Design of clone-specific probes from genome sequences for rapid PCR-typing of outbreak pathogens.

The genome sequence of one OXA-48-producing Klebsiella pneumoniae belonging to sequence type (ST) 405, and three belonging to ST11, were used to design and test ST-specific PCR assays for typing OXA-48-producing K. pneumoniae. The approach proved to be useful for in-house development of rapid PCR typing assays for local outbreak surveillance.

A single-residue mutation, G203E, causes 3-hydroxy-3-methylglutaric aciduria by occluding the substrate channel in the 3D structural model of HMG-CoA lyase.

3-Hydroxy-3-methylglutaric aciduria is a rare autosomal recessive genetic disorder that affects ketogenesis and leucine metabolism. The disease is caused by mutations in the gene coding for 3-hydroxy-3-methylglutaryl-coenzyme A lyase (HL). To date 26 different mutations have been described. A (betaalpha)(8) TIM barrel structure has been proposed for the protein, and almost all missense mutations identified so far localize in the beta sheets that define the inside cavity. We report an Italian patient who bears homozygously a novel HL mutation, c.608G > A (p. G203E) in beta sheet six. A structural model of the mutated protein suggests that glutamic acid 203 impedes catalysis by blocking the entrance to the inner cavity of the enzyme. Loss of functionality has been confirmed in expression studies in E. coli, which demonstrate that the G203E mutation completely abolishes enzyme activity. Beta sheet six and beta sheet two are the two protein regions that accumulate most missense mutations, indicating their importance in enzyme functionality. A model for the mechanism of enzyme function is proposed.

Expression of porcine CD163 on monocytes/macrophages correlates with permissiveness to African swine fever infection.

Monocytes-macrophages, the target cells of African swine fever virus (ASFV) are highly heterogeneous in phenotype and function. In this study, we have investigated the correlation between the phenotype of specific populations of porcine macrophages and their permissiveness to ASFV infection. Bone marrow cells and fresh blood monocytes were less susceptible to in vitro infection by ASFV than more mature cells, such as alveolar macrophages. FACS analyses of monocytes using a panel of mAbs specific for porcine monocyte/macrophages showed that infected cells had a more mature phenotype, expressing higher levels of several macrophage specific markers and SLA II antigens. Maturation of monocytes led to an increase in the percentage of infected cells, which correlated with an enhanced expression of CD163. Separation of CD163+ and CD163- monocytes demonstrated the specific sensitivity of the CD163+ subset to ASFV infection. In vivo experiments also showed a close correlation between CD163 expression and virus infection. Finally, mAb 2A10 and, in a lower extent, mAb 4E9 were able to inhibit, in a dose-dependent manner, both ASFV infection and viral particle binding to alveolar macrophages. Altogether, these results strongly suggest a role of CD163 in the process of infection of porcine monocytes/macrophages by ASFV.

A comparative sequence analysis to revise the current taxonomy of the family Coronaviridae.

