Author: Dr. Johnathan Kiel

The simplest mathematical description of Ro: Infectious contacts β per unit time, all assumed infected, and disease with infectious (latent incubation) period of 1/γ, (frequency), then the basic reproduction number is R0 = β/γ. Diseases have multiple latency periods; therefore, the reproduction number for the disease is the sum of reproduction numbers for each transition time (Rt). The effective reproduction number, Re, changes with time, affected populations, and circumstances. The reproduction number, as widely used and referred to, appropriately and inappropriately, hides that transmission is stochastic not deterministic, often dominated by a small number of individuals, and heavily influenced by superspreading events https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3000897. The complexities of tracking, and therefore, mitigating infectious diseases, when they get out of hand, like COVID, limit the usefulness and predictability of mathematical models. Coronaviruses, as a group and SARS-CoV-2 in particular, confound Ro values; they can be easily blocked by barriers (masks, etc), distance, quarantine, and other environmental factors but are highly efficient in infecting hosts exposed to an infectious dose; this keeps overall % of infection low, but sustains virus in the population indefinitely. All this calculating gives one the sense of potentially great precision and accuracy in detecting an infectious disease in the environment, in reservoir or primary hosts, whether the infection is apparent or not, if one only has a test that is sensitive enough, has a low false positive rate, and one collects enough samples. In practice, many infectious agents, including SARS-CoV-2, are not so homogeneously distributed or even distributed normally. They favor certain niches and circumstances. Most of what one collects are samples of “empty space, volume” in respect to the agent, where the probability of detecting it in spite of the size of the sampled volume is zero. This condition is what defeats the models and mathematics. The math and models can be beautiful and sophisticated and the calculations perfect but “garbage in, garbage out” or “empty space in, empty space out!”Testing must give the idea of prevalence of active cases, recovered resistant individuals, and their respective density distribution in a population to have any idea of the probability of effective or failed transmission or growth rate of a local epidemic or global pandemic. The Ro (Re, more realtime) value is not very valuable for predicting absolute numbers of a population which will be infected but better for predicting how to stop an epidemic or pandemic, and it is not trivial to accurately calculate https://royalsociety.org/-/media/policy/projects/set-c/set-covid-19-R-estimates.pdf. So in the simplest terms what does the Ro value tells us, especially since it continuously changes into different Rt and Re subsets? Does it tell us, without resorting to history, what conditions have to be met over time? When a disease will move from endemic to epidemic? Ideally, the answer is yes to the second question, and how it is done, the answer to the first question, theoretically, has been made quite simple and clear, as math is supposed to do after, what seems to those of us who are not mathematicians, very much a confusing and esoteric series of symbolic manipulations and calculations. To put the answer most succinctly, R0, R naught, the basic reproduction number, the average number of other people each infected person must infect in a population, who are susceptible to the disease, in order to maintain the disease endemically (without any outside introduction or change which influences the number of infected to grow) or for it to go epidemic (exponentially in number of infected people or animals), or eventually pandemic (super exponential because of the interaction in space and time of many populations). For the endemic case:

For an infectious disease to die out after introduction:

For an infectious disease to become epidemic:

For it to become pandemic:

Where n is the number of separate susceptible populations which may be connected by the infectious agent being transported from one susceptible population to another through space and p is the exponential multiplication of the original source contacts which will become sources for spread to new populations. For an infectious disease to remain in the endemic state, the basic reproduction number and the proportion of the population susceptible must be inversely related, otherwise it will disappear or become epidemic and, perhaps, pandemic. This all assumes the susceptible hosts subject to exposure are exposed under the same circumstances with the highest probability of infection. Here is where the simple math gets complicated and I will not pursue it here but will just say it brings up the problem of incident versus absorbed dose (seen with toxins and radiation effects) which is somewhat analogous in infectious disease to the infectious dose, or multiplicity of infection (MOI).

Viruses cannot be seen (usually except for a few very large ones at the limits of light microscopy) with a visible light microscope and will not grow independently in cell-free medium. Their effect on a lawn of target cells in which they have replicated is seen as the formation of plaques (clear “holes”) in an otherwise solid opaque or translucent lawn of animal or human cells or, in the case of bacteriophages, a bacterial parasitizing virus, in a lawn of bacteria on agar. Some animal and human viruses like retroviruses do not kill the cells they infect but transform them, so they do not stop growing when they form a monolayer in culture but continue to “pile up” into transforming foci representing a “viral colony”. Other effects, cytopathic effects (CPE), are even more subtle and take more training to observe and count as “colonies”. Even though cells may be shedding virus without dying or showing observable cytopathic effect, they cannot be observed or counted. The dilution to zero plaque or CPE is an inverse measure of the number of virions found in the original volume of inoculums and should be extrapolatable to zero virus particles, and thus, theoretically, one viral particle can be detected without ever seeing it. Like the bacteria this is a fiction and only theoretical. Otherwise, if one million virions are added to one million cells, the MOI should be one. If ten million virions are added, the MOI should be 10. If you add 100,000 virions, then the MOI is 0.1. However, this doesn’t happen because every target cell does not actually come in contact with a single virion. If we use k to represent the number of viral particles per cell, then we let P( k) equal the fraction of cells infected by that number of viral particles and m be the MOI. Then the fraction of target cells which remain uninfected (0 viral particles) is equal to the following:

Where e is the natural log base, approximately equal to 2.71828182846. For MOI of 1 or more, the calculation is:

In many cultures of viruses, in order to infect most of the cells a MOI of 10 is used because for a million of target cells, less than a hundred should remain uninfected, a trivial number. In fact, in a culture of one million cells, 999,500 cells receive more than one virion:

In other words, it takes a certain number of microbes to cause an infection, or an obvious apparent infection such as illness or death. For instance, the infectious agent of Q Fever, the most infectious agent in the world, requires a single organism to cause disease, and for comparison, anthrax requires about 10,000 spores to kill a human. However, they are not uniformly applied to susceptible populations and many other factors control and confound their outbreaks as detectable disease. Although it is difficult to test this simple model in an uncontrolled population like humans or animals in the wild, it can and has been demonstrated and predictive in at least one case that supports the validity of the model: the eradication of smallpox in 1977. This global experiment was based on the assumption that If the proportion of the population that is immune exceeds the “herd immunity” for the infectious disease, then the disease can no longer be sustained in that population. In the case of smallpox, this level was exceeded by vaccination and the disease was consequently eliminated. It is hoped that this can be done with other diseases by vaccination such as with polio or by isolation and depopulation in the case of animals as with brucellosis in cattle in the United States or as rabies was in the United Kingdom. The reverse has been demonstrated by refusal to vaccinate children or by inadequate immunization because of changes in the vaccine or virulence of the pathogen, as in the cases of measles and whooping cough, respectively. How would we write this in mathematical terms to determine the number of individuals which should be vaccinated to protect the population? We would use the following formula:

Where q equals the herd immunity, the portion of a population (or herd), which provides protection for individuals who have not developed immunity. As you can derive from the simple algebraic formula, the larger the reproduction number of the infectious disease the more the population has to be covered by vaccination.

A mathematical approach to increase the precision, accuracy and predictability of epidemiology, is the application of the “ROC curve” to determine if a disease can be “detected” in a population by infection and subsequent testing for that infection by some assay or observation of signs and symptoms. The “ROC curve”, Receiver Operating Characteristic was first used during World War II for the interpretation of radar signals before it was used in other types of sensors, including “sentinel” animals or humans who would indicate the presence of infectious diseases. Following the attack on Pearl Harbor in 1941, the United States Army began new research to increase the prediction of correctly detected Japanese aircraft from their radar signals, so was born the “ROC curve”. DARPA decided that the ROC curve would be the “gold standard” of detectability for biological agents. In the most general terms, the ROC curve is the fraction of true positives out of the total actual positives vs. the fraction of false positives out of the total actual negatives at various threshold settings. The ROC curve which applies to the ability of a sample to detect a positive for the presence of an infectious agent is an empirical plot of the number of infectious particles (bacteria or viruses) necessary to infect, or be “detected”, by the human or animal acting as a detector (actually measured by a clinical assay for the specific presence of the agent or the appearance of signs or symptoms which meets the case definition of the particular infectious disease) versus the false detection of such a particle. As the sensitivity of the detector is increased (equal to the number of true positives), the number of false positives increases sometimes linearly sometimes asymptotically or non-linearly depending on the characteristics of the detector. One has to decide the threshold of sensitivity vs acceptable false positives. The ROC curve is used to do this. The idea is to determine what you would expect for a given set of characteristics of the infection to be able to detect an infection. This approach tells the minimum number of samples from hosts, or the number of infected individuals in a given population that must be collected in the former and examined in the latter to detect an infection in a population, especially if it is not apparent or is endemic. In a sense, testing should tell the prevalence of the infection in the population in order to predict its Ro and growth rate of infection in that population, at least in theory. Bottom line is Ro is always being re-calculated from these data and testing results in addition to following numbers of cases over time (in realtime). It is more a result, an effect, an assessment, of where we stand in the fight against COVID and if we are approaching its end.

