VECTOR-BORNE AND ZOONOTIC DISEASES Volume 15, Number 6, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/vbz.2014.1701

A Review of Knowledge Gaps and Tools for Orbivirus Research Barbara S. Drolet,1 Piet van Rijn,2 Elizabeth W. Howerth,3 Martin Beer,4 and Peter P. Mertens 5

Abstract

Although recognized as causing emerging and re-emerging disease outbreaks worldwide since the late 1800s, there has been growing interest in the United States and Europe in recent years in orbiviruses, their insect vectors, and the diseases they cause in domestic livestock and wildlife. This is due, in part, to the emergence of bluetongue (BT) in northern Europe in 2006–2007 resulting in a devastating outbreak, as well as severe BT outbreaks in sheep and epizootic hemorrhagic disease (EHD) outbreaks in deer and cattle in the United States. Of notable concern is the isolation of as many as 10 new BT virus (BTV) serotypes in the United States since 1999 and their associated unknowns, such as route of introduction, virulence to mammals, and indigenous competent vectors. This review, based on a gap analysis workshop composed of international experts on orbiviruses conducted in 2013, gives a global perspective of current basic virological understanding of orbiviruses, with particular attention to BTV and the closely related epizootic hemorrhagic disease virus (EHDV), and identifies a multitude of basic virology research gaps, critical for predicting and preventing outbreaks. Key Words:

Orbivirus—Bluetongue virus—Epizootic hemorrhagic disease virus—Culicoides—Midges—Review.

Introduction

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revention and control of insect-transmitted orbivirus diseases is dependent upon a knowledge of virus–vector–host interactions, pathogenesis, diagnostics, epidemiology, and control strategies. Recent advances in reverse genetics, deep sequencing, animal models, genomics, monoclonal antibodies, reference sera, and expressed proteins all contribute to the growing field of orbivirus research. This field also explores at the molecular level how these viruses replicate and interplay with the host and insect vector to spread and cause disease in new individuals and populations. A better understanding of the basic virology of bluetongue virus (BTV), epizootic hemorrhagic disease virus (EHDV), and other orbiviruses is needed to help model current and future disease outbreaks and to re-examine the criteria for domestic (enzootic) versus exotic (nonenzootic or incursive) serogrouping and topotyping. It is also important for understanding virus transmission mechanisms in and between

vertebrate hosts and insect vectors to evaluate the global risks associated with orbiviruses that have the potential to emerge and threaten animal health, livestock trade, and even human health. Lack of knowledge in many areas still remains, including molecular determinants of mammalian host and insect vector specificity, factors of cell culture adaptation, and the modulation of virulence. Furthermore, viral determinants involved in receptor binding and immune responses of mammalian hosts and vectors are barely known. Basic orbivirology is of paramount importance for predicting disease outbreaks in at-risk populations of animals, understanding the mechanisms of arthropod-borne virus (arbovirus) maintenance and emergence, and developing diagnostics, vaccines, and control strategies. In this review, we first identify the gaps in our knowledge, posed as questions, in the following areas—mammalian host range, vector host range, clinical outcome and determinants of virulence, antigenic determinants and epitope mapping,

1

US Department of Agriculture, Agricultural Research Service, Arthropod-Borne Animal Diseases Research Unit, Manhattan, Kansas. Department of Virology, Central Veterinary Institute of Wageningen University (CVI), The Netherlands; Department of Biochemistry, Centre for Human Metabonomics, North-West University, South Africa. 3 Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia. 4 Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Insel Riems, Germany. 5 Vector-Borne Diseases Programme, The Pirbright Institute, Pirbright, Woking, United Kingdom. 2

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viral genome sequence and encoded proteins, receptors, transmission mechanisms in and between vertebrate hosts and invertebrate vectors, immune responses of mammalian and vector hosts, and genome analysis. We then propose, in general terms, how modern technology and research tools, such as reverse genetics, monoclonal antibodies and reference antisera, genomic sequence data, virus collections, cell culture lines, vector colonies, small animal models, and target host animal models, can help to answer those questions. Knowledge Gaps in Orbivirology Mammalian host range and clinical outcome

