ARBOROVIRUSES

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ARBOROVIRUSES

• Mohammed El-Khateeb
• 2nd April 2015

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Overview

• Etiology
• Epidemiology and history
• Pathogenesis and Pathology
• Clinical Manifestation
• Diagnosis
• Treatment
• Prevention and Control

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Arthropod-Borne Viruses

• Arthropod-borne viruses (arboviruses) are More
  Than 600 Different viruses that can be
  transmitted to man by arthropod vectors.
• The WHO definition is as follows
• Viruses maintained in nature principally, or to
  an important extent, through biological
   transmission between susceptible vertebrate
  hosts by hematophagus arthropods or through
  transovarial and possibly venereal transmission
  in arthropods.

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Arthropod-Borne Viruses

• Arboviruses belong to three families
• 1. Togaviruses e.g. EEE, WEE, and VEE
• 2. Bunyaviruses e.g. Sand fly Fever, Rift Valley
  Fever, Crimean- Congo Hemorrhagic Fever
• 3. Flaviviruses e.g. Yellow Fever, dengue,
  Japanese Encephalitis

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Arthropod-Borne Viruses

• Togaviridae
• a) Sindbis
• b) Semliki Forest
• c) Venezuelan equine encephalitis
• d) Eastern equine encephalitis
• e) Western equine encephalitis
• f) Chikungunya

• Flaviviridae
• a) Dengue
• b) Yellow fever
• c) Japanese encephalitis
• d) West Nile encephalitis
• e) St. Louis encephalitis
• f) Russian spring-summer encephalitis
• g) Powassan encephalitis

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Negatively Stained Virions of Semliki Forest Virus
Togaviridae, genus Alphavirus
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Arthropod-Borne Viruses
FAMILY
ENVELOPE Yes. Heat, detergent labile Yes,
Heat, detergent labile no
SYMMETRY Icosahedral 40-75 nm 40-45
nm Helical Icosahedral
GENOME ssRNA (ve) ssRNA
(-ve) segmented dsRNA, segmented
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VIRAL STRUCTURE

• It has three proteins
• Envelope protein
• Core protein
• Membrane protein

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General Antigenic Properties of Flaviviral
Proteins - 1

• E-glycoprotein
• Most important flaviviral antigen
• Very immunogenic
• Most serological assays detect reactivity with
  this protein
• Capsid
• Elicits primarily group-reactive antibody
• M-protein
• Very small (75 a.a.)
• Embedded in the virion envelope membrane and not
  highly immunogenic

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Replication
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Arthropod Vectors

• Mosquitoes
• Japanese encephalitis, dengue, yellow fever, St.
  Louis encephalitis, EEE, WEE, VEE etc.
• Ticks
• Crimean-Congo haemorrhagic fever, various
  tick-borne encephalitides etc.
• Sandflies
• Sicilian sandfly fever, Rift valley fever.

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Examples of Arthropod Vectors
Aedes Aegyti
Assorted Ticks
Phlebotmine Sandfly
Culex Mosquito
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Animal Reservoirs

• In many cases, the actual reservoir is not known.
  The following animals are implicated as
  reservoirs
• Birds Japanese encephalitis, St Louis
  encephalitis, EEE, WEE
• Pigs Japanese encephalitis
• Monkeys Yellow Fever
• Rodents VEE, Russian Spring-Summer
  encephalitis

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Transmission

• The most common route of infection is bite of
  infectious mosquito
• Other transmission modes where revealed in 2002
  such as
• Blood Transfusion
• Organ Transplantation
• Intrauterine
• Percutaneous exposure (occ. exposure)
• Breastmilk (probable)

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Transmission Cycles

• Man - arthropod -man
• e.g. dengue, urban yellow fever.
• Reservoir may be in either man or arthropod
  vector.
• In the latter transovarial transmission may take
  place.
• Animal - arthropod vector - man
• e.g. Japanese encephalitis, EEE, WEE, jungle
  yellow fever.
• The reservoir is in an animal.
• The virus is maintained in nature in a
  transmission cycle involving the arthropod vector
  and animal. Man becomes infected incidentally.
• Both cycles may be seen with some arboviruses
  such as yellow fever.

