Detection of Rift Valley Fever Virus RNA in Formalin-Fixed Mosquitoes by in Situ Hybridization (Rnascope®)

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Brief Report Detection of Rift Valley Fever Virus RNA in Formalin-Fixed Mosquitoes by In Situ Hybridization (RNAscope®)

Sarah Lumley 1,2,3, Laura Hunter 1, Kirsty Emery 1 , Roger Hewson 1, Anthony R. Fooks 3 , Daniel L. Horton 2 and Nicholas Johnson 2,3,*

1 Microbiology Services Division, Public Health England, Wiltshire SP4 0JG, UK; [email protected] (S.L.); [email protected] (L.H.); [email protected] (K.E.); [email protected] (R.H.) 2 School of Veterinary Medicine, University of Surrey, Guildford GU2 7XH, UK; [email protected] 3 Virology Department, Animal and Plant Health Agency (Weybridge), Woodham Lane, Surrey KT15 3NB, UK; [email protected] * Correspondence: [email protected]

Abstract: Rift Valley fever virus (RVFV) causes a zoonotic mosquito-borne haemorrhagic disease that emerges to produce rapid large-scale outbreaks in livestock within sub-Saharan Africa. A range of mosquito species in Africa have been shown to transmit RVFV, and recent studies have assessed whether temperate mosquito species are also capable of transmission. In order to support vector competence studies, the ability to visualize virus localization in mosquito cells and tissue would enhance the understanding of the infection process within the mosquito body. Here, the application of in situ hybridization utilizing RNAscope® to detect RVFV infection within the mosquito species, Citation: Lumley, S.; Hunter, L.; Culex pipiens, derived from the United Kingdom was demonstrated. Extensive RVFV replication was Emery, K.; Hewson, R.; Fooks, A.R.; Horton, D.L.; Johnson, N. Detection detected in many tissues of the mosquito with the notable exception of the interior of ovarian follicles. of Rift Valley Fever Virus RNA in Formalin-Fixed Mosquitoes by In Situ Keywords: Rift Valley fever virus; mosquito; virus detection Hybridization (RNAscope®). Viruses 2021, 13, 1079. https://doi.org/ 10.3390/v13061079 1. Introduction Academic Editors: Bas B. Oude Rift Valley fever is an economically significant zoonotic disease of livestock in sub- Munnink, Marietjie Venter and Saharan Africa. Outbreaks can occur suddenly, often driven by the onset of heavy rainfall, Luisa Barzon resulting in significant morbidity and mortality amongst livestock and humans [1]. The causative agent is Rift Valley fever virus (RVFV), classified within the family Phenuiviridae Received: 21 April 2021 and genus Phlebovirus, which has a tri-segmented genome consisting of a small (S) segment Accepted: 2 June 2021 encoding the virus nucleoprotein and a non-structural protein, a medium (M) segment Published: 5 June 2021 encoding two glycoproteins, Gn and Gc, and a large (L) segment encoding the virus RNA-dependent RNA polymerase. Rift Valley fever virus is transmitted to and between Publisher’s Note: MDPI stays neutral mammals by mosquitoes, although human infections usually result from close contact with regard to jurisdictional claims in published maps and institutional affil- with contaminated carcasses. Abortions in ruminants are often observed within livestock iations. following infection with RVFV. In addition, the movement of infected livestock has been implicated in the establishment of RVFV in areas outside of sub-Saharan Africa, such as the Arabian Peninsula [2] and islands within the Indian Ocean [3,4]. A range of mosquito species have been associated with transmission of RVFV, particu- larly those within the genera Aedes and Culex, reviewed in [5,6]. Establishing the vector Copyright: © 2021 by the authors. competence of particular species has been essential in understanding the epidemiology of Licensee MDPI, Basel, Switzerland. RVFV transmission, both during outbreaks and inter-epidemic periods. Virus detection This article is an open access article distributed under the terms and in mosquitoes usually relies on virus isolation in cell-cultures and reverse transcription conditions of the Creative Commons polymerase chain reaction (RT-PCR). However, the ability to detect the virus in the context Attribution (CC BY) license (https:// of organ structures, using immunohistochemistry, provides further information on the creativecommons.org/licenses/by/ ability of arthropod-borne viruses to infect particular mosquito species [7,8]. Formalin 4.0/). fixation, whilst inactivating the virus for safe handling outside of primary containment, can

