HERPETOVIRIDAE

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HERPETOVIRIDAE

• OBJECTIVES
• - Definition of herpsviruses
  groups
• - HSV and diseases
• - HSV Virology
• - HSV biology
• - Type of HSV and clinical
  spectrum
• - Host responeses and virus-cell
  interaction
• - Viral pathogenesis and
  establishment of latency
• - sources and routes of
  transmission
• - Diagnostic tests and
  interpretation of the results
• - Epidemiology of Herpes viruses ,
  prevention control
• - Herpes viruses and oncogenic
  potentials

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Herpesviridae

• Large family of large, complex viruses
• Infect vertebrate hosts
• Three subfamilies
• Alphaherpesvirinae, 2 genera
• Betaherpesvirinae, 3 genera
• Gammaherpesvirinae, 2 genera
• Very important as human pathogens
• Cause cold sores, genital herpes, chicken pox,
  shingles, mononucleosis and many other diseases
• Infection is for life herpesviruses become
  latent in hosts, then reactivate

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Herpesviruses

• HHV1, Herpes simplex 1
• Cold sores, epithelial and neuronal cells
• HHV2, Herpes simplex 2
• Sexually transmitted disease (STD), also as
  above, teratogenic, can be fatal in newborns
• HHV3, Varicella-zoster
• Chicken pox, shingles
• HHV4, Epstein Barr virus
• Mononucleosis, Burkitts lymphoma, lymphoid
  tissue only
• HHV5, Cytomegalovirus
• Salivary gland tropic, teratogenic, can be fatal
  in newborns
• HHV6, Roseolovirus
• Childhood rash, multiple sclerosis?
• HHV8
• Associated with Kaposis sarcoma

HSV 6,7,8 all identified after 1990, after HIV
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Cryo-EM shows regular, external structure
preserved
Irregular structures often seen in micrographs
are artifacts of distortion from negative staining
Nucleocapsid contains core of protein wrapped in
genomic DNA
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Herpesvirus particles

• Genome Single large segment of dsDNA, 3 of
  particle weight (124-235 kbp)
• Core Nucleic acid wrapped around cylindrical
  structure 25-30 nm
• Capsid T16 icosahedron composed of 162
  capsomeres (150 hexamers and 12 pentamers),
  capsid diameter 100-110
• Tegument poorly defined material between capsid
  and envelope, contains alpha trans-inducing
  factor (a-TIP) necessary for activation of a
  genes and virion host shutoff protein (VHS)
• Envelope Derived from nuclear membrane,
  surrounds tegument, has spike glycoproteins
  virion diameter 120-200 nm

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Human Herpesvirus 1 particles and genome
organization
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Biology of herpesviruses

• All specify a large array of enzymes involved in
  nucleic acid metabolism
• Virus DNA synthesis and capsid assembly in
  nucleus
• Production of infectious progeny is accompanied
  by destruction of infected cell
• A single virus can cause several diseases
• Herpesviruses remain latent in the host for life,
  and can be reactivated to cause lesions at or
  near the initial infection site

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HSV Establishes Latent Infections

• Once infection has taken place HSV can remain
  dormant for months, years, lifetime
• Cell types that HSV can infect and remain dormant
• Neurons, B-cells and T-cells
• Examples
• Shingles which can appear years after first
  chickepox infection (caused by varicella zoster,
  causes both chickenpox and shingles)
• Genital Herpes outbreaks

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Virus Latency
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Types of HSV

• The Type 1 virus causes cold sores. Most people
  get Type 1 infections during infancy or
  childhood.

• The Type 2 virus causes genital sores. Most
  people get Type 2 infections following sexual
  contact with an infected person. ii

Both types can be differentiated by biologic,
biochemical and antigenic properties
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Symptoms

• Mouth sores
• Genital lesions (male) -- may be preceded by
  burning or tingling sensation
• Genital lesions (female) -- may be preceded by
  burning or tingling sensation
• Blisters and/or ulcers -- most frequent on the
  mouth, lips and gums or genitalia
• Fever blisters
• Fever -- may be present especially during the
  first episode
• Enlargement of lymph nodes in the neck or groin

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Immunology
The constant battle between our body and invaders

• T cells can prevent HSV-1 reactivation from
  latency in sensory neurons with the help of gamma
  interferon

• we can produce two different antibodies, one
  against each type of HSV

• When both HSV 1 antibodies and HSV 2 antibodies
  are present, they can crosslink with one another
  and neither Antibody will be effective.

• HSV proteins inhibit cellular DNA synthesis and
  the virus uses its own primase and other
  replication machinery to replicate its DNA.

• .

