Simbu Serogroup Research Articles

The N-terminal domain of Schmallenberg virus envelope protein Gc is highly immunogenic and can provide protection from infection

Schmallenberg virus (SBV) is transmitted by insect vectors, and therefore vaccination is one of the most important tools of disease control. In our study, novel subunit vaccines on the basis of an amino-terminal domain of SBV Gc of 234 amino acids (“Gc Amino”) first were tested and selected using a lethal small animal challenge model and then the best performing formulations also were tested in cattle. We could show that neither E. coli expressed nor the reduced form of “Gc Amino” protected from SBV infection. In contrast, both, immunization with “Gc Amino”-encoding DNA plasmids and “Gc-amino” expressed in a mammalian system, conferred protection in up to 66% of the animals. Interestingly, the best performance was achieved with a multivalent antigen containing the covalently linked Gc domains of both, SBV and the related Akabane virus. All vaccinated cattle and mice were fully protected against SBV challenge infection. Furthermore, in the absence of antibodies against the viral N-protein, differentiation between vaccinated and field-infected animals allows an SBV marker vaccination concept. Moreover, the presented vaccine design also could be tested for other members of the Simbu serogroup and might allow the inclusion of additional immunogenic domains.

Schmallenberg Virus: A Novel Virus of Veterinary Importance.

In late 2011, unspecific clinical symptoms such as fever, diarrhea, and decreased milk production were observed in dairy cattle in the Dutch/German border region. After exclusion of classical endemic and emerging viruses by targeted diagnostic systems, blood samples from acutely diseased cows were subjected to metagenomics analysis. An insect-transmitted orthobunyavirus of the Simbu serogroup was identified as the causative agent and named Schmallenberg virus (SBV). It was one of the first detections of the introduction of a novel virus of veterinary importance to Europe using the new technology of next-generation sequencing. The virus was subsequently isolated from identical samples as used for metagenomics analysis in insect and mammalian cell lines and disease symptoms were reproduced in calves experimentally infected with both, this culture-grown virus and blood samples of diseased cattle. Since its emergence, SBV spread very rapidly throughout the European ruminant population causing mild unspecific disease in adult animals, but also premature birth or stillbirth and severe fetal malformation when naive dams were infected during a critical phase of gestation. In the following years, SBV recirculated regularly to a larger extend; in the 2014 and 2016 vector seasons the virus was again repeatedly detected in the blood of adult ruminants, and in the following winter and spring months, a number of malformed calves and lambs was born. The genome of viruses present in viremic adult animals showed a very high sequence stability; in sequences generated between 2012 and 2016, only a few amino acid substitutions in comparison to the initial SBV isolate could be detected. In contrast, a high sequence variability was identified in the aminoterminal part of the glycoprotein Gc-encoding region of viruses present in the brain of malformed newborns. This mutation hotspot is independent of the region or host species from which the samples originated and is potentially involved in immune evasion mechanisms.

Schmallenberg virus: on its way out or due for a comeback?

SCHMALLENBERG virus (SBV) emerged during the summer of 2011 in north-western Europe and was identified for the first time in dairy cattle herds in Germany (Hoffmann and others 2012). The...

Effective interference between Simbu serogroup orthobunyaviruses in mammalian cells

The Simbu serogroup of orthobunyaviruses comprises a wide range of viruses with different medical and veterinary relevance. These viruses are known to reassort, and coinfection of the same cell is one of the prerequisites for reassortment. Here, a mammalian cell line was infected with various members of this virus group, inoculated after several time points with a second Simbu serogroup virus, and analyzed by strain or species specific immunofluorescence staining. Different virus species or different strains of the same virus species were able to co-infect mammalian cells, but only for a limited time frame. After a few hours, the replication of the first virus led to a gradual inhibition of a second virus until a complete resistance to superinfection after 24h regardless whether it is another strain of the same virus species or a distinct member of the serogroup.

What can Akabane disease teach us about other arboviral diseases.

