Diferenciace Diferenciace vakcinačních a vakcinačních a
divokých kmenů divokých kmenů varicella-zoster viru varicella-zoster viru
(VZV) pomocí (VZV) pomocí molekulárně molekulárně
biologických metodbiologických metod
RNDr. RNDr. Vanda BoVanda Boštíková, Ph.D.štíková, Ph.D.
Fylogenetická analýza založená na genotypizaci celých sekvencí VZV
Loparev et al., J. Virol., Vol. 81, 2007
(rubella)
Loparev et al., J. Virol., Vol. 78, 2004
Globální distribuce VZV - prevalence 70 % a více ve vyznačených oblastech
(2011 - E1, E2, M1 – M4, J)
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 yearsHealthy People 2020 goals 2010 90.51% VZV USA
Varicella in Vaccinated Varicella in Vaccinated PersonsPersons
Should we worry about it?Should we worry about it?
Gomi et al., J. Virol., Vol. 76, 2002
VZV
Genotyping
Pst -
Bgl +
Pst -
Bgl +
Pst +
Bgl -
Msp +
Sma +
Msp -
Sma -
Msp -
Sma -
Pst +
Bgl +
Msp -
Sma -
VaccineWildtype
Japanese
Wildtype
USWildtype
VZVGenotyping
Pst -
Bgl +
Pst +/-
Bgl +
Pst +
Bgl +
Msp +
Sma +
Msp -
Sma -
Msp -
Sma -
Pst +
Bgl -
Msp -
Sma -
OKAvWildtypeJapan
Wildtype20% USAWAustraliaAfrica,Asia
WildtypeEurope,EAustralia,80% US
Pst-,Bgl- reported from Australia
Preliminary genotyping:
The implementation of routine universal varicella
vaccination in the US created a need to distinguish
wild-type infections from vaccine injuries.
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+=OKAv
Pst+Bgl-
Pst+Bgl-
Pst+Bgl-
Pst+Bgl+
Pst+Bgl+
Pst+Bgl+
Pst+Bgl+
20% Pst+Bgl+
Pst-Bgl+
Pst+Bgl-
Pst+Bgl-
37%
The differences in the melting temperature of the Sma I ORF62 PCR product discriminate Oka vaccine and wt VZV strains.
VZVgenotyp ORF 22 :
4 SNPs #37902,38055,38081,38177
European Clade A or D
Mosaic Clade CM1M2M3M4
JapaneseClade B
+
ORF 50/ORF 21#87841/#3725
Clade A (E1)Genotype like DumasC/T
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-CarnesMarlene Deleon-Carnes Antonio GonzalezAntonio Gonzalez Myron LevinMyron Levin Stephanie LiffickStephanie Liffick Vladimir LoparevVladimir Loparev Stanislav PlíšekStanislav Plíšek Miloslav SalavecMiloslav Salavec Scott D. Schmid Scott D. Schmid
