Virus-Binding Proteins Recovered from Bacterial Culture Derived from Activated Sludge by Affinity Chromatography Assay Using a Viral Capsid Peptide

The contamination of water environments by pathogenic viruses has raised concerns about outbreaks of viral infectious diseases in our society. Because conventional water and wastewater treatment systems are not effective enough to inactivate or remove pathogenic viruses, a new technology for virus removal needs to be developed. In this study, the virus-binding proteins (VBPs) in a bacterial culture derived from activated sludge were successfully recovered. The recovery of VBPs was achieved by applying extracted crude proteins from a bacterial culture to an affinity column in which a custom-made peptide of capsid protein from the poliovirus type 1 (PV1) Mahoney strain (H₂N-DNPASTTNKDKL-COOH) was immobilized as a ligand. VBPs exhibited the ability to adsorb infectious particles of PV1 Sabin 1 as determined by enzyme-linked immunosorbent assay. The evaluation of surface charges of VBPs with ion-exchange chromatography found that a majority of VBP molecules had a net negative charge under the conditions of affinity chromatography. On the other hand, a calculated isoelectric point implied that the viral peptide in the affinity column was also charged negatively. As a result, the adsorption of the VBPs to the viral peptide in the affinity column occurred with a strong attractive force that was able to overcome the electrostatic repulsive force. Two-dimensional electrophoresis revealed that the isolated VBPs include a number of proteins, and their molecular masses were widely distributed but smaller than 100 kDa. Amino acid sequences of N termini of five VBPs were determined. Homology searches for the N termini against all protein sequences in the National Center for Biotechnology Information (NCBI) database showed that the isolated VBPs in this study were newly discovered proteins. These VBPs that originated with bacteria in activated sludge might be stable, because they are existing in the environment of wastewater treatments. Therefore, a virus removal technology utilizing VBPs as viral adsorbents can be developed, since it is possible to replicate VBPs by protein cloning techniques.     

Construction of a cloning system for the mass production of a virus-binding protein specific for poliovirus type 1

In our previous study, virus-binding proteins (VBPs) demonstrating the ability to strongly bind poliovirus type 1 (PV1) were recovered from a bacterial culture derived from activated sludge. The isolated VBPs would be useful as viral adsorbents for water and wastewater treatments. The VBP gene of activated sludge bacteria was isolated, and the cloning system of the VBP was established. The isolation of the VBP gene from DNA libraries for activated sludge bacteria was achieved with the colony hybridization technique. The sequence of the VBP gene consisted of 807 nucleotides encoding 268 amino acids. Fifteen amino acid sequences were retrieved from 2,137,877 sequences by a homology search using the BLAST server at the National Center for Biotechnology Information. The protein encoded in the isolated genome was considered to be a newly discovered protein from activated sludge culture, because any sequences in protein databases were not perfectly matched with the sequence of the VBP. It was confirmed that Escherichia coli BL21 transformed by pRSET carrying the isolated VBP gene could extensively produce the VBP clones. Enzyme-linked immunosorbent assay (ELISA) revealed that the VBP clone exhibited the binding ability with intact particles of PV1. The equilibrium binding constant between PV1 and VBP in the ELISA well was estimated to be 2.1 x 10(7) (M(-1)), which also indicated that the VBP clones have a high affinity with the PV1 particle. The VBP cloning system developed in this study would make it possible to produce a mass volume of VBPs and to utilize them as a new material of the specific adsorbent in several technologies, including virus removal, concentration, and detection.     

Construction of a Cloning System for the Mass Production of a Virus-Binding Protein Specific for Poliovirus Type 1

In our previous study, virus-binding proteins (VBPs) demonstrating the ability to strongly bind poliovirus type 1 (PV1) were recovered from a bacterial culture derived from activated sludge. The isolated VBPs would be useful as viral adsorbents for water and wastewater treatments. The VBP gene of activated sludge bacteria was isolated, and the cloning system of the VBP was established. The isolation of the VBP gene from DNA libraries for activated sludge bacteria was achieved with the colony hybridization technique. The sequence of the VBP gene consisted of 807 nucleotides encoding 268 amino acids. Fifteen amino acid sequences were retrieved from 2,137,877 sequences by a homology search using the BLAST server at the National Center for Biotechnology Information. The protein encoded in the isolated genome was considered to be a newly discovered protein from activated sludge culture, because any sequences in protein databases were not perfectly matched with the sequence of the VBP. It was confirmed that Escherichia coli BL21 transformed by pRSET carrying the isolated VBP gene could extensively produce the VBP clones. Enzyme-linked immunosorbent assay (ELISA) revealed that the VBP clone exhibited the binding ability with intact particles of PV1. The equilibrium binding constant between PV1 and VBP in the ELISA well was estimated to be 2.1 × 10⁷ (M⁻¹), which also indicated that the VBP clones have a high affinity with the PV1 particle. The VBP cloning system developed in this study would make it possible to produce a mass volume of VBPs and to utilize them as a new material of the specific adsorbent in several technologies, including virus removal, concentration, and detection.     