The Coronaviridae family, comprising the Coronavirus and Torovirus genera, is part of the Nidovirales order that also includes two other families, Arteriviridae and Roniviridae. Based on genetic and serological relationships, groups 1, 2 and 3 were previously recognized in the Coronavirus genus. In this report we present results of comparative sequence analysis of the spike (S), envelope (E), membrane (M), and nucleoprotein (N) structural proteins, and the two most conserved replicase domains, putative RNA-dependent RNA polymerase (RdRp) and RNA helicase (HEL), aimed at a revision of the Coronaviridae taxonomy. The results of pairwise comparisons involving structural and replicase proteins of the Coronavirus genus were consistent and produced percentages of sequence identities that were distributed in discontinuous clusters. Inter-group pairwise scores formed a single cluster in the lowest percentile. No homologs of the N and E proteins have been found outside coronaviruses, and the only (very) distant homologs of S and M proteins were identified in toroviruses. Intragroup sequence conservation was higher, although for some pairs, especially those from the most diverse group 1, scores were close or even overlapped with those from the intergroup comparisons. Phylogenetic analysis of six proteins using a neighbor-joining algorithm confirmed three coronavirus groups. Comparative sequence analysis of RdRp and HEL domains were extended to include arterivirus and ronivirus homologs. The pairwise scores between sequences of the genera Coronavirus and Torovirus (22-25% and 21-25%) were found to be very close to or overlapped with the value ranges (12 to 22% and 17 to 25%) obtained for interfamily pairwise comparisons, but were much smaller than values derived from pairwise comparisons within the Coronavirus genus (63-71% and 59-67%). Phylogenetic analysis confirmed toroviruses and coronaviruses to be separated by a large distance that is comparable to those between established nidovirus families. Based on comparison of these scores with those derived from analysis of separate ranks of several multi-genera virus families, like the Picornaviridae, a revision of the Coronaviridae taxonomy is proposed. We suggest the Coronavirus and Torovirus genera to be re-defined as two subfamilies within the Coronavirdae or two families within Nidovirales, and the current three informal coronavirus groups to be converted into three genera within the Coronaviridae.

Antigenic and immunogenic properties of a chimera of two immunodominant African swine fever virus proteins.

A chimera of the two immunodominant African swine fever (ASF) virus proteins p54 and p30 was constructed by insertion of the gene CP204L into a Not I restriction site of E183L gene. The resulting chimeric protein p54/30, expressed by a recombinant baculovirus in insect cells and in Trichoplusia ni larvae, retained antigenic determinants present in both proteins and reacted in Western blot with a collection of sera from inapparent ASF virus carrier pigs. Remarkably, pigs immunized with the chimeric protein developed neutralizing antibodies and survived the challenge with a virulent African swine fever virus, presenting a reduction of about two logs in maximum viremia titers with respect to control pigs. In conclusion, this study revealed that the constructed chimeric protein may have utility as a serological diagnostic reagent and for further immunological studies that may provide new insights on mechanisms of protective immunity to ASFV.

Analysis of the interaction between the eukaryotic chaperonin CCT and its substrates actin and tubulin.

Two mechanisms have thus far been characterized for the assistance by chaperonins of the folding of other proteins. The first and best described is that of the prokaryotic chaperonin GroEL, which interacts with a large spectrum of proteins. GroEL uses a nonspecific mechanism by which any conformation of practically any unfolded polypeptide interacts with it through exposed, hydrophobic residues. ATP binding liberates the substrate in the GroEL cavity where it is given a chance to fold. A second mechanism has been described for the eukaryotic chaperonin CCT, which interacts mainly with the cytoskeletal proteins actin and tubulin. Cryoelectron microscopy and biochemical studies have revealed that both of these proteins interact with CCT in quasi-native, defined conformations. Here we have performed a detailed study of the docking of the actin and tubulin molecules extracted from their corresponding CCT:substrate complexes obtained from cryoelectron microscopy and image processing to localize certain regions in actin and tubulin that are involved in the interaction with CCT. These regions of actin and tubulin, which are not present in their prokaryotic counterparts FtsA and FtsZ, are involved in the polymerization of the two cytoskeletal proteins. These findings suggest coevolution of CCT with actin and tubulin in order to counteract the folding problems associated with the generation in these two cytoskeletal protein families of new domains involved in their polymerization.

Structural model of the catalytic core of carnitine palmitoyltransferase I and carnitine octanoyltransferase (COT): mutation of CPT I histidine 473 and alanine 381 and COT alanine 238 impairs the catalytic activity.

