This work was supported by the Wuhan Science and Technology Plan Project (2018201261638501).

1. Preparation of viruses and mosquitoes
<ol>
	<li>Preparation of viruses 
	NOTE: All processes were carried out in a biosafety level 2 (BSL-2) laboratory. The level of biosafety contaiment used should be&#160;determined by the pathogen&#39;s risk assessment and regulations specific to nations and regions. The process must be performed in a biosafety cabinet.
	<ol>
		<li>Inoculate 1 x 106 C6/36 cells into a T75 culture flask. Fill the flask with 10 mL of Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). 
		NOTE: Leibovitz&#39;s L-15 medium (L15) or Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) can also be used to maintain C6/36 cells.</li>
		<li>Maintain the cells in 5% CO2 at 28 &#176;C overnight and remove the cell supernatant the next day when the cells are 80%-90% confluent.</li>
		<li>Add 1 mL of virus dilution (Multiplicity of Infection (MOI) = 0.01) into the flask and incubate the cells for 1 h in 5% CO2 at 28 &#176;C. Ebinur Lake virus (EBIV)15, a mosquito-borne orthobunyavirus, was used as a model virus for this protocol.</li>
		<li>Remove the supernatant and wash the cells 3x with 5 mL of PBS. Fill the flask with 15 mL of new RPMI medium with 2% FBS. Maintain the flask in 5% CO2 at 28 &#176;C.</li>
		<li>Collect the supernatant containing virus when the required viral titer is reached (after nearly 5 days, depending on the virus used). For EBIV, this titer value was 107 plaque forming units (PFU)/mL. Sub-package them into individual 2 mL screw cap storage tubes and store the tubes containing 0.5-1 mL of virus at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Determination of viral titer 
	NOTE: Determine viral titer through the plaque assays16. BHK-21 was used to determine viral titer of EBIV, due to the obvious cytopathic effect (CPE) observed in infected cells.
	<ol>
		<li>Inoculate 1 x 105 BHK-21 cells into each well of 24-well plates. Fill each well with 1 mL of DMEM medium containing 10% FBS and 1% P/S. Maintain the cells in 5% CO2 at 37 &#176;C overnight.</li>
		<li>Make six (10-6) successive 10-fold dilutions of virus-containing supernatant with fresh DMEM medium containing 10% FBS and 1% P/S.</li>
		<li>Add 100 &#181;L of virus diluent into each well of 24-well plates containing the monolayer BHK-21. Discard the virus diluent after incubation for 1 h in 5% CO2 at 37 &#176;C. Add 500 &#181;L of DMEM containing 1.5% methylcellulose to each well.</li>
		<li>Culture cells in 5% CO2 at 37 &#176;C for 48 h (the apparent CPE was found after 48 h for EBIV). Fix the cells overnight with 750 &#181;L of 3.7% formaldehyde.</li>
		<li>Stain them with 200 &#181;L of 2% crystal violet for 15 min. Count the number of plaques to calculate the viral titer.</li>
	</ol>
	</li>
	<li>Mosquito breeding in the lab 
	NOTE: Rear mosquitoes in an arthropod containment level 1 (ACL-1) laboratory.
	<ol>
		<li>For the preparation of the dechlorinated water, fill a 20 L bucket with tap water and allow it to stand for 7-8 days without light. Do not cover the lid.</li>
		<li>Keep eggs and larvae of Ae. aegypti in a mosquito room with the following conditions: 28 &#177; 1 &#176;C with a light/dark cycle of 12 h and a relative humidity of 75% &#177; 5%.</li>
		<li>Maintain nearly 1,000 larvae in a shallow dish (22 cm x 15 cm x 6 cm) containing dechlorinated water after the eggs hatch. Feed them with nearly 1/8 teaspoon of mice feedstuff powder. Refresh the food daily and change the water every 2 days.</li>
		<li>Once the larvae pupate after 5-7 days, use a one-off dropper to transfer pupae to a plastic cup (7 oz) filled with dechlorinated water.</li>
		<li>Put five cups containing pupae (about 200 per cup) into one mesh cage (30 cm x 30 cm x 30 cm) and keep the cages in the insect incubator with the same conditions as in step 1.3.2.</li>
		<li>Put a tiny sponge containing an 8% (m/w) glucose solution on each mesh cage to feed adult mosquitoes. After adult emergence, take out the cups without pupae. Keep the mesh cages with about 1,000 adult mosquitoes per cage in the same insect incubator.</li>
	</ol>
	</li>
</ol>
2. Preparation for oral infection
<ol>
	<li>Prepare female mosquitoes for oral infection. Use the mosquito absorbing machine to collect 5-8-day-old mosquitoes. Anesthetize them at -20 &#176;C for 1 min and check to see whether the mosquitoes are dizzy. If not, anesthetize them for another minute at -20 &#176;C.</li>
	<li>Pour them quickly into the plastic cups (24 oz) at about 200 mosquitoes (female and male) per cup. Cover each cup quickly with a well-cut mosquito net, followed by a lid with a hole (approximately 6 cm in diameter) in the middle.</li>
	<li>Starve the mosquitoes for 24 h before the oral-infection and prepare infectious blood meal as follows.</li>
