Virology Review
Margie Morgan, PhD, MT(ASCP),D(ABMM)microbeswithmorgan.com
Diagnostic techniques used in the Virology Laboratory
1.Direct Staining for Antigen2. Enzyme Immunoassay3. Molecular Amplification
4. Viral Cell Culture
Detect Antigen in lesion
Direct Fluorescent antibody (DFA) stain Collect cells from base of vesicular lesion Stain with Fl antibody specific for HSV and/or VZV
Look for fluorescent cells using fluorescence microscope
Fluorescent cells = viral infected cells More sensitive & specific method than Tzanck
prep (DFA 80% vs. Tzanck 50%) Tzanck prep= Giemsa stain of lesion cells/examine
for multinucleated giant cells of Herpes virus
TzanckTzanck DFA
Detection of Viral Antigens by EIA
Enzyme immunoassay – Antigen/antibody complex formed – then
bound to a color producing substrate Used most for detection of non-culturable
viruses – such as Rotavirus from stool Detect Influenza A and B , & Respiratory
syncytial virus (RSV) from nasal/NP swab Membrane EIA Liquid/well EIA
Molecular Amplification Molecular Amplification (DNA or RNA)
Rapid/Sensitive/Specific for numerous viruses Exceeds sensitivity of culture/replacing culture
Standard of practice for detecting respiratory viruses Standard of practice for HSV and Enterovirus detection
from CSF Culture <=20% PCR >=90%
Quantitative assays in transplantation - CMV Hepatitis B and C detection and viral load HIV viral load Test of diagnosis not cure – can retain DNA/RNA
for 7 – 30 days after initial diagnosis
Viral Cell Culture
Inner wall coated with monolayer of cells lines covered with liquid maintenance media
Three basic types of cell lines: Primary cell lines – directly from animal organ
into culture tube (Rhesus monkey kidney-RMK) Diploid cell lines– Can survive 20 – 50 passes
into new vials – human diploid fibroblast cells, example: MRC-5-Microbiology Research Council 5
Continuous cell lines – can survive continuous passage into new vials, usually of tumor lineage, HEp-2 and HeLa
Viral Cell culture
Tubes/flasks read under microscope for Cytopathic effect/ CPE Appearance of cells in the monolayer
after being infected with a virus Destruction is specific for each virus type
Spin Down Shell Vial Virus Culture - •Designed to speed up virus recovery•Cells are on the round coverslip•Specimen inoculated into vial•Centrifuge vial to induce virus invasion into cell monolayer•Incubate @ 35*C, 24-72 hours•Direct fluorescent antibody stain to stain cells on coverslip – target early •antigens for virus of interest
Cover slip
Specimen collection and transport Viral transport media (VTM) - Hanks
balanced salt solution with antibiotics, Also known as Universal Transport Media needed for the transport of lesions, mucous
membranes and throats to the laboratory It is cell protective, protect the cell / protect the virus
Short term transport storage 4˚C Long term transport(>72hours) storage-70˚C VTM specimens filtered (45nm filter) to
eliminate bacteria in specimen prior to being placed on cell monolayer
Which viruses will survive the trip to the laboratory?
Most likely to survive - HSV Intermediate
Adenovirus Influenza A and B Enterovirus
Least likely to survive Respiratory Syncytial Virus (RSV) Cytomegalovirus (CMV) Varicella Zoster virus (VZV) Amplification preferred for these viruses due to survival
issues
Which viruses grow the fastest in conventional cell culture?
