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					    TOGA
GROUP A ARBOVIRUSES
       Alphaviruses (27 members)
   Serological relationships
      Cross-reactivity
          among species
          not genera
      Classification based on their antigenic properties
       (initially).
   Homologies in amino acid sequences of coat protein
    (of about 40%)
      genome of the WEE aprox. 82% homologous with
       EEE
   Aura virus              getah virus            Pixuna virus
   Babanki virus           Highlands J virus      Ross River virus
   Barmah Forest           Kyzylagach virus       Sagiyama virus
    virus                   Mayaro virus           Semliki Forest virus
   bebaru virus            Middelburg virus       Sindbis virus
   Buggy Creek virus       Mucambo virus          Una virus
   chikungunya virus       Ndumu virus            Venezuelan equine
   Eastern equine          Ockelbo virus           encephalitis virus
    encephalitis virus      o'nyong-nyong          Western equine
   Everglades virus         virus                   encephalitis virus
   Morgan virus                                    Whataroa virus.

   Rubivirus (1 member,
    Rubella).
       structure
   Spherical, 65-70nm;
   Capsid: icosahedral,
   80 trimer spikes,
   each spike = 3 x E1/E2
    heterodimers,
                      Genome
   RNA
   Single-stranded
   (+)sense,
   non-segmented
   5' cap
   3' poly-A.
   occurs in the cytoplasm
      rapid ~4h
                                            Replication
   attachment
   Cellular receptors are not known
      INFECT MANY DIFFERENT HOSTS
      Receptors widely distributed.
      May be several per virus
   Glycoprotein spikes are responsible for receptor
    binding
      Envelope proteins of virus grown in different cells
        have different net charges
      Very sensitive to pH and ionic strength-
   if virus coated with AB can attach to Fc receptor
      On Some cells of IS
   Penetration 2 mechanisms     Replication:
      accumulates in pits
            endocytosis
       direct membrane fusion


    2 rounds of translation:
    first
                                      Replication:
      (+)sense genomic RNA
      '49S' = 11.7kb
      acts directly as mRNA
      AUG codon 59 nucleotides from 5' cap is site of initiation
      partially translated (5' end) to produce N.S. proteins.
           About 2/3 of genome
           proteins are responsible for replication,
   1st rounds of translation   Replication:
   Two stop codons
   Terminate S1 get 230 K
    protein
      Cleaved to 3 NS
        proteins
   When read through to S2
    get 270 KD
      Cleaved to 4
      4th may regulate RNA
        synthesis
   S2 in 5' end of 26S RNA
      Two more stops right
        after
          Make sure stop
  TRANSCRIPTIONAL AND TRANSLATIONAL
   CONTROL OF EARLY TOGAVIRAL RNAS

  Start                             Start             (A)n
cap               Weak
                  Stop      Stop               Stop
           Occasional
           Read-through          Translation



                 Proteolytic cleavage

      Non-structural proteins

          ~90%            ~10%
   Two species of (+) RNA
    are synthesized,
                                   Replication:
      full length genomic
       RNA 49S
          Translated early into
           more NS
          Once capsid protein
           around encapsidated
      sub-genomic mRNA
       ('26S' = 4.1kb).
          Translated into
           structural proteins
          structural proteins
           from 3' end of
           genome
          Internal 21
           nucleotide junction
           site highly conserved
       3/1 Ratio of 26/49
                           Replication:
   2 rounds of
    translation:
   2ND forming a
    complementary
    (-)strand,
      template for
        further
        (+)strand
        synthesis.
      Initiate at 3"
        end of virion
        RNA
      19 nucleotides
        adjacent to poly
        A highly
        conserved
   Assembly and release
      Release and maturation
       almost simultaneous.
                                 Replication:
   In vertebrates
      occurs at the cell
       surface,
      envelope is acquired as
       the virus buds from the
       cell.
      Membrane Pulls tight so
       looks like shrink wrap
   Insects
      Buds from internal into
       vesicles
      Take to surface
   Alternate hosts, budding
    mechanisms and how enters
    (endo and fusion)
       Togavirus Replication Strategy
                       5’      Pre-NS precursor
Entry         ssRNA (+)

