Experimental BTV-3 and BTV-8 infection ofCulicoides sonorensisbiting midges

Orbivirus, bluetongue virus, BTV-3, Culicoides, biting midges, vector, vector 17 competence 18 19

20 BTV-3 emerged in the Netherlands in 2023 and spread rapidly to neighboring 21 countries. Compared to the BTV-8 outbreak in 2006, the course of the BTV-3 epizootic is 22 more severe. Experimental infection of laboratory-reared Culicoides sonorensis midges 23 showed a slightly higher replication rate of BTV-3 than of BTV-8. 24 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 6, 2024. ; https://doi.org/10.1101/2024.12.05.627042doi: bioRxiv preprint Main text 25 Bluetongue virus (BTV) is a non-contagious orbivirus that is transmitted between its 26 mammalian hosts by Culicoides biting midges, causing severe disease in ruminant livestock 27 (1). The first BTV outbreak ever recorded in central Europe in 2006 was caused by a serotype 28 8 strain (BTV-8) and led to a major epidemic (2). In 2023, a devastating BTV-3 outbreak 29 started in the Netherlands and rapidly spread to neighboring countries (3). Arthropod-borne 30 pathogens such as BTV are mainly spread by the dispersal of infected vectors and the 31 movement of infected livestock (3). During vector monitoring in late 2023 in Germany near 32 the Dutch border, BTV-3 was detected in a pool sample of Culicoides biting midges (4). 33 Comparison of the spread between farms of BTV-8 in 2006/2007 and BTV-3 in 2023 34 by transmission kernel analysis, which describes the distance-dependent probability of disease 35 transmission from an infected farm to a susceptible farm, revealed a very similar kernel shape 36 parameter of the BTV-8 and the BTV-3 outbreaks. This suggests that the mechanisms of 37 disease spread through short distance dispersal of infected midges and other modes for longer 38 distances, such as livestock movement, were similar between the two outbreaks (3). However, 39 a much higher amplitude parameter was observed for the 2023 BTV-3 epidemic, indicating a 40 faster disease spread. This could be due to higher temperatures of about 2°C above normal 41 during the observed period (September-November) of the 2023 BTV-3 outbreak compared to 42 the corresponding period of the 2006 BTV-8 outbreak. Another reason could be a higher 43 infection and transmission efficiency of the midges for BTV-3, which would also result in a 44 faster spread of the disease (3). 45 Since laboratory colonies of European biting midge vector species are not available, 46 several experimental infection studies have been conducted using field-collected midges to 47 investigate the vector competence of Culicoides species with different BTV-8 strains (5, 6). 48 The laboratory-reared colony of Culicoides sonorensis is a suitable model to study infection 49 dynamics under standardised laboratory conditions (7, 8), since this species plays a crucial 50 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 6, 2024. ; https://doi.org/10.1101/2024.12.05.627042doi: bioRxiv preprint role for BTV transmission in North America (9). Although most experimental BTV-8 51 infection studies using field-captured midges aim to calculate replication rates, the results of 52 different studies are not easily comparable due to differences in experimental design, sample 53 processing and data analysis. No studies are available for the more recent BTV-3 strain. 54 To directly compare the replication properties of BTV-8 and BTV-3 in biting midges, 55 we performed infection experiments with the laboratory colony of C. sonorensis. 56 57 The Study 58 59 A laboratory colony of C. sonorensis was reared in the BSL2 insectary of the 60 Friedrich-Loeffler-Institut (FLI), Greifswald-Insel Riems, as described previously (10). Three-61 day-old biting midges were offered caprine (trial one) or ovine (trial two) heparin blood, 62 obtained from the FLI, mixed 1:1 with BTV-8 or BTV-3 in cell culture medium (Minimum 63 Essential Medium). Virus stocks of BTV-8 (strain BH311/06, isolated from a German sheep 64 during the 2006 outbreak) and BTV-3 (4) were propagated on BHK-21 cells (RIE164, 65 Collection of Cell Lines in V eterinary Medicine (CCLV), Friedrich-Loeffler-Institut, 66 Greifswald-Insel Riems, Germany). The blood meal contained 10 6 50 % tissue culture 67 infective dose per ml (TCID50/ml), which was confirmed by back-titration after feeding. As a 68 negative control (NC), blood was mixed with virus-free cell culture medium. After pre-69 heating to 37°C, the blood meal was offered to the midges using a “Hemotek membrane 70 feeding system” (Hemotek, Blackburn, UK) for 30 minutes. Midges were sorted under short-71 term CO2-anaesthesia on a cooling plate. Clearly engorged females were transferred to a new 72 cage and kept for the course of the experiment. 16 blood-fed midges per group (BTV-8, BTV-73 3, NC) were processed immediately after feeding as uptake controls (Figure 1). Midges were 74 kept inside gaze covered cages in an incubator at 27°C and a relative humidity of 85% with an 75 8h dark/ 16h light regime and supplied with 5% glucose ad libitum. After an incubation period 76 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 6, 2024. ; https://doi.org/10.1101/2024.12.05.627042doi: bioRxiv preprint of 6 days, surviving midges were harvested. All midges were placed individually in tubes 77 containing 200 µl phosphate-buffered saline (PBS) and a 5 mm stainless steel ball (Figure 1). 78 Following homogenization using a TissueLyzer (Qiagen, Hilden, Germany) for three minutes 79 at 30 Hz, total RNA was extracted for each sample using the King Fisher 96 Flex (Thermo 80 Scientific, Braunschweig, Germany) in combination with the NucleoMag VET kit (Macherey 81 Nagel, Düren, Germany) according to the manufacturer’s instructions. The RNA extracts were 82 analyzed by a BTV specific RT-qPCR (11) with an external full virus BTV-3 standard, which 83 was used to calculate the number of BTV genome copies per midge. 84 Ingestion of virus-spiked blood (BTV-8 and BTV-3) led to PCR positivity in all 85 midges harvested directly after the blood meal (day 0). The highest BTV genome copy 86 number in an individual midge from a day-0-group was used as a cut off value to evaluate 87 virus replication in midges fed with the same blood meal but harvested only after 6 days of 88 incubation. Two consecutive trials with the same set-up were performed as biological 89 replicates and to achieve a higher number of analyzable midges. In the first trial using BTV-3, 90 89 out of 319 surviving midges tested positive by RT-qPCR at 6 dpi, and 17 of these (5.32%) 91 had viral loads higher than the day-0-group, indicating efficient virus replication. In the BTV-92 8 group of the first trial, 197 out of 330 surviving midges tested positive by RT-qPCR, and 12 93 of them (3.64%) showed efficient virus replication. In the second trial, in the BTV-3 group, 94 133 out of 250 surviving midges tested positive by RT-qPCR, and 8 of them (3.20%) showed 95 virus replication. In the BTV-8 group of the second trial, 110 out of 222 surviving midges 96 tested positive by RT-qPCR, and 7 (3.15%) of them showed virus replication. Midges of the 97 negative control group tested negative by RT-qPCR at all times (Figure 2). Overall, 4.39 % of 98 BTV-3 infected midges replicated the virus, while 3.44% of the BTV-8 infected midges 99 replicated the virus (Figure 2). Statistical analysis with a two-sided Fisher exact test showed 100 that the differences in the replication properties between BTV-3 and BTV-8 are not 101 statistically significant. 102 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 6, 2024. ; https://doi.org/10.1101/2024.12.05.627042doi: bioRxiv preprint 103

