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MEDICAL VIROLOGY

Anatoliy Petrovich Godovalov

MD, Associate professor,
Microbiology and Virology Dep.
Acad. E.A. Wagner Perm State Medical University
Human
Immunodeficiency
Virus
• Human: Infecting human beings
• Immunodeficiency: Decrease or
weakness in the body’s ability to
fight off infections and illnesses
• Virus: A pathogen having the
ability to replicate only inside a
living cell
 HIV
• Etiologic agent of Acquired
Immunodeficiency Syndrome (AIDS).
• Discovered independently by Luc
Montagnier of France and Robert Gallo of
the US in 1983-84.
 Types of HIV
HIV 1
– Most common in sub-Saharan Africa and
throughout the world
– Groups M, N, and O
– Pandemic dominated by Group M
HIV 2
– Most often found in West Central Africa, parts of
Europe and India

The pathogenicity of HIV-2 may be lower than that of

HIV-1
 HIV vs. AIDS
• HIV is the virus that causes AIDS
• Not everyone who is infected with HIV has
AIDS
• Everyone with AIDS is infected with HIV
• AIDS is result of the progression of HIV
Infection
• Anyone infected with HIV, although
healthy, can still transmit the virus to
another person
 How is HIV Transmitted?
• Unprotected sexual contact with an infected
partner
• Exposure of broken skin or wound to infected
blood or body fluids
• Transfusion with HIV-infected blood
• Injection with contaminated objects
• Mother to child during pregnancy, birth or
breastfeeding
Family: Retroviridae Genus: Lentivirus
 Viral Replication
• First step, HIV attaches to susceptible host
cell.
– Site of attachment is the CD4 antigen found
on a variety of cells
• helper T cells
• macrophages
• monocytes
• B cells
• microglial brain cells
• intestinal cells
– T cells infected later on.
 Early Phase HIV Infection
• In early phase HIV
infection, initial
viruses are M-tropic.
Their envelope
glycoprotein gp120 is
able to bind to CD4
molecules and
chemokine receptors
called CCR5 found on
macrophages
• In late phase HIV
infection, most of the
viruses are T-tropic,
having gp120 capable
of binding to CD4 and
CXCR4 found on T4-
lymphocytes.
 Viral Replication
• Reverse transcriptase produces
viral DNA from RNA.
–Becomes a provirus which
integrates into host DNA.
–Period of latency occurs.
 Natural History of HIV Infection

• Immune suppression
• HIV attacks white blood cells, called
CD4 cells, that protect body from
illness
• Over time, the body’s ability to fight
common infections is lost
• Opportunistic infections occur
 Incubation period
• Variable.

• Although the time from infection to the

development of detectable antibodies is
generally 1–3 months, the time from HIV
infection to diagnosis of AIDS has an
observed range of less than 1 year to 15
years or longer.

• The median incubation period in infected

infants is shorter than in adults
 Primary HIV Syndrome
• Mononucleosis-like, cold or flu-like symptoms may
occur 6 to 12 weeks after infection.
– lymphadenopathy
– fever
– rash
– headache
– Fatigue
– diarrhea
– sore throat
– neurologic manifestations.
– no symptoms may be present
 Clinical Latency Period
• HIV continues to reproduce, CD4 count
gradually declines from its normal value of 500-
1200.
• Once CD4 count drops below 500, HIV infected
person at risk for opportunistic infections.
• The following diseases are predictive of the
progression to AIDS:
– persistent herpes-zoster infection (shingles)
– oral candidiasis (thrush)
– oral hairy leukoplakia
– Kaposi’s sarcoma (KS)
 Spectrum of HIV Tests
• HIV diagnosis (Antibody/Antigen testing)
– Enzyme Immunoassays (EIAs)
– Rapid tests
– Western blot (WB)
• Early diagnosis in infants
– p24
• Initiation and monitoring of therapy
– CD4
– Viral Load
Current HIV technologies

Detection of antibodies
Screening tests
Enzyme immunosorbent assays (EIAs)
Simple/rapid immuno-diagnostics assays

