MICROBIOLOGY

Coronavirus, Slow Viruses and Prion Diseases

Dr. Palacpac
CORONAVIRUSES Coronavirus Infections
Structure and Classification Pathogenesis
 Enveloped  Limited knowledge
o Spike proteins resemble solar  Highly species-specific
corona or crown  Typically mild upper respiratory infections (“colds”) that remain
 120-160 nm localized
 Positive-strand RNA (27-32 kb) o Exception: SARS
 Cytoplasmic replication
 Budding into ER and Golgi Immunity is not durable
 Notoriously difficult to propagate in culture  Many people become resusceptible after a few years
 High frequency of recombination
 Cause colds and severe acute respiratory syndrome (SARS) Laboratory Diagnosis
 ELISA – may not discriminate past infections
Classification  HA
 Family Coronaviridae  PCR
o Genus Coronaviru  Virus isolation is difficult (often impossible) and requires great
o Genus Torovirus expertise

Replication 6 Coronaviruses that can infect Humans:

 Details are largely unknown because viruses are difficult to grow in
cell culture
 Mouse hepatitis virus is model for coronavirus replication
 Viral spike proteins mediate attachment
o Aminopeptidase N is a cell receptor target for many
coronaviruses
 Endocytosis is thought to mediate infection
 After uncoating, the viral genome (mRNA) is translated to produce
RNA polymerase
 Subgenomic RNAs are synthesized for each viral polypeptide
 Genomic RNA is cosynthesized with nucleocapsid
o Results in nucleocapsid binding immediately to genomic
RNA
 Progeny virus buds from ER and Golgi and are packaged into vesicles
 Vesicles travel to and fuse with plasma membrane, releasing viral
particles from cell
Corona Virus Replication
 Virus attaches to receptors on target cells by glycoprotein spikes on
viral envelope
 Receptor for human coronavirus 229E is aminopeptidase N
 Functional receptor for SARS-CoV is ACE 2
 Receptor for MERS-CoV is dipeptyl peptidase 4 aka CD26
Replication Cycle
 Virus absorbs to cells via its surface spikes (hemagglutinin)  enter
cytoplasm  uncoating
 Translation of genome occurs in 2 phases
a) Early phase - produces an RNA polymerase
b) Late phase - yields structural, non-structural protein
 Virus is assembled and obtain envelope from endoplasmic reticulum,
not plasma membrane
 Replication : cytoplasm
Transmission and Epidemiology
 MOT: respiratory droplets, contaminated surfaces, fomites
 Infection occus worldwide, occurs early in life outbreaks occur
primarily in winter
 Risk of transmission in health care setting, with documented hospital
outbreaks
 Alpha coronaviruses
o 229E
o NL63
 Beta coronaviruses
o HKU1
o OC43
o MERS-CoV
o SARS-CoV
Human coronavirus OC43 (HCoV-OC43), contain a 3rd glycoprotein (HE; 65 kDa)
that causes hemagglutination & has acetylesterase activity
Clinical Findings
 Human coronaviruses produce “common colds,” usually afebrile, in
adults
 Symptoms similar produced by rhinoviruses, nasal discharge, malaise
 IP : 2- 5 days, symptoms usually last 1 wk.
 Lower respiratory tract - seldom involved, pneumonia may occur.
 Clinical features of coronavirus-associated enteritis have not been
clearly described
o They appear to be similar to those of rotavirus infections
Novel coronaviruses have been identified as the cause of:
 Severe Acute Respiratory Syndrome (SARS)
 Middle East respiratory syndrome (MERS)
SARS-CoV
 Outbreak in 2003 characterized by : pneumonia respiratory failure
 Virus can also be detected in kidney, liver, SI, stool
 SARS virus probably originated in a nonhuman host, bats, amplified
in palm civets cats (intermediate host), transmitted to humans in
live animal markets
 Chinese horseShoe bats - natural reservoirs of SARS-like
coronaviruses
 Causes severe respiratory disease
 IP - about Six days
 Early symptoms fever, malaise, chills, headache, dizziness, cough,
sore throat, followed a few days later by SOB
 Death from progressive respiratory failure occurs in almost 10% of
cases, with the death rate highest among the elderly

