Epstein barr virus [autosaved]

SYNOPSIS
Introduction
History
Epidemiology
Route of transmission
Pathogenesis
Immune response
Clinical features
Complications
Diagnosis
Treatment and pRevention

Introduction
Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus.
EBV - heterophile-positive infectious mononucleosis in late adolescence or
early adulthood. Largely subclinical in early childhood.
benign, selflimited lymphoproliferative disorder, and is associated with the
pathogenesis of several human tumors, most commonly certain lymphomas
and nasopharyngeal carcinoma

History
In 1889, German physician “Pfeiffer”
fever
lymphadenopathy
malaise
hepatosplenomegaly
abdominal discomfort in adolescents and young adults
In the early 1900s
numerous case descriptions of illnesses epidemiologically and clinically compatible with IM.
In 1932, Paul and Bunnell
Identified heterophile antibodies in serum during acute IM.
FEIGIN et al. Textbook of Pediatric Infectious Diseases5th ed;2004:1952-1957.

In 1958, Dennis Burkitt
described 38 cases of “round-cell sarcoma” in children and adolescent living in
Uganda, Africa. (Lymphoma)
In 1964, Epstein and Barr
described the first human tumor virus in a Burkitt lymphoma cell line by EM
 In 1966 Gertrude and Werner Henle
described an immunoluorescent antibody test for the virus
and found that patients with Burkitt lymphoma as well
as most Americans had antibody to EBV

In 1968, Henle
reported the relationship between acute IM and EBV
In 1970
In 1982
non-Hodgkin lymphoma
in patients with acquired immunodeiciency syndrome
(AIDS) ,
in T-cell lymphoma in 1988, and in
Hodgkin lymphoma in 1989
the virus was found in nasopharyngeal carcinoma

Global burden
SUB-SAHARAN
AFRICA
NORTH AFRICA

Roth GA, Abate D, Abate KH, et al

In fact, 43% of all global incident cases of these four malignancies and 40% of
all deaths were in east asia
 The reasons for the high prevalence of these two malignancies in this
region is not clear. It is believed that a combination of genetic and
environmental risk factors are involved (salt foods, smoking)
 HL, this study shows that around 40% of all cases worldwide are EBV-attributed
and this fraction varies not only by gender and age, but also by geographical
region

In India
About 95% of the population has been exposed to this virus by the age of 40,
but only 15%–20% of teenagers and about 40% of those adults exposed to the virus
become infected.
70% of nasopharyngeal carcinomas in India due to EBV
Both HL and NHL -very rare
EBV 1 is most common

Family- Herpesviridae,
Subfamily - Gamaherpesvirinae,
Genus - Lymphocryptovirus
Species - Human herpesvirus 4
 EBV types-1 and 2 (also named types A and B) based on (EBNA-
LP), EBNA-2,
EBNA-3A, EBNA-3B, and EBNA-3C
 Type 1 – predominant in worldwide distribution
Type 2 – Africa

Structure
EBV has a toroid-shaped DNA core in a nucleocapsid with 162 capsomeres,
an outer envelope with
External glycoprotein spikes, and a protein tegument between the
nucleocapsid and envelope
Major capsid protein- p160
Small capsid proteins – p18,p23,p40

Genome
Linear , double-stranded DNA approximately 172 kb in length
B95-8 strain- first EBV genome completely cloned and sequenced
Mutated strains of EBV 1 –B95-8, raji ,daudi, P3HR-1
EBV has a series of 0.5 kb terminal direct repeats (TRs) and internal repeat
sequences (IRs) that divide the genome into short and long, largely unique sequence
domains.

oriP
A single contiguous fragment of EBV DNA with a length of 1.7 kbps supports
autonomous extrachromosomal replication and maintenance of recombinant
plasmids in human cells.
The viral protein EBNA1 binds to oriP site-specifically and recruits the cellular
DNA replication machinery
(Baer et al. 1984)