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Man-Arthropod-Man Cycle
Man - arthropod -man e.g. dengue, urban yellow
fever. Reservoir may be in either man or
arthropod vector. In the latter transovarial
transmission may take place.
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Animal-Arthropod-Man Cycle
Animal - arthropod vector - man e.g. Japanese
encephalitis, EEE, WEE, jungle yellow fever. The
reservoir is in an animal. The virus is
maintained in nature in a transmission cycle
involving the arthropod vector and animal. Man
becomes infected incidentally.
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Epidemiologic Feature

• The major outbreaks coincided with the heavy
  rainfall or floods.
• Seasonal more common in summer, July to October
• Infection provides life long immunity.
• Worldwide distribution
• More than 530 species, 150 pathogen to man

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Pathogenesis

• The nature of flavivirus disease is determined
  primarily by
• The specific tropisms of the individual virus
  type
• The concentration of infecting virus
• Individual host response to the infection

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Disease Syndromes of the Alphaviruses and
Flaviviruses
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Pathogenesis

• Virus
• Mononuclear Phagocyte
• Blood Circulation
• Viremia
• Adequate Weak
• Immunological Immunological
• Response Response
• Subclinical or mild Invades the CNS
• Systemic disease induce mortality

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Four stages

• A Prodromal Stage
• An Acute encephalitic Stage
• The Convalescence Stage
• A Sequela Stage

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Diseases Caused

• Fever and rash - this is usually a non-specific
  illness resembling a number of other viral
  illnesses such as influenza, rubella, and
  enterovirus infections. The patients may go on to
  develop encephalitis or haemorrhagic fever.
• Encephalitis - e.g. EEE, WEE, St Louis
  encephalitis, Japanese encephalitis.
• Haemorrhagic fever - e.g. yellow fever, dengue,
  Crimean-Congo haemorrhagic fever.

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• Pathogenesis and immunity
• Besides viral receptor, virus may attach to Fc
  receptor (macrophages and monocytes) via Ab to
  result in an increase of virus infection.
• Antibody is produced to block infection.
  However, non-neutralizing Ab may have antibody
  dependent enhancement (ADE) effect to enhance
  virus replication by hundred folds.

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Arthropod-Borne Viruses

• a) Dengue
• b) Yellow fever
• c) Chikungunya

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Fever/Rash/Arthritis

• 1. Triad of fever/rash/arthritis is
  characteristic of Chikungunya, onyong-nyong,
  Ross River, Mayaro, and Sindbis viruses
• 2. Symptoms generally appear after 2-3 days
  incubation
• a) fever, chills, myalgia
• b) polyarthralgia mainly affecting small joints
• c) maculopapular rash
• 3. Arthritis generally resolves in a few
  weeks, but may persist for months, or years in
  some cases.

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Arthropod-Borne Viruses Encephalitis

• West Nile encephalitis
• Japanese encephalitis
• St. Louis encephalitis
• Russian spring-summer encephalitis
• Powassan encephalitis
• Venezuelan equine encephalitis
• Eastern equine encephalitis
• Western equine encephalitis

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Transmission of six encephalitis arboviruses
Humanscan be infectedvia mosquito bites.
Small mammalsare hosts for VEE andCalifornia
viruses only.
Encephalitis arbovirusescan overwinter
insidemosquito eggs.
Mosquitoesare vectors.
Wild birds
Horses,and rarely other domestic mammalsare
hosts for equine viruses.
Domestic fowls
Birds are hosts forall six encephalitisarbovirus
es.
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West Nile Virus

• Flavivirus
• Primary host wild birds
• Principal arthropod vector mosquitoes
• Geographic distribution
• Africa,
• Middle East,
• Western Asia,
• Europe,
• Australia,
• North America,
• Central America

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Encephalitis

• Small proportion of individuals infected (a few
  days after the onset of fever) may develop
  drowsiness, neck rigidity, progressing to
  confusion, paralysis, convulsions and coma.
• Case-fatality rates average 10 20 (higher in
  elderly).
• 3. Survivors may be left with permanent
  neurologic sequelae such as mental retardation,
  epilepsy, paralysis, deafness, and blindness.

