BaDnOn
Forum Replies Created
-
Posts
-
“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.”
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.”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.
“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.
From “Dengue Virions and Antigens in Brain and Serum of Infected Mice” Journal of Virology, Oct. 1970, p. 500-506
https://pubmed.ncbi.nlm.nih.gov/5497897/
Much technical info follows, which I’d heretofore mostly avoided. I won’t go into any astounding detail into technical methodology at this time, but it can be addressed at a later date.
In these studies, they reference “rapidly sedimenting infectious hemagglutinin” antigen (RHA), “slowly sedimenting noninfectious hemagglutinin” antigen (SHA). You can think of hemagglutinin as the “spikes” on certain viruses that attach to red blood cells and allow them entry. The virions (viral particles) compose the “RHA” component.
[Production and purification of virus]
“Suckling mice (1- to 3-day ICR strain) were inoculated intracerebrally with 10^4 plaque-forming units (PFU) of New Guinea “C” dengue-2 virus in the 33rd mouse passage. Brains were harvested from moribund mice 5 days later and homogenized (20%, w/v) in 0.02 M tris(hydroxymethyl)aminomethane (Tris) buffer (pH 8.2). These suspensions were clarified by
the addition of 3 mg of protamine sulfate per ml and centrifugation at 9,000 X g for 1 hr at 4 C.Antigens were sedimented from the supernatant fluid at 105,000X g for 2.5 hr and resuspended in a volume of buffer effecting a 100-fold concentration.
Density gradient centrifugation- Rate zonal separation of dengue virus antigens was carried out in 27-ml gradients preformed with 5 to 25% (w/v) sucrose inTris buffer. Samples (2 ml) were layered on top of each gradient and centrifuged in a Beckman 25.1 rotor at 63,000 X g for 3 hr at 3 C.Approximately 30 fractions (1 ml) were collected dropwise through the bottom of the centrifuge tube. Under these conditions as previously described (5), RHA (virion) is found in fractions 4 to 8 near the bottom of the tube, SHA is found in fractions 18 to 22, and SCF is found in fractions 27 to 29. Equilibrium centrifugation in CsCl was accomplished by adjusting samples to approximately 1.23 g/cm3 with a saturated solution of CsCl in Tris buffer.”
After centrifugation of a 5-ml sample at 104,000 X g for 40 hr at 3 C (Beckman SW-39 rotor), fractions were collected as above. Refractive indexes of the CsCl fractions were obtained in a Bauch & Lomb refractometer and converted to density by the method of Ifft et al.”Electron Microscopy:
“Single-drop samples of appropriate sucrose gradient fractions were placed directly onto grids covered with carbon-coated collodion membranes prewashed in chloroform. Specimens were
allowed to settle for several minutes and the excess was removed by pipette. The grids were inverted on water to remove sucrose, dried, and stained with 1% aqueous uranyl acetate. Micrographs were taken at 58,000 magnification in a Hitachi 1 1B electron microscope.”There were also assays to count the antigens (viral particles in the case of RHA), and determine their infectiousness:
Antigen Assay:
“Hemagglutinating (HA) antigen were carried out in microtiter plates (Linbro Chemical Co., New Haven, Conn.) by standard methods (2) with goose erythrocytes at pH 6.2. Complement fixation tests were carried out by a microtiter modification of the method described by Kent and Fife (4) utilizing hyperimmune mouse ascitic fluid as a source of excess specific antibody (1). Infectivity titrations were carried out by plaque assay in LLC-MK2 cell cultures.”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-fundamentalsCentrifugation 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.
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…
Anyway, the papers that will soon follow describe this virus being purified from mouse brain tissue by centrifugation and give electron micrographs of the purified virions.
(I’ve replaced the deprecated term “millimicrons” and its units with “nanometers”, nm, which are equivalent and represent billionths of a meter.)
