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Basic Clinical Virology

Virus
-

Latin word for poison.


small pathogen, can replicate only inside the living cells.
size in a range of about 20 nm to about 250 nm.
ability to pass through filters that retained bacterial agents.
cannot be cultured on medium, propagate only in living cells or tissues.

m as the base unit:


1,000 pm = 1 nm
1,000 nm = 1 m
1,000 m = 1 mm
10 mm = 1 cm

Structure

virus is composed of two major parts: (i) Capsid and (ii) Nucleic acid.

virus has only one type of nucleic acid, either DNA or RNA, but not both.
Complex or irregular, e.g. bacteriophage, vaccinia.
- Glycoproteins (i.e., proteins with carbohydrates attached) that project from some
enveloped viruses.

Helical virus

A generalized structure of a virus

Icosahedral virus

I.

Classification of virus
1. Baltimore classification

The system was defined in 1971. It is a classification system that places viruses into one of
seven groups depending on a combination nucleic acid (RNA or DNA), strandedness (singlestranded or double-stranded), Senses, and method of replication.
Group I: double-stranded DNA viruses (listed only few important genus)
Adenoviridae: Adenovirus.
- non-enveloped, icosahedral
Papillomaviridae: Papillomavirus

- non-enveloped, icosahedral
Herpesviridae: Herpes simplex virus, varicella-zoster virus, cytomegalovirus, EpsteinBarr virus
- ds DNA, enveloped virus
Group II: single-stranded DNA viruses (+)sense DNA
Parvoviridae: Parvoviruses
- Non-enveloped, icosahedral
Group III: double-stranded RNA
Reoviridae: Rotavirus, Reoviruses
- Non-enveloped, icosahedral
Group IV: positive-sense single-stranded RNA viruses
Picornaviridae: Enterovirus e.g polio, coxsackie
Rhinovirus e.g common flu viruses
Cardiovirus e.g encephalomyocarditis virus
Aphthovirus e.g FMDV
- Non-enveloped, icosahedral

Caliciviridae: Norwalk virus


Hepatitis E virus
- Non-enveloped, icosahedral

Togaviridae: Genus Alphavirus: chikungunya


Genus Rubivirus: Rubella virus
- Enveloped, icosahedral

Flaviviridae:

Dengue virus
Hepatitis C virus
Yellow fever virus
- Enveloped, Icosahedral
Coronaviridae: Corona virus
- Enveloped helical
Astroviridae: Astrovirus
- Non-enveloped, icosahedral

Group V: negative-sense single-stranded RNA viruses


Arenaviridae: Lymphocytic choriomeningitis virus
- Enveloped, complex.

Orthomyxoviridae: Influenzavirus A
Influenzavirus B
Influenzavirus C
Thogotovirus
- Enveloped, helical

Paramyxoviridae: Measles virus


Mumps virus
Respiratory syncytial virus
Rinderpest virus
- Enveloped, helical

Group VI: positive sense single-stranded RNA-RT viruses with DNA intermediate in lifecycle
Retroviridae: Retroviruses
Group VII: double stranded DNA-RT viruses
Hepadnaviruses: Hepatitis B virus
- Enveloped, Icosahedral, partially circular ds
Bacterial virus: These are pathogenic viruses infecting bacteria and are called bacteriophages
or simply phages. Their nucleic acid is DNA, e.g. T2, T4, T6 bacteriophages
Bacteriophages (or phages) are viruses that infect bacteria

II.

Laboratory diagnosis of viral infections

Serological, molecular biological and tissue culture methods are used to diagnose viral
infections and to accertain the extent of viral damage and the quality and intensity of the body's
immune response.
A. Growth of the virus in Tissue Cultures
The type of cell culture used for virus cultivation depends on the sensitivity of
the cells to that particular virus. In the clinical laboratory, multiplication of the
virus can be followed by determining the following:
1. The Cytopathic effect or cytopathogenic effect (abbreviated CPE)
refers to degenerative changes in cells, especially in tissue culture,
and may be associated with the multiplication of certain viruses
e.g. HSV, VZV, Influenza A and B, Parainfluenza 1, 2, and 3, Adenovirus,
RSV, CMV, Enterovirus, and Rhinovirus
2. The appearance of a hemagglutinin (e.g mumps, influenza) or
complement-fixing antigen (e.g, poliomyelitis, varicella, measles).