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Viral Genes Block Immune Response

• Out of 84 genes only 37 involved in replication
• Some of the remainder involved in blocking immune
  response against virus
• Vhs and ICP27 block interferon effects by
  degrading cellular mRNAs
• ICP47 binds transporter proteins that aid antigen
  presentation
• Self and viral peptides are constantly being
  presented thru MHC I and provoke immune responses
  when appropriate
• ICP47 prevents transport of viral peptides on
  surface of cell
• ? no viral antigen presentation which means no
  immune response

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HSV TYPE-SPECIFIC SEROLOGY

• Subclinical genital herpes in the mother in late
  pregnancy is responsible for most neonatal herpes
  cases.
• HSV type-specific serology should be used for
  prenatal testing.
• Counseling safe sexual practices, abstinence
  should be provided to patient/partner, depending
  on results of the serologic testing.

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Diagnosis of HSV

• The appearance of HSV is often so typical that no
  testing is needed to confirm an infection, only a
  physical.
• The genital herpes sores may not be visible to
  the naked eye so a doctor may have to do a swab
  from the infected skin (culture) and send it to
  the lab for analysis.
• A viral PCR can be run on a swab of infected
  tissue.
• A blood test, can show if a person has been
  infected with HSV but cannot distinguish between
  type I and II.
• There are also newer blood tests that can tell
  whether a person has been infected with HSV 1
  and/or 2 by the patients immune response to the
  herpes infection.

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HSV DIAGNOSTIC TESTS

• Tzanck prep low sensitivity, not recommended
  as diagnostic test
• DFA stain scrapings same day, sensitive
• ELISA detection of HSV in skin lesions
• Eye consult
• EEG, MRI (temporal lobe involved)
• Histopathology

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HSV-CULTURES

• Culture at 24 - 48 hrs results in 2-7dskin,
  conjunctiva, mouth/nasopharynx, rectal, urine,
  blood, CSF
• Hi-risk /symptomatic Rx pending cultures
• Positive cultures from any site obtained gt 48hrs
  viral replication rather than colonization
• CSF cultures usually negative in encephalitis
• PCR DNA-CSF test of choice test before
  after RX

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CMV DNA PCR CMV-IgM

• CMV DNA PCR
• CSF preferred test
• positive result poor neurologic outcome.
• Blood positive active infection associated
  with hearing loss
• Newborn heel stick dried blood spots
  opportunity for universal screening?
• CMV IgM not recommended for neonates
• False-positives and false-negatives occur

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CHILD CARE CENTERS

• Plastic surfaces and toys harbor CMV for hours
• Viruria 70 infected gt18 mth old children.
• 30 seroconversion among mothers whose children
  shed CMV vs 0 if children dont shed
• Child to mother transmission confirmed.
• Young children in child care centers are
  important source of primary CMV infection for
  pregnant women.

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MATERNAL DIAGNOSIS Is it primary infection?

• IgG seroconversion ELISA
• New CMV specific IgM immunoblot
• IgG avidity index Anti CMV IgG has low avidity
  for first 14 wks after conversion
• IgG avidity index IgMimmunoblot combination
  may help identify primary inf.Guerra et al AM.
  J Ob Gyn 2000

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CMV - PRENATAL DIAGNOSISFetus infected?
Symptomatic?

• Viral culture amniotic Fluid
• Viral culture fetal blood
• DNA-PCR quantitative PCR
• CMV-IgM in cordocentesis not very sensitive
• Ultrasound
• Hematologic tests
• Guerra et al AM. J Ob Gyn 2000

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DAIGNOSIS - CMV CULTURE

• Virus must be isolated in urine /saliva lt3 wks
  of age to prove congenital infection
• If gt3 wks of age, cannot differentiate
  congenital, natal, or postnatal infection unless
  the infant previously has had a negative culture.
  This distinction is important because congenital
  infection is associated with hearing impairment.
• Shell-vial culture-helps rapid identification.

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TRANSFUSION-ACQUIRED CMV

• Characterized by a gray ashen pallor, respiratory
  distress, pneumonia, hepatosplenomegaly,
  hepatitis, atypical lymphocytosis,
  thrombocytopenia, and hemolytic anemia
• Infection in LBW infants may be severe
• 10 mortality.