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 MICROBIOLOGY
Coronavirus, Slow Viruses and Prion Diseases
Dr. Palacpac
 SARS involves a cytokine storm, with elevated levels of multiple MERS-CoV
chemokines and cytokines in peripheral circulation for about 2  Outbreak in 2012 characterized by pneumonia and respiratory
weeks failure
 Likely originated in bats, became widespread in caMels as shown by
Severe Acute Respiratory Syndrome (SARS) seropositivity in animals in region.
 Initial outbreak in SE Asia  It is likely that contact with either bats or camels leads to initial
 Hong Kong and Singapore first reported human infections, which can then be transmitted from person to
 Disease originated in China person.
 Originally thought to be from wild game markets  Causes mild to severe respiratory illness in children, adults.
o Palm civet cat (which isn’t a cat) - Paradoxurus o Px with coMorbidities are more severely affected, as
hermaphroditus elderly.
o Raccoon dog (which isn’t a dog) - Nyctereutes  IP: 2–13 days, with extended illness in some cases leading to
procyonoides pneumonia and death.
 It is a bat virus  Lab : leukopenia, lymphopenia, thrombocytopenia, elevated LDH
 Chinese horseshoe bats (Rhinolophus sinicus)
 No virus isolation Immunity
o Amplification of coronavirus RNA from anal swabs  Immunity develops but not absolute.
o Serology  Immunity against surface projection antigen is probably most
o It is highly-similar, but not identical to SARS-CoV important for protection.
o Mutations have most likely occurred in transmission from  Resistance to reinfection may last several years,
bats to civets to humans  Reinfections with similar strains - common.
o Reverse genetics of SARS-CoV and some bat viruses has
been done Laboratory Diagnosis
 No animal pathogenesis model  Coronavirus antigens in cells in respiratory secretions may be
detected using ELISA test if a high-quality antiserum is available.
Pathogenesis of SARS  Enteric coronaviruses can be detected by Examination of stool
 Virus is transmitted by samples by Electron Microscopy
respiratory and fecal routes  PCR assays - preferred method to detect coronavirus nucleic acid in
 Infection is mediated by respiratory secretions and in stool samples.
human angiotensin-  Viremia with SARS and MERS coronaviruses is detectable in plasma
converting enzyme 2 (hACE2) by PCR
receptor  Difficult virus isolation
 High expression  Serodiagnosis using acute and convalescent sera is one means of
o Lung alveolar epithelial cell confirming coronavirus infections for epidemiologic purposes
o Intestinal enterocytes H emagglutination tests
 Low expression E LISA
o Blood vessels (virtually all organs) I ndirect immunofluorescent antibody assays,
 Pneumonia
o Cause of death is lung failure Treatment
 No proven tx for coronavirus infections
 No vaccine
 Protease inhibitors used in tx of HIV infections (eg, lopinavir) have in
vitro activity against SARS coronavirus

Control Measures
 Q uarantine of those who had been exposed
 U se of gloves, gowns, goggles, masks by health care workers
 I solation of patients
 T ravel restrictions
 High suspicion for MERS-CoV in patients returning from Arabian
Peninsula
 Requires Appropriate testing, infection control precautions to
prevent further spread.

