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From the European Society for Clinical Virology 2022 Conference in Manchester UK, Vincent speaks with Richard Knight about prion diseases and the outbreak of bovine spongiform encephalitis that led to cases of variant Creutzfeldt-Jakob disease in humans.
Host: Vincent Racaniello
Guest: Richard Knight
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Trudy joins the the TWiVlords to discuss new tests for detecting prions in the blood, and evidence showing that foamy retroviruses originated in the seas with their jawed vertebrate hosts at least 450 million years ago.
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A sensitive and specific blood test has been developed that could be used to limit the risk of transmission of prion disease through the blood supply (link to papers one and two).
Prion diseases, also known as spongiform encephalopathies, are uniformly fatal, chronic degenerative neurological diseases caused by misfolding of a cellular protein, PrPC. Transmissible encephalopathies may be acquired by organ transplant, receiving contaminated blood, or the ingestion of contaminated food.
In the 1990s a new spongiform encephalopathy, variant Creutzfeld-Jakob disease or vCJD, began to appear in Great Britain. Variant Creutzfeld-Jakob disease is caused by prions acquired by the consumption of cattle with bovine spongiform encephalopathy, also a prion disease affectionately known as mad cow disease. To date 231 cases of vCJD have been reported, mainly in the UK and France.
Although the spread of BSE has been controlled by surveillance and feeding restrictions, it is estimated that millions of people were exposed to BSE prions. The concern is that some of these individuals might be infected but show no symptoms of disease. If they donate blood, they may transmit infection to others. It is known that several cases of vCJD have been transmitted from infected blood donors, so further transmission is a major concern. So far prion diseases have only been diagnosed after death, by detection of conformationally altered prion proteins in the brain.
Two sensitive and specific assays for vCJD prions have now been developed that show promise for non-invasive pre-symptomatic diagnosis of the disease. They are both based on a technology called protein misfolding cyclic amplification (PMCA, illustrated; image copyright ASM Press, 2015). A small amount of the normal human prion protein, PrPC (produced in transgenic mice) is mixed with plasma. The samples are incubated to allow formation of prion oligomers, followed by disruption by a pulse of sonication to disrupt the oligomers. The cycle is repeated multiple times, much like polymerase chain reaction (PCR) which is used to amplify small amounts of DNA. Prions are detected by western blot analysis after treatment with proteinase K. The misfolded, pathogenic prions, PrPSC , are not completely digested with this enzyme.
In one study, PMCA was used to analyze blood samples from 14 cases of vCJD and 153 controls, which included healthy individuals and those with other neurological diseases, including sporadic CJD (sCJD – not caused by ingestion of contaminated beef). All 14 samples from cases of vCJD were positive in the PMCA assay, but not any of the other samples.
In a second study, the PMCA assay was positive in samples from all 18 patients with vCJD. Of 134 control samples, just one was positive for vCDJ, from a patient with sCJD. Furthermore, the assay detected vCJD prions in archived blood samples from donors who gave blood before developing symptoms of the disease.
These findings suggest that the new assays can detect vCJD prions in the blood before the appearance of the neurological symptoms of spongiform encephalopathy. While additional samples must be analyzed to validate the results, they are nonetheless promising as a way to prevent spread of the disease via the blood supply. Unfortunately, if you are diagnosed with vCJD by one of these assays, that is the only positive outcome – there are as yet no treatments for any spongiform encephalopathy.
Spongiform encephalopathies are neurodegenerative diseases caused by misfolding of normal cellular prion proteins. A 2014 case of variant Creutzfeldt-Jacob prion disease in the United States was probably caused by eating beef from animals with bovine spongiform encephalopathy (BSE), or mad cow disease.
Human spongiform encephalopathies are placed into three groups: infectious, familial or genetic, and sporadic, distinguished by how the disease is acquired initially. In the mid 1980s, a prion disease called bovine spongiform encephalopathy appeared in cows in the United Kingdom. It is believed to have been transmitted to cows by feeding them meat and bone meal, a high protein supplement prepared from the offal of sheep, cattle, pigs, and chicken. Some of the animals prepared for feed likely had a prion disease. Cases of variant Creutzfeld-Jakob disease, a new spongiform encephalopathy of humans, began to appear in 1994 in Great Britain. They were characterized by a lower mean age of the patients (26 years), longer duration of illness, and differences in other clinical and pathological characteristics. Variant Creutzfeldt-Jakob disease (vCJD) is caused by prions transmitted by the consumption of cattle with bovine spongiform encephalopathy.
In late 2012 a male Texas resident began showing symptoms of depression and anxiety, followed by delusions, hallucinations, and other changes in behavior. Over the next 18 months the patient’s condition deteriorated, leading to inability to ambulate or speak, and after several episodes of aspiration pneumonia and sepsis the patient died. During the illness prion disease was suspected, but tests for this condition were negative. After death, examination of brain biopsies revealed typical prion plaques, and misfolded prion proteins were found in urine, confirming the diagnosis of variant Creutzfeldt-Jacob disease.
The source of the patient’s prion disease was likely consumption of contaminated beef from cows with bovine spongiform encephalopathy. The patient probably acquired the infection in Russia, Lebanon, or Kuwait, three countries that had received BSE-contaminated beef from the UK, and and where he had previously lived. He resided in the US for 14 years before developing symptoms, an incubation period consistent with models of the vCJD epidemic.
