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“Electron micrographs of dengue HA antigens isolated in sucrose gradients (145,000 magnification); bar = 50 nm. (a) Rapidly sedimenting HA (RHA) contains complete virions. (b) Slowly sedimenting native HA (SHA) treated with Tween-80 and ether contains “ring” structures. (c) Slowly sedimenting HA derived from complete virions by Tween-80-ether treatment (derived SHA) contains aggregates of small doughnut particles each measuring 7 nm in diameter.”
Summary: a) Intact dengue virus. Everything else: Pieces of viral particles, etc.
Results of the antigen and infectivity assays over time:
I’m not going to go deeply into detail here, but suffice it to say that there are more viral antigens over time, and there are more infectious viral particles over time, until the 5th day. Also, there appears to be a much higher viral concentration in the tiny mice brains than in their blood.
Next paper:
“Physical and Biological Properties of Dengue-2: Virus and Associated Antigens”
Journal of Virology, Apr. 1970, p. 524-532
https://pubmed.ncbi.nlm.nih.gov/4195055/The abstract of this one does a better job than the last. I will enumerate for clarity:
“1. Dengue virus suspensions from mouse brain and cell culture were fractionated into three components by rate zonal centrifugation in sucrose gradients.
2. “Infectious virus sedimented in a single zone and possessed hemagglutinating (HA) and complement fixing (CF) activity. “
3. “Electron micrographs showed the virion to be a spherical particle 48 to 50 nm in diameter with 7-nm spherical structures on its surface.
4 “Buoyant density in CsCl of virions from mouse brain was estimated at 1.22 g/cm3 and from cell culture at 1.24 g/cm3. During centrifugation of virions in CsCl, an additional HA component appeared with a buoyant density of 1.18 g/cm3. It was shown in electron micrographs to consist of virion fragments. “
5. “A noninfectious component with HA and CF activity sedimented in sucrose more slowly than intact virus, had a buoyant density of 1.23 g/cm3 in CsCl, and appeared as “doughnut” forms measuring 13.8 to 14 nm in diameter.”
6. “A third component,with CF activity and no HA activity, sedimented very little in sucrose gradients. Particles of the same size and shape as the spherical subunits on the surface of the virion were observed in electron micrographs.”
“FIG. 2. RHA and SHA components from 5 to 25% sucrose gradients. Bars represent 100 Mm. (a) RHA. X100,000. (b,c,d) RHA. Arrows in2dicate 7-nm subunits on the surface of the virion. X200,000. (e) SHA. X 100,000. (f,g,h) SHA. X 200,000.”
“FIG. 3. RHA, SHA, and SCF dengue virus components. Bars represent 100 nm. (a,b,c,d) RHA component from sucrose gradient after storage at 4 Cfor 10 days. (a) X 100,000. (b,c,d) arrows indicate 7-nm subunits. X 200,000. (e) SCF component from sucrose gradient. X 200,000. (f) HA component with a buoyant density of 1.22 g/cm3 after CsCI equilibrium density gradient centrifugation of RHA. X 200,000. (g) HA component with buoyant density of 1.18 g/cm3 after CsCI equilibrium density gradient centrifugation of RHA. X 200,000. (h) HA component with
buoyant density of 1.23 g/cm3 after CsCl equilibrium density gradient centrifugation of SHA. X 200,000.”Many critiques could be made of these studies, and I’m sure this will be done. For now, I’m nearing the end of my first installment in my introduction to virology.
I think there is enough here to “scientifically prove/evidence the existence of the alleged dengue virus (showing that the alleged particle exists and causes the disease that it’s alleged to cause)” and “[describe] the purification of particles that are alleged to be said virus(es), directly from bodily fluid/tissue/excrement, with purification confirmed via EM imaging (the images must be available as well)”.
I have only briefly covered 63 years of dengue virus research. There have been 53 years since the last mentioned papers, along with 53 years of advances of science, technology and methodology.