	<li>In a biosafety cabinet under BSL-2 conditions, take out the virus stock from the -80 &#176;C freezer and thaw them on ice. Dilute the virus stock to a concentration of 2 x 106 PFU/mL with RPMI 1640 medium containing 10% FBS and 1% P/S. Mix the virus dilution with an equal volume of defibrated horse blood. 
	NOTE: The processes were carried out in a biosafety cabinet in an ACL-2 laboratory.&#160;</li>
</ol>
3. Perform oral infection
NOTE: All steps were performed in an ACL-2 laboratory. The process must be performed in the glove box to prevent mosquitoes from escaping.
<ol>
	<li>Perform artificial feeding by using the artificial mosquito feeding system for 1 h.
	<ol>
		<li>Cut the collagen membranes to the appropriate size (approximately 5 cm in diameter), secure them to the reservoirs with o-rings, then add the virus-blood mixture (approximately 3 mL) to the reservoirs. Use plastic plugs to seal reservoirs and prevent leaks. 
		NOTE: These steps were performed in a biosafety cabinet.</li>
		<li>Put the plastic cups (24 oz) containing mosquitoes into the glove box. Screw the sealed reservoirs onto the FU1 Feeder and put one reservoir on one cup, then turn on the power unit. Feed the mosquitoes for 1 h</li>
	</ol>
	</li>
	<li>Anesthetize mosquitoes by CO2 for several seconds until they faint. In brief, place the carbon dioxide spray gun in the middle of the net mesh through the hole on the cup containing mosquitoes, open the value, and allow huge amounts of carbon dioxide to be spewed out to anesthetize mosquitoes.</li>
	<li>Pour them into a Petri dish placed on ice and cover the lid quickly. Pick out the engorged female mosquitoes with forceps. To identify, males have feathery antennae and females have plain antennae. Transfer them to new plastic cups at 50 female mosquitoes per cup. 
	NOTE: The engorged female mosquitoes were picked to maintain consistency as feeding amount affects the viral content.</li>
	<li>Wrap the cup with a cut mosquito net mesh and then cover it with a lid with a hole in the middle. Cut the sponge into a small piece and put it on the cup.</li>
	<li>Add 8% glucose solution onto the sponge using a plastic disposable dropper. Keep the cups containing female mosquitoes in the incubator at 27 &#176;C at 80% humidity for 10 days. Replace a new glucose-saturated sponge every 72 h.</li>
</ol>
4. Preparation for intrathoracic inoculation
<ol>
	<li>Prepare the microinjection needles as follows. Use a PUL-1000 to make microinjection needles (Figure 1A) with the following parameters in the needle puller program: Heat index = 450, force (g) = 110, distance (mm) = 1.00, and delay (s) = 0.</li>
	<li>Cut the tip of a pulled needle with tweezers under the dissecting microscope at 16x magnification (Figure 1B). Do not make the tip too big, otherwise it can harm the mosquitoes.</li>
	<li>Prepare female mosquitoes as in step 2.1. 
	NOTE: The following processes were performed in an ACL-2 laboratory.&#160;</li>
	<li>Prepare the infectious virus dilution as follows:
	<ol>
		<li>Take out the virus stock from the -80 &#176;C freezer and thaw them on ice. Dilute virus to a 100 nL virus dilution containing 100-500 PFU virus with RPMI 1640 medium containing 10% FBS and 1% P/S. Keep the virus dilution on ice.</li>
	</ol>
	</li>
	<li>Backfill the needle with mineral oil through a disposable sterile syringe. Attach the needle into the injector following the instruction on the machine.</li>
	<li>Insert the needle into a tube containing the infectious virus dilution. Do not break the tip of the needle. Press the FILL button to fill the needle with virus solution. The final volume of the virus solution in the needle is approximately 4.0 &#181;L. Once the needle is filled with the virus, be careful not to touch and damage it. 
	NOTE: The steps were performed in a biosafety cabinet.</li>
</ol>
5. Perform viral injection under a dissecting microscope
NOTE: All steps were performed in an ACL-2 laboratory.&#160;
<ol>
	<li>Anesthetize mosquitoes at -20 &#176;C for 1 min. Pour them into a Petri dish placed on ice and cover the lid quickly.</li>
	<li>Pick out nearly 50 female mosquitoes with tweezers and put them on the ice plate. Place the ice plate under the injector and insert the needle into the thoracic cavity of mosquito under the dissecting microscope (Figure 1C).</li>
	<li>Set the injection volume to 100 nL and rate to 50 nL/s. Press the INJECT button to let the virus solution flow into the mosquito. If successful injection occurs, the abdomen will slightly bulge.</li>
	<li>Put the infected mosquito into a new plastic cup (24 oz) placed on ice. When the number of the infected mosquitoes is enough, wrap the cup with a cut mosquito net mesh and then cover it with the lid.</li>
	<li>Keep the cup containing infectious mosquitoes in the incubator at 27 &#176;C at 80% humidity for 10 days. Feed the mosquitoes as in step 3.5.</li>
</ol>
6. Processing of the infected female mosquitoes