Confirmatory or supplemental tests

Western blot (WB)

Alternatives to confirmatory tests

Repetitive EIA or rapid assays
 HIV Rapid Tests
• Qualitative assay to detect HIV antibodies
• Most detect HIV 1 and HIV 2
• As reliable as EIAs
• Issues:
– Small volumes
– Validation of use
– Appropriate training
Source of False Positive Results
MULTIPLE PREGNANCY

MULTIPLE TRANSFUSION

AUTO IMMUNE DISORDER

CHRONIC HEPATITIS,

CHRONIC ALCOHOLIC

HBV VACCINATION

ANTIBODY TO POLYSTERENE
 Repeat
End; patient is
considered non-reactive Enzyme reactive EIA in
Duplicate
negative Immunoassay
2 non-
(EIA) reactive
results
(run singly)
1 or 2
reactive Patient is
results considered
Must perform one of the following negative
confirmation tests

TMA
IFA Western
transcription-
immunofluorescence Blot
mediated
assay amplification

Indeterminate
Positive: patient is HIV+ Result = inconclusive
Negative: patient is HIV-negative Re-draw in 2-4 weeks
Drugs Against HIV
– Reverse Transcriptase Inhibitors.
– Protease Inhibitors. Prevent viral
maturation.
MEDICAL VIROLOGY

Anatoliy Petrovich Godovalov

MD, Associate professor,
Microbiology and Virology Dep.
Acad. E.A. Wagner Perm State Medical University
VIRAL HEPATITIS
 Hepatitis (Liver-Attacking) Viruses
Hepatitis A – fecal/oral, contaminated food, vaccine
available
Hepatitis B – blood, semen, vertical (mother-child), vaccine
available
Hepatitis C – blood (IV drug use, transfusion, organ
donation, unsterile injecting equipment, sexual intercourse)
Hepatitis D – survives only in cells co-infected with hepatitis
B
Hepatitis E* – contaminated food or water, fecal/oral
*causes short-term disease and is not a chronic carrier state
 Viral Hepatitis
• When they occur, the signs and symptoms of
viral hepatitis can include:
– Fever
– Fatigue
– Loss of appetite
– Nausea
– Vomiting
– Abdominal pain
– Jaundice
– Dark urine
– Clay-colored stool
– Joint pain
Icteric symptoms of liver damage
Hepatitis A virus
 Taxonomy
• order Picornavirales
• family Picornaviridae
• genus Hepatovirus
• species Hepatovirus A
 Family Picornaviridae. Hepatitis A virus

• Small, non-enveloped
RNA virus with
cubical symmetry.
• One stable serotype only
 Hepatovirus A
• Is inactivated by:
9ultraviolet irradiation
9heating at 100°C for 5 min
9autoclaving 121°C for 20 min
91:4000 formalin at 37°C for 3
days
 Hepatitis A
• occurs endemically in all parts of the world
• minor and major outbreaks
• the rates of infection are high
• is usually contracted in early childhood
• 1 virion → infection
• high prevalence: lower socioeconomic status class
and females
• peak in March and April
Geographic distribution of HAV infection
Every year there are an estimated 1.4 million cases worldwide.
• Transmitted from a sick person:
• 10 days following infection and then 3-4 weeks
after the beginning of the jaundice period
• Modes of transmission – fecal-oral
• Routes of spread: stools, urine and
nasopharyngeal secretions
• Incubation period – 2-6 weeks
 I jaundice
Pathogenesis C
E
B
Ingestion E
↓ R

multiplying in intestinal G
epithelium
↓ P
H
blood E
↓ N
O
liver M
E
N
O
N
without jaundice 
 Laboratory diagnosis of hepatitis A
• Biochemical tests to study liver function:
- Bilirubin
- Alanine aminotransferase (ALT)
- Aspartate aminotransferase (AST)

