Virology Flashcards
size of viruses
20-300 nanometers
ways to classify viruses
- Nature of nucleic acid (RNA or DNA)
- Morphology/symmetry of protein shell (capsid)
- Presence or absence of a lipid membrane (envelope)
- Dimensions of the virion (viral infection particle) and capsid
viral shapes
Helical symmetry
Icosahedral symmetry
Spherical
Complex (bacteriophage)
protein coat surrounding the nucleic acid
Capsid or nucleocapsid
used to bind to cell surface
glycoprotein spikes
viral membrane the carries the viral glycoproteins
made from host cell
envelope
RNA viruses must either…
encode a nucleic acid polymerase to produce mRNA from RNA templates
Or
use a reverse transcriptase to convert RNA to DNA (retrovirus) and then produce mRNA
——– DNA can be directly translated into mRNA
——– DNA cannot be translated directly; must be copied
positive
negative
virus infectious cycle
Attachment and entry
Decoding genome information
Genome replication
Assembly and release of viral particles with genome
specimens for viral culture must contain…
cells
steps of creating a cell culture medium
- Disassociate tissue into a single cell suspension by mechanical disruption then treatment with proteolytic enzymes
- Suspend in culture medium and place in plastic flask or plate
- Cells divide in about 24 to 48 hours and cover plastic surface to form a monolayer
- Most cells retain viability after being frozen at low temperatures
- Can pass or split cells (passage) by dispersing them and reestablishing them into a new flask or plate
primary cell culture lifespan
5-20 divisions
primary cell culture sources
Chicken or mouse embryos, monkey kidneys or humas tissue such as embryonic amnion, kidney, foreskin of respiratory epithelium
diploid cell culture lifespan
50 or 100 divisions
diploid cell culture sources
Human embryo cells such as WI-38 strain, and MRC-5 derived from human embryonic lung fibroblast cells
Can be propagated or passaged indefinitely in culture
continuous cell culture
Usually derived from tumor tissue or by treating a primary cell line with a mutagenic chemical
continuous cell culture
Prior to molecular detection, viruses are visualized in infected cells
cytopathic effect (CPE)
cytpathic effect examples
Rounding up
Detachment
Cell lysis
Formation of fused mulitnucleated cells (Syncytium)
time to development of CPE
Some take 1 to 2 days while others may not produce CPE for weeks
respiratory syncytial virus (RSV) CPE
giant cells and syncytia formation
advantages of NAAT over viral culture
- Faster
- More sensitive
- Detect more viruses than culture
- Require less skill
- Can determine viral load for quantitative monitoring
- Can test as viral syndromic panels
limitations of multiplex viral syndromic panels
- Not all positive results indicate current infection
- Likelihood of at least one false positive increased in multiplex panels with many targets
- Even a positive results does not rule out the possibility of another concomitant infection or super infection
- Does not test for every viral cause of infection
- Loss of technical expertise of clinical virologist
- More expensive than using testing for key viral pathogens
3 requirements for viral infection
- Sufficient number of infectious viral particles
- Access to these particles by submissive and permissive cells
- Uneducated or dampened antiviral defenses
To be effective, the vaccine must…
induce protective immunity in a portion of the population, so it is sufficient to impede person-to-person transmission (herd immunity)
Most antivirals target viral enzymes such as…
proteases and nucleic acid synthesizing proteins
can help avoid the appearance of viruses resistant to one treatment or the other
the use of two or more drugs with distinct targets
Knowing a virus’s —— helps to reduce testing required to only a few viruses rather than a pan virus approach
season
phases of viral infection
- Prodrome – pre-symptomatic shedding
- Acute phase – shedding decreases and IgM appears
- Resolution phase – shedding ends and IgG appears
when should viral samples be collected?