Viruses of the Simbu serogroup cause lesions to foetuses that are seen at birth and that correlate with the stage of pregnancy at which the dam first contracts the virus. The Simbu serogroup comprises arboviruses known to cause outbreaks of abnormal parturitions in domestic ruminants; these abnormalities include abortion, stillbirth, and congenitally deformed neonates. Simbu serogroup members include: Akabane virus (AKAV), Aino virus, Cache Valley virus, and Schmallenberg virus. Lately, dairy herds calf malformations have been observed in Europe, where there have been reports of clinical manifestations such as diarrhoea, fever, and reduced milk yield in adult lactating cows. The Israeli dairy cattle industry has experienced 2 major episodes of abnormal parturitions that resulted from 2 arboviral Simbu serogroup episodes, which occurred 35 years apart. A wave of apparently newly introduced AKAV was noted from the beginning of January 2012. Investigations carried out throughout the period of late Summer 2011 to early Winter 2012, associated the Israeli AKAV strain with central nervous system manifestations in lactating cows. A lack of clinical/epidemiological 'uniformity' among the AKAV infections was noted during these investigations. Here we describe and discuss the clinical and spatial distribution differences found among the 3 above-mentioned outbreaks. Comparable features in the clinical presentation, spatial distribution, and target‑animal issues relating to Akabane disease are discussed.

Isolation and characterization of Oya virus a member of Simbu serogroup, family Bunyaviridae, isolated from Karnataka, India

During a study on Japanese encephalitis (JE) from Kolar district of Karnataka state, India in 1986; two virus isolates were obtained in infant Swiss albino mouse from a pig and a human serum sample. For characterization of these virus isolates, they were propagated in Vero CCL-81 cells. These virus isolates were screened for flaviviruses (Japanese encephalitis, West Nile, Dengue, Kyasanur forest disease) and Alphavirus (Chikungunya) by RT-PCR and found to be negative. Further these they were screened for bunyaviruses using genus-specific primers. A virus isolate from a human sample was sequenced using next generation sequencing; which identified it as Oya virus, Simbu group of the genus Orthobunyavirus of the family Bunyaviridae. Phylogenetic analysis of L, M, S (N and NSs) revealed its close association with Chinese strain of Oya virus in Simbu serogroup with the distance of 6.5>4.2>3.2% for nucleotides and 2.4>0.8>0.0% for the amino acid of L>M>S segments respectively. Based on the PCR results; an isolate from pig sample was also confirmed as Oya virus. This study was strengthened by findings of IgG antibody positivity against Oya virus in retrospective serum samples of suspected febrile illness cases from this area by an indigenously developed ELISA. Oya virus positivity was also recorded in human samples collected from Karnataka using nested RT-PCR. This is the first report of the presence of Oya virus in human samples. Further studies are needed to determine disease-causing potential in humans.

Activation of c-Jun N-terminal kinase by Akabane virus is required for apoptosis

Akabane virus (AKAV) belongs to the Simbu serogroup of the genus Orthobunyavirus in the family Bunyaviridae. It has been shown that AKAV induces apoptosis in mammalian cells. It is necessary to understand the signaling pathways involved in AKAV-induced apoptosis to further elucidate the molecular virology of AKAV. c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) are mediators of apoptosis; therefore, we investigated the roles of JNK and p38 MAPK cascades in AKAV-infected cells. We found that JNK and p38 MAPK as well as their downstream substrates, c-Jun and heat shock protein 27 (HSP27), were phosphorylated in response to AKAV infection. A JNK inhibitor (SP600125) inhibited AKAV-mediated apoptosis whereas a p38 MAPK inhibitor (SB203580) did not. We conclude that AKAV infection activates the JNK and p38 MAPK signaling pathways, and the JNK cascade plays a crucial role in AKAV-induced apoptosis in vitro.

Schmallenberg Virus Infection Diagnosis: Results of a German Proficiency Trial.

Since Schmallenberg virus (SBV), an orthobunyavirus of the Simbu serogroup, was detected in Central Europe in 2011 for the first time, numerous diagnostic test systems for genome or antibody detection have been established. Therefore, a laboratory proficiency trial with 28 veterinary laboratories was initiated to allow performance evaluations of the different veterinary diagnostic laboratories and the performance of the used assays. A panel of selected sera and bovine semen samples for the analysis by real-time PCR and an additional set of serum samples for serological analysis were provided. All participants were asked to investigate the samples with the test systems routinely used in their laboratory. While SBV-genome was reliably detected in serum samples, the sensitivity in semen samples seems to depend on the application of the recommended optimized nucleic acid extraction method (TRIzol® LS Reagent-based, Hoffmann etal., 2013, Vet. Microbiol., 167, 289). SBV-antibody-positive samples and sera negative for antibodies against Simbu serogroup viruses were in most cases correctly classified by the participants with the used commercial ELISA kits. However, a serum of the panel which contained antibodies against Akabane and Aino viruses, which are closely related to SBV, was repeatedly tested positive by two of four used ELISA kits. However, an excellent diagnostic sensitivity and specificity was achieved using a serum neutralization test. In conclusion, the here described German SBV proficiency test demonstrated that the available test systems allowed reliable SBV diagnostics in standard veterinary laboratories when recommended and approved assays are used.

