Virology 2014

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