A new Spotted fever was seen in 2018 and 2019 in the United States in 3 dogs which had fever and hematological abnormalities; blood samples contained Rickettsia reactive antibody and Rickettsia DNA sequences by PCR amplification. These dogs came from 3 separate states: Tennessee, Illinois (signs developed 3 days after returning from a tick-infested area in Arkansas), and Oklahoma. The tickborne Rickettsia parkeri, R. philipii (Rickettsia 364D), and R. rickettsii cause Rocky Mountain spotted fever (RMSF), human spotted fever group (SFG) rickettsioses (mortality as high as 30% without treatment, but historically, a death rate as high as 80%; however, with doxycycline treatment, that rate drops to 0.5 %). R. rickettsii is the only known cause of SFG rickettsiosis in dogs. SFG Rickettsia seroprevalence is high in dogs in the United States and Mexico. In this study, they examined 3 genes (gltA, htrA, and ompA) and 2 intergenic spacer regions (23S-5S and mmpA-purC) in Rickettsia sp. by PCR. These targetted genes of Rickettsia were 100% identical in the 3 dogs. Multilocus genetic analysis placed this new Rickettsia sp. in a clade among SFG Rickettsia between human pathogens R. heilongjiangensis and R. massiliae. They failed to culture and isolate the new Rickettsia sp. from whole blood. The tick species, in these cases, were not identified, but ticks common to the states involved include Amblyomma americanum, Dermacentor variabilis, and Rhipicephalus sanguineus sensu lato, all of which are known to transmit Rickettsia. Haemophysalis longicornis, an invasive tick species recently found in the United States, including in Tennessee and Arkansas, may also be a possible vector of Rickettsia spp. Wilson JM, Breitschwerdt EB, Juhasz NB, Marr HS, de Brito Galvão JF, Pratt CL, et al. Novel Rickettsia species infecting dogs, United States. Emerg Infect Dis. 2020 Dec. https://doi.org/10.3201/eid2612.200272. The appearance of a new tick-borne spotted fever in dogs should not be unexpected; we have seen this before.

Flinders Island Spotted Fever, caused by Rickettsia honei, first discovered on Flinders Island Australia, is transmitted by Aponomma hydrosauri (a reptile-associated tick, now Bothriocroton hydrosauri). It was first identified on Flinders Island in 1991 by Dr Robert Stewart, who suspected the clinical symptoms of his human patients indicated a rickettsial infection. Symptoms included fever, headache, myalgia, a mild cough, and a maculopapular rash. Serological analysis, including the Weil-Felix agglutination and rickettsial-specific immunofluorescence tests, indicated a member of the Spotted Fever Group (SFG) was responsible for the disease. The reptilian vertebrate hosts for the tick on Flinders Island are tiger snakes (Notechis scutatus), copperhead snakes (Austrelaps superbus), and blue tongue lizards (Tiliqua nigrolutea). In 1998, a similar, if not identical, rickettsia was found in ticks in South Texas. In 2008, our entomologists at Brooks, also found the rickettsia in Amblyomma cajennense ticks co-infected with Coxiella burnetii (Q Fever). R. honei has been found in Ixodes and Rhipicephalus ticks in Asia and in Amblyomma cajennense, as we observed, in North America. Its worldwide spread is hard to explain.

A Spotted fever type Rickettsia from Ghana, West Africa, entered the US in ticks and imported snakes through Florida in 2002. The disease was associated with the Gulf Coast Tick, Amblyomma maculatum, introduced into a snake collection after importation, and the imported Snake Tick Aponomma latum, on the premises. The latter is the most common tick parasite of large snakes in most of sub-Saharan Africa and in the pet trade worldwide. It frequently arrives in the USA on imported pet pythons especially from West African countries such as Ghana. After many snakes died, the outbreak was finally stopped by tick control on the snakes and premises and tetracycline treatment of snakes in prodromal or asymptomatic phases of the disease (otherwise it was 100% lethal after neurological signs appeared even with treatment). Its responses to tetracycline and our inability to consistently culture any free-living microbes from the blood and tissues of acute cases supported it being a Rickettsia or Ehrlichia. Various Rickettsia, Ehrlichia ruminantium (cause of Heartwater), and Orientia tsutsugamushi (cause of Scrub Typhus) target vascular endothelium (Heartwater especially targets brain endothelium) as their main tissue to be infected as did Viper Plague. It was isolated, by blind passage for 4 years, from the livers of snakes, and cultured and isolated in snake and turtle cell lines, and then, the isolates were used to infect various other mammalian cell lines, including mouse, human, and bovine. Because of clinical signs of accumulated peritoneal straw-colored fluid, pneumonitis, gastroenteritis, neurological signs, seizures and sudden death as well as a positive PCR for the genetic marker, it was assumed to be an aberrant Heartwater. The putative causative agent was isolated in viper cells and propagated in turtle cells, but also infected bovine endothelial cells, and human cells (HeLa) as well.

The Heartwater agent is of such concern that it is forbidden, even for research purposes, to be maintained on the US mainland. Snakes belonging to Viperid, Colubrid and Elapid families succumbed to this new disease from July 2002-February 2003 in a private collection into which the snakes imported from Africa through Florida were introduced along with the ticks they carried. However, in spite of being intermingled in the collection, no western diamond-backed rattlesnakes (Crotalus atrox) showed signs of infection or died. This agent(s) may have already appeared in the US as a zoonosis combined with or diagnosed as Q Fever (MMWR, September 1, 1978, V o l. 27, No. 3, 5 May 25, 1978, New York State, the Suffolk County Department of Health Services). During this 1978 outbreak, a total of 11 persons became symptomatic. All were involved in unpacking and de-ticking (Amblyomma nuttalli, Aponomma latum, and Aponomma flavomaculatum) or came in contact with a shipment of 500 ball pythons (Python regius), imported on May 3 from Accra, Ghana. The vipers, which were involved with the 2002 outbreak, were shipped to Florida from Ghana along with ball pythons (some may have been co-mingled in transport bags) from the same vendor(s). Characteristic lesions of abundant straw-colored fluid in the common body cavity, inflammation with abundant mucus in the lungs and intestines, and accompanying diarrhea as well as per acute deaths in many cases with little or no signs beforehand were observed. This unknown Rickettsia tested positive with antibody against the OX19 Proteus vulgaris antigen, the same antigen used in the Weil–Felix Test. This test uses antibody against the OX19 antigen of the Gram-negative, free-living bacterium Proteus vulgaris which cross reacts with spotted fever and typhus groups of Rickettsia. It was the first serological test for the diagnosis of these rickettsial diseases (Edmund Weil and Arthur Felix in 1916). As I have already stated, emerging spotted fever group Rickettsia has already been seen as a zoonosis from reptiles, so except for the unusual genetics, this result should not have been a surprise. What was a surprise was that a Rickettsia could have appropriated genetic material from an Ehrlichia, probably co-infecting the same ticks. Species lines in these types of microbes have become very blurred. We were just very lucky that this worked because overall sensitivity can be as low as 33% and specificity as low as 46%.