Serological screening for Schmallenberg virus in exotic and indigenous cattle in Nigeria

Schmallenberg virus (SBV), a recently emerged Orthobunyavirus, is associated with abortions, stillbirths and congenital malformations in ruminants. Considering that Culicoides species which transmit this disease have previously been identified in Nigeria as vectors of bluetongue, another livestock disease that causes abortions, it is speculated that SBV also circulates in the Nigerian ruminant population. We therefore conducted a pilot study to investigate the occurrence of anti-SBV antibodies in a limited population of cross-breed, exotic and indigenous cattle in Nigeria. Serum samples obtained from 60 Friesian-White Fulani (FWF), 7 Jersey and 53 indigenous cattle were screened for SBV antibodies using a commercial indirect ELISA kit that detects antibodies against recombinant SBV nucleoprotein in ruminant sera. An overall seropositivity of 29.2% (35/120) was obtained with antibodies being detectable in 23.3% FWF (14/60), 42.9% Jerseys (3/7) and 34.0% (18/53) indigenous cattle. All indigenous breeds of cattle tested had seropositive animals: White Fulani (13/38, 34.2%), Sokoto Gudali (2/8, 25.0%), Red Bororo (1/5, 20.0%) and Bunaji (2/2, 100.0%). The detection of antibody-positive animals among unvaccinated cattle provides evidence of possible SBV infection in Nigeria. However, there is also the probability of cross-reactivity with other Simbu serogroup viruses, especially considering that some of these viruses have previously been reported in Nigeria. Further studies to confirm these preliminary findings using serum neutralisation assay, viral isolation or detection of SBV RNA from ruminants or Culicoides are necessary.Keywords: Abortions, Antibodies, Cattle, Congenital malformations, Nigeria, Schmallenberg virus

Circulation of a Simbu Serogroup Virus, Causing Schmallenberg Virus-Like Clinical Signs in Northern Jordan.

Schmallenberg virus (SBV)-like clinical cases of abortions in northern Jordan in early 2013, together with the emergence of SBV in Europe in 2011, its rapid spread within the following years and the detection of this virus in Turkey, raised questions about the distribution of SBV or related orthobunyaviruses. To evaluate the occurrence of SBV or related members of the Simbu serogroup of orthobunyaviruses in Jordan, bulk milk (cattle) and serum samples (cattle, sheep and goat) collected in northern Jordan in 2013 were first tested by commercially available SBV antibody ELISAs. Indeed, 3 of 47 bulk milk samples and 57 of 115 serum samples provided positive results, but SBV specificity of the ELISA results could not be confirmed by virus neutralization assays. Instead, subsequent cross-neutralization tests were able to further investigate the specificity of these antibodies. Here, a significant inhibition of Aino virus was observed. Thus, the causative agent was most likely a Simbu serogroup virus closely related to Aino virus. Consequently, these results confirm that members of this group of virus are not only present in Europe, Africa or Australia, but also in the Middle East.

Detection and differentiation of Schmallenberg, Akabane and Aino viruses by one-step multiplex reverse-transcriptase quantitative PCR assay.

