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Article

Diversity of Transmission Outcomes Following Co-Infection of Sheep with Strains of Bluetongue Virus Serotype 1 and 8

by
Eva Veronesi
1,2,*,†,
Karin Darpel
1,†,
Simon Gubbins
1,
Carrie Batten
1,
Kyriaki Nomikou
1,3,
Peter Mertens
1,3 and
Simon Carpenter
1,*
1
The Pirbright Institute, Pirbright, Surrey GU24 0NF, UK
2
National Centre for Vector Entomology, Institute of Parasitology, Vetsuisse Faculty, 8057 Zurich, Switzerland
3
University of Nottingham, Sutton Bonington, Leicestershire LE12 5RD, UK
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to the studies.
Microorganisms 2020, 8(6), 851; https://doi.org/10.3390/microorganisms8060851
Submission received: 5 March 2020 / Revised: 31 May 2020 / Accepted: 2 June 2020 / Published: 5 June 2020
(This article belongs to the Special Issue Bluetongue Virus)
Browse Figures
Versions Notes

Abstract

:
Bluetongue virus (BTV) causes an economically important disease, bluetongue (BT), in susceptible ruminants and is transmitted primarily by species of Culicoides biting midges (Diptera: Ceratopogonidae). Since 2006, northern Europe has experienced multiple incursions of BTV through a variety of routes of entry, including major outbreaks of strains of BTV serotype 8 (BTV-8) and BTV serotype 1 (BTV-1), which overlapped in distribution within southern Europe. In this paper, we examined the variation in response to coinfection with strains of BTV-1 and BTV-8 using an in vivo transmission model involving Culicoides sonorensis, low passage virus strains, and sheep sourced in the United Kingdom. In the study, four sheep were simultaneously infected using BTV-8 and BTV-1 intrathoracically inoculated C. sonorensis and co-infections of all sheep with both strains were established. However, there were significant variations in both the initiation and peak levels of virus RNA detected throughout the experiment, as well as in the infection rates in the C. sonorensis that were blood-fed on experimentally infected sheep at peak viremia. This is discussed in relation to the potential for reassortment between these strains in the field and the policy implications for detection of BTV strains.

1. Introduction

Bluetongue virus (BTV) is an arboviral pathogen mainly of ruminants and deer that is transmitted primarily by biting midges of the genus Culicoides (Diptera: Ceratopogonidae) [1,2]. BTV is a member of the genus Orbivirus within the family Reoviridae and its genome comprises 10 linear segments of double-stranded RNA [3], which code for 7 structural and 4/5 non-structural proteins [4,5]. Currently, at least 27 serotypes of BTV have been identified, based on the variability of the virus outer capsid protein VP2 (encoded by genome segment 2), that exhibit only limited levels of cross protection in the ruminant host [6,7,8,9]. In endemic regions, simultaneous infections can frequently occur with multiple BTV strains and/or serotypes in a single host. In some areas, there is also an increased frequency of these events due to the use of live attenuated vaccine strains that can be transmitted by Culicoides [10,11,12]. These multiple infections may lead to a high rate of reassortment in the field and recent laboratory-based studies have also demonstrated few apparent constraints on the process of BTV genome segment exchange [13,14].
Since 2006, BTV has caused widespread and economically damaging outbreaks of bluetongue (BT) that have had a significant impact on sheep and cattle production across the Western Palearctic region, through both clinical disease and movement restrictions imposed at a regional and national level to control BTV spread [2,15]. While technologies to produce safe and efficacious inactivated vaccines are available to reduce the impact of BTV outbreaks in northern Europe [16,17], the emergence and re-emergence of strains presents an issue in terms of the proportionate policy response, as the pathogenicity between strains of BTV in different breeds and species of hosts varies significantly [18,19]. This has led to difficulties in defining scenarios under which vaccination should or should not be employed [20,21], both from the perspective of the impact of clinical disease and in reducing the burden of trade restrictions between countries [22,23,24,25].
During the period 2006–2010, the most damaging outbreaks of BT were caused by a BTV serotype 8 (BTV-8) strain, which entered northern Europe directly via an unspecified route from sub-Saharan Africa [26], and a BTV serotype 1 (BTV-1) strain that spread through Morocco into Spain and then into France [1,2]. Both BTV strains were eventually controlled through vaccination campaigns but overlapped in distribution for a significant period of time during 2008–2009 in Spain and France. Anecdotally, these BTV strains differed significantly in their phenotype, most notably in clinical severity (BTV-8 causing significant clinical signs in cattle, which were absent in BTV-1 infection, and BTV-1 having a more severe clinical impact than BTV-8 infection in sheep) and in the occurrence of transplacental transmission in cattle (observed repeatedly in animals infected with BTV-8) [27,28,29,30]. This led to discussion around the policy implications of co-circulating BTV strains in northern Europe, particularly where changes in the spread or pathogenicity of novel emerging strains of BTV had the potential to occur.
A study carried out previously in Belgium examined the co-infection of calves with BTV-1 and BTV-8 strains with a view to explore the potential for competitive exclusion of these strains [31]. Rather than aim to examine the potential for reassortant strains to emerge in northern Europe, this study used serotyping assays to define the likelihood of transmission of BTV-1 and BTV-8 from co-infected calves, as this was a priority for informing predictions of strain spread and subsequent vaccination strategy. While the initial in vivo experiment was confounded by a contaminating strain of BTV serotype 15, a second study using needle-based inoculation demonstrated that BTV-8 replicated to potentially transmissible levels in three calves, while BTV-1 was present only at the limit of detection of the real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assay used to quantify BTV RNA. The quantities of BTV-8 RNA were significantly reduced in these animals when compared to those infected with just the BTV-8 strain.
In our study, we revisited this earlier experiment, following the development of improved in vivo models for the study of BTV infection. We used colony-derived Culicoides sonorensis, intrathoracically (IT) inoculated with strains of BTV-1 and BTV-8, to co-infect four sheep with both viral strains simultaneously. We then assessed the development of viral RNA of both strains using an rRT-PCR assay that detected BTV segment 2 (which determines serotype). Following the development of viremia, we then allowed further batches of uninfected C. sonorensis to blood feed on co-infected sheep and examined the retention of the two serotypes in the insect host. The aim of the study was to examine the variation in responses to co-infection in both the ruminant and insect host.

2. Materials and Methods

2.1. Viruses, Vectors, and Hosts Used during Trials

2.1.1. Viruses

Two BTV strains were used throughout the experiments. All isolations and passages were carried out using a cell line established from embryos of Culicoides sonorensis in the USA (KC: C. sonorensis) [32,33]. Cells were grown in Schneider’s insect cell medium (Lonza, Slough, UK) and supplemented with 1% penicillin/streptomycin (Sigma-Aldrich, Dorset, UK), 1% Amphotericin B (Sigma-Aldrich, Dorset, UK), and 10% Gibco® heat-inactivated fetal calf serum (ThermoFisher Scientific, Waltham, MA, USA) (‘growth media’). A BTV-8 strain (NET2006/06) was isolated from an infected sheep detected in The Netherlands during 2006. Similarly, a BTV-1 strain (MOR2007/01) from an infected sheep detected in Morocco was also isolated on the same cell line. Both viruses were subsequently passaged once on the KC C. sonorensis cell line and used as a KC2 passage for vector infection. The titer was quantified using a serial dilution 96-well endpoint titration assay on KC cells incubated for 7 days at 28 °C. Titer of the progeny virus in the cell supernatants was determined using an indirect in-house antigen sandwich enzyme-linked immunosorbent assay (ELISA) to detect the presence of virus in a serially diluted titration of samples [34] and calculated using the method of Spearman and Kärber [35]. C. sonorensis were intrathoracically (IT) inoculated using 0.2 µL of tissue culture supernatant of BTV-8 or BTV-1 at a titer of 5.75–6 log10 TCID50/mL [34].

