VIRAL SEROLOGY

GB virus C, formerly known as hepatitis G virus

HEPATITIS VIRUS
Hepatitis
 inflammation of the liver”
 usually accompanied with fever,
nausea, vomiting and jaundice
 Causes: radiation, chemicals, disease
processes (autoimmune disease, viruses
and cancer)
Hepatitis is a general term that means
inflammation of the liver. It can be caused by
several viruses and by noninfectious agents,
including ionizing radiation, chemicals, and
autoimmune processes. The primary hepatitis
viruses affect mainly the liver. Other viruses,
such as cytomegalovirus, Epstein-Barr virus, and
herpes simplex virus, can also produce liver
inflammation, but it is secondary to other
disease processes. This section will focus on the
primary hepatitis viruses.
and also known as human pegivirus – HPgV is a
virus in the family Flaviviridae and a member of
the Pegivirus, is known to infect humans, but is
not known to cause human disease. 
Incidence
Primary hepatitis viruses account for
approximately 95% of the cases of hepatitis.
These viruses are classified as primary hepatitis
viruses because they attack primarily the liver
and have little direct effect on other organ
systems. The secondary viruses involve the liver
secondarily in the course of systemic infection
of another body system. The viruses for
hepatitis types A, B, C, D, E, and GB virus C, as
well as secondary viruses (e.g., EBV, CMV), have
been isolated and identified.
GENERAL CHARACTERISTICS

(HAV), hepatitis B virus (HBV), or hepatitis C

virus (HCV). These unrelated viruses are
transmitted via different routes and have
different epidemiologic profiles. Safe and
effective vaccines have been available for
hepatitis B since 1981 and for hepatitis A since
1995.

ETIOLOGY

2 major groups of acute viral hepatitis: SIGNS AND SYMPTOMS

 Primary Hepatitis: HAV, HBV, HCV,
HDV, HEV, and GB virus C
 Secondary Hepatitis: Epstein-Barr Virus
(EBV), Cytomagalovirus (CMV), and
Herpesvirus

