Major Streptomyces species associated with fissure scab of potato in South Africa including description of Streptomyces solaniscabiei sp. nov.

Streptomyces species are the causal agents of several scab diseases on potato tubers. A new type of scab symptom, caused by Streptomyces species, was observed in South Africa from 2010 onwards. The disease was initially thought to be caused by a single Streptomyces species, however, subsequent isolations from similar symptoms on other potato tubers revealed diversity of the Streptomyces isolates. The objective of this study was to characterise these isolates in order to determine what are the major species involved in the disease. This was done by sequencing and phylogenetic analyses of the 16S rDNA as well as five housekeeping genes, investigation of growth on different culture media, standard phenotypic tests and scanning electron microscopy of culture morphology. The presence of the pathogenicity island (PAI) present in plant pathogenic Streptomyces species was also investigated. The genomes of eight isolates, selected from the three main clades identified, were sequenced and annotated to further clarify species boundaries. Three isolates of each of the three main clades were also inoculated onto susceptible potato cultivars in order to establish the pathogenicity of the species. The results of the phylogenetic and genome analyses revealed that there are three main species involved, namely, Streptomyces werraensis, Streptomyces pseudogriseolus and a novel Streptomyces species that is described here as Streptomyces solaniscabiei sp. nov., with strain FS70T (= PPPPB BD 2226T = LMG 32103T) as the type strain. The glasshouse trial results showed that all three of the Streptomyces species are capable of producing fissure scab symptoms. None of the Streptomyces isolates from fissure scab contained the full PAI and the mechanism of disease initiation still needs to be determined. Genomic comparisons also indicated that S. gancidicus Suzuki 1957 (Approved Lists 1980) is a later heterotypic synonym of S. pseudogriseolus Okami and Umezawa 1955 (Approved Lists 1980).

Complete genome sequence of ornithogalum virus 3.

Ornithogalum thyrsoides, a widely cultivated bulbous ornamental plant endemic to South Africa, has significant commercial value as a pot plant and for the production of cut flowers. However, infection by viruses threatens the success of commercial cultivation, as symptoms negatively affect the appearance of the plant and flowers. To date, four Ornithogalum-infecting viruses have been reported. Complete genome sequence data are available for three of these viruses, but the genome of the potyvirus ornithogalum virus 3 (OV3) has not been fully sequenced. In this study, the complete sequence of OV3 was determined by high-throughput sequencing (HTS) and validated by Sanger sequencing. Based on recognition of protease cleavage patterns and multiple sequence alignments with closely related viruses, the polyprotein of OV3 was predicted to be proteolytically cleaved to produce 10 mature peptides containing domains conserved in members of the genus Potyvirus. Phylogenetic analysis and species demarcation criteria confirm the previous classification of OV3 as a member of a separate species in this genus. This is the first report of a complete genome sequence of OV3.

Complete genome sequence of a novel polerovirus in Ornithogalum thyrsoides from South Africa.

Ornithogalum thyrsoides, commonly known as chincherinchee, is an indigenous ornamental plant widely cultivated in South Africa. It is commercially valued as a flowering pot plant and for the production of cut flowers. Virus infections resulting in the development of severe necrotic mosaic symptoms threaten the success of commercial cultivation. The virome of an O. thyrsoides plant displaying necrotic mosaic symptoms was determined using high-throughput sequencing (HTS). In this plant, ornithogalum mosaic virus and ornithogalum virus 3 were identified, as well as a previously unknown virus. The full genome sequence of this virus was confirmed by Sanger sequencing using overlapping amplicons combined with rapid amplification of cDNA ends (RACE). Based on genome organisation and phylogenetic analysis, this novel virus can be classified as a polerovirus.

Evaluation of different adjuvants for foot-and-mouth disease vaccine containing all the SAT serotypes.

