Name: protocols for investigating the host-tissue distribution, transmission-mode, and effect on the host fitness of a densovirus in the cotton bollworm
Uploaded: 2017-04-12T15:00:00
Description: Watch this Scientific Journal Video about Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm at JoVE.com

This work was supported by the National Key Basic Research Program of China (No. 2013CB127602) and the Science Fund for Creative Research Groups of the National Science Foundation of China (No. 31321004).

1. Construction of the HaDV2-free Cotton Bollworm Colony
<ol>
	<li>Rear cotton bollworm larvae (H. armigera) on an artificial diet32 in a controlled growth room or an artificial climate chamber at 25 &#177; 1 &#176;C, with 14 h light/10 h dark and 60% relative humidity.</li>
	<li>Aid single-pair mating of newly eclosed moths by using a plastic cage per pair (10 cm height, 5 cm diameter) covered with cotton gauze to ensure good ventilation and to serve as an oviposition substrate. To inc...

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpesvirus+latency+confers+symbiotic+protection+from+bacterial+infection.">Herpesvirus latency confers symbiotic protection from bacterial infection.</a> Nature. 447, 326-329 (2007).</li><li>Ponferrada, V. G., Mauck, B. S., Wooley, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+envelope+glycoprotein+of+human+endogenous+retrovirus+herv-w+induces+cellular+resistance+to+spleen+necrosis+virus.">The envelope glycoprotein of human endogenous retrovirus herv-w induces cellular resistance to spleen necrosis virus.</a> Arch. Virol. 148, 659-675 (2003).</li><li>M&#225;rquez, L. M., Redman, R. S., Rodriguez, R. J., Roossinck, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+virus+in+a+fungus+in+a+plant:+three-way+symbiosis+required+for+thermal+tolerance.">A virus in a fungus in a plant: three-way symbiosis required for thermal tolerance.</a> Science. 315, 513-515 (2007).</li><li>Xu, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+genome+sequence+of+a+monosense+densovirus+infecting+the+cotton+bollworm,+Helicoverpa+armigera.">Complete genome sequence of a monosense densovirus infecting the cotton bollworm, Helicoverpa armigera.</a> J. Virol. 86, 10909-10909 (2012).</li><li>Xu, P. J., Liu, Y. Q., Graham, R. I., Wilson, K., Wu, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Densovirus+is+a+mutualistic+symbiont+of+a+global+crop+pest+(Helicoverpa+armigera)+and+protects+against+a+baculovirus+and+Bt+biopesticide.">Densovirus is a mutualistic symbiont of a global crop pest (Helicoverpa armigera) and protects against a baculovirus and Bt biopesticide.</a> PLoS Pathog. 10, e1004490 (2014).</li><li>Liang, G. M., Tan, W. J., Guo, Y. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improvement+in+the+technique+of+arti&#64257;cial+rearing+cotton+bollworm.">An improvement in the technique of arti&#64257;cial rearing cotton bollworm.</a> Plant Protec. 25, 15-17 (1999).</li><li>Zhou, W. 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In the last few decades, most studies on insect-virus interactions have focused on honey bee health34,35,36, vectors of human disease37, plant viruses38, and some insect pathogenic viruses that have great potential as biological control agents39. Little attention has been paid to covert viruses in insects, especially in lepidopteran pests. Here, we present a...