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limit the application of some techniques. RNAscope®, a form of in situ hybridization using RNA probes, was developed to detect RNA within formalin-fixed, paraffin-embedded tissue [9]. A recent report has developed RNAscope® for detection of RVFV within mam- malian liver tissue [10], a key tissue for diagnosis in livestock, using probes that target the virus polymerase gene on the L segment. In this study, we have adapted a similar approach, but using probes designed against the virus nucleoprotein gene on the S segment, to investigate the presence and distribution of RVFV RNA within an infected mosquito. Control probes targeting mosquito glyceraldehyde-3-dehydrogenase (GAPDH) gene confirmed the methodology was effective for insect tissue. Staining with RVFV probes demonstrated extensive RVFV RNA labelling in key structures such as the basal layer of the mosquito midgut and the proventriculus, suggesting points of entry for the virus following consumption of a bloodmeal.

2. Materials and Methods 2.1. Mosquitoes, Viruses and Infection Protocol A colonized, indigenous mosquito strain of Culex pipiens, Linnaeus 1758, was used as the infection model. This strain, designated Caldbeck [11], was originally trapped from a location in London (United Kingdom) in 2011. The RVFV strain used for infections was ZH501, originally isolated from a human with haemorrhagic fever in Egypt in 1977, and it was grown in Vero cells. This was diluted in horse blood to a final titre of 107 plaque forming units (pfu)/mL. Mosquito infection with RVFV was conducted as previously described [12]. A second group was fed horse blood with tissue culture media as a negative control group. Blood-fed mosquitoes were maintained at 25 ◦C for 7 days prior to processing. Briefly, individual mosquitoes were dipped in 70% ethanol to reduce hydrophobicity and then immersed in 0.5 mL 10% buffered formalin within a screw-cap tube for 5 days at room temperature. Mosquitoes were paraffin embedded, sectioned (3 µm) and mounted on glass slides.

2.2. In Situ Hybridization: RNAscope® RNAscope® (Advanced Cell Diagnostic ACD) is a commercially available RNA in situ hybridization (RNA-ISH) technology, applied here to study RVFV replication and localization with light microscopy of the whole mosquito following oral infection. Novel probes targeting the negative sense nucleoprotein-coding gene of RVFV strain ZH501 (nucleotides 915–1652; GenBank accession number DQ380148.1) and mosquito GAPDH gene from Culex quinquifasciatus, Say 1823, (nucleotides 54–810; GenBank accession number XM_001846997.1) were designed and produced by Advanced Cell Diagnostics. Mosquito sections were selected based on preliminary images of uninfected Culex pipiens stained with haematoxylin and eosin. A negative control probe, supplied with the RNAscope® 2.5 High Deﬁnition (HD)–Red Assay kit (ACD, Hayward, California, USA), was performed on parallel sections targeting the bacterial dihydrodipicolinate reductase (DapB) gene that was not expected to be present in the sample; any observed staining to this negative probe was due to non-speciﬁc binding. RNAscope® staining procedures were conducted following the manufacturer’s instructions; all steps were performed at room temperatures with reagents supplied with the kit, unless stated. Slides were dewaxed for 5 min in xylene, immersed in 100% alcohol for 2 min, then dried. Slides were treated with hydrogen peroxide for 10 min, washed in water and placed into the target retrieval solution (supplied with the kit) for 15 min at 98–100 ◦C, then washed immediately with distilled water, followed by 100% alcohol and dried. A hydrophobic barrier was drawn around the section using the pen and templates provided and dried for 20 min or overnight. Sections were treated with Protease Plus (ACD) at 40 ◦C for 15 min. Prepared sections were washed with water before applying the probes (RVFV, GAPDH, DapB) for 2 h at 40 ◦C and then washed in 1× wash buffer (twice for 2 min). After each of the subsequent hybridization steps, the sections were washed in 1× wash buffer (twice for 2 min). Solution AMP 1 (supplied by the manufacturer ACD) was applied for 30 min, Viruses 2021, 13, x FOR PEER REVIEW 3 of 7

