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RogueSolace

Condensed Notebook

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(This is a condensed version of Personal Journal/Medical Documentation/Notes of Elizabeth Smith. All notes are carefully written and kept in a private location no on person. No names are written to indicate author.  Can only take IC if I tell you that you can.

Copy of original book given to @Skinner to be given to UN/WHO in Miroslavl. Understanding was it ended up in the trash via higher authorities.)

 

The only reason this is here is because I need something to amuse me or I'm going to start freaking out like everyone else 

While this is mostly humor, it could have possible relations to what may actually be found.

 

What a Real Harvard Psychiatrist Thinks About Sci-Fi Zombies

 

“Through education Dr. Steven C. Schlozman is an assistant professor of psychiatry at Harvard Medical School and a lecturer at the Harvard School of Education. He is also an avid sci-fi and horror fan - and, apparently, the world's leading authority on the neurobiology of the living dead. He has even drafted a fake medical journal article on the zombie plague, which he calls Ataxic Neurodegenerative Satiety Deficiency Syndrome, or ANSD (the article has five authors: one living, three "deceased" and one "humanoid infected").”

Copied from an article by Mark Strauss, a senior editor at Smithsonian magazine.

According to Dr. Steven C. Schlozman, this is your brain on zombies:

 

Spoiler

The Frontal Lobe

This part of the brain is involved with "executive functioning" - enabling us to think carefully and solve problems in an abstract way. Clearly, there's not much going on there if you have the misfortune of being afflicted with living deadness. But we do know that zombies can see us and sense us. Schlozman concludes that zombies possess just enough frontal lobe activity to "listen" to the thalamus, through which sensory input is processed.

But the frontal lobe function most relevant to understanding zombie behavior is the control of "impulsivity"-the general term for when you do something and, if you had two more seconds, you might not have done it. For instance, if in a fit of rage you have the sudden urge to punch your boss in the face, the frontal lobe intervenes and allows you to consider why that might be a bad idea.

 

The Amygdala and Anterior Cingulate Cortex

Absent a properly functioning frontal lobe, a zombie is driven entirely by base emotions - such as rage - that are housed in the primitive parts of our brain, notably the amygdala. There's precedence for this in nature. A crocodile brain, for instance, is mostly driven by the amygdala. Researchers have confirmed this by introducing lesions into the amygdala of animal specimens: the result is a drop in the attack and retreat response that correlates significantly with the amount of damage that's done to that region of the brain. A crocodile without an amygdala isn't really a crocodile. As such, Schlozman argues, "you can't really be mad at zombies, because that's like being mad at a crocodile," adding that it's the delicate balance between frontal lobe and amygdala "that makes us human."

That balance is maintained by the anterior cingulate cortex, which modulates and dampens the excitability of the amygdala as it talks to the frontal lobe. So, when the amygdala gets all stirred up by fear, anger or lust, the anterior cingulate cortex steps on it a little bit, giving the frontal lobe time to think everything through before it sends signals toward the motor cortex and we act upon those impulses.

A zombie would have a dysfunctional anterior cingulate cortex, rendering it unable to modulate feelings of anger. The result? Hyper-aggression.

 

The Cerebellum and the Basal Ganglia

Science may once and for all settle the heated debate over whether "the infected" in 28 Days Later could be classified as zombies.

Schlozman says "no," observing that "the infected" possess "some sort of higher cortical function going on that allows them to hunt humans." Moreover, the fake zombies in 28 Days Later exhibit fluidity of motion. They can run, jump, climb and quickly change direction-activities that the true Romero zombies are incapable of performing.

Clearly, zombies suffer from cerebellar and basal ganglia dysfunction (duh!). Those are the parts of the brain that make fluidity of motion possible. The basal ganglia helps us with coordinated movement. The cerebellum helps us with balance. In fact, if you visit the website of the National Institutes of Health and read about cerebellar degeneration (such as ataxia), the symptoms match the familiar gait of the living dead: "a wide-legged, unsteady, lurching walk, usually accompanied by a back and forth tremor in the trunk of the body…"

 

Mirror Neurons

This is recent, cutting-edge research in the field of neuroscience. Schlozman describes mirror neuron theory as a "neurobiological model for empathy, which suggests, in a very hopeful way, that we might be wired to connect with one another." Regions of the brain are recruited in response to social interactions in which we watch and thus experience the experiences of the "other."

As a press release issued by the European Science Foundation explains it: in Just as the same mirror neurons fire when observing and doing certain tasks, so other mirror neurons may be triggered both when experiencing a particular emotion and when observing someone else with that emotion.

But, Schlozman asks, what if the things we're fighting have brains that are incapable of connecting? In response, we disconnect from each other. Schlozman quotes a veteran of the Battle of Yonkers in the book World War Z: "Shock and Awe! But what if the enemy can't be shocked and awed? Not just won't, but biologically can't?"

At the Battle of Yonkers, the humans hit the zombie horde with everything they've got. But the zombies keep coming. They don't look scared. They don't look excited. They don't look enraged. And that actually freaks out the humans more than anything else, prompting the humans to turn on each other.

Schlozman suggests that mirror neurons also help explain the popularity of the zombie genre among the living. While watching these movies, "we like the permission to look at these things that look human - but aren't human - and have utter and complete permission to blow their heads off." In other words, we get off on the thrill of guiltless violence. We enjoy a brief vacation from empathy, and take our crocodile brains out for a spin.

 

By way of example, I came across an interview with actor Mike Christopher Berhosky, who played the iconic Hare Krishna zombie in the 1978 movie, Dawn of the Dead. Berhosky describes the audience reaction to the film's screening:

I got bashed in the head and everyone CHEERED. Took the wind right outta' my sails. Everyone hated the Hare Krishna devotees for their incessant pestering and swarming them at the airports and such….Killing off my character had the effect of releasing a lot of pent up frustration….bashing in the Hare Krishna zombie's head was much more than getting rid of another pesky zombie…it was VENGEANCE.

But the fun lasts only up to a point. As the movies progress, Schlozman says, we start to feel uncomfortable with the loss of our humanity-that we are "so willing to forsake those mirror neurons."

 

The Ventromedial Hypothalamus

In the movies, zombies are always hungry, no matter how many supporting actors they consume. The most likely explanation is that zombies don't have a properly functioning ventromedial hypothalamus: the region of the brain that lets you know whether you've eaten enough. The result is hyperphagia. Zombies will eat and eat and eat, but never feel satiated.

That raises a slightly awkward question: If zombies are constantly eating, then how come they never poop?

Schlozman doesn't know for sure, but he has at least one promising theory: Maybe the living dead are constipated.

Now we know why zombies are always moaning.

 

Brain Behavior

·       While major things seem to ensure some kind of ‘death’, obviously some parts of the brain must be active for the specific actions of the body

·       Prey Drive- “In all predators the prey drive follows an inevitable sequence: Search (orient, eye); Stalk, chase; Bite (grab-bite, kill-bite); dissect, consume. In wolves, the prey drive is complete and balanced since it utilizes the whole range from search to kill and finally consumes the prey in order to survive.”

 

Predator and Prey Instinct: Yale Mice Study

Spoiler

 

Areas of the brain needed to understand results.

Central Amygdala (CeA)

 unnamed.jpg 

·       The amygdala part of the brain is responsible for our reactions both physically and mentally to outside stimuli, especially adrenaline, freeze, flight or fight, etc. This part of the brain deals with emotions and motivation.

 

Reticular Formation (PCRt)

 unnamed2.png 

·       The reticular formation has projections to the thalamus and cerebral cortex that allow it to exert some control over which sensory signals reach the cerebrum and come to our conscious attention. It plays a central role in states of consciousness like alertness and sleep.

 

The Periaqueductal Gray (PAG)

 image009.jpg 

·       The periaqueductal gray (PAG, also known as the central gray) is the primary control center for descending pain modulation. It has ncephalin-producing cells that suppress pain.

·       It can provoke defensive responses characterized by freezing immobility, running, jumping, tachycardia, and increases in blood pressure and muscle tonus. In contrast, stimulation of the caudal ventrolateral PAG can result in an immobile, relaxed posture known as quiescence, whereas its inhibition leads to increased locomotor activity.

·       Lesions of the caudal ventrolateral PAG can greatly reduce conditioned freezing, whereas lesions of the dorsal aspect can reduce innate defensive behavior, virtually “taming” the animal.

 

Yale University Notes

·       Via findings from scientists at Yale studying mice they found where in the brain the activity of hunting and killing is

 

“Highlights

•Stimulation of central amygdala (CeA) elicited hunting of live and artificial prey

•CeA projections to the reticular formation (PCRt) control biting attacks

•CeA projections to periaqueductal gray (PAG) control locomotion during pursuit

•CeA integrates craniofacial and locomotor modules during goal-directed behavior

Summary

Superior predatory skills led to the evolutionary triumph of jawed vertebrates. However, the mechanisms by which the vertebrate brain controls predation remain largely unknown. Here, we reveal a critical role for the central nucleus of the amygdala in predatory hunting. Both optogenetic and chemogenetic stimulation of central amygdala of mice elicited predatory-like attacks upon both insect and artificial prey. Coordinated control of cervical and mandibular musculatures, which is necessary for accurately positioning lethal bites on prey, was mediated by a central amygdala projection to the reticular formation in the brainstem. In contrast, prey pursuit was mediated by projections to the midbrain periaqueductal gray matter. Targeted lesions to these two pathways separately disrupted biting attacks upon prey versus the initiation of prey pursuit. Our findings delineate a neural network that integrates distinct behavioral modules and suggest that central amygdala neurons instruct predatory hunting across jawed vertebrates.”

 

The study is essentially explaining for my notes that in order for the predator and prey chase and kill instinct to be active, these parts of the brain MUST somehow be getting activated via nerve cells.

An interesting thing to note as well from the study: “The animals did not, however, attack other mice in the cage. Hunger also affected predatory behavior. Hungry mice more aggressively pursued prey during light stimulation than mice that were not hungry. “The system is not just generalized aggression,” says de Araujo. “It seems to be related to the animal’s interest in obtaining food.”

 

Major Hypothesis: The illness is caused by an unknown virus. Here are similarities in nature of possibly similar things. People always note the one 'zombie fungus', but don't realize there is so much more.

                               Symbiotic Bacteria/Virus·     

Spoiler

   For a virus to achieve a symbolic relationship with its host it has to attach itself to cells, then invade and infect a cell. Then it puts its own DNA into the host’s cells, which is then multiplied as new cells form. Spreading infection.

·        Bacteriophage (or phage) is a type of virus that is able to infect other bacteria viruses symbiotically and in some cases, human cells (virus within a virus, virus within cells)

·        Lysogeny is the steady relationship that occurs between a bacteriophage and its host.

·        Viruses can be dangerous, but in some cases can also teach the immune system how to protect the body against other viruses

·        Some bacterial infections even keep us healthy and fend off other infections

·        Bacteriophages/Phages are known to have genes that make the bacteria they invade more stable

·        Viral Symbioses (Virus’s that have a mutual beneficial relationship with the host) are known to be more parasitic to the host than Bacterial

·        Various Bacteriophages are carriers for transmitting resistance to predators

·        One virus can help mice from getting two others

Symbiotic Animal & Plant Behavior

Spoiler

Venus Fly Trap (functions without a brain)

·        Venus flytraps produce antiseptic juice along with digestive to keep pray from deteriorating

·        Without a brain the flytrap uses the water pressure in its body to move, along with nerves to signal it to create chemicals

·        Jasmonate is a touch hormone, which is released by wounds and injuries and coordinates programs of defense and repair.

·        In the Venus fly trap, jasmonate doubles as a carnivory hormone. It primes the gland cells in the trap to start making digestive enzymes. So it is able to be used differently that the original purpose most creatures use it for.

Parasitoid Wasp & Ladybug

·        The bacteria Hamiltonella defense has a phage (source of resistance) that can keep an aphid safe from its enemy. YET when this phage was removed, it made the insect vulnerable to its enemy. Meaning that the infection keeps the bug safe, and removing it negates that safety. The infection actually protects it.

 

·        Parasitoid wasp uses symbiotic RNA virus (DcPV) to take over the nervous system of a ladybug, when the pupa hatches through the ladybug, it will go and make a web between its legs to stay safe. The ladybug shows bodyguard behavior towards the pupa.

·        The body guarding behavior is a response to the RNA virus as a neurological disorder. The DcPV causes trauma within the ladybug as well as overcoming it with partial paralysis and tremors. This causes what we perceive to be ‘body guarding’ behavior.

·        Polydnaviruses (an insect virus) are used by some viruses to fight the host’s immune system.

Emerald Cockroach Wasp & Cockroach

·        Wasps that sting roaches deliver venom directly into a part of the roaches’ brain called the sub- esophageal ganglia (a main nerve that feeds other nerves). This puts the roach into a trance like state as well as makes it less fearful and lose the will to flee from predators.

·        Even though the roach is not paralyzed in any way, it will spend a week to a month not moving until the pupa eat through it.

·        The roach does have a defense against this though. It can produce Serratia marcescens bacteria that possess a suite of protein-degrading enzymes that cut apart fragile larval cells.

·        In order to get around this bacteria, the wasps sterilize their food by secreting antimicrobial compounds; mullein called called (R)-(-)-mellein, and micromolide. Both compounds showed broad-spectrum antibacterial activity, and the combination of the two was particularly effective.

Rhizocephalans Barnacle & Crabs

·        A virus can alter hormones in the male crab’s neurophysiology, it causes the male crab to act like a female one. Its abdomen flattens and widens, and it starts taking on the behavioral traits of a pregnant female.

Hairworm & Cricket

·        A parasite worm to grasshoppers has developed a way to mimic natural chemical signals in the bug’s brain. An infected brain had heightened levels of neurotransmitters and chemicals responsible for movement and orientation, particularly with relation to gravity. These proteins are similar to the ones produced by the insect, but are not naturally occurring, suggesting that the parasite is able to produce and excrete its own chemical signals to screw with the cricket’s mind.

 

Chemical Behavior

Spoiler

·        Tetradotoxin, which is found in the liver and the ovaries of some species of Puffer Fish. Is five hundred times more deadly than Cyanide. It is a potent ion channel blocker which while it can be fatal, in smaller (minuscule) doses leads to a near-death state wherein metabolic functions are so depressed that the poisoned person is thought to be dead”

·        Datura stramonium (jimson weed), and Datura metel (angel’s trumpet): both psychoactive herbs from the same family that cause delirium, disorientation, and amnesia. It is also believed if the drug was administered repeatedly, it could produce a state of extreme psychological passivity.

·        Datura has long been used for asthma symptoms. The active agent is atropine. The leaves are generally smoked either in a cigarette or a pipe.

·        The Zuni people once used datura as an analgesic to render patients unconscious while broken bones were set.

·        The Chinese also used it as a form of anesthesia during surgery.

Brain Behavior

Spoiler

Predator and Prey Instinct: Yale Mice Study

·        •Stimulation of central amygdala (CeA) elicited hunting of live and artificial prey

·        •CeA projections to the reticular formation (PCRt) control biting attacks

·        •CeA projections to periaqueductal gray (PAG) control locomotion during pursuit

·        •CeA integrates craniofacial and locomotor modules during goal-directed behavior

What a Real Harvard Psychiatrist Thinks About Sci-Fi Zombie

·        Frontal Lobe- (impulsivity, executive functioning/think & solve problems in abstract way). Believes that just enough of the frontal lobe is active to “listen” to the thalamus, through which sensory input is processed.

·        Amygdala and Cingulate Cortex- balance is maintained by the anterior cingulate cortex, which modulates and dampens the excitability of the amygdala as it talks to the frontal lobe. So, when the amygdala gets all stirred up by fear, anger or lust, the anterior cingulate cortex steps on it a little bit, giving the frontal lobe time to think everything through before it sends signals toward the motor cortex and we act upon those impulses.

·        A zombie would have a dysfunctional anterior cingulate cortex, rendering it unable to modulate feelings of anger. The result? Hyper-aggression.

·        The Cerebellum and the Basal Ganglia- Clearly, zombies suffer from cerebellar and basal ganglia dysfunction (duh!). Those are the parts of the brain that make fluidity of motion possible. The basal ganglia helps us with coordinated movement. The cerebellum helps us with balance. In fact, if you visit the website of the National Institutes of Health and read about cerebellar degeneration (such as ataxia), the symptoms match the familiar gait of the living dead: "a wide-legged, unsteady, lurching walk, usually accompanied by a back and forth tremor in the trunk of the body…"

·        Mirror Neurons- area that creates empathy

·        The Ventromedial Hypothalamus- the region of the brain that lets you know whether you've eaten enough. The result is hyperphagia. Zombies will eat and eat and eat, but never feel satiated.