BackgroundSchmallenberg virus (SBV), Akabane virus (AKAV) and Aino virus (AINV) are members of the Simbu serogroup within the genus Orthobunyavirus, family Bunyaviridae, which can cause reproductive disorders including abortion, stillbirth and congenital malformation in ruminants. Because, the clinical signs are similar, confirmatory diagnosis requires viral detection to differentiate infection between these three viruses.MethodsIn this study, a one-step multiplex reverse-transcriptase quantitative PCR (one-step mRT-qPCR) was developed for the simultaneous detection and differentiation of SBV, AKAV and AINV.ResultsThe detection limit of the one-step mRT-qPCR for SBV, AKAV and AINV were 2.4 copies (10 0.6 TCID 50/ml), 96.2 copies (10 1.5 TCID 50/ml) and 52.3 copies (10 1.2 TCID 50/ml), respectively. Various field samples such as bovine serum, bovine whole blood, bovine brain, goat serum and Culicoides were analyzed using the one-step mRT-qPCR and compared with previously published RT-qPCRs. The test results of the field samples were identical for the one-step mRT-qPCR and RT-qPCRs, which showed all samples to be negative for SBV, AKAV and AINV, except for one bovine brain sample (1/123) that was positive for AKAV.ConclusionThe one-step mRT-qPCR allows for the simultaneous detection of three viral pathogens (SBV, AKAV and AINV) that cause reproductive failure.

Broad-range detection of arboviruses belonging to Simbu serogroup lineage 1 and specific detection of Akabane, Aino and Peaton viruses by newly developed multiple TaqMan assays

TaqMan assays were developed for the broad-range detection of arboviruses belonging to Simbu serogroup lineage 1 in the genus Orthobunyavirus and also for the specific detection of three viruses in the lineage, Akabane, Aino and Peaton viruses (AKAV, AINOV and PEAV, respectively). A primer and probe set was designed for the broad-range detection of Simbu serogroup lineage 1 (Pan-Simbu1 set) mainly targeting AKAV, AINOV, PEAV, Sathuperi and Shamonda viruses (SATV and SHAV), and the forward and reverse primers of the Pan-Simbu1 set were also used for the specific detection of AKAV with another probe (AKAV-specific set). In addition, two more primer and probe sets were designed for AINOV- and PEAV-specific detection, respectively (AINOV- and PEAV-specific sets). All of the four primer and probe sets successfully detected targeted viruses, and thus broad-range and specific detection of all the targeted viruses can be achieved by using two multiplex assays and a single assay in a dual (two-color) assay format when another primer and probe set for a bovine β-actin control is also used. The assays had an analytical sensitivity of 10 copies/tube for AKAV, at least 100 copies/tube for AINOV, 100 copies/tube for PEAV, one copy/tube for SATV and at least 10 copies/tube for SHAV, respectively. Diagnostic sensitivity of the assays was tested with field-collected bovine samples, and the results suggested that the sensitivity was higher than that of a conventional RT-PCR. These data indicate that the newly developed TaqMan assays will be useful tools for the diagnosis and screening of field-collected samples for infections of AKAV and several other arboviruses belonging to the Simbu serogroup lineage 1.

Detection of Oropouche virus segment S in patients and inCulex quinquefasciatus in the state of Mato Grosso, Brazil.

This study aimed to investigate the circulation of Orthobunyavirus species in the state of Mato Grosso (MT) Brazil. During a dengue outbreak in 2011/2012, 529 serum samples were collected from patients with acute febrile illness with symptoms for up to five days and 387 pools of female Culex quinquefasciatus captured in 2013 were subjected to nested-reverse transcription-polymerase chain reaction for segment S of the Simbu serogroup followed by nucleotide sequencing and virus isolation in Vero cells. Patients (5/529; 0.9%) from Cuiabá (n = 3), Várzea Grande (n = 1) and Nova Mutum (n = 1) municipalities were positive for the S segment of Oropouche virus (OROV). Additionally, eight/387 Cx. quinquefasciatus pools were positive for the segment, with a minimum infection rate of 2.3. Phylogenetic analysis indicated that all the samples belong to the subgenotype Ia, presenting high homology with OROV strains obtained from humans and animals in the Brazilian Amazon. The present paper reports the first detection of an Orthobunyavirus, possibly OROV, in patients and in Cx. quinquefasciatus mosquitoes in MT. This finding reinforces the notion that arboviruses frequently reported in the Amazon Region circulate sporadically in MT during dengue outbreaks.

Detection of serum neutralizing antibodies to Simbu sero-group viruses in cattle in Tanzania.

