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  1. 1. Bio416 Final: State of research on Chlamydiaphage Chp2, a gokushovirus.
  2. 2. If you think back to our textbook chapter on phiX174, you'll remember that it mentions gokushovirus as a phage infecting obligate intracellular bacteria. I thought that sounded pretty cool, and I know enough to know that “gokusho” is Japanese...
  3. 3. So I had a vision of “gokushovirus,” an exotic, unique virus from Japan...
  4. 4. So I looked in a pretty wide variety of sources for articles on “gokushovirus,” but I really couldn't find anything, and I couldn't quite figure out what gokushovirus is...
  5. 5. [picture: scan of e-mail redacted ] So I e-mailed Bentley Fane, the author our textbook chapter, and he wrote back an answer that still didn't tell me exactly what gokushovirus was, but he did mention that the Chlamydiaphage Chp2 that he and his team work with is also a gokushovirus...
  6. 6. So I reduced the scope of this presentation to just Chlamydiaphage Chp2, and I went back to the databases to look again...
  7. 7. But I could still only find five articles, so I propose to summarize them all, which should give an overview of all the research done on this virus...
  8. 8. In the course of researching Chp2, I did figure out what gokushovirus is. Here you can see a phage tree with the microviridae. Gokushovirus is the subfamily whose type is represented by Chp1.
  9. 9. Here you can see the relationship of gokushovirus to the microviridae genuses. The place of our subject Chp2 and the typical bacteriophage phiX174 are indicated.
  10. 10. Here's a comparison of the genomes of Chp2 and phiX174
  11. 11. Chlamydiaphage was first described in a 1982 paper called “Chlamydia have phage, too”...
  12. 12. Why was this interesting? Because the life cycle of Chlamydia, an obligate intracellular bacteria, has properties unique from non-obligate bacteria... including “elementary bodies” and “reticulate bodies” Need slides on chlamydia life cycle
  13. 13. A second Chlamydiaphage was described in this 2000 paper.
  14. 14. Rather than being from Japan, though, it was found after researchers studying Chlamydial infections in sheep found a unexpected protein during gel electrophoresis. The protein turned out to be like Chp1 protein Vp1.
  15. 15. Culturing of these samples revealed a phage infecting the Clamydia that showed properties unlike the Chp1.
  16. 16. Sequencing of the new phage showed “Chp2” to be closely related to Chp1
  17. 17. Around the time Chp2 was discovered, new information about the Chlamydia genus caused it be divided into genus Chlamydia and genus Chlamydophila. Researchers in this 2002 paper wanted to know whether Chp2 would infect both genuses.
  18. 18. By creating hybridomas to produce antibodies against Chp2 proteins, the researchers were able to use immunofluorescence to detect the inclusions of infected Chlamydophila.
  19. 19. Then, using their developed technique, they tested Chp2's infectivity in the nine known species of Chlamydia and Chlamydophila. Chp2 infects some species of Chlamydophila but not species of Chlamydia...
  20. 20. Which means you can rest easy in the knowledge that your one-night stand didn't infect your Chlamydia with Chp2...
  21. 21. The researchers then incubated Chlamydophila in substances that degrade either saccharides or proteins, and again tried to infect with Chp2, demonstrating that Chp2 binds to protein but not saccharides, as does phiX174...
  22. 22. This 2004 paper sought to determine the nature of the identified Vp3 protein to help determine which parts of the genome would need to be conserved in any transgenic research using Chp2 on Chlamydia.
  23. 23. Remember: phiX174 uses scaffolding proteins to build a procapsid; it inserts DNA into the procapsid; then the scaffolding proteins drop off, collapsing the particle into a smaller virion...
  24. 24. The researchers centrifuged cultured cells, then divided the product into 0.1mL fractions of increasing density. They tested these with ELISA for the Vp1 (coat) protein and Vp3 (hypothesized scaffolding) protein, and they performed qPCR. The results shown here demonstrate that the heavier fractions contained Vp1 but lost the Vp3 protein and this corresponds with the appearance of DNA, suggesting that Vp3 does in fact work as a scaffolding protein...
  25. 25. A stain and electron miscroscopy revealed that the lighter fractions stained dark in the middle, suggesting empty centers that could accept stain, and that the denser fractions did not stain dark in the middle.
  26. 26. This 2007 paper attempted to create an assay for determining Chp2's infectivity without having to use the normal plaque assays used to study phages. The attempt was to find a way of calculating the infectivity of Chp2 based on counting stained inclusions.
  27. 27. The researchers cultured cells and then stained them with a Chlamydophila-specific stain and counted the number of infected cells. They then used PCR to determine the total number of Chlamydial genomes in their cultures. The count showed 1.7 x 10 7 ifu/ml, while the PCR showed 6.9 x 10 9 ifu/ml, giving a specific infectivity of 1:400.
  28. 28. Chlamydophila were infected with Chp2 and latex spheres were used to assist with counting the Chp2 procapsids and virions under electron microscope. The count determined virions to be at 3.0 x 10 12
  29. 29. The original procedure was done again with a stain specific for Chp2 instead of Chlamydophila, and inclusion bodies were counted rather than cells. Inclusions were counted at 5.8 x 10 9. This number is approximately 400 times smaller than 2.3 x 10 12 , which is close to the count performed with spheres. This suggests that an assay in which inclusions are counted and multiplied by 400 will approximate the specific infectivity of Chp2.
  30. 30. In this final paper from 2008, the researchers attempted to demonstrate the life cycle of Chp2 as it relates to the host Chlamydophila.
  31. 31. Groups of Buffalo Green Monkey Kidney cells were cultured with Chlamydophila both with and without Chp2 infection. Chp2 infection was shown to create morphological changes in the Reticulate Bodies and slow down the replication cycle of the Chlamydophila.
  32. 32. PCR analysis of bacterial and virus genomes showed that virus replication (probably stage III) follows bacterial replication, which is inhibited by the virus.
  33. 33. Western blot analysis for proposed scaffolding proteins Vp3 and ORF 5 and ELISA for coat protein Vp1 demonstrated that these appeared concurrently. Because these are less sensitive than PCR, it is suggested that the protein appearance coincides with the acceleration of virus genome appearance. Because the proteins and packaging are appearing around 48hrs into the culture, which was observed to be 7 or 8 replications of the bacteria, Chp2 must not exit its host like phiX174. How it does so is the topic of the next research project...