DARPA is planning for an airborne COVID detector — Defense Systems https://defensesystems.com/articles/2020/11/11/darpa-sensars-covid-detection.aspx?s=ds_121120&oly_enc_id=&m=1. Once again DARPA has chosen to ignore or is totally unaware of what they already had in-house in the DoD. Trying to get antibody or other anti-ligands to interact in the air is extremely difficult unless one uses nanoparticles. The molecular interaction of ligand and anti-ligand (antigen and antibody) in air was attempted in the past at U.S. Army Edgewood Chemical Biological Center and AFRL; it failed but nanoparticles carrying a water coating had promise spiedigitallibrary.org/conference-pro…

“Dr Andy Ellington of the University of Texas, one of the co-inventors of SELEX, unbeknownst to us, had received substantially more support than we had, from DARPA, to develop aptamer sensors for biological warfare agents. DARPA was not under the constraint of “forced collaboration” suffered by the Service Labs and their contractors. He went on to receive more money for developing aptamer “beacons”, with a consortium of other institutions and contractors, that were supposed to interact with the agents on the fly in a biological “attack” cloud and cause them to “light up” by fluorescence (from interaction with laser light) that would reveal their presence remotely. This project was doomed from the beginning for two reasons: (1) the Army had tried the approach with antibody in the early 1990’ s at Aberdeen and failed; and (2) As Dr Eric Holwitt always said, “Most of the volume of air that we are looking in for agents is empty space; it is not that the interaction is not sensitive enough, but that there is no agent with which to interact.” This would later also be a problem with standoff detectors that depended on lasers only; the cross section of the microbes diluted in a cloud was just too small to give a decent return signal, much less any natural spectra that were specific for the agent. They were lucky to just tell the difference between a dust cloud or sand storm and a biological, which just as well have been pollen or fungal spores.—-The Black Dragon Trilogy.https://a.co/e38es8z.

Nano droplets for in air interactions:

Recently, it was reported in Kaiser Health News (https://khn.org/news/search-for-a-snakebite-drug-might-lead-to-a-covid-treatment-too/) and the AVMA Animal Health SmartBrief that a new small molecule inhibitor of phospholipase A2, Varespladib, (https://www.nature.com/articles/s41598-019-53755-5.pdf) of snake venom for treatment of snake bite might treat COVID-19 lung disease as well because another PLA2 enzyme in human tissue is in the inflammatory cascade. The Brooks Counterproliferation Team made neutralizing aptamers against AB binary toxins: Botox, Shiga toxin, phospholipase A2 of South American rattlesnake venom, and anthrax toxins; United States Patent US 9,273,345 B2, Mar.1,2016. The snake antivenin aptamers were going to be pursued for this snake (Crotalus durissus terrificus) as well as other significantly dangerous snakes in collaboration with Administración Nacional De Laboratorios E Institutos De Salud (National Administration of Laboratories and Institutes of Health (ANLIS) ) but because of the closure of Brooks in 2011, this never happened.

Many bacterial toxins are AB toxins, that is, they contain a trans-cell membrane cell transporting component B and an affector or toxic effect generator A. Oddly, this motif is seen in the venoms of snakes. The most famous of the AB toxins is produced by the anaerobic bacterium Clostridium botulinum. It is the number one biotoxin on the Select Agent, potential biowarfare/ bioterrorism, list with an estimated human median lethal dose (LD-50) of 1.3–2.1 ng/ kg by injection or 10–13 ng/kg by inhalation. Other species of this genera also produce AB toxins which cause neurological toxicity, tetanus (tetanus toxin produced in wounds by Clostridium tetani), or necrotizing effects (iota toxin of Clostridium perfringens), gangrene. Botulinum toxin is composed of a heavy chain (equivalent to the B chain) and a light chain (equivalent to the A chain) linked together by a single disulfide bond (as many of the snake venoms and related polypeptide hormones such as insulin and nerve growth factor are linked by more than one disulfide linkage). The toxin is produced by the bacteria as an inactive pre-toxin (approximately 150k) which is activated by proteolytic cleavage into the 100kD heavy chain and the 50kD light chain. Botulinum neurotoxin exists as 7 different serotypes, A, B, C, D, E, F and G. All these various serotypes inhibit acetylcholine release from nerve endings, but by targeting different intracellular neuronal protein components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) acetylcholine transport complex. However, their potencies vary substantially, with some being more toxic and more prevalent clinically in some host species than others. Some are more likely to be associated with human disease like Type A (Western North America) and B (Eastern North America) and others with animal disease, in waterfowl Type C and Type E sporadically among fish-eating birds, such as common loons (Gavia immer) and gulls. Even though botulinum toxicity is usually considered only a toxicity, it can be produced in infants by the consumption of honey contaminated with C. botulinum spores which grow into vegetative forms and produce toxin in vivo, in situ. Occasionally this is seen in adults. An even rarer form is wound botulism related to C. botulinum which grows and produces Type A and Type B toxins, but in at least one case, Type E, in place. Another prominent AB toxin which has seen much press is Shiga (from Shigella dysenteriae shiga toxigenic group of Escherichia coli (STEC), which includes serotypes O157:H7, O104:H4, and other enterohemorrhagic E. coli (EHEC)), which produce related Shiga-like toxin, which is composed of Shiga-like toxin 1 and 2 (SLT-1 and 2 or Stx-1 and 2), with Stx-1 differing from Stx by only 1 amino acid and Stx-2 sharing 56% of its sequences with Stx-1. Shiga toxin inhibits protein synthesis by a mechanism similar to ricin toxin produced by the castor bean, Ricinus communis. After entering a cell, the affector portion of the toxin acts as a N-glycosidase, cleaving a specific adenine from the 28S RNA of the 60S subunit of the eukaryotic ribosome, stopping protein synthesis. Besides causing the gastroenteritis of O157 infection, it also is neurotoxic. The glycosphingolipid, Gb3, is the receptor for Shiga and Shiga-like toxins. It is present in great amounts on renal epithelial cells leading to renal toxicity. Gb3-type receptors are also found in central nervous system neurons and endothelium, which may explain the toxin’s neurotoxicity. Stx-2 increases the expression of its receptor Gb3 and causes neuronal dysfunctions through this positive feedback mechanism. Our group of researchers at AFRL, Brooks City-Base, discovered that curcumin, if given before exposure to the toxin, decreases the Gb3 on target cells. We also developed small synthetic oligonucleotides which bind to the toxin and prevent its action in cell culture. Therefore, Shiga toxicity is a toxigenic disease which is infectious, but not only for the human host. Its genes are encoded on a latent, temperate lambdoid prophage in Escherichia coli. The phage regulatory network is a significant contributor to toxin production and release by this pathogenic E. coli and allows the phage to be released from lysogenic E. coli to co-opt other normally non-hemorrhagic enteric E. coli. This opens another variant from Koch’s Postulates, the conversion of resident microbes which are non-pathogenic into pathogenic ones, violating the first hypothesis by having all hosts carrying potentially pathogenic microbes. This mechanism of toxin genes being conveyed to a susceptible co-opted microbial host does not end with E. coli. It exists in one of the most common bacteria which we vaccinate against in childhood with the classic DPT shot, the toxin of the diphtheria bacterium, Corynebacterium diphtheria. Diphtheria toxin was discovered in 1890 by Emil Adolf von Behring. In 1951, it was discovered that the toxin gene was not encoded on the bacterial chromosome, but by a latent temperate phage infecting all toxigenic lysogenic strains of the diphtheria bacteria. The toxin inhibits protein synthesis as does Shiga and other bacterial toxins and some antibiotics. It does this by acting as the enzyme NAD-ADP-ribosyltransferase (EC 2.4.2.36). It catalyzes the transfer of nicotinamide adenine dinucleotide to the eukaryotic cell elongation factor-2 (eEF2), inactivating this protein so that it cannot participate in the protein synthesizing function of the ribosomes. It ADP-ribosylates the unusual amino acid diphthamide in eEF2. The toxin, like Shiga toxin and snake venoms, is an AB toxin. It is structurally a single polypeptide chain of 535 amino acids composed of two subunits linked by disulfide bridges. Cancer drugs were eventually developed using the toxin, Denileukin Diftitox, which uses diphtheria toxin as an anti-neoplastic pharmaceutical, and Resimmune ™ which is an immunotoxin for cutaneous T cell lymphoma. The latter uses diphtheria toxin (truncated by the cell binding domain) coupled to anti-CD3 antibody. Cholera, which Robert Koch investigated and used the observation of its carrier state in people to disregard his first postulate, is caused by the bacterium Vibrio cholerae, which produces an AB toxin similar to diphtheria toxin and is also co-opted by a bacteriophage which conveys the toxin genes. The cholera toxin is an oligomeric protein made up of six subunits, a single copy of the A (enzymatic “affector” subunit) and five copies of the B subunit (receptor binding), AB5. Subunit B binds and delivers subunit A to the cell where it activates the G protein which activates adenylate cyclase. The five B subunits have a mass of 11 kDa each and form a five-membered ring. The A subunit is 28 kDa and has two functional substructures, the A1 portion of the chain: a globular ADP-G protein-ribosylase and the A2 chain, an extended alpha helix which sits in the center of the B subunit ring. The toxin is similar in structure and mechanism to the heat-labile enterotoxin of Escherichia coli. The subunit A enzyme activates, with different specificity, but essentially the same catalytic activity as subunit A of diphtheria toxin. All these toxins are potential targets for therapeutic neutralizing aptamers which can be selected from a large library of DNA sequences and amplified for clinical use https://patentimages.storage.googleapis.com/4d/9f/d0/ce71a48231f7c5/US9273345.pdf.

