title: Negeviruses found in multiple species of mosquitoes from southern Portugal: Isolation, genetic diversity, and replication in insect cell culture.
abstract: In this report, an RT-PCR approach based on the use of degenerate primers allowed the identification of negeviruses in four different species of mosquitoes (Ochlerotatus caspius, Culex pipiens, Cx. theileri and Cx. univittatus) collected in southern Portugal. The genomes of two of these viruses, sequenced to full completion, were shown to encode all the proteins encoded by previously described negeviruses. One of these viruses induces exuberant cytopathic effect in insect cell culture, with no obvious signs of apoptosis induction, replicating very rapidly and allowing for the detection of viral genomes in the infected culture supernatant as soon as 4h post-infection. This virus was also shown to use a dsRNA intermediate, which was found to be fully formed and active 3h after infection. Phylogenetic analysis of two products encoded by the viral ORF1 placed both viruses among Negev virus cluster, in the recently proposed Nelorpivirus taxon.
authors.affiliation: Unidade de Microbiologia Médica (Grupo de Virologia), Instituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa (UNL), Rua da Junqueira 100, 1349-008 Lisboa, Portugal. Electronic address: saracarapeta@gmail.com.
authors.forename: Sara
authors.initials: S
authors.lastname: Carapeta
authors.affiliation: Unidade de Microbiologia Médica (Grupo de Virologia), Instituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa (UNL), Rua da Junqueira 100, 1349-008 Lisboa, Portugal. Electronic address: beatrizdobem@gmail.com.
authors.forename: Beatriz
authors.initials: B
authors.lastname: do Bem
authors.affiliation: Unidade de Microbiologia Médica (Grupo de Virologia), Instituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa (UNL), Rua da Junqueira 100, 1349-008 Lisboa, Portugal. Electronic address: jammy1137@gmail.com.
authors.forename: James
authors.initials: J
authors.lastname: McGuinness
authors.affiliation: Unidade de Microbiologia Médica (Grupo de Virologia), Instituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa (UNL), Rua da Junqueira 100, 1349-008 Lisboa, Portugal; Unidade de Parasitologia e Microbiologia Médicas (UPMM), IHMT/UNL, Lisboa, Portugal. Electronic address: aidaesteves@ihmt.unl.pt.
authors.forename: Aida
authors.initials: A
authors.lastname: Esteves
authors.affiliation: Unidade de Saúde Pública Internacional e Bioestatística and Centro de Malária e Outras Doenças Tropicais (CMDT), IHMT/UNL, Lisboa, Portugal. Electronic address: ana.abecasis@ihmt.unl.pt.
authors.forename: Ana
authors.initials: A
authors.lastname: Abecasis
authors.affiliation: Unidade de Microbiologia Médica (Grupo de Virologia), Instituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa (UNL), Rua da Junqueira 100, 1349-008 Lisboa, Portugal. Electronic address: amendes@ihmt.unl.pt.
authors.forename: Ângela
authors.initials: Â
authors.lastname: Lopes
authors.affiliation: Centro de Estudos do Ambiente e do Mar (CESAM), and Centro de Investigação Interdisciplinar Egas Moniz (CiiEM), Quinta da Granja, Monte de Caparica, 2829-511 Caparica, Portugal. Electronic address: apamatos@kanguru.pt.
authors.forename: António P
authors.initials: AP
authors.lastname: de Matos
authors.affiliation: Unidade de Microbiologia Médica (Grupo de Virologia), Instituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa (UNL), Rua da Junqueira 100, 1349-008 Lisboa, Portugal; Unidade de Parasitologia e Microbiologia Médicas (UPMM), IHMT/UNL, Lisboa, Portugal. Electronic address: jp@ihmt.unl.pt.
authors.forename: João
authors.initials: J
authors.lastname: Piedade
authors.affiliation: Unidade de Parasitologia e Microbiologia Médicas (UPMM), IHMT/UNL, Lisboa, Portugal; Unidade de Parasitologia Médica, IHMT/UNL, Lisboa, Portugal. Electronic address: pauloalmeida@ihmt.unl.pt.
authors.forename: António P G
authors.initials: AP
authors.lastname: de Almeida
authors.affiliation: Unidade de Microbiologia Médica (Grupo de Virologia), Instituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa (UNL), Rua da Junqueira 100, 1349-008 Lisboa, Portugal; Unidade de Parasitologia e Microbiologia Médicas (UPMM), IHMT/UNL, Lisboa, Portugal. Electronic address: Ricardo@ihmt.unl.pt.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Parreira
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D016000:Cluster Analysis; D003465:Culex / Q000821:virology*; D003588:Cytopathogenic Effect, Viral; D014644:Genetic Variation; D008969:Molecular Sequence Data; D044969:Ochlerotatus / Q000821:virology*; D010802:Phylogeny; D011174:Portugal; D012328:RNA Viruses / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017385:Sequence Homology
chemical_list: D012367:RNA, Viral
keywords: Cytopathic effect; Mosquitoes; Negevirus; Phylogenetic analysis; Portugal; RT-PCR; Viral replication; dsRNA intermediate