as soon as possible after onset of symptoms – Likelihood of positive results usually within first 3 days
may need to place sample in ——- for transport
viral transport medium (VTM)
VTM example
Buffered salt solution such as Hanks with a neutral pH
Most useful VTM should allow simultaneous detection of viruses by…
several methods such as culture, antigen detection and NAAT
transport time
2-4 hours of collection
preferred transport temp
2-8°
Enzymes present in specimens capable of inactivating viruses are less active
Specimens can be held at 2 to 8°C for up to ———- before inoculation onto cell culture
1 to 2 days
Freezing of specimens (-70°C or lower) is not recommended for viral isolation (cell culture) unless…
specimen processing will not occur within 2 days or more
lose activity with repeated freeze-thaw cycles
RSV, CMV and VZV
very susceptible to temperature changes and has about a 90% loss of infectivity after 24 hours at 37°C and 4 days at 4°C
RSV
RNA targets (HIV or HCV) sample
EDTA-treated whole blood
DNA targets (CMV) require…
EDTA-treated blood
Whole blood, used for NAAT, needs to be processed to remove…
inhibitors of DNA and RNA polymerases such as heme and the metabolic precursors of heme
Can use ——– for NAAT for diagnosis of HIV, HBV, neonatal HSV and congenital CMV
dried blood spots
blood/BM collection for viruses
Collect in heparin
Transport at room temperature
CSF collection
- Do not centrifuge – need to maintain cells present in specimen
- Dilution in VTM can cause false negative results
- Transport at room temperature and store up to 48 hours at 2 to 8°C or freeze at -70°C if longer storage is required
must extract ——- from CSF
why?
Extract nucleic acids prior to testing to remove globulins that inhibit the activity of thermostable polymerases
cells needed for respiratory collection
ciliated epithelial cells of the posterior nasopharynx
——- respiratory specimens are not acceptable due to low levels of viruses
Anterior nasal and throats
sputum is suboptimal
——- collects more respiratory epithelial cells from posterior nasopharynx
Flocked swab
collect HSV from skin lesions without ——— because…
bleeding, since neutralizing antibody in blood can impair recovery of virus
Rectal swab is inferior to …
2 to 5 mL of liquid stool or 2 to 5 grams of formed stool
Can use specially formulated collection paper strips for NAAT for …… (enteric viruses)
rotavirus, adenovirus types 40 and 41 and norovirus
influenza antigenic differences based on…
matrix protein (M) and nucleoprotein (NP)
Influenza A are further classified into subtypes by two major surface glycoproteins:
Hemagglutinin (HA)
Neuraminidase (NA)
occurs due to small changes/mutations in the genes that lead to changes in the surface proteins of the virus
antigenic drift
major abrupt change in the flu virus resulting in new HA or new HA and NA proteins
antigenic shift
Occurs only with type A viruses and they are the only flu viruses known to cause pandemics
antigenic shift
influenza description
Enveloped, spherical, with single-strand RNA
Late November to December begins with influenza ——-
Transitions to —— in February through April
type A
type B
influenza tx
Amantadine and rimantadine (type A only)
Zanamivir, oseltamivir and peramivir
influenza CPE
destruction of cell sheet within a week of inoculation
Can perform hemadsorption using erythrocytes of different animal species (guinea pig)
influenza
Types 1 and 2 in October and November, type 3 in late March to April
Severe disease in those with immunocompromised immune systems
parainfluenza
50% of croup (barking cough) in children
parainfluenza (HPIV)
HPIV tx
Currently no FDA cleared antivirals or vaccines
HPIV CPE
rounded cells and syncytium formation in 4 to 8 days
Gets its name from the characteristic formation of multinucleated giant cells in cell cultures
RSV
Most serious cause of lower respiratory tract illness in young children and infants
Bronchiolitis, pneumonia
RSV
Cell culture is the gold standard for —– due to excellent specificity
RSV
RSV tx
Ribavirin and palivizumab (monoclonal antibody)
Presents very similar to RSV but poor or no growth in cell cultures
human metapneumovirus
Derive their name form the predominant site of replication – the nose
rhinovirus
Causes 2/3 of cases of upper respiratory syndromes known as the common cold
rhinovirus
rhinovirus CPE
Large and small rounded, refractile cells with pyknotic nuclei in 24 hours to 4 days
enterovirus description
Non-enveloped with single strand RNA
Stable in liquid environments and can survive for weeks; Water, body fluids, sewage
enteroviruses
enterovirus highest incidence
young infants and children 5 to 10 years old