2.1.2. Vectors

Culicoides sonorensis used was the PIRB-s-3 strain, originally derived from the Sorona (AA) line propagated in Denver, Colorado, USA [32]. All C. sonorensis utilized in the experiments had emerged from pupae at least four days prior to use.

2.1.3. Hosts

Female adult Dorset Poll sheep (>2 years of age) originating in the UK were used in a group of four animals during the trial, housed in a high containment animal facility at the Pirbright Institute and fed twice a day with grain pellets and with ad libitum access to hay and water throughout the experiment [29]. An uninfected ‘direct contact transmission control’ sheep was housed in the same pen as the treatment animals. All animals were screened for BTV antibodies prior to use using a commercial competitive anti-VP7 antibody ELISA (IDVet, Montpellier, France).

2.2. Detection of BTV and Typing

RNA extraction from blood samples containing ethylenediamine tetraacetic acid anticoagulant and rRT-PCR was carried out as described previously [36]. To control for inter-plate variation between independent extractions and rRT-PCR, several replicates (for a 96-well plate, a minimum of 5 replicates) of BTV RNA extracted from positive sheep blood with a known rRT-PCR Cq value were included on each extraction/rRT-PCR plate. The sample results of respective plates were only accepted if the positive control samples were within 2 Cq values of the previously determined value. Initial detection of BTV RNA was carried out using the rRT-PCR assay based on genome segment 1(Seg-1) detection as previously described and used (termed here as the detection assay) [34,37]. Serotype identification of BTV-1 and BTV-8 strains was conducted directly parallel to the BTV detection assay in potential co-infected samples (sheep blood and orally blood-fed midges) using individual typing rRT-PCR assays based on the BTV genome segment 2 (Seg-2) and quantified by the Cq value (termed typing assays) [31]. As previously validated, the separate BTV-1 and BTV-8 typing rRT-PCR assays had the same efficiency and analytical sensitivity [38]. The serotype specificity of the BTV-1/BTV-8 rRT-PCR typing assays used was also confirmed against virus samples of the BTV-1 and BTV-8 strains used in this study as well as additional closely related strains with no evidence of cross reaction.

2.3. Infection of Sheep

Techniques used to infect C. sonorensis with BTV have been described previously [34]. Briefly, over 2000 C. sonorensis were intrathoracically (IT) inoculated with 0.2 µL of either the BTV-1 or BTV-8 strain using pulled glass capillary needles (Narishige, London, UK) and a microinjector with a manual syringe driver (Sutter Instruments, California, USA). Inoculation was carried out under light CO2 anesthesia and surviving C. sonorensis were transferred to specific pill boxes separate for each BTV strain used and incubated for 5 days at 25 °C. A total of 575 C. sonorensis survived the incubation period and were used to infect the four sheep (designated sheep 1–4; an unexposed sheep was designated sheep 5 as a transmission control). The same pill boxes were used in which the C. sonorensis were stored to reduce the requirement for further anaesthetization and no attempt was made to equalize the numbers exposed to the sheep for each strain to avoid further insect mortality. The four sheep were then restrained and two pill boxes containing BTV-8 NET 2006/06 (pot 1) and BTV-1 MOR 2007/01 (pot 2) IT-inoculated C. sonorensis simultaneously placed on both inner thighs for a period of 10 min (Figure 1). Following this exposure, pill boxes were returned to the laboratory, C. sonorensis anaesthetized, and the fully disseminated BTV infection rate was quantified in those individual C. sonorensis that had taken a blood meal using the same rRT-PCR assay (as serotypes were separated and known), as defined in a previous study [34].

2.4. Monitoring of Sheep

Blood samples were taken from the jugular vein on the day before the experiment began (day 0) and then on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, and 16 following infection. Clinical signs of disease and rectal temperature were recorded for each animal daily. A postmortem of all sheep was carried out following euthanasia at 16 days post infection (dpi), or when individuals reached a humane endpoint for the protocol. Blood samples taken in the first 10 days post infection were tested for their quantity of viral RNA by rRT-PCR on the same day. Nucleic acid was extracted from 50 µL of the blood sample and the quantity of viral RNA in the sample subsequently assessed by detection and typing rRT-PCR assays. Preliminary data suggested that a blood sample with a rRT-PCR Cq value of less than 25 would result in the animal reaching peak viremia within two days.

2.5. Clinical Score Calculation

A daily clinical score was recorded for each animal. The score was based on the following 11 criteria: 1. Redness of eyes; 2. Redness of mucosal membranes (oral and nasal); 3. Facial edema; 4. Salivation; 5. Nasal discharge; 6. Cough; and 7. Ulcers (oral and/or nasal) and were all scored daily from 0.5 (very mild) to 3 (severe) (increments of 0.5 were used). 8. Temperature was incorporated into the daily score as follows: The normal body temperature of each individual sheep was determined for at least 3 days prior to infection and the average body temperature was used as the “baseline”. An increase of ≥1 °C from the baseline temperature incurred a clinical score of 1; ≥1.5 °C of 2 and ≥2 °C of 3 for each day of occurrence. 9. A behavior score was included as follows: Apathy and slowness = 1; reluctance to get up unaided and prolonged daily separation from the group = 2; reluctance to get up in response to direct stimulation/body posture indicative of pain = 3 (immediate humane endpoint). 10. A food intake score was determined as follows: Reduced food intake = 1; avoiding concentrate but eating hay = 2; no food intake = 3. The food intake was scored across two daily meals and then averaged. 11. Clinical signs of BTV infection in feet was determined separately for each foot, ranging from 0.5–3 and considering warmth, reddening, lesions, and lameness and subsequently averaged for a daily foot score.
Sheep were euthanized if they reached pre-defined humane endpoints of moderate to high severity limits defined within the study protocol in the ethics project license. These endpoints could be reached either by severity of single behavioral or clinical signs or a combination of several signs of moderate severity on a single day.

2.6. Pathology Score Calculation

The total gross pathology score was determined through a combination of a hemorrhage score, an edema score, an ulceration score across a standardized range of selected organs, and a lung lesion score; all criteria were marked from 0.5 (very mild) to 3 (severe).

2.7. Infection of Culicoides sonorensis

At peak viremia, pots of approximately 250 adult C. sonorensis were exposed to the inner thigh of each sheep. Once returned to the laboratory, blood-fed females were selected under light CO2 anesthesia and incubated for 8 days at 25 °C. Those surviving this period were selected under CO2 anesthesia and placed individually in racked 96 sample polypropylene tubes (Qiagen, Crawley, UK). A selected number of blood-fed females were also processed immediately following blood feeding without incubation to obtain BTV RNA values of uptake only (D0 midges). Individual Culicoides were then homogenized using a 3-mm stainless steel ball bearing (Dejay Distribution Ltd., UK) in 100 µL of Schneider’s media (Sigma Aldrich, Poole, UK), before the addition of a further 900 µL of Schneider’s media. Pools of 400 µL were then created in polypropylene tubes using 50 µL of homogenate from each of 8 individuals and screened using the detection rtRT-PCR assay [37]. Where BTV was detected, individual samples from that pool were retested using the detection rtRT-PCR assay and the BTV-1/BTV-8 typing rtRT-PCR assays, as for sheep blood [38].

