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Healthy rabbits are susceptible to Epstein-Barr virus infection and infected cells proliferate in immunosuppressed animals.

Khan G, Ahmed W, Philip PS, Ali MH, Adem A - Virol. J. (2015)

Bottom Line: Quantitative PCR indicated an increase in EBV viral load in PBMCs as the duration of immunosuppression increased.Western blotting indicated that EBNA-1 and EBNA-2 were also expressed in the liver and spleen of infected animals.The infected cells expressed several EBV-latent gene products which are probably driving the proliferation, reminiscent of what is seen in immunocompromised individuals.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Epstein-Barr virus (EBV) is an oncogenic virus implicated in the pathogenesis of several human malignancies. However, due to the lack of a suitable animal model, a number of fundamental questions pertaining to the biology of EBV remain poorly understood. Here, we explore the potential of rabbits as a model for EBV infection and investigate the impact of immunosuppression on viral proliferation and gene expression.

Methods: Six healthy New Zealand white rabbits were inoculated intravenously with EBV and blood samples collected prior to infection and for 7 weeks post-infection. Three weeks after the last blood collection, animals were immunosuppressed with daily intramuscular injections of cyclosporin A at doses of 20 mg/kg for 15 days and blood collected twice a week from each rabbit. The animals were subsequently sacrificed and tissues from all major organs were collected for subsequent analysis.

Results: Following intravenous inoculation, all 6 rabbits seroconverted with raised IgG and IgM titres to EBV, but viral DNA in peripheral blood mononuclear cells (PBMCs) could only be detected intermittently. Following immunosuppression however, EBV DNA could be readily detected in PBMCs from all 4 rabbits that survived the treatment. Quantitative PCR indicated an increase in EBV viral load in PBMCs as the duration of immunosuppression increased. At autopsy, splenomegaly was seen in 3/4 rabbits, but spleens from all 4 rabbit were EBV PCR positive. EBER-in situ hybridization and immunoshistochemistry revealed the presence of a large number of EBER-positive and LMP-1 positive lymphoblasts in the spleens of 3/4 rabbits. To a lesser extent, EBER-positive cells were also seen in the portal tract regions of the liver of these rabbits. Western blotting indicated that EBNA-1 and EBNA-2 were also expressed in the liver and spleen of infected animals.

Conclusion: EBV can infect healthy rabbits and the infected cells proliferate when the animals are immunocompromised. The infected cells expressed several EBV-latent gene products which are probably driving the proliferation, reminiscent of what is seen in immunocompromised individuals. Further work is required to explore the potential of rabbits as an animal model for studying EBV biology and tumorigenesis.

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Immune responses to EBV. IgM (A) and IgG (B) responses to EBV infection in 6 healthy rabbits (Rbt 415–420) over a period of 7 weeks post infection. Generally, IgM levels were elevated in the first week post-infection and then tailed off by week 5. In contrast, IgG levels gradually increased and remained high up to the 7 weeks follow up. In one rabbit (Rbt 419), both the IgM and IgG responses to EBV were delayed compared to the other animals, peaking at week 4 for IgM and week 7 for IgG (C). The data presented here is for plasma at dilution 1/100. Similar patterns were seen with plasma diluted 1/200 and 1/400, but with lower OD values.
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Fig1: Immune responses to EBV. IgM (A) and IgG (B) responses to EBV infection in 6 healthy rabbits (Rbt 415–420) over a period of 7 weeks post infection. Generally, IgM levels were elevated in the first week post-infection and then tailed off by week 5. In contrast, IgG levels gradually increased and remained high up to the 7 weeks follow up. In one rabbit (Rbt 419), both the IgM and IgG responses to EBV were delayed compared to the other animals, peaking at week 4 for IgM and week 7 for IgG (C). The data presented here is for plasma at dilution 1/100. Similar patterns were seen with plasma diluted 1/200 and 1/400, but with lower OD values.

Mentions: Six healthy male New Zealand white rabbits aged 2–4 months (0.8-1.2 kg) were exposed to EBV by intravenous injection of 1 ml of cell-free, 0.45 μm filtered culture supernatant from B95-8 cell line. Each animal was inoculated 3 times on alternative days. The average EBV copy number in each inoculum, as determined by quantitative PCR (qPCR) [39] was estimated to be 1.74 × 107 copies. The infectivity of the virus in the culture supernatant was assessed by its ability to successfully immortalize human PBMCs in vitro [40]. All 6 animals seroconverted and mounted a strong antibody response, but none developed any systemic signs of acute EBV infection. As expected, IgM was the first antibody to be triggered. In general, IgM levels were highest in week 1 and then gradually declined to background levels by week 5 (Figure 1A). IgG levels on the other hand were low to start with, but then increased and remained fairly high during the 7 weeks follow up (Figure 1B). All the OD readings were calculated relative to pre-infection plasma. Interestingly, in one rabbit (Rbt 419), both the IgG and IgM responses, albeit strong, were delayed compared to the other animals and peaked at weeks 4 for IgM and 7 for IgG (Figure 1C). The significance of this observation is not clear, however it is noteworthy that this animal subsequently developed an extensive and widespread EBV infection.Figure 1


Healthy rabbits are susceptible to Epstein-Barr virus infection and infected cells proliferate in immunosuppressed animals.

