in individual pearl oysters (N = 4). Ex vivo cultivation was performed using three tissue fragments from the marginal, pallial, and central zones of the mantle from each oyster. The copy number of PiBV in the culture seawater was quantified on days 2, 4, 6, and 9 post-infection. The white bars represent the PiBV copy number on day 2, the light gray bars represent day 4, the gray bars represent day 6, and the black bars represent day 9. Bottom panel: Box plots showing the PiBV copy numbers from the marginal, pallial, and central zones of all four oysters using a total of 12 tissue fragments. The black dots indicate the actual PiBV levels in each tissue fragment. Significant differences among the three tissue zones on the same sampling day are indicated by three lowercase letters, while significant differences within the same tissue zone across the four sampling days are denoted by different uppercase letters (Steel–Dwass test, p < 0.05). The color coding of the bars is consistent with that described for the top panel: the white bars represent the PiBV copy number on day 2, the light gray bars represent day 4, the gray bars represent day 6, and the…

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  Figure 3. Ex vivo cultivation was performed using 12 mantle fragments from both the left and right sides of four oysters. The PiBV copy number in the culture medium was measured on day 7 post-infection. The open box represents the left mantle, while the gray box represents the right mantle. The black dots indicate the actual PiBV copy numbers in each tissue culture. Different letters denote significant differences among the different groups (Steel–Dwass test, p < 0.05). The boxes in the box plots indicate the interquartile range (IQR) with the median, and the whiskers extend to the most extreme data points within 1.5 times the IQR from the lower or upper quartile.

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  Figure 4. The PiBV copy number was measured on day 7 post-infection after ex vivo cultivation at different temperatures. Top panel: The results using pearl oysters reared at 16–18 °C (N = 8). Different combinations of letters indicate significant differences, as estimated by the Steel–Dwass test (p < 0.05). Bottom panel: The results from pearl oysters reared at 23–25 °C (N = 12). White and gray represent the left and right mantle, respectively. Detailed statistical test results are provided in Supplementary Table S1. The boxes in the box plots indicate the interquartile range (IQR) with the median, and the whiskers extend to the most extreme data points within 1.5 times the IQR from the lower or upper quartile.

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  Figure 5. Representative histological images of the mantle tissue from Pearl Oyster 6 on days 0, 2, 4, 7, and 10 (D0, D2, D4, D7, D10) are shown. Panel (A) presents low-magnification cross-sectional images of the mantle, while panels (B–D) are higher magnifications of the outer layer of the mantle shown in (A). Panel (D) is a higher magnification of panel (B), and panel (E) shows a high-magnification image of the neural tissue (arrow) from panel (A). No time-dependent tissue changes were observed on day 0, day 2, or day 4. On day 7, localized detachment of the epithelial cells was noted (D7-C). The arrowhead in D7-A indicates the detached epidermis, which corresponds to nearby detached tissue. On D10, the mantle exhibited atrophy, and the three folds (inner, middle, and outer folds) at the edge of the mantle were not distinguishable (D10-A). Additionally, on day 10, the epidermis showed widespread detachment and folding (arrowheads in D10-A, D10-C-1), and an accumulation of hemocytes beneath the epidermis was observed (arrowed area in D10-C-2). However, throughout the 10-day period, the ciliary structures on the outermost layer of the epithelial cells were still recognizable (D0, D2, D4,…

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  Figure 6. The mantle tissue fragments from the marginal zones of six pearl oysters reared at 16–18 °C and six oysters reared at 23–25 °C were used in this study. The tissue samples were infected with serial dilutions of PiBV and incubated at 25 °C. PiBV infection was assayed at day 7 post-infection to determine the TCID50. The two groups were compared using the Mann–Whitney U test. The boxes in the box plots indicate the interquartile range (IQR) with the median, and the whiskers extend to the most extreme data points within 1.5 times the IQR from the lower or upper quartile.

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  Figure 7. Each point on the graph shows the ratio of the titer following UV radiation treatment to the initial titer. Tissue fragments excised from the marginal zone of the mantle of four individual pearl oysters were pooled and randomly distributed into 96-well plates to determine the TCID50.