                                  Proteolytic
                                  cleavages
                                                      Non-structural
                                                      proteins
         3’
                  3’
                                         5’
      ssRNA (-)
                       5’                                    Structural
                                 3’                          precursor
                            Subgenomic ssRNA (+)
         ssRNA (+)
 5’                                   Capsid protein
                                                           Proteolytic
                                                           cleavages

                                  Envelope proteins
         3’
Life cycle of the Semliki forest virus




             Alberts et al. Figure 6-78
CLINICAL
RUBELLA
                            Rubella
   Rubella Nearly
    Eradicated in the US
      272 cases
       reported in 1999.
      58,000 cases
       reported in 1969.
   Mild disease of
    childhood
       Infectious before
        signs and
        symptoms
                            Rubella
   maternal infection
     first 12 weeks of pregnancy
         80% probability of a fetal infection with
          serious residual defects,
     weeks 13 and 14,
         67% probability
     week 15 and 16
         26. 25%
   MMR (measles, mumps, rubella) vaccination and autism
     no evidence ssociated with an increased risk of autism
                         Rubella
   Rubella CRS
      affects virtually all organ systems.
         intrauterine growth retardation.
         nerve deafness
         cataracts
         cardiac anomalies such as ventricular and atrial
          septal defect.
         sequelae include motor and mental retardation.
encephalitis
      Eastern Equine encephalitis
   rare but deadly disease
   severe sequelae for survivors.
   enzootic cycle
      wild birds
      mosquitoes
         Culiseta melanura
             rarely bites mammals
             rare transmission to
              humans and equines
         Asian tiger mosquito
          Aedes albopictus
             in fresh water
              swamps
      Eastern Equine encephalitis
   Most infections are inapparent
      (infection/case = 23/1).
      mortality is high
          if sequelae included mortality may reach 90%.
      Mortality in horses is 90%.
      Children seem to show a higher susceptibility
      older adults at higher risk too
   vaccine for horses and for laboratory workers
  Eastern Equine Encephalitis
Human cases: 1964-2001, Avg. 5/yr




    Human Cases Reported from 20 States
    Western Equine encephalitis (WEE)
   most prevalent in the western plain states
   human cases may follow outbreaks in horses.
   vast majority of cases are inapparent
      adults case/infection = 1/1150, children 1/58
   Fatality rates can reach 3-4%
      higher mortality seen in individuals over 55.
   Five documented cases of in utero transmission.
   birds and mosquitoes
   Culex tarsalis; breeds in ditches
   inactivated vaccine for equines and human lab workers..
Western Equine Encephalitis Human cases:
         1964-2001, Avg. 17/yr