This Texas patient is the fourth worldwide since 2012 to be diagnosed with vCJD; the others were from the UK and France. There are likely to be additional cases of vCJD in the future: surveys of archived appendix tissues in the UK show that 1 in 2,000 persons born during 1941-1985 have asymptomatic vCJD infection. These individuals could transmit the misfolded prion proteins to others via transplantation, blood transfusion, or surgical instruments (prion infectivity is not destroyed by autoclaving).
The good news is that vCJD is rare: there have been 230 reported cases of vCJD worldwide caused by consumption of BSE beef. The bad news is that vCJD will probably continue to appear in humans for many years, not only from the aftermath of the BSE epidemic. Rare cows spontaneously develop BSE, and because cattle are slaughtered before disease symptoms are evident, contaminated meat could enter the food supply.
Chronic wasting disease is a prion disease of cervids (deer, elk, moose) that is potentially a threat to human health. A role for environmental prion contamination in transmission is supported by the finding that plants can take up prions from the soil and transmit them to animals.
A concern is that prions of chronic wasting disease could be transmitted to cows grazing in pastures contaminated by cervids. Consumption of infected cows would then pass the disease on to humans. When deer are fed prions they excrete them in the feces before developing clinical signs of infection, and prions can also be detected in deer saliva. In the laboratory, brain homogenates from infected deer can transmit the disease to cows.
To determine whether prions can enter plants, wheat grass roots and leaves were exposed to brain homogenates from hamsters that had died of prion disease. The plant materials were then washed and amounts of prions were determined by protein misfolding cyclic amplification. Prions readily bound these plant tissues, at low concentrations and after as little as 2 minutes of incubation. Mouse, cervid, and human prions also bound to plant roots and leaves. When living wheat grass leaves were sprayed with a 1% hamster brain homogenate, prions could attach to the leaves and be detected for 49 days.
To determine if prions in plants could infect animals, plants were exposed to brain homogenates, washed thoroughly, and then fed to hamsters. The positive control for this experiment was to feed hamsters the brain homogenates. All animals fed infected plants or brain homogenates succumbed to prion disease.
Plants can also take up prions from animal waste. This conclusion was reached by incubating leaves and roots for 1 hour with urine or feces obtained from prion-infected hamsters or cervids. Prions were readily detected in these samples, even after extensive washing.
Experiments were also done to examine whether plants could take up prions from the soil. Barley grass plants were grown on soil that had been mixed with hamster brain homogenate, and then 1-3 weeks later, stem and leaves were assayed for the presence of prions. Small amounts of prions were detected in stems from all plants, while 1 in 4 plants contained prions in leaves, at levels that should be able to infect an animal.
These results show that prions can bind to plants and be taken into the roots, where they may travel to the stem and leaves. Therefore it is possible that prions excreted by deer could pass on to other animals, such as grazing cows, or even humans consuming contaminated plants (illustrated – image credit). Cooking plants will not eliminate infectivity, just as cooking contaminated beef did not halt the spread of bovine spongiform encephalopathy. Keeping cervids out of grazing or growing fields should be considered as a way to manage the risk of prions entering the human food chain.
Transmissible spongiform encephalopathies (TSEs) are rare human neurodegenerative disorders that are caused by infectious proteins called prions. A naturally occurring variant of the human prion has been found that completely protects against the disease.
A protective variant of the prion protein was discovered in the Fore people of Papua New Guinea. Beginning in the early 1900s, the prion disease kuru spread among Fore women and children as a result of ritual cannibalism of the brains of deceased relatives. When cannibalism stopped in the late 1950s, kuru disappeared.
Survivors of the kuru epidemic are heterozygous for a prion protein gene (prnp) with a unique amino acid change not seen in other populations, a change at position 127 from glycine to valine (G127V). The G127V change was always seen together with methionine at 129. Heterozygosity for M and V at amino acid 129, which is protective against prion disease, is found in humans all over the world.
Transgenic mice were used to determine if the G127V change in prion protein protects against disease. These mice lack the murine prnp gene (which encodes the normal prion protein) and contain a copy of either the wild type human prnp gene, or one with changes at amino acids 127 and 129. The mice were then inoculated intracerebrally with brain extracts from individuals who died of kuru. Mice with wild type human prnp were susceptible to infection. In contrast, transgenic mice heterozygous for the variant prnp (G127M129/V127M129) were completely resistant to infection. The mice were also resistant to infection with prions from cases of another human TSE, Creutzfeldt-Jacob disease.
Prnp transgenic mice were also challenged with variant Creutzfeldt-Jacob disease prions. This novel TSE arose after consumption of beef from animals with the prion disease bovine spongiform encephalopathy (BSE). These mice were susceptible to infection with vCJD prions, not a surprising result given that the Fore people were never exposed to BSE prions. However, mice homogygous for the altered prnp (V127M129/V127M129) were completely resistant to infection with vCJD prions – as resistant as mice with no prnp genes.
The protective effect of the M129V polymorphism is thought to be a consequence of inhibition of protein-protein interactions during prion propagation (i.e. the conversion of normal prion to pathogenic prion). How the G127V change confers protection is unknown.
These results show that the G127V change confers resistance to kuru and was likely selected as a consequence of the epidemic. If kuru had not been stopped by the abolition of cannibalism, it likely would have been self-limiting, as individuals with resistance to the disease, caused by the G127V change, repopulated the Fore people.