Specific Orbivirus species infect certain host species and cause disease in only a subset of the permissive species. Although BTV and EHDV can infect several domestic and wildlife ruminant species, BT is primarily a disease of sheep (Verwoerd and Erasmus 2004, Maclachlan et al. 2009) and EHD a disease of deer; particularly white-tailed deer (WTD) (Odiawa et al. 1985, Stallknecht et al. 1995). The molecular basis for this specificity is poorly understood, but may be directed by the outer capsid proteins VP2 and VP5, which are involved in cell attachment and virus penetration of mammalian cells (Hassan and Roy 1999, Forzan et al. 2004). VP2 is thought to be composed of a duplicated structure (Belhouchet et al. 2010). Questions related to the molecular mechanisms involved in the observed spectrum of infection outcomes include: 1. What mammalian host cell factors determine species susceptibility? Specifically: a. What makes sheep refractory to EHDV infection, but cattle and deer susceptible? b. Why does EHDV only sporadically cause disease in cattle (Kedmi et al. 2011) and why is the disease much less severe as compared to deer? c. What is the reason for the host range restriction of the newly described putative BTV-25 (Vogtlin et al. 2013)? 2. What factors determine susceptibility differences between subpopulations within an animal species? 3. Why is it that indigenous African sheep breeds rarely demonstrate clinical signs as compared to improved European sheep breeds (Coetzee et al. 2012a)? 4. Can we define host preferences of viruses on a molecular level? 5. Why do WTD originating from different geographic locations, and of different subspecies, vary in susceptibility to EHDV (Gaydos et al. 2002b)? Vector host range and clinical outcome

The ‘‘episystem’’ hypothesis suggests that there is some restriction of transmission of newly introduced (incursive) BTV strains by indigenous Culicoides populations (Gibbs and Greiner 1994, Daniels et al. 2004, Tabachnick 2010). The spread of multiple BTV strains and serotypes into Europe, the arrival of multiple new EHDV (Allison et al. 2012) and BTV (Ostlund 2010) serotypes in the United States, and the arrival of western topotype strains in India all indicate this is not universally true. However, none of the recently introduced

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BTV serotypes isolated in the southeastern region of the United States (BTV-1, -3, -5, -6, -9, -12, -14, -19, -22, -24) have, thus far, spread across the rest of North America where the primary vector for enzootic BTV serotypes is C. sonorensis. The same is true for BTV-1, -2, -4, -8, -9, and -16 in southern Europe, except for BTV-8, which is also transmitted by Culicoides species of the Obsoletus group endemic in northern parts of Europe (Meiswinkel et al. 2007, Dijkstra et al. 2008, Mehlhorn et al. 2008). The detection of reassortants between different topotypes and serotypes in southern Europe suggests that reassortants are currently emerging that may have novel biological properties (Lorusso et al. 2013a, b). It is possible that individual genes from incursive strains will be wider spread after reassortment with enzootic strains, which may contribute to transmissibility by additional vector species or enhanced virulence. BTV-26 can be transmitted horizontally between goats and grows in mammalian cells but fails to grow in a Culicoides-derived cell line (KC) (Wechsler et al. 1989, Batten et al. 2013). This may represent an extreme example of the virus’s genetic control over vector competence. Questions related to the molecular mechanisms involved in transmission by the insect vector and their possible effects on disease include: 1. In determining vector competency, which Culicoides species or subspecies, and other arthropod species, are competent vectors for BTV, EHDV, and other orbiviruses? 2. What are the viral and environmental determinants of vector competence? 3. What are the molecular mechanisms by which vector species become infected by orbiviruses? 4. What molecular mechanisms are involved at the midgut and salivary gland infection barriers that result in a vector being competent verses refractory? Can those mechanisms be exploited to block insect infection and virus transmission? 5. Which viral genes/proteins are the key players for vector infection/transmission? Do the relative expression levels of nonstructural proteins play a role? 6. What role do the arthropod vectors play in development of clinical disease? Critical to this: What role do vector salivary proteins, delivered with virus during feeding, play in orbivirus infection? Viral determinants of virulence and clinical outcome