Viability of poliovirus/rhinovirus VPg chimeric viruses and identification of an amino acid residue in the VPg gene critical for viral RNA replication

Picornaviral RNA replication utilizes a small virus-encoded protein, termed 3B or VPg, as a primer to initiate RNA synthesis. This priming step requires uridylylation of the VPg peptide by the viral polymerase protein 3D(pol), in conjunction with other viral or host cofactors. In this study, we compared the viral specificity in 3D(pol)-catalyzed uridylylation reactions between poliovirus (PV) and human rhinovirus 16 (HRV16). It was found that HRV16 3D(pol) was able to uridylylate PV VPg as efficiently as its own VPg, but PV 3D(pol) could not uridylylate HRV16 VPg. Two chimeric viruses, PV containing HRV16 VPg (PV/R16-VPg) and HRV16 containing PV VPg (R16/PV-VPg), were constructed and tested for replication capability in H1-HeLa cells. Interestingly, only PV/R16-VPg chimeric RNA produced infectious virus particles upon transfection. No viral RNA replication or cytopathic effect was observed in cells transfected with R16/PV-VPg chimeric RNA, despite the ability of HRV16 3D(pol) to uridylylate PV VPg in vitro. Sequencing analysis of virion RNA isolated from the virus particles generated by PV/R16-VPg chimeric RNA identified a single residue mutation in the VPg peptide (Glu(6) to Val). Reverse genetics confirmed that this mutation was highly compensatory in enhancing replication of the chimeric viral RNA. PV/R16-VPg RNA carrying this mutation replicated with similar kinetics and magnitude to wild-type PV RNA. This cell culture-induced mutation in HRV16 VPg moderately increased its uridylylation by PV 3D(pol) in vitro, suggesting that it might be involved in other function(s) in addition to the direct uridylylation reaction. This study demonstrated the use of chimeric viruses to characterize viral specificity and compatibility in vivo between PV and HRV16 and to identify critical amino acid residue(s) for viral RNA replication.     

Viral composition and context in metagenomes from biofilm and suspended growth municipal wastewater treatment plants

Wastewater treatment plants (WWTPs) contain high density and diversity of viruses which can significantly impact microbial communities in aquatic systems. While previous studies have investigated viruses in WWTP samples that have been specifically concentrated for viruses and filtered to exclude bacteria, little is known about viral communities associated with bacterial communities throughout wastewater treatment systems. Additionally, differences in viral composition between attached and suspended growth wastewater treatment bioprocesses are not well characterized. Here, shotgun metagenomics was used to analyse wastewater and biomass from transects through two full-scale WWTPs for viral composition and associations with bacterial hosts. One WWTP used a suspended growth activated sludge bioreactor and the other used a biofilm reactor (trickling filter). Myoviridae, Podoviridae and Siphoviridae were the dominant viral families throughout both WWTPs, which are all from the order Caudovirales. Beta diversity analysis of viral sequences showed that samples clustered significantly both by plant and by specific sampling location. For each WWTP, the overall bacterial community structure was significantly different than community structure of bacterial taxa associated with viral sequences. These findings highlight viral community composition in transects through different WWTPs and provide context for dsDNA viral sequences in bacterial communities from these systems.     

Norovirus‐binding proteins recovered from activated sludge micro‐organisms with an affinity to a noroviral capsid peptide