Khan G, Ahmed W, Philip PS, Ali MH, Adem A - Virol. J. (2015)

Immune responses to EBV. IgM (A) and IgG (B) responses to EBV infection in 6 healthy rabbits (Rbt 415–420) over a period of 7 weeks post infection. Generally, IgM levels were elevated in the first week post-infection and then tailed off by week 5. In contrast, IgG levels gradually increased and remained high up to the 7 weeks follow up. In one rabbit (Rbt 419), both the IgM and IgG responses to EBV were delayed compared to the other animals, peaking at week 4 for IgM and week 7 for IgG (C). The data presented here is for plasma at dilution 1/100. Similar patterns were seen with plasma diluted 1/200 and 1/400, but with lower OD values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4340116&req=5

Fig1: Immune responses to EBV. IgM (A) and IgG (B) responses to EBV infection in 6 healthy rabbits (Rbt 415–420) over a period of 7 weeks post infection. Generally, IgM levels were elevated in the first week post-infection and then tailed off by week 5. In contrast, IgG levels gradually increased and remained high up to the 7 weeks follow up. In one rabbit (Rbt 419), both the IgM and IgG responses to EBV were delayed compared to the other animals, peaking at week 4 for IgM and week 7 for IgG (C). The data presented here is for plasma at dilution 1/100. Similar patterns were seen with plasma diluted 1/200 and 1/400, but with lower OD values.
Mentions: Six healthy male New Zealand white rabbits aged 2–4 months (0.8-1.2 kg) were exposed to EBV by intravenous injection of 1 ml of cell-free, 0.45 μm filtered culture supernatant from B95-8 cell line. Each animal was inoculated 3 times on alternative days. The average EBV copy number in each inoculum, as determined by quantitative PCR (qPCR) [39] was estimated to be 1.74 × 107 copies. The infectivity of the virus in the culture supernatant was assessed by its ability to successfully immortalize human PBMCs in vitro [40]. All 6 animals seroconverted and mounted a strong antibody response, but none developed any systemic signs of acute EBV infection. As expected, IgM was the first antibody to be triggered. In general, IgM levels were highest in week 1 and then gradually declined to background levels by week 5 (Figure 1A). IgG levels on the other hand were low to start with, but then increased and remained fairly high during the 7 weeks follow up (Figure 1B). All the OD readings were calculated relative to pre-infection plasma. Interestingly, in one rabbit (Rbt 419), both the IgG and IgM responses, albeit strong, were delayed compared to the other animals and peaked at weeks 4 for IgM and 7 for IgG (Figure 1C). The significance of this observation is not clear, however it is noteworthy that this animal subsequently developed an extensive and widespread EBV infection.Figure 1

Bottom Line: Quantitative PCR indicated an increase in EBV viral load in PBMCs as the duration of immunosuppression increased.Western blotting indicated that EBNA-1 and EBNA-2 were also expressed in the liver and spleen of infected animals.The infected cells expressed several EBV-latent gene products which are probably driving the proliferation, reminiscent of what is seen in immunocompromised individuals.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Epstein-Barr virus (EBV) is an oncogenic virus implicated in the pathogenesis of several human malignancies. However, due to the lack of a suitable animal model, a number of fundamental questions pertaining to the biology of EBV remain poorly understood. Here, we explore the potential of rabbits as a model for EBV infection and investigate the impact of immunosuppression on viral proliferation and gene expression.

Methods: Six healthy New Zealand white rabbits were inoculated intravenously with EBV and blood samples collected prior to infection and for 7 weeks post-infection. Three weeks after the last blood collection, animals were immunosuppressed with daily intramuscular injections of cyclosporin A at doses of 20 mg/kg for 15 days and blood collected twice a week from each rabbit. The animals were subsequently sacrificed and tissues from all major organs were collected for subsequent analysis.

Results: Following intravenous inoculation, all 6 rabbits seroconverted with raised IgG and IgM titres to EBV, but viral DNA in peripheral blood mononuclear cells (PBMCs) could only be detected intermittently. Following immunosuppression however, EBV DNA could be readily detected in PBMCs from all 4 rabbits that survived the treatment. Quantitative PCR indicated an increase in EBV viral load in PBMCs as the duration of immunosuppression increased. At autopsy, splenomegaly was seen in 3/4 rabbits, but spleens from all 4 rabbit were EBV PCR positive. EBER-in situ hybridization and immunoshistochemistry revealed the presence of a large number of EBER-positive and LMP-1 positive lymphoblasts in the spleens of 3/4 rabbits. To a lesser extent, EBER-positive cells were also seen in the portal tract regions of the liver of these rabbits. Western blotting indicated that EBNA-1 and EBNA-2 were also expressed in the liver and spleen of infected animals.

Conclusion: EBV can infect healthy rabbits and the infected cells proliferate when the animals are immunocompromised. The infected cells expressed several EBV-latent gene products which are probably driving the proliferation, reminiscent of what is seen in immunocompromised individuals. Further work is required to explore the potential of rabbits as an animal model for studying EBV biology and tumorigenesis.

Show MeSH
Related in: MedlinePlus