Genomic and Biological Characterization of a New Cypovirus Isolated from Dendrolimus punctatus

Abstract

A novel cypovirus (designated DpCPV-MC) was isolated from the pine moth Dendrolimus punctatus using serial in vivo cloning procedures. DpCPV-MC occurs in typical polyhedral occlusion bodies, containing a number of spherical virions. Laboratory bioassays indicated that the infectivity of DpCPV-MC against second-instar Spodoptera exigua larvae does not differ significantly from that of Dendrolimus punctatus cypovirus 1. Full-length amplification of the DpCPV-MC cDNAs identified 16 dsRNA genome segments. Each segment encodes one open reading frame with unique conserved terminal sequences at the 5′ and 3′ ends, which differ from those of all previously reported cypoviruses. On a phylogenetic tree based on the amino acid sequences of the polyhedrin of 19 cypovirus species, DpCPV-MC was closest to the type-4 cypoviruses. Homology searches showed that ten segments of DpCPV-MC (S1, S2, S3, S4, S5, S7, S8, S9, S12, and S13) encode putative CPV structural and nonstructural proteins, three segments (S6, S10 and S14) encode putative insect proteins or other viral proteins, and the other three segments (S11, S15, and S16) encode proteins that have no obvious sequence similarity to any known protein. Based on RNA secondary structures analysis, two segments of them (S11 and S16) were predicted to possibly transcript less efficiently than the other segments. We speculate that DpCPV-MC is composed of several genotypes. The ten CPV-related segments constantly exist in all genotypes, and one or two of the six CPV-unrelated segments co-exist with the ten CPV-related segments in one DpCPV-MC genotype, thus each virion contains no more than 12 segments. Based on our results and the literature, DpCPV-MC is a new cypovirus (Cypovirus 22, strain DpCPV-22).

Identification and Characterization of Two Novel RNA Viruses from Anopheles gambiae Species Complex Mosquitoes

“…Neighbor-joining trees show that reconstructed RdRp and polyhedrin sequences are related but distinct from known Cypoviruses. The CPVs have been classified into 16 types by the International Committee on Taxonomy of Viruses [ 34 ], or 21 types based on the electrophoretic migration patterns of their dsRNA genome segments [ 35 ]. Therefore, it is not surprising that the phylogeny of these viruses inferred from the polymerase sequence, and to a lesser extent from the polyhedrin gene, does not reflect the virus types classification.…”

“…We hypothesize that AnCPV is a segmented (10 to 11 segments) double stranded RNA virus of the genus Cypovirus , sub-family Spinareovirinae , family Reoviridae . Based on the phylogenetic tree on the polyhedrin gene, this virus could be a novel member of the Cypovirus type 17 [ 35 ].…”

Genomic analysis of a cypovirus isolated from the eucalyptus brown looper, Thyrinteina arnobia (Stoll, 1782) (Lepidoptera: Geometridae)

“…A phylogenetic analysis based on the amino acid sequences of several reovirus RdRp proteins showed that TharCPV-14 nested together in a branch containing the other isolates of Cypovirus 14 in a position closer to the hypothetical ancestor of this species. On the hand, viruses with different evolutionary origins were found infecting simultaneously the same insect host (Graham et al, 2007;Zhou et al, 2014). Interestingly, the in silico analysis showed that each of the segments of the TharCPV-14 genome contained a single ORF and the conserved terminal sequences at their 5′ and 3′ ends (5′ -AGAA…CAGCU -3′) as shown for the other two known cypoviruses from the Cypovirus 14 species.…”

A Reverse Genetics System for Cypovirus Based on a Bacmid Expressing T7 RNA Polymerase

“…After the mixture turned clear, the pH was adjusted to 7.4 with 1 mol/L Tris-HCl, pH 6.8, and centrifuged at 3800× g for 5 min to remove debris. The supernatant was treated with 15 μL of proteinase K (50 mg/mL) at 50 °C for 15 min [29]. The dsRNAs were extracted using TRIzol reagent (Invitrogen), analyzed on 1% agarose gels and stored at −70 °C.…”