Toscana virus inhibits the interferon beta response in cell cultures.

Toscana virus (TOSV) is an emerging pathogen in the Mediterranean basin where it causes summertime outbreaks of aseptic meningitis and meningoencephalitis. Many aspects of TOSV biology remain unknown including the possible implication of an amplifying mammalian host besides its vector. The three experiments described here were designed to assess the relationship between TOSV and type-I interferon (IFN) response. The main findings were as follows. First, TOSV growth in Vero cells is sensitive to an antiviral state induced by low-dose addition of exogenous IFN beta (IFN-ß) (10IU/ml). Second, no IFN-ß mRNA or IFN-ß was detectable after infection of HeLa and 293T cells by TOSV. Finally, TOSV inhibits IFN-ß production induced by Sendaï virus, a well known inducer of IFN-ß production. In addition to showing that TOSV can inhibit the IFN-ß response, these findings suggest that anti-IFN capability is maintained by regular contact with that of a mammalian host.

Long PCR Product Sequencing (LoPPS): a shotgun-based approach to sequence long PCR products.

Here we describe a practical procedure for sequencing long PCR products. The method relies on ultrasonic shearing of PCR products, resulting in fragments 700-1,000 nt long. Termini are subsequently repaired to obtain blunt ends and 3' A-overhangs are added before TA cloning. A predetermined number of clones are sequenced using an insert-independent primer to obtain an overlapping contig covering the full length of the PCR product. This method is cost effective and enables the complete sequencing of any large PCR product in a high-throughput format. Processing of amplified DNA requires 3 h handling time prior to the ligation step, and the clone library is available 2 d later. The complete sequence information is obtained approximately 5 d after the PCR step, depending on the sequencing procedure adopted.