Diagnostic Tools for Bluetongue and Epizootic Hemorrhagic Disease Viruses Applicable to North American Veterinary Diagnosticians

Abstract

This review provides an overview of current and potential new diagnostic tests for bluetongue (BT) and epizootic hemorrhagic disease (EHD) viruses compiled from international participants of the Orbivirus Gap Analysis Workshop, Diagnostic Group. The emphasis of this review is on diagnostic tools available to North American veterinary diagnosticians. Standard diagnostic tests are readily available for BT/EHD viruses, and there are described tests that are published in the World Organization for Animal Health (OIE) Terrestrial Manual. There is however considerable variation in the diagnostic approach to these viruses. Serological assays are well established, and many laboratories are experienced in running these assays. Numerous nucleic acid amplification assays are also available for BT virus (BTV) and EHD virus (EHDV). Although there is considerable experience with BTV reverse-transcriptase PCR (RT-PCR), there are no standards or comparisons of the protocols used by various state and federal veterinary diagnostic laboratories. Methods for genotyping BTV and EHDV isolates are available and are valuable tools for monitoring and analyzing circulating viruses. These methods include RT-PCR panels or arrays, RT-PCR and sequencing of specific genome segments, or the use of next-generation sequencing. In addition to enabling virus characterization, use of advanced molecular detection methods, including DNA microarrays and next-generation sequencing, significantly enhance the ability to detect unique virus strains that may arise through genetic drift, recombination, or viral genome segment reassortment, as well as incursions of new virus strains from other geographical areas.

Overview of Current Diagnostics

US animal industry concern over risks of bluetongue (BT) and epizootic hemorrhagic disease (EHD) were highlighted in the US Animal Health Association Resolution 16 resulted in a gap analysis workshop organized by the US Department of Agriculture and Department of Interior (McVey et al. 2015). This review is a compilation of discussions from the diagnostic group that included international experts but with an emphasis on North America. Initial diagnosis of BT is frequently by observed characteristic clinical signs (Elbers et al. 2008); however, diagnostic tests for BT virus (BTV) and EHD virus (EHDV) are necessary for accurate disease diagnosis, surveillance, and in support of trade. Infections of ruminants with either BTV or EHDV can be asymptomatic; therefore, the World Organization for Animal Health (OIE) Terrestrial Animal Health Code (OIE 2014a) specifies that the BTV status of a country or zone should be defined by active surveillance to detect BTV infection as determined by either virus isolation or other agent detection test, or by serology.

In North America, it is generally regarded that, with the exception of limited species of wild ruminants, BT typically exhibits severe morbidity and mortality in sheep. Other domestic ruminants, such as cattle, can, at times, also be susceptible to clinical disease, making BT of greater consequence to domestic ruminant markets. Particularly with exotic or nonendemic strains of BTV, morbidity and mortality may be unusually high in sheep and cattle (MacLachlan et al. 2009, Coetzee et al. 2014). Additional costs can come from regulatory restrictions or mandatory testing imposed on animal movement and product trade aimed at reducing the spread of disease. For countries and states where BT has not been declared eradicated, such trade restrictions can result in an ongoing cost to lost trading markets, even in the absence of active outbreaks of BT (DeHaven et al. 2004).

In contrast, EHD has historically exhibited greater morbidity and mortality among wild ruminants, particularly in white tailed deer, and has therefore had a greater impact on wildlife and the captive/domestic cervid industry (Stallknecht et al. 2002, Stallknecht and Howerth 2004). BTV and EHDV likely share common insect vectors, often co-circulate geographically, infect similar animal species, and can produce similar clinical signs of disease in susceptible animals (Ruder et al. 2012). Because these two viruses have significantly different impacts on trade regulations, it is important to distinguish between them.