Viral hepatitis is the most common liver disease

worldwide. The viral agents of acute hepatitis
can be divided into two major groups, as
follows:
As a clinical disease, hepatitis can occur in acute
or chronic forms. The signs and symptoms of
hepatitis are extremely variable. It can be mild,
transient, and completely asymptomatic or it
can be severe, prolonged, and ultimately fatal.
Many fatalities are attributed to hepatocellular
carcinoma in which hepatitis viruses B and C are
the primary causes. The course of viral hepatitis
can take one of four forms—acute, fulminant
acute, subclinical without jaundice, and chronic.
HEPATITIS A VIRUS (HAV)
 Etiology: PicoRNAvirus
 Formerly known as “infectious
hepatitis”
 MOT: fecal-oral transmission
HAV is a small, RNA-containing picornavirus and
the onlyhepatitis virus that has been
successfully grown in culture (Fig. 23-1, A). The
structure is a simple nonenveloped virus with a
nucleocapsid designated as the hepatitis A (HA)
antigen(HA Ag). Inside the capsid is a single
molecule of singlestranded ribonucleic acid
(RNA). The RNA has a positivepolarity and
proteins are translated directly from the RNA.
Replication of HAV appears to be limited to the
cytoplasm of the hepatocyte.
The highest titers of HAV are detected in acute-
phase stool samples. Human infectivity of saliva
and urine from patients with acute hepatitis A
does not pose a significant risk. Sexual contact
has been suggested as a possible mode of
transmission.
Hepatitis A virus was formerly called infectious
hepatitis or short-incubation hepatitis because
it is the most rapidly spread type of hepatitis
In developing countries, hepatitis A is primarily
a disease of young children; the prevalence of
infection, as measured by the presence of
antibody (immunoglobulin G [IgG] anti-HAV),
approaches 100% at or shortly after 5 years of
age.
Progress of infection:
 Incubation of 2-7 weeks, may be
asymptomatic or may include jaundice
  Clinical illness develop abruptly and  indicates acute infection, appears 4-5
include fever, anorexia, vomiting, weeks after exposure
fatigue and malaise  disappears in 3-6 months, replaced by
 Increase in serum transaminases (ALT IgG anti-HAV
and AST)
IgG anti-HAV
 RUQ pain, dark urine and pale stool
 Recovery 2-4 weeks, no carrier state  peaks during convalescence and may
 Mortality 0-1% remain detectable for life
Nonimmune adult patients infected with HAV Shortly after the onset of fecal shedding, an IgM
can develop clinical symptoms within 2 to 6 antibody is detectable in serum, followed within
weeks after exposure (average,≈4 weeks). a few days by the appearance of an IgG
However, hepatitis A is often a subclinical antibody. IgM anti-HA is almost always
disease, with many patients being anicteric. detectable in patients with acute HAV. IgG anti-
Clinically apparent cases show elevated serum HAV, a manifestation of immunity, peaks after
liver function enzyme and bilirubin levels, with the acute illness and remains detectable
jaundice developing several days later. Viremia indefinitely, perhaps lifelong.
and fecal shedding of virus disappear at the
The finding of IgM anti-HAV in a patient with
onset of jaundice. Atypical presentations
acute viral hepatitis is highly diagnostic of acute
include prolonged intrahepatic cholestasis,
HAV. Demonstration of IgG anti-HAV indicates
relapsing course, and extrahepatic immune
previous infection. The presence of IgG anti-
complex deposition, all of which resolve
HAV protects against subsequent infection with
spontaneously.
HAV but is not protective against hepatitis B or
Complete clinical recovery is anticipated in other viruses.
almost all patients. Hepatitis A rarely causes
Diagnostic Evaluation:
fulminant hepatitis and does not progress to
chronic liver disease. Unusual clinical variants of  Hepatitis A antibodies (total)—EIA
hepatitis A include cholestatic, relapsing, and  Hepatitis A antibody, IgM antibody
protracted hepatitis. In cholestatic hepatitis,
serum bilirubin levels may be dramatically
elevated (>20 mg/dL) and jaundice persists for
weeks to months before resolution. In relapsing
hepatitis and protracted hepatitis, complete
resolution is anticipated. A chronic carrier state
(persistent infection) and chronic hepatitis
(chronic liver disease) do not occur as long-term
sequelae of hepatitis A. Rarely, injection with
HAV may cause fulminant hepatitis, with about
0.1% mortality. Fulminant hepatitis is the most
likely complication of coinfection with other
The short period of viremia makes detection
hepatitis viruses.
difficult. Specific IgM antibody usually appears
Immunologic Manifestations: about 4 weeks after infection and may persist
for up to 4 months after onset of clinical
IgM anti-HAV
symptoms. The presence of IgG or total (IgM
and IgG) antibody indicates past infection or
immunization and associated immunity. The
total assay detects IgM and IgG antibodies but
does not differentiate between them. The
hepatitis A antibody IgM assay is appropriate
when acute HAV infection is suspected. Specific
IgG antibody apparently protects an individual
from symptomatic infection, but specific IgM
may increase with reinfection. In the acute
phase of HAV, liver function levels (e.g., serum
liver enzyme levels) will be elevated and may
aid in establishing the diagnosis.
Prevention:
A safe, highly immunogenic, formalin-
inactivated, singledose vaccine is available to
prevent HAV infection (Box 23-1). HAV vaccine
should be targeted at high-risk groups (e.g.,
staff in child care centers; food handlers;
international travelers, including military
personnel; homosexual men; institutionalized
patients).
Hepatitis B Virus (HBV)
Etiology:
 HepaDNAvirus
 Formerly known as “serum hepatitis”
and referred to as “long incubation
hepatitis”
 MOT: usually parenteral, direct
inoculation
HBV is the classic example of a virus acquired
through blood transfusion. It serves as a model
when transfusion-transmitted viral infections
are considered.
The Australia antigen, now called hepatitis B
surface antigen (HBsAg), was discovered in
1966. This discovery, and its subsequent
association with HBV, led to the biochemical
and epidemiologic characterization of HBV
infection.
Hepatitis B is a complex DNA virus that belongs
to the family Hepadnaviridae; a member of this
family is known as a hepadnavirus. Eight
different HBV genotypes with differences in
clinical outcomes have been identified. Viral
proteins of importance include the following:
1. The envelope protein—HBsAg
2. A structural nucleocapsid core protein
—hepatitis B core antigen (HBcAg)
3. A soluble nucleocapsid protein—
hepatitis B e antigen (HBeAg)
The unique structure of the DNA of HBV is one
of the distinguishing characteristics of a
hepadnavirus. The DNA is circular and double
stranded, but one of the strands is incomplete,
leaving a single-stranded or gap region that
accounts for 10% to 50% of the total length of
the molecule. The other DNA strand is nicked (3′
and 5′ ends are not joined). The entire DNA
molecule is small and all the genetic
information for producing both HBsAg and
HBcAg is on the complete strand.
During the disease process, viral DNA of HBV is
actually incorporated into the host’s DNA. HBV
relies on a retroviral replication strategy
(reverse transcription from RNA to DNA).
Eradication of HBV infection is rendered difficult
because stable, long-enduring, covalently closed
circular DNA (cccDNA) becomes established in
hepatocyte nuclei and HBV DNA become
integrated into the host genome.
MOT:
Hepatitis B virus does not seem capable of
penetrating the skin or mucous membranes;
therefore, some break in these barriers is
required for disease transmission. Transmission
of HBV occurs via percutaneous or permucosal
routes and infective blood or body fluids can be
introduced at birth, through sexual contact, or
by contaminated needles. Infection can also
occur in settings of continuous close personal
contact.
About 50% of patients with acute type B
hepatitis have a history of parenteral exposure.
Inapparent parenteral exposure involves
intimate or sexual contact with an infectious
individual. Transmission between siblings and
other household contacts readily occurs via
transmission from skin lesions such as eczema
or impetigo, sharing of potentially blood-
contaminated objects such as toothbrushes and
razor blades, and occasionally through bites.
HBV has been found in saliva, semen, breast
milk, tears, sweat, and other biological fluids of
HBV carriers. Urine and wound exudate are
capable of harboring HBV. Stool is not
considered to be infectious.
Viral Replication:
Steps of HBV replication
Replication cycle of the hepadnaviral genome.
Enveloped virions infect the cell, releasing
relaxed circular DNA (RC-DNA) containing
nucleocapsids into the cytoplasm. RC-DNA is
transported to the nucleus, and repaired to
form cccDNA. (1) Transcription of cccDNA by
RNA polymerase II. (2) produces, amongst other
transcripts (not shown), pregenome RNA
(pgRNA). pgRNA is encapsidated, together with
P protein, and reverse transcribed inside the
nucleocapsid. (3) (+)-DNA synthesis from the (-)-
DNA template generates new RC-DNA. New
cycles lead to intracellular cccDNA
amplification; alternatively, the RC-DNA
containing nucleocapsids are enveloped and
released as virions.
The hepatitis B virus (HBV) establishes
covalently closed circular DNA (cccDNA) as a
durable miniature chromosome in the host
nucleus and relies on a retroviral strategy of
reverse transcription from RNA to negative-
strand DNA. The steps of HBV replication
targeted by nucleoside and nucleotide
analogues that are used to treat chronic HBV
infection are shown. ER, Endoplasmic reticulum;
HBsAg, hepatitis B surface antigen.
Progress of infection:
 incubation period of 4-26 weeks
 Route of infection: usually parenteral,
direct inoculation
 Duration of acute infection ranges from
4-8 weeks with symptoms similar to
HAV
 10% progress to chronic
 One-third of cases is at risk of
developing chronic active hepatitis,
cirrhosis and/or hepatocellular
carcinoma

