1. HERPES VIRIDAE
WITH SPECIAL EMPHASIS ON LAB
DIAGNOSIS
PRESENTED BY: DR TABEEN MANSOOR
MODERATED BY: DR GULNAZ BASHIR-ADDITIONAL PROFESSOR
2. INTRODUCTION
■ Herpes is derived from the Greek
word “herpein” meaning to creep.
■ Historically used to describe the
spreading ulcerating lesion
associated with HSV infections.
Creeping lesion in Herpes
3. CHARACTERISTICS
■ Large(100-200nm)
■ Double stranded DNA virus
■ Consist of a 4 layered structure
Nucleic acid core
Capsid
Tegument
Envelope
■ Replicate in the host cell nucleus.
■ Cowdry type A inclusion bodies.
4. CHARACTERISTICS
■ The life cycle of all herpes viruses in their natural host can be divided into
■ LYTIC PHASE
Virus replicates
Results in the production of infectious progeny.
LATENT PHASE
Viral replication is suppressed.
Quiescent state.
The establishment of viral latency is a hallmark of all known herpesviruses.
6. CLASSIFICATION
ALPHA HERPESVIRIDAE
■ Rapid growth
■ Short replicative cycle
■ Wide host range
■ Neurotropic herpesviruses
■ Latent infection in sensory
ganglia
■ HSV-1,HSV-2,VZV(HHV-3)
BETA HERPESVIRIDAE
■ Slow growth
■ Restricted host range
■ Long replicative cycle results in carrier
state.
■ Grow best in fibroblasts
■ Latent infection in salivary glands,
lymphocytes,renal cells
■ CMV(HHV-5),HHV-6,HHV-7
7. CLASSIFICATION
GAMMA HERPESVIRIDAE
■ Grow in lymphoblastoid cells
■ Latent infection in lymphoid tissue
■ Further divided into two genera,Lymphocryptovirus and Rhadinovirus.
■ EBV(HHV-4),HHV-8
9. HSV-1 & HSV-2
■ Belong to the genus simplexvirus
■ Transmission: Direct contact with infected secretions.
■ Site of Latency: Sensory nerve ganglia.
In orofacial herpes trigeminal ganglia are mostly involved.
In genital herpes sacral root ganglia(S2-S5) mostly involved.
■ Neurovirulence: Capacity to invade and replicate in the NS.
10. HSV-1 & HSV-2
Reactivation: Induced by
Fever
Trauma
Emotional stress
Sunlight
Menstruation
Immunocompromisation.
HSV-1 reactivates more frequently
in oral regions
HSV-2 reactivates 8-10 times more
in genital regions.
12. OROFACIAL INFECTIONS
Most common presentation of
primary herpes.
More severe than Herpes labilais.
Accompanied by
fever
myalgia
irritability
tender cervical
lymphadenopathy
APT difficult to distinguish from
ACUTE GINGIVOSTOMATITIS ACUTE PHARYNGOTONSILLITIS
13. HERPES LABIALIS
■ Also called cold sores/fever blisters.
■ Most common manifestation of recurrent HSV-
1 infection.
■ Prodrome of pain, burning,tingling at site.
■ Erythrematous papules.
■ Thin walled intra epidermal vesicles.
■ Pustular and ulcerative.
■ Sores heal in 2-3 weeks.
15. GENITAL HERPES
■ More often by HSV-2
■ But HSV-1 can also cause
■ Primary genital herpes is severe.
■ Lasts upto 3 weeks.
■ Characterized by vesicoulcerative lesions of
■ Penis,Cervix,vulva,vagina,perineum.
■ Complications include extragenital site
ulcers and aseptic meningitis.
■ Person sheds the virus for 3 weeks.
■ Recurrence is common but a milder form.
16. CONGENITAL HERPES
■ Acquired in-utero, during or after birth.
■ Mother is the most common source.
■ MC mode is by coming in contact with a herpetic lesion during passage through birth canal.
■ Post natally acquired by family members or hospital staff shedding the virus.
■ 1/5000 deliveries per year.
■ Manifestation is of 3 types:
■ 1.Lesion localized to skin,eyes,mouth
■ 2.encephalitis with or without local skin involvement
■ 3.Disseminated disease.