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Diagnosis

• Materials of epidemiology
• Clinical
• Laboratory Tests
• Tentative diagnosis
• Antibody titer HI, IF, CF, ELISA
• JE-specific IgM in serum or CSF
• Definitive diagnosis
• Virus isolation Blood, CSF sample, brain

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Diagnosis

• Serology - usually used to make a diagnosis of
  arbovirus infections. Antibody titer HI, IF, CF,
  ELISA
• Culture - a number of cell lines may be used,
  including mosquito cell lines. However, it is
  rarely carried out since many of the pathogens
  are group 3 or 4 pathogens. (Blood, CSF sample,
  brain)
• Direct detection tests - e.g detection of antigen
  and nucleic acids are available but again there
  are safety issues.

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Prevention

• Surveillance - of disease and vector populations
• Control of vector - pesticides, elimination of
  breeding grounds
• Personal protection - screening of houses, bed
  nets, insect repellants
• Vaccination - available for a number of arboviral
  infections e.g. Yellow fever, Japanese
  encephalitis, Russian tick-borne encephalitis

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Treatment/Vaccines/Control measures A.
Encephalitis 1. Vaccines exist for a number of
these viruses, but are used mainly for horses, at
risk lab workers, and some fowl known to be
intermediate hosts 2. Control of mosquitoes is
major countermeasure. B. Yellow Fever
1. Live attenuated virus vaccine. Used
when going to endemic areas
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Epidemiological Triangle
The Host
Interaction
The Virus
The Vector
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Prevention

• Vector (Mosquito) control
• Eliminate mosquito breeding areas Chemical
  larvicides, Biolarvicides, Environmental
  management
• Adult and larval control Anti-larval treatment
• Vaccination
• Personal protective measures
• Avoid prime mosquito hours from dusk to dawn
• Indoor spray and fogging Use of Insecticide

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Repellent Guidance

• Skin
• DEET still gold standard
• Both new additions good for shorter term
  protection
• Picaridin
• Roughly equivalent to DEET at same concentration
• Only a 7 product currently sold in US
• Oil of lemon eucalyptus
• Plant based
• 30 product similar to low concentration DEET
• Not for kids lt3 years old
• Clothing
• Permethrin

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Arboviruses

• Structure
• Positive sense ssRNA genome, icosahedral
  nucleocapsid, enveloped
• Pathogenesis
• Transmitted by bite of insect from host species
  sylvan and urban cycles
• Replication in cytoplasm budding
• Viremia to target tissue
• Influenza-like initial symptoms different
  viruses cause encephalitis, hemorrhagic fever,
  hepatitis, rash, arthritis
• Diagnosis
• Serology and nucleic acid
• Treatment/prevention
• No human vaccines except for Yellow Fever live
  attenuated vaccine, control of insect population

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The nomenclature of arboviruses are mostly based
on endemic areas and symptoms induced by viruses,
including fever, encephalitis and hemorrhagic
fever
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RETROVIRUSES
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RetroViruses

• RNA Viruses
• DNA From RNA by Reverse Transcriptase
• Insertion of new DNA into cellular DNA
• Hijacks the cell machinery to make VIRUS
• The virus only grows on T4 cells that are
  proliferating
• in response to an immune stimulus
• Difficult to grow in culture
• Robert Gallo HTLV-3
• Luc Montagnier LAV
• Human Immunodeficiency Virus (HIV)

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Introduction to Retroviruses

• I. Overview of retroviruses
• A. History
• B. Shared characteristics
• C. Classification
• II. Function of different regions of the
  retroviral genome
• A. Cis acting elements
• B. Gag proteins
• C. Pol proteins
• D. Env proteins
• III. Details of life cycle
• A. Early stage
• B. Late stage

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General Introduction to Retroviruses

• Retroviruses
• Ubiquitous found in all vertebrates
• Large, diverse family
• Includes HIV, FIV and FeLV
• Definition and classification of retroviruses
• Common features- structure, composition and
  replication
• Distinctive life cycle RNA-DNA-RNA
• Nucleic acid is RNA in virus, and DNA in infected
  cell
• Transmission may be either
• Horizontal - by infectious virus (exogenous
  virus) or vertical- by proviruses integrated in
  germ cells (endogenous virus)
• Can transmit either as free viral particle or
  (for some retroviruses) through cell-cell contact