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 MICROBIOLOGY
Coronavirus, Slow Viruses and Prion Diseases
Dr. Palacpac
SLOW VIRUSES AND PRION DISEASES
 Some chronic degenerative diseases of the CNS in humans are caused
by “slow” or chronic, persistent infections by classic viruses
(Subacute sclerosing panencephalitis and Progressive multifocal
leukoencephalopathy)
 Transmissable spongiform encephalopathies ( ex:Creutzfeldt-Jakob
disease) are caused by unconventional transmissable agents termed
“prions”
 The progressive neurologic diseases produced by these agents may
have incubation periods of years before clinical manifestations of the
infections become evident The disorders Includes:
Subacute Sclerosing Panencephalitis (SSPE)
 Rare degenerative CNS dse characterized by behavioral and
intellectual deterioration and seizures that occurs 7-10 years after
a wild type measles virus infection
 Slowly progressive demyelination in the CNS ending in death
 Large numbers of viral nucelocapsid structures are produced in the
neurons and glial cells
 Restricted expression of viral genes that encodes envelop proteins
 The virus in persistently infected neural cells lacks proteins needed
for the production of infectious particle
 Patients with SSPE have high titers of antimeasles antibody except for
the antibody to the M protein
 Reduced efficiency of measles virus transcription in differentiated
brain cells- important in maintaining persistent infection that leads
to SSPE
Stages of SSPE:
1. Psycho-intellectual deterioration
2. Convulsive and motor changes
3. Coma
4. Death
Diagnosis:
 Clinically (Hx and neurologic manifestations)
 Laboratories: Serology: IgG levels measles serum and CSF
 EEG, MRI, Cranial CT-SCAN
 CSF studies
 Histopathology
Treatment and Prevention and Control:
• Widespread measles vaccine led to decrease significantly the
possibility of developing SSPE
• No treatment, usually fatal after a year of diagnosis
Progressive Multifocal Leukoencephalopathy
 Causative agent JC virus- member of a family Polyomaviridae
 CNS complication that occurs in some immunosuppressed individuals
 Incidence of 5% in patients with AIDS
 Anti-viral drugs slow progression of HIV infections, fewer patients
develop this disease
 Demyelination in the CNS of patients with progressive multifocal
leukoencephalopathy results from reactivation and replication of JC
virus when an immune system is compromised
Transmissible Spongiform Encephalopathies (Prion Dse)
 The causative agents are not conventional viruses
 Infectivity is associated with proteinaceous material devoid of
detectable amounts of nucleic acid
 The term “prion” is used to designate this novel class of agents
 TSE are slow progressive neurodegenerative diseases in humans and
animals
 Causes an outbreak in 1996 in UK affecting younger people (40 years
old)
Humans:
1. Kuru
2. Creutzfeldt Jakob Disease (CJD)
3. Variant CJD (VCJD)
4. Gertsmann-Straussler-Scheinker Syndrome
5. Fatal Familial Insomnia (FFI)
6. Sporadic Fatal Insomnia
Animals:
1. Scrapie (sheeps and goats)
2. Transmissible mink encephalopathy
3. Bovine Spongiform Encephalopathy (BSE)- Mad Cow Disease
4. Chronic wasting disease ( Mule, Deer, Elk)
Prion: PrPsc
 Filterable and can transmit disease but do not conform to the
standard definition of a virus
 No virion structures or genomes
 Not elicit immune response
 Resistant to inactivation by heat , disinfectants and radiations
 Sensitive to phenol(90%), household bleach, ether, NaOH, 10%
sodium dodecyl sulfate, Autoclaving
 Prion is a mutant or conformationally distinct form of a normal
cellular protein PrPс
 Small proteinaceous infectious particle which can transmit the
disease.
 Prion has long incubation period- months to decades
 Can cause damage to the CNS leading to a Subacute Spongiform
Encephalopathy
 Although the etiologic agents may be recoverable from other organs,
the diseases are confined to the nervous system.
 The diseases are always fatal with no known case of recovery.
 The host shows no inflammatory response and no immune responses
( The agents do not appear to be antigenic)
 No interferon is elicited
 No effect on T and B cell functions
 Immunosuppression of the host has no effect on pathogenesis.
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 MICROBIOLOGY
Coronavirus, Slow Viruses and Prion Diseases
Dr. Palacpac
 PrPsc is protease resistant, aggregates into amyloid rods (fibrils) and
is in extracellular
 Normal Prpc is a protease sensitive and appears on cell surface
 Genetic susceptibility to scrapie infection is associated with point
mutation in the PrPc gene

Pathogenic Characteristic of Prion:

Template Mediated Protein Refolding
 No cytopathologic effect in vitro.
 Theory on how an aberrant protein could cause disease
 Long doubling time (5.2 days)
 A linear aggregate of PrPsc binds to an anionic structure on the cell
 Long incubation period surface such as glycosaminoglycan and the normal PrPc  causing
 Cause vacuolation of neurons (spongiform)- amyloid-like plaques, the PrPc to Refold, acquire the structure of the PrPsc and join the
gliosis chain.
 Symptoms include: loose of muscle control (ataxia), shivering,  The alpha helical structure of the PrPc is changed to a more B-pleated
tremors, dementia sheet structure of the PrPsc.
 Lack of Antigenicity  When the chain of PrPsc breaks, it creates new primers in which
 Lack of inflammation more prions can be built.
 Lack of immune response  The PrPc is continued to be made by the cell as they bind to the PrPsc
 Lack of interferon production primers- cycle continues…
 The human version of PrPc is encoded on chromosome 20
Scrapie:
 The fact that these plaques consist of host protein may explain the
• Prototype of these agents
lack of an immune response to these agents in patients with the
• Can infect hamsters- scrapie infected hamsters have scrapie
SPONGIFORM ENCEPHALOPATHIES.
associated Fibrils in their brain
• Fibrils are infections and contain the prions
• Variable susceptibility in various breeds of sheep, whereas goats are
almost susceptible
• Infectivity can be recovered from lymphoid tissues early in infection,
and high titers of the agent are found in the brain, spinal cord, and
eye (where pathologic changes are seen)
• The disease is characterized by the development of amyloid plaques
in the CNS of infected animals
• Amyloid plaques are extracellular accumulation and can be stained
by Congo Red
• Prion lacks detectable nucleic acid and consist of aggregates of a
protease-resistant, hydrophobic glycoprotein PrPsc
• Human and animals encode a protein PrPc (cellular prion protein) of
unknown function that is held in the CM by linkage between its
terminal serine and a special lipid GLYCOPHOSPHATIDYLINOSITOL-
GPI-linked protein