Fast (>=24 hours) HSV
Intermediate (5 -7 days) Adenovirus Enterovirus Influenzae VZV
Slow (10 - 14 days) RSV
Slowest (14 - 21 days) CMV
Amplification methods are superior for slow growers
Herpesviridae
Herpes Viruses
Double stranded DNA virus Eight human Herpes viruses
Herpes simplex 1 Herpes simplex 2 Varicella Zoster Epstein Barr Cytomegalovirus Human Herpes 6, 7, and 8
Latent infection with recurrent disease is the hallmark of the Herpes viruses
Latency occurs within small numbers of specific kinds of cells, the cell type is different for each Herpes virus
Herpes simplex virus 1 and 2 Transmission: direct contact/secretions Latency: dorsal root ganglia Disease –
Gingivostomatitis Herpes labialis Ocular Encephalitis Neonatal Disseminated in immune suppressed
Therapy – Acyclovir, Valacyclovir (nucleoside analogs)
Herpes virus diagnosisHerpes 1 & 2 do well in culture
Grow within 24-48 hrs in Human diploid fibroblast cells (MRC-5) - Observe for characteristic CPE Antigen detection by direct fluorescent staining of cells obtained from vesicular lesions Amplification methods available for detection from lesions and bodily fluids Cytology/Histology - intra nuclear inclusions, multinucleated giant cells Serology – More helpful to detect past infection
HSV1 and HSV2 can x-react in serology
Negative fibroblast cell Culture -uninfected cells
HSV infected monolayerRounded cells throughout the monolayer in cell culture
Multinucleated Giant Cellsof Herpes Simplex in tissue histology
Varicella Zoster Virus
Transmission: close contact Latency: dorsal root ganglia Diseases:
Chickenpox (varicella) Shingles (zoster – latent infection)
Chicken pox disease has decreased due to effective vaccine program – most serious disease occurs in immune suppressed or adult patients which progresses to pneumonia and encephalitis
Histology – multi-nucleated giant cells like those of Herpes simplex
Serology useful for immune status check Amplification useful for disease diagnosis
Varicella-Zoster Diagnosis
In cell culture –Limited # of Foci in monolayerRequire 5- 7 days to developSandpaper look to the Monolayer background withscattered rounded cells -diploid fibroblast monolayer
Younger wet vesicular lesions area the best for culture and/or molecular testing
Cytomegalovirus (CMV) Transmitted by blood transfusion , vertical and horizontal
transmission to fetus, also by close contact Latency: Macrophages Disease: Infection asymptomatic in most individuals
Congenital – most common cause of TORCH Perinatal Immunocompromised – Primary disease most serious
Laboratory Diagnosis: Cell culture CPE (Human diploid fibroblast) PCR and quantitative PCR (best method)
Histopathology: Intranuclear and intracytoplasmic inclusions “Owl Eye” InclusionsTreatment:ganciclovir, foscarnet, cidofovir
CMV pneumonia with viral inclusions
CMV infected fibroblast monolayer - Focal grape like clusters of rounded cells
Epstein Barr virus (EBV) Transmission - close contact, saliva Latency - B lymphocytes Diseases include:
Infectious mononucleosis
Lymphoreticular disease Oral hairy leukoplakia Burkitt’s lymphoma Nasopharyngeal Carcinoma 1/3 Hodgkin’s lymphoma
Unable to grow in cell culture Serology and PCR methods available for
diagnosis
EBV infection withB cell transformatin
EBV Serodiagnosis using the Heterophile Antibody
Heterophile antibodies (HA) react with antigens phylogenetically unrelated to the antigenic determinants against which they were raised
HA secondary to EBV are detected by the ability to react with horse or cattle rbcs (theory of the Monospot test)
HA rise in the first 2 - 3 weeks of EBV infection, then rapidly fall at @ 4 weeks
Cannot be used to diagnose children < 4 years of age
VCA = viral capsid antibodyEBNA = Epstein Barr nuclear antigenEA = early antigen
Human Herpes virus 6, 7 & 8
HH6 Roseola [sixth disease] 6m-2yr high fever & rash
HH7 CMV like Disease
HH8 Kaposi’s sarcoma Castleman’s disease
Onion skin of Castleman disease
Adenovirus
Adenovirus DNA - non enveloped/ icosahedral virus Latent: lymphoid tissue Transmission: Respiratory and fecal-oral route Diseases:
Adenovirus type 14 – virulent respiratory strain / pneumonia
Pharyngitis (year round epidemics) Gastroenteritis in children
Adenovirus types 40 & 41 Keratoconjuctivitis – very red eyes @ 2 wks Disseminated infection in transplant patients Hemorrhagic cystitis in immune suppressed
Adenovirus Diagnosis
Conventional cell culture (CPE) 2-5 days with round cells connected by strands –
Grows best in Heteroploid continuous passage cell lines (HeLA, Hep-2)
Amplification (PCR) is best for respiratory infection Histology - Intranuclear inclusions / smudge cells Stool EIA for enteric infections Antigen detection – staining respiratory cells by DFA
for Respiratory infections Supportive treatment – no specific viral therapy
Round cells withstranding
Smudge cells- Adenovirus
Parvoviridae – ParvovirusThe smallest known viruses!