Prepared sections were washed with water before applying the probes (RVFV,

Viruses 2021, 13, 1079 GAPDH, DapB) for 2 h at 40 °C and then washed in 1× wash buffer (twice for 2 min). 3After of 7 each of the subsequent hybridization steps, the sections were washed in 1× wash buffer (twice for 2 min). Solution AMP 1 (supplied by the manufacturer ACD) was applied for 30 min, followed by AMP 2 for 15 min, AMP 3 for 30 min and AMP 4 for 15 min, all at 40 followed°C. Buffers by AMPAMP 2 5 for and 15 6 min, were AMP incubated 3 for 30 at min room and temperature AMP 4 for 15 for min, 30 alland at 15 40 min,◦C. Buffers respec- AMPtively. 5 and Fast 6 Red were was incubated then used at room to detect temperature the signal for by 30 incubating and 15 min, at respectively. room temperature Fast Red for was10 min. then Slides used towere detect washed the signal with water by incubating and counterstained at room temperature with Gills for1 haematoxylin 10 min. Slides for were2 min washed and dried, with either water andfor 1 counterstained h at 60 °C or overnight with Gills at 1 room haematoxylin temperature. for 2 A min coverslip and dried, was ◦ eitherapplied for using 1 h at EcoMount 60 C or overnight (Biocare, at California, room temperature. USA). Images A coverslip were captured was applied with usinga Pan- EcoMountnoramic 250 (Biocare, Flash California,II slide scanner USA). (3D Images HISTECH, were captured Budapest, with Hungary), a Pannoramic and image 250 Flash selec- IItion slide was scanner performed (3D HISTECH, on digitized Budapest, images Hungary),in a Pannoramic and image Viewer selection (Version was 1.15.2 performed SP 2, 3D onHISTECH). digitized images in a Pannoramic Viewer (Version 1.15.2 SP 2, 3D HISTECH). 3. Results and Discussion 3. Results and Discussion To visualize virus infection within the tissues of Culex pipiens, mosquitoes (in groups To visualize virus infection within the tissues of Culex pipiens, mosquitoes (in groups of ﬁve) were fed a blood meal containing RVFV strain ZH501 and maintained for 7 days at of five) were fed a blood meal containing RVFV strain ZH501 and maintained for 7 days 25 ◦C. Each mosquito was then ﬁxed in formalin, and preliminary sections were stained at 25 °C. Each mosquito was then fixed in formalin, and preliminary sections were stained with haematoxylin and eosin (Figure1) to identify key structures. with haematoxylin and eosin (Figure 1) to identify key structures.

Figure 1. A haematoxylin and eosin stained 3 µm transverse section through an unfed Culex pipiens Figure 1. A haematoxylin and eosin stained 3 µm transverse section through an unfed Culex pipiens mosquito, showing the position of the main organ structures. mosquito, showing the position of the main organ structures.

® ToTo validate validate the the RNAscope RNAscope® procedure procedure and and detect detect virus virus distribution, distribution, RNA RNA probes probes werewere designed designed to to detect detect a a mosquito mosquito host host target target and and RVFV RVFV virus virus genome. genome. Sections Sections were were counterstainedcounterstained to to highlight highlight tissue tissue structures. structur Thees. The negative negative control control probe probe based onbased the DapBon the geneDapB (Figure gene 2(Figure, left-hand 2, left-hand panel) showed panel) noshowed staining, no staining, whereas whereas the ubiquitously the ubiquitously expressed ex- hostpressed GAPDH host probe GAPDH showed probe extensive showed staining extensive (Figure staining2, right-hand (Figure 2, panel). right-hand This conﬁrmedpanel). This theconfirmed integrity the of RNA integrity within of theRNA tissue within sample the followingtissue sample the ﬁxation following and the embedding fixation processand em- andbedding that the process probe and detection that the method probe wasdetection successful. method was successful.