Both BT and EHD viral infections of ruminants can result in a spectrum of disease from subclinical or inapparent clinical signs to severe disease with high morbidity and mortality (MacLachlan et al. 2009, Coetzee et al. 2014b). Variability in the clinical outcome of infection is not surprising considering the innumerable interaction combinations of multiple susceptible species/breeds/ages of mammals, multiple co-circulating virus serotypes/topotypes/reassortants, and multiple competent Culicoides vectors. Identifying and characterizing the mammalian and insect factors, as listed in the previous two sections, which affect infection outcome is essential to understanding the pathobiology of orbivirus infections (Coetzee et al. 2012b). Questions related specifically to the viral factors that affect pathogenicity include:

KNOWLEDGE GAPS AND TOOLS FOR ORBIVIRUS RESEARCH

1. Which viral–insect or viral–mammalian host molecular interactions affect virulence and clinical outcome? 2. What is the role of a naı¨ve host population? 3. Why do enzootic BTV strains often cause relatively mild clinical signs, whereas incursive strains can lead to much more severe outbreaks of disease? 4. Do different BTV and EHDV topotypes, serotypes, or strains have significant differences in their abilities to be transmitted by individual vector species or populations? 5. What role does genetic bottlenecking of the virus within the vector during midgut infection/escape and salivary gland infection/escape play in virulence? 6. What is the role of the environment on virulence? Viral genome sequence and encoded proteins

Progress on elucidating the complex nature of orbivirions and their replication has been made (Roy 2008). Still, the function of individual orbivirus proteins during replication requires further research. It is still not fully understood what proteins and which genomic regions are involved in orbivirus assembly and packaging (Burkhardt et al. 2014). Recently, it was shown that the NS3/NS3a protein is not essential (van Gennip et al. 2014), whereas RNA sequences in the NS3 gene are essential for BTV replication (Feenstra et al. 2014b). Recent discovery of a novel NS4 BTV protein in those orbiviruses transmitted by Culicoides highlights the gaps in our knowledge about these viruses (Belhouchet et al. 2011, Ratinier et al. 2011). Questions related to the viral genome sequence and encoded proteins include: 1. Which proteins and RNA sequences are involved in interactions that orchestrate virus assembly? 2. Which viral genes/proteins influence vector infection/ transmission? 3. What is the role of the nonstructural proteins in insect versus mammalian cells? 4. What is the significance of differences in the relative expression levels of different BTV proteins (e.g., NS3) in insect and mammalian systems? 5. Are there other undiscovered orbivirus proteins? 6. What are the complete sets of functions of each orbiviral protein? 7. Which viral proteins cause cell cycle arrest and cell killing (apoptosis)? 8. Are there additional nonessential proteins? 9. Are there additional RNA regions that can be deleted? Receptors

Virus–cell ligand interactions are poorly understood for orbiviruses. The limited information for BTV indicates that the outer coat proteins of BTV, VP2 and VP5, are involved in the first steps of receptor-mediated interactions with the vertebrate host cell. The initial contact of BTV with the cell surface is through binding to glycoprotein receptors (Hassan and Roy 1999, Forzan et al. 2004). In contrast, VP7 is involved in attachment to Culicoides cells (Xu et al. 1997). Molecular species for cell-surface attachment of BTV and other orbiviruses need to be identified for both vertebrate hosts and insect vectors. This is also important in understanding virulence, host/vector range,

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pathogenesis, and transmissibility of BTV and other orbiviruses. Questions related to receptors include: 1. What are the receptors and attachment mechanisms? 2. Are there different receptors for different species or orbivirus serotypes? 3. Are there different entry mechanisms as suggested by initial data for BTV-1 and -10 (Gold et al. 2010)? 4. What is the distribution of receptors in the host? 5. What is the role of receptors in host range, virulence, vector range, competence, transmission, and clinical disease? Transmission mechanisms in vertebrate hosts and invertebrate vectors