NOTE: Three different tissues/organs were dissected from each female mosquito: the gut for calculating the virus infection rate (VIR), the head for calculating the virus dissemination rate (VDR), and saliva for calculating the virus transmission rate (VTR). VIR was defined as the number of mosquitoes with virus-positive guts. VDR was defined as the number of mosquitoes with virus-positive heads divided by the number of mosquitoes with virus-positive guts by 100. VTR was defined as the number of mosquitoes with virus-positive saliva divided by the number of mosquitoes with virus-positive guts by 100.
<ol>
	<li>Collection of saliva from the infected female mosquitoes 
	NOTE: The processes must be performed in an ACL-2 laboratory.&#160;
	<ol>
		<li>Anesthetize the infected female mosquitoes by carbon dioxide anesthetization as in step 3.2. Pour them into a Petri dish placed on ice and close the lid quickly.</li>
		<li>Place 10 &#181;L pipette tips filled with immersion oil side by side on the rubber mud.</li>
		<li>Pick out female mosquitoes with tweezers and put them on an ice plate. Remove their legs and wings with tweezers. Place the mouthpart of one mosquito on each pipette tip.</li>
		<li>Collect the saliva as secreted into the oil by the mosquitoes for the next 45-60 min at room temperature.</li>
		<li>Place the pipette tips in 1.5 mL tubes containing 200 &#181;L of virus diluent medium (RPMI 1640 medium containing 10% FBS and 1% P/S). Centrifuge them at 5,000 x g for 5 min at 4 &#176;C to expel saliva into tubes. Store these tubes at -80 &#176;C for subsequent determination of transmission rates.</li>
	</ol>
	</li>
	<li>Dissect the infected female mosquitoes 
	NOTE: The processes must be performed in an ACL-2 laboratory.&#160;
	<ol>
		<li>Use tweezers to cut the heads of the female mosquitoes after saliva collection. Place each head into an individual tube containing 200 &#181;L of RPMI 1640 medium.</li>
		<li>Grab the thorax with a tweezer, then the penultimate segment of abdomen with another tweezer and pull it out. The gut will be pulled out. Clean the pulled-out gut of any stray tissue and organs.</li>
		<li>Wash the gut with PBS and place each gut into an individual tube containing 200 &#181;L of RPMI 1640 medium. Store these tubes at -80 &#176;C for subsequent determination of the virus infection rate and dissemination rate.</li>
	</ol>
	</li>
</ol>
7. Data analysis
<ol>
	<li>RNA extraction
	<ol>
		<li>Grind the tissues into pieces with a low temperature tissue homogenizer grinding machine set to the following parameters: operating frequency = 60 Hz, operation time = 15 s, pause time = 10 s, cycles = 2, and setting temperature = 4.0 &#176;C.</li>
		<li>Centrifuge the samples at 12,000 x g for 10 min at 4 &#176;C. Extract total RNA of each sample by automated nucleic acid extraction system following instrument instructions.</li>
	</ol>
	</li>
	<li>Performing qRT-PCR
	<ol>
		<li>Use the one-step RT-PCR kit to quantify the viral RNA copies using a quantitative PCR instrument17. Use the following primers for the detection of EBIV S segment F: ATGGCATCACCTGGGAAAG and R: TTCCAATGGCAAGTGGATAGAA. Set up the reaction process as: stage 1: 42 &#176;C for 5 min, 95 &#176;C for 10 s; stage 2: 40 cycles of 95 &#176;C for 5 s and 60 &#176;C for 20 s.</li>
		<li>Determine the lowest virus dose for the infection in the cells through the serial 10-fold dilutions inoculated on cells. Use the BHK-21 cells to determine the lowest virus dose of EBIV, using dilutions till 10-7 as described in step 1.2.</li>
		<li>Calculate the Ct value of the lowest virus dose by qRT-PCR. Use the cutoff value for EBIV-positive by qRT-PCR as &#60; 35. Use the following equation to calculate the copies of EBIV in each sample: 
		y = -4.0312x + 3.384 [x = log (the copies of EBIV), y = Ct value, R2 = 0.9905]</li>
		<li>Calculate the infection rate, dissemination rate and transmission rate as follow: 
		Infection rate (%) = 100 x (the number of virus-positive mosquito guts / the number of total mosquitoes) 
		Dissemination rate (%) = 100 x (the number of virus-positive mosquito heads / the number of virus-positive mosquito guts) 
		&#8203;Transmission rate (%) = 100 x (the number of virus-positive mosquito saliva / the number of virus-positive mosquito guts)</li>
		<li>Analyze the differences in continuous variables and differences in mosquito infection rates, dissemination rates, and transmission rates by using the non-parametric Kruskal-Wallis analysis for multiple comparisons and Fisher&#39;s exact test where appropriate.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The goal of this method was to provide a comprehensive risk assessment of one mosquito-borne virus by evaluating vector competence through oral feeding and intrathoracic inoculation.
In the oral-feeding experiment, engorged-mosquitoes need to be picked out and transferred to a new container, posing a severe risk to the operators. The reason for this is because any mosquito, including uninfected mosquitoes, might be a source of infection19. Consequently, mosquitoes must ...