Parvovirus DNA virus Parvovirus B19
Erythema infectiosum (Fifth disease) Cause fetal infection and stillbirths Aplastic crisis in patients with chronic hemolytic
anemia and AIDS Histology - virus infects mitotically active erythroid
precursor cells in bone marrow Molecular and Serology methods
for diagnosis
Slapped face appearanceof fifth disease
PapovaviridaePapillomavirusPolyomavirus
Infectious and oncogenic or potentially oncogenic DNA
viruses
Papillomavirus
Diseases:
skin and anogenital warts, benign head and neck tumors, cervical and anal intraepithelial neoplasia and cancer
HPV types 16, 18, & 45 = 94% Cervical CA HPV types 6 and 11 = 90% Genital warts Pap Smear for detection Hybrid capture DNA probe for detection and typing PCR – FDA cleared platforms for detection/typing Gardasil vaccine = To guard against HPV 6,11,16,18
Pap smear
Polyomavirus
JC virus [John Cunningham] Cause of Progressive multifocal
leukoencephalopathy - Encephalitis of immune suppressed Destroys oligodendrocytes in brain
BK virus Causes latent virus infection in kidney Progression due to immune suppression Hemorrhagic cystitis
Histology/PCR for diagnosis
Giant Glial Cells of JCV
HepadnavirusHepatitis B
Hepatitis B virus Enveloped DNA – Hepadna virus Hepatitis B clinical disease
90% acute 1% fulminant 9% chronic
Carrier state can lead to cirrhosis and hepatic cell carcinoma
newer therapies – stops disease progression
Vaccinate to prevent
Hepatitis B Serology Surface Antigen Positive
Active Hepatitis B or Chronic Carrier Do Hep B Quantitation Do Hep e antigen – Chronic and “bad”
Core Antibody Positive Immune due to prior infection, acute infection or chronic carrierSurface Antibody Positive Immune due to prior infection or vaccine
FlaviviridaeRNA Viruses
Hepacivirus – Hepatitis CFlavivirus – West Nile, Dengue,
and Yellow Fever
Hepatitis C virus
Spread parenterally - drug abuse, blood products or organ transplants (prior to 1992), poorly sterilized medical equipment, sexual (low risk)
Effects only humans and chimpanzees Approx 3.2 mil persons in USA have chronic Hep C Seven major genotypes (1-7)
Acute self limited disease that progresses to a disease that mainly affects the liver
Infection persists in @ 75-85%/ no symptoms 5 - 20 % develop cirrhosis 1-5 % associated with hepatocellular CA Require liver transplantation
Hepatitis C Diagnosis:
Hepatitis C antibody test If antibody positive do: RNA qualitative or quantitative assay for
viral load Requires Genotyping for proper therapy
Type 1 Hep C most common in USA No vaccine – Antivirals currently in clinical trials
and/ or FDA cleared that can cure >= 85% of infected with Hepatitis C
Flaviviruses – Mosquito borne Dengue – “breakbone fever”
Aedes mosquito / Asia and the Pacific Fever, severe joint pain, rash Small % progress to hemorragic fever
West Nile Common across the US, Bird primary reservoir Fever, Headache, Muscle weakness, 80%
asymptomatic. Small % progress to encephalitis. Meningitis, flaccid paralysis
Mosquitoes – Aedes & CulexImmunoassays for Antibody & PCR
Serum and CSF
Alpha virus – Mosquito borne
Chikungunya virus – RNA virus Mosquito borne – Aedes mosquito Origin in Asia and African continents Recent migration to the Caribbean and SE
USA with mosquito migration Travel advisory to the Caribbean
Acute febrile illness with rash followed by extreme joint pain, less fatalities than Dengue / no hemorrhagic phase
Diagnosis – Serology(IgM, IgG) and PCR
Ebola Virus >20 outbreaks since discovery in 1976
current outbreak Dec 2013 - West Africa Prolonged due to area effected is high population
with limited medical facilities Transmission direct contact with bodily fluids – fatality
rate 55% Animal reservoir (?) fruit bats
Asymptomatic are not contagious Fever, weakness, myalgias, headache, travel history
Consider malaria and typhoid
Susceptible to hospital disinfectants Testing (EIA, PCR) at CDC – pos >= 4 days of illness
Coronovirus/SARS Severe Acute Respiratory Syndrome (SARS) Outbreak in China 2003 – spread to 29 countries Incubation period of 2-10 days 2-7 days by dry cough and/or shortness of breath Development of radiographically confirmed pneumonia by
day 7-10 of illness Lymphopenia in most cases Laboratory testing for SARS-CoV available at state public
health laboratories. Available tests include antibody testing enzyme immunoassay (EIA) and reverse transcription polymerase chain reaction (RT-PCR) tests for respiratory, blood, and stool specimens. In the absence of person-to-person transmission of SARS-CoV, the positive predictive value of a diagnostic test is extremely low.