■ Dissemination has poor prognosis(80% mortality).Survivors often have neurological
impairment.
17. HSV ENCEPHALITIS
■ Occurs by direct neuronal
transmission from a peripheral site
to brain via trigeminal or olfactory
nerve.
■ In adults & children>3yr-
■ localized to temporal and frontal
lobes.
Caused by HSV-1
■ In neonates generalized brain
involvement.
HSV-2 involved
■ Recurrent form called Mollaret
18. VARICELLA ZOSTER VIRUS(HHV-3)
■ Belongs to the genus Varicellavirus
■ TRANSMISSION: Droplet infection, direct contact with infected lesion.
■ SITE OF LATENCY: Dorsal root ganglia.
■ DISEASES:
Chicken pox
Shingles/Herpes zoster
Keratitis/Herpes ophthalmicus
VZV encephalitis
Myelitis
Herpes zoster oticus & Ramsay Hunt syndrome
20. SHINGLES/HERPES ZOSTER
■ Unilateral vesicular eruption
■ Dermatomal distribution.
■ Thoracic & lumbar
dermatomes MC involved.
■ Eyelids affected when the
1st or 2nd branch of the CN-
V is involved.
Herpes zoster ophthalmicus is a sight
threatening condition.
25. HHV-6&HHV-7
■ TRANSMISSION: Respiratory route
■ SITE OF LATENCY: T lymphocytes(CD4)
■ DISEASES CAUSED
Roseola (Exanthema subitum)
Fever
Malaise
Rash
Leukopenia
Interstitial pneumonitis in organ transplant
recipients. Rash in a patient with Exanthema subitum
30. HHV 8 VIRUS
■ TRANSMISSON: Not known
■ SITE OF LATENCY:
Kaposi’s Tumour Cells,
Endothelial Cells and
Tumour Infiltrating Leukocyctes.
■ DISEASE: Kaposi’s Sarcoma Kaposi’s sarcoma in a patient with AIDS
32. SPECIMEN COLLECTION & TRANSPORT
SKIN SCRAPINGS
For HSV& VZV
Requires a Tzank smear
Should be taken from
base
Virus difficult to isolate
once crusting or
ulcerations occurs
VESICLE FLUID
Vesicle fluid is
aspirated with a
tuberculin syringe.
33. SPECIMEN COLLECTION & TRANSPORT
SWABS
Endocervical
Conjunctival
Throat
Penile
Urethral
Cotton, rayon swabs can be
used.
Alginate swabs inhibit HSV.
34. SPECIMEN COLLECTION & TRANSPORT
TRACHEAL
APSIRATE
CSF & other
sterile body
fluids
AMNIOTIC FLUID
CORD BLOOD
BLOOD
CMV
HSV,VZV(rare)
Heparizined or EDTA
anticoag blood
acceptable for CMV
TISSUE
Lung,Liver,GIT: CMV
Brain: HSV
URINE
CMV can be detected
Yield increases by 2-3 samples.
10 ml of FMU to be taken
SALIVA
SEMEN
35. SPECIMEN COLLECTION & TRANSPORT
CSF/ tracheal aspirates, collected aseptically are transported without
VTM.
Urine is refrigerated before transport and once in lab is diluted 1:1 with
culture medium.
Tissue is placed in VTM in a sterile container.
36. SPECIMEN COLLECTION & TRANSPORT
Sample should be cultured within the first hour
If not possible it should be frozen at -70deg after placing it in VTM
Prolonged storage requires immersion in
Sterile 50% neutral glycerol in saline
Culture medium with 5% fetal bovine serum.
39. ELECTRON MICROSCOPY
■ Useful for detection of viruses that don’t grow in cell culture
■ Works best if 106-107 particles/ml present.
■ Immune EM allows visualization of virus particles present in small numbers.
■ Addition of specific antiserum causes viral particles to form Ag-Ab aggregates
■ EM is labour intensive and insensitive.
■ Not present in routine laboratories.
■ Restriced to research work.
41. TZANCK SMEAR
■ Named after a French dermatologist Arnault Tzanck.
■ Procedure:
■ The vesicle is deroofed.
■ Roof of the vesicle folded back.