“Particle Size of the Virus: The diameter of the virus as determined by filtration of highly infectious human serum through gradocol membranes was estimated at 12 to 25 millimicrons [nm], because all the volunteers who received the filtrates from membranes with an average pore diameter (A. P. D.) of 75 [nm] or greater developed typically severe dengue, while 2 volunteers who received the filtrate from the membrane with an A. P. D. of 50 [nm] remained well. However, since the latter volunteers exhibited a partial immunity to reinoculation several
months later, it is possible that approximately one M. I. D. [minimal infective dose] of virus might have passed the 50 [nm] membrane, and that the virus may actually be somewhat smaller than 17 to 25 [nm].The virus could be sedimented from human serum by centrifugation at 24,000 r.p.m. for 90 minutes in an 8-inch rotor of a vacuum ultracentrifuge. Examination with the electron microscope of preparations from highly infectious human dengue serum, purified by differential centrifugation, revealed dumb-bell-shaped structures (700 [nm] x 20-40 [nm]), which were not found in similar preparations from normal human serum.”Unfortunately, there are no micrographs presented in the paper, and we’ll never know exactly what Sabin and his colleagues found here. The virions to be described later are around 50 nm, alright, but what the “dumb-bell-shaped structures” were will likely remain a mystery. Possibly, there were problems with sample preparation (perhaps there were artifacts resulting from the purification process), as electron microscopy applied to viruses was still in its infancy.
Also of note from this paper the adaptation of dengue virus to mice. In short, mice are injected with dengue and further virus is extracted from those who are found to be susceptible. This continues for many iterations or “passages” until most mice are found to be susceptible (having severe symptoms).
“Extracts of the brain and spinal cord of paralyzed mice, derived from the first 6 consecutive
passages in mice, upon inoculation in human volunteers produced clinical manifestations of varying severity – relatively mild in some and fully severe and unmodified in others…. Beginning with the seventh passage in mice, however, the virus had lost its capacity to produce the severe illness and protracted fever, characteristic of the unmodified disease in human beings, but retained its capacity to produce a rash and solid immunity to the unmodified virus.”I.e. mouse-adapted dengue virus. This becomes important because now there is an animal which can be used to propagate and study the virus.
You’ll find that human volunteers become a rarity as studies move further from the military realm (as in this case and in earlier papers) and, in my opinion, the litigiousness of society increases. That said, the volunteers for these studies had cajones grandes, as dengue can be an excruciating and horrifying experience.
The next couple of papers presented should complete the re quest for studies “showing that the alleged particle exists and causes the disease that it’s alleged to cause” and “describing 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).”
The tissue may be the brain tissue of mice, but the virus is dengue, even if adapted to mice. The virus most assuredly causes disease in the infected mice, and the brains of such mice are found to have high titers (concentrations) of virus.
The virus used in the following papers was the New Guinea “C” strain of dengue-2.
The important findings:
1. That it is possible by subcutaneous injection of blood, or derivatives of blood, from a dengue patient to transmit the disease to a healthy non-immune, and from this case in a similar way to derive a series of experimental cases. In the present communication the original virus obtained from a natural case has been passed successively through four “generations” of artificial cases, with a doubtful positive case in the fifth “generation.”
2. That the virus under certain conditions may be passed through a Pasteur-Chamberland F. filter which at the same time is keeping back ordinary organisms (staphylococci, colon bacilli).
3. That the virus does not appear to be specially contained in any one element of the blood. Carefully washed corpuscles, citrated plasma, and serum free from corpuscles all contain the virus. It is not absolutely certain, however, in the case of serum that the virus may not obtain access to this by the breaking up of corpuscles.
4. That the virus is resistant to conditions outside the body for several days (99 hours).
5. That in the blood of the dengue patient the virus was present in one case 18 hours after the onset. in one case 67 hours and in another as late as 90 hours, whilst several cases show it to have been present at 57 hours and less after the onset. It was not found in a case 130 hours after the onset.
6. That the incubation period, while variable and not shorter than 41 days, is usually from 6 to 8 days, but may be as long as 15 days.
7. That immunity may be present to injection 229 days after the onset of a previous
attack of dengue.So, again, the virus can pass through filters which stop bacteria. Also, it appears to pervade the constituents of the blood, but appears to only remain for a finite time after infection, after which immunity is established for some time.