2.8. Statistical Analysis

Levels of viral RNA (Cq values) in sheep determined by the three rtRT-PCRs (i.e., segment-1 (detection), BTV-1, and BTV-8 segment-2 (typing)) were compared using a linear mixed model with Cq as the response variable, days post exposure and assay and an interaction between them as fixed effects, and sheep as a random effect. Model selection proceeded by stepwise deletion of non-significant (p > 0.05) terms (judged by likelihood ratio tests), starting from a model including all explanatory variables. The proportion of C. sonorensis positive for viral RNA of each BTV strain after incubation was analyzed using a binomial family generalized linear model with a logit link function, with the proportion of positive midges as the response variable and the virus strain (BTV-1 or BTV-8) and blood meal Cq value as explanatory variables. Model selection proceeded by stepwise deletion of non-significant (p > 0.05) terms (judged by likelihood ratio tests), starting from a model including all explanatory variables. All analyses were implemented in R (version 3.6.1) [39].

2.9. Ethics Statement

All animal experiments were carried out in accordance with the UK Animal Scientific Procedure Act (ASPA) 1986, which transposes European Directive 2010/63/EU into UK national law. All animal experiments were conducted in compliance with a national project license number PPL 70/7819 granted by the UK Home Office to Dr Karin Darpel from 1st September 2013 to 1st September 2018.

3. Results

3.1. Infection of Sheep

A total of 575 C. sonorensis that survived the extrinsic incubation period following IT inoculation were exposed to the sheep (Table 1; Supplementary Table S1). In total, 100 of the 575 C. sonorensis took at least a partial blood meal (17% of those exposed), of which 97 produced Cq values (using the detection rRT-PCR assay) indicative of full dissemination (≤ 25) [34]. In total, 57 of these fully infected C. sonorensis were infected with BTV-1 and 40 were infected with BTV-8 and the proportions feeding on each sheep are shown in Table 1 (Supplementary Table S1).

3.2. Monitoring of Sheep

Levels of BTV RNA indicative of viremia were demonstrated for all four sheep, although the peak level of viremia in sheep 4 was reduced (Figure 2; Supplementary Table S2). Cq values did not differ significantly (p = 0.97) between the detection and typing for BTV-1 assays but were significantly (p < 0.001) lower for the typing for the BTV-8 assay compared with the other two assays (estimated difference in Cq values (95% confidence interval (CI): Typing BTV-8 vs. detection = 5.5 (4.1–7.0); typing BTV-8 vs. typing BTV-1 = 5.4 (3.9–6.8)) (Figure 2). This difference was consistent across time points (i.e., there was no significant (p = 0.16) interaction between days post exposure and assay).
In all cases, BTV-1 segment-2 RNA was detected in greater quantities from sheep during the viremic period, including in sheep 2 where a greater number of IT inoculated C. sonorensis transmitting BTV-8 (5 fully fed; 5 partially fed) were successfully fed than those transmitting BTV-1 (1 fully fed; 4 partially fed). No BTV RNA was detected in the blood of the transmission control sheep 5, which remained negative for BTV RNA throughout the entire course of the study.

3.3. Clinical Scores

All BTV-infected sheep developed classical clinical signs of BTV infection, including reddening of the eyes and mucosal membranes and facial edema. Clinical signs were first recorded at 5 dpi and daily clinical scores were determined for each individual from this day onwards (Figure 3). Two sheep (sheep 1 and 3) were euthanized on 7 dpi due to apathy and reluctance to get up, as well as cessation of food intake and a more pronounced appearance of several other visible clinical signs (Figure 4). The remaining two sheep, 2 and 4, experienced moderate clinical signs characterized by small petechial ulcerations on the nose/muzzle and redness and inflammation of the feet later in infection (11–16 dpi) (Figure 3 and Figure 4). No clinical signs were recorded in the transmission control sheep 5 at any time during the study.

3.4. Pathology Scores

Gross-pathological changes were recorded at necropsy of all four infected sheep. The acute oedemic and hemorrhagic scores were higher in those two sheep euthanized during the acute phase of the disease at 7 dpi (sheep 1 and 3), although hemorrhagic lesions of selected lymph nodes were still present at 16 dpi (Table 2 and Figure 5). Both sheep reaching the study end of 16 dpi (sheep 2 and 4) had evidence of pathological changes, with sheep 2 in particular demonstrating clinical signs associated with BT, such as a hemorrhage of the tongue and pulmonary artery (Table 2 and Figure 5).

3.5. Infection of Culicoides sonorensis

A total of 688 C. sonorensis were fed on the sheep and then survived the extrinsic incubation period (Table 3). None of the C. sonorensis exposed to sheep 1 at 6 dpi imbibed a blood meal, hence no data on viral uptake and/or replication could be obtained for this time point (Table 3). In total, 22 C. sonorensis contained BTV RNA at 8 days following feeding on the infected sheep (Table 3; Supplementary Table S3). Feeding on sheep 3 produced 14 infections in C. sonorensis (11 with Cq ≤ 25; and 3 Cq ≥ 25 in the detection assay: 5.6% containing RNA in those exposed); on sheep 4, 2 infections; and on sheep 1, 6 infections (6 Cq ≤ 25: 5.1%), while no C. sonorensis became infected from feeding on sheep 2. BTV-1 Seg-2 RNA was detected in all positive C. sonorensis fed on sheep 3 and 1, and a single individual fed on sheep 1 possessed a mixed BTV-1/BTV-8 infection (typing BTV-1 Cq: 20.75; typing BTV-8 Cq: 33.79). BTV-8 Seg-2 RNA only was detected in C. sonorensis fed on sheep 4.
Assuming C. sonorensis were infected if they had any Cq value, there was no significant difference in the proportion of positive individuals between strains (p = 0.27). There was, however, a significant increase in the probability of C. sonorensis containing detectable BTV RNA following incubation with a decrease in the Cq value of the blood on which it originally fed (odds ratio (OR): 0.77, 95% confidence interval (CI): 0.70–0.86, p < 0.001). If blood meal Cq was not included as a factor in the analysis, there was a significantly higher proportion of C. sonorensis positive for the BTV-1 strain compared with the BTV-8 strain (OR for BTV-8 compared with BTV-1: 0.14, 95% CI: 0.04–0.49, p < 0.001). This suggests that the difference in the proportion of positive C. sonorensis for each strain is a consequence of higher levels of virus possessing a segment 2 sequence corresponding with BTV-1 than those corresponding to BTV-8 (Table 3). These conclusions remain the same if C. sonorensis were assumed to be positive only if they had a Cq value < 25.