BackgroundOrthobunyaviruses belonging to the Simbu sero-group occur worldwide, including the newly recognized Schmallenberg virus (SBV) in Europe. These viruses cause congenital malformations and reproductive losses in ruminants. Information on the presence of these viruses in Africa is scarce and the origin of SBV is unknown. The aim of this study was to investigate the presence of antibodies against SBV and closely related viruses in cattle in Tanzania, and their possible association with reproductive disorders.ResultsIn a cross-sectional study, serum from 659 cattle from 202 herds collected in 2012/2013 were analyzed using a commercial kit for SBV ELISA, and 61 % were positive. Univariable logistic regression revealed significant association between ELISA seropositivity and reproductive disorders (OR = 1.9). Sera from the same area collected in 2008/2009, before the SBV epidemic in Europe, were also tested and 71 (54.6 %) of 130 were positive. To interpret the ELISA results, SBV virus neutralization test (VNT) was performed on 110 sera collected in 2012/2013, of which 51 % were positive. Of 71 sera from 2008/2009, 21 % were positive. To investigate potential cross reactivity with related viruses, 45 sera from 2012/2013 that were positive in SBV ELISA were analyzed in VNTs for Aino, Akabane, Douglas, Peaton, Sabo, SBV, Sathuperi, Shamonda, Simbu and Tinaroo viruses. All 45 sera were positive for one or more of these viruses. Twenty-nine sera (64.4 %) were positive for SBV, and one had the highest titer for this virus.ConclusionsThis is the first indication that Aino, Akabane, Douglas, Peaton, Sabo, SBV, Sathuperi, Shamonda and Tinaroo viruses circulate and cause negative effect on reproductive performance in cattle in Tanzania. SBV or a closely related virus was present before the European epidemic. However, potential cross reactivity complicates the interpretation of serological studies in areas where several related viruses may circulate. Virus isolation and molecular characterization in cattle and/or vectors is recommended to further identify the viruses circulating in this region. However, isolation in cattle is difficult due to short viremic period of 2 to 6 days, and isolation in vectors does not necessarily reflect the situation in cattle.

Schmallenberg disease in sheep or goats: Past, present and future

Schmallenberg disease has emerged in North-Western Europe in 2011 and has since spread widely, even across the European borders. It has the potency to infect many, mainly ruminant, species, but seems to lack zoonotic potential. Horizontal transmission occurs through various Culicoides biting midges and subsequent trans-placental transmission causes teratogenic effects. In some small ruminants, clinical signs, including fever, decreased milk production and diarrhea occur during the viraemic phase, but infection is mostly asymptomatic. However, fetal Schmallenberg virus infection in naïve ewes and goats can result in stillborn offspring, showing a congenital arthrogryposis-hydranencephaly syndrome. The economic impact of infection depends on the number of malformed lambs, but is generally limited. There is debate on whether Schmallenberg virus has newly emerged or is re-emerging, since it is likely one of the ancestors of Shamonda virus, both Orthobunyaviruses belonging to the species Sathuperi virus within the Simbu serogroup viruses. Depending on the vector-borne transmission and the serologic status, future outbreaks of Schmallenberg disease induced congenital disease are expected.

Schmallenberg virus infection

Since Schmallenberg virus, an orthobunyavirus of the Simbu serogroup, was identified near the German-Dutch border for the first time in late 2011 it has spread extremely quickly and caused a large epidemic in European livestock. The virus, which is transmitted by Culicoides biting midges, infects domestic and wild ruminants. Adult animals show only mild clinical symptoms or none at all, whereas an infection during a critical period of gestation can lead to abortion, stillbirth or the birth of severely malformed offspring. The impact of the disease is usually greater in sheep than in cattle. Vaccination could be an important aspect of disease control.

Molecular Characterization and Seroprevalence in Pigs of SC0806, a Cat Que Virus Isolated from Mosquitoes in Sichuan Province, China.