       Human Cases Reported from 21 States
   Eight serologically distinct viruses
     subtype I, variants A/B, and C
                                                VEE
     Epizootic.
     Seem to disappear between epizootics
        Do they hide
        Re-emerge due to mutation of enzootic strains
     enzootic vs epizootic strain switch
        amino acid substitutions in the e2 envelope
        position 117 of the E2 envelope glycoprotein
        replacement of Glu by Lys
        E3 and E2 genes.
     epidemic/epizootic viruses closely related to, enzootic
      subtype
     Venezuelan Equine encephalitis
                (VEE)
   enzootic cycle
      rodents
      mosquito (Culex melanoconion).
   Mortality in humans
      .5% in adults
      4% in children.
   VEE II Everglades
      Florida
          Three CNS cases.
   Major epidemic in horses in Texas (1971)
      vaccine for equines and human lab workers
     Flavi
GROUP B ARBOVIRUS
   70 members
      many viruses with cross reactive surface Ags involved
          Cross protection
              vaccines
              multiple infections
   13 cause disease in humans.
   Most are group B arboviruses.
      Declared a separate family in 60S
   shares sequence homology with certain plant viruses
      particularly tobacco mosaic virus.
      nucleotide sequence is similar to picornavirus.
   1-yellow fever virus group (mosquito-borne):
      yellow fever virus
   2-tick-borne encephalitis virus group
      tick-borne encephalitis virus (TBEV)
           (a) European subtypes
                Hanzalova virus (HANV)
                 Hypr virus (HYPRV)
                 Kumlinge virus (KUMV)
                 Neudoerfl virus (NEUV)
           (b) Far eastern subtypes
                (Russian spring summer encephalitis virus) (RSSEV)
                 Absettarov virus (ABSV)
                 Karshi virus (KSIV)
                 Kyasanur forest disease virus (KFDV)
                 Langat virus (LGTV)
                louping ill virus (LIV)
                Negishi virus (NEGV)
                 Omsk hemorrhagic fever virus (OMSKV)
                Powassan virus (POWV)
                 Royal farm virus (RFV)
                 Sofyn virus (SOFV)
                Carey Island virus (CIV)
   3-Rio Bravo virus group (no known vector):
      Apoi virus (APOIV)
       Bukalasa bat virus (BUBV)
       Dakar bat virus (DBV)
       Entebbe bat virus (ENTV)
       Rio Bravo virus (RBV)
       Saboya virus (SABV)
   4-Japanese encephalitis virus group (mosquito-borne):
      Alfuy virus (ALFV)
       Japanese encephalitis virus (JEV)
       Kokobera virus (KOKV)
       Koutango virus (KOUV)
       Kunjin virus (KUNV)
       Murray Valley encephalitis virus (MVEV)
        St. Louis encephalitis virus (SLEV)
       Stratford virus (STRV)
        Usutu virus (USUV)
        West Nile virus (WNV)
   5-Tyuleniy virus group (tick-borne):
      Meaban virus (MEAV)
       Saumarez Reef virus (SREV)
       Tyuleniy virus (TYUV)
   6-Ntaya virus group (mosquito-borne):
      Bagaza virus (BAGV)
       Israel turkey meningoencephalitis virus (ITV)
       Ntaya virus (NTAV)
       Tembusu virus (TMUV)
       Yokase virus (YOKV)
   7-Uganda S virus group (mosquito-borne):
      Banzi virus (BANV)
      Bouboui virus (BOUV)
      Edge Hill virus (EHV)
       Uganda S virus (UGSV)
   8-Dengue virus group (mosquito-borne):
      Dengue virus 1 (DENV-1)
      Dengue virus 2 (DENV-2)
      Dengue virus 3 (DENV-3)
      Dengue virus 4] (DENV-4)
   9-Modoc virus group (no known vector):
      Cowbone Ridge virus (CRV)
        Jutiapa virus (JUTV)
        Modoc virus (MODV)
      Sal Vieja virus (SVV)
      San Perlita virus (SPV)
   3-viruses with no known vector:
      Aroa virus (AROAV)
        Cacipacore virus (CPCV)
        Hepatitic C (HCV)
      Montana myotis leukoencephalitis virus (MMLV)
        Sokuluk virus (SOKV)
        Tamana bat virus (TABV)
   Pestivirus no known human pathogens
      Type     Species
            bovine diarrhea virus (BDV)
      border disease virus (sheep) (BDV)
       bovine diarrhea virus (BDV)
       hog cholera virus (HCV)
   Tentative Species in the Genus
      1-tick-borne viruses:
         Gadget's Gully virus (GGYV)
           Kadam virus (KADV)
      2-mosquito-borne viruses:
         Bussuquara virus (BSQV)
           Ilheus virus (ILHV)
           Jugra virus (JUGV)
         Kedougou virus (KEDV)
           Naranjal virus (NJLV)
           Rocio virus (ROCV)
           Sepik virus (SEPV)
           Spondweni virus (SPOV)
           Wesselsbron virus (WSLV)
           Yaounde virus (YAOV)
           Zika virus (ZIKAV)
                       structure
   Spherical, 40-60nm;
   Capsid: Symmetry
    indistinct,
      nucleocapsid core (C )
       protein
          113 amino acid