Current diagnostic procedures for both BT and EHD rely on virus isolation, or detection of viral antigen, nucleic acid, or antibody (Afshar 1994, Savini et al. 2011). Standard diagnostic tests are readily available for BT/EHD viruses (Table 1), and described tests are published in the OIE Manual (OIE 2014a, b, c). Although commercial enzyme-linked immunosorbent assay (ELISA) test kits are available, and many laboratories are experienced in running these assays (IDEXX, Westbrook, ME; IDVet, Grabels, France; Life Technologies, Grand Island, NY; and VMRD Inc. Pullman, WA), there is considerable variation in the diagnostic format used for these viruses. Detection of viral RNA by conventional or real-time RT-PCR (real-time PCR) is useful for screening samples prior to virus isolation, with the latter being the preferred method. There are published and/or commercial real-time PCR tests for both of these viruses (Jimenez-Clavero et al. 2006, Shaw et al. 2007, Toussaint et al. 2007, Wilson et al. 2009, Hoffmann et al. 2009, Clavijo et al. 2010, Schroeder et al. 2013). There is a conventional single-tube multiplex RT-PCR assay for determining the serotype of the primary US BTV strains (Johnson et al. 2000), as well as an array of conventional RT-PCR assays able to identify and differentiate between the 26 known BTV serotypes (Maan et al. 2012). A commercial BTV real-time PCR kit is not readily available in the United States; thus, various published assays are being used. Additionally, a real-time PCR for detecting BTV has been developed and evaluated against 24 serotypes of BTV by the US Department of Agriculture, Animal and Plant Health Inspection Service (USDA-APHIS; McIntosh, unpublished data). Although there is considerable experience with the BTV real-time PCR, there has been limited comparison of protocols used by various state and federal veterinary diagnostic laboratories.

Table 1.

Orbivirus Diagnostic Tests Available Now or Under Development

Test	What does it detect?	Development status	Capability	Purpose
AGID	Antibodies to BTV and/or EHDV	Available	Inexpensive, easy to run serology assay, but lacks specificity	Primarily diagnosis, but can be used for serosurvey
Indirect ELISA	Antibodies to BTV and/or EHDV	Available	Slightly more complex serology assay to run, but increased specificity	Diagnosis and sero-prevalence survey
Competitive ELISA	Antibodies to BTV and/or EHDV	Not commercially available in the United States	Slightly more complex serology assay to run, but with internationally accepted serogroup specificity; a recommended OIE test	Diagnosis and sero-prevalence survey
Competitive ELISA using recombinant proteins	Antibodies to BTV and/or EHDV	Not commercially available in the United States	Slightly more complex serology assay to run, but with internationally accepted serogroup specificity	Diagnosis and sero-prevalence survey
Antigen cELISA	BT and/or EHD viral proteins	Available	Deployable by laboratories with ELISA technology; largely replaced by real-time PCR internationally	Detecting virus (for diagnosis and active surveillance)
Virus isolation by ECE	Infectious virus	Available	The standard recommended OIE method	Detecting virus (for diagnosis and active surveillance)
Virus isolation by cell culture	Infectious virus	Available	Published as an equally sensitive isolation method to ECE if using Culicoides cells, otherwise less sensitive	Detecting virus (for diagnosis and active surveillance)
Standard PCR	BTV or EHDV viral RNA	Available	Largely replaced by real-time PCR internationally	Detecting virus (for diagnosis and active surveillance)
Nested PCR	BTV or EHDV viral RNA	Available	Largely replaced by real-time PCR internationally	Detecting virus (for diagnosis and active surveillance)
Real-time PCR	BTV or EHDV viral RNA	Available	The most commonly accepted PCR method internationally	Detecting virus (for diagnosis and active surveillance)
Multiplex PCR	BTV and EHDV viral RNA	Published	Equivalence data relative to the real-time PCR should be available where used	Detecting virus (for diagnosis and active surveillance)
Serotype real-time RT-PCR	BTV and EHDV viral serotype specific RNA	Published for some viral episystems	Not validated in all episystems	Detecting virus (for diagnosis and active surveillance)
Serotype RT-PCR array	Viral RNA from L2	Published	Not validated in all episystems.	Detecting virus (for diagnosis and active surveillance).
Topotyping PCR, with sequencing	Likely geographic origin of a viral isolate	Published	Available but limited in use	Detecting virus (and characterizing it for diagnosis and active surveillance)

AGID, agar gel immunodiffusion; BTV, bluetongue virus; EHDV, epizootic hemorrhagic disease virus; ELISA, enzyme-linked immunosorbent assay; cELISA, competitive ELISA; ECE, embryonated chicken eggs; OIE, World Organization for Animal Health; real-time PCR, real-time RT-PCR.