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A Little Retrovirus History
1960s Howard Temin suggested DNA provirus
was part ofreplication cycle RNA ?DNA
? RNA ? Protein Won Nobel prize (with Baltimore)
in 1970 after they independently discovered RT
activity in infected cells 1980 Human T-cell
leukemia virus discovered, the first pathogenic
human retrovirus. 1982 Human immunodeficiency
virus discovered. 1990 First gene therapy trial
involving the use of retroviral-based vectors in
patient with a deficiency in adenosine deaminase
(ADA). 2006 Xenotropic murine leukemia-related
virus discovered.

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Retroviruses

• Strange Viruses ?
• At time-central dogma of molecular
  biologyDNA?RNA?Protein
• So.. RNA couldnt be template for DNA
• Unique replication cycle based on reverse
  transcription. Flow of information from RNA to
  DNA. (1971 Nobel Prize Temin / Baltimore)
• Retroviruses have been isolated from numerous
  species including chickens (RSV), mice (MLV),
  monkeys (SIV), and humans (HIV, HTLV)
• Simple Retroviruses encode only the genes gag,
  pol, and env (RSV)
• Complex Retroviruses encode in addition
  regulatory genes (HIV)
• Retroviruses are single-stranded RNA viruses that
  replicate through a double-stranded DNA
  intermediate.

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• They are association with the development of
  tumors in their host organisms.
• Study of these viruses eventually led to the
  discovery and development of the oncogene
• theory of tumorgenesis
• Some of the viruses actually contained oncogenes
  within their genomes, while others interacted
  with oncogenes in either a direct or indirect way
  to contribute to tumor formation.

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• Historically, because of their pattern of
  pathogenicity, these viruses were grouped into
  three subfamilies
• The acutely oncogenic retroviruses, or
  oncoretroviruses (such as those described above)
• The lentiviruses (associated with slow
  diseases or those with long latent periods)
• The spumaviruses (foamy viruses, named because
  of the pathogenic changes observed in infected
  cells).

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THE OLD NOMENCLATURE

• Members
• Oncogenic viruses (Oncoviruses) (endogenous)
• Avian oncoviruses RSV, AMV, AEV, RAVs RAV- 0
  RAV-1 .
• Murine oncoviruses e.g., MoMLV, A-MuLV.
• Mammalian oncoviruses e.g., FeLV, HaMSV, SSV .
• Mouse Mammary Tumor Virus (MMTV) the only B-
  type particle .
• Mason-Pfizer Monkey Virus (MPMV) one of the
  few D-type particles .
• Human T-Cell Lymphotropic Virus ( HTLV-1 2 ).
• Lentiviruses - HIV-1, HIV-2, SIV, FIV, EIAV,
  CAEV.
• Spumaviruses HFVs, SFV.
• 2 and 3 non endogenous

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Retrovirus Classification
Genus
Example
Genome
Avian leukemia virus
Alpharetrovirus
Simple
Betaretrovirus
Mouse mammary tumor virus
Simple
Murine leukemia virus Feline leukemia
virus Xenotropic murine leukemia-related virus
Gammaretrovirus
Simple
Human T-cell leukemia virus
Deltaretrovirus
Complex
Wall-eyed sarcoma virus
Epsilonretrovirus
Complex
HIV, SIV, FIV
Lentivirus
Complex
Human foamy virus
Spumavirus
Complex
Yeast TY-3
Metavirus
Drosophila melanogaster Gypsy
Errantvirus
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Retrovirus Structural Overview
Enveloped virus with lipid bilayer and viral
spike glycoproteins.
Have outer matrix protein and inner core capsid
containing viral genome.
Genome Two copies of single stranded
positive-stranded RNA (8-10kb).
All retroviruses contain gag, pol and env
genes. Simple - only gag, pol, env Complex -
additional genes involved in replication.
Reverse transcriptase to generate DNA
Viral genes are integrated into host genome.
Progeny virus produced using host cell
transcriptional and translational machinery.
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Retroviruses
Transmission EM
Scanning EM
3 D representation of HIV virion
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Retrovirus Genome (Diploid)

