PMID- 19968865
OWN - NLM
STAT- MEDLINE
DCOM- 20100120
LR  - 20211020
IS  - 1743-422X (Electronic)
IS  - 1743-422X (Linking)
VI  - 6
DP  - 2009 Dec 7
TI  - A rapid and efficient method for studies of virus interaction at the host cell 
      surface using enteroviruses and real-time PCR.
PG  - 217
LID - 10.1186/1743-422X-6-217 [doi]
AB  - BACKGROUND: Measuring virus attachment to host cells is of great importance when 
      trying to identify novel receptors. The presence of a usable receptor is a major 
      determinant of viral host range and cell tropism. Furthermore, identification of 
      appropriate receptors is central for the understanding of viral pathogenesis and 
      gives possibilities to develop antiviral drugs. Attachment is presently measured 
      using radiolabeled and subsequently gradient purified viruses. Traditional 
      methods are expensive and time-consuming and not all viruses are stable during a 
      purification procedure; hence there is room for improvement. Real-time PCR 
      (RT-PCR) has become the standard method to detect and quantify virus infections, 
      including enteroviruses, in clinical samples. For instance, primers directed to 
      the highly conserved 5' untranslated region (5'UTR) of the enterovirus genome 
      enable detection of a wide spectrum of enteroviruses. Here, we evaluate the 
      capacity of the RT-PCR technology to study enterovirus host cell interactions at 
      the cell surface and compare this novel implementation with an established assay 
      using radiolabeled viruses. RESULTS: Both purified and crude viral extracts of 
      CVB5 generated comparable results in attachment studies when analyzed with 
      RT-PCR. In addition, receptor binding studies regarding viruses with coxsackie- 
      and adenovirus receptor (CAR) and/or decay accelerating factor (DAF) affinity, 
      further demonstrated the possibility to use RT-PCR to measure virus attachment to 
      host cells. Furthermore, the RT-PCR technology and crude viral extracts was used 
      to study attachment with low multiplicity of infection (0.05 x 10(-4)TCID50/cell) 
      and low cell numbers (250), which implies the range of potential implementations 
      of the presented technique. CONCLUSION: We have implemented the well-established 
      RT-PCR technique to measure viral attachment to host cells with high accuracy and 
      reproducibility, at low cost and with less effort than traditional methods. 
      Furthermore, replacing traditional methods with RT-PCR offers the opportunity to 
      use crude virus containing extracts to investigate attachment, which could be 
      considered as a step towards viral attachment studies in a more natural state.
FAU - Jonsson, Nina
AU  - Jonsson N
AD  - School of Pure and Applied Natural Sciences, University of Kalmar, SE-391 82 
      Kalmar, Sweden. nina.jonsson@hik.se
FAU - Gullberg, Maria
AU  - Gullberg M
FAU - Israelsson, Stina
AU  - Israelsson S
FAU - Lindberg, A Michael
AU  - Lindberg AM
LA  - eng
PT  - Evaluation Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20091207
PL  - England
TA  - Virol J
JT  - Virology journal
JID - 101231645
RN  - 0 (CLMP protein, human)
RN  - 0 (Coxsackie and Adenovirus Receptor-Like Membrane Protein)
RN  - 0 (RNA, Viral)
RN  - 0 (Receptors, Virus)
SB  - IM
MH  - Animals
MH  - Cell Line
MH  - Chlorocebus aethiops
MH  - Coxsackie and Adenovirus Receptor-Like Membrane Protein
MH  - Cricetinae
MH  - Cricetulus
MH  - Enterovirus/genetics/*isolation & purification/*physiology
MH  - Humans
MH  - Polymerase Chain Reaction/*methods
MH  - RNA, Viral/*analysis/genetics
MH  - Receptors, Virus/metabolism
MH  - Time Factors
MH  - *Virus Attachment
PMC - PMC2797521
EDAT- 2009/12/09 06:00
MHDA- 2010/01/21 06:00
PMCR- 2009/12/07
CRDT- 2009/12/09 06:00
PHST- 2009/09/04 00:00 [received]
PHST- 2009/12/07 00:00 [accepted]
PHST- 2009/12/09 06:00 [entrez]
PHST- 2009/12/09 06:00 [pubmed]
PHST- 2010/01/21 06:00 [medline]
PHST- 2009/12/07 00:00 [pmc-release]
AID - 1743-422X-6-217 [pii]
AID - 10.1186/1743-422X-6-217 [doi]
PST - epublish
SO  - Virol J. 2009 Dec 7;6:217. doi: 10.1186/1743-422X-6-217.