An important technology essential to prepare for diseases caused by these Orbivirus species is the ability to rapidly characterize outbreak strains and strains detected by active surveillance using molecular sequencing. Such genotyping usually targets one of the more conserved genome sequences of the Orbivirus species concerned and allows inferences regarding its likely geographic origin to be deduced. This technology has been limited to a few research laboratories. More advanced molecular detection methods, including whole-genome next-generation sequencing (NGS) and panviral DNA microarrays have been demonstrated to be useful tools in detection and rapid genetic characterization of viruses with segmented genomes (Potgieter et al. 2009, Maan et al. 2010, Gaudreault et al. 2014, Wilson et al. 2015). The following sections will describe this overview in greater detail. The specific discussion sections are tests available for detecting the infectious agent, those available for detection of antibodies to the infectious agent, and developing technologies.

Available tests for detection of the infectious agent

This section reviews the current means for detection of the virus, viral antigen, or RNA from infected animals. The presence of the viral antigen and/or RNA is indicative of a recent infection but not necessarily the presence of replicating virus.

Virus isolation

There has been considerable discussion around this topic. The standard, as listed in the OIE Manual for many years, has been a primary passage by intravenous inoculation of embryonated chicken eggs (ECE) (Gard et al. 1988) with the inoculum prepared from the specimen, followed by a passage in mosquito cells (C6/36 cells) and one to two subsequent passages in a mammalian cell line for the detection of BTV by visible cytopathic effect (CPE). A number of cell lines to BTV infection have been evaluated and may be employed as indicators of BTV (or only “the virus”) (Afshar 1994, Savini et al. 2011). Since the OIE guidelines were developed, new cell lines derived from Culicoides sonorensis (KC cells) have been established, and these are being used for virus isolation internationally (McHolland and Mecham 2003). The original KC cell line is no longer recommended because it contains low-level contamination with BTV genomic material (Mecham et al. 2004). The newer cell lines are available through a simple material transfer agreement. Some laboratories are using immortalized or primary cultures of bovine endothelial cells for the initial isolation step. It should be noted that the ideal isolation method (ECE or cell culture) is likely viral strain dependent.

Differences in experiences with virus isolation were noted among the participants of the Orbivirus Gap Analysis Workshop Diagnostic Group. As an example, the Berrimah Veterinary Laboratory in Australia has been conducting virus isolation routinely from sentinel cattle blood samples for over three decades and recommends the use of ECE primary passage system for BTV (Weir, personal communication). They have advised that up to 25% of their BTV isolates are from nonhemorrhagic embryos. The US experience differs from that of Australia. The US labs also have decades of Orbivirus isolation work in which BTV was never isolated from an inoculum that did not yield at least one hemorrhagic egg (Ostlund, personal communication). Still other laboratories have stopped using ECE for initial BTV isolation and instead employ Culicoides cells. Unfortunately a structured comparison of the performance characteristics of the various isolation protocols being employed is lacking.

There is concern regarding the natural transmission of BTV strains derived from live, attenuated vaccines or BTV that contain reassorted gene segments that may have been derived from such vaccine strains. In some instances, such strains may retain or have acquired traits that contribute to adaptation to cell culture. Such strains may be more likely to be isolated in cell culture systems than wild-type strains.

Although virus isolation is an excellent method for detection of the pathogenic agent and is typically needed to facilitate further pathogen characterization and pathogenesis studies, it is laborious and time consuming. Other methods of obtaining BTV isolates, albeit not readily employed in diagnostics, include intracranial inoculation of suckling mice or inoculation of alpha/beta interferon receptor–deficient mice (for review, see Coetzee et al. 2014). The diagnostic group indicated that many laboratories therefore choose to run presumptive tests that detect the presence of virus antigen or nucleic acid. These tests include antigen capture enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, in situ hybridization, and/or nucleic acid amplification by conventional or real-time PCR.