In the last few decades, the development of sequencing technology such as next-generation sequencing (NGS) has facilitated the discovery of many novel DNA and RNA viruses, especially nonpathogenic viruses, but also novel isolates of previously known viruses1,2,3,4,5,6,7,8,9,10,11,12. In the model organism Drosophila melanogaster, more than 20 new partial virus genomes have been detected using metagenomic techniques13. Many viral sequences, including novel viruses, have also been identified in other insects, such as honey bees, mosquitoes, Asian citrus psyllids, dragonflies, and multiple lepidopteran species14,15,16,17,18,19,20,21.
In the future, it can be expected that more novel viruses will be discovered in insects using these advanced technologies; hence, our understanding of the virus-host interaction may change accordingly6,9. For example, virus-host interactions are considered to be more complicated than previously thought, because many novel viruses are being defined as mutualistic partners rather than strict pathogens22. For example, the mutualistic densovirus DplDNV in Dysaphis plantaginea induces the winged morph and increases mobility, facilitating the dispersal of the host as well as the virus23. Moreover, mutualistic viruses have been described with regards to mammalian health, the drought and cold tolerance of plants, and the impact of bacterial infections24. Seneca valley virus-001 is shown to mediate selective cytotoxicity towards tumor cells with neuroendocrine cancer features25. Hepatitis A virus infections suppress hepatitis C virus replication and may lead to recovery from hepatitis C26. Herpesvirus latency confers symbiotic protection from bacterial infection27. The envelope glycoprotein of human endogenous retrovirus HERV-W induces cellular resistance to spleen necrosis virus28. Curvularia thermal tolerance virus (CThTV) from a fungal endophyte is involved in the mutualistic interaction between this fungus and a tropical panic grass29. Hence, knowledge of the interactions between the newly found viruses and their hosts should generate fresh perspectives on their biology and management. However, the novel viruses, especially the covert viruses displaying no obvious signs typical of acute infection, have seldom been investigated, and we need a pipeline and protocols to investigate the impacts of the newly found viruses on their hosts.
Previously, we have reported the prevalence a new monosense densovirus Helicoverpa armigera densovirus (HaDV2) in the cotton bollworm, Helicoverpa armigera, and presented evidence of a mutualistic relationship between HaDV2 and the cotton bollworm30,31. In this paper, we will describe the laboratory protocol to study in detail the interaction between HaDV2 and its cotton bollworm host. The protocol presented here may also be highly relevant to researchers examining the role of other orally-transmitted viruses, especially in lepidopteran pests.

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			<td>24-well plate</td>
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			<td>07-200-740</td>
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			<td>DNA extraction kit</td>
			<td>TIANGEN</td>
			<td>DP304-03</td>
			<td>Multiple suppliers available.</td>
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			<td>thermal cycler</td>
			<td>Veriti; Applied Biosystems</td>
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			<td>pEASY-T Cloning Vector</td>
			<td>TransGen, Beijing, China</td>
			<td>CT301-02</td>
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			<td>Premix Ex Taq (Probe qPCR)</td>
			<td>Takara</td>
			<td>RR390A</td>
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			<td>Probes</td>
			<td>Invitrogen</td>
			<td>Custom order</td>
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			<td>Primers</td>
			<td>Invitrogen</td>
			<td>Custom order</td>
			<td></td>
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			<td>microspectrophotometry NanoDrop 2000c&#160;</td>
			<td>Thermo scientific&#160;</td>
			<td>not available</td>
			<td></td>
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			<td>7500 Real-Time PCR system</td>
			<td>Applied Biosystems</td>
			<td>not available</td>
			<td></td>
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			<td>stereomicroscope SZX-16</td>
			<td>Olympus</td>
			<td>not available</td>
			<td></td>
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			<td>sucrose</td>
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			<td></td>
			<td>Multiple suppliers available.</td>
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			<td>vitamin complex</td>
			<td></td>
			<td></td>
			<td>Multiple suppliers available.</td>
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protocols for investigating the host-tissue distribution, transmission-mode, and effect on the host fitness of a densovirus in the cotton bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Progenies from the parents that were HaDV2-free (Figure 3A) were reared as the NONINF-strain. We successfully amplified the actin gene using the same DNA templates, suggesting that the DNA templates were of good quality (Figure 3B). In addition, the randomly selected eight progenies were also free of HaDV2 (Figure 3C), HaNPV (Figure 3D), and Wolbachia (Figure 3E). Again, we concluded tha...

Watch this Scientific Journal Video about Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm at JoVE.com

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm (Video) | JoVE

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

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