4. Discussion

In this paper, we used a C. sonorensis BTV sheep infection model to explore transmission of BTV-1 and BTV-8 strains that co-circulated during the early stages of BTV incursions in northern Europe. In a previous study in which calves were co-infected with BTV-1 and BTV-8 strains using needles, high BTV-8 viremia was detected [31]. In contrast, in our study, using a natural infection and sheep host model resulted in viruses of serotype 1 predominating within all four sheep tested. This even occurred in the case of sheep 2, where more C. sonorensis fully infected with BTV-8 were fed to repletion on the sheep than those that were infected with BTV-1. The study also demonstrates that strains of serotype 8 can be recovered in C. sonorensis fed on co-infected animals despite relatively low levels of RNA of this serotype being present within the sheep. Finally, one C. sonorensis from a total of 22 that developed infections from feeding on viremic sheep developed a mixed infection of BTV-1 and BTV-8, indicating the potential for strains to co-infect the insect host as proposed in studies previously [40].
Intrathoracic infection of C. sonorensis using both BTV-1 and BTV-8 consistently led to fully disseminated infections as inferred through the use of rRT-PCR Cq values [34]. Successful feeding of infected C. sonorensis on the sheep following IT inoculation and incubation is not certain and hence is not standardized. Relatively large numbers of C. sonorensis were therefore exposed to the sheep, with numbers successfully feeding ranging from 5 to 23 individuals for infection with either BTV-8 or BTV-1. While the number of Culicoides infected with BTV blood feeding on sheep in the field has not been assessed, their populations and subsequent biting rates are among the highest in hematophagous insects [41,42,43,44]. All sheep were successfully simultaneously infected with both strains of BTV through the feeding of C. sonorensis with fully disseminated infections, illustrating the high rates of transmission obtained via this route [45,46].
Bluetongue virus RNA was first detected in sheep 1, 3, and 4 on day two post infection (Figure 2). In contrast, RNA was only detected on day 4 post infection in sheep 2 (Figure 2). Similarly, sheep 2 also reached peak BTV RNA levels in blood on day 7 post infection, later than observed in sheep 1 (day 5), sheep 3 (day 5), and sheep 4 (day 3). Sheep 2 was fed on by fewer fully infected C. sonorensis (15) than sheep 1 (32), sheep 3 (21), and sheep 4 (32), and this lower rate of inoculation may have led to this delayed onset of RNA detection as demonstrated in a previous study [45].
Co-infection with BTV-1 and BTV-8 was successfully established in all four sheep, despite sheep 2 only being exposed to one C. sonorensis infected with BTV-1 taking a full blood meal and four taking partial blood meals. In all four sheep, BTV-1 Seg-2 RNA was detected in greater quantities than BTV-8 Seg-2 RNA at all time points using the BTV typing assays and was closely correlated with Seg-1 RNA detected using the BTV detection assay. There was no visible correlation between the numbers of infected C. sonorensis feeding and the peak of RNAemia attained. In addition, the ratio between C. sonorensis infected with each serotype feeding on the sheep did not seem to influence the levels of viral RNA for the two strains of BTV.
Divergence in the levels of viral RNA, however, was recorded between sheep 1 and 3 when compared to sheep 2 and 4, with the former experiencing a very high and sustained plateau of BTV-1 Seg-2 RNA levels at < 20 Cq and the latter only achieving this for a brief period of 1 day in sheep 2 and not at all in sheep 4. The drivers for differences in the levels of viral RNA were unclear and not correlated either with differences in the number of infected C. sonorensis initially imbibing a blood meal from the sheep, or a difference in the ratio between C. sonorensis infected with either BTV-1 or BTV-8 taking a blood meal. Both sheep 1 and 3 suffered significantly greater clinical signs than sheep 2 and 4 and this may have been due to the greater quantity of circulating BTV-1 with virus titers ranging 5 to 6 Log10TCID50 (Table 3). Studies using BTV-1 and BTV-8 strains of the same phylogenetic lineage as used in the current study have reported an earlier onset of clinical disease in BTV-8-infected sheep, but with the greatest severity observed in BTV-1-infected sheep (10–15 dpi). This observation was correlated with an earlier rise in BTV-8 RNA levels but comparable BTV RNA generation detected in sheep with either virus strain at peak infection [30]. Future studies could explore if the initial progression and initiation of viral RNA production (in combination with or independent of peak levels of BTV RNA produced) is at least partially correlated to subsequent clinical disease progression [47].
The infection rates of C. sonorensis when fed on the sheep were low and similar to those obtained during previous in vivo studies [45,48] and in epidemiologically relevant Culicoides species present in northern Europe fed via membrane-based techniques in Switzerland [49] and pledglet-based methods in the UK [50]. As expected, the likelihood of being able to detect the successful uptake of infectious virus by blood feeding C. sonorensis (processed immediately after feeding without any further incubation) was largely correlated with the amount of infectious viral particles circulating in the systemic blood of the sheep on the day of feeding. In our experiments, the exception to this rule were three individuals fed on sheep 2 at 7 dpi (sheep blood Cq value of 17.56); otherwise, all individual C. sonorensis feeding on a sheep with ≤Cq21 resulted in a positive rRT-PCR detection of BTV RNA. It is currently unclear why those individuals feeding on sheep 2 did not have detectable amounts of BTV RNA in their blood meal; however, it is important to note that the viral presence in the skin periphery could differ from the level detected systemically in blood samples taken at the jugular vein [51]. Low virus RNA levels were occasionally also detected in individual C. sonorensis directly following blood feeding on sheep with less viral RNA in the blood; however, detection was less consistent and at the detection limit of the diagnostic rRT-PCR assay (Table 3 and Supplementary Material Tables S2 and S3).
Culicoides sonorensis fed on those sheep with the highest level of virus RNAemia also had the greatest probability of developing infections (Table 3, sheep 1 and 3), and in the majority of individuals, BTV-1 replicated to levels previously indicative of fully transmissible infections (≤Cq25) [34]. Nonetheless, it is interesting to note that in three individual midges, some degree of viral replication of BTV-8 occurred despite relatively low detected Seg-2 RNA levels (≤28Cq in the sheep at the time of feeding), and in the absence of any detectable BTV-8 viral RNA uptake at day 0. Hence, the probability of BTV replication within vector insects following blood feeding on infected sheep does not seem solely explainable by viral levels detected in the systemic sheep blood at the time, although as only segment 2 RNA from BTV-8 was identified it is currently unknown if the virus might have reassorted within the ruminant host.
A key aspect not addressed in the current study is the detection and potential impact of reassortment. Reassortment of BTV can occur when two strains of the virus co-infect a single cell, either in the host or vector. The BTV genome consists of 10 linear segments of double-stranded RNA, and in tissue culture, these segments have been demonstrated to be exchanged with few constraints between European strains of BTV serotype 1 (BTV-1) and BTV serotype 8 (BTV-8) [14]. Analysis of full genome sequences from across European strains of BTV has highlighted that this phenomenon occurs frequently but may also be subject to specific selective pressures that are not fully understood [13,52,53]. Reassortment of BTV strains in the ruminant host were studied in vivo in the USA during the 1980s in both ruminants and vectors [40,54,55]. These experiments highlighted differences in the rate of reassortment between sheep (95% parental strains recovered and 5% reassortant) and a bull (11% parental strains and 89% reassortant). In addition, mixed infection of colony-derived Culicoides sonorensis led to proportions of reassortant progeny that varied between 7% and 78% in 8 reassortant-yielding flies. The current study has demonstrated that BTV-1 and BTV-8 duel infections can occur in both the ruminant and insect host and further studies are required to characterize this process.
The policy implications of the current study are significant in highlighting circulation of multiple strains within the ruminant and vector hosts. It demonstrates the importance of serotype determination and full genome characterization where there is doubt regarding the identity of circulating strains. In the case of sheep 4, there was also demonstration that a strain of a serotype that was circulating only at reduced levels of RNA within the ruminant host was transmitted to and replicated within C. sonorensis. A key question arising from this observation is whether this represented a reassortant strain and at what point in the cycle of infection this reassortment took place. The study highlights, however, that simple extrapolation from Cq values to likely infection of Culicoides requires care, particularly in areas where multiple serotypes and strains of BTV co-circulate.

5. Conclusions

This experimental study has demonstrated that co-infection of bluetongue virus strains in sheep can lead to a range of outcomes in onwards transmission from sheep by Culicoides biting midges. We infected Culicoides biting midges with either BTV-1 or BTV-8 by injecting the virus into individuals and then allowing it to replicate within the insect. Then, we used these fully infected Culicoides to infect four sheep with both strains simultaneously. Both the clinical signs and the profile and peak level of viral RNA developed in each sheep varied significantly across the sheep alongside clinical disease signs to a greater degree than was expected. We then fed more unexposed Culicoides on the sheep to assess infection rates. We found that both strains persisted in the host, in contrast to a previous study, and that infection of Culicoides was dependent on being fed when the viruses were at present in the sheep at sufficient levels and a dose-dependent relationship with the specific strain. These results emphasize the importance of using a natural route of infection during BTV transmission experiments.