EBV latent genes
EBNA1
EBNA1 is a DNA-binding protein that is required for the replication and maintenance of the EBV
genome;
EBNA1 also acts as a transcriptional transactivator of ‘orip’ and and the LMP1 promoter
EBNA2
EBNA2 is a transcriptional activator of both cellular and viral genes, and up-regulates the
expression of certain B-cell antigens, including CD21 and CD23, as well as LMP1 and LMP2
EBNA3
Studies with EBV recombinants have demonstrated that EBNA3A and EBNA3C are essential for
B-cell transformation in vitro, whereas EBNA3B is dispensable . EBNA3C can induce the up-
regulation of both cellular (CD21) and viral (LMP1) gene expression

LMP1
LMP1 functions as a constitutively activated
member of the tumor necrosis factor receptor
(TNFR) superfamily
Up-regulation of anti-apoptotic proteins (Bcl-
2)
Activate NFkB transcription factor
Through MAP kinase pathway activate
JAK/STAT
Activate the phosphatidylinositol 3-kinase
(PI3-K) pathway resulting in a variety of effects
including cell survival mediated through the
Akt (PKB) kinase, actin

Inhibit tumour suppressor P53
modulates the G1-S cell-cycle checkpoint
LMP1 induces the expression of pro-inflammatory
Cytokines
LMP1 imparts resistance to apoptosis
LMP1 induces an epithelial-mesenchyme-transition (EMT)
LMP1 promotes angiogenesis

LMP2
Two distinct proteins- LMP2A and LMP2B
LMP2A can drive the proliferation and survival of B-cells in the absence of signaling through the BCR. A
role for LMP2B in regulating LMP2A function has been suggested
LMP2 in modifying the normal programme of B-cell development to favor the maintenance of EBV
latency and to prevent inappropriate activation of the EBV lytic cycle
doi: https://doi.org/10.1371/journal.pone.0183856.g001

EBV associated small rna’s (EBER1)(EBER2)
EBER1 and EBER2 were functional back-ups of viral oncoprotein LMP-
1, whichactivated the oncogenic PI3K/Akt signaling pathway .
Up-regulated expression of a Bcl-2 protein
Inhibit double stranded RNA dependent protein kinase PKR –tumour
suppressor
 EBV produces a homologue of IL-10 (vIL-10), which inhibits
macrophages and dendritic cells and suppresses antiviral T cell
responses.

Host
Over 90% of adults are infected with EBV.
Most common- 2-4 years and around 15 years
In sexually active young adults
Immunocompromised
Blood transfusion and bone marrow transplantation
Males affected more than females

Primary EBV infection
In developing countries
-80-100% of children becoming infected by 3-6 yrs of age
-clinically silent or mild disease.
In developed countries
-occurs later in life, 10-30 years of age
-induce clinically mononucleosis syndrome

Route of transmission
Oral secretion:
Kissing
Median EBV oral loads peaked at 4.8 log10 copies/mL
(63,000 copies/mL) a median of 2 months after onset of infection and
persisted for a median of 5.2 months
Blood products,Transplanted organs :
less commonly than CMV
Intrauterine
Infrequent , if infected; no adverse fetal outcomes and no viral transmission to the fetus.

Pathogenesis
Mainly Source of infection : saliva
During primary infection, EBV infects epithelial cells
in Waldeyer’s ring in the oropharynx
where it replicates and then can infect resting naïve
B cells or may infect resting, naïve B cells in the
tonsilar crypts directly,
 Virus replication has been found in rare tonsilar
epithelial cells andin epithelial cells during infectious
mononucleosis (IM)

INITIAL EVENTS:
Chandran B, Hutt-Fletcher L. 2007

Fusogen gB and gH/gL.-For entry into epithelial cells
For infection of B lymphocytes-the B-cell receptor-binding protein gp42 is required
.
The EBV membrane glycoprotein gp42 binds to its cell surface receptor major histocompatibility complex
class II (MHC-II) to initiate entry into the cell.
Also, gp350/220 binds to its cell surface receptor CD21 for entry.
 gB CTD - cellular localization, cell surface expression, fusion function, and fusion regulation
Following endocytosis, the virion and packaged tegument proteins are released into the cytoplasm
following fusion of the virion membrane with endosomal membrane
The mechanism of how receptor binding by gH/gL or gp42 triggers fusion mediated by gB is still unknown.

