Virus Genes

, Volume 47, Issue 3, pp 532–545 | Cite as

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

  • Daniela Duque Ferreira
  • Shelley Cook
  • Ângela Lopes
  • António Pedro de Matos
  • Aida Esteves
  • Ana Abecasis
  • António Paulo Gouveia de Almeida
  • João Piedade
  • Ricardo Parreira


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.


Flaviviruses Negeviruses Mosquitoes Phylogenetic analysis Cytopathic effect 



This work was partially supported by Fundação para a Ciência e a Tecnologia (Ministério da Educação e Ciência) through UPMM funds.

Supplementary material

11262_2013_960_MOESM1_ESM.pdf (64 kb)
Supplementary data 1 Molecular analysis of COI (mitochondrial cytochrome c oxidase subunit I) sequences amplified from the pools of mosquitoes from which OCFVPT strains were isolated. The phylogenetic tree (Neighbor-Joining) was constructed using genetic distances corrected with the Kimura 2 parameter formula, based on multiple alignments of nucleotide sequences (all codon positions were used). The scale bar indicates 0.5 % of genetic diversity. The COI sequences from amplicons obtained from the analyzed pools (174, 207 and 350) were unambiguously identified as Ochlerotatus caspius. All the trees showed similar topologies. The example given indicates the analysis of the COI sequence from mosquito pool #174 (indicated as unknown specimen #174). (PDF 63 kb)
11262_2013_960_MOESM2_ESM.pptx (140 kb)
Supplementary data 2 Bayesian phylogenetic analysis of flavivirus E (A), NS3 (B) and NS5 (C) proteins, based on alignments of amino acid sequences. Posterior probability values ≥ 0.90 are indicated at specific branches. The list of sequences used, denoted by viral abbreviated name and accession numbers, can be found in Supplementary Table 3. The size bar indicates 40 % (A) or 20 % (B and C) of genetic distance (PPTX 140 kb)
11262_2013_960_MOESM3_ESM.doc (68 kb)
Supplementary material 3 (DOC 68 kb)
11262_2013_960_MOESM4_ESM.docx (29 kb)
Supplementary material 4 (DOCX 29 kb)


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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Daniela Duque Ferreira
    • 1
    • 2
  • Shelley Cook
    • 3
  • Ângela Lopes
    • 1
    • 2
  • António Pedro de Matos
    • 4
    • 5
  • Aida Esteves
    • 1
    • 2
  • Ana Abecasis
    • 6
  • António Paulo Gouveia de Almeida
    • 2
    • 7
  • João Piedade
    • 1
    • 2
  • Ricardo Parreira
    • 1
    • 2
  1. 1.Unidade de Microbiologia Médica, Grupo de VirologiaInstituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa (UNL)LisbonPortugal
  2. 2.Unidade de Parasitologia e Microbiologia Médicas (UPMM), Instituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa (UNL)LisbonPortugal
  3. 3.Natural History MuseumLondonUK
  4. 4.Centro de Estudos do Ambiente e do Mar (CESAM/FCUL), Faculdade de Ciências da Universidade de LisboaLisbonPortugal
  5. 5.Centro de Investigação Interdisciplinar Egas Moniz (CiiEM)CaparicaPortugal
  6. 6.Unidade de Saúde Pública Internacional e Bioestatística and Centro de Malária e Outras Doenças Tropicais (CMDT), IHMT/UNLLisbonPortugal
  7. 7.Unidade de Parasitologia Médica and UPMM, IHMT/UNLLisbonPortugal

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