Electron microscopy

Traditionally, electron microscopy (EM) has been useful in the diagnosis of diseases of unknown etiology because identification of a virus type or family may be deduced from virus morphology (Hyatt and Eaton 1990). Negative-staining EM is extremely rapid when there is abundant virus. This may be further aided by virus isolation in cell culture in which the virus can be propagated to high titer, although this delays the reporting of results. Transmission EM of ultrathin sections of fixed infected tissues, while also requiring high concentrations of virus, may be used to not only identify the virus family but also to reveal evidence of cellular pathology caused by the infection.

More efficient molecular diagnostic techniques that provide precise identification of virus species and strain have largely replaced EM in differential diagnostic testing for known viruses. However, EM continues to provide a valuable nonpathogen-specific diagnostic approach to the detection of emerging divergent or new viruses where no prior detailed knowledge of virus identity or genetic sequence is available. Advanced molecular detection methods including panviral/panpathogen microarrays or NGS represent evolving efficient and highly sensitive alternatives to EM as nonbiased detection methods for previously unknown, divergent, or emerging viruses (for review, see Bexfield and Kellam 2011).

Antigen detection

Methods for detection of the presence of BTV protein using antigen competitive ELISA (cELISA) in whole insects (Mecham et al. 1990) and mammalian blood (Mecham 1993) are available. This method was also adapted to a western dot blot application (Nunamaker et al. 1997). These tools were used to demonstrate variation in BTV vector competence depending on virus serotype in Culicoides colonies (Mecham and Nunamaker 1994). In some laboratories, antigen detection cELISA is routinely used for monitoring the success of virus isolation procedures (Hawkes et al. 2000).

Immunohistochemistry or immunofluorescence assay (IHC/IFA) are additional established technologies, but because of the labor intensity these techniques are more often employed in experimental pathogenesis studies rather than in diagnostics. The IHC/IFA assay has diagnostic uses in specialized situations, such as linking a lesion to a detected infectious agent antigen; however, there are circumstances where the presence of the agent might be incidental to the disease. This could happen in BTV infections of cattle, in particular, where the duration of viremia can be prolonged. Hence, during the period of viremia, an animal may coincidently suffer from a different disease, but the BTV infection will also be detected. Such situations would not be easily interpretable; thus, while the technology complements the full pathological investigation, antigen detection alone should not be interpreted as a definitive diagnostic test.

Nucleic acid detection

Detection of viral RNA has been used for some time, initially by simple nucleic acid hybridization technologies (Wilson 1990). Although hybridization methods are relatively easy to perform, they lack analytical sensitivity. Hybridization assays have been enhanced by new reporter systems, e.g., PCR-based methods (Zhang et al. 2012); however, these systems are not yet readily available. Conventional PCR assays, requiring resolution of products by agarose gel electrophoresis, have been developed for BTV and EHDV (Akita et al. 1993, Katz et al. 1993, Wilson 1994, Shad et al. 1997). Analytical sensitivity is increased by the use of nested RT-PCR, which also increases the risk of laboratory contamination with amplified PCR products. The resulting risk of false-positive test results is mitigated in well-managed and constructed laboratories with rigidly enforced work practices based on separation of the steps in the test procedure. However, not every laboratory has been designed to enforce such work practices effectively, and work practices are still susceptible to human error. Therefore, the method of choice has been real-time PCR, where the assay is run within an enclosed tube.