■ Excess fluid removed by dabbing with sterile gauze.
■ Clean glass slide pressed against the base of the
ulcer.
■ Cells from the base of the ulcer will stick to the slide.
■ Making an impression smear.
42. MICROSCOPY AND STAINING
■ Methanol fixation
■ Staining by Giemsa, Wrights or Papanicolou stains.
■ Multinucleated giant cells with faceted nuclei.
■ Ground glass appearance (Tzanck cells).
■ Intranuclear eosinophilic inclusion bodies.
■ Tzanck cells are found in
HSV,VZV,CMV and Pemphigus vulgaris.
■ Cannot distinguish HSV-1 & HSV-2.
43. MICROSCOPY AND STAINING
■ Presence of intranuclear inclusion bodies is also demonstrated
Owls eye inclusion bodies of CMVCowdry A inclusion bodies
44. DIRECT FLUORESCENT ANTIGEN STAINING
■ Cells scraped from ulcer bases
■ Lesion is mixed with a HSV specific antibody.
■ If virus present forms a complex with the Ab.
■ This is identified by immuno-fluorescent or immuno-
peroxidase staining.
■ Distinguishes HSV-1& HSV-2.
■ Gives results in 2-3 hours
45. SEROLOGY
ANTIGEN DETECTION:
■ Enzyme immunoassay
Done for HSV-1 & 2.
■ ELISA
ANTIBODY DETECTION
ELISA
Done for EBV
In our lab IgM ELISA for VCA of EBV is done.
CMV& HSV ELISA done routinely as part of the TORCH test.
46. SEROLOGY FOR EBV
EBV induces :
■ Specific antibodies to
EBV
■ Unrelated non-EBV
heterophile antibodies
■ These heterophile Ab
react to antigens from
animal RBCs.
47. PAUL BUNNELL TEST
■ Paul Bunnell Ab were described in 1932 as heterophile Ab on the basis of
their ability to agglutinate sheep RBCs.
■ Ab in a patient with IM react with sheep RBCs and cause agglutination.
■ Test is performed in a series of tubes.
■ Utilizes serial two-fold dilutions of heat inactivated serum
■ Added in the presence of consant volume of sheep cells.
■ Agglutination is scored visually after 2 hours.
■ A positive titer of 224 or higher was considered adequate to support
diagnosis of IM
48. MONOSPOT TEST
■ Heterophile Ab test used for screening of
IM.
■ Patients serum placed on a slide
■ Mixed with Guinea Pig Kidney Antigen
and Preserved Horse Erythrocytes.
■ If heterophile Ab are present the blood
agglutinates.
■ It indicates infectious mononucleosis.
■ Sensitivitiy:70-90% .
■ Specificity:100%
49. SEROLOGY FOR EBV
■ Heterophile Ab.
■ Peak levels -2-6 weeks.
■ Remain positive upto a
year.
■ Although virtual 100%
specificity exists with the
Monospot test rarely false
positive results occur in:
Toxoplasmosis
Rubella
Lymphoma
Malignancies esp leukemia
50. SEROLOGY FOR EBV
ANTIBODY DETECTION:
Targeted against
1.VCA(viral capsid antigen)
2.EA (early antigen)
3.EBNA (Ebstein Barr nuclear antigen) appears after 1-2 months and
persists throughout life
51. ANTICOMPLEMENT IMMUNOFLUORESCECE
TEST (ACIF)
■ EBV infected lymphoblastic cell line
■ Source of antigen- RAJI cells
■ They express only EBNA not EA or VCA
■ These cells are fixed to a slide and
reacted with patient’s serum.
■ Presence of Ab is looked for with the
help of fluorescent microscopy.
■ Also used for CMV and seroconversion
of viral vaccines like VZV.
53. SEROLOGY FOR CMV
Detection of IgM antibody to
■ CMVNA
■ CMV-VCA
■ An anti-CMV immediate early antigen monoclonal Ab assay is
available.
■ This reacts with an early protein and can detect CMV infection 3
hours into the infection.
54. CMV ANTIGENEMIA
■ Defined as detection of CMV pp65 antigen in
leukocytes.
■ The pp65 assay measures messenger matrix
proteins on the CMV virus.
