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Virology Review 
Margie Morgan, PhD, MT(ASCP), 
D(ABMM) 
microbeswithmorgan.com
Diagnostic techniques used in 
the Virology Laboratory 
1.Direct Staining for Antigen 
2. Enzyme Immunoassay 
3. 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 
Tzanck 
Tzanck 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 diagnosis 
Herpes 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 monolayer 
Rounded cells throughout 
the monolayer in cell culture 
Multinucleated Giant Cells 
of 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 
monolayer 
Require 5- 7 days to 
develop 
Sandpaper look to the 
Monolayer background with 
scattered 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” Inclusions 
Treatment: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 with 
B 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 antibody 
EBNA = Epstein Barr nuclear antigen 
EA = 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) 
Round cells with 
stranding 
 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
Smudge cells- Adenovirus
Parvoviridae – Parvovirus 
The 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 appearance 
of fifth disease
Papovaviridae 
Papillomavirus 
Polyomavirus 
Infectious and oncogenic or 
potentially oncogenic DNA 
viruses
Papillomavirus 
 Diseases: 
Pap smear 
 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
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
Hepadnavirus 
Hepatitis 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 carrier 
Surface Antibody Positive 
 Immune due to prior infection or vaccine
Flaviviridae 
RNA Viruses 
Hepacivirus – Hepatitis C 
Flavivirus – 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 & Culex 
 Immunoassays 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.
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
Picornaviridae 
Enteroviruses 
Hepatitis 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 Enterovirus 
Teardrop and kite like cells in 
Rhesus 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 A 
Influenza 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 A 
Disease: 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 
Measles 
Parainfluenza 1,2,3,4 
Mumps 
Respiratory Syncytial Virus 
Human Metapneumovirus
Measles 
 Measles 
Measles syncytium 
 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
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 formation 
In heteroploid cell line 
Respiratory 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
Retrovirus 
RNA Virus/Reverse Transcriptase Enzyme 
Human Immunodeficiency Virus 
HIV
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 Diagnosis 
Antibody 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 B 
 Monitor 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 Virus 
Rubella
Rubella 
 Known as the “Three day measles” – German measles 
 Congenital 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 disease 
 Respirastory transmission 
 Diagnosis - Serology in combination with clinical 
symptoms – Rash, low fever, cervical 
lymphadenopathy 
 Live attenuated vaccine (MMR) to prevent
Bunyaviridae 
enveloped 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
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
Chickenpox vs Smallpox lesions 
Chicken pox – Lesions in 
different stage of development 
Smallpox – all lesions same 
stage of development
Rhabdoviruses 
bullet 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 particles 
EM showing the bullet 
shaped virus 
Negri bodies – 
Intracytoplasmic 
brain biopsy specimen

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Virology 2014

  • 1. Virology Review Margie Morgan, PhD, MT(ASCP), D(ABMM) microbeswithmorgan.com
  • 2. Diagnostic techniques used in the Virology Laboratory 1.Direct Staining for Antigen 2. Enzyme Immunoassay 3. Molecular Amplification 4. Viral Cell Culture
  • 3. 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 Tzanck Tzanck DFA
  • 4. 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
  • 5. 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
  • 6. 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
  • 7. 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
  • 8. 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
  • 9. 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
  • 10. 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
  • 11. 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
  • 13. 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
  • 14. 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)
  • 15. Herpes virus diagnosis Herpes 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
  • 16. Negative fibroblast cell Culture -uninfected cells HSV infected monolayer Rounded cells throughout the monolayer in cell culture Multinucleated Giant Cells of Herpes Simplex in tissue histology
  • 17. 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
  • 18. Varicella-Zoster Diagnosis In cell culture – Limited # of Foci in monolayer Require 5- 7 days to develop Sandpaper look to the Monolayer background with scattered rounded cells - diploid fibroblast monolayer Younger wet vesicular lesions area the best for culture and/or molecular testing
  • 19. 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” Inclusions Treatment:ganciclovir, foscarnet, cidofovir
  • 20. CMV pneumonia with viral inclusions CMV infected fibroblast monolayer - Focal grape like clusters of rounded cells
  • 21. 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 with B cell transformatin
  • 22. 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
  • 23. VCA = viral capsid antibody EBNA = Epstein Barr nuclear antigen EA = early antigen
  • 24. 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
  • 26. 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
  • 27. Adenovirus  Diagnosis  Conventional cell culture (CPE) Round cells with stranding  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
  • 29. Parvoviridae – Parvovirus The smallest known viruses!
  • 30. 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 appearance of fifth disease
  • 31. Papovaviridae Papillomavirus Polyomavirus Infectious and oncogenic or potentially oncogenic DNA viruses
  • 32. Papillomavirus  Diseases: Pap smear  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
  • 33. 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
  • 35. 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
  • 36. 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 carrier Surface Antibody Positive  Immune due to prior infection or vaccine
  • 37. Flaviviridae RNA Viruses Hepacivirus – Hepatitis C Flavivirus – West Nile, Dengue, and Yellow Fever
  • 38. 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
  • 39. 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
  • 40. 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 & Culex  Immunoassays for Antibody & PCR  Serum and CSF
  • 41. 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
  • 42. 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
  • 43. 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.
  • 44. 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
  • 46. 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
  • 47. CPE of Enterovirus Teardrop and kite like cells in Rhesus Monkey Kidney cell culture
  • 48. 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
  • 49. Orthomyxoviruses Influenza virus A Influenza virus B
  • 50. 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
  • 51. Influenzae A Disease: 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
  • 52. Paramyxoviruses – SS RNA Measles Parainfluenza 1,2,3,4 Mumps Respiratory Syncytial Virus Human Metapneumovirus
  • 53. Measles  Measles Measles syncytium  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
  • 54. 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
  • 55. 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
  • 56. 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
  • 57. Classic CPE = Syncytium formation In heteroploid cell line Respiratory syncytial virus CPE Histology
  • 58. 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
  • 60. 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
  • 62. 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
  • 63. Retrovirus RNA Virus/Reverse Transcriptase Enzyme Human Immunodeficiency Virus HIV
  • 64. 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
  • 65. HIV Laboratory Diagnosis Antibody 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 B  Monitor CD4 counts for infection severity
  • 66. 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)
  • 68. Rubella  Known as the “Three day measles” – German measles  Congenital 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 disease  Respirastory transmission  Diagnosis - Serology in combination with clinical symptoms – Rash, low fever, cervical lymphadenopathy  Live attenuated vaccine (MMR) to prevent
  • 69. Bunyaviridae enveloped RNA viruses Hantavirus
  • 70. 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
  • 71. Poxviruses Smallpox virus (Variola virus) Vaccinia virus
  • 72. 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
  • 73. Chickenpox vs Smallpox lesions Chicken pox – Lesions in different stage of development Smallpox – all lesions same stage of development
  • 74. Rhabdoviruses bullet shaped RNA virus Rabies virus
  • 75. 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
  • 76. Rabies virus particles EM showing the bullet shaped virus Negri bodies – Intracytoplasmic brain biopsy specimen