Immunologic Manifestations:
1. Hepatitis B surface antigen (HBsAg)
2. Hepatitis B e antigen (HBeAg)
3. Hepatitis B core antibody, total or IgM
(anti-HBc)
4. Hepatitis B e antibody (anti-HBe)
5. Hepatitis B surface antibody (anti-HBs)
Diagnostic Evaluation:
Laboratory diagnosis and monitoring of acute
and chronic HBV infections involve the use of
several of the following tests:
1. Hepatitis B surface antigen (HBsAg)
2. Hepatitis B e antigen (HBeAg)
3. Hepatitis B core antibody, total or IgM
(anti-HBc)
4. Hepatitis B e antibody (anti-HBe)
5. Hepatitis B surface antibody (anti-HBs)
6. Hepatitis B viral DNA by polymerase
chain reaction(PCR, qualitative and
quantitative)
Serum testing procedures may be performed by
qualitative chemiluminescent immunoassay,
qualitative EIA, quantitative real-time PCR,
quantitative real-time PCR–nucleic acid
sequencing, or real-time PCR with reflex to
genotype. Immunohistochemistry may be used
to detect HBsAg in liver tissue samples.
Hepatitis D Virus (HDV)
The hepatitis D virus (HDV), initially called the
delta agentand then the hepatitis delta virus,
was first described in 1977 as a pathogen that
superinfects some patients already infected
with HBV (see Table 23-1). Persons with acute
or chronic HBV infection, as demonstrated by
serum HBsAg, can be infected with HDV. HBV is
required as a so-called helper to initiate
infection.
The HDV is a replication defective or incomplete
RNA virus that by itself, is unable to cause
infection. HDV consists of a single-stranded,
circular RNA coated in HBsAg. HDV is interesting
because it can force the host’s RNA polymerase
to transcribe the HDV RNA genome.
Progression of infection: Hepatitis D is a severe
and rapidly progressive liver disease for which
no therapy has proven effective. Patients with
this form of hepatitis are significantly more
likely to have cirrhosis and liver failure and to
require liver transplantation than patients with
HBV infection alone. Chronic HDV infection is
responsible for more than 1000 deaths/year in
the United States. The mortality rate can be up
to 20% of infected patients.
MOT:
Hepatitis D virus is spread chiefly by direct
contact of HBsAg carriers with HDV- or HBV-
infected individuals. Family members and
intimate contacts of infected individuals are at
greatest risk. IV drug users and individuals with
multiple sex partners are two other high-risk
groups. Maternal-neonatal transmission is
uncommon.
Hepatitis D can be acquired either as a co–
primary infection (coinfection) with HBV (e.g.,
after inoculation with blood or secretions
containing both agents) or as a superinfection in
patients with established HBV infection (HBsAg
carriers or patients with chronic hepatitis B).
A superinfection can make an HBV infection
worse by transforming a mild infection into a
persistent infection in 80% of patients. In
contrast, coinfection rarely leads to a chronic
condition. Although HDV is dependent on HBV
for its expression and pathogenicity, replication
of HDV appears to be independent of the
presence of its associated hepadnavirus.
Progress of infection:
 hepatitis D infection may be benign and
brief
 fulminant hepatitis and chronic
hepatitis
 chronic HDV infection -> associated
with increased hepatic damage and a
more severe clinical course
 Superinfection may results to sequential
attacks of HBV in the same patient
Signs and Symptoms
Hepatitis D infection may be benign and brief,
but fulminant hepatitis and chronic hepatitis
have been attributed with increasing frequency
to HDV. Chronic HDV infection is associated
with increased hepatic damage and a more
severe clinical course than is expected from
chronic HBV infection alone. The occurrence of
sequential attacks of HBV in the same patient is
probably attributable in most cases to HDV
infection superimposed on a previous acute
HBV infection.
Immunologic Manifestations:
 HDAg is found early in infection but
disappears rapidly
 IgM anti-HDV and total anti-HDV (IgM
and IgG) are detected during acute
phase
 IgM anti-HDV and HBsAg with IgM
anti-HBc indicates co-infection
 IgM anti-HDV in patients with chronic
HBV infection indicates superinfection
Hepatitis C Virus (HCV)
Etiology: at risk for contracting HCV. Although most
hepatitis C patients are injectable drug abusers,
Member of the hepaci virus genus of the
many patients acquire HCV without any known
Flaviviridae family.
exposure to blood or drug use. Sporadic or
Hepatitis C, previously called non-A, non-B community-acquired infections without a
(NANB) hepatitis, was regarded as a diagnosis of known source occur in about 10% of acute
exclusion because of the absence of specific hepatitis C cases and 30% of chronic cases.
serologic markers and unknown viral origin.
HCV has now been identified, with immunologic
assays developed for its detection. No
homology exists among HAV, HBV, or HDV and
HCV.

Viral Characteristics

Hepatitis C virus is an enveloped flavivirus. It is a

small, enveloped,single-stranded RNA virus.
After binding to the cell surface, HCV particles
enter the cell by receptor-mediated
endocytosis. Because the virus mutates rapidly, Progress of infection:
changes in the envelope protein may help it
evade the immune system. There are at least six  Clinically and epidemiologically similar
major HCV genotypes and more than 50 to HBV
subtypes of HCV. The different genotypes have  60-70% of HCV patients will develop
different geographic distributions. Genotype 1 chronic hepatitis, 10-20% cirrhosis and
represents most infection in North and South 15% hepatocellular carcinoma
America, and Europe. Genotypes la and lb are  HCV and HBV may be present as co-
the most common genotypes in the United infections
States. The HCV genotype does not appear to
Disease with the same frequency
play a role in the severity of disease.
Extrahepatic immunologic abnormalities have
HCV infection is a leading cause of chronic
been shownto occur frequently in patients with
hepatitis, cirrhosis, and liver cancer and is a
chronic HCV infection.
primary indication for liver transplantation in
Western countries. HCV infection has been linked to a number of
extrahepatic conditions, including Sjögren’s
MOT:
syndrome, cryoglobulinemia, urticaria,
Viral Transmission erythema nodosum, vasculitis,
glomerulonephritis, and peripheral neuropathy.
HCV is spread primarily by percutaneous
contact with infected blood or blood products. Traditional Hepatitis C Virus Testing
Currently, injectable drug abuse is the most
 Qualitative chemiluminescent
common risk factor. Workers with needlestick
immunoassay and qualitative EIA
injuries, infants born to HCV-infected mothers,
 Qualitative recombinant immunoblot
those with multiple sexual partners, and
assay
recipients of unscreened donor blood are also
  Quantitative real-time PCR assay
 Qualitative PCR assay
 Quantitative branched chain DNA test
 polymerase chain reaction–nucleic acid
sequencing
 interleukin 28 B (IL-28B)–associated
variants test
 two single-nucleotide polymorphisms
(SNPs) method
o qualitative PCR
o qualitative fluorescence
monitoring
Western Blot/ recombinant immunoblot assay
(RIBA) - Can be used as a confirmatory test
PCR - can detect low levels of HCV RNA in serum
Hepatitis C RNA Titers in Serum
 quantitative PCR and a branched DNA
test
 an indirect assessment of viral load
Hepatitis E Virus (HEV)
 Similar to HAV in transmission and
clinical course
 Found primarily in developing
countries, Africa and Asia
 Results in acute hepatitis, no risk of
chronic hepatitis
 Pregnant women with HEV may develop
fulminant liver failure and death
 IgM anti-HEV
 IgG anti-HEV
Hepatitis E virus (HEV) belongs to the genus
Hepevirus in the Herpeviridae family.
Immunologic Manifestations:
A short-lived, IgM anti-HEV has been found in
acute-phase sera. IgG anti-HEV appears and
replaces IgM anti-HEV about 2 to 4 weeks after
symptoms subside. The duration of detectable
IgG anti-HEV remains uncertain.
Diagnostic Evaluation:
Specific serologic tests for IgM and IgG anti-HEV
are available. HEV can be diagnosed by
performing immunoelectron microscopy on a
stool specimen. Liver serum enzymelevels, if
elevated, are indicative of the acute phase of
the infection.
Hepatitis G Virus (HGV)
 Independently discovered 1995-1996 by
2 separate research groups
 RNA virus
 Transmissible by blood-borne route
 Found in patients with acute or chronic
liver disease
 Frequently occurs as a coinfection with
HCV
 ELISA and Western Blot methods have
been developed
Etiology
The cause of hepatitis G is the hepatitis G virus
(HGV), an RNA virus. HGV is almost identical to
a viral agent called GB virus type C (GBV-C). In
1995 and 1996, two independent groups
discovered and sequenced an agent with
limited homology to HCV, named GBV-C/HGV.
The viral agents have 96% amino acid identity
and represent variants of HGV. It’s very
common to find people with hepatitis C that are
co-infected with GBV-C.
Epidemiology

Hepatitis G virus is a bloodborne agent (Table

23 5). Transfusion recipients and IV drug
abusers are at risk of infection. HGV infection
frequently occurs as a coinfection with HCV.
Prevalence patterns of GBV-C/HGV suggest that
the virus is transmitted sexually. GB virus C
infection is common; 1% to 2% of U.S. blood
donors have HGV RNA detectable in their
serum. HGV is estimated to produce 900 to
2000 infections/year, most of which may be
asymptomatic. Chronic infection develops in
90% to 100% of infected persons. Chronic
disease is rare or may not occur at all.

HIV
HIV is the predominant virus which is
responsible for the Acquired Immunodeficiency
Syndrome (AIDS).
BACKGROUND:
In 1983, Researchers at the Pasteur Institute in
Paris isolated a Retrovirus, which was coined:
 Lymphadenopathy-associated virus
(LAV) - from a homosexual man with
the lymphadenopathy.
Concurrently an American research
team headed by Dr. Robert Gallo,
isolated the same Class virus, which
they labeled:
 Human-T-lymphotropic retrovirus
(HTLV) type III
In 1984, The Gallo team was able to
demonstrate conclusively through virologic and
epidemiologic evidence that HTLV Type III was
the cause of AIDS, when it was demonstrated
the LAV and HTLV Type III three were the same
virus an international commission changed both
names of the virus, HIV to eliminate the
confusion caused by the two names and to
acknowledge that the virus is the cause of HIV.
 Human immunodeficiency virus (HIV)
VIRAL CHARACTERISTICS
 member of the family Retroviridae, a
type D retrovirus that belongs to the
lentivirus subfamily.
Included in this family are oncoviruses such as
Human-T-lymphotropic retroviruses 1 and 2,
which primarily induced proliferation of infected
cells and formation of tumors. Since the
discovery of this virus much has been learned
about the impact of HIV on human cells.
Two distinct HIV viruses, namely HIV-1 and
HIV-2 cause AIDS:
 HIV-1 subtypes: (divided into at least
nine subtypes)
group M (subtypes A-H)
group N
group O
 HIV-2 subtypes: (divided into two
subtypes)
groups A and B

HIV-1 virus is composed of a lipid membrane,
structural proteins and glycoproteins that
protrude.
Viral genome:
The viral genome consists of three important
structural components. These gene components
works for various products:
 pol - Produces DNA polymerase and
endonuclease integrase (p31)
 gag - Codes for p24(main core protein)
and for proteins such as p17, p9, and p7
 env - Codes for two gp41 and gp120 Virus
Long terminal redundancies (LTRs) border
HIV-1
these three components. HIV-2 has a different
envelope and slightly different core proteins.
Cells infected with HIV can be examined with an
electron microscope. The virus may appear as
buds of the cell membrane particles. The virion
HIV-2
has a double-membrane envelope and an
electron-dense laminar crescent or semicircular
cores. An intermediate, less electron-dense
layer lies between the envelope and core. In a
mature, free extracellular virion, the core
appears as a bar-shaped nucleoid structure in
cross section. This structure appears circular
and is frequently located eccentrically. It is
composed of structural proteins and
glycoproteins that occupy the core and
envelope regions of the particle.The virion
consists of knoblike structures composed of a
protein called glycoprotein (gp) 120, which is
anchored to another protein called gp41. Each
knob includes three sets of these protein
molecules. The core of the virus includes a
major structural protein called p25 or p24
encoded for by the gag gene. After human
exposure, these and other viral components
may induce an antibody response important in
serodiagnosis.
Retroviruses contain a single, positive-stranded
ribonucleic acid (RNA) with the genetic
information of the virus and a special enzyme
called reverse transcriptase in their core.
Reverse transcriptase enables the virus to
convert viral RNA into deoxyribonucleic acid
(DNA). This reverses the normal process of
transcription in which DNA is converted to RNA
thus, the term retrovirus.
Viral gene products:
 tat, trs, sor, 3’orf gene products –
regulate viral gene expression
HIV Proteins of Serodiagnostic Importance
Protein Location Gene
gp41 envelope env
gp160/120 envelope env
p24 core gag
gp34 envelope env
gp140 envelope env
p26 core gag
These glycoproteins and proteins can be
detected using Sandwich Enzyme Immunoassay
and the Western Blot Testing. Western Blot
Testing is known as one of the most commonly
used confirmatory test for HIV.
Viral Replication

Encoding Gene Antigen

gag p55

gag p24

gag p17

pol p66

pol P51

sor p24 *
Now in this process, we have here the
env gp160 illustration which shows the mechanisms of HIV
entry into a cell. 1st step, HIV gp120 binds to the
env gp120
CD4 on the T-cell and then there will be
env gp41 conformational changes which occur in gp120
and this conformational change are would
3′ orf p27 promote binding to either CCR5 or CFCR4, and
then there will also be conformational changes
in gp41 which will expose the fusion peptides
and these fusion peptides of activated g41
The first step in this replication cycle is viral
contains hydrophobic amino acid residues that
adherence or fusion of HIV to the host cell
promote insertion into the host cell plasma
surface. Now these viral adherence involves
membrane lipid bilayer. And then there will be
receptors on the host cell, and that receptor is
subsequent fusion of viral and cell membranes.
CD4 and then aside from CD4, we also have the
After viral fusion with a host cell, 2nd step is
coreceptors we have the CCR5 and the CFCR4
insertion of viral RNA, reverse transcriptase in
both of them are receptors for Chemokines. For
the nucleus, integrase and other viral proteins
viral adherence the most involved structure of
into the host cell. 3rd step, viral DNA will be
HIV-1 is gp120 and gp41.
formed with the action of reverse transcriptase.
And then once viral DNA is made, or
synthesized. 4th step, viral DNA will be
transported across the nucleus and with the
help of endonuclease integrase, it will integrate
the viral DNA into the host DNA. 5th step, new
viral RNA is used as genomic RNA and also to
make viral proteins. 6th step, new viral RNA and
proteins move to the cell surface and a new
immature HIV will be formed. 7th step or last
step, we have the formation of the mature of
the virion wherein virus matures by protease
releasing individual HIV proteins.
MOT
HIV virus has been isolated from blood semen
vaginal secretions, saliva, tears, breast milk,
cerebrospinal fluid or CSF fluid, amniotic fluid
and urine. However,
 Only blood, semen, vaginal secretions,
and breast milk have been implicated in
the transmission of HIV.
 Sexual contact
 Percutaneous
 Vertical transmission
 Breastfeeding
HIV has been found in saliva and tears in very
low quantities from some AIDS patients. It is
important however to understand that finding a
small amount of HIV in the body fluid does not
necessarily mean that HIV can be transmitted
by the body fluid. HIV has not been recovered
from the sweat of HIV infected persons. And
contact with saliva, tears, or sweat has never
been shown to result in transmission of HIV.
Viral transmission of HIV-1 can be
cervicovaginal, penile, rectal, oral,
percutaneous, intravenous, in utero or
breastfeeding after birth. More than 80% of
adults infected with HIV-1 became infected
through the exposure of mucosal surface to the
virus; most of the remaining 20% were infected
by a percutaneous or IV route.
SIGNS AND SYMPTOMS
The CDC has a classification used to define
stages of HIV-related illness.
So in the illustration, there from the top we
have the timeline and then we have a course of
infection and then we have the corresponding
CDC classification. So, first, from the time of
exposure up to the manifestation of infection,
and then we have seroconversion or formation
of antibodies, this three fall under CDC group I
in this we have your Primary HIV infection. We
also have the asymptomatic stage. So during
asymptomatic stage, patient does not show any
signs, nor symptoms of the infection. During
asymptomatic stage, this falls under CDC group
II classification. And then we also have the
symptomatic stage for this falls under CDC
group III classification wherein this is
characterized by persistent generalized
lymphadenopathy. And then we have from the
symptomatic stage prior to the diagnosis of
AIDS, we have the CDC group IVA wherein there
are constitutional symptoms. And then lastly,
we have the CDC group IVB-E, this is the course
of infection which is associated with AIDS. So, in
this stage or this classification, we have
neurological disease, opportunistic infections,
secondary cancer, and other complications that
can be observed from the patient.
During the early period after primary infection,
widespread dissemination of the virus occurs
(Viremia) and this happen with a sharp progress resulted from improved recognition of
decrease in the number of CD4+ T cells in opportunistic disease processes, improved
peripheral blood.The early burst of virus in the therapy for acute and chronic complications,
blood, viremia, is often accompanied by flulike and introduction of chemoprophylaxis against
symptoms that can be so severe that the key opportunistic pathogens. The second
affected person may seek help at a hospital decade of the epidemic witnessed extraordinary
emergency department. An immune response progress in developing highly active
to HIV develops, with a concurrent decrease in antiretroviral therapy (HAART), as well as
detectable viremia. It was previously believed continuing progress in preventing and treating
that the human immune system could drive the opportunistic infections. HAART has reduced
AIDS virus into a latent period that kept it the incidence of opportunistic infections and
inactive for years. However, this concept has extended life. In addition, prophylaxis against
been replaced with the new vision of a virus specific opportunistic infections continues to
that is furiously creating copies of itself provide survival benefits even among patients
throughout the disease course, even when the receiving HAART.
patient appears healthy.Even when HIV cannot
The most common opportunistic infections are
be detected in the blood (viremia), it infects
P. jiroveci (P. carinii), cytomegalovirus (CMV ),
lymphatic tissues (in large quantities), including
Mycobacterium avium intracellulare,
the tonsils and lymph nodes throughout the
Cryptococcus, Toxoplasma, Mycobacterium
body.The absence of viremia generally lasts
tuberculosis, herpes simplex, and Legionella.
until the end stage of the disease. This phase is
Histoplasma capsulatum is being recognized
followed by a prolonged period of clinical
with increasing frequency. The most frequent
latency (range, 7 to 11 years; median, 10
malignancy observed is an aggressive, invasive
years). During the period of clinical latency, the
variant of Kaposi’s sarcoma, discovered in many
patient is usually asymptomatic. Differences in
cases on autopsy. Malignant B cell lymphomas
the infecting virus, host’s genetic makeup, and
are increasingly recognized in patients with or
environmental factors (e.g., concomitant
at high risk for AIDS.
infection) have been suggested as causes of the
variable duration of clinical latency in persons
not receiving antiretroviral therapy. Treatment
with inhibitors of viral reverse transcriptase
(e.g., zidovudine [Retrovir]) and prophylaxis for
pneumonia caused by Pneumocystis jiroveci
(previously called P. carinii) have increased
AIDS-free time in HIV-1–infected persons.

Opportunistic Infections in Immunosuppressed

and Immunodeficient Patients

Since AIDS was first recognized in the early

1980s, remarkable progress has been made in
improving the quality and duration of life for
HIV-infected persons in the industrialized world.
During the first decade of the epidemic, this
Immune System Alterations mechanism are the underlying factors in the
progression of HIV infection.
 Normal CD4:CD8 = 2:1

However, in cases of AIDS, this value is a

reverse. So we have: Laboratory Diagnosis of HIV Infection

 AIDS = 0.5:1 or 1:2 Methods utilized to detect HIV:

however, the reverse values in this ratio is not  Antibody – detection of the antibodies
caused by the increase in CD8 but rather due to against viral antigens
the decrease in CD4 count.  Antigen - identification or
demonstration of the viral antigens
The HIV virus is fragile and, as the virus particle
 Viral nucleic acid - the quantification of
leaves its host cell, a molecule called gp120
viral nucleic acid
frequently breaks off the outer coat of the virus.
 Virus in culture - in some practices we
Glycoprotein 120 can bind to the CD4
also have culture of the virus.
molecules of uninfected cells and, when that
1. ELISA Testing
complex is recognized by the immune system,
 first serological test developed to
these cells can be destroyed. The lysis of
detect HIV infection
infected cells and gp120-bound uninfected cells
 antibodies detected include those
leads to the gradual depletion of the CD4+
directed against p24, gp120, gp160 and
lymphocytes. Defects in immunity are related to
gp41, detected first in infection and
this T cell depletion. Progressive defects in the
appear in most individuals
immune system also include a severe B cell
 However, Eliza testing is used for
failure and defects in monocyte and
screening only and there are
granulocyte function. Although HIV-1 destroys
possibilities of false positives to occur.
CD4+ cells directly and hampers the immune
system,this process does not cause the severe
2. Western Blot Testing
immunodeficiency seen in AIDS. The severe
deficiency can be explained only if the cells are  most popular confirmatory test and
also destroyed by other means. Several indirect western blot testing should be coupled
mechanisms have been suggested. Infection by with another test usually an antibody
HIV can cause infected and uninfected cells to tests such as Elisa. So when a patient is
fuse into giant cells called syncytia,which are positive, Elisa and Western Blot testing
nonfunctional. Autoimmune responses, in then that is confirmatory for HIV.
which the immune system attacks the body’s  antibodies to p24 and p55 appear
own tissues, may also be at work. In addition, earliest but decrease or become
HIV-infected cells may send out protein signals undetectable
that weaken or destroy other cells of the  antibodies to gp31, gp41, gp120, and
immune system. It is possible that the binding gp160 appear later but are present
of HIV to a target cell triggers the release of the throughout all stages of the disease
enzyme protease. Proteases digest proteins; if
released in abnormal quantities, they might
weaken lymphocytes and other cells and
decrease cell survival. The decline in T cells and
subsequent alteration of the immune

On this illustration we have here the general
procedures involved in western blot testing. So
on the 1st picture, we have there the first step
for Western Blot we have the separation of viral
proteins using electrophoresis. And then of
course, there is the blotting or transfer of the
fraction antigen from a gel to a membrane such
as a nitrocellulose membrane and then after
that, we will add the primary antibody specific
towards the antigens of HIV. And then the
addition of secondary antibody, which is
labeled. Lastly, we have incubation of the
blotted fractions with certain labels. And then
on the 2nd picture, we have here the end result
for a western blot test. So you can see there you
have different bonds for the different viral
structures of HIV-1.
2. Western Blot Testing: Interpretation of
result
 no bands, negative
 in order to be interpreted as positive a
minimum of 3 bands directed against
the following antigens must be present:
p24, p31, gp41 or gp120/160
 CDC criteria require 2 bands of the
following: p24, gp41 or gp120/160
 indeterminate results are those samples
that produce bands but not enough to
be positive, may be due to the
following:
1. prior blood transfusions, even
with non -HIV-1 infected blood
2. prior or current infection with
syphilis
3. prior or current infection with
malaria
4. autoimmune diseases
5. infection with other human
retroviruses
6. second or subsequent
pregnancies in women
*** run an alternate HIV
confirmatory assay
3. Indirect Immunofluorescence Assay
 can be used to detect both virus and
antibody to it
 antibody detected by testing patient
serum against antigen applied to a slide,
incubated, washed and a fluorescent
antibody added
 virus is detected by fixing patient cells
to slide, incubating with antibody

So on the images, we can see there the positive
reaction or Indirect Immunofluorescence Assay,
so those gene structures they represent the cells
which are infected with the virus.
5. Polymerase Chain Reaction (PCR)
4. Detection of p24 HIV antigen by Sandwich
EIA
 p24 antigen only present for short time,
disappears when antibody to p24
appears
 In this test, we have a Sandwich
Technique wherein on the membrane
surface we have there the HIV or the
HIV-1 antibody. HIV-1 antibody serves
to capture the p24 antigen present
from the patient sample. Then, after the
formation of these immune complexes
between the capture antibody and p24
antigen will now add the second anti-
HIV-1 antibody or antibody against p24
and this time, this antibody is labeled.
So, for example, we have this
biotinylated anti-P24 polyclonal
antibody and then after that we will add
the gold nanoproteins or nanoparticles
labeled streptavidin and then of course,
there will be color formation. So, if
there are colorful formations after
testing let's adjust the presence of p24
in the sample.
 test not recommended for routine
screening as appearance and rate of
rise are unpredictable
 sensitivity lower than ELISA
 most useful for the following:
a) early infection suspected in
seronegative patient – no
antibody formation yet
b) newborns – newborn patients
do not develop these widely
against HIV antigens
c) CSF
d) monitoring disease progress
 detects HIV DNA in the WBC’s of a
person – HIV DNA not RNA because in
this process inside the infected cells, the
HIV RNA was already converted into
DNA with the help of reverse
transcriptase, and was already
integrated into the host DNA through
the endonuclease integrates.
 amplifies tiny quantities of the HIV DNA
present, each cycle of PCR results in
doubling of the DNA sequences present
 the DNA is detected by using
radioactive or biotinylated probes
 once DNA is amplified it is placed on
nitrocellulose paper and allowed to
react with a radio-labeled probe, a
single stranded DNA fragment unique
to HIV, which will hybridize with the
patient’s HIV DNA if present
  radioactivity is determined- or color monitor viral load and T-cell
formation, if there are hybridization count
reactions.  repeat 4-6 weeks after starting
or changing ART to determine
6. Virus isolation
effect on viral load
 definitive diagnosis of HIV – just like in
Usually this viral load test is performed in
bacteriology for example, a certain area
conjunction with CD4 counts and to monitor the
or bacterium is recovered and identified
effect of the antiretroviral drug. So basically if
from a culture then that is a definitive
the ART or antiretroviral therapy is effective,
diagnosis of infection. The same is true
there is a decrease in viral load of the patient.
with HIV. So once the virus or its
antigens are recovered and identified in 8. Testing of neonates
a certain culture, then that is the
 difficult due to presence of maternal
definitive diagnosis of HIV.
IgG antibodies
 best sample is peripheral blood, but can
 use tests to detect IgM or IgA
use CSF, saliva, cervical secretions,
antibodies, IgM lacks sensitivity, IgA
semen, tears or material from organ
more promising
biopsy
 measurement of p24 antigen
 cell growth in culture is stimulated,
 PCR testing maybe helpful but still not
amplifies number of cells releasing virus
detecting antigen soon enough: 38 days
 cultures incubated one month, infection
to 6 months to be positive
confirmed by detecting reverse
transcriptase or p24 antigen in
supernatant

7. Viral Load Tests

 viral load or viral burden is the quantity

of HIV-RNA that is in the blood
 measures the amount of HIV-RNA in
1mL of blood
 take 2 measurements 2-3
weeks apart to determine
baseline
 repeat every 3-6 months in
conjunction with CD4 counts to