Mosquito-borne viruses (MBVs), a heterogeneous group of RNA viruses, can persist in mosquito vectors and subsequently spread to vertebrate hosts1. The clinically important MBVs are majorly distributed in four virus families, namely Flaviviridae, Togaviridae, Reoviridae, and Peribunyavividae2,3. In recent decades, these viruses have been reported all across the globe, causing public health issues. As one of the most well-known MBVs, Dengue virus (DENV) has become the most prevalent emerging or re-emerging arbovirus in over 100 countries during the last 20 years4. Since the discovery of Zika virus (ZIKV) inland, almost all tropical and subtropical countries and territories of the continent have reported human ZIKV infections5. In order to assess the risk of virus transmission, numerous studies in recent years have focused on mosquito vector competence for these viruses6,7. As a result, it is critical to effectively prevent and control vector-borne diseases.
Aedes aegypti (Ae. aegypti), one of the most easily reared mosquitoes in the laboratory, is an important vector of DENV, ZIKV, Chikungunya virus (CHIKV), and yellow fever virus (YFV)8. For a long time, Ae. aegypti was solely found in the African continent and in Southeast Asia, but in recent years it has colonized nearly all continents9. Furthermore, the global abundance of Ae. aegypti has been continuously growing, with an estimated 20% increase by the end of the century10. From 2004 to 2009 in China, there was an evident increase in Ae. aegypti vector competence for DENV due to higher day-to-day temperatures11. The status of Ae. aegypti as the pathogenic vector has significantly risen in China. Consequently, to address these challenges, it is necessary to investigate the vector competence of Ae. aegypti's to transmit viruses.
As a haematophagous arthropod, the female mosquito pierces the skin of a vertebrate host and feeds on the blood. Mosquitoes do occasionally acquire viruses from virus-infected hosts and then transfer the viruses to a new host. As such, to determine vector competence, mosquitoes are fed an artificial bloodmeal containing arboviruses through a feeding system in the laboratory setting12. Individual mosquitoes are separated into heads, bodies, and saliva secretions several days after infection. To measure virus infection, dissemination, and transmission rates, virus titers have been detected by quantitative reverse-transcription PCR (qRT-PCR) or plaque assay. However, not all mosquitoes develop midgut infections and the capacity to transfer a virus to the next host following blood feeding. It is linked with mosquitoes' physiological barriers, which prevent pathogens from penetrating the body and play a vital role in their innate immunity13. The midgut barriers, particularly the midgut infection barrier (MIB) and midgut escape barrier (MEB), influence whether the virus could infect the vector systemically and the efficiency with which it spreads. It obstructs the analysis of other tissues' infection, such as salivary glands which also exhibit salivary gland infection and escape barriers13,14. To better characterize the infection of midguts and salivary glands in the vector, a detailed protocol for oral feeding and intrathoracic inoculation of arbovirus in Ae. aegypti is presented herein. This protocol might be applied to additional arbovirus infections in a variety of mosquito vectors, such as DENV and ZIKV infection in Aedes spp., and could prove to be a practicable procedure.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aedes aegypti&#160;</td>
			<td></td>
			<td></td>
			<td>Rockefeller strain</td>
		</tr>
		<tr>
			<td>Automated nucleic acid extraction system&#160;</td>
			<td>NanoMagBio</td>
			<td>S-48</td>
			<td></td>
		</tr>
		<tr>
			<td>BHK-21 cells</td>
			<td>National Virus Resource Center, Wuhan Institute of Virology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buckets</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C6/36 cells&#160;</td>
			<td>National Virus Resource Center, Wuhan Institute of Virology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon dioxide spray gun&#160;</td>
