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Characterization of a novel Negevirus and a novel Bunyavirus isolated from Culex (Culex) declarator mosquitoes in Trinidad.

ABSTRACT: Pools of mosquitoes were tested for insect-specific viruses using cytopathic effect (CPE) assays on Aedes albopictus (C6/36) cells. Illumina sequencing of RNA from pool TR7094, which produced extensive CPE 2 days post-infection, yielded the complete genome sequences of a previously unknown Bunyavirus, designated Cumuto virus (CUMV), and a second virus designated Wallerfield virus (WALV). WALV shared highest amino acid identity (60.1 %) with Dezidougou virus from Côte d'Ivoire, a positive-sense, single-strand RNA, insect-specific virus belonging to the newly proposed genus Negevirus associated with mosquitoes and phlebotomine sandflies. The S, M and L segments of CUMV were most closely related to those of Gouleako virus, also from Côte d'Ivoire (amino acid identities of 36 %, 38% and 54 % respectively). Neither virus produced CPE on vertebrate cells, or illness in newborn mice. Isolation and characterization of these viruses increase our knowledge of the geographical distribution, diversity and host range of mosquito-specific bunyaviruses and negeviruses.

SUBMITTER: Auguste AJ

PROVIDER: S-EPMC3917067 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Characterization of a novel Negevirus and a novel Bunyavirus isolated from Culex (Culex) declarator mosquitoes in Trinidad.

Auguste Albert J AJ Carrington Christine V F CVF Forrester Naomi L NL Popov Vsevolod L VL Guzman Hilda H Widen Steven G SG Wood Thomas G TG Weaver Scott C SC Tesh Robert B RB

The Journal of general virology 20131121 Pt 2

Pools of mosquitoes were tested for insect-specific viruses using cytopathic effect (CPE) assays on Aedes albopictus (C6/36) cells. Illumina sequencing of RNA from pool TR7094, which produced extensive CPE 2 days post-infection, yielded the complete genome sequences of a previously unknown Bunyavirus, designated Cumuto virus (CUMV), and a second virus designated Wallerfield virus (WALV). WALV shared highest amino acid identity (60.1 %) with Dezidougou virus from Côte d'Ivoire, a positive-sense, ...[more]

PMID: 24262627

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Project description:BACKGROUND:Mosquitoes are the potential vectors for a variety of viruses that can cause diseases in the human and animal populations. Viruses in the order Picornavirales infect a broad range of hosts, including mosquitoes. In this study, we aimed to characterize a novel picorna-like virus from the Culex spp. of mosquitoes from the Zambezi Valley of Mozambique. METHODS:The extracted RNA from mosquito pools was pre-amplified with the sequence independent single primer amplification (SISPA) method and subjected to high-throughput sequencing using the Ion Torrent platform. Reads that are classified as Iflaviridae, Picornaviridae and Dicistroviridae were assembled by CodonCode Aligner and SPAdes. Gaps between the viral contigs were sequenced by PCR. The genomic ends were analyzed by 5' and 3' RACE PCRs. The ORF was predicted with the NCBI ORF finder. The conserved domains were identified with ClustalW multiple sequence alignment, and a phylogenetic tree was built with MEGA. The presence of the virus in individual mosquito pools was detected by RT-PCR assay. RESULTS:A near full-length viral genome (9740 nt) was obtained in Culex mosquitoes that encoded a complete ORF (3112 aa), named Culex picorna-like virus (CuPV-1). The predicted ORF had 38% similarity to the Hubei picorna-like virus 35. The sequence of the conserved domains, Helicase-Protease-RNA-dependent RNA polymerase, were identified by multiple sequence alignment and found to be at the 3' end, similar to iflaviruses. Phylogenetic analysis of the putative RdRP amino acid sequences indicated that the virus clustered with members of the Iflaviridae family. CuPV-1 was detected in both Culex and Mansonia individual pools with low infection rates. CONCLUSIONS:The study reported a highly divergent, near full-length picorna-like virus genome from Culex spp. mosquitoes from Mozambique. The discovery and characterization of novel viruses in mosquitoes is an initial step, which will provide insights into mosquito-virus interaction mechanisms, genetic diversity and evolution.

Project description:BackgroundThe family Tymoviridae comprises three plant virus genera, including Tymovirus, Marafivirus, and Maculavirus, which are found in most parts of the world and cause severe agricultural losses. We describe a putatively novel member of the family Tymoviridae, which is isolated from mosquitoes (Culex spp.), referred to as CuTLV.Methods and resultsThe CuTLV was isolated by cell culture, which replicates and causes cytopathic effects in Aedes albopictus C6/36 cells, but not in mammalian BHK-21 or Vero cells. The complete 6471 nucleotide sequence of CuTLV was determined. The genome of CuTLV is predicted to contain three open reading frames (ORFs). The largest ORF1 is 5307 nucleotides (nt) in length and encodes a putative polypeptide of 1769 amino acids (aa), which contains the conserved motifs for the methyltransferase (MTR), Tymovirus endopeptidase (PRO), helicase (HEL), and RNA-dependent RNA polymerase (RdRp) of the replication-associated proteins (RPs) of positive-stranded RNA viruses. In contrast, the ORF1 sequence does not contain the so-called "tymobox" or "marafibox", the conserved subgenomic RNA promoter present in all tymoviruses or marafiviruses, respectively. ORF2 and ORF3 putatively encode a 248-aa coat protein (CP) and a proline-rich 149-aa polypeptide. The whole genomic nucleotide identity of CuTLV with other members of family Tymoviridae ranged from 46.2% (ChiYMV) to 52.4% (GFkV). Phylogenetic analysis based on the putative RP and CP genes of CuTLV demonstrated that the virus is most closely related to viruses in the genus Maculavirus.ConclusionsThe CuTLV is a novel virus related to members of the family Tymoviridae, with molecular characters that are distinct from those of tymoviruses, marafiviruses, and other maculaviruses or macula-like viruses. This is the first report of the isolation of a Tymoviridae-like virus from mosquitoes. Further investigations are required to clarify the origin, replication strategy, and the public health or agricultural importance of the CuTLV.

Dataset Information

Characterization of a novel Negevirus and a novel Bunyavirus isolated from Culex (Culex) declarator mosquitoes in Trinidad.

Publications

Characterization of a novel Negevirus and a novel Bunyavirus isolated from Culex (Culex) declarator mosquitoes in Trinidad.

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