Post on 16-Apr-2017
Enteric bacteria promote human and mouse norovirus infection of B cells
Melissa K. Jones,1* Makiko Wantanbe,1* Shu Zhu,1 Christina L. Graves,2,3 Lisa R. Keyes,1 Katrina R.
Grau,1 Mariam B. Gonzalez-Hernandez,4 Nicole M. Iovine,5 Christina E. Wobus,4 Jan Vinjé,6 Scott A. Tibbetts,1 Shannon M. Wallet,2,3 Stephanie M.
Karst1†
Presented by: Katherin Portwood
1
• Easily transmitted in vomit aerosols, person to person contact, and fecal-oral contamination
• Small infection dose: 10-20 viral particles• Cruise ship diarrhea • Dehydration, vomiting, nausea, diarrhea• Duration: 24-48 hours• Most prevalent strain today is GII.4 Sydney
Norovirus is a lytic virus • Attaches to host cell and inserts its genome• Utilizes host machinery and one viral protein (RNA dependent
RNA polymerase) to replicate virons• Host cells are destroyed to release virus descendants
Norovirus is an +ssRNA virus• RNA dependent
RNA polymerase (RdRp) replicates the genome – +ssRNA– -ssRNA– +ssRNA
Norovirus is a non-enveloped virus• Norovirus binds to Histo-
Blood Group Antigens (HBGAs)
• Every virus has capsid proteins that protect the genome– VP1 is the major capsid
protein in norovirus.– VP1 encodes for a P
domain capable of binding to HBGAs
Histo-Blood Group Antigens
7
Norovirus genes
3
Mouse Norovirus (MuNoV)
• Acute infection
• Protective immunity – short lived
• Persistent infections
• Attenuated
Infect macrophages and Dendritic Cells of the host immune
system
Time course of infection
MNV-1 MNV-
3
Measurement of Virus infectivity• Cytopathic effects (CPE)– Structural changes in the host cells that are caused by viral
invasion– Evidence of cell lysis (plaques)– Represents actively replicating norovirus
• Plaque forming units (PFU)– Visual detections to determine the amount of virus particles from
dead host cells• TCID-50– Represents the viral concentration necessary to induce cell death/
pathological changes in 50% of inoculated cells– Determined by a specific calculation
Figure 1A
• RAW246.7- mouse macrophage
• M12- Mature mouse B cell
• WEHI.231- immature mouse B cell
• CMT-93- intestinal epithelial cells
MuNoVs infect B cells in culture
Determining cell viability
• Propidium Iodide staining– Red fluorescent stain– Only permeates the
membranes of dead cells– Binds to DNA
Figure 1B
MuNoVs infect B cells in culture
Immunofluorescence
4
Figure 1C
MuNoVs infect B cells in culture
Figure 1D
MuNoVs infect B cells in culture
Figure 2A
MuNoVs target Peyer’s patch B cells
Figure 2B
MuNoVs target Peyer’s patch B cellsWhy was there no significant difference in infection of the colon tissue?
Peyer’s Patches
• lymphatic follicles that sample the contents of the small intestine
• “waiting room” for B cells who will soon interact with their antigen
• Once the interaction is made, the B cells travel to the mesenteric lymph nodes to continue immune responses
11
Peyer’s Patches
8
NoV
B
Peyer’s Patches
8
Peyer’s Patches
8
Peyer’s Patches
8
Peyer’s Patches
8
Peyer’s Patches
8
Peyer’s Patches
8
Peyer’s Patches
8
Peyer’s Patches
8
Peyer’s Patches
8
Peyer’s Patches
8
Peyer’s Patches
8
Peyer’s Patches
8
Peyer’s Patches
8
Peyer’s Patches
8
Peyer’s Patches
8
Peyer’s Patches
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Peyer’s Patches
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Peyer’s Patches
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Peyer’s Patches
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Peyer’s Patches
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Peyer’s Patches
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Reverse Transcriptase-Polymerase Chain Reaction
5
Figure 2C
• RT-PCR detected the presence of viral genome in CD19 marked B cells and bulk cells collected from Peyer’s Patches.