          highly basic

   matrix ('M');
   Envelope: 1 glycoprotein
    ('E').
              Flavivirus Genome
   ss (+) RNA genome
   Approximately 11 kb
      long open reading frame of approximately 10,200
        nucleotides
   5’-m7GpppAmp cap
      Lacks some internal methylation
   Lacks 3’-polyA tail
      has conserved sequences
      May hairpin to protect
   Codes for
      3 structural proteins
                                   Flavivirus
         5' end of genome
         Capsid (C),
                                   Genome
         membrane (prM/M)
         envelope (E)
      7 non-structural proteins
         at 3' end.
         NS1, NS2A, NS2B,
           NS3, NS4A, NS4B,
           NS5
   Conserved sequences at 3'
    and 5' end
      may be recognition for
       replicase
      By starting at either end
       could make + and -
       strands
               Flavivirus Genome
   Pestivirus and the Hepacivirus genus
      internal ribosomal entry sites (IRES)
         provide a site of translation initiation for
          host ribosomes.
   Flavivirus
      ribosomal scanning to commence protein
       synthesis.
   in cytoplasm
   attachment and penetration
      Endocytosis
                                                Replication:
      Also Fc receptors
           if VIRUS covered with AB
           Gets around cross reactive AB
           Targets specific cells WITH Fc
            receptors
   replication
      entire virus genome is translated as a
        single polyprotein
           cleaved into mature proteins
           Very fast - poly protein doesn’t
            accumulate
           Host enzymes
      Complementary (-)strand RNA is
        synthesized by N.S. proteins,
           used as a template for genomic
            progeny RNA synthesis.
      5’NCR Structural protein    Non-structural proteins   3’NCR         RNA
cap


       C prM E       NS1 NS2A NS2B       NS3 NS4A 2KNS4B NS5
                                                                    Polyprotein


                        Post-translational Processing

 C prM          E   NS1 NS2A NS2B NS3           NS4A 2K NS4B         NS5

  pr        M                       NS3’     NS3”

        Signal peptidase site
                                         NS3 Protease, helicase, NTPase
        Unique site
        NS2B-NS3 protease site           NS5 Methyltransferase, RNA polymerase
    Replication:
   Assembly occurs
    during budding,
       characteristically into
        cytoplasmic vacuoles
        rather than the cell
        surface
       do not see naked nucleo
        capsids in cytoplasm
       Release when cell lyses.
flavi
DISEASES OF
 INTEREST
Human SLE Case Distribution by State
           1964 - 2001

        3
                    2           19
    2                                      8                                       9
            3                   3                    5
                        1                                   21
                                                                             36        CT 1
                                           25
                                 14                               440
        3                                            695 368                           DC 9
                1       88                                              12    7        NJ 131
                                    125         75            67
  123                                                                         3        DE 1
                                                            141                        MD 9
                                      11
            29              6                   65
                                                         337 150    5
                                    970
                                                 80
                                                                             379

                                                           1990s
                                                           2000
                                                           2001
                            SLE
   The virus was first
      recognized in 1932 Paris, Illinois

      isolated in 1933 during the St. Louis epidemic from
       a human brain.
   Since 1964 there have been 4,478 reported human cases of St.
    Louis encephalitis
      1964 - 1992 the U.S. averaged 86 cases per year.

   1975 epidemic with 1800 documented infections.
   1991 outbreak in Pine Bluff, Arkansas.
           ETIOLOGIC AGENT
   St. Louis encephalitis
    virus
   flavivirus related to
    Japanese encephalitis
    virus
                          SLE
   St. Louis encephalitis virus is NOT transmitted from
    person-to-person.
   Only infected mosquitoes can transmit St. Louis
    encephalitis virus
                  SLE - BIRDS
   sparrows, finches, blue jays, robins and doves
   a bird becomes infected by the bite of a carrier
    mosquito
      produces more virus in its blood

      can infect other susceptible mosquitoes that bite

   infected bird can produce an appreciable amount of
    virus in one or two days
      the virus disappears two to three days later

      bird remains ¨infective¨ for only a few days

   Birds do not show any symptoms of disease and
    become immune
                   SLE Symptoms
   Most cases are inapparent
      case/infection = 1/100

