Virology Flashcards
Introduction – Viruses
Subcellular pathogens
DNA and RNA genomes
Cause a wide variety of disease
By far the most rapidly changing area of clinical microbiology
Classification of Viruses
Genome: RNA (14 families) and DNA (7 families); ss, ds, linear, circular, single or multiple segments Enveloped and non-enveloped Morphologic – Icosahedral – Helical
DNA Viruses
Double-stranded linear genomes except as noted.
Parvoviridae; ssDNA
– B19
Hepadnaviridae; enveloped
– Hepatitis B virus
Polyomaviridae; circular genome
– JC & BK viruses
Papillomaviridae; circular genome
– Human papillomavirus
Adenoviridae
– Many serotypes
Herpesviridae; enveloped
– EBV, CMV, VZV; HSV; HHV 6-8.
Poxviridae ; enveloped
– Smallpox, vaccinia, molluscum contagiosum
RNA Viruses
Retroviridae; enveloped
– HIV, HTLV,
dsRNA viruses
– Reoviridae - Rotavirus
Minus-strand ssRNA viruses
– Paramyxoviridae; enveloped - Parainfluenza, measles, mumps, RSV
– Rhabdoviridae; enveloped - Rabies virus, VSV
– Filoviridae ; enveloped - Ebola, Marburg
– Orthomyxoviridae; enveloped - Influenza
– Bunyaviridae; some enveloped - La crosse, hantaviruses, CCHF, RVF
– Arenaviridae; enveloped - Lymphocytic choriomeningitis virus
Plus-strand ssRNA viruses
– Picornaviridae - Enteroviruses, rhinoviruses, hepatitis A virus
– Caliciviridiae - Noroviruses, hepatitis E virus
– Astroviridae - Human astroviruses (gastroenteritis)
– Coronaviridae; enveloped - SARS, other human coronaviruses
– Flaviviridae; enveloped - Yellow fever, hepatitis C virus
– Togaviridae; enveloped - WEE, EEE, rubella
Others
– ssRNA satellite agent: hepatitis delta virus
– prions
Viral Diagnostic Methods
Viral culture methods
Antigen detection methods
Nucleic Acid testing methods
Viral Serology / Antibody Detection
Viral Culture
Requires
–Permissive cells
–Means of detecting growth
–Means of identifying virus
Types of Viral Culture
Viral tube culture
– Cell types
Primary cells, e.g. monkey kidney cells,
Diploid cell cultures, e.g. human foreskin fibroblasts, human embryonic lung
Permanent cell lines, e.g. HeLa, A549, MRC-5, HEp-2
– Detection by seeing cytopathic effect (CPE) in 1-20+ days
– Identification with specific Fluorescent Antibody (FA) testing
– Even in the molecular era, viral culture is still a primary way of detecting novel and unexpected viruses
Shell vials – targeted cultures
– Inoculation of cells on a cover slip in a small tube, a ‘shell vial’
Centrifugation often improves yield
– Short incubation (24-48 hours)
– FA or other stain for specific target viruses
Viral Tube Cultures – Detection
Cytopathic Effect (CPE)
– Early; rounding of cells
– Later: cell death, ‘plaques’
– CPE characteristics (time, morphology) relate to possible viral etiologies
Viral Conventional Culture – Cells
Different viruses, different cells Limitations – Slowgrowing viruses ex. CMV – Nongrowing viruses ex. HBV, HPV – Loss in transport
Viral Culture – Types of CPE
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Shell Vial Culture
Culture 1-2d on coverslip
Stain for specific target viruses, esp. early antigens
Slow-growing viruses e.g. CMV
Respiratory viruses
– Cell mixes and antibody cocktails
Other samples with defined targets, e.g. skin lesions
Highly sensitive and specific
Relatively rapid
Detects only the defined viruses you’re looking for.
Viral Antigen Detection
A variety of methods
– DFA
– Instrumented EIAs (esp. for hepatitis)
– Rapid tests for respiratory and GI viruses
Usually faster than culture
Some are automated; some are point-of care
Usually insensitive relative to culture or molecular methods
Viral DFA
Slide made directly from sample – Stained with fluorescent antibody Skin lesions: HSV / VZV Respiratory samples: Influenza, assorted respiratory viruses High skill required, can approach sensitivity of culture
Rapid Antigen Tests
Influenza, RSV, Adenovirus
Access vs. Quality
– Insensitive
– Available anywhere with a lab, and some places without.
Viral Molecular Diagnostics
Detection of viral DNA or RNA – PCR (many vendors) – SDA (BD) – TMA (Gen-probe) Rapidly evolving Remember a reverse transcriptase step (RT-PCR) is required to detect RNA viruses
Molecular Applications
Detection
– Rapidly becoming test of choice for many viruses
Herpesvirus and enterovirus CNS infections.
Respiratory viral infections.
