Linda K. Dixon

Application of next-generation sequencing technologies in virology

The progress of science is punctuated by the advent of revolutionary technologies that provide new ways and scales to formulate scientific questions and advance knowledge. Following on from electron microscopy, cell culture and PCR, next-generation sequencing is one of these methodologies that is now changing the way that we understand viruses, particularly in the areas of genome sequencing, evolution, ecology, discovery and transcriptomics. Possibilities for these methodologies are only limited by our scientific imagination and, to some extent, by their cost, which has restricted their use to relatively small numbers of samples. Challenges remain, including the storage and analysis of the large amounts of data generated. As the chemistries employed mature, costs will decrease. In addition, improved methods for analysis will become available, opening yet further applications in virology including routine diagnostic work on individuals, and new understanding of the interaction between viral and host transcriptomes. An exciting era of viral exploration has begun, and will set us new challenges to understand the role of newly discovered viral diversity in both disease and health.

African Swine Fever Virus Isolate, Georgia, 2007

The virus isolate introduced to the Caucasus in 2007 is closely related to a group of viruses, genotype II, circulating in Mozambique, Madagascar, and Zambia.

African Swine Fever Virus Protein p54 Interacts with the Microtubular Motor Complex through Direct Binding to Light-Chain Dynein

ABSTRACT Dynein is a minus-end-directed microtubule-associated motor protein involved in cargo transport in the cytoplasm. African swine fever virus (ASFV), a large DNA virus, hijacks the microtubule motor complex cellular transport machinery during virus infection of the cell through direct binding of virus protein p54 to the light chain of cytoplasmic dynein (LC8). Interaction of p54 and LC8 occurs both in vitro and in cells, and the two proteins colocalize at the microtubular organizing center during viral infection. p50/dynamitin, a dominant-negative inhibitor of dynein-dynactin function, impeded ASFV infection, suggesting an essential role for dynein during virus infection. A 13-amino-acid domain of p54 was sufficient for binding to LC8, an SQT motif within this domain being critical for this binding. Direct binding of a viral structural protein to LC8, a small molecule of the dynein motor complex, could constitute a molecular mechanism for microtubule-mediated virus transport.

Comparison of the genome sequences of non-pathogenic and pathogenic African swine fever virus isolates

The genomic coding sequences, apart from the inverted terminal repeats and cross-links, have been determined for two African swine fever virus (ASFV) isolates from the same virus genotype, a non-pathogenic isolate from Portugal, OURT88/3, and a highly pathogenic isolate from West Africa, Benin 97/1. These genome sequences were annotated and compared with that of a tissue culture-adapted isolate, BA71V. The genomes range in length between 170 and 182 kbp and encode between 151 and 157 open reading frames (ORFs). Compared to the Benin 97/1 isolate, the OURT88/3 and BA71V isolates have deletions of 8-10 kbp that encode six copies of the multigene family (MGF) 360 and either one MGF 505/530 copy in the BA71V or two copies in the OURT88/3 isolate. The BA71V isolate has a deletion, close to the right end of the genome, of 3 kbp compared with the other isolates. The five ORFs in this region include an additional copy of an ORF similar to that encoding the p22 virus structural protein. The OURT88/3 isolate has interruptions in ORFs that encode a CD2-like and a C-type lectin protein. Variation between the genomes is observed in the number of copies of five different MGFs. The 109 non-duplicated ORFs conserved in the three genomes encode proteins involved in virus replication, virus assembly and modulation of the hosts defences. These results provide information concerning the genetic variability of African swine fever virus isolates that differ in pathogenicity.

