A rapid and efficient method for studies of virus interaction at the host cell surface using enteroviruses and real-time PCR.
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.


CONCLUSIONS
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.
Jonsson
Nina
N
School of Pure and Applied Natural Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden. nina.jonsson@hik.se
Gullberg
Maria
M
Israelsson
Stina
S
Lindberg
A Michael
AM
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D006224:Cricetinae; D003412:Cricetulus; D004770:Enterovirus / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D011991:Receptors, Virus / Q000378:metabolism; D013997:Time Factors; D053585:Virus Attachment
C568771:CLMP protein, human; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D012367:RNA, Viral; D011991:Receptors, Virus