Development of Real-Time RT-PCR Assays for Detection and Typing of Epizootic Haemorrhagic Disease Virus.

Epizootic haemorrhagic disease virus (EHDV) is an emerging arboviral pathogen of wild and domestic ruminants worldwide. It is closely related to bluetongue virus (BTV) and is transmitted by adult females of competent Culicoides vector species. The EHDV genome consists of ten linear double-stranded (ds)RNA segments, encoding five non-structural and seven structural proteins. Genome-segment reassortment contributes to a high level of genetic variation in individual virus strains, particularly in the areas where multiple and distinct virus lineages co-circulate. In spite of the relatively close relationship between BTV and EHDV herd-immunity to BTV does not appear to protect against the introduction and infection of animals by EHDV. Although EHDV can cause up to 80% morbidity in affected animals, vaccination with the homologous EHDV serotype is protective. Outer-capsid protein VP2, encoded by Seg-2, is the most variable of the EHDV proteins and determines both the specificity of reactions with neutralizing antibodies and consequently the identity of the eight EHDV serotypes. In contrast, VP6 (the viral helicase), encoded by Seg-9, is highly conserved, representing a virus species/serogroup-specific antigen. We report the development and evaluation of quantitative (q)RT-PCR assays targeting EHDV Seg-9 that can detect all EHDV strains (regardless of geographic origin/topotype/serotype), as well as type-specific assays targeting Seg-2 of the eight EHDV serotypes. The assays were evaluated using orbivirus isolates from the 'Orbivirus reference collection' (ORC) at The Pirbright Institute and were shown to be EHDV pan-reactive or type-specific. They can be used for rapid, sensitive and reliable detection and identification (typing) of EHDV RNA from infected blood, tissue samples, homogenized Culicoides, or tissue culture supernatant. None of the assays detected RNA from closely related but heterologous orbiviruses, or from uninfected host animals or cell cultures. The techniques presented could be used for both surveillance and vaccine matching (serotype identification) as part of control strategies for incursions in wild and domestic animal species.

Segmental configuration and putative origin of the reassortant orbivirus, epizootic hemorrhagic disease virus serotype 6, strain Indiana.

In 2006, an exotic reassortant orbivirus, epizootic hemorrhagic disease virus serotype 6 (EHDV-6) [strain (Indiana)], was first detected in the United States. To characterize the reassortment configuration of this virus and to conclusively determine the parental virus of each RNA segment, the complete genome of EHDV-6 (Indiana) was sequenced, in addition to the genomes of representative EHDV-6 and EHDV-2 isolates. Based on genomic comparisons to all other EHDV serotypes, we determined that EHDV-6 (Indiana) originated from a reassortment event between the Australian prototype strain of EHDV-6 (CSIRO 753) and the North American topotype of EHDV-2 (Alberta). Additionally, phylogenetic analysis of all EHDV-6 (Indiana) isolates detected in the United States from 2006 to 2010 suggests that the virus may be undergoing continual reassortment with EHDV-2 (Alberta). In 2010, EHDV-6 (CSIRO 753) was detected in Guadeloupe, demonstrating that the parental virus of the reassortment event is circulating in the Caribbean.

A feline rotavirus G3P[9] carries traces of multiple reassortment events and resembles rare human G3P[9] rotaviruses.

The full-length genome sequence of a feline G3P[9] rotavirus (RV) strain, BA222, identified from the intestinal content of an adult cat, was determined. Strain BA222 possessed a G3-P[9]-I2-R2-C2-M2-A3-N1-T3-E2-H3 genomic constellation, differing substantially from other feline RVs. Phylogenetic analyses of each genome segment revealed common origins with selected animal and zoonotic human RVs, notably with rare multi-reassortant human G3P[9] RVs (Ita/PAI58/96 and Ita/PAH136/96). Altogether, the findings suggest that feline RVs are genetically diverse and that human RVs may occasionally originate either directly or indirectly (via reassortment) from feline RVs.

Detection of a novel reassortant epizootic hemorrhagic disease virus (EHDV) in the USA containing RNA segments derived from both exotic (EHDV-6) and endemic (EHDV-2) serotypes.