			<td>wuhan Yihong</td>
			<td>YHDFPCO2</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugal machine</td>
			<td>Himac&#160;</td>
			<td>CF16RN</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX96 Touch Real-Time PCR Detection System&#160;</td>
			<td>Bio-Rad</td>
			<td>CFX96 Touch</td>
			<td></td>
		</tr>
		<tr>
			<td>Ebinur Lake virus</td>
			<td></td>
			<td></td>
			<td>Cu20-XJ isolation</td>
		</tr>
		<tr>
			<td>Formaldehyde&#160;</td>
			<td>Wuhan Baiqiandu</td>
			<td>B0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Glove box&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Hushi</td>
			<td>10010518</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion oil&#160;</td>
			<td>Cargille</td>
			<td>16908-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Insect incubator</td>
			<td>Memmert</td>
			<td>HPP750T7</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Temperature Tissue Homogenizer Grinding Machine&#160;</td>
			<td>Servicebio</td>
			<td>KZ-III-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Virus Genome Extraction Kit</td>
			<td>NanoMagBio</td>
			<td>NMG0966-16</td>
			<td></td>
		</tr>
		<tr>
			<td>mesh cages (30 x 30 x 30 cm)</td>
			<td>Huayu</td>
			<td>HY-35</td>
			<td></td>
		</tr>
		<tr>
			<td>methylcellulose</td>
			<td>Calbiochem</td>
			<td>17851</td>
			<td></td>
		</tr>
		<tr>
			<td>mice feedstuff powder&#160;</td>
			<td>BESSN</td>
			<td>BS018</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode Puller</td>
			<td>WPI</td>
			<td>PUL-1000</td>
			<td>PUL-1000&#160;is a microprocessor controlled horizontal puller for making glass micropipettes or microelectrodes used in intracellular recording, patch clamp studies, microperfusion or microinjection.</td>
		</tr>
		<tr>
			<td>Mosquito net meshes&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoject III Programmable Nanoliter Injector</td>
			<td>Drummond</td>
			<td>3-000-207</td>
			<td></td>
		</tr>
		<tr>
			<td>One Step TB Green PrimeScript PLUS RT-PCR Kit&#160;</td>
			<td>Takara</td>
			<td>RR096A</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Gibco</td>
			<td>C10010500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>151140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic cupes (7 oz)&#160;</td>
			<td>Hubei Duoanduo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic cups (24 oz)&#160;</td>
			<td>Anhui shangji</td>
			<td>PET32-Tub-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic disposable droppers</td>
			<td>Biosharp</td>
			<td>BS-XG-O3L-NS</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerator (-80 &#176;C)</td>
			<td>sanyo</td>
			<td>MDF-U54V</td>
			<td></td>
		</tr>
		<tr>
			<td>Replacement Glass Capillaries</td>
			<td>Drummond</td>
			<td>3-000-203-G/X</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium 1640&#160;</td>
			<td>Gibco</td>
			<td>C11875500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw cap storage tubes (2 mL )</td>
			<td>biofil</td>
			<td>&#160;FCT010005</td>
			<td></td>
		</tr>
		<tr>
			<td>Shallow dishes&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sponge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile defibrillated horse blood</td>
			<td>Wuhan Purity Biotechnology</td>
			<td>CDHXB413</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 culture flask</td>
			<td>Corning</td>
			<td>430829</td>
			<td></td>
		</tr>
		<tr>
			<td>The artificial mosquito feeding system&#160;</td>
			<td>Hemotek</td>
			<td>Hemotek PS6</td>
			<td></td>
		</tr>
		<tr>
			<td>The dissecting microscope&#160;</td>
			<td>ZEISS</td>
			<td>&#160;stemi508</td>
			<td></td>
		</tr>
		<tr>
			<td>The ice plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>The mosquito absorbing machine&#160;</td>
			<td>Ningbo Bangning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>The pipette tips&#160;</td>
			<td>Axygen</td>
			<td>TF</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Dumont</td>
			<td>0203-5-PO</td>
			<td></td>
		</tr>
	</tbody>
</table>