■ By immunofluorescence assay or messenger
RNA amplification.
■ These proteins are typically expressed only
during viral replication.
55. FAMA-Fluorescent antibody to membrane antigen
for VZV
■ Gold standard for assessing immunity to
varicella.
■ Detects seroconversion after vaccination.
■ Uses unfixed VZV infected human embryonic
lung fibroblast (HELF) cells.
■ Incubated with serial 2 fold dilutions of sera.
■ Cells are then washed,incubated.
■ Fixed on a slide & observed using
fluorescence microscopy.
■ More sensitive than CFT,EIA but requires
working with a live virus
56. LATEX AGGLUTINATION FOR VZV
■ Serum is added to a
suspension of latex particles
coated with VZV antigen.
■ If human serum contains Ab to
VZV a reaction occurs causing
visible agglutination.
■ Used to assess antibody levels
to VZV.
57. WESTERN BLOT FOR HSV
■ Gold standard for detection of Ab to HSV.
■ Discriminates HSV-1 and HSV-2.
■ Sera are reacted against separated, fixed protein arrays(blots) from
HSV-1or HSV-2.
■ Patterns of binding bands are highly predictive of infection with HSV-
1 OR HSV-2.
■ Expensive, time and requires skilled interpretation.
■ Currently not available commercially.
58. CELL CULTURE
■ A-549
■ MRC-5
■ McCoy cell line
■ Human diploid fibroblast
■ Rhabdomyosarcoma
■ Mink lung
■ Primary rabbit kidney
■ CV-1
■ Vero
■ Hep-2 cells.
Isolation of the virus on cell culture is the gold standard.
Cell lines used are
59. CELL CULTURE
■ Cell culture should be kept
for atleast 4 weeks.
■ But most viruses show
characteristic cyto pathic
effect within 2 days of
inoculation
■ EBV however does not grow
in cell cultures and has no
CPE.
60. CELL CULTURE FOR HSV
■ Cell line used:Hep-2,HDF
■ CPE description:
Rounded swollen refractile cells
Occasional syncytia, esp with type 2
Rapidly involves entire monolayer.
■ Rate of growth:1-3 days (Upto 7
days)
■ Distinct CPE sufficient to identify
■ Confirm by FA
61. ELVIS(ENZYME INDUCED VIRUS INDUCIBLE
SYSTEM) FOR HSV
■ Modified version of the conventional cell culture.
■ The system employs genetically engineered baby hamster
kidney cells(BHK) called ELVIS cells.
■ These cells bear DNA and HSV inducible promoter gene
chimerically linked to an E coli Lac Z operon.
■ Sequence linked to the reporter gene B-galactosidase.
■ When HSV from patient’s swab infects these ELVIS cells,
the enzyme is switched on.
■ Cells are checked for the presence of using ONPG or X-
gal.
■ This causes infected cells to turn blue.
62. CELL CULTURE FOR VZV
■ Cell line used HDF
■ Characteristic CPE;
Discrete foci of rounded, swollen,
refractile cells
Slowly involves entire monolayer.
■ Rate of growth:5-28 days.
■ Confirm by FA
63. CELL CULTURE FOR CMV
■ Cell line used: HDF
■ Characteristic CPE:
Discrete small foci of rounded cells
■ Rate of growth: 5-28 days
■ Distinct CPE ,sufficient to diagnose
■ Confirm by FA
64. CMV SHELL VIAL ASSAY
■ The shell vial method employs
centrifugation of the patient specimen
onto a cell Monolayer contained in a
small vial called shell vial.
■ Virus may be detected by DFA or IFA
staining within hours or days of
inoculation.
■ Shell vial method is used primarily for
detection of CMV, HSV, and VZV.
■ Cell line used for shell vials is MRC-5
(Human Fibroblast cells).
CMV after grown in shell vial culture
66. MOLECULAR
POLYMERASE CHAIN REACTION
HSV 1&2:
1.HSV glycoprotein B gene.
PCR has been used to detect HSV glycoprotein B gene in HSV-2 as a cause of
recurrent meningitis(Mollaret).
Also useful in herpetic keratitis.
