A metagenomic survey of viral abundance and diversity in mosquitoes from Hubei province

Abstract

Mosquitoes as one of the most common but important vectors have the potential to transmit or acquire a lot of viruses through biting, however viral flora in mosquitoes and its impact on mosquito-borne disease transmission has not been well investigated and evaluated. In this study, the metagenomic techniquehas been successfully employed in analyzing the abundance and diversity of viral community in three mosquito samples from Hubei, China. Among 92,304 reads produced through a run with 454 GS FLX system, 39% have high similarities with viral sequences belonging to identified bacterial, fungal, animal, plant and insect viruses, and 0.02% were classed into unidentified viral sequences, demonstrating high abundance and diversity of viruses in mosquitoes. Furthermore, two novel viruses in subfamily Densovirinae and family Dicistroviridae were identified, and six torque tenosus virus1 in family Anelloviridae, three porcine parvoviruses in subfamily Parvovirinae and a Culex tritaeniorhynchus rhabdovirus in Family Rhabdoviridae were preliminarily characterized. The viral metagenomic analysis offered us a deep insight into the viral population of mosquito which played an important role in viral initiative or passive transmission and evolution during the process.

Fig 1. Taxonomic classification of viral sequences in three samples.

(A) Viral sequences were classified by host type. Percentage of Mycovirus was too low which hardly can be seen from figure. (B) Viral sequences were classified on family level. The family with reads number less than 10 did not show in figure. Different host types and families were denoted with different colors.

Fig 2. Neighbor joining phylogenetic tree of TTSuV1 based on 678 bp segment.

678 bp containeduntranslated region (UTR), the complete NS2 gene (ORF2) and 191 bp of the 5’end of capsid protein gene (ORF1). Six TTSuV1 sequences characterized in this study were marked with black arrow.

Fig 3. Alignment of PPV contigs in three samples.

ORF of reference was labeled with grey strips. Contigs of three samples filled with red, yellow and purple respectively. Stroked arrow denoted the amplified fragments through PCR.

Fig 4. Neighbor joining phylogenetic tree of PPV based on nucleic acid of NS1.

PPV sequences identified in this study were labeled with black arrows.

Fig 5. Amplification and sequence analysis of DNV in Sample III.

(A) The mapping of DNV contigs in this study. It only presented the contigs used for assembly. ORF of reference was labeled with grey strips. Stroked arrow denoted the amplified fragments through PCR. (B) The ORF prediction of the sequenced novel DNV in Sample III. Black part (279 bp to 545 bp) of ORF1 was identified as Parvo_NS1 Superfamily. (C) 119 aa highly conserved region within NS1 of novel DNV like other DNVs. Domain A was the NTP-binding part. B and C were helicase domain.

Fig 6. Phylogenetic analysis of DNV using neighbor joining method.

(A) phylogenetic trees constructed based on aa sequence of putative ORF1 of HB-3 DNV and NS1 of other DNVs. (B) Partial nucleic acid sequence of Sample I contigs located in NS1 region was used for Phylogenetic analysis. DNVs identified in this study were marked with black arrows.

Fig 7. Alignment of CTRV contigs in Sample I.

Grey strips were the contigs of CTRV in Sample I. Short line between two parts of L protein was the intron sequence.

Fig 8. Alignment and phylogenetic analysis of contigs in Family Dicistroviridae of Sample I.

(A) The mapping of contigs in Family Dicistroviridae of Sample I. ORF of reference was labeled with grey strips. Stroked arrowdenoted the amplified fragments through PCR. Triangle glyph with a line denoted the region used for phylogenetic analysis.(B) 1473 bp of the non-structure protein gene was used for phylogenetic analysis. BSRV detected in Sample I was labeled with black arrows.