A blood test for prion disease

PCMA for prionsA 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.

A case of prion disease acquired from contaminated beef

prion conversionSpongiform 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.

Resistance to prion disease in humans

prion conversionTransmissible 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.

Is chronic wasting disease a threat to humans?

Areas with CWDChronic wasting disease (CWD) is a prion disease of cervids (deer, elk, moose). It was first detected in Wyoming and Colorado, and has since spread rapidly throughout North America (illustrated; image credit). Because prions that cause bovine spongiform encephalopathy (BSE, mad cow disease) are known to infect humans, there is concern that CWD might also cross the species barrier and cause a novel spongiform encephalopathy. Recent experimental results suggest that CWD prions are not likely to directly infect humans.

The prion protein PrPC is encoded by the prnp gene, which is essential for the pathogenesis of transmissible spongiform encephalopathies (TSEs). Transgenic mice have been used to understand the species barrier to prion transmission. When mice are inoculated with human prions, few animals develop disease and the incubation periods are over 500 days. When the mouse prnp gene is replaced with the human gene, the mice become uniformly susceptible to infection with human prions and the incubation period is shorter. The species barrier to prion transmission is therefore associated with differences in the prion protein sequence between host and target species.

Mice have been used to understand whether CWD prions might be transmitted to humans. Mice are not efficiently infected with CWD prions unless they are made transgenic for the cervid prnp gene. Four different research groups have found that  mice transgenic for the human prnp gene are not infected by CWD prions. These findings suggest that CWD prions are not likely to be transmitted directly to humans. However, changing four amino acids in human prnp to the cervid sequence allows efficient infection of transgenic mice with cervid prions.

Another concern is that prions of chronic wasting disease could be transmitted to cows grazing in pastures contaminated by cervids. Prions can be detected in deer saliva and feces, and contamination of grass could pass the agent on to cows. In the laboratory, brain homogenates from infected deer can transmit the disease to cows. Therefore it is possible that cervid prions could enter the human food chain through cows.

A further worry is that BSE prions shed by cows in pastures might infect cervids, which would then become a reservoir of the agent. BSE prions do not infect mice that are transgenic for the cervid prnp gene. However, intracerebral inoculation of deer with BSE prions causes neurological disease,  and the prions from these animals can infect mice that are transgenic for the cervid prnp gene. Therefore caution must be used when using transgenic mice to predict the abilities of prions to cross species barriers.

Although the risk of human infection with CWD prions appears to be low, hunters should not shoot or consume an elk or deer that is acting abnormally or appears to be sick, to avoid the brain and spinal cord when field dressing game, and not to consume brain, spinal cord, eyes, spleen, or lymph nodes. No case of transmission of chronic wasting disease prions to deer hunters has yet been reported.

TWiV 299: Rocky Mountain virology

On episode #299 of the science show This Week in VirologyVincent visits the Rocky Mountain Laboratories in Hamilton, Montana and speaks with Marshall Bloom, Sonja Best, and Byron Caughey about their work on tick-born flaviviruses, innate immunity, and prion diseases.

You can find TWiV #299 at www.microbe.tv/twiv.

A mad cow in America

spongiform encephalopathyA dairy cow in California is the fourth known American case of mad cow disease, which is caused by prions, infectious agents composed only of protein (the story hit the press the day after my lecture on this type of illness). Unlike viruses, prions have no nucleic acid and no protective coat. But virologists know all about them because, as Stanley Prusiner once said, there was a time when only virologists believed that they existed.

Prions are found in mammals and in fungi, but only in mammals are they infectious and pathogenic. All mammals make normal forms of the prion protein (PrPc) which is found in many tissues including the nervous system. The pathogenic form, called PrPSc, is a structurally altered form of PrPc. The PrPSc protein, named after the first prion disease studied, scrapie in sheep, causes PrPc to undergo a structural transformation to the pathogenic form. The PrPSc protein becomes deposited in amyloid fibrils in the brain, leading to neurodegenerative diseases known as transmissible spongiform encephalopathies (TSE), after the sponge-like appearance of the brain observed in afflicted animals (image).

There are three different ways to acquire a TSE. One is by infection: a human consumes meat that contains PrPSc, or receives a corneal transplant from a donor with an undiagnosed TSE . The PrPSc proteins make their way to the brain where they cause the host’s PrPc to misfold and become the pathogenic PrPSc. The more PrPSc that is made, the more the normal PrPc is converted to the pathogenic form. After an incubation period of many years, the host develops an invariably fatal neurodegenerative disease characterized by dementia in humans. There is also a familial form, in which mutations in the gene encoding PrPc are inherited; these cause the PrPc protein to misfold to form the pathogenic form. In the sporadic form PrPc spontaneously converts to PrPSc without any known mutation or infection.

TSEs occur in different forms with varied symptoms and pathology. There are TSEs of humans (Creutzfeld-Jacob disease, fatal familial insomnia, Gerstmann-­Sträussler syndrome, Kuru) cows (bovine spongiform encephalopathy or mad cow disease), sheep and goats (scrapie), deer, elk, and moose (chronic wasting disease), and of a variety of other mammals.

This brings us back to the mad American cow, the first in the US since 2006. It died on a dairy farm and was tested for BSE as are 40,00o other cows each year in this country. The reason why this is big news is that back in the 1990s there was an outbreak of human TSE in the United Kingdom caused by consuming beef from animals with BSE. The cows acquired BSE by being fed processed animal byproducts as protein supplements, which unknowingly contained pathogenic prions. Bt the time the disease was detected in cows, contaminated meat had already entered the human food chain. Cows are routinely tested for BSE precisely to avoid a similar outbreak of human TSE.

The dead cow apparently had atypical BSE – that is, it was not a consequence of eating contaminated meat and it was not an inherited disease. Atypical BSE is caused by strains of prions distinct from other forms. This is good news because it means that the feed that the cow was receiving was not contaminated with pathogenic prions. Furthermore, the cow was not destined for meat production; it was a dairy cow that had died and was selected for random sampling.

Could the milk produced by this cow and consumed by humans pose a risk for transmission of a TSE to humans? It is known that ewes with scrapie shed infectious and pathogenic prions in their milk. However cows with BSE have  much less PrPSc accumulation in peripheral tissues, and in particular lymphoid tissues which include the mammary glands. It seems unlikely that cow milk contains prions, but it is a question worth revisiting. Pathogenic prions are highly resistant to heat, ultraviolet irradiation and other extreme conditions, so would certainly survive the pasteurization process.

Virology lecture #24: Unusual infectious agents

Download: .wmv (350 MB) | .mp4 (96 MB)

Visit the virology W3310 home page for a complete list of course resources.