Carnitine palmitoyltransferase I (CPT I) and carnitine octanoyltransferase (COT) catalyze the conversion of long- and medium-chain acyl-CoA to acylcarnitines in the presence of carnitine. We propose a common three-dimensional structural model for the catalytic domain of both, based on fold identification for 200 amino acids surrounding the active site through a threading approach. The model is based on the three-dimensional structure of the rat enoyl-CoA hydratase, established by x-ray diffraction analysis. The study shows that the structural model of 200 amino acids of the catalytic site is practically identical in CPT I and COT with identical distribution of 4 beta-sheets and 6 alpha-helices. Functional analysis of the model was done by site-directed mutagenesis. When the critical histidine residue 473 in CPT I (327 in COT), localized in the acyl-CoA pocket in the model, was mutated to alanine, the catalytic activity was abolished. Mutation of the conserved alanine residue to aspartic acid, A381D (in CPT I) and A238D (in COT), which are 92/89 amino acids far from the catalytic histidine, respectively (but very close to the acyl-CoA pocket in the structural model), decreased the activity by 86 and 80%, respectively. The K(m) for acyl-CoA increased 6-8-fold, whereas the K(m) for carnitine hardly changed. The inhibition of the mutant CPT I by malonyl-CoA was not altered. The structural model explains the loss of activity reported for the CPT I mutations R451A, W452A, D454G, W391A, del R395, P479L, and L484P, all of which occur in or near the modeled catalytic domain.

Escherichia coli FtsZ polymers contain mostly GTP and have a high nucleotide turnover.

The cell division protein FtsZ is a GTPase structurally related to tubulin and, like tubulin, it assembles in vitro into filaments, sheets and other structures. To study the roles that GTP binding and hydrolysis play in the dynamics of FtsZ polymerization, the nucleotide contents of FtsZ were measured under different polymerizing conditions using a nitrocellulose filter-binding assay, whereas polymerization of the protein was followed in parallel by light scattering. Unpolymerized FtsZ bound 1 mol of GTP mol(-1) protein monomer. At pH 7.5 and in the presence of Mg(2+) and K(+), there was a strong GTPase activity; most of the bound nucleotide was GTP during the first few minutes but, later, the amount of GTP decreased in parallel with depolymerization, whereas the total nucleotide contents remained invariant. These results show that the long FtsZ polymers formed in solution contain mostly GTP. Incorporation of nucleotides into the protein was very fast either when the label was introduced at the onset of the reaction or subsequently during polymerization. Molecular modelling of an FtsZ dimer showed the presence of a cleft between the two subunits maintaining the nucleotide binding site open to the medium. These results show that the FtsZ polymers are highly dynamic structures that quickly exchange the bound nucleotide, and this exchange can occur in all the subunits.

Influenza A virus NEP (NS2 protein) downregulates RNA synthesis of model template RNAs.

The influenza A virus NEP (NS2) protein is an structural component of the viral particle. To investigate whether this protein has an effect on viral RNA synthesis, we examined the expression of an influenza A virus-like chloramphenicol acetyltransferase (CAT) RNA in cells synthesizing the four influenza A virus core proteins (nucleoprotein, PB1, PB2, and PA) and NEP from recombinant plasmids. Influenza A virus NEP inhibited drastically, and in a dose-dependent manner, the level of CAT expression mediated by the recombinant influenza A virus polymerase. This inhibitory effect was not observed in an analogous artificial system in which expression of a synthetic CAT RNA is mediated by the core proteins of an influenza B virus. This result ruled out the possibility that inhibition of reporter gene expression was due to a general toxic effect induced by NEP. Analysis of the virus-specific RNA species that accumulated in cells expressing the type A recombinant core proteins and NEP showed that there was an important reduction in the levels of minireplicon-derived vRNA, cRNA, and mRNA molecules. Taken together, the results obtained suggest a regulatory role for NEP during virus-specific RNA synthesis, and this finding is discussed regarding the biological implications for the virus life cycle.

Influenza virus matrix protein is the major driving force in virus budding.

