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Jaagsiekte sheep retrovirus infection of lung slice cultures.

Cousens C, Alleaume C, Bijsmans E, Martineau HM, Finlayson J, Dagleish MP, Griffiths DJ - Retrovirology (2015)

Bottom Line: Interestingly, the JSRV Env protein is directly oncogenic and capable of driving cellular transformation in vivo and in vitro.JSRV was able to infect cells within lung slices, to produce new infectious virions and induce cell proliferation.Immunohistochemical labeling revealed that infected lung slice cells express markers of type II pneumocytes and phosphorylated Akt and ERK1/2.

View Article: PubMed Central - PubMed

Affiliation: Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Edinburgh, UK. chris.cousens@moredun.ac.uk.

ABSTRACT

Background: Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA), a transmissible neoplastic disease of sheep. OPA is an economically important veterinary disease and is also a valuable naturally occurring animal model of human lung cancer, with which it shares a similar histological appearance and the activation of common cell signaling pathways. Interestingly, the JSRV Env protein is directly oncogenic and capable of driving cellular transformation in vivo and in vitro. Previous studies of JSRV infection in cell culture have been hindered by the lack of a permissive cell line for the virus. Here, we investigated the ability of JSRV to infect slices of ovine lung tissue cultured ex vivo.

Results: We describe the use of precision cut lung slices from healthy sheep to study JSRV infection and transformation ex vivo. Following optimization of the culture system we characterized JSRV infection of lung slices and compared the phenotype of infected cells to natural field cases and to experimentally-induced OPA tumors from sheep. JSRV was able to infect cells within lung slices, to produce new infectious virions and induce cell proliferation. Immunohistochemical labeling revealed that infected lung slice cells express markers of type II pneumocytes and phosphorylated Akt and ERK1/2. These features closely resemble the phenotype of natural and experimentally-derived OPA in sheep, indicating that lung slice culture provides an authentic ex vivo model of OPA.

Conclusions: We conclude that we have established an ex vivo model of JSRV infection. This model will be valuable for future studies of JSRV replication and early events in oncogenesis and provides a novel platform for studies of JSRV-induced lung cancer.

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JSRV-infected lung slice and early experimental OPA show a similar pattern of JSRV-SU labeling. Sections of JSRV-infected tissue were labeled with a monoclonal antibody to JSRV Env (SU) by IHC. Positive labeling is shown by brown pigment. A, B) JSRV-infected lung slice 8 dpi; C, D) JSRV-infected lung slice 16 dpi; E, F) Lung tissue from lambs experimentally infected with JSRV 10 dpi; G, H) Lung slices infected with control virus JSRV-∆RT 16 dpi showed no labeling. I, J) Lung slices infected with pooled supernatant of previous JSRV-infected lung slices 12 dpi. K, L) Lung slices infected with pooled supernatant of previous JSRV-∆RT infected lung slices 12 dpi. Dashed circles in A, C, E, G, I and K indicate the regions shown at higher magnification in B, D, F, H, J and L, respectively.
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Fig3: JSRV-infected lung slice and early experimental OPA show a similar pattern of JSRV-SU labeling. Sections of JSRV-infected tissue were labeled with a monoclonal antibody to JSRV Env (SU) by IHC. Positive labeling is shown by brown pigment. A, B) JSRV-infected lung slice 8 dpi; C, D) JSRV-infected lung slice 16 dpi; E, F) Lung tissue from lambs experimentally infected with JSRV 10 dpi; G, H) Lung slices infected with control virus JSRV-∆RT 16 dpi showed no labeling. I, J) Lung slices infected with pooled supernatant of previous JSRV-infected lung slices 12 dpi. K, L) Lung slices infected with pooled supernatant of previous JSRV-∆RT infected lung slices 12 dpi. Dashed circles in A, C, E, G, I and K indicate the regions shown at higher magnification in B, D, F, H, J and L, respectively.

Mentions: We then examined JSRV-infected lung slices by IHC using an antibody to the JSRV Env (SU) protein [29]. Foci of SU-positive cells were detectable from 4 dpi onwards (Figure 3A-F), but the number of positive cells per section was highly variable even within a single experiment (i.e., replicate lung slices from a single donor animal) and the proportion of positive sections varied between experiments (i.e., lung slices from different donor animals). For example, in the experiment shown in Figure 3A-D, at 8 dpi only 1 group of SU-positive cells was detected from sections of 4 replicate lung slices whereas at 12 or 16 dpi two or three groups of cells expressing JSRV SU were visible in the sections of 3 of the 4 lung slice replicates. The histological appearance of clusters of labeled cells in JSRV-infected lung slices (Figure 3A-D) was indistinguishable from early nodules in OPA-E (Figure 3E-F). SU-labeling of cells was localized in the cytoplasm and at the cell surface with stronger labeling at the apical pole. SU-labeling was not seen in any of the uninfected lung slices nor in JSRV-∆RT-treated lung slices (Figure 3G, H), thus confirming the specificity of the anti-SU antibody in IHC.Figure 3


Jaagsiekte sheep retrovirus infection of lung slice cultures.

