Herpesviruses are a leading cause of human viral diseases and include herpes simplex virus types 1 and 2, varicella zoster virus, cytomegalovirus, Epstein-Barr virus, and human herpesvirus 8. They are capable of causing overt disease during primary infection or remaining latent in sensory ganglia or lymphocytes. Herpesviruses can be reactivated from latency to cause recurrent disease. Laboratory diagnosis involves virus isolation in cell culture, antigen or antibody detection, PCR, and histopathological examination of clinical samples. Treatment options include acyclovir, valacyclovir, and ganciclovir depending on the infecting virus.

1. HERPESVIRUSES
Dr. Pendru Raghunath Reddy

2. Introduction
• Herpes viruses are a leading cause of human viral
diseases, second only to influenza and cold viruses
• Are capable of causing overt disease or remaining silent for
many years only to be reactivated
• Name Herpes comes from the Latin herpes which, in
turn, comes from the Greek word herpein which means to
creep

3. Morphology
 100 – 200 nm in diameter, contains an icosahedral capsid containing
linear double stranded DNA genome
 Surrounded by a lipid envelope containing peplomers
 Between capsid and envelope is tegument

4. Classification of Human Herpesviruses
Subfamily
Scientific name
Common name
Alphaherpesvirinae
Human herpesvirus 1
Human herpesvirus 2
Human herpesvirus 3
Herpes simplex virus type 1
Herpes simplex virus type 2
Varicella-zoster virus
Betaherpesvirinae
Human
Human
Human
Human
Gammaherpesvirinae
Human herpesvirus 4
Human herpesvirus 8
herpesvirus
herpesvirus
herpesvirus
herpesvirus
5
6
6a
7
Cytomegalovirus
Epstein-Barr (EB) virus
Kaposi’s sarcoma associated
virus

5. Herpes Simplex Virus (HSV)
 These are very large viruses and their genome (150 kb)
encodes at least 80 proteins
 Almost any human cell type can be affected by HSV
 The genome of HSV-1 and HSV-2 share 50 - 70% homology
 Man is the only natural host
 HSV-1 is usually associated with oral and occular lesions
 HSV-2 is responsible for the majority of genital infections

6. Pathogenesis
Source of infection
Saliva, skin lesions or respiratory secretions
from patients or carriers
Mode of transmission
Direct contact with virus containing secretions or
with lesions
HSV 1: Kissing or saliva contaminated fingers
HSV 2 : Sexual intercourse, congenital infection

7.  HSV is shed in saliva, tears, genital and other secretions
 The primary infection occurs through the skin, oral mucous
membranes, or eyes resulting in a vesicle formation
 The virus spreads to draining lymphnodes producing lymphadenitis
 After primary infection the virus travels by retrograde intra-axonal flow
to sensory root ganglia
 They settle within the neurons in the ganglia (trigeminal-HSV-1;
sacral-HSV-2)
 The Herpesvirus DNA gets integrated into the host cell genome
 These get reactivated when provoked by various stimuli
(common cold, fever, pneumonia, stress, exposure to sunlight etc)

9. Diseases caused by HSV-1
1. Acute gingivostomatitis
2. Herpes labialis
3. Keratoconjunctivitis
4. Eczema herpeticum
5. Encephalitis
6. Dendritic keratitis

10.  Acute gingivostomatitis is the
commonest manifestation of primary
herpetic infection
 The patient experiences pain and
bleeding of the gums
 1 - 8 mm ulcers with necrotic bases
are present
 Neck glands are commonly enlarged
accompanied by fever
 Usually a self limiting disease which
lasts around 13 days
Gingivostomatitis

11.  Following primary infection, 45% of
orally infected individuals will
experience reactivation
 Herpes labialis (cold sore) is a
recurrence of oral HSV
 A prodrome of tingling, warmth or
itching at the site usually heralds the
recurrence
 About 12 hours later, red-ness
appears followed by papules and
then vesicles
Herpes labialis
(cold sore)

12.  This is found in children with
active eczema
 The virus can spread to other
organs such as the liver
and adrenals
Eczema herpeticum