Aims: Transmission routes of noroviruses, leading aetiological agents of acute gastroenteritis, are rarely verified when outbreaks occur. Because the destination of norovirus particles being firmly captured by micro‐organisms could be totally different from that of those particles moving freely, micro‐organisms with natural affinity ligands such as virus‐binding proteins would affect the fate of viruses in environment, if such microbial affinity ligands exist. The aim of this study is to identify norovirus‐binding proteins (NoVBPs) that are presumably working as natural ligands for norovirus particles in water environments. Methods and Results: NoVBPs were recovered from activated sludge micro‐organisms by an affinity chromatography technique in which a capsid peptide of norovirus genogroup II (GII) was immobilized. The recovered NoVBPs bind to norovirus‐like particles (NoVLPs) of norovirus GII, and this adsorption was stronger than that to NoVLPs of norovirus genogroup I. The profile of two‐dimensional electrophoresis of NoVBPs showed that the recovered NoVBPs included at least seven spots of protein. The determination of N‐terminal amino acid sequences of these NoVBPs revealed that hydrophobic interactions could contribute to the adsorption between NoVBPs and norovirus particles. Conclusions: NoVBPs conferring a high affinity to norovirus GII were successfully isolated from activated sludge micro‐organisms. Significance and Impact of the Study: NoVBPs could be natural viral ligands and play an important role in the NoV transmission.     

Peptides derived from HIV-1, HIV-2, Ebola virus, SARS coronavirus and coronavirus 229E exhibit high affinity binding to the formyl peptide receptor

Peptides derived from the membrane proximal region of fusion proteins of human immunodeficiency viruses 1 and 2, Coronavirus 229 E, severe acute respiratory syndrome coronavirus and Ebola virus were all potent antagonists of the formyl peptide receptor expressed in Chinese hamster ovary cells. Binding of viral peptides was affected by the naturally occurring polymorphisms at residues 190 and 192, which are located at second extracellular loop-transmembrane helix 5 interface. Substitution of R190 with W190 enhanced the affinity for a severe acute respiratory syndrome coronavirus peptide 6 fold but reduced the affinity for N-formyl-Nle-Leu-Phe by 2.5 fold. A 12 mer peptide derived from coronavirus 229E (ETYIKPWWVWL) was the most potent antagonist of the formyl peptide receptor W190 with a K(i) of 230 nM. Fluorescently labeled ETYIKPWWVWL was effectively internalized by all three variants with EC(50) of approximately 25 nM. An HKU-1 coronavirus peptide, MYVKWPWYVWL, was a potent antagonist but N-formyl-MYVKWPWYVWL was a potent agonist. ETYIKPWWVWL did not stimulate GTPgammaS binding but inhibited the stimulation by formyl-NleLeuPhe. It also blocked beta arrestin translocation and receptor downregulation induced by formyl-Nle-Leu-Phe. This indicates that formyl peptide receptor may be important in viral infections and that variations in its sequence among individuals may affect their likelihood of viral and bacterial infections.     

Poly(rC) binding proteins and the 5' cloverleaf of uncapped poliovirus mRNA function during de novo assembly of polysomes

Poliovirus (PV) mRNA is unusual because it possesses a 5'-terminal monophosphate rather than a 5'-terminal cap. Uncapped mRNAs are typically degraded by the 5' exonuclease XRN1. A 5'-terminal cloverleaf RNA structure interacts with poly(rC) binding proteins (PCBPs) to protect uncapped PV mRNA from 5' exonuclease (K. E. Murray, A. W. Roberts, and D. J. Barton, RNA 7:1126-1141, 2001). In this study, we examined de novo polysome formation using HeLa cell-free translation-replication reactions. PV mRNA formed polysomes coordinate with the time needed for ribosomes to traverse the viral open reading frame (ORF). Nascent PV polypeptides cofractionated with viral polysomes, while mature PV proteins were released from the polysomes. Alterations in the size of the PV ORF correlated with alterations in the size of polysomes with ribosomes present every 250 to 500 nucleotides of the ORF. Eukaryotic initiation factor 4GI (eIF4GI) was cleaved rapidly as viral polysomes assembled and the COOH-terminal portion of eIF4GI cofractionated with viral polysomes. Poly(A) binding protein, along with PCBP 1 and 2, also cofractionated with viral polysomes. A C24A mutation that inhibits PCBP-5'-terminal cloverleaf RNA interactions inhibited the formation and stability of nascent PV polysomes. Kinetic analyses indicated that the PCBP-5' cloverleaf RNA interaction was necessary to protect PV mRNA from 5' exonuclease immediately as ribosomes initially traversed the viral ORF, before viral proteins could alter translation factors within nascent polysomes or contribute to ribonucleoprotein complexes at the termini of the viral mRNA.     

Rescue of functional replication origins from embedded configurations in a plasmid carrying the adenovirus genome.