Epizootic hemorrhagic disease virus (EHDV) is a Culicoides-transmitted orbivirus that infects domestic and wild ruminants and is provisionally thought to be distributed throughout Africa, North America, Australia, East Asia and the Middle East. Historically, of the seven proposed serotypes of EHDV, only EHDV-1 and EHDV-2 have been reported from North America. In 2006, EHDV isolates were recovered from moribund or dead white-tailed deer (Odocoileus virginianus) in Indiana and Illinois that could not be identified as either EHDV-1 or EHDV-2 by virus neutralization tests or by serotype-specific RT-PCR. Additional serological and genetic testing identified the isolates as EHDV-6, a serotype that, although originally described from Australia, has recently been recognized as an emerging pathogen of cattle in Morocco, Algeria and Turkey. In 2007 and 2008, EHDV-6 was isolated again from white-tailed deer, this time in Missouri, Kansas and Texas, suggesting that the virus is capable of overwintering and that it may become, or already is, endemic in a geographically widespread region of the USA. Genetic characterization of the virus indicates that it is a reassortant, such that the outer capsid proteins determining serotype specificity (VP2 and VP5) are derived from exotic EHDV-6, whilst the remaining structural and non-structural proteins are apparently obtained from indigenous EHDV-2 (Alberta).

Emergence of bluetongue serotypes in Europe, part 2: the occurrence of a BTV-11 strain in Belgium.

An EDTA-blood sample from a cow without clinical signs, which gave early birth to a newborn calf that died soon after delivery, was shown to be positive for bluetongue virus (BTV)-RNA using a group-specific real-time RT-PCR (RT-qPCR). In-house serotype-specific RT-qPCR assays for bluetongue virus serotype 1 (BTV-1), -6 and -8 all gave negative results. Subsequent assays were carried out using conventional (gel-based) RT-PCR primers for all 25 BTV serotypes and only two primer sets, both specific for BTV-11, gave bands of the expected size. The cDNAs generated were sequenced and comparisons of the genome segment 2 sequence with that of the modified 'live' vaccine strain of BTV-11 from South Africa showed 100% identity. A survey of all ruminants in a 1-km area around the first positive farm using a BTV-11 serotype-specific RT-qPCR revealed five other holdings with in total nine BTV-11 positive animals. A cross-sectional monitoring of dairy cattle in Belgium showed an overall prevalence of 3.8% on herd level and 0.2% on animal level. A BTV-11 has been introduced into the Belgian cattle herd during the 2008 vector season. The source of the infection and the way by which the virus was introduced are unknown.

Improved strategies for sequence-independent amplification and sequencing of viral double-stranded RNA genomes.

This paper reports significant improvements in the efficacy of sequence-independent amplification and quality of sequencing of viruses with segmented double-stranded RNA (dsRNA) genomes. We demonstrate that most remaining bottlenecks in dsRNA virus genome characterization have now been eliminated. Both the amplification and sequencing technologies used require no previous sequence knowledge of the viral dsRNA, there is no longer a need to separate genome segments or amplicons and the sequence-determined bias observed in cloning has been overcome. Combining very efficient genome amplification with pyrophosphate-based 454 (GS20/FLX) sequencing enabled sequencing of complete segmented dsRNA genomes and accelerated the sequence analysis of the amplified viral genomes. We report the complete consensus sequence of seven viruses from four different dsRNA virus groups, which include the first complete sequence of the genome of equine encephalosis virus (EEV), the first complete sequence of an African horsesickness virus (AHSV) genome determined directly from a blood sample and a complete human rotavirus genome determined from faeces. We also present the first comparison between the complete consensus sequence of a virulent and an attenuated strain of AHSV1. Ultra-deep sequencing (>400-fold coverage) of the AHSV1 reference and attenuated strains revealed different ratios of reassortants in the reference strain and allowed quasispecies detection in the plaque-purified attenuated strain of AHSV1. This approach amounts to a paradigm shift in dsRNA virus research, since it is sensitive and specific enough for comprehensive investigations of the evolution and genetic diversity in dsRNA virus populations.

Design of primers and use of RT-PCR assays for typing European bluetongue virus isolates: differentiation of field and vaccine strains.