Altered shape
Detachment from substarte
Lysis
Altered membrane
permeability
Inclusion bodies
apoptosis

B. Chick Embryos inoculation


Virus growth in an embryonated chick egg may result in the death of the embryo
(e.g, encephalitis virus), the production of pocks or plaques on the
chorioallantoic membrane (e.g, herpes, smallpox, vaccinia), the development of
hemagglutinins in the embryonic fluids or tissues (e.g influenza), or the
development of infective virus (eg, Poliovirus type 2).
C. Detection of virus-specific antibodies in the blood.
two types of antibodies are produced:
IgM: first antibody, highly effective at neutralizing viruses during the early
infection.
IgG: second antibody, produced indefinitely, may offer immunity.
IgM in the blood of the host is used to test for acute infection, IgG indicates an
past infection.
Both types of antibodies are measured by ELISA.

Manual ELISA system

Architect: automated system

D. Detection of virus antigens


- The virus antigens in the blood or other body fluids or tissues can be detected by
immunoassays e.g ELISA. This type of assay can be useful for certain
infections, e.g. parvovirus B19, HIV, hepatitis B virus (HBV) and human
herpesvirus-6 (HHV-6).
Other techniques are: immunofluorescence

Apple-green fluorescent particles

E. Qualification of virus particles by electron microscopy.

Hepatitis B virus
Papillomavirus

F. Detection of virus nucleic acids


Detection of virus encoded DNA and RNA is done with polymerase chain reaction.
Nucleic acid hybridization with virus-specific probes detects specific viruses. For
additional reading, refer to Wikipedia.

G. Hemagglutination assay and Haemagglutination inhibition test


Haemagglutination inhibition test used to detect the presence of a certain
haemagglutinating virus or other haemagglutinin antigen based on whether the red blood
cells ability to clump together when the antibody to the virus or other antigen is added to
it.
If the virus or antigen is present, the antibody kills it and thereby stops it from being able
to stick the red blood cells to each other.

III.

Anti-viral agents
Nomenclature of currently approved antiviral agents
(excluding antiretriviral agents)

1. Acyclovir
A synthetic analog of the purine nucleoside, guanosine, with potent antiviral
activity against herpes simplex viruses type 1 and 2, varicella-zoster virus and
other viruses. After conversion in vivo to the active metabolite acyclovir
triphosphate, acyclovir competitively inhibits viral DNA polymerase, incorporates
into and terminates the growing.

Metabolized by kidney, decrease interval in renal Impairment to q12-24h.


Indications
Primary or Recurrent Herpes Simplex Virus Infection
Oral Herpes (Herpes Labialis)
Genital Herpes
Route: oral, IV, topical
2. Valacyclovir (valtrex)
Prodrug of acyclovir
For zoster, genital herpes, chicken pox
Applications: for recurrences of mucocutaneous and genital herpes
3. Famciclovir (Prodrug of penciclovir)
- treatment of zoster and genital herpes,
- suppresion of recurrences
4. Penciclovir cream (topical)
- recurrent orolabial HSV.
IV.

Medically important virus

A. Human herpesvirus :
-

Herpes simplex virus I & II

HSV infection of some neuronal cells does not result in cell death, instead maintained by
cell in a repressed state called latency. Subsequently activation of the viral gene release
is called reactivation
Capsid (icosahedral)

Envelop

Clinical features
1) Oral-Facial Infections: Gingivostomatitis and pharyngitis
- most frequently HSV-1
- vesicles on the hard and soft palate, gingival, tongue and lip.

2) Genital Infections
- most frequently HSV-2

3)

Herpetic Whitlow
HSV infection of the finger

4)

Central Peripheral Nervous System Infections


cause of acute, sporadic viral encephalitis in children and young adults.

5)

Neonatal HSV Infection


neonates (infants less than 6 weeks) have the highest frequency of visceral
and/or CNS nfection of any HSV-infected patient population

Investigation/Laboratory diagnosis

Viral isolation and identification


Direct immunofluorescence of vesicular scrapping
Culture (swab specimen) on cell line +/- immunofluorescence

Serology
IgM & IgG titre from blood & CSF

B. Varicella-zoster virus
-

Causes two distinct clinical entities: varicella or chicken pox and herpes zoster or
shingles.
Varicella is the primary illness usually in childhood extremely contagious by an
exanthematous vesicular rash
Zoster is a recurrent manifestaion of latent infection from the dorsal root ganglia
presents dermatomal vesicular rash

Chicken pox

herpes zoster

Investigation/Laboratory diagnosis
1) Mainly clinical
2) Serology EIA using serum by detection of IgM antibodies

C. Cytomegalovirus
-

can affect almost every organ of the body, resulting in fever of unknown origin,
pneumonia, hepatitis, encephalitis, myelitis, colitis, uveitis, retinitis, and neuropathy.
infections are frequently associated with salivary glands, infection can also be life
threatening in immunocompromised patients (e.g. patients with HIV, organ transplant
recipients).
Neonates: congenital cytomegalovirus infection

Investigation/Laboratory diagnosis
-

Enzyme-linked immunosorbent assayfor IgM & IgG in blood.