MuNoVs target Peyer’s patch B cells
Flow Cytometry
6
Figure 2D
• N-term is a non-structural protein that activates host cell apoptosis
• CD19 and B220 are B cell surface markers
• Flow cytometry detected the presence of viral replication in diverse types of B cells
MuNoVs target Peyer’s patch B cells
Figure 3A
• GII.4 Sydney is the current dominate HuNoV strain
• Human Burrkits lymphoma cells (BJABs) were the test B cells
• viral genome growth in B cells at 3 and 5 dpi
HuNoVs productively infect B cells in culture
Figure 3B
• UV light acts as a mutagen -inactivates the viral replication process
HuNoVs productively infect B cells in culture
Figure 3C-D
HuNoVs productively infect B cells in culture
Figure 3E-F
HuNoVs productively infect B cells in culture
Figure 4 A
Intestinal bacteria facilitate NoV infections
Figure 4 B
Intestinal bacteria facilitate NoV infections
Figure 4 C
Intestinal bacterial facilitate NoV infections
Figure 4 D
Intestinal bacteria facilitate NoV infections
Conclusion
• Murine Norovirus infects B cells in culture• MuNoVs target Peyer’s Patch B cells • HuNoVs productively infect B cells in culture• Intestinal bacteria facilitate NoV infections
Bibliography1. Madigan, M. T., Martinko, J. M., Bender, K. S., Buckley, D. H., Stahl, D. A. (2015). Brock biology of microorganisms
fourteenth ed. Pearson Education Inc. 249; 266-267; 909.2. Kaiser, G. E., (2007). Doc Kaiser’s Microbiology Home Page. http://
faculty.ccbcmd.edu/courses/bio141/lecguide/unit4/viruses/ssplusRNA_lc.html. 3. Hyde, J. L., Sosnovtsev, S. V., Green, K. Y., Wobus, C., Virgin, H. W., Mackenzie, J. M., (2009). Mouse Norovirus
Replication Is Associated with Virus-Induced Vesicle Clusters Originating from Membranes Derived from the Secretory Pathway. Journal of Virology, 83, (19).9709-9719.
4. University of Queensland (2015). Immunofluorescence- Background. http://www.di.uq.edu.au/sparqcbeifbackground.
5. Optimization.gene-quantificatio.info. http://www.gene-quantification.de/optimization.html. 6. Jahan-Tigh, R. R., Ryan, C., Obermoser, G., Schwarzenberger, K. (2012). Flow Cytometry. Journal of Investigative
Dermatology, 132, 1-6.7. Miura T, Sano D, Suenaga A, Yoshimura T, Fuzawa M, Nakagomi T, Nakagomi O, Okabe S. (2013). Histo-blood group
antigen-like substances of human enteric bacteria as specific adsorbents for human noroviruses. Journal of virology 87 (17).
8. Allan McI. Mowat (2003). Anatomical basis of tolerance and immunity to intestinal antigens Nature Reviews Immunology 3, 331-341 http://www.nature.com/nri/journal/v3/n4/images/nri1057-f1.gif
9. Kang Rok Han,1 Yubin Choi,1 Byung Sup Min,1 Hyesook Jeong,2 Doosung Cheon,2 Jonghyun Kim,3 Youngmee Jee,2 Sungho Shin1 and Jai Myung Yang1 (2010). Murine norovirus-1 3Dpol exhibits RNA-dependent RNA polymerase activity and nucleotidylylates on Tyr of the VPg. Journal of General Virology (2010), 91, 1713–1722
10. http://en.wikipedia.org/wiki/Peyer%27s_patch11. http://www.ncbi.nlm.nih.gov/gv/rbc/xslcgi.fcgi?cmd=bgmut/systems_info&system=abo