Some caution is needed in interpretation of RT-PCR and real-time PCR results because BTV nucleic acid can be detected in blood from infected animals long after viable virus is no longer present in a specimen (MacLachlan et al. 1994). In experimentally infected calves, viral RNA was detected by RT-PCR at 16–20 weeks after infection as compared to only 2–8 weeks after infection by virus isolation (MacLachlan 1994). This suggests extended persistence of viral RNA without the presence of infectious virus or continued active virus replication. A positive test result will indicate that an animal has been infected or vaccinated at some time prior to the collection of the specimen but the animal may not necessarily still be infectious to Culicoides insects and therefore may no longer be capable of sustaining transmission of the virus.

A potential use for molecular detection tests such as PCR is for the detection of viruses in insect vectors. Methods for detecting BTV or EHDV RNA have been available for some time (Wilson 1991, Wilson and Chase 1993). Virus group or serotype specific tests can be used, depending on the purposes of the study. The approach has applications in surveillance and in research; however, there can be situations where there is Orbivirus transmission with low virus prevalence in the biting midge (Culicoides) vectors (Wieser-Schimpf et al. 1993). One study was able to use PCR to detect and serotype BTV from vector species demonstrating a changing pattern of its maintenance over a 6-month observation period in an endemic area (Melville et al. 1996).

Quantitative/real-time RT-PCR (real-time PCR)

Several real-time PCR assays have been developed for BTV (Wilson et al. 2004, Orru et al. 2006, Shaw et al. 2007, Toussaint et al. 2007, Hoffmann et al. 2009, Schroeder et al. 2013, Maan et al. 2014). There are fewer real-time PCR assays available for EHDV (Wilson et al. 2009, Clavijo et al. 2010, Eschbaumer et al. 2012). These PCR-based diagnostic procedures are exquisitely analytically sensitive and theoretically can be configured to any desired specificity if the sequence of the target viral gene of interest is known.

A review of available real-time assays for BTV in 2009 summarizes the design and strategies of the assay at that time (Hoffmann et al. 2009). Since then, new assays have been developed, including an improved multiplex real-time PCR assay that detects and distinguishes between BTV and EHDV (Wilson et al. 2009). Additionally, an as yet unpublished real-time PCR for detecting BTV has been developed and evaluated on field diagnostic samples and virus isolates of 24 serotypes of BTV by the Foreign Animal Diseases Laboratory (McIntosh et al., unpublished). The described method in the OIE Manual is the method of Hofmann et al (OIE 2014a, b).

An aspect often overlooked in evaluation of real-time PCR is selection of the sample and nucleic acid extraction method. Nucleic acid extraction methods must be evaluated for the animal source (Brito et al. 2011) and the specimen chosen (Vanbinst et al. 2010). EDTA blood represents the most common sample type for molecular detection of BTV (MacLachlan et al. 2009); however, PCR inhibitors, such as immunoglobulin G and hemoglobin, can have significant negative effects on the sensitivity of RT-PCR–based tests (Al-Soud and Radstrom 2001). In this regard, molecular test validation with regard to sample type and species is important. Likewise, improved RNA purification methods are needed to reduce the impact of such inhibitors and to minimize the potential for variable test performance between different species and tissue types.

The reduced contamination and ability to run the real-time PCR in a high-throughput format has resulted in it becoming a standard test for the detection of RNA viruses in biological (diagnostic) specimens in many laboratories. To increase robustness of the assay and to ensure detection of all the BTV strains from different episystems, it has been recommended that two conserved genes be targeted in parallel (Toussaint et al. 2007) or simultaneously (Wilson et al. 2009). The National Veterinary Services laboratory has completed conventional and real-time PCR comparisons and contributed to the selection of a specific PCR for the May, 2014, updated OIE chapter on BTV (Ostlund, unpublished data).