The Simbu serogroup currently consists of a highly diverse group of related arboviruses that infect both humans and economically important livestock species. Cat Que virus (CQV), a Simbu serogroup virus of the genus Orthobunyavirus (family Bunyaviridae), was first isolated in 2004 from mosquitoes during surveillance of arbovirus activity in acute pediatric encephalitis in northern Vietnam. We report here the complete genome sequence of SC0806 isolated from mosquitoes (Culex tritaeniorhynchus) in Sichuan Province, China. Consistent with the genomic organization of Simbu serogroup viruses, the SC0806 genome comprises three RNA segments-a large (L) segment (6928 nucleotides) that encodes the 2261-amino-acid RNA-dependent RNA polymerase, a medium (M) segment (4481 nucleotides) that encodes the 1433-amino-acid polyprotein, and a small (S) segment (984 nucleotides) that encodes a 234-amino-acid nucleocapsid protein and a 95-amino-acid nonstructural protein. The respective lengths of the 5'-untranslated region (UTR) and 3'-UTR of L, M, and S are 56 and 86, 43 and 136, and 44 and 238 nucleotides. Sequence (nucleotide and deduced amino acid) comparison and phylogenetic analysis revealed that SC0806 was closely related to the reported Vietnam isolate CQV. This is the first time that CQV has been isolated in Sichuan Province, China. Anti-SC0806 immunoglobulin M (IgM) and IgG antibodies were found in pigs reared locally, indicating that CQV has formed a natural cycle in the local area. Surveillance of the distribution and pathogenicity of SC0806 should be strengthened.

Schmallenberg virus infection of ruminants: challenges and opportunities for veterinarians.

In 2011, European ruminant flocks were infected by Schmallenberg virus (SBV) leading to transient disease in adult cattle but abortions and congenital deformities in calves, lambs, and goat kids. SBV belonging to the Simbu serogroup (family Bunyaviridae and genus Orthobunyavirus) was first discovered in the same region where bluetongue virus serotype 8 (BTV-8) emerged 5 years before. Both viruses are transmitted by biting midges (Culicoides spp.) and share several similarities. This paper describes the current knowledge of temporal and geographical spread, molecular virology, transmission and susceptible species, clinical signs, diagnosis, prevention and control, impact on ruminant health, and productivity of SBV infection in Europe, and compares SBV infection with BTV-8 infection in ruminants.

A novel panel of monoclonal antibodies against Schmallenberg virus nucleoprotein and glycoprotein Gc allows specific orthobunyavirus detection and reveals antigenic differences.

A panel of monoclonal antibodies (mAbs) specific for the nucleocapsid (N) protein or the glycoprotein Gc of Schmallenberg virus (SBV), a novel member of the Simbu serogroup (genus Orthobunyavirus, family Bunyaviridae), was produced and used to analyze antigenic differences among members of this serogroup. Reactivity with various SBV-isolates and other Simbu serogroup viruses was assessed by an indirect immunofluorescence test and by immunoblotting. The Gc-specific mAbs detected different SBV isolates as well as two closely related members of the Simbu serogroup. In addition, one mAb showed a highly specific reactivity with the homologous SBV strain only. Based on their differing reactivity with different SBV-strains, these antibodies represent a valuable novel tool to rapidly determine the phenotype of new SBV isolates. In contrast, the N-specific mAbs showed a broad reactivity spectrum and detected not only all the tested SBV-isolates, but also several other viruses of the Simbu serogroup. One out of these mAbs even recognized all of the tested Simbu serogroup viruses in the indirect immunofluorescence assay. In order to further characterize the N-specific antibodies, PepScan analysis was performed and a specific epitope could be identified. In summary, the newly generated mAbs showed differing pan-Simbu virus-, pan-SBV- as well as SBV-isolate-specific reactivity patterns. Thus, they represent valuable tools for the development of novel antigen and antibody detection systems either specific for SBV or, in a broader approach, for the pan-Simbu serogroup diagnostics.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0165-4) contains supplementary material, which is available to authorized users.

Evaluation of in vitro methods for assessment of infection of Australian Culicoides spp. with bluetongue viruses.

Biting midges from the genus Culicoides (Diptera: Ceratopogonidae) are the vectors of several globally important arboviruses that affect livestock. These include orbiviruses from the bluetongue virus (BTV) and African horse sickness virus (AHSV) groups and members of the Simbu serogroup of orthobunyaviruses, such as the recently emerged Schmallenberg virus. In this article, the authors evaluate several methods for feeding wild‑caught Australian Culicoides on BTV infected preparations of blood and sucrose. Feeding Culicoides on the membrane of embryonated chicken eggs was identified as the preferred feeding method. Although, cotton wool pads soaked in either virus‑infected blood or virus‑sucrose mixtures were also successful. A non‑destructive nucleic acid extraction technique for the detection of viral RNA in Culicoides was also evaluated as it allows for readily differentiating infected from non‑infected Culicoides.