experimental viral infection in adult mosquitoes by oral feeding and microinjection

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public health. Once ingested, the viruses must overcome the mosquito midgut barrier to reach the hemolymph, from where they might potentially spread to the salivary glands. When a mosquito bites, these viruses are spread to new vertebrate hosts. Similarly, the mosquito may pick up different viruses. In general, only a tiny portion of viruses may enter the salivary glands via the gut. The transmission efficiency of these viruses to the glands is be affected by the two physical barriers found in different mosquito species: midgut barriers and salivary glands barriers. This protocol presents a method for virus detection in salivary glands of Aedes aegypti&#39;s&#160;following oral feeding and intrathoracic injection infection. Furthermore, determining whether the guts and/or salivary glands hinder viral spread can aid in the risk assessments of MBVs transmitted by Aedes aegypti.

To examine EBIV distribution in the infected mosquitoes via artificial blood feeding (the viral final titer was 6.4 x 106 PFU/mL) and intrathoracic injection (the viral dose was 340 PFU), viral RNAs in saliva, heads, and guts of the mosquitoes at 10 days post infection (dpi) were determined.
For Ae. aegypti, virus titer of EBIV in the guts, heads, and saliva of the intrathoracically inoculated female mosquitoes were much higher than that in the oral-infected female...

Watch this Scientific Journal Video about Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection at JoVE.com

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

This methodology, which included oral feeding and intrathoracic injection infection, could effectively assess the influence of midgut and/or salivary gland barriers on arbovirus infection.

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public ...

experimental-viral-infection-adult-mosquitoes-oral-feeding

Research

JoVE Journal

Immunology and Infection

2.0K Views.  Chinese Academy of Sciences. This methodology, which included oral feeding and intrathoracic injection infection, could effectively assess the influence of midgut and/or salivary gland barriers on arbovirus infection. 

Video: Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and composition 1, 2. In general, biofilms develop from initial microbial attachment on a surface followed by formation of cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which occur in a complex extracellular matrix. The majority of biofilm matrices harbor exopolysaccharides (EPS), and dental biofilms are no exception; especially those associated with caries disease, which are mostly mediated by mutans streptococci 3. The EPS are synthesized by microorganisms (S. mutans, a key contributor) by means of extracellular enzymes, such as glucosyltransferases using sucrose primarily as substrate 3.
Studies of biofilms formed on tooth surfaces are particularly challenging owing to their constant exposure to environmental challenges associated with complex diet-host-microbial interactions occurring in the oral cavity. Better understanding of the dynamic changes of the structural organization and composition of the matrix, physiology and transcriptome/proteome profile of biofilm-cells in response to these complex interactions would further advance the current knowledge of how oral biofilms modulate pathogenicity. Therefore, we have developed an analytical tool-box to facilitate biofilm analysis at structural, biochemical and molecular levels by combining commonly available and novel techniques with custom-made software for data analysis. Standard analytical (colorimetric assays, RT-qPCR and microarrays) and novel fluorescence techniques (for simultaneous labeling of bacteria and EPS) were integrated with specific software for data analysis to address the complex nature of oral biofilm research.
The tool-box is comprised of 4 distinct but interconnected steps (Figure 1): 1) Bioassays, 2) Raw Data Input, 3) Data Processing, and 4) Data Analysis. We used our in vitro biofilm model and specific experimental conditions to demonstrate the usefulness and flexibility of the tool-box. The biofilm model is simple, reproducible and multiple replicates of a single experiment can be done simultaneously 4, 5. Moreover, it allows temporal evaluation, inclusion of various microbial species 5 and assessment of the effects of distinct experimental conditions (e.g. treatments 6; comparison of knockout mutants vs. parental strain 5; carbohydrates availability 7). Here, we describe two specific components of the tool-box, including (i) new software for microarray data mining/organization (MDV) and fluorescence imaging analysis (DUOSTAT), and (ii) in situ EPS-labeling. We also provide an experimental case showing how the tool-box can assist with biofilms analysis, data organization, integration and interpretation.