Supplementary Materials

The following are available online at https://www.mdpi.com/2076-2607/8/6/851/s1, Table S1: Levels of bluetongue virus RNA (Cq) detected in intrathoracically inoculated Culicoides sonorensis following incubation for 5 days at 25 °C and exposure to uninfected sheep. Table S2: Levels of bluetongue virus RNA (Cq) detected in sheep following exposure to Culicoides sonorensis intrathoracically inoculated with either a strain of BTV serotype 1 or serotype 8. Table S3: Feeding of uninfected Culicoides sonorensis on sheep infected with bluetongue virus. Culicoides sonorensis successfully taking a blood meal were processed immediately following feeding and following incubation for 8 days at 25 °C.

Author Contributions

Conceptualization, S.C., K.D., S.G. and P.M.; Methodology, E.V., K.D., K.N. and S.G.; Validation, E.V., K.D., K.N., S.G. and C.B.; Formal Analysis, E.V., K.D., S.C. and S.G.; Writing—Original Draft Preparation, S.C.; Writing—Reviewing and Editing, S.C., K.D., P.M., E.V., K.N. and S.G.; Visualization, S.C., S.G. and K.D.; Supervision, S.C. and P.M.; Project Administration, S.C.; Funding Acquisition, S.C. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by Defra project SE: 2618 “Bluetongue virus reassortment and overwintering in the Culicoides host” awarded to SC. Additional support was provided by UKRI projects BBS/E/I/00001701, BBS/E/I/00001737, BBS/E/I/00007039 and BBS/E/I/00001736.

Acknowledgments

The authors would like to acknowledge the animal technicians at The Pirbright Institute, without whom this work would not be possible. We would also like to thank Eric Denison for production of Culicoides sonorensis used during the trials.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