This blog is their legacy and the book Notes from the Shadows in The Black Dragon Trilogy their history (see Amazon eBooks).

In an earlier post, I discussed autoantibodies against Interferon I and how this interfered with the first line of defense against SARS-CoV-2. Now recent data has revealed that the virus induces other autoantibodies against other targets which explains the variety of symptoms and perhaps chronic disease following initial COVID recovery. Autoantibodies are not unprecedented in viral diseases. The exclusion of cells in the immune system that produce antibody or cellular immune responses directly against self is a central tenet of immunology. Sir Frank McFarlane Burnet was awarded the Nobel Prize for Medicine and Physiology in 1960 for his work on the immunological recognition distinguishing non-self from self. He coined the term for immune cells’ generating antibodies against self the “forbidden clone”, and formulated the hypothesis, of the same name, that in autoimmunity there exist immune cells able to make antibodies against a variety of self-antigens. He proposed autoimmune disease develops because of the escape of self-reactive clones of lymphocytes’, which are normally deleted during development to generate immune tolerance of self. These autoreactive clones can multiply and cause immune-mediated organ damage through peripheral self-antigen immune recognition and direction of effector cells to kill targets cell in a variety of tissues and organs. He thought these clones might arise as a result of somatic mutation early in lymphoid development. The proposal pre-dated knowledge of the roles of T and B lymphocytes. During his time, the role of autoantibodies was recognized in diseases such as Grave’s Disease, associated with thyroid stimulating antibodies, directed against the human thyroid stimulating hormone receptor. Later in the 20th century, T cells came to be central in theories of autoimmunity and B cells fell off the map. The truth lies somewhere in between. https://pmj.bmj.com/content/postgradmedj/88/1038/226.full.pdf.

However, autoimmunity may persist not only because of detrimental mutation, but also because it serves an evolutionary, beneficial purpose. It has been counterbalanced by apoptosis, which suppresses immune signals while still getting rid of unwanted or unneeded cells, without triggering inflammation. When necrotic tissue infected by pathogens or cells undergoing disease processes contain intracellular pathogens hidden from antibodies or immune surveillance, the tiniest departure from normal surface structure or exposure of internal cell structures to the immune response may be essential to elimination of the pathogen. Various forms of programmed cell death may lead to various levels of immune response exposure: autophagic cell death, necroptosis, elimination by shedding, keratinocyte death by cornification,and cell–cell cannibalism by entosis https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2994033/. These various self- killing mechanisms and autoimmunity may also be important in preventing the development of cancer, or allowing it to proceed, when these countermeasures are out of balance.

The autoimmune response is not new to viral infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6723519/pdf/viruses-11-00762.pdf. Examples of infections that yield autoantibodies are those with enteric viruses such as Coxsackie B virus and rotavirus and other types such as influenza A viruses, herpesviruses, measles, mumps, and rubella. Autoimmune B cell responses contribute to mixed cryoglobulinemia, glomerulonephritis, and vasculitis, of hepatitis B and C virus infection. Another example in animals is antibody to feline oncornavirus-associated cell membrane antigen (FOCMA) on the surface of feline lymphosarcoma cells in sera of cats relatively resistant to development of FeLV (feline leukemia virus), Lymphosarcoma, and FeSV (feline sarcoma virus) fibrosarcoma. The antibodies do not exclusively attack viral antigens but consistently bind to 70-kDa proteins on the surface of lymphosarcoma cells https://pubmed.ncbi.nlm.nih.gov/6318434/. Therefore, resistance to cancer, caused by these viruses, is associated with this anti-host cell antibody, not necessarily direct antiviral antibody.

Other chronic disease conditions triggered by viruses and caused by autoimmunity include Type 1 juvenile diabetes and Type 1.5 adult onset autoimmune diabetes. Type 1 diabetes is a lifelong chronic disease characterized by the loss of or severely reduced number of insulin-producing β cells in the islet of Langerhans, presence of islet autoantibodies, and, especially in younger individuals, insulitis consisting of infiltration of the islets predominantly by CD8+ T cytotoxic cells and macrophages. In explanted islets cultured from coxsackievirus B-1-infected islets, the expression of the coxsackie–adenovirus receptor gene was increased and was 10-fold higher in endocrine compared with exocrine cells of the pancreas. There is high suspicion of viral causality because of these observed experimental results https://drc.bmj.com/content/4/1/e000219.

Another autoimmune disease where virus is suspected but not immediately linked is multiple sclerosis (MS). The comparison of chronic canine distemper, which leads to spinal cord and CNS nerve process demyelination, has led to this suspicion by analogy, but incrimination of the canine distemper virus being the cause in human MS has not withstood scientific examination. T cells have been considered the main (MS) protagonists in the pathogenesis of multiple sclerosis. Only recently has evidence arose supporting B cells as major contributors. Rather than merely producing autoantibodies, they collaborate with T cells by priming and regulating T cells, and mediate both pro- and anti-inflammatory interactions. However, despite intensive searching, the target antigen(s) of B cells in MS have not been identified https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4519967/pdf/ijms-16-16576.pdf. The original viral instigator may have only left a ghost antigen behind long after it was eliminated.

Such examples make the autoimmune pathogenesis of COVID not unexpected. Autoantibodies which recognize cell membrane phospholipids cause an autoimmune disease called antiphospholipid syndrome (APS). In APS patients, autoantibodies activate clot-forming cells, putting those patients at higher risk of blood clot formation. These antibodies may also be present in a variety of bacterial or viral infections. However, cause and effect of blood clotting during infection is difficult to prove. Severely ill COVID-19 patients can have high levels of neutrophils, and some have phospholipid-binding antibodies in their blood. Of the patients studied, of those who developed blood clots, only half had the auto antibodies https://stm.sciencemag.org/content/scitransmed/early/2020/11/02/scitranslmed.abd3876.full.pdf. Additionally, the immunological response in severe COVID-19 infection, including TLR7 activation by SARS-CoV2 single-stranded RNA, is sufficient to drive new autoimmunity without the patient’s response against a variety of self-antigens being genetically pre-determined. https://www.medrxiv.org/content/10.1101/2020.10.21.20216192v2.full.pdf. All this being said, the immune system has several possible pathways to follow in COVID-19, some of which I have discussed in earlier posts. The good and bad news is that the progression has these choices. This means that chemically programmable immunity using antigen-aptamer conjugates has a significant probability of re-directing the response down one such pathway that effectively eliminates the SARS-CoV-2 virus without triggering pathogenic autoimmunity or chronic autoimmune disease, which would persist after the virus is gone. There is even a possibility that redirection can ameliorate such residual autoimmunity or even, under ideal conditions, end it. However, until research is supported toward testing these hypotheses, we will never know.

Viruses are such good vectors of engineered genetic material because they are near perfect deliverers of nucleic acids from durable, protective packages into host cells. The nucleic acids delivered take over the replicative machinery of the host cells to reproduce themselves and express proteins which automatically re-package the new nucleic acids. The new virions have the means, by virtue of their coat proteins, to extrude from the cell by fusing with the outer cell membrane, enveloping with cell membrane lipids, by passing through pores formed by transmembrane proteins, or by bursting the cell to be released. How could these wonderfully elegant devices be duplicated without protein to assemble nucleic acids to direct the host cells to build more vectors? Polymers or lipid containers or combinations thereof could duplicate the delivery of nucleic acids across cell membranes to provide expressible genetic elements, but the vectors could not be biosynthetically reproduced, like viruses, to carry on the process to new host cells. This was an advantage to prevent inadvertent uncontrollable transfer, but limited the number of cells producing the new trait or protein. How could both be achieved without loss of control? Could a polymer be made from existing biological feedstock synthetically as well as biosynthetically? Could sufficient but minimal genetic information be transferred to drive host cells to make more vector as well as transfer the designed expressible genes included? Diazoluminomelanin (DALM) is key to the answers. The whole story of this wonderful, amazing material is told in 4 books I wrote and published between 1994 and 2018, inclusive, and previous posts. The three main reasons for the discovery of DALM was (1) to replace the enzyme peroxidase in the hydrogen peroxide and luminol interaction in the chemiluminescent reaction with a single component, (2) find a chemical indicator of radio frequency radiation and microwave absorption, thermochemiluminescence, (3) find a potential application(s) that would justify funding the basic research. The latter made for a moving target of potential applications. The polymer was first synthesized by the following simple reaction:

Solving the structure of the resulting polymer and explaining it’s extraordinary properties would be far more difficult and take much longer.

What was most remarkable was that by cloning a subunit of a plant (barley) nitrate reductase gene through plasmids into bacterial to even human cells, endogenous nitrate reductase in bacteria and nitric oxide synthase in animal and human cells could be greatly enhanced in their production of nitrite. They also became capable of endogenously synthesizing DALM when fed nitrate, 3-amino-L-tyrosine and luminol. They produced it in a spheroid nanoparticle form.

In a previous post, I showed how our last experiments at Brooks showed plasmids containing the nitrate reductase gene subunit could significantly inhibit the replication of vaccinia virus (close vaccine relative of smallpox) and surprisingly how certain arrangements of the gene, with one that produces iRNA, which blocks a viral gene that inhibits apoptosis, actually increases host cell lytic plaques (following viral infection), but did not necessarily increase the subsequent yield of virus. Unfortunately, because of the closing of Brooks further confirmation was not possible, particularly of the latter. What has been confirmed over and over again with many experiments is that DALM produced by these cells could be activated by pulsed microwaves which will destroy cells bearing DALM nanoparticles, external or internal, as a fail safe against transfected or transformed cells, and destroy viral or bacterial pathogens contained within or as bystanders, but spare cells that are not in the immediate vicinity. This would have been a remarkable achievement that makes DALM nanoparticles superior to any gene vector currently available for research or therapy or for vaccine delivery.

mRNA vaccines, like those being clinically tested, and possibly alternate DNA vaccines, discussed in an earlier post, not only must lead to the synthesis of the correct protein in situ to yield the appropriate immune response, but also not trigger innate immune responses directly that are pathological. We saw this in our previous research on artificial nucleic acids for treatment of infectious diseases. The toxicity of aptamers noted in our earlier studies may have been influenced by their interactions with the innate immune system, either TLR-9 receptors or other unknown ones that trigger adverse responses. In vitro studies have demonstrated that, in autoimmune-prone mice, dual signaling via the B cell receptor and non-CpG (cytosine/guanine base coupled) DNA results in synergistic B cell activation in a TLR9-independent manner. These results suggest that engagement of a TLR9-independent DNA activation pathway may trigger autoimmunity. TLR9 can mediate either efficient Th1- or Th2-dominated effects depending on whether innate immunity is stimulated by CpG or other certain non-CpG oligodeoxynucleotides (ODN).

Lymphocyte produced Th1-type cytokines produce pro-inflammatory responses responsible for killing intracellular parasites and for promoting autoimmune responses. Interferon gamma is the main Th1 cytokine. Excessive proinflammatory responses can lead to uncontrolled tissue damage. The Th2-type cytokines, including interleukins 4, 5, and 13, promote IgE and eosinophilic responses in atopy, and in addition, interleukin-10, as an anti-inflammatory response. IL-10, produced by CD4 + lymphocytes of the Th-2 subset, not only inhibits synthesis of gamma-interferon by both T cells and NK cells, but also inhibits the synthesis of NOS by mouse macrophages. Th2 responses counteract Th1 mediated anti-microbicidal actions. The optimal scenario is well balanced Th1 and Th2 responses, appropriate for the immune challenge. Allergy may be a Th2 weighted imbalance with Th-1. Immunologists have been seeking ways to redirect allergic Th2 responses toward Th1 responses to try to reduce the occurrence of atopy.

The non-CpG sequences that can be found in aptamers could cause this Th1 to Th2 shift, the latter is usually considered less inflammatory. Therefore, subsets of sequences must be carefully selected in therapeutic or vaccine nucleic acids to avoid these untoward undesirable effects. The following chart shows that some of the aptamers in our studies had these non-CpG sequences:

The following types of Toll-like receptors control the off-target consequences of nucleic acid therapeutics and vaccines; the secret is finding a combination with the targeted function of the nucleic acids that is mutually beneficial: TLR3 against Double-stranded RNA of Viruses; Poly (I:C) Synthetic analog of double-stranded RNA ; TLR7 and TLR8 against Single-stranded RNA from Viruses; and TLR9 against Unmethylated CpG DNA of Bacteria, Protozoa, Viruses, and Mitochondrial DNA (possibly pathogenic).

In addition to insights into how the immune system might respond to synthetic artificial nucleic acids, we must also consider how off-target effects might manipulate host nucleic acid functions, protein expression and physiology, in general. There are natural examples (viroids) of such manipulation, some pathological. However, their discovery also led to new approaches to gene therapy such as RNA interference to effect gene expression changes (translation into functional proteins by affecting other genes but not being transcribed themselves). RNA interference (RNAi) inhibits gene expression or translation by neutralizing targeted mRNA molecules. Historically, RNAi has also been known as co-suppression, post-transcriptional gene silencing (PTGS), and quelling.

“Viroids are plant pathogens and one human pathogen, Hepatitis D “virus”, which are made solely of nucleotides composing a single-stranded RNA. Viroid genomes range in size from 246 to 467 nucleotide bases, smaller than any known virus. They were discovered by Theodor Otto Diener, plant pathologist at the Agricultural Research Service in Maryland, in 1971. They do not code for protein and they use RNA polymerase II, a host cell enzyme which mediates messenger RNA (mRNA) synthesis from DNA, for replication, using the viroid RNA for a template. Some viroids are ribozymes, being catalysts which self-cleavage and ligate viroid genomes from larger replication intermediates. The first viroid identified was Potato Spindle Tuber Viroid. Approximately 33 “species” are known. The viroids replicate through a double-stranded intermediate RNA. They are then cleaved by a dicer enzyme into siRNAs (small interfering RNA), which are then incorporated into an RNA-induced silencing complex. The viroid siRNAs hybridize with the plant’s mRNA and cause disease by degradation or inhibition of translation of the mRNA into plant proteins, causing viroid diseases.”— The Black Dragon Trilogy by JOHNATHAN KIEL
https://a.co/3iXtFwv

“Human disease caused by a viroid is hepatitis D, or Delta Agent. Delta Agent is a viroid contained in the Hepatitis B Virus capsid. So, this virus and the Delta Agent simultaneous infect the patient. The hepatitis D viroid RNA complements and hybridizes with human liver cell 7S RNA, a small cytoplasmic RNA component of the signal recognition particle, the structure involved in the translocation of secretory and membrane-associated proteins. The hepatitis D viroid causes liver cell death by binding to and preventing the function of the 7S RNA and perhaps cleaving it.”— The Black Dragon Trilogy by JOHNATHAN KIEL
https://a.co/iUKBaeW

This examination is not meant to discourage taking advantage of nucleic acids for vaccine or therapeutic development but only to encourage the appropriate care needed before proceeding to the clinic.

The initial vaccine candidates from Pfizer and Moderna are both mRNA vaccines, if approved they would be the first mRNA vaccines ever approved. mRNA is an unstable molecule that breaks down when exposed to significant temperature fluctuations, even at ultra-low temperatures. mRNA vaccines require ultra-low temperatures of -70˚ to -80 for storage and shipment. In vaccine form, the importance of mRNA’s temperature stability is greatly amplified. This is an example of advanced biotechnology leading medical practicability: a demanding cold chain difficult to achieve even in the US and advanced countries but impossible in developing countries, not even considering that these requirements will drive the expected cost through the roof. https://www.laboratoryequipment.com/569702-Hurdle-1-Develop-COVID-19-Vaccine-Hurdle-2-Deliver-it-at-Ultra-low-Temperatures/.

Because of the mRNA vaccine ULT demands, Pfizer designed temperature-controlled shipping containers which use dry ice to keep the vaccine vials at -75˚C for 10 days. These are roughly the size of a suitcase and weigh about 70 pounds when fully loaded. However, a shortage of ultra-low temperature (ULT) freezers is anticipated because they are not usually available in most clinics, creating an added expense and probable loss, waste of vaccine, especially since federal distribution of cold-storage vaccines is anticipated to be sent in 1,000-dose shipments. The rush to purchase ULT freezers could lead to a national shortage, hampering distribution of such vaccines. AstraZenca and Oxford’s vaccine, which may be released next, needs -20˚C temperatures to ensure efficacy, a little better.

The developing world will need vaccine volume provided by 2 to 3 approved vaccines. They may even have to wait for a liquid vaccine, or at least a -20˚C vaccine that is widely available (still problematic in many countries and remote regions). The logistics of the frozen mRNA vaccines are nearly impossible for developing countries. The Associated Press estimates vaccine storage issues could leave 3 billion people in developing countries without access to a coronavirus vaccine. Are there other subunit nucleic acid vaccines that are more stable? Yes. DNA vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1986720/.Messenger RNA (mRNA) molecules provide the templates in the cytoplasm of a cell for translation by the ribosome and tRNA (amino acid transfer RNA) into protein, making multiple copies of the protein from each mRNA template. This amplification provides a multiplication of the vaccine immunogen per molecule compared to providing individual protein molecules. However, offsetting that advantage, in addition to the instability of mRNA, only about one of 10,000 molecules of mRNA will escape into the cytoplasm after being taken up by a cell. Amplification by translation of mRNA into protein has to overcome the losses and the inefficiencies of degradation and the translation to protein. Unlike plasmid DNA, which must enter the nucleus of the cell, mRNA only needs to be present in the cytoplasm, which eliminates the additional barrier, the nuclear membrane, that plasmid DNA has to cross. However, plasmid DNA is more stable than mRNA, and each DNA molecule results in the production of multiple mRNA molecules, thus the theoretical advantages of one over the other is found in the net stability of plasmid DNA versus mRNA, as well as differences in efficiencies of targeting the proper cells, movement to the cytoplasm or nucleus followed by transcription of the plasmid DNA (transcription from DNA to mRNA), and the translation of mRNA, whether transcribed from DNA or translated from mRNA, to protein https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6631684/. The Brooks Counterproliferation Team had to address these problems and others to achieve field durability for DNA aptamers, their vectors, and resistance to degradation in transit in the field and even methods to manufacture them in place in the field as necessary on demand.

In the military, we always considered durability and survivability of all technology used against biological agents so it could be used under adverse conditions with little or no logistical tail, minimal technical skill to operate or maintain safely under the most adverse conditions. Of particular concern was the area of NBC (Nuclear, Biological, Chemical) defense, aircraft and WMD (Weapons of Mass Destruction) neutralization (that is decontamination of aircraft and preventing the Special Forces aircraft from becoming contaminated in the first place by using appropriate NBC neutralization, and NBC sensor, monitoring, technologies). All these detection, isolation and identification technologies had to answer these questions: Is the agent present? What might it be? And is it safe to bring back a sample without contaminating the Special Forces aircraft so that it can’t land in permissive territory after exiting non-permissive territory with the agent in hand? Aptamers, used to isolate, identify and neutralize biological agents in place of antibodies and to turn infectious agents into autogenous vaccines by neutralizing their pathological mechanisms in an infected individual therapeutically, could be self-delivered by inhalation. This approach would have extreme operational utility when an attack occurred and could save many military by safe administration of the aptamers, quickly, that required no special handling or even cold storage. All the technologies were measured by their ease of use under adverse conditions, an approach which seems to be lost on SARS-CoV-2 vaccine developers, but which is absolutely necessary to distribute and effectively apply vaccines quickly worldwide to quell this pandemic.

A “system of systems”: The following are examples of military ruggidized technologies for use against pathogens under adverse, limited field conditions:

Controlling the spread of SARS-CoV-2 within buildings is tied to air quality, flow, dilution with outside air, filtration, and active decontamination. A recent webinar was held discussing how buildings can be made safer in the presence of SARS-CoV-2: Heating, Ventilation, and Air Conditioning Strategies for COVID-19 by Federal Facilities Council, Wed, October 28, 2020, 1:00-2:30 pm CST. The bottom line was that dilution of internal air with frequent external air intake exchanges is the “gold standard” to reduce levels of indoor infectious virus, but depending on outside air temperature and humidity characteristics, conditioning could prove expensive, especially for old systems in aging buildings. Measures which achieve comparable air quality to dilution were discussed. Filtration is the next best and cost effective, if the proper filter is chosen, installed in the right part of the HVAC system and installed properly so air doesn’t go around it. It effectively reduces numbers of airborne infectious particles which, in turn, reduces the exponential decay effect and increases the efficacy of active decontamination methods. The most cost effective filter is rated MERV 13, as long as it has not achieved this rating by being electrostaticly charged (when the charge is lost as with condensation, the filter rating drops to 6-8). Particles that were of most concern were around 500 nm because, as I discussed in the post on aerosol vs airborne, these are not subject to the described mechanisms of deposition and can enter deep into the respiratory system. The presentation stated these made up 90% of the air suspended particles for up to 41 hours, but did not indicate what portion of original expelled particles these represented (since the range in absolute numbers is indeterminate). Previously reported averages in that post were around 800 nm aerodynamic diameter.

According to the American Society of Heating, Refrigerating and Air-Conditioning Engineers (ASHRAE), this filtration helps provide healthier indoor air quality.

Minimum Efficiency Reporting Values, or MERVs (Minimum Efficiency Reporting Values)

UVC (254 nm wavelength) was considered the most effective active decontamination technology. The presenters stated that UVC should be positioned downstream of the cooling coils, to kill 90 % of the virus and to keep clean condensation pans from Legionella bacteria. UV lamps were also recommended to be placed in ceilings out of eye and skin damage range (radiation drops off 1/re2 with distance from the source). My experience with such lamps is they continue to visibly shine even after they have lost much of their UVC output, and that output is drastically reduced by dust accumulated on the bulbs. Besides the hazard and that not enough UVC for the required dwell time may reach infectious particles to fully inactivate a dose in transit, in spite of controlled optimized laboratory experiments demonstrating high efficacy, they fall short in prolonged field use unless closely monitored and maintained.

Examining UV dose of disinfection in a precise way, the inactivation ratio is determined by the applied UV dose through the disinfecting unit. The UV dose (mJ cm−2) is calculated by the delivered irradiance or fluence rate to microbial cells (mW cm−2) multiplied by the exposure time (s). Therefore, for UV- induced reactions, the most accurate report of the kinetic data is related to UV dose rather than time. If there were no confounding factors, the disinfection of biological agent contaminated air and surfaces with UVC could be straightforward and predictable. The required UV dose for 90% viral inactivation is increased by 1.5−2 times for a surface compared with air due to microscopic aggregation on surfaces. Other surfaces have microscopic irregularities, crevices, and roughness which counter the line of sight killing of the virus or microbe on that surface. A similar shadowing effect in air treatment can be caused by the presence of larger particles and dust in the air stream. In addition, the microbial cells may be attached and agglomerated on dust, which require higher UV doses for inactivation. One study indicated 120 mJ cm−2 of UVC dose is required to reduce the tissue culture infective dose (TCID) from 3.8 × 10e7 to 180 TCID50/mL, which is a more than 99.999% reduction. Based on this observation, SARS-CoV-1 (and most likely SARS-CoV-2) is similar to influenza virus and hepatitis A virus in being eliminated by UV radiation https://pubs.acs.org/doi/pdf/10.1021/acsphotonics.0c01245.

Another active decontamination method discussed at the webinar was Photocatalytic Oxidation (PCO), commercially available hydroxy-free radical generating systems that are 95% effective and “smoke eaters” which destroy VOCs, volatile organic compounds. in general. At Brooks, we tested our own in-lab-manufactured device under field conditions to efficiently destroy biological warfare stimulants.

Since hydroxyls result from water vapor, hydroxyl generation requires significant relative humidity in the air. One such commercial system, HYDRA PRO XL solves the low-humidity problem by generating and transferring water vapor to a direct injection manifold inside the machine which evenly distributes the vapor over UV lamps to maximize hydroxyl production https://www.boie.us/hydra-pro-xl.php.

In addition to this PCO mode, the HYDRA PRO XL’s has three AOP modes which can double to triple hydroxyl free radical production. They are low concentration Hydrogen Peroxide Vapor (HPV), low concentration UV Ozone (O3/UV) and a combination process. In the PCO mode (Safe Mode), the HYDRA PRO XL uses water, UV light and a catalyst, titanium oxide, to safely generate hydroxyl free radicals which remove organics causing odors, kill bacterial spores and pathogens. PCO mode can be used with or without the included water vapor injection system, depending on site conditions. The resulting byproducts are harmless carbon dioxide and water.

PCO hydroxyl generation, originally developed by NASA, is ozone-free and safe to use indoors for continuous operation in unventilated spaces for as long as it takes to remove odors and break down contaminants.

Contaminated air is drawn through an odor-neutralizing pre-filter and a HEPA filter to trap most particulates. Then, the unit uses six germicidal (253-nm UVC) lamps in a 2-layer crosshatch pattern along with 3 layers of nano-catalyst to produce hydroxyl radicals which breakdown volatile organic compounds (VOCs) and other contaminants that pass through the unit’s air flow. The next mode, AOP Mode 1, uses vaporous hydrogen peroxide in a direct injection system instead of pure water. The resulting low-concentration hydrogen peroxide vapor (HPV) breaks down into hydroxyl free radicals when exposed to the unit’s germicidal UV light without requiring the catalyst. To initiate AOP Mode 1, one adds a commercially-available hydrogen peroxide solution to the unit’s direct injection reservoir and runs the Advance in PCO mode. PhotoCatalytic reactions will occur simultaneously. No ozone is generated in Safe Mode, however, HPV levels in the air must be monitored if workers are present. This AOP is the simplest method to generate hydroxyl radicals using UV light: Hydrogen Peroxide / UV Light:
H₂O₂ + UV —› 2 •OH

To maintain safe air quality they must be tested routinely for ozone production. Ionizing air systems from producing airborne ions to plasma-induced active (singlet) oxygen and monoatomic oxygen species are also effective but also must not produce ozone. Ozone is a poor decontamination component in the output of these systems when compared with hydroxyl radicals, singlet oxygen or monoatomic oxygen or thermal electrons generated at the plasma source.

As noted in an earlier post, the initial amount of an infectious agent greatly influences significantly surviving amounts of the agent, regardless of method of active decontamination, based on the conversion of the deactivation of the agent from linear to exponential decay. The following graphs show such a conversion, not with amount of anthrax spores, although this has been seen with large amounts of anthrax spores exposed to ionizing radiation, but with the addition of other wavelengths of light (visible) to the UV exposure. This is not biological repair but chemical passive repair which could occur in viruses or by the viral genome being reassembled from damaged pieces into infectious virus once they enter host cells. The damage by UV or higher energy electromagnetic radiation, like X-rays and gamma radiation, is dependent on unobscured line of sight and intensity, partially dependent on the particulate nature of all electromagnetic radiation, photons. Also, target theory plays a role; the smaller the RNA or DNA genome the more photons, intensity, required to destroy or damage individual viral genomes, to inactivate potential infectious doses in the environment.

In our investigations at Brooks, we discovered some viruses’ ability to reconstruct themselves from broken pieces, albeit from high numbers of original virus, even following UV damage. “The small and simple VEEV (Venezuelan Equine Encephalitis Virus, a previous biological warfare agent) has one more trick which it shares with the plant Tobacco Mosaic Virus; it can resurrect itself from broken pieces. The nsP4 sequence in VEEV is very similar to the replicases of three plant viruses, including Tobacco Mosaic Virus. The Brooks Lab performed a critical experiment using the RetroXpress(TM) system of Clontech derived from a retrovirus which could transfer genes but could not infect and replicate, my group at Brooks constructed a new virus that contained the nsP4 sequence inserted into this platform and expected that the new virus could infect cells but not replicate. It not only infected (transfected) marker genes into the target mammalian cells but also formed plaques of transformed cells (those which resembled cancer cells and formed uninhibited growth mounds of cells). These grew and multiplied in the cell culture. A new complete infectious virus had formed. How? Tobacco Mosaic Virus has been shown to repair and reactivate after exposure to UV with the help of an unknown plant nucleic acid repair system. Since we inserted only the nsP4 protein into the host cells co-infected with the defective retrovirus, we proposed that the new virus used cellular RNA dependent RNA polymerase in conjunction with the expressed nsP4 to form a repair complex which constructed a chimera virus from the retroviral pieces and other unknown viral nucleic acid components inherently present. Perhaps, the infections by killed virus from killed vaccines previously reported were not just because of contamination with whole live virus but with components which survived the formalin damage and were reassembled into whole restored infectious virus, albeit at a less effective infectious dose of original broken pieces. Intact VEEV will produce a transmissible infection with as little as 12 PFU (plaque forming units equivalent to 10 virions for a multiplicity of infection of 1 but more likely 10X10 = 100 virions). I have speculated that these broken viruses, if reconstituted from broken virions or free RNA would require 10e6 units to make one intact virion.”

As discussed in an earlier post, cold plasma devices, which generate active oxygen species from air, require no additional chemicals only energy, but at present, are not as developed for commercial use (Sloan, M.A., Vivekananda, J., Holwitt, E.A., and Kiel J.L. U.S. Patent 7,892,484. Methods and Compositions for Neutralizing Anthrax and Other Bioagents, 22 Feb 2011).Water vapor or humidity does favor plasma gas production from the nanoparticle/pulsed microwave version. The humidity levels, which favor PCO and certain cold plasma generation, have also been shown to shorten the environmental life of SARS-CoV-2 , alone, (40-60% relative humidity), but humidifying a building holds its own unique set of problems—big challenges are the condensation problem, thermal breaks and vapor barrier.

Much confusion has arisen about when to test, what are the best tests, what do the results mean for SARS-CoV-2. The biggest lie is that they are the reason the numbers have risen so fast lately. If we don’t see it, we can’t report it, nor can we even begin to control it. The highest level of separation of what tests tell us is the distinction between incidence and prevalence of infection. The frequency (number of health events in a population and it’s relationship of that number to the size of the population is prevalence) and pattern (the occurrence of health-related events by time, place, and person is incidence) of health events in a population. COVID is a dynamic moving target for diagnostics and when target levels are measured in the course of the infection is critical. When the USAF Biological Counterproliferation Team at Brooks was still active, we told commanders and general officers that the only way to make an informed decision about a biological attack was to run three orthogonal tests (measuring different aspects of a biological agent by different methods). If only two were run, then they should have a coin to flip to make a decision. The third test was a tie breaker in case one of the other tests were positive and the other negative. The fundamental tests are (1) isolation, culture and identification, (2) PCR (polymerase chain reaction, for SARS-CoV-2, real time reverse transcriptase PCR, RNA to DNA to complete the reactions), (3) antigen testing (measuring parts of virus other than RNA with specific indicator labeled antibody), and finally, (4) measuring antibody levels in serum with a specific viral antigen, and sometimes, measuring type of antibodies with second labeled antibody https://www.nature.com/articles/d41586-020-02661-2. The last one is the least useful for early diagnosis, except if IgA or IgM antibody is measured. As noted in an earlier post, IgA, and/or IgM decline within 3 months. It is the IgG, particularly IgG3, that lasts longer: 7 months and the memory B cells even longer.

Culture is dependent on taking samples from actively shedding sites and blocking contamination, especially bacterial with antibiotics, and isolating specific plaques showing cytopathic effects (CPE) on at least partially selective host cells. However, such CPE may vary from very subtle host cellular changes to out right lysis, leaving holes in a single continuous mono layer of host cells in culture. The difficulties are obvious, and multiplying virus in the lab is hazardous https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036342/pdf/jkms-35-e84.pdf and https://www.protocols.io/view/culture-of-the-severe-acute-respiratory-syndrome-c-bcduis6w.

PCR is easier and only requires isolation of the RNA in this case, removing inhibitors or performing viral lysis and release of intact RNA in the process. However, it measures genomic equivalents of virus, if quantitative, but for most commercially available tests, only threshold of detection, but not necessarily infectious virus. Fortunately, most viral RNA is resistant to ubiquitous environmental RNA degrading enzymes. The threshold for a positive is the number of PCR replication cycles to produce detectable DNA copies, usually less than 35 to 30 cycles (Ct, cycle threshold) of the PCR machine. Cts < 29 are strong positive reactions indicative of abundant target nucleic acid in the sample. Cts of 30-37 are positive reactions indicative of moderate amounts of target nucleic acid. Cts of 38-40 are weak reactions indicative of minimal amounts of target nucleic acid which could represent an infection state or environmental contamination.

In a PCR study of 396 patients, time until negative conversion was significantly shorter in the asymptomatic group than in the symptomatic group: median 14.5 days (11.0–21.0 days) and 18.0 days (15.0– 22.0 days), respectively. Rebound of thermal cycle threshold of detection values (Ct) was seen in 78 patients (19.7%). Time until negative conversion is shorter in asymptomatic COVID-19 than in symptomatic COVID-19. Rebound of Ct values was common https://www.ijidonline.com/action/showPdf?pii=S1201-9712%2820%2930620-2.

The antigen tests measure the outer proteins of the virus and must be sampled, isolated from binding inhibitors, and detected in a tube or chromatographic surface, by trapping in a precipitate or along a diffusion path and labeling it specifically, usually with a second antibody in addition to the trapping one, with a colorimetric or fluorescent reagent. This test can be quantitative in a micro titer well plate (replacing tubes) by making dilutions to extinction, or below threshold of detection. The bed-side immunodiffusion rapid tests are only positive or negative tests. The course of an infection or the quality of the sample and its quantitative viral content control the outcomes https://www.ijidonline.com/action/showPdf?pii=S1201-9712%2820%2930658-5. One study showed a high sensitivity and specificity in samples mainly obtained during the first week of symptoms and with high viral loads. The rapid antigen assay has the potential to become important for early diagnosis of SARS-CoV-2, especially in situations with limited access to molecular methods. Sensitivity and specificity were 93.9% (86.5–97.4%) and 100% (92.1– 100%), respectively, with a diagnostic accuracy of 96.1%. Sensitivity was significantly higher in samples with high viral loads https://www.ijidonline.com/action/showPdf?pii=S1201-9712%2820%2930405-7.

Seroprevalence is most useful in determining the vulnerability of a population as opposed to the incidence, a rate, showing the most immediate infection progression.

To show how difficult the gold standard for viruses, isolation and culture, and converting these numbers into quantitative PCR is, I give work we performed at Brooks on adenovirus, a human virus causing everything from mild, cold-like symptoms to death and with asymptomatic human carriers, depending on the strain, as an example. It is a much more durable virus than SARS-CoV-2 and can easily be spread by fomites and airborne secretions. We investigated its transmission by shared gas masks used for training and supposedly disinfected, because of some deaths from the virus amongst recruit trainees (Establishment of Optimal Parameters for United States Air Force MCU-2/P Mask Sanitization Procedures to Effectively Neutralize Adenovirus Serotype 14).

From the initial copy numbers generated during the RT-PCR reaction, the titer (ge/mL: genomic equivalents per milliliter) of each unknown sample was calculated as shown below:
Copy Number 1 X 10e3 μL Titer = 9 μL x 10 x Dilution Factor x mL
Titration methods fall into one of two categories: determination of either the infectious or the particle (infectious plus noninfectious) titer. To overcome this problem, the current RT-PCR assay was developed to detect only encapsidated viral genomes. Nonencapsidated and/or incomplete genomes were eliminated during the DNase treatment (to destroy free DNA not extracted from intact virus) step, more accurately reflecting the infectious titer.

Adenoviral genome equivalents/mL following elution from mask pieces exposed to 181 ppm of bleach and/or detergent. M=mask, V=virus, D=detergent, B=bleach, W=water

How bad is prevalence of SARS-CoV-2, COVID, in highly vulnerable populations? The kidney hemodialysis population has a higher proportion of older people, men, and people living in majority Black and Hispanic neighbourhoods than in the US adult population. Seroprevalence of SARS-CoV-2 was 8.0% , 8.3% when standardized to the US dialysis population, and 9.3% (8.8–9.9) when standardized to the US adult population https://www.thelancet.com/action/showPdf?pii=S0140-6736%2820%2932009-2. Even for the most vulnerable, the highest prevalence argues against natural herd immunity and more for the typical “animal” coronavirus endemic sustainment strategies that these viruses have evolved with their hosts. For the least at risk populations, the samples examined in a recent study, showed 250 were positive of 33,041 children (age of 0-18 years) without symptoms who were tested at 28 hospitals for SARS-CoV-2 through May 29, 2020. Across the hospitals represented by these children, prevalence varied from 0% to 2.2%, with a pooled prevalence of 0.65% (0.47%-0.83%). Asymptomatic pediatric prevalence was associated with weekly incidence of COVID-19 in the general population during the 6-week period over which testing of asymptomatic children occurred. No other factor (population, number of tests performed, region, testing indication, or sample collection site) demonstrated a significant association with prevalence in asymptomatic infected children. Later data from 15,612 children were compared with prevalence in an asymptomatic pediatric population calculated from concurrent Johns Hopkins University weekly incidence data using a best-fit equation derived from this correlation, and the results showed the correlation persisted at this later time (coefficient, 0.86). Therefore, the incidence in the local general population and contacts should drive testing in children.https://jamanetwork.com/journals/jamapediatrics/fullarticle/2769878.

The general US population incidence has been determined by various methods. As of April 4, 2020, the estimated case count was 5 to 50 times higher than the official positive test counts across the different states. Nationally, the estimates of COVID-19 symptomatic cases as of April 4 were likely in the range of 2.2 to 4.9 million, with possibly as many as 8.1 million cases, up to 26 times greater than the cumulative confirmed cases of about 311,000. When these methods were extended to May 16, 2020, it was estimated that cumulative symptomatic incidence ranged from 6.0 to 10.3 million, as opposed to 1.5 million positive test counts https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310656/pdf/nihpp-2020.04.18.20070821.pdf. How tests are applied is critical to the value of the data to making true numbers and distribution of the virus and cases of COVID-19.

“What would the simplest and purest form of a Nanobe which could take over another life form and make it a new, different one? The following is such a model of such a transforming synthetic life form which could be made from basic chemicals:”— The Black Dragon Trilogy by JOHNATHAN KIEL
https://a.co/136glXE

Multiple potential applications:

1) Simulants for microbial detection and neutralization, using the Nanobes’ “internal” diagnostics. (2) Test platform for nanotoxicological research. (3) Transfection vector for biomedical research and therapeutics (superior to just re-directing the immune response, as we had previously tried, because Nanobes can be turned “on” and “off” with nonionizing electromagnetic radiation (NEMR)). (4) Against antibiotic resistant bacteria, especially intracellular bacteria (in vivo and ex vivo, in self–sterilizing materials). (5) Against viruses and other parasites (in vivo and ex vivo, self-sterilizing materials). (6) Against cancer (7) RFR nano-directed ablation surgery (8) RFR controlled gene expression (9) Transformation vector for agriculture (10) RFR (and other NEMR) controlled pesticides (11) Transient persistent transformation of crops (For one season; self-limiting desirable trait transfer) (12) Transformation vector for bio energy (13) Enhancing cyanobacterial production of biofuels (14) Enhancing microbial production of materials and feedstocks.

Although CRISPR (clustered regularly interspaced short palindromic repeats) gene editing technology has come like a storm with great promise for therapeutic gene editing ( 2020 Nobel Prize in Chemistry: Emmanuelle Charpentier, at the Max Planck Unit for the Science of Pathogens in Berlin, and Jennifer Doudna, at the University of California, Berkeley), it still has delivery, off- target effect, and control problems (can’t readily be turned “on and off”). Many researchers are vigorously pursuing solutions. Nanobes provide an alternative that is electromagnetically controlled with a fail-safe system. Nanobes could even provide a fail-safe delivery system for CRISPR.

There are also plant agricultural applications

“We tried to address a major problem in agriculture, Greening Disease of Citrus Fruit caused by the bacterium Candidatus Liberibacter asiaticus transmitted by the insect vector the Asian Citrus Psyllid, which has invaded citrus farms of Florida and threatens those of Texas.”… “Aside from using Nanobes to deliver CRISPR in the field, which might be quite complicated, the alternative was to use the microwave and radio frequency absorbing properties of Nanobes with DALM to do the job, combining physics and engineering with biology. Heat treatment eliminates Candidatus Liberibacter asiaticus from infected citrus trees under controlled conditions. This approach would have provided wireless control (using light, microwaves, or magnetic induction to activate the Nanobe heating) which could have been turned on and off with an external switch controlling the energy source. Even if transformation was used as a method as well, if something went wrong, the transformed cells could be killed by raising the energy level to a lethal level, but not high enough to destroy surrounding non-transformed cells in the plant.”— The Black Dragon Trilogy by JOHNATHAN KIEL. https://a.co/0ApfKej

Non-biological applications of Nanobes:

(1) Nano-plasma cleaning of surfaces by E field focusing (2) Nano-plasma etching of surfaces for device construction (3) Ultrawide-band-emitting nano-transmitters/ receivers for RFIDs (4) Nano-magnetically controlled switches and circuits (5) Taggants to track materials (in conjunction with people?)”

“Nanobes not only facilitate this biohacking but provide a convenient delivery system for the products. No longer will this type of biotechnology be confined to highly sophisticated and elite well-funded and institutionally supported labs of the Northeast or California coast of the USA, but they will also be available to all as the personal computer made computer technology and software development availabe to all. There is inherent freedom, progress and danger in such accessibility.”— The Black Dragon Trilogy by JOHNATHAN KIEL
https://a.co/fLu1BHE