Abstract
BackgroundHearMNPV, a nucleopolyhedrovirus (NPV), which infects the cotton bollworm, Helicoverpa armigera, comprises multiple rod-shaped nucleocapsids in virion(as detected by electron microscopy). HearMNPV shows a different host range compared with H. armigera single-nucleocapsid NPV (HearSNPV). To better understand HearMNPV, the HearMNPV genome was sequenced and analyzed.MethodsThe morphology of HearMNPV was observed by electron microscope. The qPCR was used to determine the replication kinetics of HearMNPV infectious for H. armigera in vivo. A random genomic library of HearMNPV was constructed according to the “partial filling-in” method, the sequence and organization of the HearMNPV genome was analyzed and compared with sequence data from other baculoviruses.ResultsReal time qPCR showed that HearMNPV DNA replication included a decreasing phase, latent phase, exponential phase, and a stationary phase during infection of H. armigera. The HearMNPV genome consists of 154,196 base pairs, with a G + C content of 40.07%. 162 putative ORFs were detected in the HearMNPV genome, which represented 90.16% of the genome. The remaining 9.84% constitute four homologous regions and other non-coding regions. The gene content and gene arrangement in HearMNPV were most similar to those of Mamestra configurata NPV-B (MacoNPV-B), but was different to HearSNPV. Comparison of the genome of HearMNPV and MacoNPV-B suggested that HearMNPV has a deletion of a 5.4-kb fragment containing five ORFs. In addition, HearMNPV orf66, bro genes, and hrs are different to the corresponding parts of the MacoNPV-B genome.ConclusionsHearMNPV can replicate in vivo in H. armigera and in vitro, and is a new NPV isolate distinguished from HearSNPV. HearMNPV is most closely related to MacoNPV-B, but has a distinct genomic structure, content, and organization.
Highlights
HearMNPV, a nucleopolyhedrovirus (NPV), which infects the cotton bollworm, Helicoverpa armigera, comprises multiple rod-shaped nucleocapsids in virion(as detected by electron microscopy)
Electron microscopy observation Scanning electron microscopy revealed that the purified occlusion bodies (OBs) of NPV originating from infected cotton bollworm have irregular shapes, with diameters of about 2 ± 0.3 μm (Figure 1A)
Transmission electron microscopy showed multiple rod-shaped nucleocapsids of about 230 nm in length and 50 nm in width embedded in each OB, with multiple nucleocapsids packaged within the envelope of the virion (Figure 1B)
Summary
Viruses and insects HearMNPV was originally isolated from a naturally infected H. armigera in the Shanghai city, China in the 1970s. Sections were cut, stained with uranyl acetate and lead citrate, and examined under a JEM-1230 transmission electron microscope (TEM) at an accelerating voltage of 80 kV. Time zero of the infection was defined as the time when the diet soaked OBs or ddH2O was removed from the culture boxes Larvae used in this experiment were sacrificed at various time points ranging from 4 to 96 h postinoculation (p.i.). HearMNPV DNA copy number was determined by real-time qPCR with primers specific to the rr2b gene. Three independent qPCRs were performed using rr2b or actin specific primers, and standard curves were generated. For each larval DNA extract, three independent qPCRs were performed using rr2b and actin specific primers. Bootstrap analyses were performed to evaluate the robustness of the phylogenies using 1000 replicates for both NJ and MP analyses
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