RSUSSH 2020

IN20-034 Upregulation of Noxa and p53 upregulated modulator of apoptosis (PUMA) in response to Zika virus infection

Presenter: Miss Diksha Pokhrel
Mahidol University, Thailand

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Abstract

      Zika virus (ZIKV), an arthropod borne arbovirus is a positive sense single-stranded RNA virus that belongs to the genus Flavivirus within the family Flaviviridae. The symptoms associated with ZIKV are normally a mild fever, headache, rash and conjunctivitis, but after 2015, ZIKV became a public health concern worldwide because of the occurrence of microcephaly in newborns from mothers infected with Zika virus during pregnancy. Virus replication inside host cells causes accumulation of misfolded or unfolded proteins and exploitation of ER membranes can cause ER stress that leads to anti-viral defense mechanisms and cell death. Noxa and p53 upregulated modulator of apoptosis (PUMA) belong to Bcl-2 homology domain (BH3) family and induction of both Noxa and PUMA is mediated through p53 in prolonged ER stress conditions. As a recently emerging virus, little is known about strain specific manifestations of this virus and whether ZIKV associated with cases of microcephaly are functionally distinct from normal circulating virus remains unclear. Therefore, this study aimed to analyze expression of Noxa and PUMA in A549 cells after infection with two different Thai strains of Zika virus, namely SV0010/15 isolated from a Thai case of Zika fever, and MU1-2017 isolated from the brain tissue of a fetus aborted for reasons of abnormal development including microcephaly. Results shows significant increase in expression of Noxa and PUMA in A549 cells infected by both viruses, when compared with mock infected cells. This shows that ZIKV infection in A549 cell induces p53 mediated ER stress that may lead to apoptosis.

Keywords: ZIKV; ER-stress; NOXA; PUMA; Apoptosis

Citation format:

Pokhrel, D., Hitakarun, A., Roytrakul, S., Wikan, N., & Smith, D.. (2020). Upregulation of Noxa and p53 upregulated modulator of apoptosis (PUMA) in response to Zika virus infection. Proceeding in RSU International Research Conference, May 1, 2020. Pathum Thani, Thailand.

QUESTIONS & ANSWERS

Asst. Prof. Dr. Thanet Sophonnithiprasert (Chairperson)

Thank you for sharing your works. I have a question about the result of semi-quantitative PCR as showed in your VDO clip presentation. NOXA and PUMA are ER stress mediators that lead to ER-stress induced apoptosis. So, the ER stress inducer, such as Tunicamycin (I think it abbreviated to "TM" in your picture), should be increase NOXA and PUMA expression, but both genes were no changed significantly in expression when treated with TM. Can you explain why? Sorry, if I misunderstand about abbreviation TM, it may be not Tunicamycin. Thanks in advance for your response.

Tadsanee Punjanon (Visitor)

What are the reasons for choosing these two strains of Zika virus?

vorachai sirikulchayanonta (Visitor)

Dr.Vorachai Sirikulchayanonta, MD

     Thank you very much for your interesting topic. From your study, both strains of Zikv  produced  P53 mediated ER stress that could lead to apoptosis in A549 cells which are pulmonary type2. Can this finding explain the microcephaly which effects the neurones or glia cells? Or some other cells which deal with cytokine releases that cause fever?

Miss Diksha Pokhrel (Presenter)
  1. Thank you for sharing your works. I have a question about the result of semi-quantitative PCR as showed in your VDO clip presentation. NOXA and PUMA are ER stress mediators that lead to ER-stress induced apoptosis. So, the ER stress inducer, such as Tunicamycin (I think it abbreviated to "TM" in your picture), should be increase NOXA and PUMA expression, but both genes were no changed significantly in expression when treated with TM. Can you explain why? Sorry, if I misunderstand about abbreviation TM, it may be not Tunicamycin. Thanks in advance for your response.            We appreciate this comment.  Commentator is indeed correct in that TM represents tunicamycin, and tunicamycin has been shown to induce expression of Noxa and PUMA.  The commentator should look at the actin bands as these are reduced in intensity in the TM treated cells as compared to the non-treated cells.  Thus, although it looks as though there is no up-regulation in response to tunicamycin, normalization against actin shows that they are both indeed up-regulated by tunicamycin. 

      2.  What are the reason for choosing two different strains of ZIKV?

Thank you for the question.

In this study, we have chosen two Thai strains of ZIKV that is Zika SV0010/15 (ZIKV-15)- isolated from the serum of Thai with self-limiting Zika fever and ZIKV-MU1-2017: isolated from brain tissues of an aborted fetus with Zika virus-associated microcephaly. The main objective behind using two different strains is that we want to see whether ZIKV associated with cases of microcephaly are functionally distinct from the normal circulating ZIKV that causes fever.

  

  1. Thank you very much for your interesting topic. From your study, both strains of Zikv  produced  P53 mediated ER stress that could lead to apoptosis in A549 cells which are pulmonary type2. Can this finding explain the microcephaly which effects the neurones or glia cells? Or some other cells which deal with cytokine releases that cause fever.

Thank you for the question.

There are several cell lines of a human origin (HEK293T/17, HEK293, HeLa, Hep3B, and  A549) and of a primate origin (LLC-MK2 and Vero) that are susceptible to  ZIKV infection (Diteepeng et al., 2019). We used A549 cells in this study as a higher percentage of infection is seen in A549 cells from both strains of viruses ZIKV-15 and ZIKV-MU1.

This finding does not explain the pathophysiology behind microcephaly or how the different strains of ZIKV infects neurons or glia cells. This study mainly focuses on understanding the mechanism of apoptosis induced by two different strains of ZIKV that will helps us to identify if ZIKV associated with microcephaly is functionally distinct from the regular circulating virus. However, future studies investigating a more suitable cell type (such as neuronal cells) are planned.