Bluetongue virus (BTV) is the causative agent of bluetongue, a disease of ruminant livestock that occurs almost worldwide between latitudes 3 degrees S and 5 degrees N. There are 24 serotypes of BTV (currently identified by serum neutralization assays). Since 1998, eight strains of six BTV serotypes (1, 2, 4, 8, 9 and 16) have invaded Europe. The most variable BTV protein is major outer-capsid component VP2, encoded by segment 2 (Seg-2) of the double-stranded RNA virus genome. VP2 represents the major target for neutralizing (and protective) antibodies that are generated in response to BTV infection, and is therefore the primary determinant of virus serotype. RT-PCR primers and assays targeting Seg-2 have been developed for rapid identification (within 24 h) of the six European BTV types. These assays are sensitive, specific and show perfect agreement with the results of conventional virus-neutralization methods. Previous studies have identified sequence variations in individual BTV genome segments that allow different isolates to be grouped on the basis of their geographical origins (topotypes). The assays described in this paper can detect any of the BTV isolates of the homologous serotype that were tested from different geographical origins (different Seg-2 topotypes). Primers were also identified that could be used to distinguish members of these different Seg-2 topotypes, as well as field and vaccine strains of most of the European BTV serotypes. The serotype-specific assays (and primers) showed no cross-amplification when they were evaluated with multiple isolates of the most closely related BTV types or with reference strains of the remaining 24 serotypes. Primers developed in this study will be updated periodically to maintain their relevance to current BTV distribution and epidemiology (http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/rt-pcr-primers.htm).

VP2-segment sequence analysis of some isolates of bluetongue virus recovered in the Mediterranean basin during the 1998-2003 outbreak.

The complete nucleotide sequences of the VP2 segments of bluetongue virus (BTV) isolates recovered from Italy, Greece and Israel, from 1998 to 2003, were determined. Phylogenetic analysis of these sequences, those from related viruses and the South African vaccine strains, were used to determine the probable geographic origin of BTV incursions into Italy. Results indicated that viruses from each of the four serotypes isolated in Italy (2, 4, 9 and 16) possibly had a different origin. Analysis of the bluetongue virus serotype 2 (BTV-2) isolates gave evidence that this serotype probably moved from Tunisia. BTV-4 results showed probable incursion from the southwest and not from Greece or Israel. BTV-9 isolates clearly have an eastern origin (most probably Greece), whereas BTV-16 isolates are indistinguishable from the BTV-16 live attenuated vaccine strain. The phylogenetic findings were supported by polyacrylamide gel electrophoresis (PAGE) analysis of the complete amplified genome of each isolate except for BTV-16 Italian field isolate, which showed a slightly different PAGE profile. A combination of the complete VP2 sequencing and PAGE analysis of complete genomes, allowed not only phylogenetic analysis, but also vaccine detection and assessment of reassortment events.

Serogroup-reactive and type-specific detection of bluetongue virus antibodies using chicken scFvs in inhibition ELISAs.

Two bluetongue virus (BTV) serotype 10-specific single-chain Fv chicken antibody fragments (scFvs) were evaluated in a competitive ELISA. The binding of one (F3) to purified BTV was only inhibited by antibodies against the homologous serotype. The binding of the other (F10) was blocked by antisera to each of the 24 BTV serotypes. F10 recognised VP7, a major structural protein of the BTV core, but not if the protein was directly adsorbed to a plastic surface. It did, however, bind to recombinant VP7 that had been captured from suspension by rabbit IgG. This made it possible to develop an scFv based inhibition ELISA for BTV antibodies using recombinant VP7 without prior purification. The resulting immunoassay detected antibodies to 24 BTV serotypes, but not those directed against three serotypes of the related epizootic haemorrhagic disease virus. A phage library displaying fusion peptides expressed by fragments of the BTV genome segment 7 cDNA was constructed and screened using F10. Comparing selected peptides with the amino acid sequence of VP7 showed that recognition by the scFv required at least 131 residues representing the protein's upper domain. By providing well-characterised immunological reagents, recombinant antibody technology can contribute to the development of improved immunoassays for BTV diagnosis.