  1. Purse, B.V.; Carpenter, S.; Venter, G.J.; Bellis, G.; Mullens, B.A. Bionomics of temperate and tropical Culicoides midges: Knowledge gaps and consequences for transmission of Culicoides-borne viruses. Annu. Rev. Entomol. 2015, 60, 373–392. [Google Scholar] [CrossRef] [PubMed]
  2. Carpenter, S.; Wilson, A.; Mellor, P.S. Culicoides and the emergence of bluetongue virus in northern Europe. Trends Microbiol. 2009, 17, 172–178. [Google Scholar] [CrossRef] [PubMed]
  3. Drolet, B.S.; van Rijn, P.; Howerth, E.W.; Beer, M.; Mertens, P.P. A Review of Knowledge Gaps and Tools for Orbivirus Research. Vector Borne Zoonotic Dis. 2015, 15, 339–347. [Google Scholar] [CrossRef] [PubMed]
  4. Ratinier, M.; Caporale, M.; Golder, M.; Franzoni, G.; Allan, K.; Nunes, S.F.; Armezzani, A.; Bayoumy, A.; Rixon, F.; Shaw, A.; et al. Identification and Characterization of a Novel Non-Structural Protein of Bluetongue Virus. PLoS Pathog. 2011, 7, e1002477. [Google Scholar] [CrossRef] [Green Version]
  5. Stewart, M.; Hardy, A.; Barry, G.; Pinto, R.M.; Caporale, M.; Melzi, E.; Hughes, J.; Taggart, A.; Janowicz, A.; Varela, M.; et al. Characterization of a second open reading frame in genome segment 10 of bluetongue virus. J. Gen. Virol. 2015, 96, 3280–3293. [Google Scholar] [CrossRef]
  6. Breard, E.; Schulz, C.; Sailleau, C.; Bernelin-Cottet, C.; Viarouge, C.; Vitour, D.; Guillaume, B.; Caignard, G.; Gorlier, A.; Attoui, H.; et al. Bluetongue virus serotype 27: Experimental infection of goats, sheep and cattle with three BTV-27 variants reveal atypical characteristics and likely direct contact transmission BTV-27 between goats. Transbound. Emerg. Dis. 2018, 65, e251–e263. [Google Scholar] [CrossRef]
  7. Erasmus, B.; Potgieter, A.C. The History of Bluetongue. In Bluetongue; Mellor, P.S., Baylis, M., Mertens, P.C., Eds.; Elsevier: Amsterdam, The Netherlands, 2009; pp. 7–21. [Google Scholar] [CrossRef]
  8. Hofmann, M.A.; Renzullo, S.; Mader, M.; Chaignat, V.; Worwa, G.; Thuer, B. Genetic Characterization of Toggenburg Orbivirus, a New Bluetongue Virus, from Goats, Switzerland. Emerg. Infect. Dis. 2008, 14, 1855–1861. [Google Scholar] [CrossRef]
  9. Maan, S.; Maan, N.S.; Nomikou, K.; Veronesi, E.; Bachanek-Bankowska, K.; Belaganahalli, M.N.; Attoui, H.; Mertens, P.P.C. Complete Genome Characterisation of a Novel 26th Bluetongue Virus Serotype from Kuwait. PLoS ONE 2011, 6, e26147. [Google Scholar] [CrossRef] [Green Version]
  10. Van den Bergh, C.; Coetzee, P.; Venter, E.H. Reassortment of bluetongue virus vaccine serotypes in cattle. J. S. Afr. Vet. Assoc. 2018, 89, e1–e7. [Google Scholar] [CrossRef]
  11. Veronesi, E.; Hamblin, C.; Mellor, P.S. Live attenuated bluetongue vaccine viruses in Dorset Poll sheep, before and after passage in vector midges (Diptera ceratopogonidae). Vaccine 2005, 23, 5509–5516. [Google Scholar] [CrossRef]
  12. Batten, C.A.; Maan, S.; Shaw, A.E.; Maan, N.S.; Mertens, P.P.C. A European field strain of bluetongue virus derived from two parental vaccine strains by genome segment reassortment. Virus Res. 2008, 137, 56–63. [Google Scholar] [CrossRef] [PubMed]
  13. Nomikou, K.; Hughes, J.; Wash, R.; Kellam, P.; Breard, E.; Zientara, S.; Palmarini, M.; Biek, R.; Mertens, P. Widespread Reassortment Shapes the Evolution and Epidemiology of Bluetongue Virus following European Invasion. PLoS Pathog. 2015, 11, e1005056. [Google Scholar] [CrossRef] [PubMed]
  14. Shaw, A.E.; Ratinier, M.; Nunes, S.F.; Nomikou, K.; Caporale, M.; Golder, M.; Allan, K.; Hamers, C.; Hudelet, P.; Zientara, S.; et al. Reassortment between two serologically unrelated bluetongue virus strains is flexible and can involve any genome segment. J. Virol. 2013, 87, 543–557. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  15. Pascall, D.J.; Nomikou, K.; Bréard, E.; Zientara, S.; da Silva Filipe, A.; Hoffmann, B.; Jacquot, M.; Singer, J.B.; De Clercq, K.; Bøtner, A.; et al. “Frozen evolution” of an RNA virus suggests accidental release as a potential cause of arbovirus re-emergence. PLoS Biol. 2020, in press. [Google Scholar] [CrossRef]
  16. Oura, C.A.; Wood, J.L.; Sanders, A.J.; Bin-Tarif, A.; Henstock, M.; Edwards, L.; Floyd, T.; Simmons, H.; Batten, C.A. Seroconversion, neutralising antibodies and protection in bluetongue serotype 8 vaccinated sheep. Vaccine 2009, 27, 7326–7330. [Google Scholar] [CrossRef]
  17. Moulin, V.; Noordegraaf, C.V.; Makoschey, B.; van der Sluijs, M.; Veronesi, E.; Darpel, K.; Mertens, P.P.; de Smit, H. Clinical disease in sheep caused by bluetongue virus serotype 8, and prevention by an inactivated vaccine. Vaccine 2012, 30, 2228–2235. [Google Scholar] [CrossRef]
  18. Coetzee, P.; Stokstad, M.; Venter, E.H.; Myrmel, M.; Van Vuuren, M. Bluetongue: A historical and epidemiological perspective with the emphasis on South Africa. Virol. J. 2012, 9, 198. [Google Scholar] [CrossRef] [Green Version]
  19. Maclachlan, N.J.; Drew, C.P.; Darpel, K.E.; Worwa, G. The pathology and pathogenesis of bluetongue. J. Comp. Pathol. 2009, 141, 1–16. [Google Scholar] [CrossRef]
  20. Sumner, T.; Burgin, L.; Gloster, J.; Gubbins, S. Comparison of pre-emptive and reactive strategies to control an incursion of bluetongue virus serotype 1 to Great Britain by vaccination. Epidemiol. Infect. 2013, 141, 102–114. [Google Scholar] [CrossRef]
  21. Szmaragd, C.; Wilson, A.J.; Carpenter, S.; Wood, J.L.; Mellor, P.S.; Gubbins, S. The spread of bluetongue virus serotype 8 in Great Britain and its control by vaccination. PLoS ONE 2010, 5, e9353. [Google Scholar] [CrossRef] [Green Version]
  22. Rushton, J.; Lyons, N. Economic impact of Bluetongue: A review of the effects on production. Vet. Ital. 2015, 51, 401–406. [Google Scholar] [CrossRef]
  23. Sok, J.; Hogeveen, H.; Elbers, A.R.; Velthuis, A.G.; Oude Lansink, A.G. Expected utility of voluntary vaccination in the middle of an emergent Bluetongue virus serotype 8 epidemic: A decision analysis parameterized for Dutch circumstances. Prev. Vet. Med. 2014, 115, 75–87. [Google Scholar] [CrossRef] [PubMed]
  24. Velthuis, A.G.; Mourits, M.C.; Saatkamp, H.W.; de Koeijer, A.A.; Elbers, A.R. Financial evaluation of different vaccination strategies for controlling the bluetongue virus serotype 8 epidemic in The Netherlands in 2008. PLoS ONE 2011, 6, e19612. [Google Scholar] [CrossRef]
  25. Velthuis, A.G.; Saatkamp, H.W.; Mourits, M.C.; de Koeijer, A.A.; Elbers, A.R. Financial consequences of the Dutch bluetongue serotype 8 epidemics of 2006 and 2007. Prev. Vet. Med. 2010, 93, 294–304. [Google Scholar] [CrossRef] [PubMed]
  26. Maan, S.; Maan, N.S.; Ross-smith, N.; Batten, C.A.; Shaw, A.E.; Anthony, S.J.; Samuel, A.R.; Darpel, K.E.; Veronesi, E.; Oura, C.A.; et al. Sequence analysis of bluetongue virus serotype 8 from the Netherlands 2006 and comparison to other European strains. Virology 2008, 377, 308–318. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  27. Sanchez-Cordon, P.J.; Perez de Diego, A.C.; Gomez-Villamandos, J.C.; Sanchez-Vizcaino, J.M.; Pleguezuelos, F.J.; Garfia, B.; del Carmen, P.; Pedrera, M. Comparative analysis of cellular immune responses and cytokine levels in sheep experimentally infected with bluetongue virus serotype 1 and 8. Vet. Microbiol. 2015, 177, 95–105. [Google Scholar] [CrossRef] [PubMed]
  28. Darpel, K.E.; Batten, C.A.; Veronesi, E.; Williamson, S.; Anderson, P.; Dennison, M.; Clifford, S.; Smith, C.; Philips, L.; Bidewell, C.; et al. Transplacental transmission of bluetongue virus 8 in cattle, UK. Emerg Infect. Dis. 2009, 15, 2025–2028. [Google Scholar] [CrossRef]
  29. Darpel, K.E.; Batten, C.A.; Veronesi, E.; Shaw, A.E.; Anthony, S.; Bachanek-Bankowska, K.; Kgosana, L.; bin-Tarif, A.; Carpenter, S.; Muller-Doblies, U.U.; et al. Clinical signs and pathology shown by British sheep and cattle infected with bluetongue virus serotype 8 derived from the 2006 outbreak in northern Europe. Vet. Rec. 2007, 161, 253–261. [Google Scholar] [CrossRef] [Green Version]
  30. Sanchez-Cordon, P.J.; Pleguezuelos, F.J.; de Diego, A.C.P.; Gomez-Villamandos, J.C.; Sanchez-Vizcaino, J.M.; Ceron, J.J.; Tecles, F.; Garfia, B.; Pedrera, M. Comparative study of clinical courses, gross lesions, acute phase response and coagulation disorders in sheep inoculated with bluetongue virus serotype 1 and 8. Vet. Microbiol. 2013, 166, 184–194. [Google Scholar] [CrossRef]