The mechanisms involved in vertical transmission and teratogenic effects in the vertebrate host are poorly understood. BTV has been used as a model of virus-induced teratogenesis and abortion in ruminants (MacLachlan et al. 2000). Although placental crossing of BTV-4 was identified in Cyprus in the 1977 epizootic (P.A. Gibbs, pers. comm.), the ability of BTV to cross the placenta has historically been most strongly associated with laboratory-acquired cell adaptation. Thus, it was surprising to discover that the BTV-8 strain causing the recent BT outbreak in Europe was capable of crossing the placenta efficiently (Santman-Berends et al. 2010), resulting in a high incidence of congenital brain defects in cattle depending on the time of infection during gestation (Vercauteren et al. 2008). Because the origin of this BTV-8 strain is still unknown, we cannot speculate whether it might have originated from a laboratory. The mechanism of vertical transmission of BTV in both vertebrate and invertebrate hosts is poorly understood. Interestingly, in both mammalian and Culicoides cells, cellular release of BTV is related to NS3 expression, suggesting that virus distribution within a host or a vector is mediated by NS3 (Feenstra et al. 2014a, van Gennip et al. 2014). Minor routes of virus transmission independent of the insect vector may be important in overwintering and critical in designing control strategies. Questions related to virus dissemination and transmission among vertebrates include: 1. What are the host determinants for viral dissemination? 2. What are the host determinants for efficient vertical and horizontal transmission? 3. What is the potential of transmission by other routes such as mechanical or oral, and what is the potential of these routes for field persistence and epidemiology of the virus, in particular, in overwintering? Questions related to virus dissemination and transmission among invertebrates include: 1. What are the determinants for virus replication in the vector? 2. What are the determinants for salivary gland infection and subsequent secretion in saliva? 3. Can transovarial transmission (TOT) occur in any/all of the orbivirus vectors? How frequently? What are the determinants? 4. What role would TOT play in transmission, persistence, and epidemiology of the virus, especially in overwintering?

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Both humoral and cell-mediated responses are elicited in BTV-infected animals (MacLachlan 1994, Maclachlan et al. 2014). The serotype-specific outer capsid viral protein VP2, as well as VP5, are the only BTV proteins shown to induce neutralizing antibodies (Lobato et al. 1997, Schwartz-Cornil et al. 2008) and confer protection against reinfection with the same strain/serotype (Roy et al. 1992, Schwartz-Cornil et al. 2008). Still, there is evidence for partial cross-protection and the existence of cross-reactive immune responses, including neutralizing antibodies (Neitz 1948, MacLachlan et al. 1992, Calvo-Pinilla et al. 2014). Serogroup-reactive antibodies are induced by the core protein VP7 and nonstructural proteins, but they are not protective (Huismans and Erasmus 1981, MacLachlan et al. 1987). Cell-mediated immune responses are cross-reactive between serotypes (Ghalib et al. 1985) and limit viral spread early in the infection; however, these only provide short-term protection and cannot completely clear the infection ( Jeggo et al. 1984, Andrew et al. 1995). It would be very useful to identify viral proteins and epitopes that could generate a protective and cross-reactive cell-mediated immune response and possibly lead to improved vaccines. Reverse genetic technologies may assist in these studies. Insects have several antiviral defense strategies, including RNA interference (RNAi), Toll pathway, defensins, phagocytosis, and the antiviral cell signaling molecule VAGO, as reviewed by Merkling and van Rij (2013). The way orbiviruses control or evade immune responses of their mammalian and insect vector hosts is poorly understood. Questions related to immunogenic determinants and epitope mapping include: 1. What are the viral factors modulating adaptive and innate immune responses to orbiviruses? 2. Which epitopes are type-specific and which are responsible for cross-neutralizing antibodies following multiple sequential infections with different serotypes? Questions related to host immune responses to viral antigens include: 1. How do immune responses affect protection of the host against orbivirus infection, the clinical outcome, and onward transmission? 2. How is partial cross-protection (e.g., decreased clinical disease) following infection with a second orbivirus type (Gaydos et al. 2002a) mediated? 3. What are the mechanisms of cellular and humoral immune defenses? 4. Would cross-reactive cell-mediated immune responses be sufficient for protection, or only a useful component in protection? 5. What are the immune/antiviral responses of arthropod vector species to orbivirus infection, and are these related to vector competence? 6. Are there specific viral antisilencing genes? 7. What are the potential roles for microRNA responses? Genome analyses

Identifying orbivirus strains by serotype is most relevant for implementing vaccination strategies, but completely insufficient for characterizing and identifying virus lineages