So, again, the virus can pass through filters which stop bacteria. Also, it appears to pervade the constituents of the blood, but appears to only remain for a finite time after infection, after which immunity is established for some time.
But what of the particles?
The first reference I found to the viral particles (virions) themselves was in a paper from 1952:
“Research on Dengue During WWII”
https://www.ajtmh.org/view/journals/tpmd/1/1/article-p30.xml“Experimental Investigations Regarding the Etiology of Dengue Fever”
…The findings of which were:
1. No organism, either bacterium or protozoon (sic), can be demonstrated in either fresh or stained specimens of [infected] blood with the microscope.
2. The red-blood count in dengue is normal.
3. There occur no characteristic morphological changes in the red or white corpuscles in this disease.
4. Dengue is characterized by a well-marked leucopenia, the polymorphonuclear leucocytes being decreased, as a rule, while there is a marked increase in the small lymphocytes.
5. No organism of etiological significance occurred in broth or citrated blood cultures.
6. The intravenous inoculation of unfiltered dengue blood into healthy men is followed by a typical attack of dengue.
7. The intravenous inoculation of filtered dengue blood into healthy men is followed by a typical attack of the disease.
8. The cause of the disease is, therefore, probably ultramicroscopic in size.
9. Dengue can be transmitted by the mosquito, Culex jatigans, and this is probably the most common method of its transmission.
10. The period of incubation in experimental dengue average three days and fourteen hours.
11. Certain individuals are absolutely immune to dengue, as proven by our experiments.
12. Dengue is not a contagious disease, but is infectious in same manner as is yellow fever and the malarial fevers.Other important remarks on this paper are how well they documented every case of experimentally induced dengue fever. There are even quantitative indications of the disease (well-marked leucopenia and marked increase in small lymphocytes).
Out of 11 volunteers, 7 injected with dengue-infected blood became ill with the disease, one was “doubtful”, and 3 appeared immune. Both volunteers given filtered blood became ill with the disease. The filtration method was shown to remove 2 species of bacteria.
So far, from this 116 year old paper, we have arguably begun to satisfy Massey’s requirement that we find papers that “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)”.
While there are no particles to be found yet, there is SOMETHING in the blood of dengue patients that is evidenced to cause dengue in others, even when specific agents could not be found and were excluded by filtration.
This will be further supported by the next paper to be mentioned:
“Further Experiments in the Etiology of Dengue Fever”https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2206837/
Let us return to the dengue virus and Christine Massey’s request.
To reiterate, she requested:
“1. All studies/reports in the possession, custody or control of the … (CDC) and/or the Agency for Toxic Substances and Disease Registry (ATSDR) that 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);
Or…
2. If the CDC has no studies responsive to #1 above, then please indicate such explicitly, and provide all studies and/or reports in the possession, custody or control of the … (CDC) and/or the Agency for Toxic Substances and Disease Registry (ATSDR) describing 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’d like one thing to be kept in mind regarding science: Hypotheses aren’t really proven. What is actually done in science is to disprove all alternative hypotheses. The remaining hypothesis is then “proven”.
With that said, let us begin to investigate the virus responsible for dengue fever.
First, here is an excellent and detailed paper from 1907:
https://www.jstor.org/stable/30073165
“A Filterable Virus, The Cause of Infectious Laryngotracheitis of Chickens”
The summary and conclusions are given here:
“1. Experiments have shown that tracheal exudate from two strains of laryngotracheitis of chickens from New Jersey and two from California when suspended in bouillon and passed through Berkefeld V filters will produce the disease. Two of six Berkefeld N filters allowed
the etiological agent to pass, whereas four did not. Attempts to produce the disease with Seitz filtrates were unsuccessful. These results demonstrate that laryngotracheitis is caused by a filtrable virus that because of its size or some other property does not pass readily through the finer filters.
2. It has been shown that the sera from fowls that have recovered from an infection with one of the New Jersey viruses will neutralize the same strain and also the one California strain tested. In order to demonstrate neutralization conclusively it was necessary to titrate samples of dried virus and in the tests to use approximately ten infecting doses.