Qualitative and quantitative PCR testing

D. Influenzavirus A
-

Influenza A viruses are classified, based on the viral surface proteins hemagglutinin (HA
or H) and neuraminidase (NA or N)

The type A viruses are the most virulent human pathogens among the three influenza
types and causes the most severe disease. The serotypes that have been confirmed in
humans, ordered by the number of known human pandemic deaths, are:
H1N1 caused "Spanish Flu" in 1918, "Swine flu" in 2009.
H2N2 caused "Asian Flu".
H3N2 caused "Hong Kong Flu".
H5N1 is a pandemic threat.
H7N7 has unusual zoonotic potential.
H1N2 is endemic in humans and pigs.
H9N2, H7N2, H7N3, H10N7.

Investigation/Laboratory diagnosis
Nasopharyngeal specimens are typically more effective than throat swab specimens for
viral isolation, polymerase chain reaction (PCR) and immunofluorescence assays.
E. Human Papillomavirus
-

HPV are typically transmitted through sexual contact and infect the anogenital region.
Two vaccines are available to prevent infection by some HPV types, Gardasil, marketed
by Merck and Cervarix, marketed by GlaxoSmithKline.
Infections associated with HPV types are listed in Table.
Disease

HPV type

Common warts
Plantar warts
Flat warts
Anogenital warts
Genital cancers

2, 7
1, 2, 4, 63
3, 10
6, 11, 42, 44 and others
Highest risk: 16, 18, 31, 45

Investigation/Laboratory diagnosis
-

Papanicolaou smears from cervical scrapings often show cytologic evidence HPV
infection

F. Rotavirus
-

the most common cause of severe diarrhoea among infants and young children.

by the age of five, nearly every child in the world has been infected with rotavirus at
least once.
adults are rarely affected.
transmitted by the faecal-oral route.
Investigation/Laboratory diagnosis

detection of virus stool by enzyme immunoassay, latex agglutination.

G. Coxsackievirus
-

transmitted by the fecal-oral route.


leading causes of aseptic meningitis, divided into group A and group B viruses based on
early observations of their pathogenicity in mice.
infect the skin and mucous membranes, causing acute hemorrhagic conjunctivitis,
herpangina, (AHC), and hand-foot-and-mouth (HFM) disease.
Herpangina
seen most commonly at the back of the mouth and throat.
spread by the faecal-oral route.
caused mainly by coxsackie A16 virus and is highly contagious

H. Group B Coxsackie virus


- infect the heart, pleura, pancreas, and liver, causing pleurodynia, myocarditis,
pericarditis, and hepatitis.
- coxsackie B infection of the heart can lead to pericardial effusion.
- coxsackievirus B pancreatitis is associated with the development of insulin-dependent
diabetes (IDDM).
Investigation/Laboratory diagnosis
-

Culture: A throat swab or stool specimen.

I.

Enterovirus 71

one of the major causative agents for hand, foot and mouth disease (HFMD), and is
sometimes associated with severe central nervous system diseases.

Investigation/Laboratory diagnosis

Diagnosis of enterovirus infections is often clinical.


Viral isolation: The virus can be isolated from CSF, blood, or feces, depending on the site
affected, and the yield is increased if multiple sites are sampled.

J. Dengue virus
-

caused dengue fever may lead to dengue hemorrhagic fever (DHF) and dengue shock
syndrome .(DSS)
a mosquito-borne flavivirus. Mosquitos Aedes aegypti and Aedes albopictus
four distinct serotypes of the dengue virus( dengue-1, dengue-2, dengue-3 and dengue
4)
Investigation/Laboratory diagnosis

The diagnosis of dengue is usually made clinically. The classic picture is high fever with
no localising source of infection, a rash with thrombocytopenia, low platelet and white
blood cell count.
Serology: detection of dengue IgM & IgG in the blood by ELISA.
Culture: only for early stage of infection.

K. Chikungunya virus
-

an insect-borne virus, transmitted to humans by virus-carrying aedes


mosquitoes, causes an acute febrile phase of the illness (2-5 days) followed by
a prolonged arthralgic disease that affects the joints of the extremities. The joint
pain persists for weeks or months, or in some cases years.

Investigation/Laboratory diagnosis
-

ELISA assay to measure Chikungunya-specific IgM levels.

L. Rubella virus
-

commonly known as German measles.


transmitted by the respiratory route and replicates in the nasopharynx and lymph
nodes.
capable of crossing the placenta and infecting the fetus.
Infection of the mother by Rubella virus during pregnancy within the first 20
weeks of pregnancy, the child may be born with congenital rubella syndrome
(CRS).
primary symptom is the appearance of a rash (exanthem) on the face which
spreads to the trunk and limbs. Other symptoms include suboccipital & posterior
cervical lymphadenopathy and conjunctivitis.