PrPc and PrPsc

 The PrPc is identical to PrPsc in its protein sequence but differs in
tertiary structure because of the differences in the folding of the
proteins
 The level of PrPsc is increased in infected brain because the protein
becomes resistant to degradation

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 MICROBIOLOGY
Coronavirus, Slow Viruses and Prion Diseases
Dr. Palacpac
 Different strains of PrPsc occur because of mutations in the PrPc as
because of self pertuating alternative folding patterns of the protein.
o e.g: specific mutation at codon 129 determine the severity
of VCJD dse. Conformational rather than genetic mutation
is another property that distinguishes prions from viruses.
 The PrPsc acts as a template to transmit its conformation onto each
new PrPsc analogous to a genetic template (DNA or RNA)
transmitting its sequence onto a new viral genome.
Pathogenesis
Spongiform Encephalopathy:
 Describes the appearance of vacuolated neurons as well as their loss
of functions and the lack of an immune response or inflammation.
 Vacuolation of the neurons, formation of amyloid containing plaques
and fibrils, proliferation and hyperthropy of astrocytes, vacuolation
of neurons and adjacent glial cells are observed.
 The PrPsc is taken up by the neurons and phagocytic cells, leading to
the vacuolation of the brain tissue which contributes to tissue
damage.
 Prion can also be isolated from other tissue other than the brain.
 But only the brain shows any pathologic changes.
 No inflammation or immune response to the agent which it is
distinguished from viral encephalitis.
 A protein marker (14-3-3 brain protein) can be detected in the CSF of
symptomatic person (not specific for prion dse).
 The incubation period for CJD and KURU  30 years and progresses
rapidly once symptoms become evident and leads to death in a year.
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Prion Diseases of Animals (Animal Transmissible Spongiform
Encephalopathies)
1. Scrapie
 Prototype prion- First animal spongiform encephalopathy to be
described
 Endemic disease of sheep and goats- European Continent
 MOT: Vertically from EWE to Lamb
Natural transmission- occur during parturition or scarification
or by oral route
 Incubation period: 2 years
 S/S: affected animals become wasted, ataxic, and irritable
 Develop intense pruritus or rub or scrape their bodies against trees
and rocks
 The animal develops paralysis and dies
 Placenta of EWE- an important source of infection (contaminate the
farm)
Pathogeneis:
 The scrapie agent have been adapted- it can infect hamsters and mice
 Infected hamsters have scrapie associated fibrils in the brain. The
fibrils contain the prion.
 The scrapie agent (prion) enters the body thru skin and in sheep
intact intestinal tract.
 The prion replicates first in the brain another lymphoid organs.
 B lymphocytes are probably required for its spread thru the
circulation to the brain
2. Bovine Spongiform Encephalopathy (BSE)
 Mad Cow Disease- emerged in cattle in UK 1986 and affects 1.5
million cases by 1995.
 This outbreak was resulted from oral ingestion of scrapie agent via
contaminated bone meal from scrapie- infected sheep carcasses and
recycling of BSE infected carcasses.
 In 1996, a new variant form of CJD was recognized in the UK that
occurred in younger people- under 30 years old and with longer
duration of illness than the classic form of CJD.
 New variant form of CJD had the same pathologic characteristics to
those of BSE.
 The new variant forms of CJD and BSE are caused by a common agent,
indicating that the BSE agent had infected humans
3. Transmissible Mink Encephalopathy (TME):
 Resembles scrapie except that it occurs in minks
 Represents a strain of sheep scrapie agent acquired from feeding of
contaminated beef to mink.
Human Prion Disease
1. Creutzfeldt-Jacob Dse:
 Chronic degenerative dse of the CNS, rapidly progressive and fatal
 Typically affects middle aged adults
 Caused by TSE agents (prion)- characterized by spongy appearance
of the cerebral gray matter- resulting from degeneration of the
neurons and axons.
 Spongiosis, Gliosis and Neuronal loss
 Abnormal proteins (Prion protein, PrP) accumulate in the brain both
as diffuse deposits and in the form of amyloid plaques.
 Prp can be isolated from tissues other than the brain, but only the
brain shows any pathological changes.
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Coronavirus, Slow Viruses and Prion Diseases
Dr. Palacpac
 Patient develops progressive dementia, ataxia, myoclonus, memory
loss, behavioral changes, visual abnormalities, pyramidal and EPS
and cerebellar signs. Patient die less than a year after the onset.
 Forms of CJD: Sporadic, Familial and Acquired (Iatrogenic CJD and
variant CJD)
 Sporadic CJD which occur throughout the world with a frequency of
one case per million population per year. Accounts for 85% of CJD.
Most common form of the human prion dse. Spontaneous
conversion from PrPc to PrP CJD
 Two familial forms of CJD are Gerstmann-Straussler Scheinker
Syndrome and Fatal Familial Insomnia- Dse are rare (10-15% of CJD
cases) and are due to inheritance of mutations in PrP gene.
 Iatrogenic CJD has been transmitted accidentally by contaminated
growth hormone from human cadaver pituitary glands, corneal
transplant, contaminated surgical instruments, cadaveric human
dura mater grafts.
 vCJD is from ingestion of beef infected with BSE; it is now known that
vCJD can also be acquired through blood transfusion from a person
incubating vCJD. Only about 211 cases of vCJD have ever been
identified, and most of those are in the United Kingdom, followed by
France

