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					Diferenciace vakcinačních a
 divokých kmenů varicella-
 zoster viru (VZV) pomocí
 molekulárně biologických
           metod
    RNDr. Vanda Boštíková, Ph.D.
Fylogenetická analýza založená na genotypizaci celých sekvencí
                            VZV




     Loparev et al., J. Virol., Vol. 81, 2007
(rubella)
Globální distribuce VZV - prevalence 70 % a více ve vyznačených
                         oblastech
                (2011 - E1, E2, M1 – M4, J)




     Loparev et al., J. Virol., Vol. 78, 2004
VZV Oka live-attenuated vaccine developed 1974 in Japan by Takahashi and
   others by extensive passage of a clinical isolate named “Oka” through hu and
   non-hu cells in vitro
  A series of clinical trials (11 000 healthy adults and children) demonstrated the
   safety and efficacy of the vaccine in the USA in 1995: approx. 85% of
   individuals had complete protection
  Immunity persists for at least 20 years
Healthy People 2020 goals
2010 90.51% VZV USA
Varicella in Vaccinated Persons
  Should we worry about it?
Gomi et al., J. Virol., Vol. 76, 2002
                                                               The implementation of routine universal
   Preliminary                                                                  varicella
                                                                vaccination in the US created a need to
   genotyping:                                     VZV
                                                   VZV
                                                Genotyping
                                                Genotyping
                                                                              distinguish
                                                                   wild-type infections from vaccine
                                                                                injuries.

                -                                                  +                               +
          Pst                          -
                                  Pst +/-                    Pst                             Pst
                +                                                  +
                                                                   -                               +
                                                                                                   -
          Bgl                     Bgl +                      Bgl                             Bgl


                    +                   -                              -                               -
         Msp                      Msp                        Msp                             Msp
                    +                       -                              -                               -
         Sma                      Sma                        Sma                             Sma



                                                Wildtype
                                                 Wildtype
                                                                               Wildtype
                                                                               Wildtype                        Wildtype
                         OKAv
                        Vaccine
                                                Japan
                                                Japanese
                                                                               20% USA
                                                                                    US
                                                                                                               Europe,EAustralia,
                                                                                                               Wildtype
                                                                               WAustralia
                                                                                                               80% US
                                                                               Africa,Asia


Pst-,Bgl- reported from Australia
Our lab developed LightCycler real-time PCR hybridization probe-based assays to detect VZV(PCR)
and genotype VZV strains based on the presence or absence of a BglI, PstI, or MspI restriction sites
within open reading frames (ORFs) 54, 38, and 62, respectively.
                                         Pst+Bgl-

    Pst+Bgl-       Pst+Bgl-
                              Pst+Bgl-
    20% Pst+Bgl+
                                            Pst+Bgl+     Pst+ Bgl+
Pst-Bgl+                                                 Pst-Bgl+=OKAv
                                          Pst+Bgl+                 37%

                        Pst+Bgl+

                                              Pst+Bgl+
                                                           Pst+Bgl-
The differences in the melting temperature of the Sma I
ORF62 PCR product discriminate Oka vaccine and wt
VZV strains.
                        VZV
                       genotyp22 :
                          ORF
                           4 SNPs
                   #37902,38055,38081,38177




                      Mosaic Clade C
 European                    M1               Japanese
Clade A or D                 M2                Clade B
                             M3
                             M4
     +

                      Clade A (E1)
ORF 50/ORF 21   Genotype like DumasC/T
 #87841/#3725

                     Clade D (E2)
                         T/C
Loparev et al., Virology, 2008
              Virology. 2009 Jan 20;383(2):216-25. Epub 2008 Nov 20.

Distribution of varicella-zoster virus (VZV) wild-type genotypes in northern and
  southern Europe: evidence for high conservation of circulating genotypes.


   Loparev VN, Rubtcova EN, Bostik V, Tzaneva V, Sauerbrei A, Robo A, Sattler-
   Dornbacher E, Hanovcova I, Stepanova V, Splino M, Eremin V, Koskiniemi M,
                          Vankova OE, Schmid DS.
   Marlene Deleon-Carnes
   Antonio Gonzalez
   Myron Levin
   Stephanie Liffick
   Vladimir Loparev
   Stanislav Plíšek
   Miloslav Salavec
   Scott D. Schmid

				
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