  31. Dal Pozzo, F.; Martinelle, L.; Thys, C.; Sarradin, P.; De Leeuw, I.; Van Campe, W.; De Clercq, K.; Thiry, E.; Saegerman, C. Experimental co-infections of calves with bluetongue virus serotypes 1 and 8. Vet. Microbiol. 2013, 165, 167–172. [Google Scholar] [CrossRef] [Green Version]
  32. Nayduch, D.; Cohnstaedt, L.W.; Saski, C.; Lawson, D.; Kersey, P.; Fife, M.; Carpenter, S. Studying Culicoides vectors of BTV in the post-genomic era: Resources, bottlenecks to progress and future directions. Virus Res. 2014, 182, 43–49. [Google Scholar] [CrossRef] [Green Version]
  33. Wechsler, S.J.; McHolland, L.E.; Tabachnick, W.J. Cell lines from Culicoides variipennis (Diptera: Ceratopogonidae) support replication of bluetongue virus. J. Invertebr. Pathol. 1989, 54, 385–393. [Google Scholar] [CrossRef]
  34. Veronesi, E.; Antony, F.; Gubbins, S.; Golding, N.; Blackwell, A.; Mertens, P.P.; Brownlie, J.; Darpel, K.E.; Mellor, P.S.; Carpenter, S. Measurement of the infection and dissemination of bluetongue virus in Culicoides biting midges using a semi-quantitative rt-PCR assay and isolation of infectious virus. PLoS ONE 2013, 8, e70800. [Google Scholar] [CrossRef] [PubMed]
  35. Finnay, D.J. Statistical Methods in Biological Assay; Charles Griffin & Co. Ltd.: London, UK, 1964. [Google Scholar]
  36. Darpel, K.E.; Barber, J.; Hope, A.; Wilson, A.J.; Gubbins, S.; Henstock, M.; Frost, L.; Batten, C.; Veronesi, E.; Moffat, K.; et al. Using shared needles for subcutaneous inoculation can transmit bluetongue virus mechanically between ruminant hosts. Sci. Rep. 2016, 6, 20627. [Google Scholar] [CrossRef] [Green Version]
  37. Shaw, A.E.; Monaghan, P.; Alpar, H.O.; Anthony, S.; Darpel, K.E.; Batten, C.A.; Guercio, A.; Alimena, G.; Vitale, M.; Bankowska, K.; et al. Development and initial evaluation of a real-time RT-PCR assay to detect bluetongue virus genome segment 1. J. Virol. Methods 2007, 145, 115–126. [Google Scholar] [CrossRef] [PubMed]
  38. Maan, S.; Maan, N.S.; Belaganahalli, M.N.; Potgieter, A.C.; Kumar, V.; Batra, K.; Wright, I.M.; Kirkland, P.D.; Mertens, P.P.C. Development and Evaluation of Real Time RT-PCR Assays for Detection and Typing of Bluetongue Virus. PLoS ONE 2016, 11, e0163014. [Google Scholar] [CrossRef] [Green Version]
  39. R Core Team. R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2019; Available online: https://www.R-Project.org/ (accessed on 5 June 2020).
  40. Samal, S.K.; el-Hussein, A.; Holbrook, F.R.; Beaty, B.J.; Ramig, R.F. Mixed infection of Culicoides variipennis with bluetongue virus serotypes 10 and 17: Evidence for high frequency reassortment in the vector. J. Gen. Virol. 1987, 68, 2319–2329. [Google Scholar] [CrossRef]
  41. Hope, A.; Gubbins, S.; Sanders, C.; Barber, J.; Stubbins, F.; Baylis, M.; Carpenter, S. Sheep breed and shearing influences attraction and blood-feeding behaviour of Culicoides (Diptera: Ceratopogonidae) on a UK farm. Parasites Vectors 2018, 11, 11. [Google Scholar] [CrossRef]
  42. Carpenter, S.; Szmaragd, C.; Barber, J.; Labuschagne, K.; Gubbins, S.; Mellor, P. An assessment of Culicoides surveillance techniques in northern Europe: Have we underestimated a potential bluetongue virus vector? J. Appl. Ecol. 2008, 45, 1237–1245. [Google Scholar] [CrossRef]
  43. Viennet, E.; Garros, C.; Gardes, L.; Rakotoarivony, I.; Allene, X.; Lancelot, R.; Crochet, D.; Moulia, C.; Baldet, T.; Balenghien, T. Host preferences of Palaearctic Culicoides biting midges: Implications for transmission of orbiviruses. Med. Vet. Entomol. 2013, 27, 255–266. [Google Scholar] [CrossRef]
  44. Elbers, A.R.W.; Meiswinkel, R. Culicoides (Diptera: Ceratopogonidae) and livestock in the Netherlands: Comparing host preference and attack rates on a Shetland pony, a dairy cow, and a sheep. J. Vector Ecol. 2015, 40, 308–317. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Baylis, M.; O’Connell, L.; Mellor, P.S. Rates of bluetongue virus transmission between Culicoides sonorensis and sheep. Med. Vet. Entomol. 2008, 22, 228–237. [Google Scholar] [CrossRef]
  46. Foster, N.M.; Jones, R.H.; Luedke, A.J. Transmission of attenuated and virulent bluetongue virus with Culicoides variipennis infected orally via sheep. Am. J. Vet. Res. 1968, 29, 275. [Google Scholar]
  47. Caporale, M.; Di Gialleonorado, L.; Janowicz, A.; Wilkie, G.; Shaw, A.; Savini, G.; Van Rijn, P.A.; Mertens, P.; Di Ventura, M.; Palmarini, M. Virus and host factors affecting the clinical outcome of bluetongue virus infection. J. Virol. 2014, 88, 10399–10411. [Google Scholar] [CrossRef] [Green Version]
  48. Jennings, D.M.; Mellor, P.S. The vector potential of british Culicoides species for bluetongue virus. Vet. Microbiol. 1988, 17, 1–10. [Google Scholar] [CrossRef]
  49. Paslaru, A.I.; Mathis, A.; Torgerson, P.; Veronesi, E. Vector competence of pre-alpine Culicoides (Diptera: Ceratopogonidae) for bluetongue virus serotypes 1, 4 and 8. Parasites Vectors 2018, 11, 466. [Google Scholar] [CrossRef]
  50. Carpenter, S.; McArthur, C.; Selby, R.; Ward, R.; Nolan, D.V.; Luntz, A.J.; Dallas, J.F.; Tripet, F.; Mellor, P.S. Experimental infection studies of UK Culicoides species midges with bluetongue virus serotypes 8 and 9. Vet. Rec. 2008, 163, 589–592. [Google Scholar] [CrossRef]
  51. Melzi, E.; Caporale, M.; Rocchi, M.; Martin, V.; Gamino, V.; di Provvido, A.; Marruchella, G.; Entrican, G.; Sevilla, N.; Palmarini, M. Follicular dendritic cell disruption as a novel mechanism of virus-induced immunosuppression. Proc. Natl. Acad. Sci. USA 2016, 113, E6238–E6247. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  52. Jacquot, M.; Rao, P.P.; Yadav, S.; Nomikou, K.; Maan, S.; Jyothi, Y.K.; Reddy, N.; Putty, K.; Hemadri, D.; Singh, K.P.; et al. Contrasting selective patterns across the segmented genome of bluetongue virus in a global reassortment hotspot. Virus Evol. 2019, 5, 14. [Google Scholar] [CrossRef]
  53. Schirtzinger, E.E.; Jasperson, D.C.; Ostlund, E.N.; Johnson, D.J.; Wilson, W.C. Recent US bluetongue virus serotype 3 isolates found outside of Florida indicate evidence of reassortment with co-circulating endemic serotypes. J. Gen. Virol. 2018, 99, 157–168. [Google Scholar] [CrossRef] [PubMed]
  54. Samal, S.K.; Livingston, C.W., Jr.; McConnell, S.; Ramig, R.F. Analysis of mixed infection of sheep with bluetongue virus serotypes 10 and 17: Evidence for genetic reassortment in the vertebrate host. J. Virol. 1987, 61, 1086–1091. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  55. Oberst, R.D.; Stott, J.L.; Blanchardchannell, M.; Osburn, B.I. Genetic reassortment of bluetongue virus serotype 11 in the bovine. Vet. Microbiol. 1987, 15, 11–18. [Google Scholar] [CrossRef]
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Figure 1. Bluetongue virus (BTV) infection of sheep via blood feeding of infected Culicoides sonorensis. C. sonorensis were infected with BTV by IT inoculation and following an incubation period of 5–6 days at 25 °C allowed to feed directly on the inner thigh of a sheep as described in the methodology. Each sheep was simultaneously exposed to one pot containing C. sonorensis infected with BTV serotype 1 and one pot containing C. sonorensis infected with BTV serotype 8 for 10 min. The sheep was placed onto its rear and held in the sitting position (similar to the position adopted for shearing).
Figure 1. Bluetongue virus (BTV) infection of sheep via blood feeding of infected Culicoides sonorensis. C. sonorensis were infected with BTV by IT inoculation and following an incubation period of 5–6 days at 25 °C allowed to feed directly on the inner thigh of a sheep as described in the methodology. Each sheep was simultaneously exposed to one pot containing C. sonorensis infected with BTV serotype 1 and one pot containing C. sonorensis infected with BTV serotype 8 for 10 min. The sheep was placed onto its rear and held in the sitting position (similar to the position adopted for shearing).
Microorganisms 08 00851 g001
Microorganisms 08 00851 g002 550
Figure 2. Levels of viremia (measured by rRT-PCR) in sheep co-infected with BTV-1 and BTV-8. Each plot shows the Cq values for the detection assay (black circles), the BTV-1 typing assay (red down-triangles), and the BTV-8 typing assay (blue up-triangles). The grey shaded areas indicate when uninfected Culicoides sonorensis were fed on the sheep.