Caliciviruses
HPV
Many others
– Optimized for sensitivity.
– Use of internal controls to detect inhibition.
– Real-time methods to limit contamination and save labor.
– Automated extraction systems
Molecular Applications
Viral load (HCV, HIV, others)
– Use of internal calibration standards to control for
extraction efficiency.
– Standardization needed.
Viral genotyping
– HCV: viral subtype affects duration and intensity of therapy, prognosis
– HIV: drug susceptibility testing by sequencing
Viral Serology
Detect antibody to viral antigens Automated, other EIA, rapid tests Primary diagnosis – HIV, HCV, HBV, EBV Immune Status Testing – HBV, VZV, MMR, etc. Issues – Screening vs confirmation – Cutoff values – Western blots and RIBAs and other confirmatory strategies
Viral Histopathology
Selected examples of viral cytopathic effects
in tissues, with inclusion bodies (when present)
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Adenovirus
ds DNA, non-enveloped, replicates in nucleus
– >47 serotypes, six sub-genera ( A to F )
Wide variety of syndromes
– Pneumonia in immunocompromised patients & military
recruits
– Acute gastroenteritis children (40,41)
– Pharyngitis, pharyngoconjunctival fever
– Keratoconjunctivitis
– Hemorrhagic cystitis
– Cervicitis, urethritis
– Disseminated disease
•Alveolar lining cells contain large, homogeneous, very
characteristic intranuclear inclusions: “Smudge cells”
Pearls from the inclusion body:
• Nuclear only, not cytoplasmic
• Large, deeply basophilic (blue) with NO halo
• Nucleo-cytoplasmic blurring, small amount of peripheral
cytoplasm left
• No cytomegalia
• No multinuclearity
• No smooth ground-glass appearance
• Very coarse, irregularlooking, “smudgy” material
• “SMUDGE CELLS”
HSV 1 & 2
All herpes family viruses are enveloped ds DNA
viruses.
Three subfamilies:
– Alphaherpesviruses - HSV-1, HSV-2, VZV
– Betaherpesviruses - CMV, HHV-6, HHV-7
– Gammaherpesviruses - EBV, HHV-8
Set up latent or persistent infection following primary
infection ( ie., “they never divorce you!!!” )
Reactivations are more likely to take place during
periods of immunosuppression
Varicella-zoster Virus (VZV)
Chickenpox (Primary varicella); vesicular, disseminated
Herpes zoster (Reactivation); vesicular, dermatomal
Beware! In an immunocompromised patient, a disseminated rash may be a case of:
– Primary Varicella
– Disseminated Zoster
– Disseminated HSV
Herpes simplex virus (HSV) & Varicella-Zoster Virus
(VZV) produce inclusions that are practically identical
Multinucleated giant cells with ‘molded’ nuclei
Intranuclear inclusion with marginated chromatin
CMV (Human Cytomegalovirus)
Large (‘megalic’) blood-vessel lining cell (‘endothelial’) ballooning into the lumen
Intranuclear ‘Owl’s-eye’ inclusion with a halo
Abundant pink (eosinophilic) intracytoplasmic inclusions
Paramyxoviruses
may produce syncytia in tissue: RSV, Parainfluenza, measles
Rabies
Negri bodies: intracytoplasmic eosinophilic inclusion bodies
Hepatitis Serology
Used for diagnosis, prognosis, and immune status monitoring.
Antigen is bad:
– Implies viral activity, associated with:
Active disease (HBsAg)
Poor prognosis (HBeAg).
Positive antibody result is associated with resolved disease (HBsAb) or better prognosis (HBeAb).
Markers of acute infection are core IgM (HBcAb-IgM) and surface antigen (HBsAg):
– Antigen produced before immunity
– IgM, like always, is the first antibody subtype made.
Core antibody (HBcAb) is the test for exposure to HBV:
– Screening marker for blood
– Almost invariably present in infected patients past the acute phase
– Doesn’t reflect disease activity.
Positive surface antibody (HBsAb) alone means immunization:
– The HBV vaccine contains surface antigen but no core antigen.
Immunized people will be HBsAb positive but HBcAb negative.
Hepatitis C
Mostly blood-borne
– sexual transmission rare.
– perinatal transmission risk is <25% of infected persons
– Fulminant HCV hepatitis is very rare except in Japan.
Chronic hepatitis C
– 50-85% of persons infected with HCV develop persistent, viremic infections
– Asymptomatic or only mildly symptomatic
– Persists for decades, with fluctuating ALT / AST levels.