Most common enteric cause of viral meningitis
poliovirus (enterovirus)
Hand-foot and mouth disease
coxsackievirus (enterovirus)
enterovirus CPE
limitation
shrinkage and rounding of cells and nuclei exhibit pyknosis
Culture has no real application due to prolonged time for viral isolation
enterovirus tx
No FDA approved antiviral agents
coronavirus description
Enveloped RNA virus
Spikes of glycoproteins (peplomers)
Shedding of COVID peaks in —– week of disease so patient is often in hospital by then and it is easier to spread virus
2nd
Coronavirus isolation allowed only in a —– facility
BSL 3
Culture should not be attempted
Does not grow in most common cell lines
CPE is generally non-specific
Requires a BSL3 facility and procedures
Accidental transmission to laboratory workers has been described
coronavirus
rotavirus description
Nonenveloped, double-stranded RNA
norovirus description
Nonenveloped, single stranded RNA
enteric adenovirus description
Nonenveloped, double stranded DNA
Prior to vaccination, leading cause of severe dehydrating diarrhea in infants and kid less than 2 years
rotavirus
Cause large outbreaks: Hospitals, child-care centers, schools, restaurants, nursing homes, cruise ships, military camps
norovirus
Fever, diarrhea, vomiting, abdominal cramps, lethargy dehydration
gastroenteritis viruses
gasteroenteritis virus tx
No effective antiviral agents
rotavirus now has a vaxx
gastroenteritis specimen
stool or rectal swab
major challenge to gastroenteritis NAAT
the presence of inhibitors in sample
Could lead to false negative results
gastroenteritis cell culture
Time consuming and does not contribute to management of gastroenteritis
HSV description
Double stranded DNA with lipid rich envelope
Readily inactivated by lipid solvents (e.g., ethanol), Lysol, bleach, temperatures >56°C, and pH <5 or >11 with 30 minutes of exposure
HSV
—— associated with gingivostomatitis, and —— associated with genital lesions
HSV1
HSV2
HSV becomes latent in …
sensory ganglia
Most common cause of fatal sporadic encephalitis in immunocompromised
HSV
not acceptable for diagnosis of HSV encephalitis
Culture of CSF
Most common viral cause of corneal infection
HSV
HSV cell lines
Mink lung cells, human diploid fibroblasts (MRC-5 and WI-38), human epidermoid carcinoma lines such as Hep-2 and A549
HSV CPE
cell develop cytoplasmic granulation and become large, round (balloon)and refractile
Clusters of infected cells then lyse and detach
HSV tx
Acyclovir, valacyclovir penciclovir, and famciclovir
results from reactivation of VZV
Herpes zoster (shingles)
VZV samples collected
skin vesicular fluid
blood for dissemination
Infects humans of all ages with no seasonal transmission
Often acquired early in life and up to 100% of individuals can be seropositive
CMV
Prolonged fever, malaise, atypical lymphocytosis and mild hepatitis
Known as heterophile-negative mononucleosis
CMV
mimics EBV
heterophile-negative
CMV
Most common viral infection with transplants
CMV
Large characteristic cells with basophilic intranuclear inclusions of infected tissue
Nuclear inclusion has appearance of “owl’s eyes”
CMV
replaced cell cultures for CMV due to increased sensitivity
Shell Vial assays
Human fibroblasts best support growth
CPE and can develop within 24 hours and progress rapidly
Can resemble CPE of adenovirus and varicella-zoster virus
CMV
serology used to screen blood and organ donors and recipients for…
CMV
- Centrifuge specimen onto MRC-5 fibroblasts cells growing on a round cover slip
- Virus is embedded into the cell layer to begin the replication process faster
- Incubate 37°C for 16 to 24 hours then apply immunofluorescent antibody stain
shell vial assay for CMV
CMV tx
Ganciclovir, valganciclovir, foscarnet and cidofovir
Isolation from urine, respiratory secretions or other bodily fluids within first 3 weeks of life is traditional means of confirming diagnosis of congenital infection in newborns
urine preferred
CMV
HPV description
Nonenveloped, double-stranded DNA
HPV —- has the highest risk for cancer
16
hand and foot warts are common in children and adolescents
Walking barefoot in pool or showers
HPV
Anogenital warts
Cauliflower-like growths
Majority caused by HPV types 6 and 11
Oral and upper respiratory tract warts
Potential damage to vocal cords and possibility of respiratory obstruction
HPV
Three vaccines that target certain types
HPV
samples for HPV
Tissue samples, cervical samples which include exfoliative cells as used for a Pap test, first-void urine, swabs or brushes to ensure dislodged cells, oral rinse or gargle using mouthwash or saline
HPV culture
cannot be cultured
Use for girls who are ≥ 15 years old and those who are HIV positive
HPV vaccine