Investigation/Laboratory diagnosis
-

Detection of Rubella virus specific IgM antibodies in the blood by ELISA.


For screening, detection of Rubella virus specific IgG antibodies in the blood.

Note: The CRS comprises cardiac, cerebral, ophthalmic and auditory defects. Rubella may also
cause prematurity, low birth weight, and neonatal thrombocytopenia, anaemia and hepatitis.
The risk of major defects or organogenesis is highest for infection in the first trimester.

M. Human Immunodeficiency Virus


-

Reverse transciptase : catalyses the reverse transcription of the RNA genome into a
DNA copy, the resulting DNA is called the provirus.
Provirus can integrate into host DNA and remain dormant for weeks, months or even
years without being expressed i.e. it remains latent
reverse transcriptase makes errors relatively frequently and does not have a proofreading mechanism to check for errors, resulted the emergence of HIV mutants .
HIV Classification:
HIV-1
Group M: A, B, C, D, F, G, H, J, K, CRF, URF
Group O: west-central Africa
Group N : rare, a strain discovered in 1998 in Cameroon.
Group P: discovered in a Cameroonian woman in 2009, closely relating to gorilla
simian immunodeficiency virus.
HIV-2

Investigation/Laboratory diagnosis

SEROLOGICAL WINDOW
HIV Antibody

Serological window

HIV RNA
HIV p 24 Antigen
10

20

30

40

50

Days

Infection
* M.P. Bush. AABB 1992
J.B. Jackson. Transfusion.1995

Window period 3-6 weeks


ELISA, detection of anti-HIV 1 / 2 antibodies in the blood.
HIV diagnosis is complicated; the diagnostic algorithm will not be discussed at this
stage.
Nearly all infants born to HIV-infected mothers passively acquire maternal antibody and,
antibody will remain positive until age 18 months regardless of whether they are
infected.
Definitive diagnosis of HIV infection in early infancy requires: nucleic acid amplification
(e.g., polymerase chain reaction [PCR]) or viral culture.
Report: scenario 1.
HIV Ag/Ab Combo
HIV 1 / 2 Rapid test
HIV 1 / 2 Particle Agglutination

Detected (Cut off value 1.8)


Negative
Negative

HIV Ag/Ab Combo


HIV 1 / 2 Rapid test
HIV 1 / 2 Particle Agglutination

Detected (Cut off value 10.8)


Reactive
Reactive

N. Hepatitis B virus
-

In humans HBV infection is associated with a wide spectrum of clinical presentations:


Healthy carrier state

Acute or chronic hepatitis

Liver cirrhosis

chronic hepatitis B infection is involved in the development of hepatocellular carcinoma.


Usually the cancer does not develop until a 30-50 year latency period.
Persistent of HBsAg in the blood >6 months is considered as HBV carrier.

Investigation/Laboratory diagnosis
-

ELISA, detection of serological markers for HBV infection.


Serological markers used:
HBsAg ( hepatitis B surface antigen )
Anti-HBs ( antibody to hepatitis B surface antigen)
HBeAg ( hepatitis B e antigen )
anti-HBe ( antibody to hepatitis B e antigen )
anti-HBc ( antibody to core antigen )
anti-HBc IgM
anti-HBc IgG
HBV DNA
For routine diagnosis, clinicians normally will request HBsAg
and anti-HBs.
HBV DNA is a quantitative test to count the viral particles in the
blood.

Reports
HBsAg:
not detected
Anti-HBS:
not detected
i.e. no history of exposure, vaccination needed
HBsAg:
detected
HBeAg:
detected
i.e. HBV infection. If persisted for >6months, hepatitis

B carrier

HBsAg:
not detected
anti-HBs:
>100 mIU/ml
i.e. ? vaccination or natural acquired infection. Anti-HBs 10 mIU/mL is considered as
immunity to HBV infection.
HBsAg:
not detected
anti-HBc:
detected
i.e. in the window period, previous exposure to HBV infection
HBsAg:
detected
anti-HBs:
15 mIU/ml
- concurrently circulating HBsAg & anti-HBs
- anti-HBs has no clinical significant, low level
- found in chronic HBV carrier poor prognosis

N.

Hepatitis C virus
-

HCV is believed to be more prevalent than HBV


The virus has still not been cultured in vitro or visualized by EM.
Replication cycle is not established yet but it has been suggested that replication
probably occurs in the cytoplasm.

HCV has a highly variable nucleotide sequence, and is as susceptible to


mutation as HIV.
HCV can be divided into at least 6 genotypes:
Genotype 1: subtype 1a, 1b, 1c
Genotype 2: subtype 2a, 2b, 2c
Genotype 3: subtype 3a, 3b
Genotype 4: subtype 4a
Genotype 5: subtype 5a
Genotype 6: subtype 6a