Typing PCR (with sequencing of the amplicon)

Rapid detection of BTV or EHDV is a necessary first step in an outbreak investigation or a surveillance program, but is usually not sufficient. The detection, by PCR or possibly by virus isolation, is combined with a need to characterize the agent by determining the serotype and/or the genotype. This information can be used to establish the epidemiological relationship between the agent and previously transmitted viruses in the study area. There are serotype-specific standard RT-PCR assays for BTV (Johnson et al. 2000, Maan et al. 2012) and EHDV (Aradaib et al. 1995a, b, Aradaib et al. 2005). Serotyping can also be addressed by the combination of PCR and sequencing that permits molecular epidemiological assessments (Gould and Pritchard 1990, Aradaib et al. 1997, Potgieter et al. 2009, Maan et al. 2010, Lee et al. 2011, Gaudreault et al. 2014). In this regard, a reverse-transcription loop-mediated isothermal amplification test has been developed and applied to the specific detection of BTV serotype 8 in clinical samples (Mulholland et al. 2014). At present, genotyping of US isolates for epidemiology purposes is limited to a few research laboratories, but is becoming more prevalent with the increased adoption of PCR and sequencing technologies in diagnostic laboratories.

In situ hybridization

This technology may be considered the molecular equivalent of IHC, in that it permits the detection of viral genome in tissues and potentially in pathological lesions. In contrast, IHC permits the detection of viral antigen in host tissues. It may be useful in diagnostic situations in linking a detected infectious agent to the pathological lesions, as discussed more fully under IHC/IFA above. In situ hybridization has been used to detect Orbivirus RNA in diagnostic specimens, although it has been more widely useful for research into the pathology of BTV infections (Dangler et al. 1990, Schoepp et al. 1991, Brodie et al. 1998).

DNA microarrays

Multipathogen or panviral DNA microarrays suitable for detection of orbiviruses have been developed (Palacios et al. 2007, Quan et al. 2007, Barrette et al. 2009, Jack et al. 2009, Gardner et al. 2010, Thissen et al. 2014). These DNA microarrays capture viral DNA from a sample using many different oligonucleotide features targeting conserved genetic loci that are cross-reactive within or among a specific virus family, genus, or species. Because of this broad detection capability that may include both pathogenic and nonpathogenic agents, multiple-pathogen or panviral DNA microarrays are more suited to pathogen detection rather than diagnostics or genetic characterization. These design features, including diversity and multiplicity of well-conserved genetic detection targets, combined with random amplification of target cDNA or total nucleic acid, make DNA microarrays well suited for detection of emerging or previously unknown pathogens. DNA microarrays are also well suited to simultaneous detection of multiple agents that may be associated with disease syndromes. The ability to capture and positively select virus-derived nucleic acid from host nucleic acid also serves as a useful companion tool to conventional or NGS analysis. DNA microarrays that target all segments of segmented viral genomes, such as the USDA panviral microarray (Barrette et al., 2009), are particularly useful companion tools to sequence analyses. Mapping of positive microarray features during data analysis may further be exploited to identify candidate primer and probe binding sites for the design of new or modified conventional or real-time PCR methods that may be employed to survey and/or diagnose newly detected or divergent orbiviruses.

Available Tests for Detection of Antibodies to Infectious Agents

Serological tests have been used extensively for diagnostic and surveillance investigations. Serological tests are useful to determine if an animal has been exposed to a specific pathogen but do not necessarily indicate presence of the antigen. The most common are the agar gel immunodiffusion (AGID) and ELISA, where positive results indicate only antibody or seropositive status of an animal that may have arisen from vaccination or previous exposure or have been acquired maternally. BTV/EHDV cross-reactivity with the AGID test also adds significant complexity to the diagnosis of BT and EHD. Complement fixation tests have been used, but like the AGID, these suffer from cross-reactions between antibodies to BTV and EHDV species or serogroups (Afshar 1994). A number of ELISA tests have been developed for the detection of antibody specific to BTV (Afshar et al. 1987a, b, Drolet et al. 1990, EnMin and Chan 1996, Mecham and Wilson 2004). A novel approach was the use of single-chain Fv fragments of chicken antibodies in inhibition ELISAs for detection of BTV serogroup-specific antibodies (Fehrsen et al. 2005). These tests have been shown to be more specific than the AGID test in that they show no significant cross-reactivity to antibodies directed against EHDV (Afshar et al. 1989, 1991, 1993).