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BREAST MILK TRANSMISSION

• Seropositive mothers shed virus 20 70 of
  the time in BM
• 60 term infants fed virus positive breast milk
  develop inf but it is benign due to mat(Ig)G.
• 15-25 preterm infants (no mat IgG) develop
  symptoms, hepatosplenomegaly, pallor,
  neutropenia, thrombocytopenia, elevated LFT.
  Neurologic sequelae ??
• Co-infection with HIV - risk fctor

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TRANSPLACENTAL TRANSMISSION

• PRIMARY MATERNAL INFECTION
• 2-6 mothers/yr seroconvert in pregnancy
• Transmission 40 50
• Earlier mat. inf. more severe the fetal inf.
• RECURRENT MATERNAL INFECTIONTransmission
  0.5-1.5most infants asymptomatic
• REINFECTION new CMV strain

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EPIDEMIOLOGY

• In developing countries, almost 100.
• After an active replication stage, CMV enters a
  latent stage in leukocytes and other tissues.
  Like other herpes viruses, CMV reactivates during
  relative immuno-compromise, such as pregnancy

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CONGENITAL CMV INFECTION

• Most common congenital infection 1 newborns
  infected 40,000 /yr in US
• Leading infectious cause of
• Mental Retardation, Cerebral Palsy, or,
  most commonly, hearing impairment
• involving gt 8,000 infants/ yr in the US

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PREVENTION

• Transfusion-acquired infectionCMV
  antibody-negative blood products, leukofiltration
  of blood to remove WBC,frozen deglycerolized
  RBCs as they lack viable leukocytes.
• Breast Milk acquired CMVFreezing human milk
  20C for 3-7d. Pasteurization (62.5C) not
  routinely available.
• CMV vaccine - investigational.

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PREVENTION

• Meticulous hand hygiene after exposure to urine
  or saliva from infants and toddlers and
  immunocompromised patients
• Standard precautions only.
• Routine screening is not recommended for women of
  childbearing age as no interventions are
  available.
• Pregnant women are not excluded from caring for
  infants who are infected with CMV.

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Treatment of HSV

• The combined effects of acyclovir and human
  interferon-alpha as drug therapy are being
  investigated now.
• Hsv 1 protective surface glycoprotein gD
  expressed in CHOs and protected mice
• To keep from spreading the infection
• Keep the infected area clean and dry to prevent
  other infections from developing.
• Try and avoid touching the sores.
• Wash your hands after contact with the sores.
• Avoid sexual contact from the time you first feel
  any symptoms until the sores are completely
  healed.

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VARICELLA

• Chickenpox in pregnancy rare 5 in 10,000
• Maternal Inf lt 20 weeks gest
• Varicella embryopathy in 2 infants
• Maternal Inf gt 20 weeks gest
• Inapparent varicella, Zoster in early childhood 
• Maternal Inf 5d before to 2d after delivery
• Neonatal VZV inf/ pneumonia (can be fatal)

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HORIZONTAL TRANSMISSION-VARICELLA

• Unusuall
• Most neonates protected by mat. antibody
• 30 wk, lt 1 kg. neonates could be susceptible -
  VZIG
• Screen rapidly for VZV antibodyVZIG to
  susceptible neonates
• VZIG to all neonates following exposure ?

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VARICELLA IN PREGNANCY
Maternal Inf Potential consequences
lt20 wks gest Spont abortion Fetal Varicella Syn - 2
any stage Fetal death, herpes zoster 1st yr of life
Near term5dltdelivery 2dgtdelivery Cong disseminated varicella Varicella pneumonia (can be fatal)
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EBV Viral Structure

• A core containing a linear, dsDNA molecule of
  about 175 kbp.
• An icosahedral capsid, approximately 100-110 nm
  in diameter, containing 162 capsomeres with a
  hole running down the long axis.
• An amorphous, sometimes asymmetric material that
  surrounds the capsid, designated as the tegument
• An envelope containing viral glycoprotein spikes
  on its surface.

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Infection and Replication
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Replication

• EBV replicates in the epithelial cells of the
  mouth, tongue, salivary glands, and oral cavity
  (few symptoms may be present, but a person can
  still be infectious)
• EBV infects B cells in the lymph nodes of the
  oral cavity
• Once inside B cells, EBV expresses proteins
• Nucleus EBNA (Epstein-Barr Virus Nuclear
  Antigens)
• Plasma Membrane LMP (Latent Membrane Proteins)
• Expression of these proteins stimulates B cell
  replication in lymph nodes producing clones
• Since many B cells are infected, polyclonal
  B-cell growth occurs which allows the disease to
  begin a long time after initial exposure to EBV

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Site of Infection

• Infection of Epithelial Cells by EBV in vitro
• Active replication, production of virus, lysis of
  cell
• Infection of B cells by EBV in vitro
• Latent infection, with immortalization
  (proliferate indefinitely) of the virus-infected
  B cells
• Linear EBV genome becomes circular, forming an
  episome, and the genome usually remains latent in
  these B cells
• Viral replication is spontaneously activated in
  only a small percentage of latently infected B
  cells.
• Signal transduction pathways can reactivate EBV
  from the latent state

