Virology Review

Just the basics!

Diagnostic techniques used in the Virology Laboratory

Laboratory diagnosis Direct antigen detection from lesions

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

Can provide a HSV and VZV diagnosis More sensitive and specific than Tzanck prep (DFA 80% vs. Tzanck 50%) Tzanck prep= Giemsa stain/examine for multinucleated giant cells of Herpes virus

Tzanck

Rapid detection of viral antigens

Enzyme immunoassay – Antigen/antibody complex formed – then

combined with color forming compound Detection of non-culturable viruses – such

as Rotavirus Detection of Influenza A and B , and

Respiratory syncytial virus (RSV) Membrane EIA Liquid/well EIA

Molecular Detection Amplification of DNA or RNA

Rapid results Exceed sensitivity of culture

Standard of practice for detecting respiratory viruses Standard of practice for HSV and Enterovirus

detection in CSF Culture <=20% PCR >=90%

CMV quantitative assays in transplantation 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

Viral cell culture Inner wall coated with monolayer of cells

covered with liquid maintenance media Primary cell lines – directly from animal

into tube (Rhesus monkey kidney-RMK) Diploid cell lines– Can survive 20 – 50

passes into new vials – fibroblast cells MRC-5-(Microbiology Research Council 5)

human diploid fibroblasts Continuous cell lines – can survive

continuous passage into new vials, usually of tumor lineage, HEp-2 and HeLa

Viral Cell culture

Cytopathic effect – CPE Appearance of cell monolayer after being

infected with a virus Specific for each virus type

Spin Down Shell Vial MethodDesigned to speed up virus recoveryCells are on the round coverslipSpecimen put into vialCentrifugation to induce virus invasionIncubate 24 - 72 hoursDFA stain cells on coverslip with early antigen for virus of interest

Cover slip

Specimen collection and transport Viral transport media- Hanks balanced salt solution with antibiotics, 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

Viral specimens are filtered prior to being placed on cell monolayer to eliminate bacteria

Which viruses will survive the trip to the laboratory?

Most likely viable - HSV Intermediate

Adenovirus Influenza A and B Enterovirus

Least likely to survive Respiratory Syncytial Virus (RSV) Cytomegalovirus (CMV) Varicella Zoster virus (VZV) Amplification preferred due to survival issue

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

Molecular 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

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 for diagnosis Cytology/Histology - intra nuclear inclusions, multinucleated giant cells Serology – Most 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 +/- eliminated due to effective vaccine program – most serious disease occurs in immune suppressed or adult patient – can progress 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 Foci5- 7 days to developSandpaper look to the BackgroundScattered rounded cells

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, close contact Latency: Macrophages Disease: Asymptomatic in most individuals infected

Congenital – most common cause Perinatal Immunocompromised – Primary disease most

serious Diagnosis: Cell culture CPE (Human diploid fibroblast ,

PCR and quantitative PCR Histopathology: Intranuclear and intracytoplasmic inclusions “Owl Eye” Inclusions

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 for diagnosis

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 in 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 – new virulent respiratory strain / pneumonia

Pharyngitis (year round epidemics) Gastroenteritis in children

Adenovirus types 40 & 41 Keratoconjuctivitis 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 Histology - Intranuclear inclusions / smudge cells Stool EIA for enteric infections Antigen detection – staining respiratory cells by DFA

for Respiratory infections PCR – has become the standard of practice 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) fetal infection and stillbirths aplastic crisis in patients with chronic hemolytic anemia and AIDS Histology - virus effects 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 and 18 = 70% Cervical CA HPV types 6 and 11 = 90% Genital warts Pap Smear for detection Hybrid capture DNA probe for detection and typing PCR – FDA approved platforms for detection/typing Guardasil vaccine = To guard against HPV 6,11,16,18

Pap smear

Polyomavirus JC virus [John Cunningham]

Cause of Progressive multifocal leukoencephalopathy - E

ncephalitis 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 Virion called Dane particle Surface Ag (HBsAg)- Australian Ag Clinical Disease

90% acute 1% fulminant 9% chronic

Carrier state …….Hepatic cell carcinoma

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

FlaviviridaeRNA Viruses

HepacivirusHepatitis C

FlavivirusWest NileDengueYellow Fever

Hepatitis C virus

Spread parenterally - drug abuse, blood products, poorly sterilized medical equipment, sexual

Effects only humans and chimpanzees Seven major genotypes

Acute self limited disease to start with progressive disease that mainly affects the liver

Infection persists in 80% 20 - 30 % develop cirrhosis Associated with hepatocellular CA Require liver transplantation

Hepatitis C

Diagnosis: Hep C antibody test If antibody positive do: RNA qualitative or quantitative assay for

viral load Requires Genotyping for proper therapy

Type 1 most common No vaccine – Antivirals currently in clinical trials

that can cure a large % of infected

Flaviviruses – Mosquito borne

St. Louis Dengue – breakbone fever Yellow fever West Nile

Fever, Headache, Muscle weakness

Various species Mosquitoes

Serology / PCR

PicornaviridaeEnterovirusesHepatitis A

Enteroviruses Diverse group of > 60 viruses

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)

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 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 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) 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 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

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 Diagnosis

Antibody EIA with Western Blot confirmation Antibody test alone is NOT sufficient Western blot detects gp160/gp120 (envelope

proteins), p 24 (core), and p41(reverse trans) Must have at least 2 bands on Western blot to

confirm the diagnosis of HIV

Positive patients 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 C. neoformans and Histoplasma TB/Mycobacterium avium complex Microsporidia and Cryptosporidium Hepatitis B Hepatitis C STD’s – Syphilis, GC, Chlamydia

Syphilis rate high (mucosal contact)

Togaviridae

RNA VirusRubella

Rubella

Respiratory transmission Known as the “Three day measles” – German measles Congenital rubella – occurs in a developing fetus of a

pregnant women who has contracted Rubella, highest % in the first trimester pregnancy

Diagnosis - Serology in combination with clinical symptoms – Rash, low fever, cervical lymphadenopathy

Live 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

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 = vaccine strain used in Smallpox

vaccine, it is immunologically related to smallpox, Vaccinia can cause disease in the immune suppressed, which prevents vaccinated this population

Last case of Smallpox - Somalia in 1977 Disease begins as maculopapular rash and progresses

to vesicular rash - all lesions in same stage on a body area – rash moves from central body outward

Potential agent of Bioterrorism Any potential cases directly reported to public health

department – they will investigate and diagnose

Chicken pox – Lesions of 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

Public health department should be contacted to assist with diagnosis

Rabies virus particles EM showing the bullet shaped virus

Negri bodies – intracytoplasmic