I’m certain a further critique will be made regarding the “bodily fluid/tissue/excrement” not being human. As mentioned before, it appears this was attempted, possibly unsuccessfully, by Sabin in his 1952 “Dengue Studies in WWII” paper.
To illustrate the difficulty in extracting this and some other viruses from human tissue, consider this point: In Sabin’s WWII paper, he stated that there were at least 1 million “infectious doses” per milliliter of blood. A study from 1916 estimated that concentration to be twice that (from Schlesinger, “Dengue Viruses”, 1977).
So, let’s just say it’s 2 x 10^6 (2 million infectious viruses per milliliter). There are probably more, as not all will be infectious, but let’s go with that number. That sounds like a lot, but…
The virus (one virion) is about 50 nm in diameter. Its volume is then (50nm)(4/3)pi*r^3 = 53,051 nm^3. A grain of salt is said to measure 1.6 x 10^16 nm^3 (0.016 cubic millimeters) (#https://is.gd/shhj1X). Given the maximum packing fraction is 74% (densest packing of spheres, i.e. 74% of a space is filled with viruses), the effective volume is about 1.2 x 10^16 nm^3. To fill that with infectious virus, we’ll need 2.2 x 10^11 virions.
(2.2 x 10^11 virions)/(2 x 10^6 virions/mL ) = 111,590 mL
That’s 111.6 LITERS of blood to make a virus pellet the size of a grain of sand. That’s over 22 people sucked dry!
Of course, you don’t need all of that for electron microscopy. But do consider that, do the electron microscopy work on cell-cultured virus in that last paper (“Physical and Biological Properties of Dengue-2: Virus and Associated Antigens”), they used “Eighteen 32-oz (ca. 900 ml) bottle cultures of BS-C-1 or LLC-MK2 cells”, whose supernatant fluid (cell media fluid over the cells that produced the virus) contained about 5 x 10^5 PFU/mL (PFU is “plaque forming units” and is the estimated # of infectious virions per unit volume).
So, if we were to try and reproduce these results with human blood from dengue patients, it would require (5 x 10^5PFU/mL)*18*900mL = 8.1 x 10^9 PFU
(8.1 x 10^9 PFU)/(2 x 10^6 PFU/mL) = 4,050 mL, which is 4 liters of blood.
So you still need multiple dengue victims to donate copious amounts of blood… Hence the utility of cell culture. Also, I can attest that removing rodent brains is already a difficult and gruesome procedure, so anytime cell culture can be used, it facilitates things nicely.
Lastly, in case I didn’t address enough here regarding dengue fever, its causative agent, or its purification, isolation, propagation, etc., feel free to check:
https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/9780471729259.mc15d02s27
Or…
https://link.springer.com/protocol/10.1007/978-1-4939-0348-1_4
(Hint: There is a site called Sci-Hub that may just allow for easy access.)
Also, if you’d like to conduct your own dengue research, feel free to order some dengue virus!
https://www.atcc.org/products/vr-1856
Alright. Enough for now. I’ll return, at some point, with more virology research for discussion, and hopefully we’ll begin to address Koch’s Postulates and Dr. Bailey’s isolation requirements.
Addendum:
It appears I was accused of “sophistry” in this comment from an EP Autos patron:
“Pure sophistry. For example, you say:
[Indentations added for clarity]
‘Funny enough, much data for this virus can easily be found… At CDC’s virus arbovirus catalog:
https://wwwn.cdc.gov/arbocat/VirusDetails.aspx?ID=129
…You know, where they have no records of dengue virus causing disease, being purified
or imaged by electron microscopy…’Now, here’s what the CDC says (or perhaps doesn’t say) about the “method of isolation of Dengue 2”:
Section III – Method of Isolation
Inoculation Date 6/12/1944
Animal (Details will be in Section 6)
Man*
Route Inoculated
Intracutaneous + iv Reisolation
No
Other Reasons
Classical dengue with rash after appropriate incubation in first and subsequent volunteers
and Ae aegypti transmission.**
Homologous Antibody Formation by Source Animal
Not tested
Test(s) Used
Footnotes
Where’s the evidence of its isolation??????? Come on now.”(“Sophistry” seems to indicate that I’m deliberately lying and/or attempting to mislead for some reason, which I don’t much appreciate, but let’s let that go for now.)