3. The virus dried over calcium chloride for 10 days and then stored in the refrigerator for 60 days produced disease. Kept over calcium chloride for a month it was still active and when dried by Swift’s method it remained alive for 5 months.”Essentially, they made chickens sick with artificial infection with previously collected virus, took exudate from the tracheae of the sick chickens, and purified that with centrifugation and various filters. They then took those filtrates and attempted to infect other chickens, with some success, depending on the filter. The control bacteria they added to the centrifuged fluid, however, were always filtered. No bacteria could be cultured from the filtrates.
Berkefeld filters, by the way, are simply made from diatomaceous earth, with the “N” variety having smaller pores than the “V” variety. They are still used to remove bacteria and purify water. (*Mycoplasma, which is a very small bacterium, may sometimes pass through these filters.)
Now, studies of this era are historical and usually not as rigorous, comprehensive or definitive as later work. There would be no claimed electron micrographs of a virus until 1941 (the first commercial transmission electron microscope became available in 1938).
Studies such as this, however, demonstrate, at very least, successful attempts to purify viruses (in some capacity) from diseased organisms, show that they cause specific diseases, and that they differ from bacteria and other causes of illness in their properties.
At the same time, Friedrich Loeffler and Paul Frosch were conducting experiments on foot-and-mouth disease with the following findings (this according to secondary sources, as I have yet to find the original papers):
“1. The disease can be artificially transferred in lymph from epidermal vesicles; bacteria that these sometimes contain do not reproduce the disease.
2. Lymph, filtered for isolation of an agent responsible for immunity developing soon after infection, is still infectious.
3. The infectious agent cannot be grown in artificial media.
4. The filtrate does not contain a toxin responsible for the disease but `as yet undetectable disease agents so small that they were able to pass the filter pores retaining the smallest bacteria’ including those of Bacillus fluorescens previously added as a control.
5. The infectious agent must be so small that it would indeed escape visible detection by microscopy (according to calculations by Professor Abbe, Jena, about the limit of resolution of the microscope used).
6. The agent is not soluble but ‘corpuscular’ because it is retained by a one-pored Kitasato filter.”I’d like to get back to foot-and-mouth disease later, but for now it is evident what the implications of these early studies were. These researchers had found minute pathogens, smaller than any bacterium, and they weren’t toxins. They also couldn’t proliferate without the help of living cells. Other than that, they really had no idea what a “virus” actually was.
This would be the case for decades. Enter the era of the “filterable virus”.
An excellent example of a paper typical for this time is given here:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2180297/That paper contains a great summary of Beijerinck’s findings:
“1. Crude extracts from diseased plants passing through porcelain filter candles do not show bacterial growth during three months of storage, but remain infective. Subsequent plant inoculation by injection readily leads to infection and reproduction of the characteristic
symptoms.
2. Unlike bacteria, the infectious agent diffuses laterally into agar for at least 2 mm.
3. The agent multiplies in plants, as shown by serial transfers from plant to plant, and cannot be a toxin.
4. The agent multiplies only in actively growing tissues. It is not able to grow by itself but is carried away by the growth of dividing cells where multiplication in the living protoplasm is enormous.
5. Transport is `through the phloem’, upwards and downwards according to laws directing the movement of nutrients; in stems it is primarily vertical with little lateral spread.
6. The agent resembles living cells in that it is killed at 90oC.
7. The agent may be dried in infected leaves (in an herbarium) and in filter paper soaked in infectious sap.
8. The agent may remain in dry soil during winter and infect plants from the soil; it can also be transferred in potting soil.”
9. The agent retains infectivity after alcohol precipitation from sap and subsequent desiccation at 40 oC.Beijerinck didn’t believe his virus to be “corpuscular”, but instead referred to it as “contagium vivum fluidum”, or some kind of living fluid contagion.
A translation of that original paper can be found here:
https://www.apsnet.org/edcenter/apsnetfeatures/Documents/1998/