• Ranges from 7-10 kb in size (1 copy)

• Diploid 2 copies/virion

• Important in high recombination rate

From Flint et al. Principles of Virology (2000),
ASM Press
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PPT
y
( Packaging Signal)
PBS- primer binding site PPT- polypurine tract
AAAA 3
5m7GpppG
R
U5
U3
R
gag
pol
env
R - repeat sequence
PBS
U3 - promoter/enhancer
U5 - reverse transcription/ integration.
CA
SU
TM
MA
CA
NC
PRO
RT
IN
MA-Matrix CA- Capsid NC- Nucleocapsid
PRO- Protease RT- Reverse transcriptase IN-
Integrase
SU- surface envelope protein TM- transmembrane
envelope protein.
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Retroviral Structural genes
Gene Proteins Function gag group specific
antigen (internal structural proteins) Matrix
(MA), binds envelope, organization Capsid
(CA), protects genome and enzymes Nucleocapsid
(NC) chaperones RNA, buds pol
polymerase enzymes Reverse transcriptase RNA
to DNA RNAase H (RT) degrades template
RNA Protease (PR) maturation of
precursors Integrase (IN) provirus
integration env envelope proteins Surface
glycoprotein (SU) receptor binding
Transmembrane protein (TM) virus-cell fusion
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Viral life cycles

• Virulent these viruses lyse (kill) their host
  cell after infection.
• Temperate these viruses can replicate their
  genome along with the host cell genome without
  killing the host cell.
• These viruses are also capable of lyzing the host
  cell

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Distinct Steps in the Retroviral Life Cycle

• Attachment, Fusion, and Entry
• Reverse Transcription
• Integration
• Transcription
• Translation
• Assembly, Budding, and Maturation

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Multiplication of a Retrovirus
Capsid
Reverse transcriptase
DNA
Virus
Two identical stands of RNA
1
Retrovirus penetrates host cell.
Host cell
DNA of one of the host cells chromosomes
5
Mature retrovirus leaves host cell, acquiring an
envelope as it buds out.
Reverse transcriptase
2
Virion penetrates cell and its DNA is uncoated
Viral RNA
Identical strands of RNA
4
Transcription of the provirus may also occur,
producing RNA for new retrovirus genomes and RNA
that codes for the retrovirus capsid and envelope
proteins.
Viral proteins
RNA
3
The new viral DNA is tranported into the host
cells nucleus and integrated as a provirus. The
provirus may divide indefinitely with the host
cell DNA.
Provirus
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Retrovirus budding from a cell
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After Budding, Virus Goes from Immature to Mature
Form
Mature Form (after budding)-Core becomes more
dense-Different retroviruses have different
morphology in mature form
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Retroviruses May Transduce Cellular Sequences

• Transforming retroviruses are generated by a
  recombination event in which a
• cellular RNA sequence replaces part of the
  retroviral RNA.

• Transducing virus carries part of the host
  genome in place of part
• of its own sequence. The best known examples
  are retroviruses in
• eukaryotes and DNA phages in E. coli.
• Replication-defective virus cannot sustain an
  infective cycle by
• itself, because some of the necessary genes
  are absent (replaced by
• host DNA in a transducing virus) or mutated.
  It can, however, be
• perpetuated in the company of a helper virus.
  
• Helper virus provides missing viral functions
  to a defective virus,
• enabling to complete the infective cycle
  during a mixed infection.
• Transformation (oncogenesis) the ability to
  transform cultured
• cells so that the usual regulation of growth
  is released to allow
• unrestricted division.

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Transmission of Pathogenic Retroviruses
Virus Primary modes of transmission Range Preventative measures
ALV feces, saliva, skin, contact mother to offspring via egg worldwide common in untreated commercial flocks lacking endogenous viruses that limit infection removal of infected dams breeding resistant strains
REV feces, saliva, skin, contact mother to offspring via egg worldwide common in commercial flocks contaminant in Marek's disease vaccine none taken due to low incidence of disease
MLV mother to offspring via milk rare Lake Casitas, CA La Puente, CA none
MMTV mother to offspring via milk most inbred strains none