 

All of that means-

  • Possible way of transmission
Spoiler
  •  Bacteriophage/Phage is a virus that can infect other viruses, and in some cases human cells.
  •  Viral makes more sense than Bacterial as its fiercer
  •  
  • Possible direction of cure
Spoiler
  •  B/Phage, some can have the opposite effect and help the body protect itself against predators including other viruses
  •  While B/Phage is considered an infection, it can give the organism something it needs to defend itself against something else (virus, predator).
  • If the helpful infection is removed, the organism then loses the protection the infection gave it
  •  Polydnaviruses (an insect virus) are used by some viruses to fight the host’s immune system.
  •  To get around various bacteria, some create antimicrobial compounds to kill the bacteria before it enters the system. (Particularly effective in this case (R)-(-)-mellein, and micromolide)

 

  • Body chemistry possibilities
Spoiler
    • Body or virus could produce antiseptic secretions that keep the body from deteriorating
    • A touch hormone used to usually heal the body, can be manipulated into priming glands to excrete substances.
    • There are bacteria that can be produced to destroy specific types of cells like larval, to protect the host
    • Can alter the neurophysiology (a branch of physiology and neuroscience that is concerned with the study of the functioning of the nervous system.) In this case forcing the hosts hormones to change to the opposite gender and making the animal act as such
    • Tetradotoxin in smaller (minuscule) doses leads to a near-death state wherein metabolic functions are so depressed that the poisoned person is thought to be dead. (Ruled out, would show in blood tests)
    • Datura stramonium (jimson weed), and Datura metel (angel’s trumpet): both psychoactive herbs from the same family that cause delirium, disorientation, and amnesia. It is also believed if the drug was administered repeatedly, it could produce a state of extreme psychological passivity. (Ruled out, would show in blood tests)
    • Datura was used carefully in humans for analgesic (painkiller) and anesthesia (temporary loss of sensation or awareness). (possibly good to know)

 

  • Movement
Spoiler
  • Movement without muscle cells push/pull by changing the water pressure inside the body
  • RNA Virus like DcPV could be overpowering the body using the brain, to cause trauma, partially paralyze the body, and cause tremors that can be dangerous to others who get too close
  • A parasite is able to create a chemical that it can inject into a host, which mimic chemicals signals the host already produces. Messing with neurotransmitters and chemicals responsible for movement and orientation, particularly with relation to gravity

 

  • Brain/Behavioral Patterns
Spoiler

Possibly a virus that destroys the part of the brain or connections that induce fear

 

  • In order for predator/prey instinct to kick in, these parts of the brain MUST be functioning and able to communicate to each other
Spoiler
  • Central Amygdala
  • Reticular Formation
  • Periaqueductal gray
  • Central Amygdala is able to stimulate craniofacial (facial) and locomotor modules (movement).
  • Structures in the brain that could be damaged/altered
Spoiler
    • Amygdala (the part that responds to stimulus and decides how to react- panic/fear/anger/lust/etc.)
    • Frontal lobe (thought process)- must be active enough to receive direction from the thalamus (sensory input)
    • Motor Cortex- must be active with the frontal lobe to correspond to movement
    • Thalamus (sensory input) then must be active and able to communicate with the frontal lobe (thought process)
    • Anterior cingulate cortex (excites or calms the amygdala)- must be dysfunctional, rendering it unable to modulate feelings of anger. (THIS EQUALS HYPER AGGRESSION WHICH WE ARE SEEING. Highly plausible.)
    • Cerebellum (balance) and Basal Ganglia (coordination) together = (make motions smooth/fluid)- degeneration of this area (ataxia) can cause "a wide-legged, unsteady, lurching walk, usually accompanied by a back and forth tremor in the trunk of the body…"
    • Mirror Neurons (area that creates empathy)- if damage would not care about violence done to others
    • The Ventromedial Hypothalamus- (the region of the brain that lets you know whether you've eaten enough.) If damaged the result is hyperphagia. You will eat and eat and eat, but never feel satiated.

 

 

More Science Notes

Coma

Spoiler

 ·         In the vegetative state patients can open their eyelids occasionally and demonstrate sleep-wake cycles, but completely lack cognitive function.

 ·         The vegetative state is also called a "coma vigil". The chances of regaining awareness diminish considerably as the time spent in the vegetative state increases

 ·         Most PVS patients are unresponsive to external stimuli and their conditions are associated with different levels of consciousness. Some level of consciousness means a person can still respond, in varying degrees, to stimulation. A person in a coma, however, cannot. In addition, PVS patients often open their eyes in response to feeding, which has to be done by others; they are capable of swallowing, whereas patients in a coma subsist with their eyes closed (Emmett, 1989).

 ·         PVS patients' eyes might be in a relatively fixed position, or track moving objects, or move in a disconjugate (i.e., completely unsynchronized) manner. They may experience sleep-wake cycles, or be in a state of chronic wakefulness. They may exhibit some behaviors that can be construed as arising from partial consciousness, such as grinding their teeth, swallowing, smiling, shedding tears, grunting, moaning, or screaming without any apparent external stimulus.

·         Individuals in PVS are seldom on any life-sustaining equipment other than a feeding tube because the brainstem, the center of vegetative functions (such as heart rate and rhythm, respiration, and gastrointestinal activity) is relatively intact (Emmett, 1989).

 

 

·         most patients who do recover consciousness experience significant disability. The longer a patient is in a PVS, the more severe the resulting disabilities are likely to be. Rehabilitation can contribute to recovery, but many patients never progress to the point of being able to take care of themselves.

 ·         There are two dimensions of recovery from a persistent vegetative state: recovery of consciousness and recovery of function. Recovery of consciousness can be verified by reliable evidence of awareness of self and the environment, consistent voluntary behavioral responses to visual and auditory stimuli, and interaction with others. Recovery of function is characterized by communication, the ability to learn and to perform adaptive tasks, mobility, self-care, and participation in recreational or vocational activities. Recovery of consciousness may occur without functional recovery, but functional recovery cannot occur without recovery of consciousness (Ashwal, 1994).

·         Currently no treatment for vegetative state exists that would satisfy the efficacy criteria of evidence-based medicine. Several methods have been proposed which can roughly be subdivided into four categories: pharmacological methods, surgery, physical therapy, and various stimulation techniques. Pharmacological therapy mainly uses activating substances such as tricyclic antidepressants or methylphenidate. Mixed results have been reported using dopaminergic drugs such as amantadine and bromocriptine and stimulants such as dextroamphetamine.[25] Surgical methods such as deep brain stimulation are used less frequently due to the invasiveness of the procedures. Stimulation techniques include sensory stimulation, sensory regulation, music and musicokinetic therapy, social-tactile interaction, and cortical stimulation.[26]

 

 

·         There is limited evidence that the hypnotic drug zolpidem has an effect.[27] The results of the few scientific studies that have been published so far on the effectiveness of zolpidem have been contradictory.[28][29]

·         The first report of a zolpidem awakening came from South Africa, in 1999. A patient named Louis Viljoen, who, three years before, was declared vegetative after he was hit by a truck, had taken to clawing at his mattress during the night. Thinking he was suffering from insomnia, his family doctor suggested zolpidem to help him sleep. But 20 minutes after his mother ground the tablet up and fed it to him through a straw, Viljoen began to stir. His eyes, which normally wandered the room, vacant and unfocused, flickered with the light of consciousness. And then he began to talk (his first words were “Hello, Mummy”), and move (he could control his limbs and facial muscles). A few hours later he became unresponsive. But the next day, and for many days after that, zolpidem revived him, a few hours at a time.

·         Until recently, most doctors believed that recovering from this condition was not possible. Vegetative states were considered permanent after three months if the injury was caused by oxygen deprivation, or one year if it was caused by blunt trauma.

·         In 2003, an Arkansas man named Terry Wallis emerged, after 19 years, from a minimally conscious state. Neuroimaging suggested that his brain had essentially reconfigured itself — surviving neurons bypassed dead ones and forged new connections to one another. In a 2007 Nature paper, Nicholas Schiff, a neurologist from Weill Cornell Medical College, and his colleagues showed that deep brain stimulation — surgically implanting a “brain pacemaker” that sends electrical impulses to specific regions of the brain — can help some severely injured patients recover the ability to speak and eat, years after the injury. And just this month, Adrian Owen, a British neuroscientist, reported in the journal The Lancet that the brains of some patients who seemed vegetative responded to basic commands: their bodies didn’t move, but distinct patterns of neuronal firing were detected on EEG scans when these patients were told to make a fist (which triggered one region of the premotor cortex) or wiggle their toes (which triggered another).

·         of zolpidem as a treatment for disorders of consciousness. (Amantadine, a drug used to treat Parkinson’s disease, and the anti-anxiety medication Ativan also show promise in increasing awareness in minimally conscious patients.) So far, the evidence suggests that less than 10 percent of brain-injured patients will experience the drug’s paradoxical effects, and that among those, only a few will respond as profoundly as Viljoen did

·         Chris’s behaviors were entirely reflexive, he said; they were produced by his brainstem, which regulates basic functions like breathing and body temperature, not by his cortex, the region responsible for higher-order thinking.

·         “Every couple days, the doc would stop in the doorway and shout Chris’s name to see if Chris responded,” Judy said. “But he wouldn’t come in the room and look at Chris up close. So one day, I practically grabbed his arm and dragged him into the room, over to Chris’s bed.” She told Chris to blink his eyes. He did. Then she made the doctor walk across the room and told Chris to keep his eyes on the doctor. He did. Finally, with the doctor standing across the room, eyes fixed on Chris, she asked Chris to give her a thumbs up. When he wiggled his thumb, just the tiniest bit, the doctor’s jaw dropped. Chris was not in a vegetative state after all. He was minimally conscious.

·         Still, there was little that the community hospital could do for him. It had neither the resources nor the expertise to tease out a prognosis or chart a course of therapy. The same was true of local nursing homes, which is where many patients like Chris end up.

·         Until recently, most doctors believed that recovering from this condition was not possible. Vegetative states were considered permanent after three months if the injury was caused by oxygen deprivation, or one year if it was caused by blunt trauma.

 

 

·         In 2003, an Arkansas man named Terry Wallis emerged, after 19 years, from a minimally conscious state. Neuroimaging suggested that his brain had essentially reconfigured itself — surviving neurons bypassed dead ones and forged new connections to one another. In a 2007 Nature paper, Nicholas Schiff, a neurologist from Weill Cornell Medical College, and his colleagues showed that deep brain stimulation — surgically implanting a “brain pacemaker” that sends electrical impulses to specific regions of the brain — can help some severely injured patients recover the ability to speak and eat, years after the injury. And just this month, Adrian Owen, a British neuroscientist, reported in the journal The Lancet that the brains of some patients who seemed vegetative responded to basic commands: their bodies didn’t move, but distinct patterns of neuronal firing were detected on EEG scans when these patients were told to make a fist (which triggered one region of the premotor cortex) or wiggle their toes (which triggered another).

·         zolpidem as a treatment for disorders of consciousness. (Amantadine, a drug used to treat Parkinson’s disease, and the anti-anxiety medication Ativan also show promise in increasing awareness in minimally conscious patients.) So far, the evidence suggests that less than 10 percent of brain-injured patients will experience the drug’s paradoxical effects, and that among those, only a few will respond as profoundly as Viljoen did.

·         Chris’s behaviors were entirely reflexive, he said; they were produced by his brainstem, which regulates basic functions like breathing and body temperature, not by his cortex, the region responsible for higher-order thinking

·         “Every couple days, the doc would stop in the doorway and shout Chris’s name to see if Chris responded,” Judy said. “But he wouldn’t come in the room and look at Chris up close. So one day, I practically grabbed his arm and dragged him into the room, over to Chris’s bed.” She told Chris to blink his eyes. He did. Then she made the doctor walk across the room and told Chris to keep his eyes on the doctor. He did. Finally, with the doctor standing across the room, eyes fixed on Chris, she asked Chris to give her a thumbs up. When he wiggled his thumb, just the tiniest bit, the doctor’s jaw dropped. Chris was not in a vegetative state after all. He was minimally conscious.

·         One hallmark of the minimally conscious state is a rapid fluctuation between levels of awareness. Spend 10 or 20 minutes with Chris Cox, and you might conclude that there is nothing going on upstairs. But spend a full hour, and at some point you’ll see his puppy-dog eyes come into focus. They will appear to search for one of his parents, or to settle quizzically on the new person in the room. Ask him to say something, and he’ll smack his lips frantically before leaning forward and tapping his feet in apparent frustration. You’ll swear that he is there with you and that only his physical infirmities (he cannot quite swallow or control his jaw) prevent him from describing the netherworld from which he has just emerged. And then, a few minutes later, he’ll slip away again.

·         It’s not entirely surprising that Ambien would arouse instead of sedate. The pill has long been linked to reports of bizarre sleepwalking behavior (not to mention sleepeating, sleeptalking, even sleepdriving). Some scientists call this phenomenon “paradoxical excitation.”

·         “There are degrees of awareness, and it’s not always clear what the threshold should be. What we’re really looking for here is evidence of comprehension and will.”

·         According to Whyte, most responders fall into one of two categories: those who can take zolpidem daily, with no appreciable loss of efficacy, and those for whom the “awakenings” wane with continued use. The latter type, he says, may be the most common

·         Ativan every day; it has a similar though less profound effect on his behavior. But zolpidem they hoard like pixie dust, giving it only on special occasions, when friends and family can be there.

·         According to several studies, about 40 percent of patients who have been declared vegetative are actually minimally conscious.

·         To help eliminate this bias, Whyte developed what he calls the single-subject assessment, in which doctors design a set of tests specific to each patient’s idiosyncrasies to determine whether the patient is vegetative or minimally conscious. It is painstaking work, but the information it yields is significant. “Patients who achieve minimal consciousness early tend to have a better prognosis,” Whyte says. “And you can at least try to build a communication system with them, because you have a foundation to work from.”

·         It’s not entirely surprising that Ambien would arouse instead of sedate. The pill has long been linked to reports of bizarre sleepwalking behavior (not to mention sleepeating, sleeptalking, even sleepdriving). Some scientists call this phenomenon “paradoxical excitation.”

·         . “There are degrees of awareness, and it’s not always clear what the threshold should be. What we’re really looking for here is evidence of comprehension and will.”

·         “Once a patient progresses to minimal consciousness, we can’t predict what’s going to happen. The only way to know the outcome is to give the patient time.

·         “We still don’t understand which drugs should work on which patients, or at what dosage, or at what point in their recovery,”

 

 

 

Wounds

Spoiler

·         As all wounds are contaminated, with or without necrotic tissue, they will have an odor. Bacteria of different varieties have varying odors, colors and consistencies, and dead tissue in a wound introduces additional bacteria to the affected area. An increase in amount or consistency of exudate along with a change in color signals a cause for concern, as well as changes in odor.

 

·         Exudate or wound drainage is the result of dilation of the blood vessels during the early inflammatory stage of healing possibly caused by the presence of certain bacteria. In an attempt to heal the wound, the body is creating and maintaining an optimal moist wound environment. Exudate is a derivative of serum with a high protein content and various additives which assist with healing; exudate consists of dead cells and liquefied necrotic debris, active white blood cells, growth factors, and natural enzymes that stimulate autolysis and healing, at least when present in an acute wound.

 

 

Bleeding From the Eyes

Spoiler

  The route of transmission varies by specific virus. Some viral hemorrhagic fevers are spread by mosquito or tick bites. Others are transmitted by contact with infected blood or semen. A few varieties can be inhaled from infected rat feces or urine.

·         If you travel to an area where a particular hemorrhagic fever is common, you may become infected there and then develop symptoms after you return home. It can take up to 21 days for symptoms to develop.

·         Each of these hosts typically lives in a specific geographic area, so each particular disease usually occurs only where that virus's host normally lives. Some viral hemorrhagic fevers also can be transmitted from person to person, and can spread if an infected person travels from one area to another.

·         Haemolacria is a physical condition that causes a person to produce tears that are partially composed of blood. It can manifest as tears that are anything from merely red-tinged to appearing to be entirely made of blood.

·         Haemolacria is a symptom of a number of diseases,[1] and may also be indicative of a tumor in the lacrimal apparatus. It is most often provoked by local factors such as bacterial conjunctivitis, environmental damage or injuries.[2]

·         Acute haemolacria can occur in fertile women and seems to be induced by hormones,[2] similarly to what happens in endometriosis.

·         Ebola virus causes blood vessels to burst leading to leaks throughout the body this is primarily caused by hemorrhagic fevers. Patients can develop bruising, bleeding mucous membranes, and bloody tears as a result of infections.

 

 

Cellular Transformation

 

Spoiler

IV. DISEASE PRODUCTION BY VIRUSES

·         The ability of viruses to cause disease can be viewed at 2 distinct levels: (1) the changes that occur within individual cells, and (2) the process that takes place in the infected patient. (see Reading Assignment (A): Fig. 30-1)

 

·         A. How Viral Infections Affect Individual Cells

·         1. No apparent morphologic or functional change ( = disease).

