The genome and occlusion bodies of marine Penaeus monodon nudivirus (PmNV, also known as MBV and PemoNPV) suggest that it should be assigned to a new nudivirus genus that is distinct from the terrestrial nudiviruses

Abstract

Background: Penaeus monodon nudivirus (PmNV) is the causative agent of spherical baculovirosis in shrimp (Penaeus monodon). This disease causes significant mortalities at the larval stage and early postlarval (PL) stage and may suppress growth and reduce survival and production in aquaculture. The nomenclature and classification status of PmNV has been changed several times due to morphological observation and phylogenetic analysis of its partial genome sequence. In this study, we therefore completed the genome sequence and constructed phylogenetic trees to clarify PmNV's taxonomic position. To better understand the characteristics of the occlusion bodies formed by this marine occluded virus, we also compared the chemical properties of the polyhedrin produced by PmNV and the baculovirus AcMNPV (Autographa californica nucleopolyhedrovirus).

Results: We used next generation sequencing and traditional PCR methods to obtain the complete PmNV genome sequence of 119,638 bp encoding 115 putative ORFs. Phylogenetic tree analysis showed that several PmNV genes and sequences clustered with the non-occluded nudiviruses and not with the baculoviruses. We also investigated the characteristics of PmNV polyhedrin, which is a functionally important protein and the major component of the viral OBs (occlusion bodies). We found that both recombinant PmNV polyhedrin and wild-type PmNV OBs were sensitive to acid conditions, but unlike the baculoviral OBs, they were not susceptible to alkali treatment.

Conclusions: From the viral genome features and phylogenetic analysis we conclude that PmNV is not a baculovirus, and that it should be assigned to the proposed Nudiviridae family with the other nudiviruses, but into a distinct new genus (Gammanudivirus).

Figure 1

Circular map of the PmNV genome. Purple represents the 60 forward strand ORFs and blue represents the 55 reverse strand ORFs. Red represents the 10 direct repeat (dr) regions, which are dispersed around the genome. The innermost circle shows GC skew, which indicates possible locations of the DNA leading strand, lagging strand, replication origin, and replication terminal during DNA replication. Below average GC skew is light orange and above average dark orange. The next innermost circle is a GC plot, with light green representing below average GC content, and dark green indicating above average GC content.

Figure 2

Phylogenetic analysis of selected DNA viruses and PmNV. Multiple alignments of (A) DNA polymerase, (B) Helicase, (C) LEF-4, LEF-5, LEF-8, LEF-9 and P47 (D) P74, PIF-1, PIF-2 and PIF-3 protein sequences were performed using BioEdit. The trees were inferred using MEGA5.2 and the neighbor-joining method. The robustness of each tree was tested using bootstrap (1000) analysis. The percent values are indicated at the nodes. Organisms included in this analysis, with abbreviated names: Autographa californica multiple nucleopolyhedrovirus (AcMNPV), Bombyx mori nucleopolyhedrovirus (BmNPV), Lymantria xylin nucleopolyhedrovirus (LyxyMNPV), Cydia prmonella granulovirus (CpGV), Agrotis segetum granulovirus (AgseGV), Spodoptera litura granulovirus (SpliGV), Neodiprion sertifer nucleopolyhedrovirus (NeseNPV), Neodiprion lecontei nucleopolyhedrovirus (NeleNPV), Neodiprion Abietis nucleopolyhedrovirus (NeabNPV), Culex nigripalpus nucleopolyhedrovirus (CuniNPV), Heliothis zea virus 1 (HzNV-1), Helicoverpa zea nudivirus 2 (HzNV-2), Oryctes rhinoceros nudivirus (OrNV), Gryllus bimaculatus nudivirus (GbNV), Musca domestica salivary gland hypertrophy virus (MdSGHV), Glossina pallidipes salivary gland hypertrophy virus (MdSGHV), African swine fever virus (ASFV). Accession numbers of all the sequences are listed in Additional file 8: Table S6.

Figure 3

Transmission electron micrographs of PmNV-infected postlarval Penaeus monodon hepatopancreatic cells. (A) Occlusion bodies (OBs) and free virions (FV) in the nucleus. (B) Occluded virus (OV) in the occlusion body which consists of a polyhedrin matrix. (C) Free virions in the nucleus. (D) Mature released OBs in the tubule lumen of the hepatopancreas.

Figure 4

The polyhedrin protein matrix and occlusion bodies (OBs) production of recombinant AcMNPV- polh (PmNV) and wild-type AcMNPV in Sf9 at 3 days post-infection. (A) Photomicrographs of Sf9 cells infected with recombinant AcMNPV-polh (PmNV), wild-type AcMNPV, recombinant AcMNPV-vp28 (WSSV) as non-occluded control and non-infected cells (Normal cells). Examples of cells producing polyhedrin protein matrix and OBs are indicated by arrows. (B) Photomicrographs of purified polyhedrin protein matrix and AcMNPV OBs. Scale bars are 20 μm.

Figure 5

Effect of DAS buffer on purified recombinant polyhedrin matrix and AcMNPV OBs. (A) Photomicrographs of resuspended pellets after centrifugation showing intact PmNV polyhedrin matrix but no AcMNPV OBs. Scale bar represents 20 μm. (B) SDS-PAGE analysis of the denatured and partially denatured supernatant and the denatured pellet. The results show that DAS buffer did not solubilize the recombinant PmNV polyhedrin (S lanes 1 & 2) but did solubilize polyhedrin from AcMNPV OBs (S lanes 4 & 5). S: supernatant; P: pellet.

Figure 6

Solubility of purified recombinant PmNV polyhedrin and wild-type AcMNPV. (A) Photomicrographs of pellet samples after indicated treatments showing presence or absence of PmNV polyhedrin matrix or AcMNPV OBs. Scale bars 50 μm. (B) SDS-PAGE analysis of pellets and supernatant solutions from treatments in (A). Lane 1: TE buffer (negative control); Lane 2: 2% SDS; Lane 3: 8 M urea; Lane 4, 5 and 6: phosphate buffer adjusted to pH 2, pH 7 and pH 10, respectively.

Figure 7

Solubility of purified wild-type PmNV OBs in various buffers and different concentrations of NaCl. (A) SDS-PAGE of supernatant solutions (soluble) and pellets (insoluble) after PmNV OBs were subjected to the treatments indicated. Black arrows indicate partially denatured PmNV OBs. (B) Western blots of supernatant solutions (soluble) and pellets (insoluble) from PmNV OBs treated with various concentrations of NaCl and showing increasing solubility as salinity decreases.

Figure 8

Phylogenetic analysis of the concatenated protein sequences of LEF-4, LEF-5, DNA polymerase and Ac81 from PmNV, the nudiviruses, and four baculoviruses. This analysis follows Jehle et al.[5]. The tree was inferred using MEGA 5.2 and the neighbor-joining method. Robustness was tested using bootstrap (1000) analysis. Percent values are indicated at the nodes. Jehle et al. [5] proposed that the nudiviruses should be assigned to one of two new genera. Alphanudivirus and Betanudivirus. We propose here that PmNV should be assigned to a third new genus, Gammanudivirus.