Cousens C, Alleaume C, Bijsmans E, Martineau HM, Finlayson J, Dagleish MP, Griffiths DJ - Retrovirology (2015)

JSRV-infected lung slice and early experimental OPA show a similar pattern of JSRV-SU labeling. Sections of JSRV-infected tissue were labeled with a monoclonal antibody to JSRV Env (SU) by IHC. Positive labeling is shown by brown pigment. A, B) JSRV-infected lung slice 8 dpi; C, D) JSRV-infected lung slice 16 dpi; E, F) Lung tissue from lambs experimentally infected with JSRV 10 dpi; G, H) Lung slices infected with control virus JSRV-∆RT 16 dpi showed no labeling. I, J) Lung slices infected with pooled supernatant of previous JSRV-infected lung slices 12 dpi. K, L) Lung slices infected with pooled supernatant of previous JSRV-∆RT infected lung slices 12 dpi. Dashed circles in A, C, E, G, I and K indicate the regions shown at higher magnification in B, D, F, H, J and L, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4419405&req=5

Fig3: JSRV-infected lung slice and early experimental OPA show a similar pattern of JSRV-SU labeling. Sections of JSRV-infected tissue were labeled with a monoclonal antibody to JSRV Env (SU) by IHC. Positive labeling is shown by brown pigment. A, B) JSRV-infected lung slice 8 dpi; C, D) JSRV-infected lung slice 16 dpi; E, F) Lung tissue from lambs experimentally infected with JSRV 10 dpi; G, H) Lung slices infected with control virus JSRV-∆RT 16 dpi showed no labeling. I, J) Lung slices infected with pooled supernatant of previous JSRV-infected lung slices 12 dpi. K, L) Lung slices infected with pooled supernatant of previous JSRV-∆RT infected lung slices 12 dpi. Dashed circles in A, C, E, G, I and K indicate the regions shown at higher magnification in B, D, F, H, J and L, respectively.
Mentions: We then examined JSRV-infected lung slices by IHC using an antibody to the JSRV Env (SU) protein [29]. Foci of SU-positive cells were detectable from 4 dpi onwards (Figure 3A-F), but the number of positive cells per section was highly variable even within a single experiment (i.e., replicate lung slices from a single donor animal) and the proportion of positive sections varied between experiments (i.e., lung slices from different donor animals). For example, in the experiment shown in Figure 3A-D, at 8 dpi only 1 group of SU-positive cells was detected from sections of 4 replicate lung slices whereas at 12 or 16 dpi two or three groups of cells expressing JSRV SU were visible in the sections of 3 of the 4 lung slice replicates. The histological appearance of clusters of labeled cells in JSRV-infected lung slices (Figure 3A-D) was indistinguishable from early nodules in OPA-E (Figure 3E-F). SU-labeling of cells was localized in the cytoplasm and at the cell surface with stronger labeling at the apical pole. SU-labeling was not seen in any of the uninfected lung slices nor in JSRV-∆RT-treated lung slices (Figure 3G, H), thus confirming the specificity of the anti-SU antibody in IHC.Figure 3

Bottom Line: Interestingly, the JSRV Env protein is directly oncogenic and capable of driving cellular transformation in vivo and in vitro.JSRV was able to infect cells within lung slices, to produce new infectious virions and induce cell proliferation.Immunohistochemical labeling revealed that infected lung slice cells express markers of type II pneumocytes and phosphorylated Akt and ERK1/2.

View Article: PubMed Central - PubMed

Affiliation: Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Edinburgh, UK. chris.cousens@moredun.ac.uk.

ABSTRACT

Background: Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA), a transmissible neoplastic disease of sheep. OPA is an economically important veterinary disease and is also a valuable naturally occurring animal model of human lung cancer, with which it shares a similar histological appearance and the activation of common cell signaling pathways. Interestingly, the JSRV Env protein is directly oncogenic and capable of driving cellular transformation in vivo and in vitro. Previous studies of JSRV infection in cell culture have been hindered by the lack of a permissive cell line for the virus. Here, we investigated the ability of JSRV to infect slices of ovine lung tissue cultured ex vivo.

Results: We describe the use of precision cut lung slices from healthy sheep to study JSRV infection and transformation ex vivo. Following optimization of the culture system we characterized JSRV infection of lung slices and compared the phenotype of infected cells to natural field cases and to experimentally-induced OPA tumors from sheep. JSRV was able to infect cells within lung slices, to produce new infectious virions and induce cell proliferation. Immunohistochemical labeling revealed that infected lung slice cells express markers of type II pneumocytes and phosphorylated Akt and ERK1/2. These features closely resemble the phenotype of natural and experimentally-derived OPA in sheep, indicating that lung slice culture provides an authentic ex vivo model of OPA.

Conclusions: We conclude that we have established an ex vivo model of JSRV infection. This model will be valuable for future studies of JSRV replication and early events in oncogenesis and provides a novel platform for studies of JSRV-induced lung cancer.

Show MeSH
Related in: MedlinePlus