·         2. Cytocidal Infection – an infection that results in cell death either from cell lysis, apoptosis, or the accumulation of cytopathic effects.

·         a. Cell lysis- viral replication causes dissolution of the cell. What viruses depend on lysis of the cell to be released?

·         b. Apoptosis – viral infections can trigger premature programmed cell death.

·         c. Cytopathic effects – cellular injury caused by virus infection. (2nd meaning: the effects of virus infection on cultured cells, visible by microscopic or direct visual examination).

 

·         Infection by cytocidal viruses is usually associated with changes in cell morphology, in cell physiology and sequential biosynthetic events. Many of these changes are necessary for efficient virus replication.

 

·         Morphologic Effects: The changes in cell morphology caused by infecting virus are called cytopathic effects (CPE). Common examples are rounding of the infected cell, fusion with adjacent cells to form a syncytia (polykaryocytes), and the appearance of nuclear or cytoplasmic inclusion bodies. Inclusion bodies may represent either altered host cell structures or accumulations of viral components.

 

·         Effects on Cell Physiology: The interaction of virus with the cell membrane and/or subsequent events, (for example, de novo synthesized viral proteins) may change the physiological parameters of infected cells, including movement of ions, formation of secondary messengers, and activation cascades leading to altered cellular activities.

 

·         Effects on Cell Biochemistry: Many viruses inhibit the synthesis of host cell macromolecules, including DNA, RNA, and protein. Viruses may also change cellular transcriptional activity, and protein-protein interactions, promoting efficient production of progeny virus. For some viruses, specific cellular biochemical functions may be stimulated in order to enhance virus replication.

 

·         Genotoxic Effects: Following virus infection, breakage, fragmentation, rearrangement and/or changes in the number of chromosomes may occur.

 

·         Biologic Effects: Virus-specified proteins may alter the cell's antigenic or immune properties, shape, and growth characteristics.

 

·         DNA or RNA tumor viruses may mediate multiple changes that convert a normal cell into a malignant one. RNA tumor viruses usually transform cells to a malignant phenotype by integrating their own genetic material into the cellular genome and may also produce infectious progeny. DNA tumor virus infections are often cytocidal; thus transformation is associated with abortive or restrictive infections in which few viral genes are expressed.

 

·         Stages of Transformation: Transformation involves at least two processes: first, the cell gains the capacity for unlimited cell division (immortalization), and second, the immortalized cells acquire additional heritable genetic changes by which the cell is able to produce a tumor in an appropriate host.

 

·         Mechanisms of Oncogenic Transformation: There are two general patterns by which cell transformation may be accomplished: 1) the tumor virus may introduce and express a so-called transforming gene in the cells or 2) the tumor virus may alter the expression and (or) coding capacity of preexisting cellular genes. After development of a malignant phenotype the relevant segment(s) of the viral genome may or may not be retained in the transformed cells, depending on the mechanism of transformation. These mechanisms are not mutually exclusive, and both may occur in the same cell.

 

·        

·         Table 1: Cellular effects of viral infections[1]

 

 

Genetic

Cell Fate

Morphological

Biochemical

Physiological

Cytocidal

Productive
-----------------

Abortive

DNA degradation
-----------------

Possible mutation

Death
-----------------

Usually death

Rounding of the cell
Fusion with adjacent cells
Appearance of inclusion bodies

Inhibit DNA, RNA, and protein synthesis
Interfere with sub-cellular interactions

Insufficient movement of ions
Formation of secondary messengers
Activation of cellular cascades

 

Persistent

Latent
-----------------

Chronic
-----------------

Slow

Possible Mutation
-----------------

Possible Mutation
-----------------

Possible Mutation

Survival
-----------------

Variable
-----------------

Variable

Fusion with adjacent cells
Appearance of inclusion bodies
Budding

Immune responses limit viral spread
Antigen-antibody complexes can incorporate
viral 
antigens causing inflammation

Rare until stimulated

 

Transforming

DNA viruses
-----------------

RNA viruses

Mutation
-----------------

Mutation

Survival
-----------------

Survival

Unlimited cell replication

Inactivates tumor suppressor proteins
Impairs cell cycle regulation

Unlimited cell replication

 

Hepatitis B

The Hepatitis B viral protein X is believed to cause hepatocellular carcinoma through transformation, typically of liver cells. The viral DNA is incorporated into the host cell's genome causing rapid cell replication and tumor growth.

 

Papillomaviruses

Papillomaviruses typically target epithelial cells and cause everything from warts to cervical cancer. When human papillomavirus (HPV) transforms a cell, it interferes with the function of cellular proteins while degrading other cellular proteins.

 

Herpesviruses

The herpesvirusesKaposi's sarcoma-associated herpesvirus and Epstein-Barr virus, are believed to cause cancer in humans, such as Kaposi's sarcoma, Burkitt's lymphoma, and nasopharyngeal carcinoma. Although genes have been identified in these viruses that cause transformation, the manner in which the virus transforms and replicates the host cell is not understood.

 

Retroviruses

The retroviruses include T-cell Leukemia virus type IHIV, and Rous Sarcoma Virus (RSV). The viral gene tax is expressed when the T-cell Leukemia virus transforms a cell altering the expression of cellular growth control genes and causing the transformed cells to become cancerous. HIV works differently by not directly causing cells to become cancerous but by instead making those infected more susceptible to lymphoma and Kaposi's sarcoma. Many other retroviruses contain the three genes, gagpol, and env, which do not directly cause transformation or tumor formation.

 

HIV

Human immunodeficiency virus is a viral infection that targets the lymph nodes. HIV binds to the immune CD4 cell and reverse transcriptase alters the host cell genome to allow integration of the viral DNA via integrase. The virus replicates using the host cell's machinery and then leaves the cell to infect additional cells via budding.

·         Type I interferons (IFNs) are used to treat a wide variety of medical conditions including hepatitis C, cancers, viral and inflammatory diseases. IFNs can either be extracted from a natural source, such as cultured human cells or blood leukocytes, or they can be manufactured with recombinant DNA technologies. Most of these IFN treatments have a low response rate.[16]

·         The use of viral transformation of the Epstein-Barr virus (EBV) has been recommended to create personalized IFNs. In this process, primary B lymphocytes are transformed with EBV. These cells can then be used to produce IFNs specific for the patient from which the B lymphocytes were extracted. This personalization decreases the likelihood of an antibody response and therefore increases the effectiveness of the treatment.

Edited by RogueSolace

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//From WHO website- Rapid advice guidelines- A rapid advice guideline is produced in response to a public health emergency (such as pandemic influenza) in which WHO is required to provide rapid global leadership and guidance. This type of document needs to be produced within 1–3 months and will be evidence-informed, but it may not be supported by full reviews of the evidence. It will be prepared mainly by the responsible WHO staff members with external consultation and peer review. It must be published with a review-by date that indicates when the guidance will become invalid, or when it will be updated or converted to a standard guideline.//

Rough Draft

Wendigo Virus Rapid Advance Guideline

 

Submitted by Dr. Elizabeth Smith to W.H.O.

8/6/2017

Spoiler

Update on virus ‘Wendigo’ and protection to the world.

 

The virus nicknamed by W.H.O. doctors here, as ‘wendigo virus’, is or has already become a global pandemic. A great deal is still needed to be understood about the virus, these facts are the best advice for protection that we can currently give you.

 

Virus

·         Current belief is this virus is transmitted via body-fluids: blood, saliva, semen, vaginal fluids, and breastmilk. PROVED TRUE BY WHO RESEARCH 

·         People who have had the virus for up to a 16 hour period develop a ‘trance-like’ state. They stop all forms of communication, simply moaning. They will appear aloof and distant, eyes unfocused, wandering around and not responding to any attempted communication. Any physical damage to the body appears to not bother them.

·         It is believed that those in full stage of the virus are in a persistent vegetative state (PVS) or unresponsive wakefulness syndrome. Probable brain damage has left the body in a state of arousal, without actual awareness. Essentially the ill person is able to move around, but has no actual consciousness of themselves, their actions, or anything around them. ***** SAID SOMETHING ABOUT FRONTAL LOBE OF BRAIN, INIQUITY AND STUDY FURTHER, STILL PLAUSIBLE

·         Stay far away from these people and ensure every protection for yourself. Unfortunately this includes use of deadly force. Predator prey instinct is active and at spotting uninfected people or animals the person will violently attack with intent to kill and eat its victim, not specifically in that order.

·          If put in a situation where use of deadly force is needed, aim with an attempt to smash the head and the neck.

·         Use all precautions from being scratched, bitten, and any contact with body fluids. 

 

Symptoms

·         Bleeding from the eyes

·         Rash that excretes a sap-like puss

·         Infection at wound site ; irritation, redness, itching, swelling and draining fluids

·         The one thing we are positive of is that the only way to tell if a person is truly infected is to look in the mouth for any signs of a black bile-like substance.

 

Critical care

·         If a person throws up this black bile substance or has any in their mouth, they need to be put in immediate seclusion and cared for as anyone would care for someone in their last hours. At this point there is nothing we can do. The virus will cause the body to die (loss of brain function, heart stops) and then almost immediate restart itself. At this point PVS is active and it is suggested to use deadly force before they attack.

·         There have been rumors of delayed reactions to the virus for up to a week. Any victim should be places in quarantine for at least a week and a half. Have still heard no other accounts of this happening as of 12/17

·         There have been reports of people being bitten and scratched who have not developed any symptoms. This leads us to believe some humans have immunity to the virus. True. More people have been discovered with immunity. 

·         Please seek immediate medical help if you come into contact with body fluids.

·         Please understand that being bitten or scratched is not a death sentence. The virus may not react, or simply need to be professionally cleaned off of skin by trained hazmat.

·         Most body-fluid viruses can live for up to a week on any surface, thus the urgency to find help.

  

Safety 

Makeshift Bio-hazard Suit has proven effective as long as seals are not torn or broken.

·         Light and noise activate the predatory behavior. Keep light minimal and be as quiet as possible.

·         It is possible if not spotted, to lay on the ground in high grass or other barriers, and not move (wait for infected to wander off) or army crawl away. Stop moving as soon as they show an indication of hearing sound, wait until they wander off and repeat.

·         Thrown objects that make noise upon landing can also be used as a short distraction to run away.

·          Do not share any drinks, food, or anything that has come into contact with a body fluid of any sort.

·         I recommend that anyone coming into near contact with anyone infected or thought to be needs to be wearing full hazmat clothing or as close as possible.

o   Duct tape any openings to the skin such as wrist and gloves, boots to pants, shirt and pants etc.

o   Layering of clothing especially waterproof is highly encouraged.

o   Try to stay away from fabrics that absorb liquid.

o   Ensure the face is fully covered, all of it. Full face shield masks or gas masks are approvable if something is also covering the head area.

o   Gasmasks are advisable, not for the virus itself but for other pathogens or dangerous chemicals that could be in the air from decomposition or other sources.

o   Upon exiting the area of exposure, be sure to spray down all clothing with disinfectant or water with 0.03-0.05% bleach combination. This will be sure to kill any virus on the topical layers.

o   Carefully remove tape and dispose. I do not recommend burning as we don’t know if the pathogen can be released into the air this way.

o   Carefully remove all layers and again spray down the majority of the person and clothing again with disinfectant (avoid eyes, mouth and sensitive areas).

o   Wash clothing in a bleach solution and allow to dry. Properly dispose of anything such as rubber gloves, disposable masks, etc. in a marked biohazard container.

 

 

Phages

AKA possible "Base of Virus Wendigo, or possible ideas for a vaccine/cure"

Spoiler

·         Bacteriophages isolated and purified from the wild have long been used to treat infections in people, particularly in Eastern Europe. These viruses infect only specific species or strains of bacteria, so they have less of an impact on the human body’s natural microbial community, or microbiome, than antibiotics do. They are also generally thought to be very safe for use in people.

·         But the development of phage therapy has been slow, in part because these viruses are naturally occurring and so cannot be patented. Bacteria can also quickly evolve resistance to natural phages, meaning researchers would have to constantly isolate new ones capable of defeating the same bacterial strain or species. And it would be difficult for regulatory agencies to continually approve each new treatment.

·         Virus Trained to Kill Bacteria. Scientists have stumbled upon a new way for coping with drug-resistant bacteria. The discovery involves bacteriophages, viruses that infect bacteria. ... This means that phages can be engineered to treat bacterialinfections that have become resistant to antibiotics

·         The phage preparation was ingested by d'Herelle, Hutinel, and several hospital interns in order to confirm its safety before administering it the next day to a 12-year-old boy with severe dysentery. The patient's symptoms ceased after a single administration of d'Herelle's antidysentery phage, and the boy fully recovered within a few days. The efficacy of the phage preparation was “confirmed” shortly afterwards, when three additional patients having bacterial dysentery and treated with one dose of the preparation started to recover within 24 h of treatment

·         Richard Bruynoghe and Joseph Maisin, who used bacteriophages to treat staphylococcal skin disease. The bacteriophages were injected into and around surgically opened lesions, and the authors reported regression of the infections within 24 to 48 h. Several similarly promising studies followed and encouraged by these early results, d'Herelle and others continued studies of the therapeutic use of phages (e.g., d'Herelle used various phage preparations to treat thousands of people having cholera and/or bubonic plague in India. In addition, several companies began active commercial production of phages against various bacterial pathogens.

·         The medical team obtained emergency approval from the U.S. Food and Drug Administration to try the experimental treatment, which must be tailored to suit an individual patient’s case. For Patterson, the phage was administered intravenously and through catheters into his abdomen. Within 48 hours of receiving the phage therapy, Patterson woke up from the coma. “We began to see his blood pressure stabilize, his white [blood cell] count begin to come down, and it was very clear that he was having a clinical response to the bacteria phage,” Schooley said. Three months after receiving the phage therapy, there was no evidence of the harmful bacteria in Patterson’s body. By the fifth month, he was sent home from hospital.

·         At first, he had tested the safety of phage suspension on himself, his co-workers, and family, then on patients suffering from “bacillary dysentery” and cholera (since 1919). After that, phages were applied as a therapy to wound recovery. Another experiment that focused on the healing value of phages investigated Salmonella gallinarum as an infectious agent of avian typhosis (published in 1926). This test also confirmed phage protection, as well as it did against other species, like Pasteurella multocida (bovine hemorrhagic septicaemia, published in the same year). Nevertheless, the first publication about phage therapy described the work of Bruynoghe and Maisin. Their results were published in 1921.

·         example, Soothill et al. (63–65) reported the utility of phages in preventing and treating experimental disease in mice and guinea pigs infected with Pseudomonas aeruginosa and Acinetobacter, and they suggested that phages might be efficacious in preventing infections of skin grafts used to treat burn

          

Interactions Between Bacteriophages and Host Immune System During Phage Therapy

Spoiler

Phage therapy may carry a risk of immunological reactions, that is why studies about interactions between phages and immunity are very important for the rational use of this treatment. Immune response against bacteriophages depends on the localization of bacterial infection and the injection site of therapeutic phages. Under physiological conditions, some phages are associated with the eukaryotic component of a gut microbiota and ingested food [35]. High frequency of natural contact of animals/humans with various types of phages is evidenced by the anti-phage antibodies detected in the sera of different species (e.g. human) [44].

Furthermore, an oral administration of phages during phage therapy of bacterial infection caused by Staphylococcus, Klebsiella, Escherichia, Proteus and Pseudomonas induces the production of antibodies as well [7]. There is no evidence of immunological complications after the consumption of large amounts of phages [36]. Moreover, the topical applications of phages have not shown any side effects [57]. A different situation is observed in the other internal organs and blood stream, which are not natural environment for phages. Intravenous administration of bacterial pathogens strongly stimulates both innate and adaptive immunity [28]. Furthermore, studies show that phages can penetrate into the circulation, regardless of the route of administration [7]. If there are no host bacteria for specific phages, they are rapidly removed from the blood and internal organs by phagocytic cells [28]. Moreover, bacterial predators are internalized and eliminated by cells of the reticuloendothelial system of liver and spleen.

Interestingly, Kupffer cells (specialized macrophages which are located in the liver) can phagocyte phages four times faster than spleen macrophages, which suggests that arrested bacterial pathogens in spleen may stimulate lymphocytes to produce antibodies [714]. Innate immunity, known as the first line of defence, is often sufficient to eliminate pathogens before the activation of adaptive immune response. Studies have shown that patients subjected to phage therapy were characterized by the decreasing number of mature neutrophils and the increasing number of neutrophil precursors in the peripheral blood [55]. These results indicated that phage preparations can activate innate immune response, which is helpful in the clearance of bacterial infection. On the other hand, phages can affect immune cells’ metabolic activities as well. For instance, studies have shown that bacteriophages markedly inhibit ROS production in response to pathogenic bacteria and suggest that phages decrease antibacterial innate immunity [32]. However, the relevance of these findings in relation to clinical situations is discussed.