2.Glycoprotein D gene
3.Glycoprotein G gene
4.TK(Thymidine kinase)
5.DNA polymerase POL gene
67. MOLECULAR
CMV:
pp65
Commercially available quantitative PCR assays include COBAS Amplicor CMV
Monitor test.
It measures viremia in the range of 600-100,000 copies/ml
CMV Hybrid Capture
Quantitative assay.
■ Amount of data generated is proportional to amount of target present.
■ Viral load can be calculated.
68. MOLECULAR
EBV
Genes targeted are
■ EBNA1(Ebstein Barr Nuclear Antigen)
■ LMP1(Latent membrane protein 1)
■ LMP2(Latent membrane protein 2)
■ BZLF1(Trancriptional activator)
69. EGG INOCULATION
■ Chorioallantoic membrane (CAM) is mainly employed for HSV.
■ Virus growth and replication indicated by visible lesions-
pocks.
70. ANIMAL INOCULATION
■ Many animal models have been used.
■ Intracranial inoculation of HSV-2 resulted in
neurological manifestations, dissemination and
death of mice.
■ HSV-1 inoculated in anterior chamber of eye
results in ocular disease in mice.
■ Cotton top tamarins inoculated with EBV
frequently develop malignant lymphomas.
Ocular lesions in AC of eye due to HSV-1 inoculation
71. TREATMENT
HSV
■ Antiviral therapy:
Supresses clinical manifestations.
Shortens duration of illness.
Prevents complications like post herpetic neuralgia,
Prevents recurrence.
Latency however still occurs.
Acyclovir, Penciclovir, Famaclovir given for HSV infections.
Ocular herpes: Topical trifluridine, idoxuridine.
HSV encephalitis: IV acyclovir
72. TREATMENT
■ CMV
■ Drug of choice for CMV infections is Ganciclovir and Valgancyclovir.
■ Acyclovir is not given as CMV lacks the enzyme thymidine kinase required to
activate it.
■ EBV
■ No effective antiviral therapy available.
■ Acylovir reduces EBV shedding but is ineffective clinically.
■ Adoptive transfer of EBV reactive T cells has shown promise in
lymphoproliferative disease.
73. TREATMENT
■ HHV-8
■ Introduction of HAART has reduced incidence of KS.
■ Local radiation therapy,cryotherapy and intralesional therapy with
Vinca alkaloids has been used.
■ HHV-6,HHV-7
■ No antiviral therapy available for Roseola infantum.
■ Treatment is supportive.
74. PREVENTION
■ Prevention against most of the herpes viruses includes avoiding contact
with the virus.
■ Vaccination is only available for VZV.
■ Live attenuated vaccine OKA-strain is FDA approved.
■ VariZIG (Varicella zoster immune globulin) is given for passive
immunization.
75. PREVENTION
■ Prevention against CMV infections consists of proper screening of blood
before transfusion.
■ Use CMV negative blood and tissue for blood transfusion and organ
transplantation.
■ CMVIg also used for prophylaxis in transplant patients.
■ Leflunomide is an antimetabolite given off label.
Editor's Notes
Roseola classica presentation:9-12 month infant who develops high fever with febrile seizure.After 3 days rapid defervescence occurs with appearance of morbilliform rash.
Burkitts lymphoma:Malignancy of B cells mc affects jaw in African children.Form of NHL
//Kaposi sarcoma is a spindle cell tumour derived from endothelial cell lineage.It has 4 types:Epidemic of Aids related,Immunocompromised,classic/sporadic,endemic/African.AIDS related variety most aggressive and most common.Involves skin,oral mucosa,ln,visceral organs
*In many respects, β-galactosidase is best recognized for its reaction with X-gal (5-bromo-4-chloro-3-indoyl-β-d-galactopyranoside), a soluble colorless compound consisting of galactose linked to a substituted indole. β-Galactose has high specificity for the galactose part of its substrates but low specificity for the remainder. Thus, it hydrolyzes X-gal, releasing the substituted indole that spontaneously dimerizes to give an insoluble, intensely blue product. On growth medium containing X-gal, colonies of E. coli that have an active β-galactosidase become blue because of this reaction.
*An elvis reporter system uses green fluorescent protein(GFP) which is detected by fluorescent microscopy.