13. Diseases caused by HSV-2
1. Genital herpes (penis, urethra, cervix, vulva, vagina)
2. Neonatal herpes
3. Aseptic meningitis
Note: Besides above mentioned primary infections, herpesviruses may
present as latent infection, reactivation and recrudescence

14. Herpes Simplex Viruses (HSV)
Characteristic
Urogenital infections
Nongenital infections:
Labialis
Keratitis
Whitlow (hand)
Encephalitis (adult)
Neonatal infection
Transmission
HSV-1
10 - 30%
HSV-2
70 - 80%
80 - 90 %
+
+
+
30 %
primarily
non-genital
10 - 20 %
+
70 %
primarily
genital

15. HSV-1 and HSV-2 cross react serologically. They can be differentiated
by the following features
 By using specific monoclonal antibodies
 HSV-2 forms larger pocks on chick embryo CAM
 HSV-2 replicates well in chick embryo fibroblast cells
 HSV-2 is more neurovirulent in laboratory animals
 The infectivity of HSV-2 is more temparature sensitive than that
of HSV-1
 HSV-2 is more resistant to antiviral agents
 Restriction endonuclease analysis of viral DNA

16. Laboratory diagnosis
Specimens
Vesicle fluid, skin swab, saliva, corneal scrapings, brain biopsy and CSF
according to the site of invovlement
1. Direct examination
Smears prepared from base of vesicles are stained with toludine blue.
multinucleated giant cells with faceted nuclei and homogeneously
stained chromatin (Tzanck cells) are present in a positive smear

17. Cowdry type A intranuclear inclusion bodies may be seen in
Giemsa stained smears
Herpes virions may be demonstrated in specimens by
electron microscopy
Viral antigens can also be demonstrated in the scrapings from the
base of the lesions and tissue preparation by immunofluorescence

18. 2. Cell culture
 Virus can be isolated on human fibroblasts, HEp-2 cells, Vero cells
and CAM of embryonated egg
 Swollen, rounded cells may appear within 1-5 days
 Diagnosis can be confirmed by immunofluorescent staining of
infected cell culture
Cytopathic effect of HSV
in cell culture: Note the
ballooning of cells

19. 3. Serology
Primary infection can be diagnosed by detection of virus specific
IgM antibody or by a rising titre of antibody
Various tests like CFT, neutralisation, immunofluorescence, ELISA
and RIA have been employed for antibody detection
4. PCR
Chemotherapy
 HSV infection can be treated with acyclovir (acycloguanosine)
 Valaciclovir and famciclovir are more effective oral agents
 When resistance to these drugs develop, drugs like trisodium
-phosphonoformate (Foscarnet) may be useful

20. Varicella-Zoster
 Varicella (chickenpox) and herpes zoster (shingles) are caused
by a single virus
 Chickenpox follows primary infection in a non-immune individual,
whereas herpes zoster is a reactivation of the latent virus
 VZV is similar to the HSV in its morphology
 Only one antigenic type of VZV is known

21. Epidemiology
• Primary varicella is an endemic disease. Varicella is one of the
classic diseases of childhood, with the highest prevalence occurring
in the 4 - 10 years old age group
• Varicella is highly communicable, with an attack rate of 90% in
close contacts
• Most people become infected before adulthood but 10% of young
adults remain susceptible
• Herpes zoster, in contrast, occurs sporadically and evenly
throughout the year

22. Varicella
Pathogenesis
Source of infection
Patient with varicella or zoster
Mode of transmission
Inhalation of respiratory droplets
Sometimes through conjunctiva

23. Pathogenesis
• The virus is thought to gain entry via the respiratory tract and
spreads shortly after to the lymphoid system
• After an incubation period of 14 days, the virus arrives at its main
target organ, the skin
• Following the primary infection, the virus remains latent in the
cerebral or posterior root ganglia. In 10 - 20% of individuals, a single
recurrent infection occurs after several decades
• The virus reactivates in the ganglion and tracks down the sensory
nerve to the area of the skin innervated by the nerve, producing a
varicellaform rash in the distribution of a dermatome