Figure 2. Levels of viremia (measured by rRT-PCR) in sheep co-infected with BTV-1 and BTV-8. Each plot shows the Cq values for the detection assay (black circles), the BTV-1 typing assay (red down-triangles), and the BTV-8 typing assay (blue up-triangles). The grey shaded areas indicate when uninfected Culicoides sonorensis were fed on the sheep.
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Figure 3. Daily clinical score of sheep co-infected with two BTV strains. The daily cumulative score across 11 clinical criteria are shown for each sheep (S1–S4). Clinical progression was markedly more rapid and severe in sheep 1 and 3 and the total clinical score accumulated on a single day was a clear indicator for overall severity requiring euthanasia (*). Sheep 2 and 4 also developed disease of overall moderate severity, but this was more reflected in the duration and chronic manifestation (ulcers, feet pathology) without affecting any behavior or feeding responses.
Figure 3. Daily clinical score of sheep co-infected with two BTV strains. The daily cumulative score across 11 clinical criteria are shown for each sheep (S1–S4). Clinical progression was markedly more rapid and severe in sheep 1 and 3 and the total clinical score accumulated on a single day was a clear indicator for overall severity requiring euthanasia (*). Sheep 2 and 4 also developed disease of overall moderate severity, but this was more reflected in the duration and chronic manifestation (ulcers, feet pathology) without affecting any behavior or feeding responses.
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Figure 4. Clinical manifestation of BTV co-infection in Dorset Poll sheep. All four sheep developed typical clinical signs indicative of BTV infection, such as reddening of the eyes and mucosal membranes, facial edema, nasal discharge, and salivation. However, clinical progression was quicker and more pronounced in sheep 1 and 3 (e.g., Panel A, sheep 3) and both sheep were euthanized at 7 dpi on reaching the humane endpoints of the study protocol. Sheep 2 and 4 developed moderate clinical BT (e.g., panel B sheep 2) and developed more chronic disease features, such as ulcers and petechial bleeding of the nostrils, later in infection (12–16 dpi).
Figure 4. Clinical manifestation of BTV co-infection in Dorset Poll sheep. All four sheep developed typical clinical signs indicative of BTV infection, such as reddening of the eyes and mucosal membranes, facial edema, nasal discharge, and salivation. However, clinical progression was quicker and more pronounced in sheep 1 and 3 (e.g., Panel A, sheep 3) and both sheep were euthanized at 7 dpi on reaching the humane endpoints of the study protocol. Sheep 2 and 4 developed moderate clinical BT (e.g., panel B sheep 2) and developed more chronic disease features, such as ulcers and petechial bleeding of the nostrils, later in infection (12–16 dpi).
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Figure 5. Hemorrhages observed across different organs in BTV co-infected sheep. BTV-specific pathological features observed in sheep 1 and 3 euthanized at day 7 post-infection included hemorrhages, petechial bleeding, and ulceration in the oral mucosa membrane (A), tongue (B), pre-scapular lymph node (C), and pulmonary artery (D).
Figure 5. Hemorrhages observed across different organs in BTV co-infected sheep. BTV-specific pathological features observed in sheep 1 and 3 euthanized at day 7 post-infection included hemorrhages, petechial bleeding, and ulceration in the oral mucosa membrane (A), tongue (B), pre-scapular lymph node (C), and pulmonary artery (D).
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Table
Table 1. Number of intrathoracically (IT) inoculated Culicoides sonorensis blood fed on sheep. Each C. sonorensis was inoculated with either a bluetongue virus serotype 1 (BTV-1) or BTV-8 strain and then incubated in pill boxes at 25 °C for five days prior to feeding on sheep.
Table 1. Number of intrathoracically (IT) inoculated Culicoides sonorensis blood fed on sheep. Each C. sonorensis was inoculated with either a bluetongue virus serotype 1 (BTV-1) or BTV-8 strain and then incubated in pill boxes at 25 °C for five days prior to feeding on sheep.
Sheep NumberTotal Inoculated Culicoides ExposedCulicoides Fully EngorgedCulicoides Partially EngorgedTotal Blood Fed (% Total Exposed)Number ≤ 25 Cq * (% Total Blood-Fed on Sheep)
BTV-1 MOR2007/0115012517 (34)16 (52)
BTV-8 NET2006/0615911415 (25)15 (48)
BTV-1 MOR2007/01271145 (7)5 (33)
BTV-8 NET2006/062685510 (15)10 (67)
BTV-1 MOR2007/0135814115 (26)13 (68)
BTV-8 NET2006/06384516 (7)6 (32)
BTV-1 MOR2007/01411216723 (21)23 (72)
BTV-8 NET2006/06473369 (12)9 (28)
Total-5756733100 (17)-
* rRT-PCR Cq value ≤ 25 indicates C. sonorensis containing a fully disseminated infection with bluetongue virus [27].
Table
Table 2. Pathology score of all sheep at time point of euthanasia. Sheep 1 and 3 were euthanized as they exceeded clinical limits for the study at day 7 post-infection with bluetongue virus (BTV). Clinical signs exhibited by sheep 2 and 4 were observed to the end of the study (day 16 post infection with BTV).
Table 2. Pathology score of all sheep at time point of euthanasia. Sheep 1 and 3 were euthanized as they exceeded clinical limits for the study at day 7 post-infection with bluetongue virus (BTV). Clinical signs exhibited by sheep 2 and 4 were observed to the end of the study (day 16 post infection with BTV).
Sheep 1 Sheep 2 Sheep 3 Sheep 4
Day of necropsy7 dpiEnd of Study7 dpiEnd of Study
Clinical Scoring
Edema2020
Face2020
Body0000
Lung0000
Body cavities0000
Hemorrhage1210146
Lymph nodes (Pre-scapular + Pharyngeal + Femoral) + tonsil1 + 3 + 0 + 32 + 2 + 0 + 22 + 3 + 0 + 31 + 3 + 0 + 2
Mucosal membranes3130
Tongue2130
Muscles and subcutaneous0000
Heart vessels0200
Ulcer4213
Mouth/Nose22 (mouth and nose 1)12
Feet2001
Pneumonia0000
Total Score1812179
Table
Table 3. Culicoides sonorensis blood feeding on sheep co-infected with BTV-1 and BTV-8. C. sonorensis were fed at day 6 and day 7 post infection of sheep with bluetongue virus and then stored for 8 days at 25 °C prior to processing.
Table 3. Culicoides sonorensis blood feeding on sheep co-infected with BTV-1 and BTV-8. C. sonorensis were fed at day 6 and day 7 post infection of sheep with bluetongue virus and then stored for 8 days at 25 °C prior to processing.
Sheep (Day)Sheep BTV Detection Assay CqSheep BTV-1 Typing Assay CqSheep BTV-8 Typing Assay CqSheep Virus ISOLATION (Log10TCID50)Culicoides sonorensis (n) Fed on SheepC. sonorensis Positive on Detection Assay (% total) Following 8-Day Incubation *Positive C. sonorensis BTV Typing Assays for BTV-1; BTV-8
1 (6)17.4917.87 27.37 ----
1 (7)17.0717.4828.475.75886 (6.8); 65 BTV-1; 1 BTV-1 +BTV-8
2 (6)24.08 24.61 28.4708000
2 (7)17.5617.1129.726.03200
3 (6)19.5119.7230.496.01127 (6.2); 6All BTV-1
3 (7)20.9719.1534.365.0967 (7.3); 5All BTV-1
4 (6)24.9325.7529.75017600
4 (7)27.1026.5332.3601042 (1.9); 0All BTV-8
* rRT-PCR Cq value ≤25 indicates C. sonorensis fully disseminated infection [27].

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Veronesi, E.; Darpel, K.; Gubbins, S.; Batten, C.; Nomikou, K.; Mertens, P.; Carpenter, S. Diversity of Transmission Outcomes Following Co-Infection of Sheep with Strains of Bluetongue Virus Serotype 1 and 8. Microorganisms 2020, 8, 851. https://doi.org/10.3390/microorganisms8060851

AMA Style

Veronesi E, Darpel K, Gubbins S, Batten C, Nomikou K, Mertens P, Carpenter S. Diversity of Transmission Outcomes Following Co-Infection of Sheep with Strains of Bluetongue Virus Serotype 1 and 8. Microorganisms. 2020; 8(6):851. https://doi.org/10.3390/microorganisms8060851

Chicago/Turabian Style

Veronesi, Eva, Karin Darpel, Simon Gubbins, Carrie Batten, Kyriaki Nomikou, Peter Mertens, and Simon Carpenter. 2020. "Diversity of Transmission Outcomes Following Co-Infection of Sheep with Strains of Bluetongue Virus Serotype 1 and 8" Microorganisms 8, no. 6: 851. https://doi.org/10.3390/microorganisms8060851

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