Up to 10% of B cells in the blood early in infection are EBV positive. Most
of these cells are thought to be killed by NK cells, antibody-dependent
cell-mediated cytotoxicity (ADCC), or cytotoxic T cells.
Latent replication
Differentiation into plasma cells and
either shed virus into the saliva directly
or infect epithelial cells
Lytic replication
Ebv infected B cells

REPLICATION CYCLE
Cohen JI. Epstein-Barr virus infection. N Engl J Med 2000;343[7]:481–492.)

Latent and lytic cycle
NEJM;343:481-492.

Contact with
infected saliva
Memory B cells
GC- centroblasts to
centrocytes
Daughter memory
cells
proliferating B
blasts
Plasma cells
Shedding via saliva
Infect naïve B cells
Lytic cycle begins
Latency 3
Latency 2
Latency 0
Latency 1

Viral replication
Lymphoreticular system
Liver
Spleen
B lymphocytes in peripheral blood
People with X-linked agammaglobulinemia, who lack B cells, do not
become latently infected with EBV or shed virus, suggesting that B cells
are the main reservoir of infection.
While EBV is present in B cells in the blood of healthy Individuals.EBV
has been detected in the blood in cells other than B cells including CD4
and CD8 T cells in patients with HIV and NK cells in patients with chronic
active EBV disease

oncogenesis
Pathogenesis of EBV-associated carcinoma involves a complex
interplay between different patterns of viral gene expression and cellular
genetic changes
Activation of
proto-oncogene
Defect in DNA
repair
Carcinogenesis
Inhibition of
tumour
suppressor gene
Affecting gene
regulating
apoptosis

Activation of proto oncogene
C-MYC activation by translocation:
MYC is a master transcriptional regulator that increases the expression
of genes that are required for aerobic glycolysis, the so-called Warburg
effect.
The translocation partner for MYC is usually the IgH locus [t(8;14)
Activation of NFkB:
 EBV+ tumor cells express latent membrane protein-1(LMP-1), a protein
encoded by the EBV genome that transmits signals that up-regulate NF-
κB result of acquired loss-of-function mutations in IκB

Inhibition of tumour suppressor gene
Inhibit P53 tumour suppressor
EBERs inhibit the activity of the double-stranded RNA-dependent protein kinase PKR,
which is act as a tumor suppressor
Apoptosis dysregulation
Upregulation of BCl-2 ,BCl6
Cell cycle dysregulation
Induce signalling pathways of JAK/STAT & PI3K/AKT

Lung RW, Hau PM, Yu KH, Yip KY, Tong JH, Chak WP et al
(2018)

Immune response
Innate immune response
Toll-like, RIG-I-like, NOD-like, and
AIM2-like receptors as well as cyclic
GMP-AMP synthase and JAK-STAT
Pontejo, S.M.; Murphy, P.M.; Pease, J.E. Chemokine subversion by human herpesviruses. J.
Innate Immun.2018

Antibody response
Acute EBV infection induces polyclonal B-cell activation with elevated levels of IgG, IgM, and IgA.
In addition to heterophile antibody, IgM and IgG antibodies
to the viral capsid antigen (VCA) are usually present at
the onset of symptoms of IM
Later in infection antibody to EBNA is detected and IgM
antibody to VCA disappears. IgG antibody to VCAand
EBNA persists for life.
Antibodies to EBV early antigens (EAs)-diffuse (EA-D,
diffusely in the nucleus and cytoplasm) or restricted (EA-
R, restricted to the cytoplasme). EA-D antibodies are often
present 3 to 4
weeks after the onset of IM, especially in patients with
severe
illness, and are frequently detected in patients with
nasopharyngeal carcinoma (NPC) or chronic active EBV
(CAEBV).
EA-R antibodies are often detected in African BL.
From Cohen JI. Epstein-Barrvirus. In: Young NS, Gerson SL, High KA, eds.Clinical
Hematology. Philadelphia: Elsevier,2006:956–966, copyright © 2006 Elsevier

Cellular immunity
CD4 cells,
CD8 cells,
NK cells
( Küppers, 2003; Kutok and Wang, 2006; Hislop et al., 2007; Rickinson et al., 2014; Cohen, 2015a; Taylor
et al., 2015; Thorley-Lawson, 2015).