To get insights into the role played by each of the influenza A virus polypeptides in morphogenesis and virus particle assembly, the generation of virus-like particles (VLPs) has been examined in COS-1 cell cultures expressing, from recombinant plasmids, different combinations of the viral structural proteins. The presence of VLPs was examined biochemically, following centrifugation of the supernatants collected from transfected cells through sucrose cushions and immunoblotting, and by electron-microscopic analysis. It is demonstrated that the matrix (M1) protein is the only viral component which is essential for VLP formation and that the viral ribonucleoproteins are not required for virus particle formation. It is also shown that the M1 protein, when expressed alone, assembles into virus-like budding particles, which are released in the culture medium, and that the recombinant M1 protein accumulates intracellularly, forming tubular structures. All these results are discussed with regard to the roles played by the virus polypeptides during virus assembly.

Rescue of synthetic RNAs into thogoto and influenza A virus particles using core proteins purified from Thogoto virus.

The ribonucleoprotein (RNP) complexes of Thogoto virus (THOV), a tick-borne orthomyxovirus, have been purified from detergent-lysed virions. The purified RNPs were then disrupted by centrifugation through a CsCl-glycerol gradient to obtain fractions highly enriched in nucleoprotein (NP) and virtually devoid of viral genomic RNA. When these NP-enriched fractions were incubated with a synthetic THOV-like RNA, and the mixtures were transfected into THOV-infected cells, the synthetic RNA was expressed and packaged into THOV particles. Similarly, hybrid mixtures containing purified THOV NP and influenza A virus synthetic RNAs (either a model CAT RNA or a gene encoding the viral neuraminidase), were prepared and transfected into influenza A virus-infected cells. The synthetic CAT RNA, was shown to be expressed and packaged into virus particles, and the neuraminidase gene was rescued into influenza virions. These data are discussed in terms of the similarities observed between THOV and influenza A virus and the potential application of the THOV purified proteins for rescuing synthetic genes into infectious viruses.

Several protein regions contribute to determine the nuclear and cytoplasmic localization of the influenza A virus nucleoprotein.

A systematic analysis was carried out to identify the amino acid signals that regulate the nucleo-cytoplasmic transport of the influenza A virus nucleoprotein (NP). The analysis involved determining the intracellular localization of eight deleted recombinant NP proteins and 14 chimeric proteins containing the green fluorescent protein fused to different NP fragments. In addition, the subcellular distribution of NP derivatives that contained specific substitutions at serine-3, which is the major phosphorylation site of the A/Victoria/3/75 NP, were analysed. From the results obtained, it is concluded that the NP contains three signals involved in nuclear accumulation and two regions that cause cytoplasmic accumulation of the fusion proteins. One of the karyophilic signals was located at the N terminus of the protein, and the data obtained suggest that the functionality of this signal can be modified by phosphorylation at serine-3. These findings are discussed in the context of the transport of influenza virus ribonucleoprotein complexes into and out of the nucleus.

In vitro inhibition of the replication of haemorrhagic septicaemia virus (VHSV) and African swine fever virus (ASFV) by extracts from marine microalgae.

We have screened for in vitro inhibition of viral replication with extracts from the following marine microalgae: Porphyridium cruentum, Phaeodactylum tricornutum, Tetraselmis suecica, Chlorella autotrophica, Dunaliella tertiolecta, Dunaliella bardawil, Isochrysis galbana, Isochrysis galbana var Tiso, Ellipsoidon sp. and Tetraselmis tetrathele. We have used as viral models two enveloped viruses of significant economic importance, the viral hemorrhagic septicemia virus (VHSV) of salmonid fish and the African swine fever virus (ASFV). The aqueous extracts from P. cruentum, C. autotrophica and Ellipsoidon sp., produced a significant inhibition of the in vitro replication of both viruses in a dose-dependent manner. That this inhibition could be due to sulfated polysaccharides was suggested because the same pattern of viral inhibition was obtained by using exocellular extracts from microalgae enriched in these compounds and/or dextran sulfate of high molecular weight. However, the inhibition of viral replication did not correlate with the percentage of sulfatation of the exocellular polysaccharides. Extracts from marine microalgae may have prophylactic utility against fish and mammalian viral diseases.