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and new isolates. For example, full-genome sequencing shows that there is a high degree of segment reassortment resulting in a spectrum of BTV strains circulating in the field. A new classification/identification system (possibly barcoding) is needed to account for each segment, and, as an example, will contribute to a better understanding of the relatedness of exotic and enzootic serotypes. Critical to this classification/identification is a much-needed global open access database for full-genome sequence data. This would facilitate analyses to determine relatedness, as well as genetic drift and shift. It could also be useful for predicting important epidemiological information such as expected host and vector range, and together with disease reports, virulence. An important outcome of whole genome sequence comparisons is the ability to determine ‘‘exotic’’ segments in reassortants based on enzootic viruses and have informed policy on the classification of ‘‘exotic’’ on the level of genome segments. Questions related to the genome include: 1. How significant are the observed differences in %GC content of orbiviruses from different vector species? 2. Are there differences in the codon usage of different orbiviruses? 3. What are the molecular mechanisms involved in genetic shift, including segment selection for packaging, virus assembly, and more fit reassortants that may result in the emergence of a novel (more virulent) strain? 4. What is the significance of genetic drift and shift for these viruses? 5. How long does a specific genotype (containing a specific combination of genome segments) persist in the field? 6. Do certain segments form reassortment groups that move together between strains (e.g., genome segments encoding the two outer capsid proteins)? 7. Can reassortment lead to novel antigenic properties/ serotypes? 8. What can segment sequence comparisons tell us about important domains and epitopes that confer virulence, transmissibility, and vector/vertebrate host range? 9. What effect do genetic bottlenecks and quasi-species selection within and between the vector and vertebrate hosts have on virulence and transmissibility? 10. Are there quasi-species in the individual animal, the investigated population, and during transmission (Caporale et al. 2014)? What is their significance? 11. Is there selection of quasi-species during vertical transmission or during reassortment? 12. Do selective pressures favor more virulent strains? Questions related to the enzootic/nonenzootic/incursive topotype characterization criteria include: 1. What is the genetic variability and molecular basis for antigenic variation between different Orbivirus species, serotypes and topotypes? 2. What are the characteristics and distribution of novel Orbivirus species and serotypes? What is our current capability to detect them? 3. What is the significance of reassortment (e.g., between live vaccine strains and wild type viruses, or between enzootic and exotic virus strains)?

KNOWLEDGE GAPS AND TOOLS FOR ORBIVIRUS RESEARCH

4. How should exotic or incursive versus enzootic classification be determined? Should it continue to be based on the VP2 segment, which has yet to show any link to virulence? Or should it be based on a wholegenome percent similarity of genes known, thus far, to play a role in virulence? Or, since reassortment, genetic drift, natural selection pressures, and quasispecies/founder effects all contribute to a spectrum of viruses circulating and emerging, should a 10-digit bar code be used to indicate serotype relatedness/origin of each of the 10 segments individually? 5. With the exception of one recently discovered BTV serotype, both BTV and EHDV require an infected Culicoides midge for transmission. For EHDV, both enzootic and exotic serotypes can be studied in Biosafety Level 2 (BSL-2) facilities. However, exotic serotypes of BTV are considered high risk and can only be studied in BSL-3 and BSL-3Ag facilities. What is the reason for this discrepancy? 6. How many repeated isolations of incursive serotypes must be demonstrated before it is reclassified as enzootic, allowing research at BSL-2 facilities? Tools for Basic Orbivirus Research Reverse genetics

The recent development of reverse genetic technologies for BTV (Boyce et al. 2008) has opened new opportunities for research to explore the detailed genetic and molecular basis for the properties of the virus. Several European research groups have implemented these technologies to generate reassortants and mutants of BTV for fundamental and applied research. These groups were partners in the European network, EMIDA-OrbiNet, which was funded from 2011 to 2014 with the objectives of using and disseminating these technologies. A similar reverse genetics collaboration network is needed in the United States for North American BTV and EHDV serotypes. Reverse genetics allows regeneration of virulent and vaccine-related BTV (van Gennip et al. 2012b, Shaw et al. 2013). These technologies can be used to directly create reassortants in the laboratory (van Gennip et al. 2012b, Shaw et al. 2013, Coetzee et al. 2014a), as well as site-specific modifications of the viral RNAs and proteins, both for vaccine development and fundamental research (van Gennip et al. 2012a, van Gennip et al. 2012b, Celma et al. 2013, Shaw et al. 2013, Feenstra et al. 2014a). Recent studies have included generation of BTV reassortants of newly discovered serotypes (van Rijn, pers. comm.), the original isolates of which (BTV-25) cannot yet be grown in cell culture (Planzer et al. 2011, Vogtlin et al. 2013). In addition, by using tagged recombinant viruses generated by reverse genetic technologies, it is possible to follow their distribution and dissemination in infected cells or tissues (Shaw et al. 2013). Therefore, it will be important to establish collaborations to create and share these reagents to enhance global orbivirus research. Monoclonal antibodies and reference antisera