During phage therapy, phages are able to induce specific antibodies (neutralizing antibodies) against them, which usually inhibit phage effectiveness to lyse the targeted bacteria in vivo [15162546]. In fact, neutralizing antibodies are defined as antibodies that bind epitopes within those parts of the virion essential for infecting the host cells [13]. However, it is not clear how long this type of antibodies will remain in circulation. Concentration of neutralizing antibodies depends on many aspects, for instance, (a) the route of phages administration (topical and oral administration cause a small increase of antibodies) and (b) the dosage protocol [47].

Studies have shown that anti-phage neutralizing antibodies are probably one of the most important factors responsible for the efficacy limitation of phage therapy [44]. However, Sulakvelidze et al. [46] suggested that the development of neutralizing antibodies should not be a significant problem during the initial treatment of acute infections, because the kinetics of phage action are much faster than the host’s production of neutralizing antibodies. Nevertheless, anti-phage antibodies can be an obstacle if they are still present at the time the second course of treatment is administered. There are three ways to solve that problem. It might be envisaged to (a) repeat phage administration, (b) increase the phage concentration or (c) use different phages because resistance is different from one phage to another [46]. Despite the fact that anti-phage neutralizing antibodies occur during phage therapy, there is also an increasing level of non-neutralizing antibodies, IgM and later IgG, and enhancement of immune response after subsequent injections of some types of phages [35].

Apart from the humoral immune response, cellular immunity also plays an important role against phages. Langbeheim has shown that subcutaneous injection of MS-2 phages resulted in a strong hypersensitivity reaction in guinea pigs. Similar results have been obtained in vitro [21]. However, some other studies indicated that cellular immune responses play only a slight role in phage inactivation. They showed that the clearance of T7 phage in T cell-deficient mice was similar to that observed in the wild-type mice [45]. In view of the contradictory results, this issue requires further study. Interestingly, some studies have shown that phages can exert immunosuppressive activity. Over the study upon the role of bacteriophages in the development of transplantation tolerance, Górski observed that phages can inhibit the activation of T cells [17]. Moreover, Kniotek indicated that after phage administration the humoral immunity is decreased as well [20].

Altogether results suggest that it is very important to test the immunological response of every single phage, particularly if intravenous therapy is being considered. However, previous clinical and animal trials have not resulted in serious immunologic reactions during phage therapy [28].

  

Phage Therapy

 possible ideas for a vaccine/cure

 Collection

Spoiler

The simplest method of phage treatment involves collecting local samples of water likely to contain high quantities of bacteria and bacteriophages, for example effluent outlets, sewage and other sources.[7]They can also be extracted from corpses.[citation needed] The samples are taken and applied to the bacteria that are to be destroyed which have been cultured on growth medium.

If the bacteria die, as usually happens, the mixture is centrifuged; the phages collect on the top of the mixture and can be drawn off.

The phage solutions are then tested to see which ones show growth suppression effects (lysogeny) or destruction (lysis) of the target bacteria. The phage showing lysis are then amplified on cultures of the target bacteria, passed through a filter to remove all but the phages, then distributed.

 Treatment

Spoiler

Phages are "bacterium-specific" and it is therefore necessary in many cases to take a swab from the patient and culture it prior to treatment. Occasionally, isolation of therapeutic phages can require a few months to complete, but clinics generally keep supplies of phage cocktails for the most common bacterial strains in a geographical area.

Phages in practice are applied orally, topically on infected wounds or spread onto surfaces, or used during surgical procedures. Injection is rarely used, avoiding any risks of trace chemical contaminants that may be present from the bacteria amplification stage, and recognizing that the immune system naturally fights against viruses introduced into the bloodstream or lymphatic system.

The direct human use of phages is likely to be very safe; in August and October 2006 the United States Food and Drug Administration approved spraying meat and cheese with phages. The approval was for ListShield and Listex (phage preparations targeting Listeria monocytogenes). These were the first approvals granted by the FDA and USDA for phage-based applications. This confirmation of safety within the worldwide scientific community opened the way for other phages applications for example against Salmonella and E-coli (http://www.fsis.usda.gov/wps/portal/fsis/topics/regulations/directives/fsis-directives)

Phage therapy has been attempted for the treatment of a variety of bacterial infections including: laryngitis, skin infections, dysenteryconjunctivitisperiodontitisgingivitissinusitisurinary tract infections and intestinal infections, burns, boils,[7] poly-microbial biofilms on chronic wounds, ulcers and infected surgical sites.[citation needed]

In 2007 a Phase 1/2 clinical trial was completed at the Royal National Throat, Nose and Ear Hospital, London, for Pseudomonas aeruginosa infections (otitis).[38][39][40] Documentation of the Phase-1/Phase-2 study was published in August 2009 in the journal Clinical Otolaryngology.[41]

Phase 1 clinical trials have now been completed in the Southwest Regional Wound Care Center, Lubbock, Texas for an approved cocktail of phages against bacteria, including P. aeruginosa, Staphylococcus aureus and Escherichia coli (better known as E. coli).[42] The cocktail of phages for the clinical trials was developed and supplied by Intralytix.

Reviews of phage therapy indicate that more clinical and microbiological research is needed to meet current standards.[43]

Administration

Spoiler

Phages can usually be freeze-dried and turned into pills without materially reducing efficiency.[7] Temperature stability up to 55 °C and shelf lives of 14 months have been shown for some types of phages in pill form.[7]

Application in liquid form is possible, stored preferably in refrigerated vials.[7]

Oral administration works better when an antacid is included, as this increases the number of phages surviving passage through the stomach.[7]

Topical administration often involves application to gauzes that are laid on the area to be treated.[7]

IV phage drip therapy was successfully used to treat a patient with MDR Acinetobacter baumannii in Thornton Hospital at UC San Diego.[44]

 

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Phage-pharming (how to make the phage stocks)

Thanks to Brendan Bohannan for the following protocol on preparing phage lysates!


© 1999 by Brendan Bohannan

Spoiler

Protocol for Phage Lysate Preparation

Note: this protocol is optimized for the production of high-titer T4 lysates (10e10 -10e12 pfu per ml). It can also be used for phages T2, T7, T5 and T6 but titers tend to be lower with these phages (10e7 - 10e9 per ml).

1.     Prepare overnight culture of permissive host (e.g., REL607 for T4,T5,T2, T7; LS371 for T6).

2.     Prepare a fresh plating of phage using rich media (M9supp plates and top agar are optimum media, but phage plates and soft agar can be used, or even LB). Phage should be diluted such that plaques are not touching.

3.     Transfer 1 ml of overnight culture to 4 ml of rich media in a 50-ml flask (M9supp is optimum media, but LB will also work).

4.     Stick a toothpick into an isolated plaque (use sterilized forceps to handle toothpick). Drop the toothpick into the host culture. Titer can sometimes be improved for phages other than T4 if you wait 30 minutes after diluting bacterial culture before adding toothpick.

5.     Shake at 37C. The culture should completely lyse in 6-8 hours (it will still be slightly turbid due to cell debris).

6.     Add 10 drops of chloroform and mix thoroughly to kill remaining bacteria.

7.     Spin at 5000 rpm for 5 minutes to remove cell debris. If the lysate is of very high titer it should appear slightly "milky" (similar to a DM25 bacterial culture).

8.     Decant supernatant (including a few drops of chloroform).

9.     Titer phage on rich media (M9 is ideal, others can be used, see above). Be careful to let chloroform settle to bottom of tube before removing aliquot of lysate for titering.

10. Store lysates at 4C as is, and/or in 20% glycerol at -80C.

 

Recipe for M9supp Media (makes 100 ml)

10X M9 Salts

 

10 ml

10% Glucose

 

5 ml

0.1M MgSO4

 

1 ml

20% Casamino Acids

 

5 ml

10-mg-per-ml B1

 

0.02 ml

10-mg-per-ml Tryptophan

 

0.02 ml

sterile dH2O

 

79 ml

M9 Agar is same as above but with 15g agar per liter; M9 Top Agar is same as above but with 7 g agar per liter. Autoclave agar and dH2O, then add sterile supplements.

 

Recipe for 10X M9 Salts (makes 1 L)

Na2HPO4

 

60 g

KH2PO4

 

30 g

NaCl

 

5 g

NH4Cl

 

10 g

dH2O

 

999 ml

Autoclave; add 1 ml of 1M CaC12

 

Phage on tap–a quick and efficient protocol for the preparation of bacteriophage laboratory stocks

Natasha BonillaMaria Isabel RojasGiuliano Netto Flores CruzShr-Hau HungForest Rohwer, and Jeremy J. Barr

Academic Editor: Siouxsie Wiles

Author information ► Article notes ► Copyright and License information ►

Spoiler

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Abstract

A major limitation with traditional phage preparations is the variability in titer, salts, and bacterial contaminants between successive propagations. Here we introduce the Phage On Tap (PoT) protocol for the quick and efficient preparation of homogenous bacteriophage (phage) stocks. This method produces homogenous, laboratory-scale, high titer (up to 1010–11 PFU·ml−1), endotoxin reduced phage banks that can be used to eliminate the variability between phage propagations and improve the molecular characterizations of phage. The method consists of five major parts, including phage propagation, phage clean up by 0.22 μm filtering and chloroform treatment, phage concentration by ultrafiltration, endotoxin removal, and the preparation and storage of phage banks for continuous laboratory use. From a starting liquid lysate of > 100 mL, the PoT protocol generated a clean, homogenous, laboratory phage bank with a phage recovery efficiency of 85% within just two days. In contrast, the traditional method took upwards of five days to produce a high titer, but lower volume phage stock with a recovery efficiency of only 4%. Phage banks can be further purified for the removal of bacterial endotoxins, reducing endotoxin concentrations by over 3,000-fold while maintaining phage titer. The PoT protocol focused on T-like phages, but is broadly applicable to a variety of phages that can be propagated to sufficient titer, producing homogenous, high titer phage banks that are applicable for molecular and cellular assays.

Keywords: Bacteriophage, Endotoxin, Cesium chloride, Top agar, Ultrafiltration, Dialysis, Speed vacuum, Phage bank

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Introduction

Due to increasing interest for the use of bacteriophage (phage) in medical, industrial, and molecular settings, new approaches are required to quickly and efficiently produce high titer, homogenous, and purified phage stocks. It is desirable that these stocks be free of bacteria, molds, debris, culture medium, and bacterial endotoxins (Adams, 1959). Typically the ability to produce high titer phage stocks is largely dependent on the particular phage and host cell under consideration, yet certain principles and methodologies can be broadly applied. Traditional techniques used for the concentration and purification of phage involve centrifugation, filtration, ultrafiltration, precipitation with Polyethylene Glycol (PEG), ultracentrifugation in cesium chloride (CsCl) gradients, and dialysis (Adams, 1959Yamamoto et al., 1970Seeley & Primrose, 1982Suttle, Chan & Cottrell, 1991Carlson, 2005Bourdin et al., 2014). Yet many of these techniques are time consuming and affect phage recovery and/or viability.

 

Phage preparations are often contaminated by macromolecules derived from the host bacteria and culture media, with the major pyrogen being the lipid A moiety of lipopolysaccharide (endotoxin) from the outer membrane of Gram-negative bacteria (Raetz et al., 2007). Endotoxins are amphipathic molecules; the lipid component is linked to a core polysaccharide and as a result they can form large aggregates greater than 1,000 kDa in solution (Magalhães et al., 2007). Endotoxin elicits a wide variety of pathophysiological effects in the body. Exposure to even small amounts can result in toxic shock, cell injury, cytokine production and the activation of immune responses (Morrison & Ulevitch, 1978Rietschel et al., 1994Alexander & Rietschel, 2001Pabst et al., 2008). Due to these effects it is important that endotoxins be removed from phage preparations when studying or applying phage in the context of eukaryotic systems. The amount of endotoxin is defined as an endotoxin unit (EU), which corresponds to the activity of 100 pg of E. coli lipopolysaccharide. The endotoxin content of distilled water is estimated at 20 EU·ml−1, with the allowed limit for intravenous and oral administration set at 5 EU·kg·hr−1 and < 20 EU·ml−1 respectively (Bruttin & Brüssow, 2005Gorbet & Sefton, 2005Abedon et al., 2011).

 

A further limitation with traditional phage preparations is the variability of titer, salts, and bacterial contaminants produced between successive propagations. Eliminating this variability has proven critical for accurate analysis for the molecular interactions of phage within the context of eukaryotic hosts (Barr et al., 2013Barr et al., 2015). Here we present the Phage On Tap (PoT) protocol as a fast and efficient way to produce homogenous laboratory phage stocks. Phage stocks were sterilized by centrifugation, 0.22 μm filtration, and chloroform treatment, before concentration and washing using ultrafiltration, and storage at 4 °C for four months with minimal degradation. However, the PoT protocol was not able to effectively reduce bacterial endotoxins, likely due to our ultrafiltration-based approach and the large aggregate size of endotoxins. Numerous endotoxin removal procedures and commercial kits are available (Boratyński et al., 2004Merabishvili et al., 2009Oślizło et al., 2011Magalhães et al., 2007Branston, Wright & Keshavarz-Moore, 2015), yet many of these methodologies lack generality, are time consuming, or are cost-prohibitive. Recently, the successful reduction of endotoxins (< 20 EU·ml−1) from phage lysates was reported by extraction with organic solvents (Szermer-Olearnik & Boratyński, 2015). We corroborate this organic solvent-based method for the removal of bacterial endotoxin from phage lysates and adapt this methodology, with reduced processing time through the use of speed vacuum, in the PoT protocol. The PoT protocol can purify phage lysates, with volumes ranging between 50–300 ml (or greater if required), producing homogeneous phage stocks for further laboratory testing. The method takes two days to purify and concentrate a final phage lysate of 10 ml of with a titer > 1010 PFU·ml−1, and a significant reduction in bacterial endotoxin levels. Our method has been tested on a variety of phages, including T4 (E. coli), T3 (E. coli), T5 (E. coli), and Spp1 (B. subtilis), and is broadly applicable to other tailed phages.

 

Materials and Methods

Spoiler

Reagents

·         Luria-Bertani (LB) broth (cat. no. DF0446; Fisher Scientific)

·         Agar (cat. no. BP1423; Fisher Scientific)

·         Calcium Chloride Dihydrate (CaCl2·2H2O) (cat. no. C69; Fisher Scientific)

·         Magnesium Chloride Hexahydrate (MgCl2·6H2O) (cat. no. BP214; Fisher Scientific)

·         Sodium Chloride (NaCl) (cat. no. S671; Fisher Scientific)

·         Magnesium Sulfate Heptahydrate (MgSO4·7H2O) (cat. no. M63; Fisher Scientific)

·         Trizma Hydrochloride (Tris HCl pH 7.4) (cat. no. 93313; Sigma-Aldrich)

·         Chloroform (cat. no. BP1145; Fisher Scientific) CAUTION: Chloroform is toxic and should only be used in a fume hood and with personal safety equipment, such as gloves and goggles.

·         Pierce™ LAL Chromogenic Endotoxin Quantitation Kit (cat. no. 88282; Thermo Fisher)

·         Note: Endotoxin Quantitation Kit is optional and is only required for the quantitation of endotoxins in phage lysates.

·         Ethanol, Absolute (C2H5OH) (cat. no. BP2818; Fisher Scientific)

 

·         Glycerol (cat. no. G31; Fisher Scientific)

 

Equipment

·         37 °C incubator with a rocker

·         Centrifuge with swinging bucket rotor

·         Microtube centrifuge

·         Centrifugal vacuum concentrator

·         Stir plate

·         Petri dish with disposable lid (cat. no. 09-720; Fisher Scientific)

·         1.7 ml microcentrifuge tubes (cat. no. 02-681; Fisher Scientific)

·         Falcon 50 ml conical centrifuge tubes (cat. no. 14-432; Fisher Scientific)

·         Small glass test tubes 13 × 100 mm (cat. no. 14-958; Fisher Scientific)

·         Serological pipettes (cat. no. 07-200; Fisher Scientific)

·         0.22 μm Sterivex filter units (cat. no. SVGP; Millipore)

·         Whatman Anotop 0.02 μm sterile syringe filters. (cat. no. 09-926-13; Fisher Scientific)

·         Amicon® Ultra-15 centrifugal filter units, Ultracel 100 kDa membrane (cat no. UFC910008; Millipore)

·         Note: 100 kDa membrane pore size is equivalent to a spherical particle with a diameter of 3 nm and is therefore sufficient for the capture of all known bacteriophages (Erickson, 2009).