VP2 gene sequence analysis of some isolates of bluetongue virus recovered in the Mediterranean Basin during the 1998-2002 outbreak.

Since 1998, five serotypes of bluetongue virus (BTV), BTV-1, BTV-2, BTV-4, BTV-9 and BTV-16, have been reported in countries surrounding the Mediterranean Basin. Preliminary data on the sequencing analysis of the VP2-genes of BTV isolates recovered during the 1998-2002 epizootic of BT in Italy, Greece and Israel were studied. The VP2-genes of the Italian BTV-2 and BTV-9, Greek BTV-4 and BTV-9, Israeli BTV-4 and BTV-16 and South African BTV-2, BTV-4, BTV-9 and BTV-16, together with those of their corresponding South African serotype reference and vaccine strains, were cloned and the sequences of their terminal ends determined. These sequences, as well as those of all BTV VP2-gene sequences currently available on GenBank, were used to compile a phylogenetic tree to determine the probable geographic origins of the BTV incursions into Europe. The Italian isolates included in this study were from different regions, animal hosts and years (2000-2002). The results demonstrated that sequencing of the terminal end of the VP2-gene of BTV can be used for topotyping. According to the phylogenetic analysis, the Italian BTV-2 and BTV-9 isolates were stable across all species, irrespective of geographic origin and year of isolation. The sequencing data of the Italian isolates were identical to those of a BTV-2 isolate from Corsica. There was 97% homology between the Italian and Corsican BTV-2 isolates and the BTV-2 vaccine and reference isolates from South Africa. Italian BTV-9 isolates were also identical to the Greek BTV-9 isolates (99% homology). Surprisingly these BTV-9 isolates had only 67% homology with the reference BTV-9 isolate from South Africa. Conversely, BTV-9 field isolates from Australia and elsewhere in Europe had 89% homology with the Italian isolate at the nucleic acid level. Greek and Israeli BTV-4 isolates were almost identical (98% homology) and shared a 90% homology with the BTV-4 South African reference and vaccine strains. Israeli BTV-16 and South African BTV-16 reference strains were also similar. From these results, it may be concluded that Italian and Corsican BTV-2, Israeli and Greek BTV-4, and South African and Israeli BTV-16 had a common origin. The Greek BTV-9 isolate had more than 99% homology with the isolates from Italy, indicating these isolates to have had a common origin. The European BTV-9 isolates, grouped as 'eastern isolates', were more similar to the Australian isolates than to the South African reference strains.

A first full outer capsid protein sequence data-set in the Orbivirus genus (family Reoviridae): cloning, sequencing, expression and analysis of a complete set of full-length outer capsid VP2 genes of the nine African horsesickness virus serotypes.

The outer capsid protein VP2 of African horsesickness virus (AHSV) is a major protective antigen. We have cloned full-length VP2 genes from the reference strains of each of the nine AHSV serotypes. Baculovirus recombinants expressing the cloned VP2 genes of serotypes 1, 2, 4, 6, 7 and 8 were constructed, confirming that they all have full open reading frames. This work completes the cloning and expression of the first full set of AHSV VP2 genes. The clones of VP2 genes of serotypes 1, 2, 5, 7 and 8 were sequenced and their amino acid sequences were deduced. Our sequencing data, together with that of the published VP2 genes of serotypes 3, 4, 6 and 9, were used to generate the first complete sequence analysis of all the (sero)types for a species of the Orbivirus genus. Multiple alignment of the VP2 protein sequences showed that homology between all nine AHSV serotypes varied between 47.6 % and 71.4 %, indicating that VP2 is the most variable AHSV protein. Phylogenetic analysis grouped together the AHSV VP2s of serotypes that cross-react serologically. Low identity between serotypes was demonstrated for specific regions within the VP2 amino acid sequences that have been shown to be antigenic and play a role in virus neutralization. The data presented here impact on the development of new vaccines, the identification and characterization of antigenic regions, the development of more rapid molecular methods for serotype identification and the generation of comprehensive databases to support the diagnosis, epidemiology and surveillance of AHS.

Variation in the NS3 gene and protein in South African isolates of bluetongue and equine encephalosis viruses.