Foot-and-mouth disease (FMD) is an economically important disease of cloven-hoofed animals that is primarily controlled by vaccination of susceptible animals and movement restrictions for animals and animal-derived products in South Africa. Vaccination using aluminium hydroxide gel-saponin (AS) adjuvanted vaccines containing the South African Territories (SAT) serotypes has been shown to be effective both in ensuring that disease does not spread from the endemic to the free zone and in controlling outbreaks in the free zone. Various vaccine formulations containing antigens derived from the SAT serotypes were tested in cattle that were challenged 1 year later. Both the AS and ISA 206B vaccines adjuvanted with saponin protected cattle against virulent virus challenge. The oil-based ISA 206B-adjuvanted vaccine with and without stimulators was evaluated in a field trial and both elicited antibody responses that lasted for 1 year. Furthermore, the ISA 206 adjuvanted FMD vaccine protected groups of cattle against homologous virus challenge at very low payloads, while pigs vaccinated with an emergency ISA 206B-based FMD vaccine containing the SAT 1 vaccine strains were protected against the heterologous SAT 1 outbreak strain.

Foot-and-mouth disease control using vaccination: South African experience.

South Africa has zoned status from the Office International des Epizooties (OIE) with the largest part of the country being foot-and-mouth disease (FMD)-free without vaccination. Outbreaks in this zone are handled differently from outbreaks in the control zones, which do not affect the export status of the country. However, the different socio-economic groupings need to be considered when reaching control decisions and in this regard, the country has been challenged with unique foot-and-mouth disease (FMD) control options. Vaccination has been shown to be effective both in ensuring that disease does not spread from the endemic to the free zone, as well as controlling outbreaks in the free zone. New adjuvants that claim to illicit longer lasting immunity have been tested with antigens derived from the SAT serotypes and animals were challenged one year post vaccination to determine the level of protection. However, even with vaccines that provide immunity for more than a year, an annual vaccination campaign will most probably not be acceptable in the buffer zone where calving occurs throughout the year.

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.

Protein aggregation complicates the development of baculovirus-expressed African horsesickness virus serotype 5 VP2 subunit vaccines.

This paper describes the expression of a cloned African horsesickness virus (AHSV) serotype 5 VP2-gene by a baculovirus recombinant that was generated by the BAC-TO-BAC system. Immunization of horses with crude cell lysates containing recombinant baculovirus-expressed AHSV5 VP2 did induce neutralizing antibodies, but afforded only partial protection against virulent virus challenge. Further analysis of partially protective crude cell lysates revealed that baculovirus-expressed AHSV5 VP2 was predominantly present in the form of insoluble aggregates. Only approximately 10% of VP2 was present in a soluble form. Immunization of guinea-pigs with aggregated and soluble forms of AHSV5 VP2 established that only soluble VP2 was capable of inducing neutralizing antibodies. This finding adds a new dimension to the development of AHSV VP2s as subunit vaccines. Further investigation is needed to limit formation of insoluble aggregates and optimize conditions for producing VP2 in a form capable of inducing protective immunity.

Sequence-independent amplification and cloning of large dsRNA virus genome segments by poly(dA)-oligonucleotide ligation.

A strategy was developed for sequence-independent synthesis and amplification of full-length cDNA of 3-4 kb genes of dsRNA viruses. The method of single primer amplification (Lambden et al., 1992) was adapted by the inclusion of a 3' poly(A) tail to an oligonucleotide ligated to dsRNA genome segments as a template for oligo(dT)-primed cDNA synthesis. Full-length copies of the largest genome segments, 1 (4 kb) and 2 (3 kb), of African horse sickness virus (AHSV) have been cloned, terminally sequenced and expressed in vitro.

Vaccinia virus expression of the VP7 protein of South African bluetongue virus serotype 4 and its use as an antigen in a capture ELISA.

Recombinant vaccinia viruses expressing the VP7 core protein of South African bluetongue virus serotype 4 (SA-BTV4) were identified by polymerase chain reaction amplification. Expression of VP7 was verified by radio-immunoprecipitation and a F(ab')2-based ELISA. Antibodies to VP7 were detected in sera from sheep that had been infected with 20 different virulent BTV serotypes by using the vaccinia virus (VV) expressed VP7 as antigen in a capture ELISA. F(ab')2-immobilised VV-expressed SA-BTV4 VP7 cross-reacted with sera directed against all 9 African horsesickness virus serotypes and epizootic haemorrhagic disease virus serotype 2.

The genetic relatedness of a number of individual cognate genes of viruses in the bluetongue and closely related serogroups.

