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About viruses and viral disease
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.
You can find TWiV #424 at microbe.tv/twiv, or listen below.
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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.
Prions are not viruses – they are infectious proteins that lack nucleic acids. Nevertheless, virologists have always been fascinated by prions - they appear in virology textbooks (where else would you put them?) and are taught in virology classes. I’ve written about prions on this blog (five articles, to be exact – look under P in the Table of Contents) and I’m fascinated by their biology and transmission. That’s why the newly solved structure of an infectious prion protein is the topic of the sixth prion article at virology blog.
Spongiform encephalopathies are neurodegenerative diseases caused by misfolding of normal cellular prion proteins. Human spongiform encephalopathies are placed into three groups: infectious, familial or genetic, and sporadic, distinguished by how the disease is acquired initially. In all cases, the pathogenic protein is the host-encoded PrPC protein with an altered conformation, called PrPsc. In the simplest case, PrPSc converts normal PrPC protein into more copies of the pathogenic form (illustrated).
The structure of the normal PrPC protein, solved some time ago, revealed that it is largely alpha-helical with little beta-strand content. The structure of PrPSc protein has been elusive, because it forms aggregates and amyloid fibrils. It has been suggested that the PrPSc protein has more beta-strand content than the normal protein, but how this property would lead to prion replication was unknown. Clearly solving the structure of prion protein was needed to fully understand the biology of this unusual pathogen.
The structure of PrPSc protein has now been solved by cryo-electron microscopy and image reconstruction (link to paper). The protein was purified from transgenic mice programmed to produce a form of  PrPSc protein that is not anchored to the cell membrane, and which is also underglycosylated. The protein causes disease in mice but is more homogeneous and forms fibrillar plaques, allowing gentler purification methods.
The structure of this form of the PrPSc protein reveals that it consists of two intertwined fibrils (red in the image) which most likely consist of a series of repeated beta-strands, or rungs, called a beta-solenoid. The structure provides clues about how a pathogenic prion protein converts a normal PrPC into PrPSc . The upper and lower rungs of beta-solenoids are likely the initiation points for hydrogen-bonding with new PrPC molecules – in many proteins with beta-solenoids, they are blocked to prevent propagation of beta-sheets. Once added to the fibrils, the ends would serve to recruit additional proteins, and the chain lengthens.
The authors note that the molecular interactions that control prion templating, including hydrogen-bonding, charge and hydrophobic interactions, aromatic stacking, and steric constraints, also play roles in DNA replication.
The structure of PrPSc protein provides a mechanism for prion replication by incorporation of additional molecules into a growing beta-solenoid. I wonder if incorporation into fibrils is the sole driving force for converting PrPCprotein into PrPSc, or if PrPC is conformationally altered before it ever encounters a growing fibril.
Here is a follow-up to last week’s article that described a case of variant Creutzfeldt-Jacob disease in a Texas resident caused by ingestion of BSE-contaminated beef 14 years ago.
A 59 year old male patient was admitted to the trauma unit in Lancaster, PA with a self-inflicted gunshot wound to the head. There was substantial bleeding and brain tissue extrusion from the bullet exit wound. While the patient was intubated, examination of his electronic health records revealed a previous diagnosis of Creutzfeldt-Jacob disease (CJD). After discussion with his family, the breathing tube was removed and the patient expired.
After discovering that the patient had CJD, TSE (transmissible spongiform encephalopathy) decontamination protocols were initiated. Equipment and surfaces that had been exposed to highly infectious brain tissues were identified. Because prions are extremely difficult to destroy, it was decided to incinerate many pieces of equipment costing tens of thousands of dollars. This decision was taken to protect workers in the trauma unit and future hospital patients from hospital-acquired CJD.
The usual sterilization conditions (121 degrees Celsius for 20 minutes under high pressure) do not destroy prion protein infectivity. Consequently the World Health Organization recommends incineration of potentially contaminated materials. While environmental transmission of prion diseases has not been reported, WHO suggests rinsing surfaces with sodium hydroxide or sodium hypochlorite for 1 hour, followed by flooding with water, to remove prions.
This case illustrates the problems associated with an unusual infectious agent, the prion, that is difficult to inactivate. It also shows the value of electronic health records. Without such readily accessible information, the discovery that the patient had CJD would have been substantially delayed, leading to further contamination.
Creutzfeldt-Jacob associated deaths have increased slowly but steadily in the US since 1979. The number of cases will likely continue to increase until early diagnosis tests become routinely available, and drugs are developed that can cure the disease.
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.
On episode #343 of the science show This Week in Virology, the TWiVerinoes discuss the potential for prion spread by plants, global circulation patterns of influenza virus, and the roles of Argonautes and a viral protein in RNA silencing in plants.
You can find TWiV #343 at www.microbe.tv/twiv.