Works cited1.1 Lin,X., Thorne, L., Jin, Z., Hammad, L. A., Li, S., Deval, J., Goodfellow. I. G., Kao, C. C. (2014). Subgenomic promoter recognition by the norovirus RNA-dependent RNA polymerases Nucleic Acids Res., 43(1), 1.1.2 Croci, R., Pezzullo, M., Tarantino, D., Milani, M., Tsay, S.-C., Sureshbabu, R., Tsai, Y., Mastrangel, E., Rohayem, J.,Bolognesi, M., Hwu, J. R. (2014). Structural Bases of Norovirus RNA Dependent RNA Polymerase Inhibition by Novel Suramin-Related Compounds. PLoS ONE, 9(3), 1.1.3 Hyde, J. L., Gillespie, L. K., & Mackenzie, J. M. (2012). Mouse Norovirus 1 Utilizes the Cytoskeleton Network To Establish Localization of the Replication Complex Proximal to the Microtubule Organizing Center. Journal of Virology, 86(8), 4110.1.4 Sosnovtsev, S. V., Belliot, G., Chang, K.-O., Prikhodko, V. G., Thackray, L. B., Wobus, C. E., Karst, S. M., Virgin, H. W., Green, K. Y. (2006). Cleavage Map and Proteolytic Processing of the Murine Norovirus Nonstructural Polyprotein in Infected Cells . Journal of Virology, 80(16), 7816, 7826, 7828-7830. 1.5 Bok, K., Prikhodko, V. G., Green, K. Y., & Sosnovtsev, S. V. (2009). Apoptosis in Murine Norovirus-Infected RAW264.7 Cells Is Associated with Downregulation of Survivin . Journal of Virology, 83(8), 3647, 3650, 3654. 1.6 Herod, M. R., Salim, O., Skilton, R. J., Prince, C. A., Ward, V. K., Lambden, P. R., & Clarke, I. N. (2014). Expression of the Murine Norovirus (MNV) ORF1 Polyprotein Is Sufficient to Induce Apoptosis in a Virus-Free Cell Model. PLoS ONE, 9(3), 1-2.1.7 Caddy, S., Breiman, A., le Pendu, J., & Goodfellow, I. (2014). Genogroup IV and VI Canine Noroviruses Interact with Histo-Blood Group Antigens. Journal of Virology,88(18), 10377–10378. 1.8 Shanker, S., Czako, R., Sankaran, B., Atmar, R. L., Estes, M. K., & Prasad, B. V. V. (2014). Structural Analysis of Determinants of Histo-Blood Group Antigen Binding Specificity in Genogroup I Noroviruses. Journal of Virology, 88(11), 6168–6176. 1.9 Reeck, A., Kavanagh, O., Estes, M. K., Opekun, A. R., Gilger, M. A., Graham, D. Y., & Atmar, R. L. (2010). Serologic Correlate of Protection against Norovirus-Induced Gastroenteritis. The Journal of Infectious Diseases, 202(8), 1212,1218. 1.10 Chachu, K. A., Strong, D. W., LoBue, A. D., Wobus, C. E., Baric, R. S., & Virgin, H. W. (2008). Antibody Is Critical for the Clearance of Murine Norovirus Infection. Journal of Virology, 82(13), 6610, 6614, 6615. 1.11 Hirneisen, K. A, Kniel, K. E. (2013). Norovirus Attachment: Implications for Food Safety. Food Protection Trends, 33 (5). 290-291.
2.1 Wobus, C. E., Karst, S. M., Thackray, L. B., Chang, K.-O., Sosnovtsev, S. V., Belliot, G., Krug, A., Mackenzie, J. M., Green, K. Y., Virgin, H. W. (2004). Replication of Norovirus in Cell Culture Reveals a Tropism for Dendritic Cells and Macrophages. PLoS Biology, 2(12), 2076-2077, 2079, 2081. 2.2 Zhu, S., Regev, D., Watanabe, M., Hickman, D., Moussatche, N., Jesus, D. M., Kahan, S. M., Napthine, S., Brierly, I., Hunter III, R. N., Devabhaktuni, D., Jones, M. K., Karst, S. M. (2013). Identification of Immune and Viral Correlates of Norovirus Protective Immunity through Comparative Study of Intra-Cluster Norovirus Strains.PLoS Pathogens, 9(9), 1, 7, 12.2.3 Mumphrey, S. M., Changotra, H., Moore, T. N., Heimann-Nichols, E. R., Wobus, C. E., Reilly, M. J., Reilly, M. J., Moghadamfalahi, M., Shukla, D., Karst, S. M. (2007). Murine Norovirus 1 Infection Is Associated with Histopathological Changes in Immunocompetent Hosts, but Clinical Disease Is Prevented by STAT1-Dependent Interferon Responses . Journal of Virology, 81(7), 3251-3252, 3257, 3261. 