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Real-time PCR techniques are now commonly used for the detection of viral genomes in various human specimens and require for validation both external and internal controls (ECs and ICs). In particular, ICs added to clinical samples are necessary to monitor the extraction, reverse transcription, and amplification steps in order to detect false-negative results resulting from PCR-inhibition or errors in the technical procedure. Here, we performed a large scale evaluation of the use of bacteriophages as ICs in routine molecular diagnosis. This allowed to propose simple standardized procedures (i) to design specific ECs for both DNA and RNA viruses and (ii) to use T4 (DNA) or MS2 (RNA) phages as ICs in routine diagnosis. Various technical formats for using phages as ICs were optimised and validated. Subsequently, T4 and MS2 ICs were evaluated in routine real-time PCR or RT-PCR virological diagnostic tests, using a series of 8,950 clinical samples (representing 36 distinct specimen types) sent to our laboratory for the detection of a variety of DNA and RNA viruses. The frequency of inefficient detection of ICs was analyzed according to the nature of the sample. Inhibitors of enzymatic reactions were detected at high frequency in specific sample types such as heparinized blood and bone marrow (>70%), broncho-alveolar liquid (41%) and stools (36%). The use of T4 and MS2 phages as ICs proved to be cost-effective, flexible and adaptable to various technical procedures of real-time PCR detection in virology. It represents a valuable strategy for enhancing the quality of routine molecular diagnosis in laboratories that use in-house designed diagnostic systems, which can conveniently be associated to the use of specific synthetic ECs. The high rate of inhibitors observed in a variety of specimen types should stimulate the elaboration of improved technical protocols for the extraction and amplification of nucleic acids.

The alphaviruses were amongst the first arboviruses to be isolated, characterized and assigned a taxonomic status. They are globally very widespread, infecting a large variety of terrestrial animals, insects and even fish, and circulate both in the sylvatic and urban/peri-urban environment, causing considerable human morbidity and mortality. Nevertheless, despite their obvious importance as pathogens, there are currently no effective antiviral drugs with which to treat humans or animals infected by any of these viruses. The EU-supported project-VIZIER (Comparative Structural Genomics of Viral Enzymes Involved in Replication, FP6 PROJECT: 2004-511960) was instigated with an ultimate view of contributing to the development of antiviral therapies for RNA viruses, including the alphaviruses [Coutard, B., Gorbalenya, A.E., Snijder, E.J., Leontovich, A.M., Poupon, A., De Lamballerie, X., Charrel, R., Gould, E.A., Gunther, S., Norder, H., Klempa, B., Bourhy, H., Rohayemj, J., L'hermite, E., Nordlund, P., Stuart, D.I., Owens, R.J., Grimes, J.M., Tuckerm, P.A., Bolognesi, M., Mattevi, A., Coll, M., Jones, T.A., Aqvist, J., Unger, T., Hilgenfeld, R., Bricogne, G., Neyts, J., La Colla, P., Puerstinger, G., Gonzalez, J.P., Leroy, E., Cambillau, C., Romette, J.L., Canard, B., 2008. The VIZIER project: preparedness against pathogenic RNA viruses. Antiviral Res. 78, 37-46]. This review highlights some of the major features of alphaviruses that have been investigated during recent years. After describing their classification, epidemiology and evolutionary history and the expanding geographic distribution of Chikungunya virus, we review progress in understanding the structure and function of alphavirus replicative enzymes achieved under the VIZIER programme and the development of new disease control strategies.

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