The African swine fever virus proteins p54 and p30 are involved in two distinct steps of virus attachment and both contribute to the antibody-mediated protective immune response.

The nature of the initial interactions of African swine fever (ASF) virus with target cells is only partially known, and to date only the ASF virus protein p12 has been identified as a viral attachment protein. More recently, antibodies to viral proteins p54 and p30 have been shown to neutralize the virus, inhibiting virus binding and internalization, respectively. Therefore, we investigated the role of these proteins in the receptor-mediated ASF virus endocytosis in swine macrophages, the natural host cells. Proteins p54 and p30, released from ASF virus particles after treatment of virions with a nonionic detergent, bound to virus-sensitive alveolar pig macrophages. Binding of these proteins was found to be specifically inhibited by neutralizing antibodies obtained from a convalescent pig or from pigs immunized with recombinant p54 or p30 proteins. The baculovirus-expressed proteins p54 and p30 retained the same biological properties as the viral proteins, since they also bound specifically to these cells, and their binding was equally inhibited by neutralizing antibodies. Binding of 35S-labeled recombinant p54 and p30 proteins to macrophages was specifically competed by an excess of unlabeled p54 and p30, respectively. However, cross-binding inhibition was not observed, suggesting the existence of two different saturable binding sites for these proteins in the susceptible cells. In addition, protein p54 blocked the specific binding of virus particles to the macrophage, while protein p30 blocked virus internalization. Both proteins independently prevented virus infection and in a dose-dependent manner, suggesting that binding interactions mediated by both proteins are necessary to give rise to a productive infection. The relevance of blockade of virus-cell interactions mediated by p54 and p30 in the protective immune response against ASF virus was then investigated. Immunization of pigs with either recombinant p54 or p30 proteins induced neutralizing antibodies which, as expected, inhibited virus attachment or internalization, respectively. However, immunized pigs were not protected against lethal infection and the disease course was not modified in these animals. In contrast, immunization with a combination of p54 and p30 proteins simultaneously stimulated both virus neutralizing mechanisms and modified drastically the disease course, rendering a variable degree of protection ranging from a delay in the onset of the disease to complete protection against virus infection. In conclusion, the above results strongly suggest that proteins p54 and p30 mediate specific interactions between ASF virus and cellular receptors and that simultaneous interference with these two interactions has a complementary effect in antibody-mediated protection.

Blocking antibodies inhibit complete African swine fever virus neutralization.

A persistent non-neutralized African swine fever virus (ASFV) fraction is found with most convalescent swine sera in in vitro neutralization assays. To study this phenomenon, antisera from convalescent pigs infected with different virus isolates and showing complete or incomplete virus neutralization were used. Different experiments determined that incomplete neutralization of ASFV is caused neither by virus aggregation, nor low affinity or stability of virus-antibody complexes. Additionally, attempts to purify antigenic escape mutant viruses from the persistent fraction was also unsuccessful. Nevertheless, competition experiments between sera demonstrated that antibodies present in sera showing persistent fraction inhibited the complete neutralization mediated by antibodies present in sera which neutralize 100% of virus infectivity. These results suggest that induction of blocking antibodies during ASFV infection could represent the main cause for the persistent surviving virus fraction observed in neutralization assays and could also explain the persistent infections observed in some convalescent pigs.

High level expression of the major antigenic African swine fever virus proteins p54 and p30 in baculovirus and their potential use as diagnostic reagents.