Associations
– Cryoglobulinemia
– Kidney disease (membranoproliferative glomerulonephritis)
– Other autoimmune diseases
– Lymphomas and hepatocellular carcinoma
Hepatitis C Tests
Hepatitis C antibody - Anti-HCV
Becomes positive 6-8 weeks after infection. Marker of HCV infection
Hepatitis C recombinant immunoblot - HCV RIBA Used to confirm borderline HCV antibody test
Hepatitis C RNA - HCV RNA, HCV viral load, etc. Viral load (amount of virus in blood) by quantitative PCR is used to decide on and monitor therapy. For diagnosis, these are supplemental tests
Hepatitis C Genotype - Provides information on viral strains used for prognostic purposes and to guide treatment decisions. Strain types 2 and 3 are more likely to respond
to treatment than strain type 1
HIV Tests
- HIV Screening EIA 1st gen: crude viral lysate 2nd gen: recombinant antigens 3rd gen: improved IgM detection 4th gen: antibody and antigen detection
-HIV Confirmatory Tests
Western blot
IFA
- HIV DNA PCR
For neonatal diagnosis - HIV RNA PCR
for primary screening of blood products
for diagnosis
for viral load testing - HIV genotyping
drug resistance monitoring by DNA sequencing; RT
and PR mutations
HIV Western Blot
Positive is:
– CDC/ASTPHLD criteria;
any two of the following bands: p24, gp41, and gp120/160
Influenza Testing
Culture
– Newer shell vial methods 24-48h
– ‘Gold standard’
DFA
– Rapid (1-2h)
– Sensitivity from top centers approaches culture.
– Requires fluorescent scope and highly trained technical staff.
Molecular Diagnostics
– Real-time methods can be ~1h or so.
– In some cases real-time PCR methods exceed culture in sensitivity
(probably due to viral loss in transport)
– High skill and equipment requirements
– Emerging ‘gold standard’ – overall probably better than culture
Rapid antigen tests
– 50-70% sensitivity, despite publications to the contrary
Lower in adults
– Rapid, simple, no equipment needed.
Decisions Based on Rapid Flu
Positive Test – Do not start antibacterials – Start antivirals – Droplet precautions – Don’t do other tests Consequences of error: potentially severe Specificity is essential!
Negative Test – Consider antibacterial therapy – Do not start antivirals – No droplet precautions – Further diagnostic studies Consequences of error: moderate Insensitivity can be tolerated
It’s All About the Flu
Two major considerations for influenza
testing:
–When to test?
When do you start / stop testing for flu?
–What specimens to collect?
How do you train your clinical staff in
specimen collection?
When to test for flu?
When?
Remember – false-positives have potentially
severe consequences, e.g. non-treatment of a serious bacterial infection.
Test during the flu season, only.
Potential strategies:
– Seasonal: test Oct-Dec→March or so.
Early season – retain specimen for confirmatory testing!
– Incidence-based testing – monitor regional
influenza per CDC and State systems, begin testing only when influenza reported in the area.
Influenza Specimen Collection
Specimen collection is the critical step in
influenza testing
Washes are somewhat better than swabs*
Important to get ciliated epithelial cells
Children shed more virus than adults;
tests tend to be more sensitive in kids.
Respiratory Viruses
Upper / Colds – Rhinovirus – Respiratory syncytial virus – Adenovirus – Parainfluenza – Influenza A,B – Enterovirus – EBV
Lower / Pneumonia – Influenza A, B – Adenovirus – Respiratory syncytial virus – Human metapneumovirus – Parainfluenza – Rhinovirus – Cytomegalovirus – Varicella-zoster – Herpes simplex – Hantavirus* – SARS CoV*
Gastroenteritis Viruses
Rotavirus Norovirus Adenovirus Enterovirus CMV (compromised patients; colitis)
Meningitis / Encephalitis Viruses
Meningitis – Enterovirus – Herpes simplex virus – Varicella-zoster – EBV – Mumps – WNV – Jamestown Canyon – Lymphocytic choriomeningitis virus (LCMV)
Encephalitis – Herpes simplex virus – Cytomegalovirus – Varicella-zoster – EBV – Arbovirus (EEE, WEE, SLE,West Nile, POW, etc) – Adenovirus – Measles, Rubella, Mumps – Influenza – Enterovirus – HIV – BKV – HHV-6 – Rabies – LCMV (transplant)
Skin
Vesicular – HSV, VZV, Enterovirus, Vaccinia, CMV, Adenovirus Papillomas – HPV, molluscum contagiosum Exanthems – Measles. Rubella, Enterovirus, Parvovirus B19, HHV-6, Dengue, West Nile, EBV, Adenovirus, CMV
Hepatitis Viruses
Hepatitis A, B, C, D, E HSV, especially in newborns EBV CMV Adenovirus
Other Syndromes
Mononucleosis
– EBV, CMV, Adenovirus, HIV, HHV-6
Ocular
– Enterovirus, Adenovirus, HSV, VZV, CMV,
Vaccinia, Measles
Marrow Suppression
– EBV, MV, HHV-6, Hepatitis A, B, C, Parvovirus B19, Influenza, Adenovirus, HIV