Biofilms formed on tooth surfaces are highly complex and exposed to constant innate and exogenous environmental challenges, which modulate their architecture, physiology and transcriptome. We developed a toolbox to examine the composition, structural organization and gene expression of oral biofilms, which can be adapted to other areas of biofilm research.

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and ...

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water environment. Developing effective methods for oral delivery of immunostimulants or vaccines, which activate the immune system against infectious diseases, is highly desirable. In devising prophylactic tools, good experimental models are needed to test their performance. Here, we show a method for oral intubation of adult zebrafish and a set of procedures to dissect and prepare the intestine for cytometry, confocal microscopy and quantitative polymerase chain reaction (qPCR) analysis. With this protocol, we can precisely administer volumes up to 50 &#181;L to fish weighing approximately 1 g simply and quickly, without harming the animals. This method allows us to explore the direct in vivo uptake of fluorescently labelled compounds by the intestinal mucosa and the immunomodulatory capacity of such biologics at the local site after intubation. By combining downstream methods such as flow cytometry, histology, qPCR and confocal microscopy of the intestinal tissue, we can understand how immunostimulants or vaccines are able to cross the intestinal mucosal barriers, pass through the lamina propria, and reach the muscle, exerting an effect on the intestinal mucosal immune system. The model could be used to test candidate oral prophylactics and delivery systems or the local effect of any orally administered bioactive compound.

The protocol describes intubating adult zebrafish with a biologic; then dissecting and preparing the intestine for cytometry, confocal microscopy and qPCR. This method allows administration of bioactive compounds to monitor intestinal uptake and the local immune stimulus evoked. It&#160;is relevant for testing the intestinal dynamics of oral prophylactics.

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water ...

Studying Cryptosporidium Infection in 3D Tissue-derived Human Organoid Culture Systems by Microinjection

Cryptosporidium parvum is one of the major causes of human diarrheal disease. To understand the pathology of the parasite and develop efficient drugs, an in vitro culture system that recapitulates the conditions in the host is needed. Organoids, which closely resemble the tissues of their origin, are ideal for studying host-parasite interactions. Organoids are three-dimensional (3D) tissue-derived structures which are derived from adult stem cells and grow in culture for extended periods of time without undergoing any genetic aberration or transformation. They have well defined polarity with both apical and basolateral surfaces. Organoids have various applications in drug testing, bio banking, and disease modeling and host-microbe interaction studies. Here we present a step-by-step protocol of how to prepare the oocysts and sporozoites of Cryptosporidium for infecting human intestinal and airway organoids. We then demonstrate how microinjection can be used to inject the microbes into the organoid lumen. There are three major methods by which organoids can be used for host-microbe interaction studies&#8212;microinjection, mechanical shearing and plating, and by making monolayers. Microinjection enables maintenance of the 3D structure and allows for precise control of parasite volumes and direct apical side contact for the microbes. We provide details for optimal growth of organoids for either imaging or oocyst production. Finally, we also demonstrate how the newly generated oocysts can be isolated from the organoid for further downstream processing and analysis.

Studying Cryptosporidium Infection in 3D Tissue-derived Human Organoid Culture Systems by Microinjection

We describe protocols to prepare oocysts and purify sporozoites for studying infection of human intestinal and airway organoids by Cryptosporidium parvum. We demonstrate the procedures for microinjection of parasites into the intestinal organoid lumen and immunostaining of organoids. Finally, we describe the isolation of generated oocysts from the organoids.

Cryptosporidium parvum is one of the major causes of human diarrheal disease. To understand the pathology of the parasite and develop efficient drugs, an ...

Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution ...