Characterization of an insect-specific flavivirus (OCFVPT) co-isolated from Ochlerotatus caspius collected in southern Portugal along with a putative new Negev-like virus

Abstract

We describe the isolation and characterization of an insect-specific flavivirus (ISF) from Ochlerotatus caspius (Pallas, 1771) mosquitoes collected in southern Portugal. The RNA genome of this virus, tentatively designated OCFVPT, for O. caspius flavivirus from Portugal, encodes a polyprotein showing all the features expected for a flavivirus. As frequently observed for ISF, the viral genomes seems to encode a putative Fairly Interesting Flavivirus ORF (FIFO)-like product, the synthesis of which would occur as a result of a -1 translation frameshift event. OCFVPT was isolated in the C6/36 Stegomyia albopicta (= Aedes albopictus) cell line where it replicates rapidly, but failed to replicate in Vero cells in common with other ISFs. Unlike some of the latter, however, the OCFVPT genome does not seem to be integrated in the mosquito cells we tested. Phylogenetic analyses based on partial ISF NS5 nucleotide sequences placed OCFVPT among recently published viral strains documented from mosquitoes collected in the Iberian Peninsula, while analyses of ORF/E/NS3/or NS5 amino acid sequences cluster OCFVPT with HANKV (Hanko virus), an ISF recently isolated from O. caspius mosquitoes collected in Finland. Taking into account the genetic relatedness with this virus, OCFVPT is not expected to be overtly cytopathic to C6/36 cells. The cytopathic effects associated with its presence in culture supernatants are postulated to be the result of the replication of a co-isolated putative new Negev-like virus.

Insect-specific flaviviruses, a worldwide widespread group of viruses only detected in insects

“…A number of ISFVs could be described in several mosquito species around the world; some of these viruses have already been isolated, whereas in other cases, only sequences detected in field-collected mosquitoes are available. These sequences strongly suggest the presence of other putative viruses not yet isolated, as in the case of OcFV, isolated after the detection of field sequences (Calzolari et al 2007, Ferreira et al 2013. Several of these sequences have been obtained in Aedes mosquitoes in Europe (Calzolari et al, 2012;Cerutti et al, 2012;Vázquez et al, 2012;Osório et al, 2014) and…”

“…Other viruses related to CFAV were subsequently isolated directly from field-collected mosquitoes, such as Kamiti River virus (KRV) in Kenya ; Culex flavivirus (CxFV) in Japan, Guatemala, USA, Mexico, Uganda, Caribbean, China, Brazil, and Argentina (Hoshino et al, 2007;Morales-Betoulle et al, 2008;Blitvich et al, 2009;Bolling et al, 2011;Cook et al, 2009;Kim et al, 2009;Saiyasombat et al, 2010;Huanyu et al, 2012;Machado et al, 2012;Goenaga et al, 2014); Aedes flavivirus (AeFV) in Japan ( Hoshino et al, 2009), Italy (Roiz et al, 2012;Grisenti et al, 2015), and USA (Haddow et al, 2013); Quang Binh virus (QBV) in Vietnam (Crabtree et al, 2009); Nakiwogo virus in Uganda (Cook et al, 2009); Calbertado virus in North America Tyler et al, 2011); Hanko virus in Finland (Huhtamo et al, 2012); Culex theileri flavivirus in Portugal (Parreira et al, 2012) (Ferreira et al, 2013).…”

Wide detection of Aedes flavivirus in north-eastern Italy – a European hotspot of emerging mosquito-borne diseases

“…Since then, it has been detected in wild-caught mosquitoes in Puerto Rico (Cook et al, 2006), Thailand (Kihara et al, 2007) and Mexico (Espinoza-Gó mez et al, 2011). Over the past 40 years, many other ISFs have been isolated and identified from field-collected mosquitoes, belonging to different species, in several locations (Ferreira et al, 2013;Haddow et al, 2013;Papa et al, 2014). These include Kamiti River virus (KRV), Culex flavivirus (CxFV), Spanish Culex flavivirus, Culex theileri flavivirus, Aedes flavivirus (AeFV), Aedes vexans flavivirus (AeveFV), Czech Aedes vexans flavivirus, Aedes galloisi flavivirus, Aedes cinereus flavivirus, Ochlerotatus flavivirus (OcFV), OcFV from Portugal, Spanish OcFV, Quang Binh virus, Nakiwogo virus, Calbertado virus, Chaoyang virus and Hanko virus.…”

“…mosquitoes (Crochu et al, 2004;Cook et al, 2009;Roiz et al, 2009;Calzolari et al, 2010a;Sánchez-Seco et al, 2010;Vázquez et al, 2012;Ferreira et al, 2013;Haddow et al, 2013;Papa et al, 2014).…”

“…Since then, it has been detected in wild-caught mosquitoes in Puerto Rico (Cook et al, 2006), Thailand (Kihara et al, 2007 and Mexico (Espinoza-Gó mez et al, 2011). Over the past 40 years, many other ISFs have been isolated and identified from field-collected mosquitoes, belonging to different species, in several locations (Ferreira et al, 2013;Haddow et al, 2013; Papa et al, 2014 Calzolari et al, 2010a Calzolari et al, , b, 2012aCalzolari et al, , 2013aCerutti et al, 2012;Ravanini et al, 2012;Pautasso et al, 2013). Moreover, DNA sequences related to AeFV were identified in wild Aedes albopictus mosquito collected in 2007(Roiz et al, 2009).…”