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FigureFigure 2. 2.Whole WholeCulex Culex pipiens pipiensmosquito mosquitomosquito sections sectionssections were werewere stained stainedstained with withwith (A (()A a) negativea negative control control probe probe that that hybridizedhybridized to to the the bacterial bacterial DapB DapB RNA RNA or or (B ()B a) positivea positive control control probe probe that that bound bound to to mosquito mosquito host host GAPDH RNA. The bound probe was stained with Fast Red, and sections were counterstained with GAPDHGAPDH RNA. RNA. The The bound bound probe probe was was stained stained with with Fast Fast Red, Red, and and sections sections were were counterstained counterstained with with haematoxylin. haematoxylin.haematoxylin.

SpeciﬁcSpecific staining staining with with the the RVFV RVFV probe probe was wa detecteds detected in in 20% 20% of of infected infected mosquitoes, mosquitoes, similarsimilar to to that that observed observed inin previousprevious vector competence competence studies studies [12]. [12]. Whole Whole mosquito mosquito sec- sectionstions stained stained with with the the RVFV RVFV probe probe demonstrat demonstrateded specific speciﬁc binding binding (Figure (Figure 3, right-hand3, right- handpanel). panel). This This detected detected RVFV RVFV RNA RNA within within the co thentext context of particular of particular tissues tissues within within the themos- mosquitoquito body. body.

Figure 3. Culex pipiens mosquitomosquito sectionssections treatedtreated withwith anan RVRVFV-specific probe that binds to nucleo- Figure 3. Culex pipiens mosquito sections treated with an RVFV-speciﬁc probe that binds to nucleopro- protein RNA. Probe binding is shownshown byby redred staining.staining. ((A) A mosquito from the mock-infected con- teintrol RNA. group. Probe (B) bindingAn RVFV-infected is shown by mosquito. red staining. Organs (A) Alabe mosquitolledlled areare fromCG,CG, cerebralcerebral the mock-infected ganglion;ganglion; JO, controlJO, John-John- group.ston’s( organ;B) An RVFV-infectedO, ommatidia (compound mosquito. Organseye); L, labelledlegs; FG, are fore-gut; CG, cerebral DLM, dorsal ganglion; longitudinal JO, Johnston’s muscle; organ;MG, midgut; O, ommatidia OF, ovarian (compound follicle. eye); L, legs; FG, fore-gut; DLM, dorsal longitudinal muscle; MG, midgut; OF, ovarian follicle.

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AtAt higherhigher magnification, magniﬁcation, the the extent extent of RVFV of RVFV replication replication could could be assessed be assessed within within in- individualdividual tissues tissues (Figure (Figure 4).4). Images Images of of mock-infected mock-infected sections sections are are shown shown on onthe the left-hand left-hand panel,panel, whilstwhilst RVFV-infected RVFV-infected images images are are shown shown on the on theright-hand right-hand panel. panel. The principal The principal ob- observationsservations are are described described below: below:

Figure 4. Higher magnification of tissues sections from Figure 2. Panels 1–4, midgut; panels 5 and Figure 4. Higher magniﬁcation of tissues sections from Figure2. Panels 1–4, midgut; panels 5 and 6, legs; panels 7 and 8, thorax; panels 9 and 10, head; panels 11 and 12, Johnston’s organ, including 6,second legs; panels segment 7and of the 8, thorax;antennae panels containing 9 and sensor 10, head;y cells; panels panels 11 and13 and 12, 14, Johnston’s ommatidia organ, within including the secondcompound segment eye; panels of the 15 antennae and 16, containingovarian follicle. sensory Abbreviations: cells; panels AMG, 13 and anterior 14, ommatidia midgut; BM, within base- the compoundment membrane; eye; panels Ch, chorion; 15 and CL, 16, corneal ovarian lens; follicle. DLM, dorsal Abbreviations: longitudinal AMG, muscle; anterior FB, fat midgut;body, FE, BM, basementfollicular membrane;epithelium; G, Ch, ganglion chorion; JO, CL, Johnson’s corneal lens;organ; DLM, L, lumen; dorsal O, longitudinal ommatidia; PPM, muscle; pharyngeal FB, fat body, FE, follicular epithelium; G, ganglion JO, Johnson’s organ; L, lumen; O, ommatidia; PPM, pharyngeal pump muscle; Pr, proventriculus; OF, ovarian follicle; R, rhabdom surrounded by retinular cells; Sc, scolopophores; YG, yolk granules.