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Primary Infection Diseases

• Infectious Mononucleosis (glandular fever) -
  fever, lymphadenopathy, and pharyngitis

• Chronic active EBV infection - severe illness of
  more than six months, histologic evidence of
  organ disease, and demonstration of EBV antigens
  or EBV DNA in tissue (mimics chronic fatigue
  syndrome)

• X-Linked Lymphoproliferative Disease - inherited
  disease of males, absence of functional SAP gene
  impairs the normal interaction of T and B cells
  resulting in unregulated growth of EBV-infected B
  cells.

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Symptoms

• The classic triad of mononucleosis is
• Inflammation of the pharynx (or tonsils) --
  usually the severest symptom
• Fever (higher in the evening)
• Lymphadenopathy (usually in the neck, groin or
  under the arms)

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Symptoms

• Other symptoms include
• Fatigue and malaise
• Rash (associated with the use of ampicillin)
• Headache
• Muscle aches
• Abdominal pain
• Occasional jaundice
• Enlargement of the spleen and liver

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Diagnosis of EBV

• Clinical diagnosis- Classic triad of symptoms
  lasting 1-4 weeks
• Serologic test- Shows elevate white blood cell
  count, an increased number of lymphocytes,
  greater than 10 atypical lymphocytes, and a
  positive reaction to a mono spot test
• Someone who appears to have infectious mono, a
  positive Paul-Bunnell heterophile antibody test
  can be done
• Serologic testing is the method of choice for
  primary infection
• EBV specific lab tests can be performed, testing
  patient for EBV antibodies.

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Immune System to the Rescue! (or not)

• Epithelial cells and polyclonal B cells express
  the viral-encoded LMP glycoprotein in their
  plasma membranes
• Killer T cells recognize the LMP glycoprotein and
  kill the EBV-infected cells
• While T cells are mounting an attack on B cells,
  the immune response of a person is abnormal
  producing atypical T cells and antibodies that
  can confirm diagnosis of infectious mononucleosis

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Treatment of EBV

• Infectious Mononucleosis
• No specific therapy just nonaspirins and rest
• Oral Hairy Leukoplakia
• Acyclovir inhibits EBV replication
• EBV Lymphoproliferative Disease
• reduction in the dose of immunosuppressive
  medication
• Surgical removal or irradiation of localized
  lymphoproliferative lesions

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Prevention and Vaccines

• EBV lymphoproliferative disease
• infusion of B-celldepleted marrow to offset the
  proliferation of donor B cells
• Removal of donor B cells along with T cells
• Vaccination studies underway but no vaccine found
  so far

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Cancers Associated with EBV

• Nasopharyngeal Carcinoma
• Southern China, Northern Africa, and Alaskan
  Eskimos
• Elevated titers of IgA antibody to EBV structural
  proteins
• Burkitt's Lymphoma
• Found in equitorial Africa and associated with
  malaria which doesnt allow T-cells to control
  proliferation of EBV-infected B cells
• Tumors present in jaw
• Hodgkin's Disease
• EBV DNA detected in tumors
• Lymphoproliferative Disease
• Impaired T-cell immunity and cannot control
  proliferation of EBV-infected B cells

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Complications

• Meningitis-- infection of the sheaths and
  membranes (meninges) covering the brain and the
  spinal cord.
• Encephalitis-- acute inflammation of the brain,
  commonly caused by a viral infection by insect
  bites or food and drink
• Eczema herpetiform-- widespread herpes across the
  skin)
• Keratoconjunctivitis-- Infection of the eye
• Prolonged, severe infection in immunosuppressed
  individuals
• Pneumonia
• Infection of the trachea
• Keratitis-- Corneal infection, irritations, and
  inflammations

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CONCEPT QUESTIONS

• -Herpesviruses are the causative agents of
• -Mention the types of HSV and their related
  diseases.
• - List the structural components their
  characteristic
• of HSV.
• - Mention the biology of HSV.
• - Define virus latency .
• - Describe latency in HSV
• - Tabulate differences between HSV-1 X HSV-2
• - What are the main syndromes of HSV infections?
• - Describe the HSV gene expression.
• - What are the immunologic pattern of HSV
  infection?