• Serology: ELISA
– Acute infection: IgM
– Past Infection i.e. immunity: IgG

• Direct demonstration:
– ELISA (detection of antigen in stool),
– RT-PCR of faeces
 Hepatitis A Host Defenses
• antibodies develop late in incubation period
• IgM
– within a week of dark urine
– peaks a week later
– lasts 40-60 days
• IgG
– after IgM
– peaks 60-80 days
– lasts many years
 Vaccination
1. Live attenuated Hepatitis A vaccine
2. Inactivated Hepatitis A vaccine comprises of formalin
inactivated virions grown in human fibroblasts or monkey
kidney cell line

• provide active immunity against a future infection

• protects against HAV in more than 95% of cases
• is given by injection
• an initial dose provides protection lasting 1 year
• the second booster dose, given 6-12 months later, provides
protection for over 20 years.
• required in sewage workers and for visitors visiting HAV
endemic regions.
Hepatitis B virus
Geographic distribution of Chronic HBV Infection
400 million carriers worldwide
 Primary Infection

™Incubation Period 4-10 weeks

™During the prodromal period, patient may have
a serum sickness-like syndrome
™Constitutional symptoms, anorexia, nausea and
jaundice
™70% develop anicteric or subclinical hepatitis
™0.5-1% develop fulminant liver failure
™Symptoms and jaundice disappear in 1-3
months, though fatigue may persist
 Hepatitis B (serum hepatitis)
• 1) Transmission:
–parenterally,
–sexually,
–vertically (from mother to fetus),
–close contact (hemocontact).
• 2) chronic hepatitis in 10% to 15% of adult
patients, 30-90% of young patients
• 3) is causally associated with primary
hepatocellular carcinoma and cirrhosis.
Concentration of hepatitis B virus in body fluids

Low/Not
High Moderate Detectable
blood semen urine
serum vaginal fluid feces
wound exudates saliva tears
breast milk
Concentration HBV in the blood 109 virions per 1 ml
 Hepatitis B
• Hepatitis B is caused by infection with the
Hepatitis B virus (HBV), the prototype member
of the hepadnavirus family
– HBV is the only human representative of this
family.
– It has a circular DNA genome
• Currently, eight genotypes (A−H) are identified
by a divergence of >8% in the entire genome
 Hepatitis B Structure
• Hepadnavirus
• dsDNA, circular, 3200 nucleotides
• enveloped icosahedral virus
• 42 nm
 Hepatitis B

Source: J Viral Hepat. 2010 Apr;17(4):229-35. Hepatitis B virus: origin and evolution.
 Family Hepadnaviridae. Hepatitis B virus
The hepatitis B virion (Dane particle):
- outer lipid envelope with the surface antigen (HBsAg).
- an electron-dense core (nucleocapsid): ds circular DNA
and polymerase surrounded by the core antigen (HBcAg).

The HBsAg is produced in excess by the infected

hepatocytes and is secreted in the form of spherical
and filamentous particles.

HBeAg is the soluble

component of the core, with is
released during active
infection.
HBxAg is a transactivator of
viral transcription that appears
to contribute to HCC by
altering patterns of host gene
expression.
Dane Particles
 Hepatitis B virus
HbsAg
Envelope glycoprotein
DNA genome
Anti-HBs
Lipid membrane

HBc
HbeAg Capsid protein
Viral polymerase Anti-HBc
Anti-HBe IgM, IgG
 HBsAg
• First specific marker

• Detectable during incubation, peaks in acute

stage

• Declines upon recovery

• Elevated in carriers

• Screening test for donor blood

 HBeAg
• Appears shortly after HBsAg and parallels
HBsAg
• Present during active replication of virus
(most infectious phase)
 Anti-HBc
• First detectable antibody

• IgM present in the interval between

disappearance of HBsAg and
appearance of Anti-HBs (core window)

• IgG produced during convalescense and

persists for life
 Anti-HBe
• Appears after disappearance of HBeAg
• Indicates resolution
 Anti-HBs
• Appears during recovery and lasts for
years
• Indicates immunity (also produced as a
result of vaccination)
 Serum HBV DNA assay
• Clearance used as an endpoint in therapy -
30-40% respond

• Rarely, to identify HBV as the etiology of liver

disease in HBsAg negative patients,
especially in patients with fulminant hepatitis
B, who may have cleared HBsAg by the time
they present or in patients with AIDS
Markers for different phases of infection
HBsAg HBeAg IgM IgG Anti- Anti- HBV
Anti- Anti- HBs HBe DNA
HBc HBc

+ + + + Early

+ + + + Chr, repl

+ + Window

Low, non-rep
+ + +
Flare-up
+ + + + of chronic

+ + + Core mutants

+ + + Recovery
Timeline for Acute Hepatitis B Virus
Infection
Timeline for Chronic Hepatitis B Virus
Infection
 Hepatitis B
Host Defenses
• Cell mediated Immunity
– important for recover in acute phase
– autoimmune liver damage in chronic infections
• Humoral Immunity
– not always protective
– HBsAg for Vaccines
• Interferon
– not detected during infection
– exogenous application effective
 Hepatitis B Treatment
• For acute infection, no medication is
available; treatment is supportive.
• For chronic infection, several antiviral drugs
(interferon alfa-2b, pegylated interferon alfa-
2a) are available.
 Hepatitis B
Control
• No specific control
• Passive Immunization
– HBV immunoglobulin
– 250-500 IU within 48 hours
– neonates of infected mothers - immediately
after birth
• Active Immunization
– HBsAg
– recombinant DNA in yeast
 Vaccines
• Against Hepatitis A
Havrix (inactivated)
• Against Hepatitis B (recombinant)
- Monovaccines (HBsAg – genetic engineering):
Engerix B
H-B-Vax
Recombivax B
- Divaccines:
Twinrix (inactivated HAV + HBsAg)
Hepatitis C virus
 Hepatitis C
• Hepatitis C virus (HCV) infection is the most
common chronic blood-borne infection
• By contrast to Chronic HBV, patients with
chronic hepatitis C almost always develop
HCC in the presence of established cirrhosis
 Hepatitis C Characterisitcs
• Flavivirus – small, enveloped, single-stranded
RNA virus, six genotypes
• Replicates in liver cells, lymphocytes and
monocytes
• Replicates >1 trillion progeny per day
• Mutates rapidly (error-prone RNA polymerase)
• Down-regulates stimulatory receptors on NK cells
• Increases inhibitory receptors on NK and CD8+
killer cells
• Can blocks activation of T cells and inhibits
production of IFN-gamma
 Family Flaviviridae
Hepatitis C viruses
Enveloped ss+RNA viruses, 55-65 nm diameter
14 genotypes of HCV

Electron micrograph of
the hepatitis C viruses
 Hepatitis C Epidemiology
• Transmission of HCV occurs through:
– Percutaneous
• Injecting drug use
• Clotting factors before viral inactivation
• Transfusion, transplant from infected donor
• Therapeutic (contaminated equipment, unsafe
injection practices)
• Occupational (needlestick)
– Permucosal
• Perinatal
• Sexual
 Hepatitis C
Prevalence of chronic infection with hepatitis
C virus
Hepatitis C Diagnosis
Hepatitis C Diagnosis
 Hepatitis C Diagnosis
• Sixty to 70% of persons newly infected with
HCV typically are usually asymptomatic or
have a mild clinical illness.
• HCV RNA can be detected in blood within 1–
3 weeks after exposure.
 Hepatitis C Treatment
• Interferon-based therapy is currently the
standard of care for patients with chronic
HCV, and has been proven to be effective in
eliminating HCV.
 Hepatitis C Prevention
• Unlike HBV, there is currently no vaccine for
HCV.
• However, with the screening of HCV in
blood transfusion services, transfusion-
related HCV infection has been lowered to
almost zero.
 Laboratory diagnosis of viral hepatitis
• Biochemical methods: Detection of the level of
bilirubin and enzymes ALT, AST in serum.
• Serology:
ELISA - Detection of the Ag
and antibodies

• Detection of a viral genome: PCR

• Rapid immunochromatographic test
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