Detection and identification of Toscana and other phleboviruses by RT‐nested‐PCR assays with degenerated primers

Abstract

Phleboviruses are a large and widespread group of viruses that are transmitted by arthropods. Toscana virus is one of the principal agents that causes meningitis in humans during the summer in Italy and, possibly, in other Mediterranean countries. Rift Valley Fever virus can cause serious illness in both animals and humans, leading to high morbidity and mortality, and is considered to be a potential agent for epizootics and human epidemics. Since information on this group of viruses is still scant, reliable laboratory tools for diagnosis and epidemiological surveillance must be developed, in order to ascertain their real impact on Public Health. Sequence data obtained from Spanish isolates of Toscana virus and other phleboviruses confirmed that natural genome variability may hamper the diagnosis of these agents by molecular methods, so this must be borne in mind when developing reliable assays. In view of the above, a novel and useful protocol has been developed for the detection and specific identification of every member of the phlebovirus genus present in a sample, including Toscana virus, based on a generic RT-nested-PCR, followed by sequencing of the amplified fragment. A change in this method also allowed specific direct detection and identification of wild isolates of Toscana virus of different geographical origin, using newly designed primers. Testing clinical samples with these assays confirmed the role of Toscana virus as an agent that causes acute aseptic meningitis in the central region of Spain.

Toscana Virus in Spain

“…Briefly, 500 μL lysis buffer was added to the tubes containing the phlebotomine sandflies, tubes were vortexed and centrifuged at 13,000 rpm for 10 min, and 250 μL of the supernatant was used for RNA extraction. A generic RT-nested-PCR method was used to detect TOSV RNA as described previously ( 17 ), which amplifies a 244-bp fragment of the L gene of phleboviruses. Sequences of the primers are shown in Table 1.…”

“…Specific TOSV detection was achieved by sequencing PCR products, as described previously ( 17 ). Briefly, PCR products were purified from a 2% low-melting-point agarose gel with the QIAquick PCR purification kit (Qiagen).…”

“…TOSV-specific sequences were confirmed by BLAST (basic local alignment search tool) search against GenBank databases. Sequences of the L gene from positive pools were compared with the corresponding sequence of the Italian strain ISS Phl.3 and with 1 Spanish TOSV isolate (STI) obtained previously ( 17 ) with the ClustalW Multiple Sequence Alignment program (1.82 version; European Bioinformatics Institute, Cambridge, UK).…”

“…Subsequently, different combinations of primers were selected to amplify 1 STI. Finally, the obtained sequence and the 1 from the Italian strain ISS Phl.3 were aligned, and specific primers were redesigned to amplify STIs recovered from sandflies and previous STIs from patients ( 17 ) (Table 1). RT-PCR conditions are available on request.…”

“…Later, cases of TOSV infections were detected in other areas of Spain ( 15 ). Phylogenetic analysis of short polymerase chain reaction (PCR) products from the L segment showed that nucleotide sequences of TOSV isolates from Granada differ significantly from the Italian strain ISS Phl.3 ( 17 ). …”