2.4 Miura, T., Sano, D., Suenaga, A., Yoshimura, T., Fuzawa, M., Nakagomi, T.,Nakagomi, O., Okabe, S. (2013). Histo-Blood Group Antigen-Like Substances of Human Enteric Bacteria as Specific Adsorbents for Human Noroviruses. Journal of Virology, 87(17), 9441, 9444-9445,9448. 2.5 Herbst-Kralovetz, M. M., Radtke, A. L., Lay, M. K., Hjelm, B. E., Bolick, A. N., Sarker, S. S., Atmar, R. L., Kingsley, D. H., Arntzen, C. J., Estes, M. K., Nickerson, C. A. (2013). Lack of Norovirus Replication and Histo-Blood Group Antigen Expression in 3-Dimensional Intestinal Epithelial Cells. Emerging Infectious Disease journal, 19 (3), 431, 434.2.6 Jones, M. K., Watanabe, M., Zhu, S., Graves, C. L., Keyes, L. R., Grau K. R., Gonzalez-Hernandez, M. B., Iovine, N. M., Wobus, C. E., Vinjé, J., Tibbetts, S. A., Wallet, S. M., Karst, S. M. (2014). Supplementary Material for Enteric bacteria promote human and mouse norovirus infection of B cells. Sciencemag, 346 (755), 1-16. 2.7 J. May, B. Korba, A. Medvedev, P. Viswanathan, Enzyme kinetics of the human norovirus protease control virus polyprotein processing order. Virology 444, 218–224 (2013).3.1 H. L. Koo, N. Ajami, R. L. Atmar, H. L. DuPont. (2010). Norovirus: The Principal Cause of Foodborne Disease Worldwide. Discov Med, 10(50), 61,64. 3.2 (2014).Murine Norovirus. Biomedical Diagonostics. 1-2.3.3 Jahan-Tigh, R. R., Ryan, C., Obermoser, G., Schwarzenberger, K. (2012). Flow cytometry. Journal of Investigative Dermatology, 132, 1-2.
3.4 Shirato, H. (2011). Norovirus and Histo-Blood Group Antigens. Department of Virology II, National Institute of Infectious Disease, 65, 95,99.3.5 Sestak, K. (2014). Role of histo-blood group antigens in primate enteric calcivirus infections. World journal of Virology, 3(3), 18-19.3.6 Prey, L., (2008). The Biotechnology Revolution: PCR and the Use or Reverse Transcriptase to Clone Expressed Genes. Nature Education 1 (1), 1-2.3.7 Odell, I. D., Cook, D. (2013). Immunofluorescence Techniques. Journal of Investigative Dermatology, 133(4), 1-2.4.1 Cytospring. PBS( Phosphate-Buffered Saline). http://www.researchgate.net/publictopics.PublicPostFileLoader.html?id=52f88bfacf57d727448b45de&key=e0b4952f88bfa0e8d84.2 Washington State Department of Health. (2013). Norovirus. http://www.doh.wa.gov/Portals/1/Documents/4400/332-083-Norovirus.pdf4.3 Madigan, M. T., Martinko, J. M., Bender, K. S., Buckley, D. H., Stahl, D. A. (2015). Brock biology of microorganisms fourteenth ed. Pearson Education Inc. 41-43,246-247, 248, 266-267, 909.4.4 Encyclopedia Britannica. (2014). http://www.britannica.com/EBchecked/topic/148948/cytopathic-effect-CPE4.5 Life Technologies (2015). https://www.lifetechnologies.com/order/catalog/product/P1304MP4.6 University of Queensland (2015). Immunofluorescence- Background. http://www.di.uq.edu.au/sparqcbeifbackground4.7 (2013). An Overview of virus quantification techniques. https://virocyt.com/wp-content/uploads/2013/04/VirusQuantificationWhitePaper.pdf4.8 Peyer patch. (2015). Encyclopædia Britannica. Retrieved fromhttp://www.britannica.com/EBchecked/topic/454716/Peyer-patch4.9 (2014). Western Blot. http://www.nature.com/scitable/definition/western-blot-2884.10 (2010). Protocols Online- Phosphate Buffered Saline.http://protocolsonline.comrecipes.phosphate-buffered-salin-pbs4.11 Johnson, M. Loading Controls for Western Blots. (2014). Labome. http://www.labome.com/method/Loading-Controls-for-Western-Blots.html
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