24. • Primary infection results in varicella (chickenpox)
• Incubation period of 7-23 days
• Presents fever, a widespread vesicular rash mostly on the trunk
• The features are so characteristic that a diagnosis can usually be
made on clinical grounds alone
• Complications are rare but occurs more frequently and with greater
severity in adults and immunocompromised patients
• Most common complication is secondary bacterial infection of the
vesicles
• Severe complications which may be life threatening include viral
pneumonia, encephalititis, and haemorrhagic chickenpox

25. Rash of Chickenpox

26. Congenital Varicella Infection
• 90% of pregnant women already immune, therefore
primary infection is rare during pregnancy
• Primary infection during pregnancy carries a greater risk
of severe disease, in particular pneumonia
First 20 weeks of Pregnancy
• Up to 3% chance of transmission to the fetus, recognised
congenital varicella syndrome;
• Scarring of skin
• Hypoplasia of limbs
• CNS and eye defects
• Death in infancy normal

27. Herpes Zoster (Shingles)
 Herpes Zoster mainly affect a single dermatome of the skin
 It may occur at any age but the vast majority of patients are more
than 50 years of age
 The latent virus reactivates in a sensory ganglion and tracks
down the sensory nerve to the appropriate segment
 The reactivation is associated with the inflammation of the nerve
which leads to neuritic pain that often precedes the skin lesions
 There is a characteristic eruption of vesicles in the dermatome
which is often accompanied by intensive pain which may last for
months (postherpetic neuralgia)

28.  Herpes
zoster affecting the eye (ophthalmic zoster) and face
(Ramsay Hunt syndrome affecting the facial nerve) may pose
great problems
 As with varicella, herpes zoster in a far greater problem in
immunocompromised patients in whom the reactivation occurs
earlier in life and multiple attacks occur as well as complications
 Complications are rare and include encephalitis and disseminated
herpes zoster
Shingles

29. Laboratory diagnosis
The clinical presentations of varicella or zoster are so characteristic
that laboratory confirmation is rarely required. Laboratory diagnosis is
required only for atypical presentations, particularly in the
immunocompromised
1. Direct Microscopy
 Toludine blue or Giemsa stained smears from the base of early
vesicles show multinucleated giant cells and type A intranuclear
inclusion bodies under light microscope
 Eelectron microscopy may be used but cannot distinguish between
HSV and VZV
 Immunofluorescense using monoclonal antibody on skin scrappings
can distinguish between the two

30. 2. Virus isolation
 VZV isolated in human fibroblast cells, human amnion, HeLa or
Vero cells
 Cytopathic effect is focal with refractile ballooned cells
 Virus antigen can be demonstrated in nuclear inclusions by
immunofluorescence using monoclonal antibody
Cytopathic effect
of VZV

31. 3. PCR
4. Serology
specific IgM antibody in patient’s serum can be
detected by ELISA
 Varicella-zoster
 Other methods such as CFT, neutralisation test and
immunofluorescence can be used for detection of
VZV-specific antibodies
Treatment
Acyclovir and vidarabine are effective in the treatment of severe
varicella and zoster

32. Cytomegalovirus
• Belong to the betaherpesvirus subfamily of herpesviruses
• Double stranded DNA enveloped virus and 150-200 nm in size
• The virus exhibits strict host-specificity
• Cytomegalovirus infections are almost always inapparent,
leading to prolonged latency, with occasional reactivation
• Cytomegalovirus disease is rare but infection with the virus is
extremely common

33. Pathogenesis
Mode of transmission
• The virus may be transmitted from person to person in several
ways (in utero, perinatally, or postnatally)
• Perinatal infection is acquired mainly through infected genital
secretions, or breast milk. Overall, 2 - 10% of infants are infected
by the age of 6 months worldwide. Perinatal infection is thought to
be 10 times more common than congenital infection
• Postnatal infection mainly occurs through saliva. Sexual
transmission may occur as well as through blood and blood
products and transplanted organ

34. • Initial replication of the virus occurs in the epithelial cells of
the respiratory and GI tracts which is followed by viraemia
resulting in infections of all organs of the body
• In symptomatic cases, renal tubular epithelium, liver and
CNS are also affected
• The virus establishes lifelong latent infection in
monocytes, B lymphocytes, epithelial cells and stromal
cells of bone marrow and in some organs like kidneys and
heart
• Once infected, the virus remains in the person for life and
may be reactivated from time to time, especially in
immunocompromised individuals

35. Clinical Manifestations
• Congenital infection - may remain inapparent at birth or may lead to
cytomegalic inclusion disease which is often fatal
• The disease is characterised by hepatosplenomegaly, jaundice,
thrombocytopenic purpura, microcephaly and chorioretinitis
• Perinatal infection - usually asymptomatic
• Postnatal infection - usually asymptomatic. However, in a minority of
cases, the syndrome of infectious mononucleosis may develop
which consists of fever, malaise, fatigue, and splenomegaly. The
heterophil antibody test is negative although atypical lymphocytes
may be found in the blood

36. • Immunocompromised patients such as transplant recipients and
AIDS patients are prone to severe cytomegalic inclusion disease
characterised by hepatosplenomegaly, jaundice, thrombocytopenic
purpura, pneumonia and micro cephaly
• Reactivation or reinfection with CMV is usually asymptomatic
except in immunocompromised patients

37. Laboratory diagnosis
Specimens
CMV can be isolated from urine, saliva, breast milk, semen,
cervical secretions and blood leucocytes
Demonstration of Cytomegalic cells
Enlarged cells with large intranuclear “owl’s eye” appearance
inclusions (cytomegalic cells) can be demonstrated in the
centrifuged deposits from urine or saliva

38. Isolation of virus
 Virus can be grown in human fibroblast cultures
 The virus replicates very slowly, therefore, cytopathic effects
(swollen refractile cells with cytoplasmic granules)
may take 2-3 weeks to appear
 For precise identification, these cultures may be stained
by immunofluorescence using monoclonal antibody
Cytopathic effect
of CMV

39. Antigen detection
CMV antigen can be detected from blood leucocytes using
monoclonal antibodies
PCR
Serology
CMV-specific IgM can be detected in the serum by ELISA

40. Treatment
For treatment of severe CMV infections, ganciclovir is the drug of
choice
Prophylaxis
 Indicated only in high risk cases such as organ transplants,
premature infants, immunodeficient persons
 Screening of blood and organ donors
 Administration of CMV immunoglobulins and Acyclovir
 No vaccine is available

41. Epstein-Barr virus
 Belong to the gammaherpesvirus subfamily of herpesviruses
 EBV has affinity for lymphoid tissue. The B lymphocytes of
human beings have receptors (CD21 molecules) for EBV
 EBV infected B lymphocytes are transformed in such a way
that they multiply continuously

42. Pathogenesis
Source of infection
Saliva of infected persons
Mode of transmission
Intimate oral contact, as in kissing appears to be main mode
of transmission

43.  The virus enters the pharyngeal epithelial cell through CD21
(or CR2) receptors
 It multiplies locally, enters the blood stream and infects
B lymphocytes
 In most cases, the virus remains latent inside the lymphocytes,
which become transformed
 The activated B lymphoblast, along with the antigen of EBV,
matures into a long-lived memory lymphocyte in the
germinal centre of the lymph node
This leads to polyclonal activation of the infected B lymphocytes
resulting in production of many kinds of antibodies to many antigens
-accompanied by nonspecific increase in total IgM, IgG and IgA

44.  EB virus antigens are expressed on the surface of infected
B lymphocytes. T lymphocytes will be activated in response
to such neoantigens
 In immunocompetent persons, proliferation of transformed
B lymphocytes is kept in check by activated T lymphocytes
 In the immunodeficient persons, lymphomas may occur because
of unchecked replication of B lymphocyte clones

45. Clinical manifestations
 Most EBV infections are inapparent
 Once infected, the virus is present in the individual for life
 The following clinical manifestations may result from EBV infection
1. Infectious mononucleosis
2. Infections in immunocompromised hosts
3. EBV-associated malignancies

46. Infectious Mononucleosis
(Glandular fever)
It is an acute self-limiting disease of children and young adults
characterised by fever, sore throat, lymphadenopathy and
splenomegaly
In some patients jaundice may be seen which is due to hepatitis
Abnormal lymphocytes are present in the blood
The incubation period is 4-7 weeks and infection is believed to occur
through respiratory route by close contact with patients
Complications occur rarely but may be serious e.g. splenic
rupture, meningoencephalitis, and pharyngeal obstruction

47. In some patients, chronic IM may occur where eventually
the patient dies of lymphoproliferative disease or lymphoma
EBV activates B lymphocytes and leads to secretion of
immunoglobulins (IgM antibodies)
2. Infections in immunocompromised hosts
EBV may cause progressive lymphoproliferative disease in
immunodeficient children, transplant recipients and AIDS patients

48. 3. EBV-associated malignancies
a) Burkitt’s lymphoma
 It is a malignant neoplasm of B-lymphocytes (tumour of jaw)
which occurs in regions of Africa and New Guinea
 The disease occurs in endemic or sporadic type
 Cells of Burkitt’s lymphoma carry multiple copies of EBV genome
b) Nasopharyngeal carcinoma
c) B-cell lymphoma

49. Burkitt’s lymphoma
affecting jaw
Burkitt's lymphoma showing
disruption of teeth and partial
obstruction of airway

50. Laboratory diagnosis
1. White blood cell count
 During the initial phase, patient develops leucopenia
 Later there is leucocytosis with a predominance of abnormal or
atypical lymphocytes
2. Paul-Bunnell test
 During infectious mononucleosis, heterophile antibodies (IgM)
appear in the serum of the patient
 These antibodies agglutinate sheep erythrocytes
 These antibodies appear in 85-90% of patients sera during
the acute phase of illness

51. Procedure
 Inactivated serum in doubling dilutions is mixed with equal
volumes of 1% sheep erythrocytes suspension
 These tubes incubated at 370C for 4 h and examined for
agglutination
 A titre of 100 or above is suggestive of infectious mononucleosis

52. Confirmation
Absorption test for Paul – Bunnell antibody
Absorption with
Guinea pig kidney
Ox red cells
Normal serum
Absorbed
Not absorbed
Antibody after
serum injections
Absorbed
Absorbed
Infectious
mononucleosis
Not absorbed
Absorbed

53. 3. EBV – specific antibodies
 The IgM antibody to EBV viral capsid antigen (VCA) appears
soon after primary infection and disappears in 1 – 2 weeks
 The IgG antibody to VCA persists throughout life and is
an indication of past or recent infection
 These can be demonstrated by indirect immunofluorescence
or ELISA
 Antibody to the EBV nuclear antigen (EBNA) is also a reliable
marker for primary infection
 Antibodies to early antigens (EA) can be demonstrated in
EB – associated lymphomas

54. 4. Antigen detection
 EBV antigen can be detected by immunofluorescence using
monoclonal antibodies
 EBNA1 is very important antigen
5. Virus isolation
 Saliva or throat washings and peripheral blood cells can be
inoculated onto lymphocytes
 If specimen contains EBV, it produces a lymphoblastoid cell line
6. Nucleic acid hybridisation
7. PCR

55. Human Herpesvirus 6
 HHV – 6 infects dividing CD4+ T lymphocytes
 Saliva is the main route of transmission
 Most HHV – 6 infections appear to be asymptomatic
 They may, however cause exanthem subitum or roseola infantum
and infectious mononucleosis – like disease with cervical
lymphadenopathy
 HHV – 6 can be isolated from peripheral blood mononuclear cells
in early febrile stage of the illness by co – cultivation
with lymphocytes

56.  Primary HHV-6 or HHV-7 infection is associated with
Roseola Infantum, which is a classical disease of childhood
 Most cases occur in infants between the ages of 4 months
and two years
 A spiking fever develops over a period of 2 days followed by
a mild rash
 The fever is high enough to cause febrile convulsions
 There are reports that the disease may be complicated by
encephalitis
Roseola infantum

57.  Human Herpesvirus 8 is associated with Kaposi’s sarcoma,
which is the commonest tumour in HIV infected individuals
Kaposi’s sarcoma