Antibodies represent vital tools for diagnosis and for research to identify and track individual proteins and epitopes

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at cellular, tissue, and whole animal/vector levels. A registry, and ideally a repository, of reference antisera of either polyclonal or monospecific-single target proteins, is needed. Characterized monoclonal antibodies to specific orbiviruses, specific proteins, and known epitopes would also be important resources. This would support many aspects of fundamental research such as analysis of antigenic variation, diagnostic assay development, confocal microscopy, and transmission studies. Genomic sequence data

Recent advances in sequencing technologies have led to an explosion in the data that are available for different pathogens. The genomes of over 300 BTV strains have already been fully sequenced. This is rapidly becoming the accepted standard for the characterization of novel outbreak strains (Maan et al. 2008, Maan et al. 2010) and has helped to identify new BTV serotypes and new Orbivirus species (Hofmann et al. 2008, Maan et al. 2011a, Belaganahalli et al. 2012). Further developments have included the generation of representative sequence data for each of the different recognized orbivirus species. Reported genomic sequence data have led to many serogroup-specific real-time PCR assays (Hoffmann et al. 2009, Leblanc et al. 2010, van Rijn et al. 2012), including a multiplex real time PCR assay (Wilson et al. 2009), and a conventional reverse transcription PCR (RT-PCR) diagnostic assay (Anthony et al. 2004), as well as the identification of newly emerging serotypes (Chaignat et al. 2009, Allison et al. 2010, Maan et al. 2011a, b, Maclachlan et al. 2013, Gaudreault et al. 2014) and novel orbiviruses (Cowled et al. 2009; Vieira Cde et al. 2009; Al’khovskii et al. 2013, Kapoor et al. 2013, Cooper et al. 2014, Li et al. 2014). A suitable website including a data exchange portal would facilitate comparisons of data for novel isolates to help identify new virus strains more rapidly on the level of each genome segment. This would also greatly facilitate rapid detection of reassortment events in the evolution of novel viruses and tracking virus strain movements (molecular epidemiology, phylogeography). Virus collections

As references for molecular epidemiology, vaccine development, and diagnostic validations, orbivirus strains should be collected and well documented in terms of their date, location, host species, and the clinical signs at point of collection from the field. Ideally these isolates should be centrally stored and available for further study, vaccine development, and as challenge strains from one or more standardized reference collections, such as that established at the Pirbright Institute, United Kingdom (www.reoviridae.org/dsRNA_virus_ proteins/ReoID/virus-nos-by-country.htm). In an effort to make shipping logistics simple, expeditious, and low in cost, one of these standardized reference virus collections should be located in a country that is free from foot and mouth disease. Similarly, virus strains for which the biological characteristics have subsequently been determined should also be stored and made more widely available. In addition to the field history and disease report, the number of cell culture passages of isolates and details of the cells used for these passages is extremely important. BTV and other orbiviruses can be adapted to a range of mammalian cell lines (including BHK

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21 cells, BSR cells, and Vero cells), likely changing the virus properties (particularly virulence). The use of endothelial cell cultures could be more widely explored to avoid adaptation (DeMaula et al. 2002a, b, McLaughlin et al. 2003). Cell culture lines

A collection of suitable cell cultures is crucial for orbivirus research. The C. sonorensis (KC) cell line, for example, originally developed by the Agricultural Research Service/ Arthropod-Borne Animal Disease Research Unit (ARS/ ABADRU) in Laramie, Wyoming, from colonized midges (Wechsler et al. 1989), is highly efficient in isolating Culicoides-borne orbiviruses (usually within a single passage and with minimal loss of virulence). The KC cell line is also valuable to explore a basis for full-genome sequencing, transcriptomics, and proteomics of a known Culicoides vector species (C. sonorensis). It also forms the initial basis for the first full-genome sequencing of any Culicoides species. Two additional cell lines (W3 and W8) were developed at the Laramie lab from wild-caught C. sonorensis. All three Culicoides cell lines are currently available from the ABADRU in Manhattan, Kansas, USA. Vector colonies

