João V. Costa

Two-dimensional analysis of african swine fever virus proteins and proteins induced in infected cells

Two-dimensional (2D) analysis of African swine fever (ASF) virus purified by Percoll gradient centrifugation resolves 54 structural proteins, 30 in conventional IEF gels and 24 in NEPHGE gels, while only 26 structural proteins are separated by SDS-PAGE. The two main bands separated by SDS-PAGE, with mol wt 150K and 72K, correspond to single spots in 2D gels. Other bands, including major bands of 38K, 35K, 24K, 17K, and 15.5K mol wt, correspond to multiple proteins of the same molecular weight but different pI. One hundred six virus-specific proteins were resolved by 2D analysis, 59 in conventional IEF gels and 47 in NEPHGE gels. Thirty-five of the virus-specific proteins are early proteins, synthesized before DNA replication, and the remaining 71 proteins are late proteins. Early proteins belong to two groups: 11 transient early proteins are synthesized only early in infection and the other 24 are persistent early proteins, synthesized at both early and late phases. Treatment with cytosine arabinoside prevents the synthesis of late proteins and blocks the shut-off of the synthesis of transient early proteins. Eleven structural proteins are major early proteins and 28 are late proteins. The remaining 15 structural proteins migrate in 2D gels like cellular proteins. Three of these cellular proteins, with mol wt 58K, 56K, and 45K were identified by immunoblotting as alpha-tubulin, beta-tubulin, and actin, respectively.

Lipase from Chromobacterium viscosum: biochemical characterization indicating homology to the lipase from Pseudomonas glumae

Previous purification of a commercial lipolytic preparation from Chromobacterium viscosum using gel filtration chromatography yielded two enzymatically active fractions, named lipases A and B. Characterization of these fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that lipase A consisted of a high molecular weight aggregate of lipase protein with lipopolysaccharides. This complex could be dissociated by treatment with EDTA-Tris buffer containing the non-ionic detergent n-octyl-beta-D-glucopyranoside and subsequent isoelectric focusing in an agarose gel containing the same detergent. Both lipases A and B revealed a major peak corresponding to an isoelectric point of 7.1. SDS-PAGE analysis of lipases A and B after purification by gel filtration or by IEF revealed one major protein band of M(r) of 33 K. Determination of N-terminal amino acid sequences confirmed that both fractions A and B contained the same lipase protein. Furthermore, the N-terminal amino acid sequence of the C. viscosum lipase was identical to the one of Pseudomonas glumae lipase.

IFNAR1 Controls Progression to Cerebral Malaria in Children and CD8+ T Cell Brain Pathology in Plasmodium berghei–Infected Mice

Development of cerebral malaria (CM), a severe and fatal form of clinical Plasmodium falciparum infection, results from a damaging cascade of vascular, inflammatory, and immunological host responses that leads to brain injury. Progression to CM can be modified by host genetic factors. Our case-control study in Angolan children aimed at highlighting the role of IFN (α, β) receptor 1 (IFNAR1) in progression to CM. We report a robust association between IFNAR1 and CM protection, as well as detailed studies showing analogous protection from experimental CM in Ifnar1−/− mice infected with P. berghei ANKA. We developed a novel cell-transfer protocol that enables spleen cell priming in the absence of disease. This led to the discovery that IFNAR1 expression in CD8+ T cells is crucial and can abrogate resistance to experimental CM in Ifnar1−/− mice. Splenic CD8+ T cells from Ifnar1−/− mice are functionally activated upon infection, yet are unable to mediate experimental CM development within the brain tissue. Our findings prove that IFNAR1 signaling unleashes CD8+ T cell effector capacity, which is vital for CM, and raises the hypothesis that the cohesive role of IFNAR1 in both human and mouse CM operates through CD8+ T cell triggering.

Genotyping with Sequenom.

