DS1-4_JVDI_10.1177_1040638718773810 – Supplemental material for Development of in situ hybridization for detection of elephant endotheliotropic herpesvirus in Asian elephants

<p>Supplemental material, DS1-4_JVDI_10.1177_1040638718773810 for Development of in situ hybridization for detection of elephant endotheliotropic herpesvirus in Asian elephants by Varankpicha Kochakul, Kittikorn Boonsri, Saruda Tiwananthagorn, Chalermchart Somgird, Chatchote Thitaram, Kidsadagon Pringproa in Journal of Veterinary Diagnostic Investigation</p

The virus kinetic study from the supernatants of H5N1-infected hNPCs at different time points post infection.

<p>The hNPCs supported H5N1 virus infection as determined by a statistical significant increase of virus titers from 10² TCID₅₀/mL at 6 hpi, which reached a maximum titer of 10⁷ TCID₅₀/mL at 48 hpi. It subsequently declined to about 10⁴ TCID₅₀/mL at 72 hpi. Results were obtained from three separate experiments. Data represented are the mean ± standard error. Asterisks indicated statistically significant differences (<i>p</i>-value<0.05). Dotted line represents detection limit of the assay.</p

The expression of the mRNA levels of IFN-α2, IFN-β1, IFN-γ, TNF-α, and IL-6 in the hNPCs following infection with and without H5N1 virus by quantitative RT-PCR.

<p>The mRNA expressions of IFN-α2, IFN-β1, IFN-γ, and IL-6 were not significantly difference, while the mRNA level expression of TNF-α was observed to be significantly up-regulated at 48 hpi, compared to that of the control groups. Data are expressed as mean fold changes with standard error compared to untreated controls after normalization with GAPDH mRNA expressions. Asterisks indicated statistically significant differences (<i>p</i>-value<0.05).</p

The cytopathic effects (CPEs), electron micrographs, and average cell number of hNPCs following H5N1 virus infection.

<p>At 24 hpi, 48 hpi, and 72 hpi, H5N1-infected hNPCs revealed the pronounced CPEs, such as cell rounding up, vacuolation, short processes of the cells, and cell detachment, while no CPEs were observed in the mock-infected control cells (<b>A</b>). Transmission electron micrographs (<b>B)</b> showed the changes in hNPC induced by H5N1 virus by shrinkage and condensation of the nucleus and chromatin, disruption of the cytoplasmic membrane and increased in cellular vacuolation (N = nucleus; V = vacuolation; M: mitochondria). The detachment of H5N1-infected hNPCs was observed by the significant decreased of average cell number per field at 24–72 hpi compared to the UV-inactivated H5N1- and mock-infected controls (<b>C</b>). Scale bar in A ≈100 μm, scale bar in B ≈ 1μm.</p

The immunostaining and mRNA expression of specific genes of hNPCs after 7 days <i>invitro</i>.

<p>Human NPCs displayed a bi- to multipolar morphology (<b>A</b>). The mRNA expression of hNPCs by RT-PCR (<b>B</b>) revealed strong positivity for Nestin (lane 2) and Pax6 (lane 3), slight positivity for Sox1 (lane 4) genes, compared to that of GAPDH gene (Lane 1). Characterization of the cells with antibodies against Nestin, Pax6, and Sox1 revealed more than 85% were immunostaining positive for the Nestin (<b>C-E</b>), Pax6 (<b>F-H</b>) and Sox1 (<b>I-K</b>) antibodies. Scale bar ≈ 100 μm.</p

Immunostaining characterization and percentage of H5N1-positive cells of hNPCs following virus infection.

<p>Virus antigens were distributed within the cytoplasm and the nucleus of the infected cells (<b>A-C</b>), with peak percentage of virus-positive cells about 75% at 24 hpi, then declined to about 30% at 72 hpi (<b>D</b>). Scale bar in A-C ≈ 30 μm. Data represented are the mean ± standard error. Asterisks indicated statistically significant differences (<i>p</i>-value<0.05).</p

The differentiated phenotypic characterization of the hNPCs following infection with the H5N1, UV-inactivated H5N1, and mock-infected control.

<p>At 6–72 hpi, the expression of Nestin mRNA level was observed to be significantly down-regulated following virus infection, while the mRNA level expressions of β-III tubulin and GFAP were not found to show a significant difference compared to the mock infected control (<b>A</b>). Immunostaining of hNPCs at 72 hpi with antibodies against Nestin, GFAP and β-III tubulin in the H5N1-infected, UV-inactivated H5N1-infected, and mock-infected groups indicated that more than 80% of the cells were expressed Nestin, while stained negative for GFAP and β-III tubulin (<b>B, C</b>). Scale bar in B≈100 μm. Data are expressed as mean fold changes with standard error relative to mock-infected groups. Asterisks indicated statistically significant differences (<i>p</i>-value<0.05).</p