Bluetongue virus (BTV) and equine encephalosis virus (EEV) are agriculturally important orbiviruses transmitted by biting midges of the genus Culicoides. The smallest viral genome segment, S10, encodes two small nonstructural proteins, NS3 and NS3A, which mediate the release of virus particles from infected cells and may subsequently influence the natural dispersion of these viruses. The NS3 gene and protein sequences of South African isolates of these viruses were determined, analysed and compared with cognate orbivirus genes from around the world. The South African BTV NS3 genes were found to have the highest level of sequence variation for BTV (20 %), while the highest level of protein variation of BTV NS3 (10 %) was found between South African and Asian BTV isolates. The inferred NS3 gene phylogeny of the South African BTV isolates grouped them with BTV isolates from the United States, while the Asian BTV isolates grouped into a separate lineage. The level of variation found in the NS3 gene and protein of EEV was higher than that found for BTV and reached 25 and 17 % on the nucleotide and amino acid levels, respectively. The EEV isolates formed a lineage independent from that of the other orbiviruses. This lineage segregated further into two clusters that corresponded to the northern and southern regions of South Africa. The geographical distribution of these isolates may be related to the distribution of the Culicoides subspecies that transmit them.

Cloning of complete genome sets of six dsRNA viruses using an improved cloning method for large dsRNA genes.

Cloning full-length large (>3 kb) dsRNA genome segments from small amounts of dsRNA has thus far remained problematic. Here, a single-primer amplification sequence-independent dsRNA cloning procedure was perfected for large genes and tailored for routine use to clone complete genome sets or individual genes. Nine complete viral genome sets were amplified by PCR, namely those of two human rotaviruses, two African horsesickness viruses (AHSV), two equine encephalosis viruses (EEV), one bluetongue virus (BTV), one reovirus and bacteriophage Phi12. Of these amplified genomes, six complete genome sets were cloned for viruses with genes ranging in size from 0.8 to 6.8 kb. Rotavirus dsRNA was extracted directly from stool samples. Co-expressed EEV VP3 and VP7 assembled into core-like particles that have typical orbivirus capsomeres. This work presents the first EEV sequence data and establishes that EEV genes have the same conserved termini (5' GUU and UAC 3') and coding assignment as AHSV and BTV. To clone complete genome sets, one-tube reactions were developed for oligo-ligation, cDNA synthesis and PCR amplification. The method is simple and efficient compared to other methods. Complete genomes can be cloned from as little as 1 ng dsRNA and a considerably reduced number of PCR cycles (22-30 cycles compared to 30-35 of other methods). This progress with cloning large dsRNA genes is important for recombinant vaccine development and determination of the role of terminal sequences for replication and gene expression.

Development of probes for typing African horsesickness virus isolates using a complete set of cloned VP2-genes.

A set of cloned full-length VP2-genes from the reference strain of each of the nine serotypes of African horsesickness virus (AHSV) was used to develop probes for typing AHSV isolates. The VP2-gene probes hybridised serotype-specific to purified viral dsRNA from its corresponding serotype. No cross-hybridisation was observed between the different AHSV serotypes or with RNA from equine encephalosis virus or bluetongue virus (BTV) which are related viruses within the genus Orbivirus that co-circulate with AHSV in South Africa. The probes were able to detect AHSV isolates from recent field cases of AHS in South Africa, despite being derived from historical reference strains. With regard to sensitivity and time considerations: radioactive 32P-labelling resulted in a marginal increase in sensitivity over digoxigenin-labelled probes. By infecting cell cultures at different multiplicities of infection (m.o.i.) and harvesting at various times post infection, it was established that AHSV RNA could be detected 16 h post infection (p.i.) at a m.o.i. of 1.00 pfu per cell and 48 h p.i. at a m.o.i. of 0.01 pfu per cell. Typing of AHSV isolates by means of VP2-gene probe hybridisation can be completed in 4 days, which is less than half the time required for conventional isolation and serotyping. This report on the use of a complete set of cloned AHSV VP2-gene probes is the first demonstration of typing for a whole specie (serogroup) in a genus of the family Reoviridae.