Colonies of a nonvector (incompetent vector) species C. nubeculosus, and a known BTV vector species, C. sonorensis (originally derived from the colony developed at ARS/ ABADRU in Laramie) are held at the Pirbright Institute. Three C. sonorensis colonies are now maintained at the ABADRU in Manhattan, Kansas: the AK colony, established from field-caught midges in Idaho; Ausman colony, established from field-caught midges in Colorado; and van Ryn colony, originally from Dr. Brad Mullens, University of California, Riverside, California. These colonies provide a vitally important resource for virus infection, replication, and transmission studies in the insect vector. By selective breeding, the C. sonorensis colony that is now maintained at Pirbright was previously used to generate more highly susceptible and refractory lines. These colonies are now in the process of being re-evaluated. The development of colonies of other Culicoides species would very much help to support comparative studies of vector competence of different vectors for individual orbiviruses and different serotypes and topotypes. One such example is C. imicola, a major vector species for BTV, EHDV, and African horse sickness virus (AHSV) from Africa, the Middle East, and Far East, that has recently expanded its territory to include most of the northern shore of the Mediterranean. Other important (competent) vectors from North America, Africa, Australia, or Europe include C. insignis, C. brevitarsis, C. wadai, C. bolitinos, and members of the C. obsoletus group. Small-animal models

Small-animal models provide cost-effective, simplified systems to generate in vivo data that facilitate development and initial testing of orbivirus vaccine candidates for sheep, cattle, deer, horses, as well as other large-animal target species. Recently, interferon receptor–negative (IFNAR) mouse

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models developed for both BTV and AHSV have been used in vaccinology and vaccine challenge studies (Calvo-Pinilla et al. 2009, Caporale et al. 2011, Castillo-Olivares et al. 2011, Jabbar et al. 2013). Current data indicate that the development of neutralizing antibodies and protective responses after vaccination in these mice show good correlation with protective responses against AHSV in horses. An important aspect of vaccine-challenge studies in any system is the availability of virulent challenge strains for each virus serotype that will reliably generate severe or lethal clinical disease in the animal model. Small-animal models need to be optimized for EHDV (Eschbaumer et al. 2012a, b) and developed for other orbiviruses. Target-host animal models

Target-host animal models are also needed for pathology, vaccinology, and immunology studies. These models must be used for proof-of-concept studies to confirm vaccine safety and efficacy in the target species and to explore vertebrate– host interactions. To improve the comparability of in vivo studies, it would be valuable to have a standardized animal model, including dose and route of infection, as well as a scoring system for clinical signs after challenge to measure the efficacy of vaccination. A system calculating a clinical reaction index has been developed (Huismans et al. 1987) and was slightly modified and used in trials with several ruminant species, including cattle, sheep, and white-tailed deer (Backx et al. 2009, Moulin et al. 2012, van Gennip et al. 2012a, b, Drolet et al. 2013, Feenstra et al. 2014a). This scoring system was also used for cattle and sheep trials by the European network EMIDA-OrbiNet (Celma et al. 2013). Summary

It is obvious that more questions than answers exist in orbivirology. Although progress has been made in molecular tools for studying orbiviruses, many critical basic virology gaps exist, especially in terms of virus–host and virus–vector interactions. These multifaceted, complex systems are difficult due to logistics and lack of commercial reagents, and they are expensive, as is all animal research requiring BSL-2 and BSL-3 containment conditions. In addition, animal models, small and large, as well as further vector models are needed to fully utilize newer molecular techniques, such as reverse genetics, to address fundamental questions and for vaccine development. There is a general consensus of a need for greater international collaboration, and the possibility of a collaborative global network of researchers/organizations to combat these important transboundary diseases. This would include potential repositories of monoclonal antibodies, reference sera, expressed viral proteins, genomic sequence data, virus isolates including recombinants, cell lines, and vector colonies to be shared. Author Disclosure Statement

No competing financial interests exist. References

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Address correspondence to: Barbara S. Drolet US Department of Agriculture Agricultural Research Service Arthropod-Borne Animal Diseases Research Unit 1515 College Avenue Manhattan, KS 66535 E-mail: [email protected]