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Virology Review
Just the basics!
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Diagnostic techniques used in the Virology Laboratory
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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
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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
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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
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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
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Viral Cell culture
Cytopathic effect – CPE Appearance of cell monolayer after being
infected with a virus Specific for each virus type
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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
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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
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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
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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
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Herpesviridae
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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
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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
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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
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Negative fibroblast cell Culture -uninfected cells
HSV infected monolayerRounded cells throughout the monolayer in cell culture
Multinucleated Giant Cellsof Herpes Simplex in tissue histology
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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
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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
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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
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CMV pneumonia with viral inclusions
CMV infected fibroblast monolayer - Focal grape like clusters of rounded cells
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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
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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
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VCA = viral capsid antibodyEBNA = Epstein Barr nuclear antigenEA = early antigen
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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
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Adenovirus
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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
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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
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Smudge cells- Adenovirus
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Parvoviridae – ParvovirusThe smallest known viruses!
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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
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PapovaviridaePapillomavirusPolyomavirus
Infectious and oncogenic or potentially oncogenic DNA
viruses
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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
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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
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HepadnavirusHepatitis B
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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
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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
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FlaviviridaeRNA Viruses
HepacivirusHepatitis C
FlavivirusWest NileDengueYellow Fever
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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
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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
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Flaviviruses – Mosquito borne
St. Louis Dengue – breakbone fever Yellow fever West Nile
Fever, Headache, Muscle weakness
Various species Mosquitoes
Serology / PCR
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PicornaviridaeEnterovirusesHepatitis A
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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)
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CPE of EnterovirusTeardrop and kite like cells inRhesus Monkey Kidney cell culture
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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
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Orthomyxoviruses
Influenza virus AInfluenza virus B
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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
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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
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Paramyxoviruses – SS RNA
MeaslesParainfluenza 1,2,3,4
MumpsRespiratory Syncytial VirusHuman Metapneumovirus
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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
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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
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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
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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
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Classic CPE = Syncytium formationIn heteroploid cell lineRespiratory syncytial virus CPE
Histology
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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
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Reoviridae
Rotavirus
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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
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Calciviruses
Norovirus
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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
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RetrovirusRNA Virus/Reverse Transcriptase Enzyme
Human Immunodeficiency VirusHIV
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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
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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
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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)
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Togaviridae
RNA VirusRubella
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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
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Bunyaviridaeenveloped RNA viruses
Hantavirus
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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
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Poxviruses
Smallpox virus (Variola virus)Vaccinia virus
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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
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Chicken pox – Lesions of different stage of development
Smallpox – all lesions same stage of development
Chickenpox vs Smallpox lesions
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Rhabdovirusesbullet shaped RNA virus
Rabies virus
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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
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Rabies virus particles EM showing the bullet shaped virus
Negri bodies – intracytoplasmic