At present, the eradication of African swine fever (ASF) in affected countries is based only on an efficient diagnosis program because of the absence of an available vaccine. The highly antigenic ASF virus proteins p54 and p30, encoded by genes E183L and CP204L respectively, were expressed in baculovirus for diagnostic purposes. A sequence comparison analysis of these genes from different field virus strains which are geographically diverse and isolated in different years, revealed that both genes are completely conserved among the isolates. Partially purified baculovirus-expressed proteins were used in ELISA and Western blot for ASF antibody detection in sera from experimentally inoculated pigs and field sera from ASF innaparent carriers. These comparative analyses showed that p54 presents better reactivity than p30 in Western blot. However, recombinant p30 was more efficient for antibody detection by ELISA, improving the discrimination between positive and negative sera by this technique. These data suggest the convenience of using p30 as ELISA antigen, while p54 should be the selected antigen for ASF virus antibody detection by Western blot. The combined use of both antigens for serodiagnosis of ASF disease will improve the sensitivity of innaparent carriers detection, facilitating also the interpretation of the tests, and eliminating the use of ASF virus in antigen production.

Neutralization susceptibility of African swine fever virus is dependent on the phospholipid composition of viral particles.

In this study we have investigated the generation of African swine fever (ASF) virus variants resistant to neutralizing antibodies after cell culture propagation. All highly passaged ASF viruses analyzed were resistant to neutralization by antisera from convalescent pigs or antibodies generated against individual viral proteins which neutralized low-passage viruses. A molecular analysis of neutralizable and nonneutralizable virus isolates by sequencing of the genes encoding for neutralizing proteins revealed that the absence of neutralization of high-passage viruses is not due to antigenic variability of critical epitopes. A comparative analysis of phospholipid composition of viral membranes between low- and high-passage viruses revealed differences in the relative amount of phosphatidylinositol in these two groups of viruses, independent of the cells in which the viruses were grown. Further purification of low- and high-passage viruses by Percoll sedimentation showed differences in the phospholipid composition identical to those found with the partially purified viruses and confirmed the susceptibility of these viruses to neutralization. The incorporation of phosphatidylinositol into membranes of high-passage viruses rendered a similar neutralization susceptibility to low-passage viruses, in which this is a major phospholipid. In contrast, other phospholipids did not interfere with high-passage virus neutralization, suggesting that phosphatidylinositol is essential for a correct epitope presentation to neutralizing antibodies. Additionally, the removal of phosphatidylinositol form a low-passage virus by a specific lipase transformed this virus from neutralizable to nonneutralizable. These data constitute clear evidence of the importance of the lipid composition of the viral membranes for the protein recognition by antibodies and may account in part for the past difficulties in reproducibly demonstrating ASF virus-neutralizing antibodies by using high-passage viruses.

Neutralizing antibodies to different proteins of African swine fever virus inhibit both virus attachment and internalization.

African swine fever virus induces in convalescent pigs antibodies that neutralized the virus before and after binding to susceptible cells, inhibiting both virus attachment and internalization. A further analysis of the neutralization mechanisms mediated by the different viral proteins showed that antibodies to proteins p72 and p54 are involved in the inhibition of a first step of the replication cycle related to virus attachment, while antibodies to protein p30 are implicated in the inhibition of virus internalization.

The structural protein p54 is essential for African swine fever virus viability.

Protein p54, one of the most antigenic structural African swine fever virus (ASFV) proteins, has been localized by immuno-electron microscopy in the replication factories of infected cells, mainly associated with membranes and immature virus particles. Attempts to inactivate the p54 gene from ASFV by targeted insertion of beta-galactosidase selection marker was uniformly unsuccessful, suggesting that this gene is essential for virus viability. To demonstrate that, we inserted in the TK (thymidine kinase) locus of the virus a construction containing a second copy of the p54 gene and beta-glucuronidase selection marker under the control of p54 and p73 promoters, respectively. Virus mutant clones expressing a second copy of p54 and beta-glucuronidase were used to achieve deletion mutants of the original copy of the gene. Virus mutants expressing only the second inserted copy of p54 and the two selection markers mentioned above were successfully obtained. Therefore, we have demonstrated that the p54 gene product plays an essential role in virus growth, characterizing for the first time in ASFV an essential virus gene.