Often in evolutionary genetics research, one needs to analyze polymorphisms in populations for which cost-efficient high-throughput arrays are nonexistent, either because the species is not a model organism or because the populations have been subjected to such specific conditions that their base variation is almost unique. In this situation, custom-made genotyping assays are required. Sequenoms MassARRAY(®) genotyping platform is a powerful and flexible method for assaying up to a few thousand markers and up to thousands of individuals. It is based on distinguishing allele-specific primer extension products by mass spectrometry (MALDI-TOF). Most stages of the experimental protocol reflect adaptations of established PCR protocols to multiplexing, which allows the simultaneous amplification and detection of multiple markers per reaction.

IN VITRO DNA REPLICATION BY CYTOPLASMIC EXTRACTS FROM CELLS INFECTED WITH AFRICAN SWINE FEVER VIRUS

Abstract A cell-free system for the study of transcription of African swine fever virus (ASFV) mRNA was developed from cytoplasmic extracts of infected cells permeabilized with lysolecithin. Extracts prepared from infected cells early and late after infection incorporated [α-32P]UTP into acid-insoluble material that was resistant to DNase and sensitive to RNase. The incorporation was inhibited by actinomycin D but not by a-amanitin. The presence of the nuclei was not required. In vitro transcription was optimal at pH 7.9 and at concentrations of 100 mM NH4Ck, 5 mM magnesium acetate, and 250 μM MnCl2. Early infected cell extracts transcribed from endogenous viral DNA a set of RNAs similar in electrophoretic migration to that observed in intact infected cells. Late infected cell extracts seemed to be unable to transcribe new RNA species besides those transcribed early after infection. The activity of the extracts could be made dependent on exogenous templates by digestion with micrococcal nuclease. RNAs transcribed after addition of native or denatured viral DNA to nuclease-treated extracts were indistinguishable from those transcribed from endogenous viral DNA. Late infected cell extracts digested with micrococcal nuclease were also active in transcribing virus-specific RNA from p2SB21, a recombinant plasmid containing the Sall B fragment of ASFV DNA.

Mycobacteriophage D29 contains an integration system similar to that of the temperate mycobacteriophage L5

A mycobacteriophage D29 DNA fragment cloned in pRM64, a shuttle plasmid that transforms Mycobacterium smegmatis, was sequenced. The determined sequence was 2592 nucleotides long and had a mean G+C content of 63.7 mol%, similar to that of mycobacterial DNA. Four ORFs were identified: one with strong homology to dCMP deaminase genes; one homologous to mycobacteriophage L5 gene 36, whose function is unknown; one encoding a possible excisase; and one encoding an integrase. The intergenic region between the putative excisase gene and the integrase gene had a lower than average G+C content and showed the presence of the same attP core sequence as mycobacteriophage L5. Transformation experiments using subclones of pRM64 indicated that the integrase gene and all the intergenic region were essential for stable transformation. A subclone containing the integrase gene and the core attP sequence was able to transform but recombinants were highly unstable. Southern analysis of total DNA from cells transformed with pRM64 and its derivatives showed that all the plasmids were integrated at one specific site of the bacterial chromosome. A recombinant exhibiting a high level of resistance to the selective drug kanamycin had two plasmids integrated at different sites. These results demonstrated that the D29 sequences contained in pRM64 were integrative, indicating that the generally hold view of D29 as a virulent phage must be reviewed.

A solid-phase enzyme linked immunosorbent assay using monoclonal antibodies, for the detection of african swine fever virus antigens and antibodies

An improved solid-phase enzyme linked immunosorbent assay (ELISA) using monoclonal antibodies was developed to detect an African swine fever virus protein (VP73) in pig samples. The use of monoclonal antibodies against VP73 allowed a sensitive and specific sandwich ELISA. This assay detected a limiting antigen concentration of 0.05 microgram/ml of VP73, lower than the detection limit of 0.6 microgram/ml obtained by using polyclonal antibodies by the same ELISA. The whole virus particle was detected with this method to a limit of 2.3 x 10(2) PFU/ml. At the same time, an indirect ELISA was developed to detect ASFV antibodies. The results also indicate that this method may be a useful technique for epidemiological surveys.