This is a DATA SHEET. It’s not meant to contain great detail. This comment is a little like decrying a book’s table-of-contents for its lack of detail.
If you want detailed information about viral isolations or any of the other information, it is contained in the references, listed at the end:
“1. SABIN, A.B. 1950. Bact. Reviews 14:225-232.
2. SABIN, A.B. and SCHLESINGER, R.W. 1945. Science 101:640-642.
3. SMITH, T.J., et al. 1970. J. Virology 5:524-532.
4. STOLLAR, V. 1969. Virology 39: 426-438.
5. STEVENS, T.M. and SCHLESINGER, R.W. 1965. Virology 27:103-112.
6. HOTTA, S. and EVANS, C.A. 1956. Virology 2:704-706.
7. SATHER, G.E. and HAMMON, W. McD. 1958. Unpublished data.
8. MEIKLEJOHN, G., et al. 1952. Am. J. Trop. Med. Hyg. 1:51-58.
9. BRANDT, W.E., et al. 1970. J. Virol. 6:500-506.
10. BRANDT, W.E., et al. 1970. J. Immunol. 105:1565-1568.
11. CARDIFF, R.D., et al. 1971. J. Virol. 7:15-23.
12. HAMMON, W.McD., et al. 1960. Science 131:1102-1103.
13. HAMMON, W.McD., et al. 1961. SEATO Med. Res. Monograph No. 2:30-35.
14. HAMMON, W.McD. and SATHER, G.E. 1964. Mil. Med. 129:130-135.
15. IBRAHIM, A.N. and HAMMON, W.McD. 1968. J. Immunol. 100:86-92
16. IBRAHIM, A.N. and HAMMON, W.McD. 1968. J. Immunol. 100:93-98.
17. McCLOUD, T.G., et al. 1971. Am. J. Trop. Med. Hyg. 20:964-968.
18. RUDNICK, A. and CHAN, Y.C. 1965. Science 149:683-689.
19. RUDNICK, A. 1971. Unpublished data.
20. ATCHISON, R.W., et al. 1971. Unpublished data.
21. CRAGIHEAD, J.E., et al. 1966. Arch. Path. 81:232-239.
22. CHASTEL, C. and FOURQUET, R. 1972. Rev. Epid. Med. Soc. Sante Publ. 20:499.
23. UC ICMR, San Francisco, CA. 1973. Unpublished data.
24. MORALES, A., et al. 1973. Am. J. Trop. Med. Hyg. 22:785-787.
25. HALSTEAD, S.B. 1984. Rev. Inf. Dis. 6:251-264.
26. GUZMAN, M.G., et al. 1984. PAHO Bull. 18:213-20.
27. DE MADRID, A.T. and PORTERFIELD, J.S. 1974. J. Gen. Virol. 23:91-96.
28. CALISHER, C.H., et al. Personal communication. 1983.
29. Rudnik, A. et al. 1975. Unpublished data.
30. Khin, M.M. and Than, K.A. 1983. Am. J. Trop. Med. Hyg. 32:590-594.”Much of the info in the data sheet is annotated with a number giving the reference from which it was derived.
I included some of these papers in my research (as well as some that weren’t listed), but some papers listed here aren’t easily accessed. The point being, it is evident that the CDC DID have access to them, and likely much more information than I could access, and they could’ve provided, at least, SOME information with Massey’s FOIA request.
Centrifugation could be its own specialization, and mastering its techniques could take some time, so I won’t elaborate greatly about what is being done, but centrally, it’s what is known as “density gradient” or “isopycnic” centrifugation, which separates components of a suspension based on the their density.
The above photo is such a technique used on bacteriophages, with the arrow denoting the band containing the virus.
Here is fairy decent summary primer for centrifugal virus purification, in case anyone is interested:
#https://www.beckman.com/resources/reading-material/case-studies/virus-purification-fundamentals
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