Raquel Portugal

Development of a nested PCR and its internal control for the detection of African swine fever virus (ASFV) in Ornithodoros erraticus

Summary.A nested PCR assay, with an internal control, was developed to detect African swine fever virus (ASFV) DNA in Ornithodoros erraticus. The assay revealed a better analytical sensitivity than virus isolation and the OIE PCR protocol. All ticks collected from the field, which were positive by virus isolation, were also positive by PCR. Viral DNA was detected in a further 19 out of 60 ticks from which no virus was isolated. Our results show that this assay is reliable and can easily be used to screen large tick populations collected in the field for the presence of ASFV.

Related strains of African swine fever virus with different virulence: genome comparison and analysis

Two strains of African swine fever virus (ASFV), the high-virulence Lisboa60 (L60) and the low-virulence NH/P68 (NHV), which have previously been used in effective immunization/protection studies, were sequenced. Both were isolated in Portugal during the 11-year period after the introduction of ASFV to the European Continent in 1957. The predicted proteins coded by both strains were compared, and where differences were found these were also compared to other strains of known virulence. This highlighted several genes with significant alterations in low-virulence strains of ASFV that may constitute virulence factors, several of which are still uncharacterized regarding their function. Phylogenetic analysis grouped L60 and NHV closest to other P72 genotype I ASFV strains from Europe and West Africa, consistent with the assumed West African origin of all European strains. Interestingly, a relatively lower genomic identity exists between L60 and NHV, both isolated in a similar geographical location 8 years apart, than with other European and west African strains isolated subsequently and in more distant locations. This may reflect the intensive passage in tissue culture, during the early 1960s, of a Portuguese isolate to obtain an attenuated vaccine, which may have led to NHV. This study contributes to a better understanding of the evolution of ASFV, and defines additional potential virulence genes for future studies of pathogenesis towards the development of effective vaccines.

A novel bromodeoxyuridine-resistant wild boar lung cell line facilitates generation of African swine fever virus recombinants

Manipulation of African swine fever virus (ASFV) genomes, in particular those from field strains, is still a challenge. We have shown recently that generation of a green-fluorescent-protein-expressing, thymidine-kinase-negative (TK−) mutant of the low-pathogenic African swine fever virus field strain NHV was supported by a TK− Vero cell line. Since NHV, like other ASFV field strains, does not replicate well in Vero cells, a bromodeoxyuridine (BrdU)- resistant cell line derived from wild boar lung (WSL) cells, named WSL-Bu, was selected. WSL cells were used because they are suitable for productive replication of NHV and other ASFV field strains. Here, we show that WSL-Bu cells enable positive selection of both TK− and TK+ ASFV recombinants, which allows for novel strategies for construction of ASFV mutants. We further demonstrate for a low-pathogenic ASFV strain that TK expression is required for infectious replication in macrophages infected at low multiplicity and that vaccinia TK fully complements ASFV TK in this respect.

Characterization of African swine fever virus IAP homologue expression in porcine macrophages infected with different virulence isolates.

Genes modulating apoptosis are encoded by many viruses and have an important role in viral evasion mechanisms. Our objective was to characterize the expression of the IAP homologue gene of African swine fever virus (ASFV), 4-CL, during in vitro infection of porcine macrophages, the preferential target cell for viral replication. Expression was compared along parallel infections by two naturally occurring ASFV isolates of different virulence: highly virulent ASFV/L60 (L60) and low virulent non-hemadsorbing ASFV/NH/P68 (NHV). Efficiency of macrophage infection by both isolates was similar, as observed both by the percentage of infected cells in culture and by virus progeny production. Our results showed that transcription of 4-CL initiates very early after infection with both isolates, since specific mRNAs were observed and quantified at 1.5h post-infection (p.i.). However, the protein was produced later, from 4 to 8h p.i., around the same time when viral DNA replication is reported to occur. 4-CL protein was more abundant in L60 than NHV infected cells, at both 8 and 16h p.i. The mRNA levels, however, did not correlate with those of protein expression. Overall our results suggest the existence of a post-transcriptional step in the regulation of 4-CL gene expression.