Sequence and Organization of the Left Multigene Family 110 Region of the Vero-adapted L60V Strain of African Swine Fever Virus

Sequencing of the left variable region of the L60V Vero cell-adapted strain of African swine fever virus (ASFV) showed the presence of three genes belonging to multigene family 110 (MGF110) and of a fourth unrelated gene. This gene was separated from the MGF110 genes by a region rich in direct repeats. The first MGF110 gene, V1L, with 104 codons, was only moderately related to the other two, W1L and W2L, with 124 and 80 codons, respectively. These two genes were closely related, W2L being a truncated duplication of W1L. Homology matrix analysis of the sequence against itself showed the existence of a repeated block corresponding to the central conserved domain of the three genes, flanked by two other repeated blocks in W1L and W2L. The comparison of the organization of the left variable region of ASFV L60V with that of field isolates and other adapted viruses revealed that adaptation of unrelated viruses resulted in similar large deletions that map, in their right boundaries, exactly at the same positions in the intergenic repeat-rich region.

NOS2 Variants Reveal a Dual Genetic Control of Nitric Oxide Levels, Susceptibility to Plasmodium Infection, and Cerebral Malaria

ABSTRACT Nitric oxide (NO) is a proposed component of malaria pathogenesis, and the inducible nitric oxide synthase gene (NOS2) has been associated to malaria susceptibility. We analyzed the role of NOS2 polymorphisms on NO bioavailability and on susceptibility to infection, Plasmodium carrier status and clinical malaria. Two distinct West African sample collections were studied: a population-based collection of 1,168 apparently healthy individuals from the Príncipe Island and a hospital-based cohort of 269 Angolan children. We found that two NOS2 promoter single-nucleotide polymorphism (SNP) alleles associated to low NO plasma levels in noninfected individuals were also associated to reduced risk of pre-erythrocytic infection as measured anti-CSP antibody levels (6.25E–04 < P < 7.57E–04). In contrast, three SNP alleles within the NOS2 cistronic region conferring increased NO plasma levels in asymptomatic carriers were strongly associated to risk of parasite carriage (8.00E–05 < P < 7.90E–04). Notwithstanding, three SNP alleles in this region protected from cerebral malaria (7.90E–4 < P < 4.33E–02). Cohesively, the results revealed a dual regimen in the genetic control of NO bioavailability afforded by NOS2 depending on the infection status. NOS2 promoter variants operate in noninfected individuals to decrease both NO bioavailability and susceptibility to pre-erythrocytic infection. Conversely, NOS2 cistronic variants (namely, rs6505469) operate in infected individuals to increase NO bioavailability and confer increased susceptibility to unapparent infection but protect from cerebral malaria. These findings corroborate the hypothesis that NO anti-inflammatory properties impact on different steps of malaria pathogenesis, explicitly by favoring infection susceptibility and deterring severe malaria syndromes.

SNP typing reveals similarity in Mycobacterium tuberculosis genetic diversity between Portugal and Northeast Brazil

Human tuberculosis is an infectious disease caused by bacteria from the Mycobacterium tuberculosis complex (MTBC). Although spoligotyping and MIRU-VNTR are standard methodologies in MTBC genetic epidemiology, recent studies suggest that Single Nucleotide Polymorphisms (SNP) are advantageous in phylogenetics and strain group/lineages identification. In this work we use a set of 79 SNPs to characterize 1987 MTBC isolates from Portugal and 141 from Northeast Brazil. All Brazilian samples were further characterized using spolygotyping. Phylogenetic analysis against a reference set revealed that about 95% of the isolates in both populations are singly attributed to bacterial lineage 4. Within this lineage, the most frequent strain groups in both Portugal and Brazil are LAM, followed by Haarlem and X. Contrary to these groups, strain group T showed a very different prevalence between Portugal (10%) and Brazil (1.5%). Spoligotype identification shows about 10% of mis-matches compared to the use of SNPs and a little more than 1% of strains unidentifiability. The mis-matches are observed in the most represented groups of our sample set (i.e., LAM and Haarlem) in almost the same proportion. Besides being more accurate in identifying strain groups/lineages, SNP-typing can also provide phylogenetic relationships between strain groups/lineages and, thus, indicate cases showing phylogenetic incongruence. Overall, the use of SNP-typing revealed striking similarities between MTBC populations from Portugal and Brazil.