104 In our study, orally infected midges were processed individually to calculate 105 replication rates of BTV-3 and BTV-8, based on PCR-determination of genome copies per 106 midge. In our laboratory colony of C. sonorensis, oral BTV-3 infection resulted in a slightly 107 higher percentage of virus-positive biting midges with demonstrated replication than BTV-8 108 infection, with a total of 4.39% of BTV-3 infected midges and 3.44% of BTV-8 infected 109 midges replicating the virus. The higher proportion of midges with BTV-3 replication may be 110 a factor contributing to the observed faster outbreak progression of the current BTV-3 111 outbreak in comparison to the BTV-8 outbreak in 2006/2007. 112 113 114 Acknowledgments 115 We would like to thank Uday Gottam and Ulrike Neumann for their excellent technical 116 assistance. Culicoides sonorensis were originally developed and supplied by The Pirbright 117 Institute under BBSRC project code: BBS/E/I/00007039. The study was funded by the 118 German Federal Ministry of Food and Agriculture (BMEL) through the Federal Office for 119 Agriculture and Food (BLE), grant number 28N207601. 120 121 Conflict of Interest 122 The authors declare that the research was conducted in the absence of any commercial 123 or financial relationships that could be construed as a potential conflict of interest. 124 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 6, 2024. ; https://doi.org/10.1101/2024.12.05.627042doi: bioRxiv preprint Figures 125 126 127 Figure 1: C. sonorensis after blood meal (left). The biting midge was placed individually in a 128 tube containing 200 µl phosphate-buffered saline (PBS) and a 5 mm stainless steel bead for 129 further processing. Engorged females in new netted cardboard cage after blood feeding 130 (right). 131 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 6, 2024. ; https://doi.org/10.1101/2024.12.05.627042doi: bioRxiv preprint 132 133 Figure 2: RT-qPCR results of midges experimentally infected with BTV-3 or BTV-8 spiked 134 blood meal and midges fed with virus-free blood (negative control, NC). Individual midges 135 were tested for BTV genome immediately after ingestion of the blood meal (day 0) or six 136 days after the blood meal (day 6). Horizontal black lines indicate the highest BTV copy 137 number measured in any of the midges of the respective group immediately after blood meal 138 ingestion. The experiment was performed in two subsequent trials, the trial number is given in 139 the label of the x-axis. 140 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted December 6, 2024. ; https://doi.org/10.1101/2024.12.05.627042doi: bioRxiv preprint

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