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• - How viral genes blocks host immune responses?
• - What are the uses of HSV type-specific serology
  ?
• - Mention the basis for HSV diagnosis .
• - What are the diagnostic test for HSV.
• - For what purposes HSV culture is required?
• - What are the significance of CMV ?
• - Mention the diagnostic methods for CMV
  detection.
• - What is the impact of day-care centers on CMV
  spread
• -Why maternal diagnosis of CMV infection is
  important?
• - What types of tests used for CMV prenatal
  diagnosis ?
• - CMV culture is useful in what ?
• - What are the modes of CMV transmission ?

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• -What are the epidemiological features of CMV?
• - Define congenital CMV infections?
• - What are the preventive measure for CMV
  infections?
• -Why acyclovir can be considered as anti-herpes ?
• - Define VZV .
• - What is the impact of VZV infections near term?
• - What are the expected outcome of varicella in
  pregnancy ?
• - Describe the structure of EBV.
• -Describe EBV infection and replication.
• - Mention the pathogenesis of EBV .
• - What is the sites of EBV infections?
• - What are the primary infections of EBV?
• - In what way EBV can evade host immune system?
• - What is the diagnostic test for infectious
  mononucleosis?
• - What are the preventive measure to EBV
  infections?
• - Mention the canccers associated with EBV
  infections.
• - What are the major complications associated
  with herpesviruses infections?

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Family Poxviridae (poxviruses) The family name
is taken from the major disease symptom caused by
these organisms, the pox. The pox is an elevated
lesion of the skin. The members of this family
are the largest of all the viruses and are
considered to be an evolutionary intermediate
between the viruses and the bacteria. The viral
particles (sometimes called elementary bodies)
are somewhat rounded, brick-shaped, or ovoid, and
have a complex structure consisting of an
internal central mass, the nucleoid, surrounded
by two membrane layers. The surface is covered
with ridges which may be tubules or threads. All
poxviruses are related immunologically by a
common internal antigen. They are divided into
genera on the basis of their more specific
antigens, nucleic acid homology, morphology and
natural hosts. The entire replication cycle
occurs in the cytoplasm. The viruses causing
human disease include
Family Poxviridae (poxviruses) The family name
is taken from the major disease symptom caused by
these organisms, the pox. The pox is an elevated
lesion of the skin. The members of this family
are the largest of all the viruses and are
considered to be an evolutionary intermediate
between the viruses and the bacteria. The viral
particles (sometimes called elementary bodies)
are somewhat rounded, brick-shaped, or ovoid, and
have a complex structure consisting of an
internal central mass, the nucleoid, surrounded
by two membrane layers. The surface is covered
with ridges which may be tubules or threads. All
poxviruses are related immunologically by a
common internal antigen. They are divided into
genera on the basis of their more specific
antigens, nucleic acid homology, morphology and
natural hosts. The entire replication cycle
occurs in the cytoplasm. The viruses causing
human disease include
Family Poxviridae (poxviruses)
The family name is taken from the major disease
symptom caused by these organisms, the pox. The
pox is an elevated lesion of the skin. The
members of this family are the largest of all the
viruses and are considered to be an evolutionary
intermediate between the viruses and the
bacteria. The viral particles (sometimes called
elementary bodies) are somewhat rounded,
brick-shaped, or ovoid, and have a complex
structure consisting of an internal central mass,
the nucleoid, surrounded by two membrane layers.
The surface is covered with ridges which may be
tubules or threads. All poxviruses are related
immunologically by a common internal antigen.
They are divided into genera on the basis of
their more specific antigens, nucleic acid
homology, morphology and natural hosts. The
entire replication cycle occurs in the cytoplasm.
The viruses causing human Variola virus - the
agent of smallpox, an infection of
reticuloendothelial, vascular endothelial and
epithelial cells Monkeypox virus - the agent of
monkeypox, a disease similar to smallpox but with
the additional symptoms of cervical and inguinal
lymphadenopathy disease include
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Variola virus - the agent of smallpox, an
infection of reticuloendothelial, vascular
endothelial and epithelial cells Monkeypox virus
- the agent of monkeypox, a disease similar to
smallpox but with the additional symptoms of
cervical and inguinal lymphadenopathy Vaccinia
virus - used to vaccinate against smallpox. It
causes a localized exanthem through epithelial
cell infection. Cowpox virus - the agent of
cowpox, a self-limiting disease resulting in
vesicles and pustules of the hands Orf virus -
the agent of contagious pustular dermatitis, an
epithelial cell infection Pseudocowpox virus -
the agent of pseudocowpox (Milker's nodules,
paravaccinia). This is an epithelial cell
infection. Molluscum contagiosum virus - this
causes molluscum contagiosum, a self-limiting
infection of epithelial cells Yaba monkey tumor
virus - this causes a histiocytoma of the head or
limbs Tanapox virus - causes tanapox, a
self-limiting epithelial cell infection