A variant of the adenovirus type 5 genome which lacks EcoRI sites has been cloned in a bacterial plasmid after the addition of EcoRI oligonucleotide linkers to its ends. Closed circular forms of the recombinant viral genome were not infectious upon their introduction into permissive eucaryotic cells. The linear genome released by digestion of the 39-kilobase recombinant plasmid (pXAd) with EcoRI produced infectious virus at about 5% of the level of wild-type controls. The viruses which arose were indistinguishable from the parental strain, and the normal termini of the viral genome had been restored. Marker rescue experiments demonstrate that provision of a DNA fragment with a normal viral end improves infectivity. When a small fragment carrying a wild-type left end (the 0 to 2.6% ClaI-B fragment) was ligated to ClaI-linearized pXAd, virus was produced with efficiencies comparable to a similar reconstitution of the two ClaI fragments of the wild-type genome. These viruses stably carry the left-end fragment at both ends, leaving the normal right end embedded in 950 base pairs of DNA. The embedded right origin is inactive. The consensus of the analyses reported here is that a free end is a necessary configuration for the sequences which make up the adenovirus origin of replication.     

Isolation of Recombinant Phage Antibodies Targeting the Hemagglutinin Cleavage Site of Highly Pathogenic Avian Influenza Virus

Highly pathogenic avian influenza (HPAI) H5N1 viruses, which have emerged in poultry and other wildlife worldwide, contain a characteristic multi-basic cleavage site (CS) in the hemagglutinin protein (HA). Because this arginine-rich CS is unique among influenza virus subtypes, antibodies against this site have the potential to specifically diagnose pathogenic H5N1. By immunizing mice with the CS peptide and screening a phage display library, we isolated four antibody Fab fragment clones that specifically bind the antigen peptide and several HPAI H5N1 HA proteins in different clades. The soluble Fab fragments expressed in Escherichia coli bound the CS peptide and the H5N1 HA protein with nanomolar affinity. In an immunofluorescence assay, these Fab fragments stained cells infected with HPAI H5N1 but not those infected with a less virulent strain. Lastly, all the Fab clones could detect the CS peptide and H5N1 HA protein by open sandwich ELISA. Thus, these recombinant Fab fragments will be useful novel reagents for the rapid and specific detection of HPAI H5N1 virus.     

Interactions of Cryptosporidium parvum, Giardia lamblia, vaccinal poliovirus type 1, and bacteriophages phiX174 and MS2 with a drinking water biofilm and a wastewater biofilm

Biofilms colonizing surfaces inside drinking water distribution networks may provide a habitat and shelter to pathogenic viruses and parasites. If released from biofilms, these pathogens may disseminate in the water distribution system and cause waterborne diseases. Our study aimed to investigate the interactions of protozoan parasites (Cryptosporidium parvum and Giardia lamblia [oo]cysts) and viruses (vaccinal poliovirus type 1, phiX174, and MS2) with two contrasting biofilms. First, attachment, persistence, and detachment of the protozoan parasites and the viruses were assessed with a drinking water biofilm. This biofilm was allowed to develop inside a rotating annular reactor fed with tap water for 7 months prior to the inoculation. Our results show that viable parasites and infectious viruses attached to the drinking water biofilm within 1 h and persisted within the biofilm. Indeed, infectious viruses were detected in the drinking water biofilm up to 6 days after the inoculation, while viral genome and viable parasites were still detected at day 34, corresponding to the last day of the monitoring period. Since viral genome was detected much longer than infectious particles, our results raise the question of the significance of detecting viral genomes in biofilms. A transfer of viable parasites and viruses from the biofilm to the water phase was observed after the flow velocity was increased but also with a constant laminar flow rate. Similar results regarding parasite and virus attachment and detachment were obtained using a treated wastewater biofilm, suggesting that our observations might be extrapolated to a wide range of environmental biofilms and confirming that biofilms can be considered a potential secondary source of contamination.     

Reprint of: Virus-binding proteins and their roles in shrimp innate immunity

Disease outbreaks caused by viral pathogens constitute a major limitation to development of the shrimp aquaculture industry. Many research have been conducted to better understand how host shrimp respond to viral infections with the aim of using the gained knowledge to develop better strategies for disease management and control. One approach has been to study the interactions between host and viral proteins, and particularly host virus-binding proteins that might play an important role in the viral infection process. Within the past five years, increasing numbers of virus-binding proteins (VBPs) have been reported in shrimp. Characterization of these molecules has emphasized on their potential therapeutic applications by demonstrating their activities in inhibition of viral replication via in vivo neutralization assay. However, signaling to induce innate antiviral immune responses as a consequence of binding between viral proteins and VBPs remain to be fully elucidated.     