Genome segments 2, 4, 6, 7, 8, 9, and 10 of bluetongue virus (BTV) serotype 10 were cloned in pBR322. The 2926-bp S2 gene, which codes for the serotype-specific antigen, was cloned as two overlapping 2.4-kb inserts. The relatedness of cognate S2 genes among different isolates of BTV10 was investigated by hybridization, restriction enzyme mapping, and sequencing of the terminal ends. Hybridization under high stringency conditions indicated a genetic diversity between isolates of BTV10 from South Africa and the United States. This was confirmed by a comparison of the restriction map of the cloned S2 gene of a BTV10 isolate from South Africa to that of the S2 gene of the BTV10 strain of the United States which has been cloned and sequenced by Purdy et al. (1985). The part of the genome that was sequenced indicated, however, that this variation was confined to an approximately 10% sequence divergence in the coding region. Very few of the nucleotide substitutions resulted in an amino acid change. The genetic variation of cognate BTV genes within the BTV serogroup as well as among different members of closely related serogroups was also investigated. DNA probes from cloned BTV10 segments were hybridized to dsRNA from 24 different BTV serotypes. Genome segments S2 and S6 were found to be almost equally serotype specific. The stringency of the wash solutions after hybridization can be manipulated to determine an order of relatedness of different cognate genes. This was illustrated by the hybridization of a sensitive RNA probe of S7 to different BTV serotypes as well as to dsRNA from closely related orbiviruses. The results confirmed a relatedness between BTV and members of the epizootic hemorrhagic disease virus (EHDV) serogroups.

A comparison of different cloned bluetongue virus genome segments as probes for the detection of virus-specified RNA.

The seven largest double-stranded (ds) RNA genome segments of bluetongue virus (BTV) serotype 4 as well as genome segment 8 of BTV10 have been cloned into pBR322. The length of the cloned genes indicates that, with the exception of genome segment 1, the entire gene has been cloned in each case. A method is described for isolating different sized cDNA transcripts on alkaline sucrose gradients with very good recovery. The eight cloned genes were compared as 32P-labeled probes for the detection of dsRNA from 21 different BTV serotypes. The S1, S3, S4, S5, and S8 genes were identified as being highly conserved. Of these, S5, which codes for nonstructural protein NS1, gave the best hybridization signal with all the dsRNA isolates. All the probes hybridized significantly weaker with the dsRNA of BTV isolates from Australia and Pakistan than with dsRNA from other serotypes. Genome segment 7, which codes for the group-specific antigen P7, was not highly conserved. Even more variation was shown by genome segment 6 which codes for outer capsid polypeptide P5. S2 which codes for protein P2 is the obvious choice for a serotype-specific probe. Hybridization of this probe with dsRNA from other serotypes reflects the cross-neutralization between BTV4 and these serotypes. The hybridization results can also be used to define the relatedness of BTV4 to other serotypes. None of the probes hybridized with dsRNA from any of the other orbiviruses investigated.

Isolation of a capsid protein of bluetongue virus that induces a protective immune response in sheep.

A method to purify the neutralization specific antigen of bluetongue virus P2 in large amounts has been developed. The purified protein is free from virus-specified or cellular contaminants and its immunological specificity has been preserved. The purification is based on the observation that protein P2 can be dissociated from the virion by treatment with monovalent or divalent salts. The salt concentration required to solubilize the outer capsid proteins is pH dependent and in general decreases with a decrease in pH. P2 purified by extraction from polyacrylamide gels does not induce immune-precipitating or neutralizing antibodies. The response against P5, on the other hand, is much less conformational dependent and P5 purified from gels readily induces P5-precipitating antibodies in rabbits. These antibodies do not neutralize the virus. Purified P2, immunoabsorbed with anticore serum to remove trace amounts of P7, was injected into sheep. An initial dose of 50 micrograms of P2 was sufficient to induce P2-precipitating antibodies as well as neutralizing and hemagglutination-inhibiting antibodies. These sheep were fully protected against challenge with a virulent strain of the same BTV serotype. Lower doses of P2 still provided a significant level of protection even though no neutralizing antibodies could be detected.