Coronovirus/SARS
MERS CoV- Middle East Respiratory Syndrome Coronavirus Isolated to Arabian peninsula (2012)
Close human to human contact can spread infection – no outbreaks
2 unrelated cases in US from travel Fever, rhinorrhea, cough, and malaise
followed by shortness of breath – 30% fatality rate
NP, Lower respiratory specimen and serum for PCR at CDC
PicornaviridaeEnterovirusesHepatitis A
PicornaviridaeEnterovirusesHepatitis A
Enteroviruses Diverse group of > 60 viruses – SS RNA
Infections occur most often in summer and fall Polio virus - paralysis
Salk vaccine Inactive Polio Vaccine (IPV)** Sabine vaccine Live Attenuated Vaccine (OPV)
Coxsackie A – Herpangina Coxsackie B – Pericarditis/Myocarditis Enterovirus – Aseptic meningitis in children,
hemorrhagic conjunctivitis Echovirus – various infections, intestine Rhinoviruses – common cold Grow in cell culture (Diploid mixed cell – Primary
Monkey Kidney) PCR superior for diagnosis of meningitis (CSF) and
more rapid and sensitive for all sites
CPE of EnterovirusTeardrop and kite like cells inRhesus Monkey Kidney cell culture
Hepatitis A Fecal – oral transmission Can be cultured but not reliably Usually – short incubation, abrupt onset, low mortality,
no carrier state Travel Diagnosis – serology, IgM positive in early infection Vaccine available
Orthomyxoviruses
Influenza virus AInfluenza virus B
Influenza A Segmented RNA genome Hemagglutinin and Neuraminidase glycoproteins spikes
on outside of viral capsid Give Influenza A the H and N designations – such as H1N1
and H3N2 Antigenic drift - minor change in the amino acids of
either the H or N glycoprotein Cross antibody protection will still exist so an
epidemic will not occur Antigenic shift - genome re assortment with a “new”
virus created/usually from a bird or animal/ this could create a potential pandemic H5N1 = Avian Influenza H1N1 = 2009 Influenza A
Influenzae ADisease: fever, malaise …. death
Diagnosis Cell culture obsolete [RMK] Enzyme immunoassay (EIA) on paper membrane can be
used in outpatient setting – Rapid but low sensitivity (60%) and can have specificity issues in off season.
Amplification (PCR) gold standard for Influenza detection
Treatment: Amantadine and Tamiflu (Oseltamivir) Seasonal variation in susceptibility
Vaccinate to prevent Influenza B
Milder form of Influenza like illness Usually <=10% of cases /year
Paramyxoviruses – SS RNA
MeaslesParainfluenza 1,2,3,4
MumpsRespiratory Syncytial VirusHuman Metapneumovirus
Measles
Measles Fever, Rash, Dry Cough, Runny Nose,
Sore throat, inflamed eyes (photosensitive) Can invade lung (see HE of Lung)
Respiratory spread - very contagious Koplik’s spots – bluish discoloration inner lining
of the cheek Subacute sclerosing panencephalitis [SSPE]
Rare chronic degenerative neurological disease Persistent infection with mutated measles virus due
to lack of immune response Diagnosis: Clinical symptoms and Serology Vaccinate – MMR (Measles, Mumps, Rubella) vaccine Treatment: Immune globulin, vitamin A
Measles syncytium
Parainfluenzae Types 1,2,3, and 4 Person to person spread Disease:
Upper respiratory tract infection in adults – more serious in immune suppressed
Croup, bronchiolitis and pneumonia in children
Heteroploid cell lines (Hep-2) for culture PCR methods are the gold standard Supportive therapy
Mumps Person to person contact Classic infection is Parotitis, but can cause infections in other sites:
Testes/ovaries, Eye, Inner ear, CNS
Diagnosis: clinical symptoms, serology available Prevention: MMR vaccine No specific therapy, supportive
Respiratory Syncytial Virus Transmission:
Hand contact and respiratory droplets Respiratory disease - from common cold to
pneumonia, bronchiolitis to croup, serious disease in immune suppressed
Classic disease: Young infant with bronchiolitis
Specimen: Naso-phayrngeal, nasal swab, nasal lavage
Diagnosis: EIA, cell culture (heteroploid cell lines), PCR is standard practice
Treatment: Supportive, ribavirin
Classic CPE = Syncytium formationIn heteroploid cell lineRespiratory syncytial virus CPE
Histology
Human Metapneumovirus 1st discovered in 2001 – community acquired
respiratory tract disease in the winter Common in young children – but can be seen in all
age groups @95% of cases in children <6 years of age Upper respiratory tract disease 2nd only to RSV in the cause of bronchiolitis
Will not grow in cell culture Amplification (PCR) for detection
Specimen: Nasal swab or NP Treatment: Supportive
Reoviridae
Rotavirus
Rotavirus