Agrobacterium VirD2-binding protein is involved in tumorigenesis and redundantly encoded in conjugative transfer gene clusters

Agrobacterium tumefaciens can transfer oncogenic T-DNA into plant cells; T-DNA transfer is mechanistically similar to a conjugation process. VirD2 is the pilot protein that guides the transfer, because it is covalently associated with single-stranded T-DNA to form the transfer substrate T-complex. We used the VirD2 protein as an affinity ligand to isolate VirD2-binding proteins (VBPs). By pull-down assays and peptide-mass-fingerprint matching, we identified an A. tumefaciens protein designated VBP1 that could bind VirD2 directly. Genome-wide sequence analysis showed that A. tumefaciens has two additional genes encoding proteins highly similar to VBP1, designated vbp2 and vbp3. Like VBP1, both VBP2 and VBP3 also could bind VirD2; all three VBPs contain a putative nucleotidyltransferase motif. Mutational analysis of vbp demonstrated that the three vbp genes could functionally complement each other. Consequently, only inactivation of all three vbp genes highly attenuated the bacterial ability to cause tumors on plants. Although vbp1 is harbored on the megaplasmid pAtC58, vbp2 and vbp3 reside on the linear chromosome. The vbp genes are clustered with conjugative transfer genes, suggesting linkage between the conjugation and virulence factor. The three VBPs appear to contain C-terminal positively charged residues, often present in the transfer substrate proteins of type IV secretion systems. Inactivation of the three vbp genes did not affect the T-strand production. Our data indicate that VBP is a newly identified virulence factor that may affect the transfer process subsequent to T-DNA production.     

Analysis of the Glycyrrhizic Acid-Binding Sites with Phage Display

Phages that expose peptides specifically interacting with glycyrrhizic acid (GA) were selected from a phage peptide library by affinity selection and ELISA. Amino acid sequence analysis of the selected peptides and human proteins with the SIM program revealed homology to tyrosine protein kinases, serine/threonine protein kinases, tyrosine phosphatases, and some receptors. Analysis of the peptide and virus protein sequences with the BLAST program showed that GA has affinity for various surface proteins of several human viruses such as HIV-1, hepatitis C virus, and herpesviruses.     

The influenza A virus M2 cytoplasmic tail is required for infectious virus production and efficient genome packaging

The M2 integral membrane protein encoded by influenza A virus possesses an ion channel activity that is required for efficient virus entry into host cells. The role of the M2 protein cytoplasmic tail in virus replication was examined by generating influenza A viruses encoding M2 proteins with truncated C termini. Deletion of 28 amino acids (M2Stop70) resulted in a virus that produced fourfold-fewer particles but >1,000-fold-fewer infectious particles than wild-type virus. Expression of the full-length M2 protein in trans restored the replication of the M2 truncated virus. Although the M2Stop70 virus particles were similar to wild-type virus in morphology, the M2Stop70 virions contained reduced amounts of viral nucleoprotein and genomic RNA, indicating a defect in vRNP packaging. The data presented indicate the M2 cytoplasmic tail plays a role in infectious virus production by coordinating the efficient packaging of genome segments into influenza virus particles.     

Massive peptide sharing between viral and human proteomes

Thirty viral proteomes were examined for amino acid sequence similarity to the human proteome, and, in parallel, a control of 30 sets of human proteins was analyzed for internal human overlapping. We find that all of the analyzed 30 viral proteomes, independently of their structural or pathogenic characteristics, present a high number of pentapeptide overlaps to the human proteome. Among the examined viruses, human T-lymphotropic virus 1, Rubella virus, and hepatitis C virus present the highest number of viral overlaps to the human proteome. The widespread and ample distribution of viral amino acid sequences through the human proteome indicates that viral and human proteins are formed of common peptide backbone units and suggests a fluid compositional chimerism in phylogenetic entities canonically classified distantly as viruses and Homo sapiens. Importantly, the massive viral to human peptide overlapping calls into question the possibility of a direct causal association between virus-host sharing of amino acid sequences and incitement to autoimmune reactions through molecular recognition of common motifs.     

