Name: protocols for investigating the host-tissue distribution, transmission-mode, and effect on the host fitness of a densovirus in the cotton bollworm
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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>IDEAL-TEK</td>
			<td>2.SA</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...

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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

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.

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

The overall goal of this protocol is to study the effect of a Densovirus, HaDV2, on its host, Cotton Bollworm. This method can help answer key questions in the virus host interaction field such as the effect a now path-standing viruses on their hosts. The main advantage of this technique that larvae use to construct virus positive and negative strains are derived from the same par-ne-co-hosts, ensuring the same genetic background.To begin the experiment, eight single-pair mating of newly enclosed moths by using a plastic cage per pair covered with cotton gauze to ensure good ventilation and to serve as an ovum position substrate. To increase the likelihood of obtaining a virus-free colony, utilize at least 30 pairs of single-pair matings. Feed adult moths with a 10%sugar and 2%vitamin solution-soaked cotton ball, and replenish the solution daily.Today's post-mating, as female adults lay their eggs on the cotton gauze, begin to change the gauze daily. Collect and maintain gauze-containing eggs in the same growth room as the adult moths until the eggs hatch. Immediately transfer newly hatched larvae to new 24-well plates with one individual per well.Five days after the first egg-laying date, collect the parent moths and store them in liquid nitrogen. Check for HaDV2 of the parents using PCR with specific primers, and keep the HaDV2-free individuals as the HaDV2-free noninfected strain. Collect adult moths individually and immediately store them in liquid nitrogen.Completely grind collected individual moths in liquid nitrogen using a sterilized mortar and pestle. Transfer around 10 micrograms of debris into a clean 1.5 milliliter tube. Then transfer the remaining debris into a new tube then immediately store them at 80 degrees Celsius.Extract DNA from the 10 micrograms of debris following the protocols provided by the manufacturer. Once the moth DNA has been isolated, check for HaDV2 using the protocol. According to the results, divide the remaining debris into two groups:HaDV2 positive and HaDV2 negative.Add one milliliter of phosphate-buffered saline, or PBS, to each individual to completely dissolve the remaining larval debris. Pre-cool the centrifuge four degrees Celsius, then spin the homogenate. On ice, filter the liquid supernatant with the 0.22 micron membrane filter.Collect 200 microliters per tube of the filtered liquids, and immediately store them at 80 degrees Celsius. To determine the oral infection efficiency with the HaDV2 infected filtered liquids of different concentration by diluting the fluid containing HaDV2. In a fume hood evenly spread 200 microliters of HaDV2 containing filtered liquids for each concentration on the surface of the solid artifical diet.Next, begin to transfer 100 freshly hatched larvae to the HaDV2 containing artificial diet in a petri dish. Gently knock the cotton gauze including freshly hatched larvae to transfer the larvae from the gauze to a sheet of white paper. Knock the paper gently so that the larvae spin silk and balloon off the paper.Use sterilized forceps to touch the silk and move the larvae to a petri dish. After 48 hours, transfer one individual larvae per well on the artificial diet to 24-well plates. Re-ar the larvae inoculated with HaDV2, using the same concentrations to the adult stage.Check for HaDV2 infection status of the adults using the protocol. Adults inoculated with 10 to the eighth copy number per microliter of HaDV2 yielded 100%infection and are kept as HaDV2 positive strain. To prevent contamination, sterilize the forceps and scissors in 75%ethanol three times and then heat them in the flame of an alcohol lamp.Using an analytical balance weigh the empty tubes that are to be used to store the tissues. Next place the larvae and adult moths from the infected strain on ice for approximately five minutes. Fix the frozen larvae on cystosepiment using two insect pins inserted in the intersegmental membranes near the head and the last abdomen segment.Then in the posterior abdomen, cut the proleg of the larvae so that the hemolymph flows out. Use a pipette to pipette about 10 microliters of larval hemolymph within about 10 seconds to prevent browning and coagulating of the hemolymph. With a pair of scissors make an incision in the cuticle ranging from the last intersegmental membrane to the head.Using two forceps, dissect foregut, midgut, hindgut, fat body, and malpighian tubules. To prevent the contamination of the remaining tissues with the hemolymph during the dissection, wash the dissected tissues in PBS buffer. Mix tissue samples from three individuals together as one replicant.Immediately transfer each tissue to the tubes in liquid nitrogen. In a clean petri dish containing PBS buffer, dissect the tissues of female and male adults and immediately transfer each tissue to the tubes in liquid nitrogen. Weigh the tubes and the above tissues together separately and calculate the mass of tissues.Extract DNA from these tissues and perform kPCR to quantify the viral titer of each tissue. Place at least 100 pupae or newly emerged adult moths derived from the noninfected strain in a cotton gauze-covered 5-liter wire mesh cage. After two days, mated adult females will start to lay eggs on the cotton gauze.Collect and maintain the cotton gauze containing the eggs. Proceed with neonate larvae inoculation by transferring the neonate larvae of the noninfected strain to the artificial diet containing HaDV2 to establish the infected strain. Similarly, transfer neonate larvae to the artificial diet covered with HaDV2-free filtered liquid to establish the control groups, the noninfected strain.Include at least 240 larvae in each treatment. After 48 hours transfer one individual larva per well to 24-well plates. Sequentially number each larva in each well, and record the instar stage and survival status daily.About five days later, when the artificial diet deteriorates, simultaneously transfer these individuals to a new 24-well plate containing fresh artificial diet. Weigh the larvae from day seven to 11 using an analytical balance. After weighing, individually return the larvae to the 24-well plate.Upon molting to the fifth instar, around ten day after hatching, transfer the larvae to glass tubes. Transfer the individuals to the glass tube at the same time each day. Fifth instar larvae can also be identified by the width of the head capsule.Next, code each larva with its original reference number on the glass tube using paint or markers. Record the larval status daily. Record the pupae and eclosion date for each individual in the glass tube, and record the mass of three-day old pupae.After eclosion, place one female and one male into a single cage with 10%sucrose and 2%vitamin solution. Replenish the sucrose solution every day. Record the date of death for each moth.When eggs are laid, change the cotton gauze daily. Count the number of eggs immediately. Finally, count newly hatched offspring while they hatch.Tri-als were run to ensure the quality of the specimens used in the experiment. Progenies from the male and female parents that we HaDV2-free were reared as the noninfected strain. We successfully amplified the actin gene in parents and progenies using the same DNA template suggesting that the DNA templates were of good quality.The randomly selected eight progenies were also free of HaDV2, HaNPV, and Wolbachia. A standard curve defining the relationship between the log of the quality of starting material and the gained CT values shows a strong linear correlation between the CT values and the log of the initial gene copy number of HaDV2. The HaDV2 copy number was calculated per microgram of each tissue, and the percentage of the total HaDV2 titer for the specific tissue revealed that HaDV2 was mainly distributed in the fat body.While attempting this procedure, it is important to remember to handle the delicate larvae during the first five days after they hatch during the en-tire procedure.

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