During primary EBV infection in IM patients, the absolute number of peripheral
blood CD8+ T cells increases 5–10-fold compared with asymptomatic individuals
Activated CD8+ T cells are elevated serum levels of proinflammatory and
immunoregulatory cytokines (IFN-γ, TNF, IL-6, IL-10, and TGF-β
Asymptomatic - 15% of all CD8+ T cells & ∼2–5% of memory CD8+ T cells
EBV-specific CD4+ T cells can comprise up to 1% of all circulating CD4+ T cells in
IM patients,

Immune escape
International Journal of Biological Sciences 14: 0565 image No. 002

Clinical features
Incubation period : 30 – 50 days
Silent, nonspecific infections : in children
prolonged low-grade fever + lymphadenopathy
cough
rhinorrhea
pharyngitis

Infectious mononucleosis (IM)
prodrome
2 – 5 days malaise, fatigue, possibly fever
acute phase
fever (last 4–5 wks), lymphadenopathy (2–4 wks),
tonsillopharyngitis, splenomegaly, hepatomegaly, rash,
abdominal pain, eyelid edema 15%

Pharyngitis is the most consistent physical finding.
1/3 of patients : exudative pharyngitis.

25-60% of patients : petechiae at the junction of the hard and soft palates.
Tonsillar enlargement can be massive, and occasionally it causes airway
obstruction

Lymphadenopathy : 90%
symmetrical enlargement.
mildly tender to palpation and not fix.
posterior cervical lymph nodes.
anterior cervical and submandibular nodes.
axillary and inguinal nodes.
Enlarged epitrochlear nodes are very suggestive of infectious mononucleosis.

Maculopapular rash : 15%
usually faint, widely scattered, and erythematous
occurs in 3-15% of patients and is more common in young children.
80% of patients, treatment with amoxicillin or ampicillin is
associated with rash
Circulating immunoglobulin G (IgG) and
immunoglobulin M (IgM) antibodies to ampicillin are demonstrable NEJM;343:481-492.

Luzuriaga K, Sullivan JL. Infectious mononucleosis. N Engl J Med. 2010;362:1993-
2000.

EBV 90% of acute IM
Etiology of most EBV-negative IM : unknown
Other Herpesviruses :
 Cytomegalovirus (CMV)
 herpes simplex 1 and simplex 2
 human herpesvirus 6
Other viruses :
 adenovirus
 hepatitis A, hepatitis B, or hepatitis C
 rubella
 primary human immunodeficiency virus in adolescents or young adults.

EBV-associated lymphoproliferative disease
congenital or acquired immunodeficiency,
severe combined immunodeficiency, patients with AIDS,
and recipients of bone marrow or organ transplants who are receiving immunosuppressive drugs
Outcome:
Most patients with this syndrome die of acute IM. Others
develop hypogammaglobulinemia, malignant B cell lymphomas,
aplastic anemia, or agranulocytosis.
Oral hairy leukoplakia
Early manifestation of infection with HIV in adults
Most patients present with raised, white corrugated lesions on the tongue (and occasionally on
the buccal mucosa) that contain EBV DNA.

Complications
Hepatitis
Splenic rupure
Airway compromise
Rheumatoid arthritis
Multiple sclerosis

EBV induced multiple sclerosis

Sample: serum, plasma, blood, saliva,tissue
Diagnosis
Atypical
lymphocytosis
serology
Heterophile antibody
test
Molecular

Complete blood count
80-90% of patients have lymphocytosis during 2-3 weeks of illness and lasts for 2-6 weeks
20-40% of the lymphocytes : atypical
Atypical lymphocytosis greater than 10% seen in up to 90% cases, but is not specific for Epstein
Barr virus (EBV).