Winter - spring season 6m-2 yrs of age, Gastroenteritis with vomiting and fluid loss –
most common cause of severe diarrhea in children
Fecal – oral spread Major cause of death in 3rd world Diagnosis – cannot grow in cell culture
Enzyme immunoassay, PCR Vaccine available
Calciviruses
Norovirus
Norovirus Spread by contaminated food and water, feces
& vomitus – takes <=20 virus particles to cause infection – so highly contagious
Tagged the “Cruise line virus” – numerous reported food borne epidemics on land and sea
Leading cause of epidemic gastroenteritis – more virulent GII.4 Sydney since spring 2012 Fluid loss from vomiting can be debilitating
Disease course usually limited, 24-48 hours PCR for diagnosis
Cannot be grown in cell culture
RetrovirusRNA Virus/Reverse Transcriptase Enzyme
Human Immunodeficiency VirusHIV
Human Immunodeficiency virus
CD4 primary receptor to gain entry for HIV into the lymphocyte Reverse transcriptase enzyme converts genomic RNA into DNA Transmission - sexual, blood and blood product
exposure, perinatal Non infectious complications:
Lymphoma, KS, Anal cell CA, non Hodgkins Lymphoma
HIV Laboratory DiagnosisAntibody EIA with Western Blot confirmation (old way)
Antibody test alone is NOT sufficient – all positive must be confirmed with a western blot test
Western blot detects gp160/gp120 (envelope proteins), p 24 (core), and p41(reverse trans)
Must have at least 2 solid bands on Western blot to confirm as a positive result
New test - Antigen/antibody combination (4th generation) immunoassay* that detects HIV-1 and HIV-2 antibodies and HIV-1 p24 antigen to screen for established infection with HIV-1 or HIV-2 and for acute infection
Positive patients on either test require additional testing: HIV RNA/DNA quantitation >= 100 copies Resistance Testing – report subtype
Most isolates in USA type BMonitor CD4 counts for infection severity
HIV infectious complications Non-compliant patients or newly diagnosed
Pneumocystis Cryptococcus neoformans & Histoplasma
(disseminated) TB/Mycobacterium avium complex
(disseminated) Microsporidia and Cryptosporidium (stool) Hepatitis B Hepatitis C STD’s – Syphilis, GC, Chlamydia
Syphilis rate high (mucosal contact)
Togaviridae
RNA VirusRubella
Rubella
Known as the “Three day measles” – German measlesCongenital rubella – occurs in a developing fetus of a pregnant women who has contracted Rubella, highest % (50%) in the first trimester pregnancy
Deafness, eye abnormalities, congenital heart diseaseRespirastory transmissionDiagnosis - Serology in combination with clinical symptoms – Rash, low fever, cervical lymphadenopathyLive attenuated vaccine (MMR) to prevent
Bunyaviridaeenveloped RNA viruses
Hantavirus
Hantavirus
USA outbreak in four corners (NM,AZ,CO,UT) Indian reservation in 1993 brought attention to this virus
Source - Urine and secretions of wild field mice Deer mouse and cotton rat most implicated
Myalgia, headache, cough and respiratory failure
Found in states west of the Mississippi River Diagnosis by serology/ no therapy
Poxviruses
Smallpox virus (Variola virus)Vaccinia virus
Poxviruses
Smallpox Smallpox virus is also known as the Variola virus Vaccinia virus is the strain used in Smallpox vaccine, it is
immunologically related to smallpox, Vaccinia can cause disease in the immune suppressed, which prevents vaccination of this population
Last case of Smallpox - Somalia in 1977 Disease begins as maculopapular rash and progresses to
vesicular rash - all lesions in same stage of developemnt in body area – rash moves from central body outward
Category A Bioterrorism agent (can maim or kill) Requires BSL4 laboratory (self contained lab) Any potential cases directly reported to public health
department – they will investigate and diagnose
Chicken pox – Lesions in different stage of development
Smallpox – all lesions same stage of development
Chickenpox vs Smallpox lesions
Rhabdovirusesbullet shaped RNA virus
Rabies virus
Rabies
Worldwide in animal populations Bat and raccoons primary reservoir in US Dogs in 3rd world countries
Post exposure shots PRIOR to the development of symptoms prevent infection
Rabies is a neurologic disease – classic sympton is salivation, due to paralysis of throat muscles
Detection of viral particles in the brain by Histologic staining known as Negri bodies is diagnostic
Public health department should be contacted to assist with diagnosis
Rabies virus particlesEM showing the bullet shaped virus
Negri bodies – Intracytoplasmicbrain biopsy specimen