2. Gerstmann-Straussler Scheinker Syndrome (GSSS):

 Inherited human TSE- transmitted as a autosomal dominant trait.
 Affects middle aged adults
 Prominent clinical features: Cerebellar Ataxia, Nystagmus, Dementia
 Associated with mutation of PrP gene, a codon 109 proline to
leucine mutation
 First identified in 1989

3. Kuru
 Occurs mainly in the members of one cannibalistic Forbe Tribe in
Eastern Highlands of Papua New Guinea
 Associated with the tribal practice of eating brains of relatives after a
non- sterilizing ritual cooking.
 KURU ( shivering tremors) – progressive cerebellar syndrome
 Incubation Period: 4-30 years
 The disease is virtually extinct from 1950- abandonment of
cannibalism

4. Fatal Familial Insomnia

 Rare disorder- progressive severe insomnia, disturbances of
autonomic function, ataxia, myoclonus and other signs and
symptoms are resembling of CJD.
 Associates with unique PrP genotype codon 178 asparagine, 129
methionine and neurological changes mainly of the thalamus

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 MICROBIOLOGY
Coronavirus, Slow Viruses and Prion Diseases
Dr. Palacpac
Laboratory Diagnosis REFERENCES
 No methods of directly detecting prions in tissues- microscopy, Ag  PPT
detection or nucleic acid probes, no serological test.  Microbiology Manual (2019)
 Initial diagnosis must be made on Clinical grounds  Dr. Palacpac Recordings
 Confirmation of diagnosis- detection of a proteinase-K-resistant
form of PrP in the Western Blot- using Ab to PrP in tonsillar biopsy.
 Autopsy- characteristic amyloid plaques, spongiform vacuoles and
immunohistologically detected PrP.
 PMCA (protein misfolding cyclic assay)- amplify the number of PrPsc
units to detect prions- polymerization of normal PrP.
 Positive 14-3-3 CSF assay in patient with dse duration of less than 2
years.
 Other useful studies include EEG, MRI, lumbar puncture with
cerebrospinal fluid (CSF) analysis, and brain biopsy.

Treatment, Prevention and Control

 No treatment for CJD, interleukins and other drugs may help slow the
progression of the disease.
 Medications to help treat the symptoms of CJD (anti-epileptics, anti-
psychotics and anti-depressants).
 Referral to Neurologist, Neuro-psychiatrist and Infectious Disease
Specialist
 Anti-malarial drug Quinacrine and anti-psychotic drug
Chlorpromazine are currently evaluated in treatment trials.
 Autoclaving at 15psi for one hour or treatment with 1% hypochlorite
 or 1N sodium hydroxide- used for decontamination of surgical
instruments or brain electrodes.
 Careful monitoring of cattle in livestock industry

What is important in the Study of Coronaviruses….

 Know the receptors site (e.g. ACE 2, CD26)
 Important features of Coronavirus
 Target cell sites of Coronavirus (aminopeptidases)
 Know alpha and beta coronaviruses (beta is more important)
 SARS and MERS (differences)
 Laboratory Diagnosis and Management

What is important in the Study of Slow Viruses..

 Main mechanism of action why slow viruses and prions happen
 Know the difference of PrPc and PrPsc
 Know the human and animal transmissible agents
 Know the different clinical implications

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