Heterophile antibody
50% in first week of illness
60-90% in the second or third weeks
Begins to decline during the fourth or fifth week and often is less than 1:40 by 2-3 months after
symptom onset
20% of patients have positive titers 1-2 years after acquisition
Heterophile Ab test
Paul Bunnell test Mono spot test
The Paul-Bunnell
Daividsohn Differential

Paul Bunnell-davidsohn test
A modification of the Paul-Bunnell test that differentiates among three types of heterophile
sheep agglutinins: those associated with infectious mononucleosis and serum sickness, and
natural antibodies against Forssman antigen.
Dis adv: TIME consuming procedure

Mono spot
Qualitative detection of IM heterophil antibodies in human serum,
plasma and whole blood using direct solid-phase immunoassay
technology.
A band of bovine (Ox) erythrocyte extracts are impregnated in the test
membrane.
If IM-specific heterophil antibody is present in the sample, it will be
captured by the bovine erythrocyte extracts.

Though the monospot test is considered to be a very specific test, the
sensitivity falls in the range of 70 to 90% .
Disadvantages :
False positive;
IM heterophil has been associated with disease states such as:
Burkitt’s Lymphoma, viral hepatitis, adenovirus, leukemia,
cytomegalovirus, rheumatoid arthritis and Toxoplasma gondii.

Serology
EAs (early antigens) :
Early in the lytic cycle
EA-D (diffuse-staining component of EA) : 80% , Positive nasopha ca
EA-R (restricted component of early antigens):Burkitt lymphoma
EA is rising at symptom onset : rise for 3-4 weeks, then quickly decline to undetectable levels by
3-4 months, although low levels may be detected intermittently for years.
VCA (Viral capsid antigen)
late in the lytic cycle
N- terminal of full length p23 and the carboxy half of p18
VCA-IgM usually is measurable at symptom onset, peaks at 2-3 weeks, then declines and unmeasurable by
3-4 months.
VCA-IgG rises shortly after symptom onset, peaks at 2-3 months, then drops slightly but persists for life.

EBNA (Epstein-Barr nuclear antigen) :
Latent infection
EBNA-1 protein is expressed in all EBV infected cells, and IgG against this protein is a late
marker of EBV infection
EBNA-1 IgG antibodies appear late, 3 to 6 months after the time of disease, then they decline
but continue to be present in a detectable level for life
Indicates past or recovering EBV infection .
 because EBNA-IgG is never developed in around 5–10% of EBV infected healthy individuals,
and this percentage is higher in immunocompromised patients
They are measured with enzyme immunoassays and indirect
immunofluorescence assays

Gieß RM, Pfuhl C, Behrens JR, Rasche L, Freitag E, Khalighy N, et al. Epstein-Barr virus antibodies in
serum

Molecular
Real time PCR
Real-time PCR is when the amplified DNA is detected as the reaction
progresses in real time.
Test has 95% sensitivity and 97% specificity for primary EBV infection.
Is expensive and not commonly used in clinical practice.

Treatment
Medical Care :
self-limited illness : not require specific therapy. Bed rest ,analgesics & good hydration.
Inpatient therapy of medical and surgical complications may be required
Corticosteroids -swelling of pharyngitis, airway obstruction, severe thrombocytopenia, and hemolytic anemia.
prednisolone (40–60 mg/d or 2–3 days, with subsequent tapering o the dose over 1–2 weeks)
Acyclovir (10 mg/kg/dose IV q8h for 7-10 d)
inhibit viral shedding from the oropharynx
Effective for oral hairy leukoplakia

Prevention
Avoid contact with saliva
Do not kiss children on the mouth.
Maintain clean conditions : avoid sharing toys
Vaccines : not available currently
The first EBV vaccine trial in humans used live recombinant vaccinia virus
expressing gp350 (Gu et al. 1995)

Epstein barr virus [autosaved]

Epstein barr virus [autosaved]

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