Competitive ELISA

The BTV cELISA has been in use for over two decades and has been evaluated extensively (Afshar et al. 1991, Reddington et al. 1991, Afshar et al. 1992, Gould and Martyn 1992, Mecham and Wilson 2004). Although variations, based on different monoclonal antibodies, exist in different countries, it is accepted that most have equivalent performance characteristics that importantly allow the detection of antibodies to BTV and not to EHDV. The specificity of the BTV cELISA for this purpose is widely accepted. BTV cELISAs are commercially available and some are licensed for use within the United States. In this regard, the USDA-APHIS requires that state diagnostic laboratories conducting BTV ELISA testing for export purposes use a standardized USDA licensed test kit.

EHDV cELISA tests have also been published, but according to the diagnostic group are not as available as the BTV cELISA (White et al. 1991, Gould and Martyn 1992, Nagesha et al. 1996, Afshar et al. 1997, Mecham and Jochim 2000). Recently a commercially available EHDV ELISA test has become available (Life Technologies, Grand Island, NY), but little information on this test is in the literature and at present there is no USDA-APHIS–licensed EHDV ELSIA test. The EHDV cELISA tests have not been validated or evaluated to the same extent as BTV tests and have therefore not been used or made widely available in the United States.

Virus neutralization test

The virus neutralization test (VNT) is the traditional approach for detecting serotype-specific antibody for both the BTV and EHDV species. The cost and labor associated with this test have resulted in diminished use in veterinary diagnostic laboratories. The VNT is useful in epidemiological studies and in measuring immune response to vaccination as a method to track vaccine deliveries and effectiveness. No direct correlation of the VNT titer with the level of protection from either vaccinated or naturally exposed animals has been established. The detection of neutralizing antibody in a VNT is usually interpreted to reflect a robust, if not protective, immune response in a given animal. In the OIE Terrestrial Code Chapter on BTV (OIE 2014a, b, c), detection of BTV-specific antibody is taken as evidence of infection, but it is emphasized that antibody-positive animals are not necessarily infectious for vectors. In fact, reference is made to the movement of animals under certain circumstances, providing they have detectable antibody to the serotypes known to be present in the country or zone of origin. This is a measure that facilitates trade and the movement of live animals under some farming systems, although it is not currently relevant to the United States.

Interpretation of antibody responses to specific serotypes can be somewhat problematic, particularly where animals have been infected with more than one serotype. Such animals may develop neutralizing antibody to a serotype(s) different from the infecting serotype, and so results must be interpreted with respect to the epidemiological situation.

Because the VNT requires the propagation of live virus in culture, there are biological risk management issues for laboratories offering the procedure. This risk could be minimized by using vaccine strains or nonspreading virus that can be generated using reverse genetics (Celma et al. 2014, Feenstra et al. 2014). It is common practice for laboratories in national networks to offer the VNT only for serotypes that are transmitted in the geographic area they service. This removes the requirement to have facilities for the handling of foreign animal disease agents. Sera requiring further evaluation for reactivity to other strains or species are typically sent to a national reference laboratory.

Developing Technologies

There are several technologies that are currently being considered for future use in diagnostics for orbiviruses. These include fluorescent microsphere assays for antigen or nucleic acid detection, NGS or high-throughput sequencing, alternative amplification techniques, and pen-side tests.

Fluorescent microsphere assays for nucleic acid, antibody, and antigen detection

Fluorescent microsphere assays are an approach to multiplexing diagnostic testing that is being investigated experimentally in a number of laboratories (Johnson et al. 2005, Balasuriya et al. 2006, Perkins et al. 2006, Hindson et al. 2008, Clotilde et al. 2011). Where research teams have developed appropriate expertise, particularly in designing and producing the reagents needed for multiple simultaneous detection reactions, the results have been encouraging (Perkins et al. 2007, Hindson et al. 2008). Fluorescent microsphere assays are easily adapted to testing for the presence of antibodies by indirect immune-fluorescence, or nucleic acids by in-solution hybridization in multiplexed formats allowing for simultaneous testing for distinct antibody responses or disease agents, and they are ideal for investigation of animal disease syndromes. Importantly, approaches to the validation of such assays are now being developed (McNabb et al. 2014).