African swine fever virus replication and genomics

African swine fever virus (ASFV) is a large icosahedral DNA virus which replicates predominantly in the cytoplasm of infected cells. The ASFV double-stranded DNA genome varies in length from about 170 to 193 kbp depending on the isolate and contains between 150 and 167 open reading frames. These are closely spaced and read from both DNA strands. The virus genome termini are covalently closed by imperfectly base-paired hairpin loops that are present in two forms that are complimentary and inverted with respect to each other. Adjacent to the termini are inverted arrays of different tandem repeats. Head to head concatemeric genome replication intermediates have been described. A similar mechanism of replication to Poxviruses has been proposed for ASFV. Virus genome transcription occurs independently of the host RNA polymerase II and virus particles contain all of the enzymes and factors required for early gene transcription. DNA replication begins in perinuclear factory areas about 6h post-infection although an earlier stage of nuclear DNA synthesis has been reported. The virus genome encodes enzymes required for transcription and replication of the virus genome and virion structural proteins. Enzymes that are involved in a base excision repair pathway may be an adaptation to enable virus replication in the oxidative environment of the macrophage cytoplasm. Other ASFV genes encode factors involved in evading host defence systems and modulating host cell function. Variation between the genomes of different ASFV isolates is most commonly due to gain or loss of members of multigene families, MGFs 100, 110, 300, 360, 505/530 and family p22. These are located within the left terminal 40kbp and right terminal 20kbp. ASFV is the only member of the Asfarviridae, which is one of the families within the nucleocytoplasmic large DNA virus superfamily.

African Swine Fever Virus Protein A238L Interacts with the Cellular Phosphatase Calcineurin via a Binding Domain Similar to That of NFAT

ABSTRACT The African swine fever virus protein A238L inhibits activation of NFAT transcription factor by binding calcineurin and inhibiting its phosphatase activity. NFAT controls the expression of many immunomodulatory proteins. Here we describe a 14-amino-acid region of A238L that is needed and sufficient for binding to calcineurin. By introducing mutations within this region, we have identified a motif (PxIxITxC/S) required for A238L binding to calcineurin; a similar motif is found in NFAT proteins. Peptides corresponding to this domain of A238L bind calcineurin but do not inhibit its phosphatase activity. Binding of A238L to calcineurin stabilizes the A238L protein in cells. Although A238L-mediated suppression of NF-κB-dependent gene expression occurs by a different mechanism, the A238L-calcineurin interaction may be required to stabilize A238L.

Protection of European domestic pigs from virulent African isolates of African swine fever virus by experimental immunisation

African swine fever (ASF) is an acute haemorrhagic disease of domestic pigs for which there is currently no vaccine. We showed that experimental immunisation of pigs with the non-virulent OURT88/3 genotype I isolate from Portugal followed by the closely related virulent OURT88/1 genotype I isolate could confer protection against challenge with virulent isolates from Africa including the genotype I Benin 97/1 isolate and genotype X Uganda 1965 isolate. This immunisation strategy protected most pigs challenged with either Benin or Uganda from both disease and viraemia. Cross-protection was correlated with the ability of different ASFV isolates to stimulate immune lymphocytes from the OURT88/3 and OURT88/1 immunised pigs.

Genomic analysis of highly virulent Georgia 2007/1 isolate of African swine fever virus.

Sequence information will facilitate research on vaccine development.

Molecular epidemiology of African swine fever virus studied by analysis of four variable genome regions

Summary.Variable regions of the African swine fever virus genome, which contain arrays of tandem repeats, were compared in the genomes of isolates obtained over a 40-year period. Comparison of the size of products generated by polymerase chain reaction (PCR) from four different genome regions, within the B602L and KP86R genes and intergenic regions J286L and BtSj, placed 43 closely related isolated from Europe, the Caribbean, West and Central Africa into 17 different virus sub-groups. Sequence analysis of the most variable fragment, within the B602L gene, from 81 different isolates distinguished 31 sub-groups of virus isolates which varied in sequence and number of a tandem repeat encoding 4 amino acids. Thus, each of these analysis methods enabled isolates, which were previously grouped together by sequencing of a more conserved genome region, to be separated into multiple sub-groups. This provided additional information about strains of viruses circulating in different countries. The methods could be used in future to study the epidemiology and evolution of virus isolates and to trace the sources of disease outbreaks.

Current strategies for subunit and genetic viral veterinary vaccine development

Developing vaccines for livestock provides researchers with the opportunity to perform efficacy testing in the natural hosts. This enables the evaluation of different strategies, including definition of effective antigens or antigen combinations, and improvement in delivery systems for target antigens so that protective immune responses can be modulated or potentiated. An impressive amount of knowledge has been generated in recent years on vaccine strategies and consequently a wide variety of antigen delivery systems is now available for vaccine research. This paper reviews several antigen production and delivery strategies other than those based on the use of live viral vectors. Genetic and protein subunit vaccines as well as alternative production systems are considered in this review.