Effect of Cecropin B and a Synthetic Analogue on Propagation of Fish Viruses In Vitro

Abstract Cecropins and other natural antimicrobial peptides are widely distributed in animals from insects to mammals. These proteins have been shown to be major constituents of the innate immune systems of animals for nonspecific defense of the host against various bacteria and parasites. Therefore, exploitation of this natural innate defense system may lead to the development of effective methods for protecting fish from invasion by microbial pathogens. Recently, we have demonstrated that the introduction of cecropin transgenes into Japanese medaka (Oryzias latipes) conferred resistance to infection by fish bacterial pathogens. Aside from a few reports documenting the antiviral effect of antimicrobial peptides including cecropins against mammalian viruses, there is no evidence for the effect of these peptides against fish viruses. In this article we present results of in vitro characterization of native cecropin B and a synthetic analogue, CF17, against several important fish viral pathogens—namely, infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus (VHSV), snakehead rhabdovirus (SHRV), and infectious pancreatic necrosis virus (IPNV). Upon coincubation of these peptides and viruses, the viral titers yielded in fish cells were reduced from several fold to 104-fold. Direct disruption of the viral envelope and disintegration of the viral capsids may be involved in the inhibition of viral replication by the peptides. Results of our studies demonstrate the potential of manipulating antimicrobial peptide genes by transgenesis to combat viral infection in fish.     

A peptide from autoantigen La blocks poliovirus and hepatitis C virus cap-independent translation and reveals a single tyrosine critical for La RNA binding and translation stimulation

La, a 52-kDa autoantigen in patients with systemic lupus erythematosus, was one of the first cellular proteins identified to interact with viral internal ribosome entry site (IRES) elements and stimulate poliovirus (PV) and hepatitis C virus (HCV) IRES-mediated translation. Previous results from our laboratory have shown that a small, yeast RNA (IRNA) could selectively inhibit PV and HCV IRES-mediated translation by sequestering the La protein. Here we have identified an 18-amino-acid-long sequence from the N-terminal "La motif" which is required for efficient interaction of La with IRNA and viral 5' untranslated region (5'-UTR) elements. A synthetic peptide (called LAP, for La peptide) corresponding to this sequence (amino acids 11 to 28) of La was found to efficiently inhibit viral IRES-mediated translation in vitro. The LAP efficiently enters Huh-7 cells and preferentially inhibits HCV IRES-mediated translation programmed by a bicistronic RNA in vivo. The LAP does not bind RNA directly but appears to block La binding to IRNA and PV 5'-UTR. Competition UV cross-link and translation rescue experiments suggested that LAP inhibits IRES-mediated translation by interacting with proteins rather than RNA. Mutagenesis of LAP demonstrates that single amino acid changes in a highly conserved sequence within LAP are sufficient to eliminate the translation-inhibitory activity of LAP. When one of these mutations (Y23Q) is introduced into full-length La, the mutant protein is severely defective in interacting with the PV IRES element and consequently unable to stimulate IRES-mediated translation. However, the La protein with a mutation of the next tyrosine moiety (Y24Q) could still interact with PV 5'-UTR and stimulate viral IRES-mediated translation significantly. These results underscore the importance of the La N-terminal amino acids in RNA binding and viral RNA translation. The possible role of the LAP sequence in La-RNA binding and stimulation of viral IRES-mediated translation is discussed.     

Generation of replication-defective virus-based vaccines that confer full protection in sheep against virulent bluetongue virus challenge

The reverse genetics technology for bluetongue virus (BTV) has been used in combination with complementing cell lines to recover defective BTV-1 mutants. To generate a potential disabled infectious single cycle (DISC) vaccine strain, we used a reverse genetics system to rescue defective virus strains with large deletions in an essential BTV gene that encodes the VP6 protein (segment S9) of the internal core. Four VP6-deficient BTV-1 mutants were generated by using a complementing cell line that provided the VP6 protein in trans. Characterization of the growth properties of mutant viruses showed that each mutant has the necessary characteristics for a potential vaccine strain: (i) viral protein expression in noncomplementing mammalian cells, (ii) no infectious virus generated in noncomplementing cells, and (iii) efficient replication in the complementing VP6 cell line. Further, a defective BTV-8 strain was made by reassorting the two RNA segments that encode the two outer capsid proteins (VP2 and VP5) of a highly pathogenic BTV-8 with the remaining eight RNA segments of one of the BTV-1 DISC viruses. The protective capabilities of BTV-1 and BTV-8 DISC viruses were assessed in sheep by challenge with specific virulent strains using several assay systems. The data obtained from these studies demonstrated that the DISC viruses are highly protective and could offer a promising alternative to the currently available attenuated and killed virus vaccines and are also compliant as DIVA (differentiating infected from vaccinated animals) vaccines.