Next-generation sequencing or high-throughput sequencing

There are many diagnosticians who consider this methodology to be the primary diagnostic platform of the future. This technology for orbiviruses is established (Potgieter et al. 2009, Gaudreault et al. 2014, Wilson et al. 2015). NGS provides a rapid means for topotyping that is important for the molecular characterization of Orbiviruses detected during outbreak investigations or by routine surveillance programs. The basis for these molecular analyses is in the observed localized evolution of different Orbivirus strains between differing epidemiological environments. Thus, when the molecular sequences of apparently similar serotypes are elucidated from different geographical locations and compared, the genetic differences are revealed. Once the genome sequences of geographically distinct virus strains are known, it becomes possible to postulate whether or not a newly sequenced virus belongs to a locally circulating virus population, or whether it might have been introduced from a different geographic area.

It is likely that NGS will become more readily accessible, and this has led the OIE to establish a working group to develop approaches to validate NGS methods for diagnostic work. However, despite the power of NGS to not only detect but to simultaneously characterize known and unknown viruses, the complexity of data analysis, detection of endogenous or inconsequential viruses, and the potential for contamination either by environmental viruses or by extraneous contaminating nucleic acids present challenges to implementing NGS as a stand-alone diagnostic test. Traditional methods that link a disease agent to a clinically sick animal or population are still required to provide diagnostic confirmation when a disease agent is detected by advanced molecular methods such as NGS and panpathogen microarrays. In this regard, advanced molecular detection methods are becoming more widely employed as detection and characterization tools rather than stand-alone diagnostic tests.

Alternative amplification techniques and pen-side tests

There are alternative amplification techniques, such as loop-mediated isothermal amplification (Le Roux et al. 2009, Aebischer et al. 2014), recombinase polymerase amplification (Piepenburg et al. 2006), and linear after the exponential phase PCR (LATE-PCR) (Sanchez et al. 2004), that are promising alternative methods but have not been applied to orbiviruses. In particular, a number of isothermal amplification methods have been developed for RNA or DNA that have the potential to advance the development of pen-side diagnostic testing (Yan et al. 2014). Similarly, lateral flow or pen-side tests for both antibody and antigen detection have been useful for other pathogens, but have yet to be widely applied to orbiviruses. Such tests have recently become available for EHDV (Bioo Scientific, Austin, TX). Because of the high economic impact of BTV and EHDV, and limited resources for test validation, veterinary diagnosticians remain cautious in adopting new technologies.

Summary

Standard diagnostic tests are readily available for BTV and EHDV (Table 1), and described tests are published in the OIE Manual. There is considerable variation in the diagnostic approach to these viruses. Commercial BTV or EHDV diagnostic test kits are available, and many laboratories are experienced in running these assays. Detection of viral RNA by conventional or real-time PCR is useful for screening samples prior to virus isolation. Viral RNA detection alone is often used as a presumptive test due to the labor and time involved in virus isolation. There are many serological assays available for BTV and EHDV, and they are important components of the diagnostician's tool chest. The AGID is still commonly used for EHDV; however, there are published cELISAs and recently a commercial EHDV ELISA. The National Veterinary Services Laboratory does perform certification of veterinary diagnostic laboratories performing BTV and EHDV serology for export purposes on an annual basis.

Advanced molecular detection methods, including NGS and pan-viral DNA microarrays, have been demonstrated to be useful tools in rapid genetic characterization of viruses with segmented genomes, including BTV. Applications of these methods are presently limited due to costs of implementation, but could benefit detection of highly divergent emerging strains or species of orbiviruses. The clinical history and combination of diagnostic tools reviewed here will lead to the proper diagnosis of the observed disease.

Footnotes

Acknowledgements

The authors thank Drs. Natasha Gaudreault and Estelle Venter for early review of this manuscript.

Author Disclosure Statement

No competing financial interests exist. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the US Department of Agriculture or Commonwealth Scientific and Industrial Research Organization.

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