   Incidence and symptoms are highly correlated with age.
      During epidemics

          people over 60 are 5 to 40 times more likely to be
           symptomatic than those under 10
      encephalitis increase from approximately 50% in those 20 or
       younger, to close to 90% for those over 60.
      Mortality

          less than 5% for those persons under 50 years of age

          7% to 25% in those over 50.
                    SLE Symptoms
   Mild infections
       fever with headache.
   severe infection
       headache, high fever, neck stiffness, stupor, disorientation,
        coma, tremors, occasional convulsions (especially in infants)
        and spastic (but rarely flaccid) paralysis.
   sometimes misdiagnosed as stroke
   residual neurological damage ("sequelae'),
       Paralysis
       memory loss
       deterioration of fine motor skills.
                       Overview of West Nile virus
•Transmission Cycle
         Mosquito vector
                                      Incidental
                                      infections




     West Nile virus




         Bird reservoir hosts     Incidental
                                  infections
                        Outbreak Timeline

         Encephalitis                     Equine Disease
      Condition Identified                  Identified




 Uganda            Israel Israel France     South Africa   Romania 1996
  1937            1951-54 1957 1962            1974        Italy 1998
                                                           Russia 1999
febrile adult woman                                        US 1999-2002
West Nile District of                                      Israel 2000
Uganda                                                     France 2000
    WEST NILE VIRUS - FACTS
    most people who were
    infected with West Nile virus
    have no symptoms or
    experience mild illness such
    as a fever, headache and
    body aches before fully
    recovering.
   In the elderly, West Nile
    virus can cause serious
    disease that affects brain
    tissue. At its most serious, it
    can cause permanent
    neurological damage and can
    be fatal.
       WNV Human Infection “Iceberg” Revisited

1 CNS disease case           <?%           ~10% fatal
         =                   CNS           (<0.1% of total infections)
~?? total infections       disease

                                                   Febrile illness alone
                             ~?%                   less frequent?
                       “West Nile Fever”




                           ~80%
                        Asymptomatic
WEST NILE VIRUS - FACTS
   One Documented case of spread to fetus
   There is no specific therapy.
        In more severe cases, intensive supportive
        therapy is indicated, i.e., hospitalization,
        intravenous (IV) fluids and nutrition, airway
        management, ventilatory support (ventilator) if
        needed, prevention of secondary infections
        (pneumonia, urinary tract, etc.), and good nursing
        care.
   There is no vaccine for humans
                   Yellow Fever
   Yellow Fever: (Latin
    'flavus' = yellow).
   One virus, of invariant
    serotype,
   Transmitted by
    mosquitoes.
      Aedes aegypti

   Urban and sylvatic cycles
      Different vectors
             Dengue and Dengue
             Hemorrhagic Fever
•   Dengue virus and its
    mosquito vectors are
    distributed worldwide in
    tropical environments

•   Dengue is an increasing
    problem globally and is
    now the most important
    vectorborne viral disease
                               Dengue
   Dengue Fever:
   4 serotypes;
   Primary infection produces a (relatively) mild,
    self-limited, febrile illness.
   Re-infection with a different antigenic type of the virus
           dengue haemorrhagic fever:
           high fever, haemorrhagic shock, myocarditis, encephalitis;
           mortality ~15% -
           probably autoimmune mediated.
           Used AB and Fc receptors to get into cell
   .
                                           DV-specific
                                                              bystander lysis
                                          T lymphocyte
         dengue virus                                                           hepatocyte
                                                           ? interaction
                              cytolysis
DV-specific Ab
                 direct                    activation
                                                                      ?
                 entry                    recruitment

                                                   cytokine
                                                  production
                          DV-infected
                                                                                    plasma
                          myeloid cell
              Ab-enhanced                                                           leakage
   virus-Ab      uptake (mono, MF, DC)           ? cell-cell interaction
   complex

                        complement
                         activation
                                                                       vascular
                                                                      endothelial
                                                                         cell