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Midgut (Figure4, panels 1–4). No staining was observed in the mock-infected mosquitoes. Rift Valley fever RNA was detected within the basal layer of the midgut epithelium, and extensive staining was observed in the proventriculus of the foregut. This suggests that by day 7, the virus had effectively crossed the midgut barrier [13]. The presence of RVFV infection of the proventriculus has been reported previously [14] and could represent a location for virus entry across the midgut epithelium. Legs (Figure4, panels 5 and 6). Rift Valley fever RNA was present within cross- sections of leg tissue from infected, but absent from mock-infected, mosquitoes. This confirms the utility of using leg samples to demonstrate virus dissemination in vector competence studies and suggests that there are cells capable of supporting virus replication within the leg structure. Thorax (Figure4, panels 7 and 8). Low levels of RVFV RNA were detected in the thoracic ganglia (G) and dorsal longitudinal muscles (DLM). This identifies potential virus replication sites in the close proximity to the salivary glands that were not located in these sections. Head (Figure4, panels 9 and 10). Rift Valley fever virus RNA was detected in the cerebral ganglion (G) relative to the rest of the head and thorax. Previous studies have suggested that RVFV infection of the ganglion could affect the mosquito regulatory func- tions [15] and, potentially, the behaviour of the infected mosquito. Johnston’s organ (Figure4, panels 11 and 12). The Johnston’s organ is found at the base of the antenna and detects vibration. Rift Valley fever virus replication was observed within the sensory scolopophores (Sc), with trace amounts of signal located at the cell bodies of the neurons (G). Ommatidia (Figure4, panels 13 and 14). Faint non-specific staining was observed on the cornea (CL) in the mock-infected section, but much stronger staining was observed in the RVFV-infected section, suggesting presence of RVFV RNA. Ovarian follicles (Figure4, panels 15 and 16). Rift Valley fever virus staining was observed on the exterior of the follicular epithelium but not within the follicles. This suggests that either the virus had not yet penetrated the follicle by day seven, or it is unable to enter the follicle of this species of mosquito. If the latter is the case, this tentatively implies that transovarial transmission does not occur. Further time-course investigations would be required to confirm this.

4. Conclusions The results reported within this study complement those reported by [10], where L segment probes were used to detect RVFV in ruminant hepatic tissue as a diagnostic test. Here, we showed that a probe directed against the S segment designed to target the RVFV nucleoprotein-coding sequence detects RVFV RNA throughout a range of mosquito tissues, and this provides further evidence for the susceptibility of Culex pipiens to support virus escape from the midgut and replication at secondary sites, as recently reported by [12]. These data also provide proof of concept for the application of RNAscope® in mosquito infection studies, demonstrating a novel highly sensitive method to detect virus RNA localization within the mosquito vector.

Author Contributions: Conceptualization, A.R.F., D.L.H., R.H. and N.J.; methodology, S.L., L.H. and K.E.; formal analysis, S.L. and N.J.; resources, N.J. and A.R.F.; writing—original draft preparation, S.L. and N.J.; writing—review and editing, all authors; supervision, A.R.F., D.L.H., R.H. and N.J.; project administration, N.J.; funding acquisition, N.J. and A.R.F. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Department for Environment Food and Rural Affairs (Defra) and the Scottish and Welsh Governments through grants SE4113, SE4116 and SE0550. Sarah Lumley was funded by the University of Surrey, Public Health England and Animal and Plant Health Agency through grant SC1402. Institutional Review Board Statement: Not applicable. Viruses 2021, 13, 1079 7 of 7

Informed Consent Statement: Not applicable. Data Availability Statement: All data has been presented in the article. Acknowledgments: Mosquitoes used in this study were provided by the Pirbright Institute under UK grant code BBS/E/I/0007039, awarded to Simon Carpenter as part of funding received from the Biotechnology and Biological Science Research Council (United Kingdom Research and Innovation). The authors thank Dan Hicks and Alejandro Nuñez (both APHA) for their assistance in preparing and sectioning samples for microscopy. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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