·         500 ml PYREX® screw cap storage bottle with plastic seal (cat. no. 13-700-446; Fisher Scientific)

·         Spectra-Por® Float-A-Lyzer® G2 Dialysis membrane, 10 mL, MWCO 3.5–5 kDa (cat. no. Z726273; Sigma-Aldrich)

·         Nalgene™ General Long-Term Storage Cryo Tubes (cat. no. 03–337; Fisher Scientific)

Reagent setup

  • LB broth: 25 g LB broth in 1 liter dH2O.
  • LB top agar: 25 g LB broth, 7.5 g Agar, in 1 liter dH2O.
  • LB agar plates: 25 g LB broth, 15 g Agar, in 1 liter dH2O.
  • SM buffer: 5.8 g NaCl, 2.0 g MgSO4·7H2O, 50 ml 1 M Tris-HCl pH 7.4, in 1 liter dH2O. Autoclave, 0.02 μm filter-sterilize before use, and store at room temperature.
  • Calcium chloride (CaCl2): Prepare a 1 M stock solution and add a final concentration of 0.001 M to desired volume of the LB broth that will be used for the liquid lysate. Autoclave, 0.02 μm filter-sterilize before use, and store at room temperature.
  • Magnesium chloride (MgCl2): Prepare a 1 M stock solution and add a final concentration of 0.001 M to desired volume of the LB broth that will be used for the liquid lysate. Autoclave, 0.02 μm filter-sterilize before use, and store at room temperature.

 

Phage on Tap (PoT) Protocol

Spoiler

Notes:

  • The procedure for phage propagation is largely specific for each phage and bacterial host. Here we use propagation conditions for T4 phage and Escherichia coli B bacterial host. It is recommended to use appropriated growth and propagation conditions for your choice of phage and host.
  • Once a sufficiently high titer phage lysate is obtained please proceed to step 3.
  • It is recommended to only propagate and purify one phage at a time to prevent cross-contamination.

1| Phage plaque assay for determination of titer (Adams, 1959)

  1. Grow E. coli B bacterial host in LB broth overnight at 37 °C.
  2. Dilute phage stock or isolate in LB broth down to the desired dilution (e.g., for a phage stock of 108PFU·ml−1 serially dilute down to 10−6 and 10−7 to obtain countable plaques).
  3. Heat LB top agar in microwave until completely molten, then allow top agar to cool in a 56 °C water bath or until it is warm to the touch.
  4. Add 1 ml of the overnight bacterial host and 1 ml of the phage dilution to a glass test tube and mix.
  5. Add 3 ml of molten top agar to the glass test tube.
  6. Quickly pour molten mixture onto an LB agar plate and tilt the plate to evenly distribute the agar. Let sit undisturbed until the agar has gelled.
  7. Once plate has gelled (5 min), invert and incubate overnight at 37 °C.
  8. Count phage plaques and determine phage titer in plaque-forming units (PFU·ml−1) using the following formula:

PFU per ml = plaques per plate × volume plated in ml × dilution factor

e.g., if there are 20 plaques when you plated out 1 ml from the 106 dilution, the titer of the phage stock is 2.0 × 107 PFU·ml−1.

 

 

2A| Phage isolation and propagation via plate lysate

  1. From a plate lysate plate pick a single phage plaque using a sterile Pasteur pipet.
  2. Resuspend the plaque into a microcentrifuge tube containing 1 ml of filter-sterilized phage diluent (SM buffer) and vortex for 5 min.
  3. Centrifuge at 4,000 × g for 5 min to remove any remaining debris.
  4. Perform plate lysate as described above using the resuspended phage.
  5. After incubation the entire plate should be lysed. Pour 5 ml of SM buffer on top of plate and shake gently for 15 min at room temperature.
  6. Collect buffer from top of plate and centrifuge at 4,000 × g for 5 min.
  7. Collect phage lysate and store at 4 °C until clean up.

Optional: Titer the lysate via plaque assay to ensure initial high titer.

2B| Phage propagation via liquid lysate

  1. Grow E. coli bacterial host in LB broth overnight at 37 °C.
  2. Prepare and autoclave 100 ml of LB broth supplemented 0.001 M CaCl2 and MgCl2 added in a 250 ml PYREX® screw cap storage bottle and save for Step 3.
  3. Spike LB broth supplemented with CaCl2 and MgCl2 with 0.1 volumes of overnight bacterial host.

Note: For a phage with a high burst size, such as T3, you may need to double volume of host added.

  1. Incubate with agitation for 1 h at 37 °C.
  2. Add 100 μl of high titer phage lysate (> 108 PFU·ml−1).
  3. Incubate at 37 °C with agitation for 5 h or until lysate clears.
  4. Collect phage lysate and store at 4 °C until clean up.

Optional: Titer the lysate via plaque assay to ensure initial high titer.

 

3| Phage cleanup (0.22 μm filtering and chloroform)

  1. Aliquot phage lysate into 50 ml sterile falcon centrifuge tubes and centrifuge at 4,000 × g for 20 min.
  2. Carefully collect supernatant using a serological pipette and transfer into properly labeled sterile falcon tube.
  3. Filter-sterilize the phage supernatant using a 0.22 μm filter to yield a bacterial cell-free phage lysate.
  4. Add 0.1 volumes of chloroform to the supernatant, vortex, and incubate at room temp for 10 min.

Note: Lipid enveloped phages are sensitive to chloroform and titer can be significantly reduced. If a drop in titer is observed skip this step.

  1. Centrifuge at 4,000 × g for 5 min and transfer supernatant into 250 ml PYREX® screw cap storage bottle and store at 4 °C until concentration.

Optional: Titer the lysate via plaque assay to ensure initial high titer.

 

 

4| Phage concentration and wash via ultrafiltration

  1. Add 15 ml of phage lysate into the upper reservoir of Amicon filter device.
  2. Centrifuge Amicon at 4,000 × g for 5 min.

Note: Centrifugation times will vary based on phage type and titer. It is important not to spin the device dry. If unsure about centrifugation times select a shorter spin time, check the lysate level, and adjust spin times appropriately.

  1. Carefully discard the filtrate into a waste bucket and add another volume of phage lysate to the sample filter cup and repeat centrifugation.

Note: The same device can be used to concentrate large volumes of phage lysate (> 100 ml) achieving an approximate 90% decrease with minimal loss in phage titer.

  1. Repeat step until all phage lysate has been concentrate to < 10 ml.
  2. Add 15 ml of SM buffer into the upper reservoir containing concentrate phage lysate and centrifuge at 4,000 × g for 5 min to wash phage lysate.

Note: SM buffer was chosen as it is suitable for the long-term storage of T-phage, but any appropriate storage buffer can be used. It is important not to spin the device dry, adjust centrifugation times accordingly.

  1. Repeat wash step and concentrate washed phage lysate to < 10 ml.
  2. Using a pipette, carefully collect phage lysate from the upper reservoir and gently wash the surface of the upper reservoir.

Note: Alternatively the entire device can be vortexed to assist with phage detachment from filter.

  1. Collect < 10 ml of concentrated and purified phage lysate. Titer phage concentrate and record PFU·ml−1.

 

5| Endotoxin removal (Morrison & Leive, 1975Szermer-Olearnik & Boratyński, 2015)

Notes:

  • This method is adapted from Szermer-Olearnik & Boratyński (2015), which demonstrates the efficient removal of endotoxins from bacteriophage lysates using water immiscible solvents that are subsequently removed via dialysis. For detailed explanation of the methodology please see Morrison & Leive (1975) and Szermer-Olearnik & Boratyński (2015).
  • Our adapted method uses a speed vacuum to remove residual organic solvent from phage lysates, instead of the lengthy dialysis washes with similar efficiency.
  • This step is optional. If you do not require removal of bacterial endotoxins from your phage preparations please go to step 7.
  1. Add 0.4 volumes of 1-octanol to phage concentrate and shake at room temp for 1 h.
  2. Incubate phage concentrate at 4 °C for 1.5 h.

Note: Alternatively concentrate can be chilled in a ice bath for 15 min.

  1. Centrifuge at 4,000 × g for 10 min.
  2. Using a syringe, pierce the bottom of the tube and collect the aqueous phase (bottom layer) that contains your phage, and transfer to a sterile 50 ml falcon tube. Do not collect the organic phage (top layer) or interface as this contains endotoxins.

Note: It is best to leave a small residual amount of phage concentrate behind to reduce the transfer of contaminating endotoxins.

Note: If endotoxin removal is not sufficient the method can be repeated to further reduce endotoxins.

 

 

6A| Dialysis removal of organic solvent (Szermer-Olearnik & Boratyński, 2015)

Notes:

  • This method is adapted from Szermer-Olearnik & Boratyński (2015) and describes the removal of residual organic solvents from phage lysates by dialysis.
  • Residual organic solvents disable downstream Pierce™ LAL Chromogenic Endotoxin Quantitation assays and must be removed in order to accurately quantify endotoxin concentrations.
  • Due to the ionic concentration of phage SM buffer used you may end up with greater than the starting volume.
  1. Pre-wet Spectra-Por® dialysis membrane with sterilized dH2O according to manufacturers instructions, being careful not to contaminate the inside of the tubing.
  2. Load maximum of 10 ml of phage concentrate inside of dialysis tubing and seal tightly.
  3. Dialyze phage concentrate against 2 liters of 25% (v/v) ethanol at 4 °C on a stirring plate for 24 h to remove residual 1-butanol, replacing the 25% ethanol solution four times at 15, 18, 21, and 24 h.
  4. Dialyze phage concentrate against 2 liters of 0.15 M NaCl solution at 4 °C on a stirring plate 24 h to remove residual ethanol, replacing the 0.15 M NaCl solution three times after 15, 19, and 24 h.
  5. Carefully collect phage concentrate by washing the inside of the dialysis tubing. Store concentrate at store 4 °C until phage bank preparation.

Optional: Titer the lysate via plate lysates to ensure high titer. Perform the Pierce™ LAL Chromogenic Endotoxin Quantitation according to manufacturers instructions to obtain quantitative endotoxin levels.

 

 

6B| Speed vacuum removal of organic solvent

Notes:

  • This method is a faster alternative to the dialysis method for the removal of residual organic solvents from phage concentrates.
  1. Aliquot 1 ml of the phage concentrate equally into microcentrifuge tubes.
  2. Place tubes into a speed vacuum 4 °C, open lids, and centrifuge at 4,000 × g for 3 h.
  3. After speed vacuum, phage concentrate volume should be reduced by approximately 30% and residual 1-octanol evaporated.
  4. Collect phage concentrate and store 4 °C until phage bank preparation.

Optional: Titer the lysate via plaque assay to ensure high titer. Perform the Pierce™ LAL Chromogenic Endotoxin Quantitation according to manufacturers instructions to obtain quantitative endotoxin levels from phage concentrates.

7| Phage bank storage

  1. Dilute phage lysate in SM buffer to generate a high-titer working stock.

Note: This step largely depends on desired concentration and volume of the phage bank. If you require higher titer phage stocks then dilute less or omit dilution, if greater volume is desired then dilute more.

  1. Titer diluted phage concentrate and record PFU·ml−1.
  2. Aliquot phage working stocks into labeled cryo tubes and store at 4 °C.
  • You now have a bank of homogenous, high titer (up to 1010–11 PFU·ml−1) phage bank for laboratory testing.
  • From our experience the use of homogenous and standardized phage banks can significantly reduce the experimental variability for the molecular characterization of phages.
Spoiler

"From the beginning of my study of bacteriophagy I have been struck by the fact that the appearance within the body of the patient of the principle which leads to bacteriophagy coincides with the time when the symptoms ameliorate. Absent during the disease, bacteriophage appears constantly in convalescents. Bacteriophagy is thus contemporaneous with recovery. In vitro bacteriophagy consists of the following: Let us take a few drops of stool derived from a convalescent from bacillary dysentery. Let us emulsify this in about 20 c.c. of sterile bouillon and filter it through a porous porcelain filter, such as the Chamberland, or through a silica candle such as the Berkefeld.

 

Let us add to a young bouillon culture of the dysentery bacillus a drop of this filtrate, and place the tube in the incubator. At first the bouillon appears cloudy, but after a few hours we note that it becomes more and more clear, and, finally after about 12 hours, sometimes more quickly, it becomes perfectly limpid. At this time all of the bacilli are dissolved. Let us take, then, a new, fresh culture of dysentery bacilli and add to it a drop of the limpid fluid which remains after the disappearance of the bacilli from the first mixture. Let us place this second tube in the incubator. We will find that the phenomenon repeats itself, for after a few hours all of the bacilli are again dissolved and the liquid is clear. We may then remove a drop of the second dissolved culture and introduce it into a third culture of dysentery bacilli. Once more the phenomenon of dissolution takes place."

 

Phage stock notes:

Out of my realm of understanding for the most part, though made sense to a fellow familiar with pharmaceuticals. Tried to get information with procedure to WHO.

  • personally took samples of bacteria from the stomach, lower intestine, and anus areas of just killed infected. 
  • Samples put onto petri dishes and clearly labeled. time/date/from/area of body
  • Petri dishes protected, kept in regulator at clinic with small generator, allowed to grow for about 4 days
  • Given to trusted person to give to WHO forces in Miroslavl along with data.

Apparently the higher ups decided to also ignore all of this completely and throw it away.... 

 

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Trying to keep on top of things listening to the news....

 Is Russia and the Soviet Union the same thing? The Soviet Union and Russia are not the same things but are very close to each other. Both terms are also informal labels. 2. “Soviet Union” represented the “Union of Soviet Socialist Republics,” a collection of 15 states that existed from 1922 to 1991.

 Base 0 = Old storage base north of Severograd, easternmost base in a chain west along Black Mountains. Mentioned that it feels like it has been there forever.

 Probable Virus Timeline

1927-1953- Soviet Union- Base 0 sealed to the public.

1991- Fall of the USSR and Chernarussian independence. Base 0 handed over to CDF. All activities forgotten.

2009 –Chernarus Civil War Ends

NAPA disbanded by Josef Prizak, most members join CDF. Chedaki hunted and executed for crimes during war.

Chernarussian & Russia were peaceful, then tensions rose.

 

2017- May 3- Martial Law declared in South Zagoria

 

2017- July 7- CDF troops set up checkpoints and turn back all traffic  from base.

2017- July 7 PM- Video of a Russian helicopter flying to the base posted on twitter

2017- July 8- Residents of Nagornoe rounded up. Nagornoe escorted south to Severograd to local school. Only reason given “for their personal safety”

2017- July 8- Residents of Kamensk rounded up then (witnesses to the helicopter visit) disappeared completely. Rumor that they were interned (held prisoner) inside Base 0.

2017- July 8- midnight- apparent disagreement between two sides of Base 0 (Russian/Cherno) resulted in the departure of the Russians back across the border.

2017- July 10- early morning- Russian Air Force annihilates Base 0, leaving a few ruins. 50 Cherno died. Cherna retaliated.

                Rabid, unresponsive soldiers appeared in Severograd. Severely burned and dismembered, did not care nor respond to pain. First call located north of city,                    3 soldiers staggering from forest and attacking livestock. Appear to originate from Base 0. 

                Russian artillery launched bombardment of Kamensk, Nagornoe & Severograd. Local was interviewed and stated “The Russian bombardment was a                            distraction from whatever went on in the base that night. They had some stake in the course of events there, after all the bombardment did little damage                    to any civilian or military structure. The goal was to distract the international press for as long as possible.”

                Wave of infected hit Severograd from the north. People brought in from the attack were bleeding from the eyes, and had developed rashes similar to                            poison oak that secreted a semi clear and bloody pus like substance.

2017- July- 11 – All people exposed to virus had turned

2017- July 12- People began fleeing en mass. Two batallions of CDF from Miroslavl arrived to Cherno, using checkpoints etc to help the chaos. All military operations directed from Balota Airfield.

                  Chaos from NE spilled into Berezino and Solnichniy

                  Air base by Vybor rushed by infected at N and E Perimeter. CAF tried bombing, but only attracted more infected.

                  West Chern having outbreaks.

                  People fleeing via Novigrad and Miroslavl airports to Europe triggered outbreaks in the areas, creating pandemic.

                  Russia and Takistan shut down borders.

                  Elektro private boat and military vessles from Miroslavl attempted to evacuate the city, mostly successful. This moved the outbreak to Miroslavl as the                        CDF had not set up virus screening areas.

                  Cherno, Elektro and Zelen only major cities left.

2017- July 17- abroad reinforcements arrive to Miroslavl and detachment sent to Chapaevesk. Deployed troops to help CDF combat outbreaks.  US Army- Chernogorsk, US Marines- Elektrozavodsk. Russian strikes target border area.

2017- July 17 -19- bombings

2017- July- 19- CDF/NATO lines falter, infected horde towards Chernogorsk, US retreats from city due to infected and severe riots. CDF withdrew to countryside.      Citizens set Cherno set ablaze, ransacked, and partied.

2017- July- 19 – Midnight- International Hotel struck by cargo plane w relief supplies, all on board killed.

2017- July- 20 – Chernogorsk in shambles, helicopters looked for refugees

2017- July- 20- Right before noon- Second wave of NATO/Russian/CDF bombings begin. Infected chase fleeing people all over the country.

2017- July- 21 – Civilizations gave up, foreigners trapped fled. Night massive storm, ship w 3,000 sunk.

2017- July 21-24- Storm rages into early morning

2017- July 24- Russia creates no fly zone over South Zagoria

2017- July 25- Some kind of nuclear bomb drops or explodes in the ocean, wiping out a NATO fleet. 

2017- July 26- Ukraine and Turkey suffering total societal breakdown. Europe and US containing major outbreaks.

 

 

 

 

Apparently some kind of military base near Kamensk. Fits more with accounts as to where Base 0 could be.

Showed hypothesis to Dr. Tyrus Mason of WHO. Nearly dragged to show Col. Ripley of UN. 

Col. Ripley shocked at finding. Everyone seems to otherwise believe that Tisy Military Base is the point of origin. 

Later sends team of WHO/UN to Kamensk Military Base

CONFIRMED: Kamensk Military Base, (NOT Tisy Military Base), is confirmed as "Ground 0", location of origin for spread of virus.

wup0h07.jpg

Will add WHO findings of base to notes. 

 

Edited by RogueSolace

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Rogue, just go get a medical degree already!! You always put so much effort and time into researching things like this, <3 Good job.

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I love me some zombie science!!! I wish I had met Elizabeth Smith when I was on Doctor West, we would have done zombie science RP for hours and hours.

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Symptom: Rash and thick secretion of puss

Possible causes:

  • Spoiler

    Cellulitis

    • Typically caused by a staph infection, skin is tender, swollen and irritated
    • May form large blisters that drain clear yellowish fluid
    • If spreads to the bloodstream, results in fever and chills
    • Can occur in areas that have been damaged or an inflamed injury

    Dermatitis:

    • An allergic reaction where the body/skin forms a rash that leaks fluid
    • The fluid is infectious and will spread the reaction
    • Streptococci bacteria can then cause Impetigo or a bacterial infection.

     

    Urticaria:

    • Hives, also a chronic condition where the body produces too much histamine, then behaves erratically to anything it perceives as a threat, making the body or an area break out in hives
    • While Urticaria itself does not fit the symptoms, it could make sense if a similar histamine reaction was taking place


     

Secretion:

Spoiler

Moderate to copious Purulent exudate consists of a thick secretion from a wound, also called pus. Mostly dead cells and often described as ‘milky’. The color can be green, yellow, brown, or white in color and is a thick liquid. Purulent drainage is almost always a sure sign of infection.

 

 

 

Symptom: Cyst’s in esophagus (Faith Capella-Hawk)

Spoiler

Faith Hawk mentioned finding the esophagus filled with cysts.

  • She said when she pierced them, the smell was if the pus had been there for a long time, even if the cysts didn’t look that old.
  • She theorized thyroglossal duct cysts, but ruled it out due to cysts not following into the lungs
  • She said cysts are formed by infection, so she does not believe this to be a symptom of a chemical agent

Personal thoughts:

Spoiler

There are three other types of cysts that are related to the throat that I found. Mucus retention cysts, epidermoid cysts, and esophageal cysts. All have the possibility to affect the vocal chords and in some cases breathing.

  1. Retention cysts tend to be around the sinus area, typically filled with mucus. Due to location I am ruling this out.
  2. Epidermoid cysts. Common, grow slowly, do not cause other symptoms. Size can range between ¼ - 2 inches across. Appear as small bump, color ranges from tan to yellow. Filled with thick, smelly matter. Usually caused by a buildup of Keratin, a type of protein, trapped under the skin near a hair follicle. Can also develop in response to HPV and skin trauma. Rarely cancerous.  
  3. Esophageal cyst. Can also refer to an esophageal duplication (embryologic duplication of a portion of the muscle and submucosa without epithelial duplication). Usually located in the neck, chest and abdomen. Lining of cyst can vary. If gastric mucosa is present in the cyst it presents hemorrhage. Other mucosa can form but are not associated with any symptoms. Usually in the lower third of the esophagus, where difficulty swallowing from compression causes them. Some occur in the upper third, they signify respiratory difficulty from the tracheobronchial tree. The last bit occur in the middle third, with retrosternal chest pain and difficulty swallowing are the most common symptoms. 

Other possible though. When reviewing definitions…

  • Cyst- sac filled with fluid
  • Abscess- an always infected sac filled with fluid. Can form anywhere in the body.
  • However, a cyst can become infected and turn into an abscess. 

 

Edited by RogueSolace

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Symptoms: Haemolacria

Spoiler

Where the tear ducts produce either fluid with blood in it, or blood instead of tears

Can be caused by

  • Hormones, eye injuries, tear in renal duct, tumor, bacterial conjunctivitis, inflammation, laceration, infection of the eyelids or nasolacrimal system, etc.
  • “In most cases of haemolacria, a head injury, tumor, blood clot, a tear in the tear duct, or a common infection, such as conjunctivitis, causes the bloody tears.”
  • Henoch-Schonlein purpura-
    • Possibility of Henoch-Schonlein purpura- “a disorder that causes inflammation and bleeding in the small blood vessels in your skin, joints, intestines and kidneys. Usually has a purplish rash, typically on the lower legs and buttocks. Henoch-Schonlein purpura can also cause abdominal pain and aching joints. Rarely serious kidney damage can occur.”
    • “In Henoch-Schonlein purpura, some of the body's small blood vessels become inflamed, which can cause bleeding in the skin, abdomen and kidneys. Why this initial inflammation develops isn't clear. It may be the result of the immune system responding inappropriately to certain triggers.”
    • “Nearly half the people who have Henoch-Schonlein purpura developed the disease after an upper respiratory infection, such as a cold. Infectious triggers may include chickenpox, strep throat, measles and hepatitis. Other triggers may include certain medications, food, insect bites or exposure to cold weather.”

Symptoms: Subconjunctival Hemorrhage

Spoiler
  • Could be from multiple reasons
  • Possibly via trauma to veins, bursting in eyes
  • Via damage of the eye itself

 

  

Symptoms: Possible Hemorrhagic Fever      

Spoiler

   While no fever matches the actual symptoms, several have similar symptoms.

  •  Crimean-Congo Hemorrhagic Fever (CCHF)
    • Transmitted via ticks, wild animals
    • Other humans via blood or body fluids
    • Onset is sudden
    • Headache, high fever, back pain, joint pain, stomach pain, vomiting. Red eyes, flushed face, red throat, petechiae (red spots) on palate.
    • May also include jaundice
    • In severe cases may change mood and sensory perception
    • 4 days into the fever, for two weeks, symptoms shift to also having: severe bruising, severed nosebleeds and uncontrolled bleeding at injection sites.
    • Care should include supportive care- careful attention to fluid balance and correction of electrolyte abnormalities, oxygenation and hemodynamic support, and appropriate treatment of secondary infections.
    • The virus is sensitive in vitro to the antiviral drug ribavirin 

 

  • Ebola
    • Possibly, other things match more symptoms imo

 

  • Nipah Virus (NiV)
    • Transmission of Nipah virus to humans may occur after direct contact with infected bats, infected pigs, or from other NiV infected people.
    • Fever, headache, inflammation of the brain, drowsiness, disorientation, mental confusion. Can progress to a coma within 24-48 hours. Some patients have respiratory illness, half of patients showing severe neurological signs also showed pulmonary signs (lungs).
    • Longer term problems have been seen, including persistent convulsions and personality changes. Can also reactive after months or even years.
    • Treated by supportive care- careful attention to fluid balance and correction of electrolyte abnormalities, oxygenation and hemodynamic support, and appropriate treatment of secondary infections.
    • The drug ribavirin has been shown to be effective against the viruses in vitro
    • Passive immunization using a human monoclonal antibody targeting the Nipah G glycoprotein has been evaluated in the post-exposure therapy in the ferret model and found to be of benefit.

 

 

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Symptoms: Frontal lobe damage

Brain Behavior

Predator and Prey Instinct: Yale Mice Study

Spoiler
  • Stimulation of central amygdala (CeA) elicited hunting of live and artificial prey
  • CeA projections to the reticular formation (PCRt) control biting attacks
  • CeA projections to periaqueductal gray (PAG) control locomotion during pursuit
  • CeA integrates craniofacial and locomotor modules during goal-directed behavior

 

What a Real Harvard Psychiatrist Thinks About Sci-Fi Zombie

Spoiler
  • In order for predator/prey instinct to kick in, these parts of the brain MUST be functioning and able to communicate to each other
  • Central Amygdala
  • Reticular Formation
  • Periaqueductal gray
  • Central Amygdala is able to stimulate craniofacial (facial) and locomotor modules (movement).

 

Structures in the brain that could be damaged/altered

Spoiler
  • Amygdala (the part that responds to stimulus and decides how to react- panic/fear/anger/lust/etc.)
  • Frontal lobe (thought process)- must be active enough to receive direction from the thalamus (sensory input)
  • Motor Cortex- must be active with the frontal lobe to correspond to movement
  • Thalamus (sensory input) then must be active and able to communicate with the frontal lobe (thought process)
  • Anterior cingulate cortex (excites or calms the amygdala)- must be dysfunctional, rendering it unable to modulate feelings of anger. (THIS EQUALS HYPER AGGRESSION WHICH WE ARE SEEING. Highly plausible.)
  • Cerebellum (balance) and Basal Ganglia (coordination) together = (make motions smooth/fluid)- degeneration of this area (ataxia) can cause "a wide-legged, unsteady, lurching walk, usually accompanied by a back and forth tremor in the trunk of the body…"
  • Mirror Neurons (area that creates empathy)- if damage would not care about violence done to others
  • The Ventromedial Hypothalamus- (the region of the brain that lets you know whether you've eaten enough.) If damaged the result is hyperphagia. You will eat and eat and eat, but never feel satiated.

 

Dr. Tyrus Mason theorized that the virus killed parts of the brain, or shut them down. I believe this is true.

Edited by RogueSolace
won't stop merging :p

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Symptoms: Infection at wound site: irritation, redness, itching, swelling and draining fluids

  • Each area of suspected infection origin (Bite, scratch) has shown extreme signs of being infected.

 

Symptoms: Coma/Persistent vegetative state/coma vigil/unresponsive wakefulness syndrome

Spoiler
  • Body is in state of arousal, without actual awareness.
  • Most PVS patients are unresponsive to external stimuli and their conditions are associated with different levels of consciousness. Some level of consciousness means a person can still respond, in varying degrees, to stimulation. A person in a coma, however, cannot. In addition, PVS patients often open their eyes in response to feeding, which has to be done by others; they are capable of swallowing, whereas patients in a coma subsist with their eyes closed (Emmett, 1989).
  • PVS patients' eyes might be in a relatively fixed position, or track moving objects, or move in a disconjugate (i.e., completely unsynchronized) manner. They may experience sleep-wake cycles, or be in a state of chronic wakefulness. They may exhibit some behaviors that can be construed as arising from partial consciousness, such as grinding their teeth, swallowing, smiling, shedding tears, grunting, moaning, or screaming without any apparent external stimulus.
  • Individuals in PVS are seldom on any life-sustaining equipment other than a feeding tube because the brainstem, the center of vegetative functions (such as heart rate and rhythm, respiration, and gastrointestinal activity) is relatively intact (Emmett, 1989).

 

People in a persistent vegetative state

CAN

Spoiler
  • Some residual cognitive function. An external stimulation, such as pain, still activates ‘primary’ sensor cortices in the patient. These areas are functionally disconnected from ‘higher order’ associative areas needed for awareness. Thus part of the cortex is still functioning.
  • That means= senses still pick up signals such as pain, but the sensors to have the body be aware (feel and respond) are disconnected. Thus they feel nothing.
  • Open their eyes and move eyelids
  • Have relatively regular sleeping and waking patterns (not necessarily related to day and night)
  • Can breathe, suck, chew, cough, gag, swallow, make guttural sounds
  • May be startled by loud noises and appear to smile or frown (involuntary basic reflex, NOT a conscious action.) They may be aware of their surroundings. They may, like a baby, instinctively grab an object when it touches their hand

CANNOT

Spoiler
  • Make conscious movements
  • Do anything that requires thought of conscious intention
  • Speak
  • Follow commands
  • Move limbs purposefully
  • Move to avoid painful stimulus

 

Caused by

Spoiler
  • Head injury
  • Cardiac or respiratory arrest that deprives the brain of oxygen
  • Severe damage to brain such as bleeding and infection

 

  • Most patients who do recover consciousness experience significant disability. The longer a patient is in a PVS, the more severe the resulting disabilities are likely to be. Rehabilitation can contribute to recovery, but many patients never progress to the point of being able to take care of themselves
  • There are two dimensions of recovery from a persistent vegetative state: recovery of consciousness and recovery of function. Recovery of consciousness can be verified by reliable evidence of awareness of self and the environment, consistent voluntary behavioral responses to visual and auditory stimuli, and interaction with others. Recovery of function is characterized by communication, the ability to learn and to perform adaptive tasks, mobility, self-care, and participation in recreational or vocational activities. Recovery of consciousness may occur without functional recovery, but functional recovery cannot occur without recovery of consciousness (Ashwal, 1994).

 

 

Edited by RogueSolace

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Conclusions

 

Rash and thick secretion of puss

Spoiler
  • The skin is obviously responding as an allergic reaction to something.
  • The thick drainage shows a major infection, probably what the skin is reacting to.
  • Possible that the virus could be causing the body to overproduce histamine, then attacking anything it deems as a threat, causing major breakouts.
  • Fluid from pus is considered contagious.
  • Doubtful it’s responding to Staph bacteria.

 

Throat cysts findings via Faith

Spoiler
  • Sounds like an epidermoid cyst to me.
  • At the same time, it sounds due to the smell, like they are filled with pus and thus infected. This would make them abscesses.
  • So, did they start off as epidermoid cysts in the neck, and then all become infected and turn into abscesses?  
  • Could the infection in the abscess degrade faster than a normal cyst, thus causing the pus to be more infected and appear older?
  • Indicates that the virus is actually dormant in the body for a length of time, slowly building up the infection.

Both

Spoiler
  • Both the skin itself and the lining of the esophagus, appear to be having physical reactions to an internal infection. Is this forming from the inside-out?
  • Or is it simply a response of all tissues? If it was though, cysts would be all over other organs. Why just the skin and esophagus?

 

Haemolacria

Probable damage via weakened blood vessels, straining from illness, injury due to illness, or reaction to an infection

See Hemorrhagic Fever below

 

Hemorrhagic Fever

Spoiler
  • Viral fevers, group of illnesses caused by multiple distinct families of viruses
  • Multiple organs in the system affected
  • Overall vascular system is damaged and the body’s ability to regulate itself

Shares similarities between Crimean-Congo Hemorrhagic Fever (CCHF) and Nipah Virus (NiV)

CCHF

  • Transmitted to other humans via blood or body fluid
  • Sudden onset
  • Flu like symptoms including red eyes and flushed face and throat
  • May have Jaundice (don’t know if that’s important)
  • Severe changes in neurological functioning and processing
  • Severe bleeding
  • Ribavirin shown somewhat effective

NiV

  • Contact by infected animals or other infected humans
  • Flu like symptoms along with rapid neurological deterioration.
  • Can progress to coma in 24-48 hours
  • Some patients have respiratory illness
  • Usually when patients had neurological symptoms, they also had pulmonary symptoms
  • Can cause permanent personality change
  • Can reactivate
  • Ribavirin shown somewhat effective

 

Frontal lobe damage

Areas of the brain

Spoiler

eNQBfCu.jpg 

 

 

 

Via Yale and Harvard studies, brain function should be similar to this

Spoiler

RwfT6b5.jpg 

 

Combining study areas with what we see hypothetically as working and not working via the first picture, we can assume the brain may look something like this.

Spoiler

bH6g2Rf.jpg 

Brain function

  • Prey Instinct is only thing working (biting attacks, control locomotion during pursuit, craniofacial and locomotor modules during goal-directed behavior)
  • Otherwise brain is in a persistent coma
  •  Hyper-aggression due to dysfunctional anterior cingulate cortex (unable to modulate feelings of anger)
  • Damaged/dysfunctional/destroyed Ventromedial Hypothalamus- turns ‘off’ the ability to feel like you’ve eaten enough (full).

 In humans called hyperphagia- a medical sign meaning excessive hunger and abnormally large intake of solids by mouth.

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Symptoms mimicking  
Viral Encephalitis
Naegleria fowleri
Rabies
Viral Encephalitis (including measles and rubella) 
 

Spoiler

 

An inflammation of the brain caused by a virus. 
An be triggered by microorganisms, bacteria, fungi and parasites
Also triggered by enteroviruses
herpes simplex virus
varicella zoster virus
Epstein-Barr virus
cytomegalovirus
adenovirus
rubella
measles
Murray Valley encephalitis (MVE) virus and Kunjin virus
Japanese encephalitis virus.

Virus moves to brain, body responds to protect, brain swells


Travels via blood or nerve cells to brain and through blood-brain barrier


Certain viruses prefer certain areas (herpes – temporal lobe by each ear)


 

Treatment for viral encephalitis

Spoiler

Various medicine for various symptoms (pain, vomiting, seizures, fever)
Antiviral medicine, intravenously
Fluids to prevent dehydration, but not too much as this can worsen cerebral oedema (swelling of the brain).
Medications or actions such as induced hypothermia for brain swelling

 


Naegleria fowleri

Spoiler

 

Humans become infected when water containing Naegleria fowleri enters the nose and the ameba migrates to the brain along the olfactory nerve, NOT from drinking water


Trophozoites can turn into a temporary, non-feeding, flagellated stage (10-16 µm in length) when stimulated by adverse environmental changes such as a reduced food source. They revert back to the trophozoite stage when favorable conditions return


Naegleria fowleri trophozoites are found in cerebrospinal fluid (CSF) and tissue, while flagellated forms are occasionally found in CSF


Cysts are not seen in brain tissue
When a cyst comes into contact with an inviting host, it sprouts tentacle-like pseudopods and turns into a form known as a trophozoite. Once it’s transformed, the trophozoite heads straight for the host’s central nervous system, following nerve fibers inward in search of the brain.
Once it’s burrowed into its host’s brain tissue — usually the olfactory bulbs — N. fowleri sprouts a “sucking apparatus” called an amoebostome and starts chowing down on juicy brain matter. As the amoeba divides, multiplies and moves inward, devouring brain cells as it goes, its hosts can go from uncomfortable to incoherent to unconscious in a matter of hours.


miltefosine has shown some promise in combination with some of these other drugs. Miltefosine has shown ameba-killing activity against free-living amebae, including Naegleria fowleri, in the laboratory. Miltefosine has also been used to successfully treat patients infected with Balamuthia and disseminated Acanthamoeba infection 

 


Rabies
 

Spoiler

 

But rabies viruses don’t just settle down anywhere in the brain, they specifically seek out the hippocampus, amygdala and hypothalamus, brain structures that play central roles in memory, fear and emotion.

And they don’t just devour brain cells indiscriminately, either; instead, they alter the ways these cells release neurotransmitters like serotonin, GABA, and endogenous opioids. In other words, they turn their hosts’ own brain chemistry against them.

In the altered states brought on by a rabies infection, animals often lash out at any nearby living thing, but this may be more out of fear than anger. Human rabies patients become terrified of water and puffs of air, both of which make them flinch and twitch uncontrollably.

To date, fewer than 10 people have survived a clinical-stage rabies infection — ever, in history. Many doctors consider the disease untreatable. The better news, though, is that it’s easily preventable with a vaccine.

 


 

Fighting back

Spoiler

 

In a turnabout, a biochemical self-destruct trigger found in many other types of cells appears to guard the lives of brain cells during an infection with West Nile virus
The self-destruct trigger, a protein called RIPK3 (pronounced rip-3), is better known for activating a certain type of cell death during infection or damaging events in other parts of the body. The death of infected cells in this manner is a protective mechanism that helps the body eliminate the infection.


During a West Nile virus infection, however, the activation of RIPK3 in brain cells doesn't cause them to die. That's because its signaling within the central nervous system is not the same as in cell types elsewhere in the body. Its brain-specific role implies that there are central nervous system functions for RIPK3 not observed in other tissues.


"RIPK3 acts as part of the milieu of signals that support anti-viral inflammation in the brain,"
RIPK3 responds to the presence of West Nile virus in the brain by placing an order for chemokines
Chemokines attract an influx of infection-fighting white blood cells
In a different cell type, such as a fibroblast, the entry of a West Nile virus would result in the cell initiating its own demise.
Too much interference with RIPK3 in the brain could make it prone to certain viral infections.
    

The rabies virus, transmitted largely through the bites of infected animals, has evolved over thousands of years to hijack nerve cells, which it uses to climb from infected muscle tissue into the brain. That allows it to bypass a major hurdle: the blood-brain barrier, a selective membrane that keeps out most pathogens that travel through the bloodstream. But the barrier also prevents treatments—like cancer drugs—from reaching infected cells, limiting options for patients.


Nanoparticle expert Yu Seok Youn and his team have engineered gold particles so that they have the same rodlike shape and size as the virus. The nanoparticle’s shape gives it more surface area than spherical particles, improving the surface protein’s ability to bind with receptors on nerve cells that serve as a gateway to the nervous system. The particles don’t carry any drugs, but the tiny gold rods readily absorb laser light, which heats them up and kills surrounding tissue.


To see whether these new particles could be effective against tumors, Youn and his team first injected them into the tail veins of four mice with brain tumors. The nanoparticles quickly traveled to the brain, where they accumulated near the tumor sites. The team then fired a near infrared laser at the nanoparticles, heating them to nearly 50°C. The light harmlessly passed through skin and bone, but heat from the gold particles radiated outward, effectively cooking nearby cancer cells.

The treatment greatly reduced the size of the tumors, Youn and his colleagues report this month in Advanced Materials.
In another experiment, the researchers used the same treatment on mice with tumor cells that had been injected into their flanks. Tumors on two of the mice disappeared after 7 days, whereas the other tumors shrank to about half their original size.


Still uncertain is just how the nanoparticles reached the tumor cells. Youn says they likely traveled the same path as the rabies virus—through the central nervous system.

 

 

Edited by RogueSolace

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[None of the following has been seen by anyone and if taken into game without my permission will be considered and pursued as metagaming.]


Rabies vaccine

 

Spoiler

http://www.who.int/vaccine_safety/initiative/tools/Rabies_Vaccine_rates_information_sheet.pdf
Made in a chicken egg (chicken fibroblast)
The virus is inactivated with ß-propiolactone



IMOVAX
 

Spoiler

Uses MRC-5 (human skin cells and virus is inactivated with beta-propiolactone.
Ingredients= human albumin (blood plasma protein), neomycin (antibiotic), phenol red indicator (PH levels)



RabAvert
 

Spoiler

Medium and process
o Chicken egg, synthetic cell culture medium (with added human albumin), gelatin, antibiotics, potassium glutamate (vitamin)




Haemaccel (a registered trademark) is a type of intravenous colloid used in the prevention or treatment of shock associated with reduction in effective circulating blood volume due to hemorrhage, loss of plasma (burns, peritonitis, pancreatitis, crush injuries), or loss of water and electrolytes from persistent vomiting and diarrhea. Haemaccel contains degraded gelatin.

 

Spoiler

Haemaccel (also marketed as Emagel in Italy, "Polygeline" in China and Solucel in Venezuela) was originally brought to market by Hoechst AG and later acquired by Piramal Enterprises.
Ingredients
o Chicken egg, synthetic cell culture medium (with added human albumin), gelatin, sodium EDTA (chemical), bovine serum, Antibiotics (chlortetracycline, neomycin), Antifungal (Amphotericin B), water for injection, haemaccel, centrifuged


IMOVAX® 3 RABIES
WISTAR RABIES VIRUS STRAIN PM-1503-3M
GROWN IN HUMAN DIPLOID CELL CULTURES


 

Spoiler

The Imovax® Rabies Vaccine produced by Sanofi Pasteur SA is a sterile, stable, freeze-dried suspension of rabies virus prepared from strain PM-1503-3M obtained from the Wistar Institute, Philadelphia, PA
The virus is harvested from infected human diploid cells, MRC-5 strain, concentrated by ultrafiltration and is inactivated by beta-propiolactone. One dose of reconstituted vaccine contains less than 100 mg human albumin, less than 150 mcg neomycin sulfate and 20 mcg of phenol red indicator. Beta-propiolactone, a residual component of the manufacturing process, is present in less than 50 parts per million.
The finished, freeze-dried vaccine is provided for intramuscular administration in a single dose vial containing no preservative. After reconstitution, immediately administer the full 1.0 mL amount of vaccine. If it cannot be administered promptly, discard.
The potency of one dose (1.0 mL) of Imovax Rabies vaccine is equal to or greater than 2.5 international units of rabies antigen.

Post-exposure immunization
Post-exposure efficacy of Imovax Rabies vaccine was successfully proven during clinical experience in Iran in which six 1.0 mL doses were given on days 0, 3, 7, 14, 30, and 90, in conjunction with antirabies serum. Forty-five persons severely bitten by rabid dogs and wolves received Imovax Rabies vaccine within hours of and up to 14 days after the bites. All individuals were fully protected against rabies.

Studies conducted by the United States Centers for Disease Control and Prevention (CDC) have shown that a regimen of 1 dose of Rabies Immune Globulin (RIG) and 5 doses of HDCV induced an excellent antibody response in all recipients. Of 511 persons bitten by proven rabid animals and so treated, none developed rabies.
Do not inject Imovax Rabies vaccine in the gluteal area as there have been reports of possible vaccine failure when the vaccine has been administered in this area.

For adults and older children, Imovax Rabies vaccine should be administered in the deltoid muscle. For infants and younger children, the anterolateral aspect of the thigh is also acceptable, depending on age and body mass (see DOSAGE AND ADMINISTRATION).

Type of exposure
Rabies is transmitted by introducing the virus into open cuts or wounds in skin or via mucous membranes. The likelihood of rabies infection varies with the nature and extent of exposure. Two categories of exposure should be considered, bite and nonbite

Bite: Any penetration of the skin by teeth.
Nonbite: Scratches, abrasions, open wounds, or mucous membranes contaminated with saliva or other potentially infectious material, such as brain tissue, from a rabid animal. Casual contact, such as petting a rabid animal, (without a bite or nonbite exposure as described above), does not constitute an exposure and is not an indication for prophylaxis. Rare reports of airborne rabies have been received from laboratory and bat-infested cave settings.
No human to human spread, except in organ transplant
Two suggested one via route of salivary contact (bite and kiss)


Pre-exposure prophylaxis is administered for several reasons. First, although pre-exposure vaccination does not eliminate the need for additional medical evaluation after a rabies exposure, it simplifies management by eliminating the need for Rabies Immune Globulin (RIG) and decreases the number of doses of vaccine needed. This is particularly important for persons at high risk for being exposed to rabies in areas where modern immunizing products might not be available or where cruder, less safe biologics might be used, placing the exposed person at increased risk for adverse events. Second, pre-exposure prophylaxis might offer partial immunity to persons whose post-exposure prophylaxis is delayed. Finally, pre-exposure prophylaxis might provide some protection to persons at risk for unrecognized exposures to rabies.
PRE-EXPOSURE RABIES PROPHYLAXIS GUIDE Pre-exposure prophylaxis consists of three 1.0 mL doses of Imovax Rabies vaccine administered intramuscularly, using a sterile needle and syringe, one injection per day on Days 0, 7, and 21 or 28. In adults and older children, the vaccine should be administered in the deltoid muscle. In infants and small children, the anterolateral aspect of the thigh may be preferable, depending on age and body mass.



PRECAUTIONS
 

Spoiler

• IN ADULTS AND CHILDREN THE VACCINE SHOULD BE INJECTED INTO THE 10 DELTOID MUSCLE. IN INFANTS AND SMALL CHILDREN, THE ANTEROLATERAL 11 ASPECT OF THE THIGH MAY BE PREFERABLE.
• When a person with a history of hypersensitivity must be given rabies vaccine, antihistamines may be given. Epinephrine (1:1000) and other appropriate agents should be readily available to counteract anaphylactic reactions, and the person should be carefully observed after immunization.
• While the concentration of antibiotics in each dose of vaccine is extremely small, persons with known hypersensitivity to any of these agents, or any other component of the vaccine, could manifest an allergic reaction. While the risk is small, it should be weighed in light of the potential risk of contracting rabies.

The package contains a vial of freeze-dried vaccine, a syringe containing 1.0 mL of diluent, a plunger for the syringe, and a sterile needle for reconstitution. Cleanse the vaccine vial stopper with a suitable germicide. Do not remove the stopper or the metal seal holding it in place. Attach the plunger and reconstitution needle to the syringe and reconstitute the freeze-dried vaccine by injecting the diluent into the vaccine vial. Gently swirl the contents until completely dissolved and withdraw the total contents of the vial into the syringe. Remove the reconstitution needle and discard. Attach a sterile needle of your choice that is suitable for intramuscular injection of your patient.
NOTE: The freeze-dried vaccine is creamy white to orange. After reconstitution, it is pink to red
Post-exposure dosage
Post-exposure dosage for previously unimmunized persons: Dose: Previously unvaccinated persons should receive 5 intramuscular doses (1 mL each) of Imovax Rabies vaccine, one dose immediately after exposure (Day 0) and one dose 3, 7, 14, and 28 days later.
RIG: Rabies immune globulin (RIG) 20 IU/kg on Day 0 in conjunction with the first vaccine dose. If possible, the full calculated dose of RIG should be used to infiltrate the wound(s). If it is not possible to do so, any remaining portion of the dose should be administered intramuscularly at a site different from the site used to administer the vaccine.
Because the antibody response following the recommended vaccination regimen with HDCV has been satisfactory, routine post-vaccination serologic testing is not recommended. Serologic testing is indicated in unusual circumstances, as when the patient is known to be immunosuppressed. Contact local or state health department or CDC for recommendations.


 


Post-exposure dosage for previously immunized persons:
 

Spoiler

When an immunized person who was vaccinated using the recommended pre-exposure regimen or a prior post-exposure regimen with a cell culture vaccine or who had previously demonstrated rabies antibody is exposed to rabies, that person should receive two intramuscular doses (1.0 mL 15 each) of Imovax Rabies vaccine, one dose immediately after the exposure and one dose 3 days 16 later. RIG should not be given in these cases. If the immune status of a previously vaccinated person who did not receive the recommended HDCV regimen is not known, full primary post-exposure antirabies treatment (RIG plus 5 doses of HDCV) may be necessary. In such cases, if antibody levels of greater than 1:5 dilution by a RFFIT can be demonstrated in a serum sample collected before vaccine is given, treatment can be discontinued after at least two doses of HDCV.

The freeze-dried vaccine is stable if stored in the refrigerator between 2°C and 8°C (35°F to 46°F). Do not freeze.


Human rabies immune globulin (HRIG) is administered only once, at the beginning of anti-rabies prophylaxis, to previously unvaccinated persons. This will provide immediate antibodies until the body can respond to the vaccine by actively producing antibodies of its own.




RabAvert, Rabies Vaccine, produced by Novartis Vaccines and Diagnostics GmbH

Spoiler

is a sterile freeze-dried vaccine obtained by growing the fixed-virus strain Flury LEP in primary cultures of chicken fibroblasts. The strain Flury LEP was obtained from American Type Culture Collection as the 59th egg passage. The growth medium for propagation of the virus is a synthetic cell culture medium with the addition of human albumin, polygeline (processed bovine gelatin) and antibiotics. The virus is inactivated with β-propiolactone, and further processed by zonal centrifugation in a sucrose density-gradient. The vaccine is lyophilized after addition of a stabilizer solution which consists of buffered polygeline and potassium glutamate. One dose of reconstituted vaccine contains less than 12 mg polygeline (processed bovine gelatin), less than 0.3 mg human serum albumin, 1 mg potassium glutamate and 0.3 mg sodium EDTA. Small quantities of bovine serum are used in the cell culture process. Minimal amounts of chicken protein may be present in the final product; ovalbumin content is less than 3 ng/dose (1 mL), based on ELISA Antibiotics (neomycin, chlortetracycline, amphotericin B) added during cell and virus propagation are largely removed during subsequent steps in the manufacturing process. In the final vaccine, neomycin is present at < 1 µg, chlortetracycline at < 20 ng, and amphotericin B at < 2 ng per dose. RabAvert is intended for intramuscular (IM) injection. The vaccine contains no preservative and should be used immediately after reconstitution with the supplied Sterile Diluent for RabAvert (Water For Injection).



 

Spoiler

RabAvert is a white, freeze-dried vaccine for reconstitution with the diluent prior to use; the reconstituted vaccine is a clear to slightly opaque, colorless suspension.


Postexposure Treatment
RabAvert, when used in the recommended postexposure WHO program of 5 to 6 IM injections of 1 mL (days 0, 3, 7, 14, 30, and one optionally on day 90) provided protective titers of neutralizing antibody (> 0.5 IU/mL) in 158/160 patients (8, 9, 13-16) within 14 days and in 215/216 patients by day 28 – 38. Of these, 203 were followed for at least 10 months. No case of rabies was observed (8, 9, 13-20)

No postexposure vaccine failures have occurred in the United States since cell culture vaccines have been routinely used (1). Failures have occurred abroad, almost always after deviation from the recommended postexposure treatment protocol (21-24). In two cases with bites to the face, treatment failed although no deviation from the recommended postexposure treatment protocol appeared to have occurred (25).

One newborn has received RabAvert on an immunization schedule of days 0, 3, 7, 14 and 30; the antibody concentration on day 37 was 2.34 IU/mL. There were no clinically significant adverse events.
Children and infants receive the same dose of 1 mL, given IM, as do adults.

The pre-exposure schedule for rabies vaccination is 3 doses, given at the following times:
Dose 1: As appropriate
Dose 2: 7 days after Dose 1
Dose 3: 21 days or 28 days after Dose 1
For laboratory workers and others who may be repeatedly exposed to rabies virus, periodic testing for immunity is recommended, and booster doses should be given as needed. (Testing or booster doses are not recommended for travelers.) Ask your doctor for details.

Vaccination after an exposure
Anyone who has been bitten by an animal, or who otherwise may have been exposed to rabies, should clean the wound and see a doctor immediately. The doctor will determine if they need to be vaccinated.
A person who is exposed and has never been vaccinated against rabies should get 4 doses of rabies vaccine – one dose right away, and additional doses on the 3rd, 7th, and 14th days. They should also get another shot called Rabies Immune Globulin at the same time as the first dose.
A person who has been previously vaccinated should get 2 doses of rabies vaccine – one right away and another on the 3rd day. Rabies Immune Globulin is not needed.


What are the risks from rabies vaccine?
A vaccine, like any medicine, is capable of causing serious problems, such as severe allergic reactions. The risk of a vaccine causing serious harm, or death, is extremely small. Serious problems from rabies vaccine are very rare.
Mild problems
soreness, redness, swelling, or itching where the shot was given (30% – 74%)
headache, nausea, abdominal pain, muscle aches, dizziness (5% – 40%)
Moderate problems
hives, pain in the joints, fever (about 6% of booster doses)
Other nervous system disorders, such as Guillain Barré syndrome (GBS), have been reported after rabies vaccine, but this happens so rarely that it is not known whether they are related to the vaccine.
NOTE: Several brands of rabies vaccine are available in the United States, and reactions may vary between brands. Your provider can give you more information about a particular brand.
Top of Page
What if there is a serious reaction?
What should I look for?
Look for anything that concerns you, such as signs of a severe allergic reaction, very high fever, or behavior changes.
Signs of a severe allergic reaction can include hives, swelling of the face and throat, difficulty breathing, a fast heartbeat, dizziness, and weakness. These would start a few minutes to a few hours after the vaccination.



The Vaccines
 

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There are three main types of rabies vaccine, the outdated nerve tissue vaccines, cell culture vaccines, and embryonated egg vaccines. Cell culture vaccines and embryonated egg vaccines have replaced nerve tissue vaccines in industrialized countries and are the ones recommended for use by WHO. They are considered safe and well tolerated (Briggs et al., 2000, WHO, 2010 B). In comparison, nerve tissue vaccines can induce severe adverse reactions including a potential risk of rabies from incomplete virus inactivation (Plotkin, 2008) and are less immunogenic. They are still used in a limited and decreasing number of developing countries.


Nerve tissue vaccines
 

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Semple rabies vaccine contains a phenol or ß-propiolactone-inactivated homogenate of rabies virus-infected goat or sheep brain tissue. The vaccine contains myelin basic protein and is used in an ever decreasing number of countries in Asia and Africa.
Fuenzalida rabies vaccine is prepared from suckling mouse brain tissue and has decreased myelin content (Nogueira, 1988). This vaccine is used in a small number of Latin American countries.


Cell Culture Vaccines and Embryonated egg-based vaccines (CCEEV)
Cell Culture Vaccines (CCV)

 

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Human diploid cell vaccine (HDCV) contains the Pitman-Moore L503 or Flury strain of rabies virus grown on MRC-5 human diploid cell culture, concentrated by ultrafiltration and inactivated with ß-propiolactone (Wiktor et al., 1980). This vaccine is licensed for intra-muscular use. It contains no preservative or stabilizer
Purified chick embryo cell vaccine (PCECV) is a sterile lyophilized vaccine obtained by growing the fixed rabies virus strain Flury LEP-25 in primary cultures of chick fibroblasts. The virus is inactivated with ß-propiolactone, purified and concentrated by zonal centrifugation (CDC, 1998; Dreesen, 1997).
Purified Vero cell rabies vaccine (PVRV) contains inactivated and lyophilized Wistar strain of rabies virus grown on Vero cell cultures in fermenters allowing mass cultivation. These are inactivated by ß-propriolactone and purified by ultracentrifugation (Jaiiaroensup et al., 1998).
Primary Hamster Kidney Cell vaccine (PHKCV) uses the Beijing strain and is inactivated with formalin and adsorbed to aluminum hydroxide. It also contains 0.01% thiomersal and 10 mg human albumin.
Embryonated egg-based vaccines (EEV)
Purified duck embryo vaccine (PDEV) uses duck embryo cells as substrate. These are inactivated by ß-propiolactone and purified by ultracentrifugation. PDEV contains thiomersal.

 

 

Edited by RogueSolace

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• 2-Phenoxyethanol

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is a glycol ether used as a preservative in vaccines.



• Aluminum

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is used in vaccines as an adjuvant, which helps the vaccine work more quickly and more powerfully. •


• Bovine casein-

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A casein is a family of phosphoproteins commonly found in mammalian milk. 80% of the proteins in cow's milk are casein.



• Bovine serum-

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Bovine "erum is the centrifuged fluid component of either clotted or defibrinated whole blood. Bovine serum comes from blood taken from domestic cattle. Serum from other animals is also collected and processed but bovine serum is processed in the greatest volume."
"Bovine serum is a by-product of the meat industry. Bovine blood may be taken at the time of slaughter, from adult cattle, calves, very young calves or (when cows that are slaughtered are subsequently found to be pregnant) from bovine fetuses. It is also obtained from what are called 'donor' animals, which give blood more than once.
Blood is available from bovine fetuses only because a proportion of female animals that are slaughtered for meat for human consumption are found (often unexpectedly) to be pregnant.
Blood is available from very young calves because calves, especially males from dairy breeds, are often slaughtered soon, but not necessarily immediately, after birth because raising them will not be economically beneficial. Older animals are, of course, slaughtered for meat.
Only donor cattle are raised for the purpose of blood donation. Donor cattle are invariably kept in specialized, controlled herds. Blood is taken from these animals in a very similar way to that used for human blood donation.
Irrespective of whether blood is taken at slaughter or from donors, the age of the animal is an important consideration because it impacts the characteristics of the serum.
Bovine serum is categorised according to the age of the animal from which the blood was
collected as follows:
•'Fetal bovine serum' comes from fetuses
•'Newborn calf serum' comes from calves less than three weeks old
•'Calf serum' comes from calves aged between three weeks and 12 months
•'Adult bovine serum' comes from cattle older than 12 months
Serum processed from donor blood is termed 'donor bovine serum'. Donor animals can be up to three years old."



• Chicken Eggs-

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Viruses can be grown in chicken eggs before being used in vaccinations



• Dulbecco's Modified Eagle's Serum-

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glucose, sodium bicarbonate, L-glutamine, pyridoxine HCl, pyridocal HCl, folic acid, phenol red, HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), L-methionine, L-cystine, sodium phosphate mono-basic, sodium pyruvate, vitamins

o DMEM (Dulbecco's Modified Eagle's medium) ... Among the most widely used of these modifications is Dulbecco's Modified Eagle's medium (DMEM). DMEM is a modification of Basal Medium Eagle (BME) that contains a four-fold higher concentration of amino acids and vitamins, as well as additional supplementary components.

o Many modifications of Eagle's Medium have been developed since the original formulation appeared in the literature. Among the most widely used of these modifications is Dulbecco's Modified Eagle's medium (DMEM). DMEM is a modification of Basal Medium Eagle (BME) that contains a four-fold higher concentration of amino acids and vitamins, as well as additional supplementary components. The original DMEM formula contains 1000 mg/L of glucose and was first reported for culturing embryonic mouse cells. A further alteration with 4500 mg/L glucose has proved to be optimal for cultivation of certain cell types

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.

• Earle's Balanced Salt Medium-

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inorganic salts, D-glucose, phenol red, calcium, magnesium salts

o Earle’s salts are designed for the short–term maintenance of cells in a CO2 environment.  GIBCO® EBSS is manufactured in our state-of-the-art cGMP, ISO certified facilities, so you can rely on the highest quality and consistency.

o Although there have been many modifications to the original formulas in efforts to produce fully defined media, salt solutions still play an important role in tissue culture. A salt solution's basic function, to maintain the pH and osmotic balance in the medium and to provide the cells with water and essential inorganic ions, is as valuable today as when it was first developed a century ago. EARLE'S BALANCED SALTS [EBSS], Product No. E6132 is one of the balanced salts available from Sigma. The selection of a balanced salt is strongly influenced by 1] type of cell, 2] type of culture [monolayer, suspension, clonal] and 3] degree of chemical definition necessary. It is important to review the literature for recommendations concerning medium, supplementation and physiological parameters required for a specific cell line.



• Fenton Medium-

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bouvine extract



• Formaldehyde-

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Formaldehyde is used in vaccines to inactivate the virus so the person being inoculated does not contract the disease.



• Human albumin-

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Human albumin is a blood plasma protein produced in the liver that, among other functions, transports hormones, fatty acids, and other compounds, and buffers pH.



• Insect Cells-

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Cabbage moth and fall armyworm cells are used to grow viruses for vaccines.


• Latham Medium-

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bovine casein (protein)



• MRC-5-

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Medical Research Council 5, human diploid cells (cells containing two sets of chromosomes) derived from the normal lung tissues of a 14-week-old male fetus aborted for "psychiatric reasons" in 1966 in the United Kingdom, Eagle's Basal Medium in Earle's balanced salt solution with bovine serum.



• Mueller Hinton Agar-

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beef extract, acid hydrolysate of casein, starch, agar



• Mueller-Miller Medium-

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glucose, sodium chloride, sodium phosphate dibasic, monopotassium, phosphate, magnesium sulfate hydrate, ferrous sulfate heptaphydrate, cystine hydrochloride, tyrosine hydrochloride, urasil hydrochloride, Ca-pantothenate in ethanol, thiamine in ethanol, pyridoxin-hydrochloride in ethanol, riboflavin in ethanol, biotin in ethanol, sodium hydroxide, beef heart infusion (de-fatted beef heart and distilled water), casein solution



• Polysorbate 80-

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Also called Tween 80, Alkest 80, or Canarcel 80 (brand names). Polysorbate 80 is used as an excipient (something to basically thicken a vaccine for proper dosing) and an emulsifier (something to bond the ingredients).



• Stainer-Scholte Liquid Medium-

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tris hydrochloride, tris base, glutamate (monosodium salt), proline, salt, monopotassium phosphate, potassium chloride, magnesium chloride, calcium chloride, ferrous sulfate, ascorbic acid, niacin, glutathione



• Thimerosal-

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Thimerosal is an organomercury compound used as a preservative.

• WI-38 human diploid cells-

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Winstar Institute 38, human diploid lung fibroblasts derived from the lung tissues of a female fetus aborted because the family felt they had too many children in 1964 in the United States


• Phenol Red-

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Most living tissues prosper at a near-neutral pH; that is, a pH close to 7. The pH of blood ranges from 7.35 to 7.45, for instance. When cells are grown in tissue culture, the medium in which they grow is held close to this physiological pH. A small amount of phenol red added to this growth medium will have a pink-red color under normal conditions. Typically, 15 mg/l are used for cell culture.


In the event of problems, waste products produced by dying cells or overgrowth of contaminants will cause a change in pH, leading to a change in indicator color. For example, a culture of relatively slowly dividing mammalian cells can be quickly overgrown by bacterial contamination. This usually results in an acidification of the medium, turning it yellow. Many biologists find this a convenient way to rapidly check on the health of tissue cultures. In addition, the waste products produced by the mammalian cells themselves will slowly decrease the pH, gradually turning the solution orange and then yellow. This color change is an indication that even in the absence of contamination, the medium needs to be replaced (generally, this should be done before the medium has turned completely orange).


Since the color of phenol red can interfere with some spectrophotometric and fluorescent assays, many types of tissue culture media are also available without phenol red.

 




https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1635529/
Dynamics of recurrent viral infection

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Viruses are obligate intracellular parasites that rely on endless, sequential transmission from host to host for continued existence. Animal viruses have adopted two life styles to ensure this continuity: acute/highly contagious or persistent infections (Villarreal et al. 2000). The spread of acutely infectious viruses, such as measles, Ebola or influenza virus, depends on efficient and rapid transmission during the short window of time before the host immune responses clear the infection or before the virus kills its host. Other viruses, including herpesviruses, human and simian immunodeficiency viruses (HIV), hepatitis viruses and human papillomaviruses, establish persistent, long-lasting infections that allow viral spread during intermittent bouts of activity occurring throughout the host's life. For these viruses, primary infection is followed by the establishment of the virus in either a chronic or latent state.
In both chronic and latent infection, long periods of relative quiescence are typically punctuated by short bursts of high viral production and release. In latent infection, no viral replication can be observed during quiescent periods, but the viral genome is preserved in a completely or partially silent state within infected cells. This non-replicative state can periodically be reversed, with production of new virus occurring during reactivation episodes. In chronic viral infection, in contrast, viral replication and infectious particle production are continually maintained. For many such infections, recurrent episodes of high viral production and release interrupt relatively long periods of low-level viral replication. We note, however, that recurrent episodes are not common to all chronic infections (HIV, for example). Likewise, although most persistent viruses can easily be classified as chronic or latent, some viruses simultaneously use both strategies. Hepatitis B virus, for instance, can maintain low levels of replication in hepatocytes, but be truly latent in peripheral blood lymphocytes or bone marrow cells.

In figure 3a, we see that T sensitively depends on the efficacy of the antibody response, k. As the antibody strength increases, so does the time between recurrences.
In figure 3b, we find that the maximum number of infected cells during the recurrent episode, ymax, also increases with antibody efficacy when k is small. For larger values of k, the peak number of infected cells cannot increase further, but this peak broadens, as illustrated by the insets. Since the lifetime of infected cells has not increased, a broader peak implies that more cells in total are infected during each recurrent episode.
Thus, if the antibody response is strong, recurrent infections are less frequent but more severe.

Herpes
Herpes viruses establish lifelong infections, and the virus cannot yet be eradicated from the body. Treatment usually involves general-purpose antiviral drugs that interfere with viral replication, reduce the physical severity of outbreak-associated lesions, and lower the chance of transmission to others. Studies of vulnerable patient populations have indicated that daily use of antivirals such as aciclovir[42] and valaciclovir can reduce reactivation rates.

Multiplicity reactivation[edit]
Multiplicity reactivation (MR) is the process by which viral genomes containing inactivating damage interact within an infected cell to form a viable viral genome. MR was originally discovered with the bacterial virus bacteriophage T4, but was subsequently also found with pathogenic viruses including influenza virus, HIV-1, adenovirus simian virus 40, vaccinia virus, reovirus, poliovirus and herpes simplex virus.[49]
When HSV particles are exposed to doses of a DNA damaging agent that would be lethal in single infections, but are then allowed to undergo multiple infection (i.e. two or more viruses per host cell), MR is observed. Enhanced survival of HSV-1 due to MR occurs upon exposure to different DNA damaging agents, including methyl methanesulfonate,[50] trimethylpsoralen (which causes inter-strand DNA cross-links),[51][52] and UV light.[53] After treatment of genetically marked HSV with trimethylpsoralen, recombination between the marked viruses increases, suggesting that trimethylpsoralen damage stimulates recombination.[51] MR of HSV appears to partially depend on the host cell recombinational repair machinery since skin fibroblast cells defective in a component of this machinery (i.e. cells from Bloom’s syndrome patients) are deficient in MR.[53] These observations suggest that MR in HSV infections involves genetic recombination between damaged viral genomes resulting in production of viable progeny viruses. HSV-1, upon infecting host cells, induces inflammation and oxidative stress.[54] Thus it appears that the HSV genome may be subjected to oxidative DNA damage during infection, and that MR may enhance viral survival and virulence under these conditions.


HSV-1 and -2 persist in the body by becoming latent and hiding from the immune system in the cell bodies of neurons. After the initial or primary infection, some infected people experience sporadic episodes of viral reactivation or outbreaks. In an outbreak, the virus in a nerve cell becomes active and is transported via the neuron's axon to the skin, where virus replication and shedding occur and cause new sores.

Haemaccel

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(a registered trademark) is a type of intravenous colloid used in the prevention or treatment of shock associated with reduction in effective circulating blood volume due to hemorrhage, loss of plasma (burns, peritonitis, pancreatitis, crush injuries), or loss of water and electrolytes from persistent vomiting and diarrhea. Haemaccel contains degraded gelatin.

Haemaccel (also marketed as Emagel in Italy, "Polygeline" in China and Solucel in Venezuela) was originally brought to market by Hoechst AG and later acquired by Piramal Enterprises.
• Ingredients
o Chicken egg, synthetic cell culture medium (with added human albumin), gelatin, sodium EDTA (chemical), bovine serum, Antibiotics (chlortetracycline, neomycin), Antifungal (Amphotericin B), water for injection, haemaccel, centrifuged

 

Edited by RogueSolace

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